U.S. patent application number 09/896095 was filed with the patent office on 2003-11-27 for directed evolution of novel binding proteins.
Invention is credited to Guterman, Sonia Kosow, Kent, Rachel Baribault, Ladner, Robert Charles, Ley, Arthur Charles, Markland, William, Roberts, Bruce Lindsay.
Application Number | 20030219886 09/896095 |
Document ID | / |
Family ID | 27399321 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030219886 |
Kind Code |
A1 |
Ladner, Robert Charles ; et
al. |
November 27, 2003 |
Directed evolution of novel binding proteins
Abstract
In order to obtain a novel binding protein against a chosen
target, DNA molecules, each encoding a protein comprising one of a
family of similar potential binding domains and a structural signal
calling for the display of the protein on the outer surface of a
chosen bacterial cell, bacterial spore or phage (genetic package)
are introduced into a genetic package. The protein is expressed and
the potential binding domain is displayed on the outer surface of
the package. The cells or viruses bearing the binding domains which
recognize the target molecule are isolated and amplified. The
successful binding domains are then characterized. One or more of
these successful binding domains is used as a model for the design
of a new family of potential binding domains, and the process is
repeated until a novel binding domain having a desired affinity for
the target molecule is obtained. In one embodiment, the first
family of potential binding domains is related to bovine pancreatic
trypsin inhibitor, the genetic package is M13 phage, and the
protein includes the outer surface transport signal of the M13 gene
III protein.
Inventors: |
Ladner, Robert Charles;
(Ijamsville, MD) ; Guterman, Sonia Kosow;
(Belmont, MA) ; Roberts, Bruce Lindsay; (Milford,
MA) ; Markland, William; (Milford, MA) ; Ley,
Arthur Charles; (Newton, MA) ; Kent, Rachel
Baribault; (Boxborough, MA) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
624 Ninth Street, N.W.
Washington
DC
20001
US
|
Family ID: |
27399321 |
Appl. No.: |
09/896095 |
Filed: |
June 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09896095 |
Jun 29, 2001 |
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08993776 |
Dec 18, 1997 |
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08993776 |
Dec 18, 1997 |
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08415922 |
Apr 3, 1995 |
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5837500 |
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08415922 |
Apr 3, 1995 |
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08009319 |
Jan 26, 1993 |
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5403484 |
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08009319 |
Jan 26, 1993 |
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07664989 |
Mar 1, 1991 |
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5223409 |
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07664989 |
Mar 1, 1991 |
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07487063 |
Mar 2, 1990 |
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07487063 |
Mar 2, 1990 |
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07240160 |
Sep 2, 1988 |
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Current U.S.
Class: |
435/184 ;
435/7.1 |
Current CPC
Class: |
C07K 14/805 20130101;
C07K 14/8117 20130101; C12N 15/1037 20130101; C12N 7/00 20130101;
C07K 14/43522 20130101; C12N 2795/14143 20130101; C07K 14/8114
20130101; C07K 1/047 20130101; C07K 1/107 20130101; A61K 38/00
20130101; C12N 2795/14122 20130101; C40B 40/02 20130101; C12N
2795/14111 20130101 |
Class at
Publication: |
435/184 ;
435/7.1 |
International
Class: |
C12N 009/99 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 1, 1989 |
US |
PCT/US89/03731 |
Claims
1. A non-naturally occurring or purified protein which inhibits
human neutrophil elastase, and which is a protein comprising a
mutant Kunitz domain, said domain being characterized by Cys at
positions corresponding to bovine pancreatic trypsin inhibitor
(BPTI) positions 5, 30, 51 and 55, Gly at a position corresponding
to BPTI position 12, and Phe at a position corresponding to BPTI
33; where, in said mutant Kunitz domain, the residue corresponding
to BPTI position 18 is Phe, and where the residues corresponding to
BPTI positions 39-42 are not charged residues.
2. The protein of claim 1 in which the residues corresponding to
BPTI positions 14 and 38 are Cys, and the residue corresponding to
BPTI position 37 is Gly.
3. The protein of claim 2 in which the residue corresponding to
BPTI position 45 is Phe and the residue corresponding to BPTI
position 43 is Asn.
4. The protein of claim 1 where said mutant domain is at least 30%
identical in amino acid sequence to BPTI.
5. The protein of claim 3 where said mutant domain is at least 30%
identical in amino acid sequence to BPTI.
6. The protein of claim 1 where said mutant domain is at least 30%
identical in amino acid sequence to ITI-D1.
7. The protein of claim 3 where said mutant domain is at least 30%
identical in amino acid sequence to ITI-D1.
8. The protein of claim 2 where the residue corresponds to BPTI
position 15 is Val, Ile or Leu.
9. The protein of claim 2 where the residue corresponding to BPTI
position 15 is Val.
10. The protein of claim 2 where the residue corresponding to BPTI
position 15 is Ile.
11. The protein of claim 2 where, the residue corresponding to BPTI
position 15 is Leu.
12. The protein of claim 1 in which the residue corresponding to
BPTI position 15 is Val and the residue corresponding to BPTI
position 17 is Met.
13. The protein of claim 1 where the residue corresponding to BPTI
position 15 is Leu, Ile or Val, the residue corresponding to BPTI
position 16 is Ala or Gly, the residue corresponding to BPTI
position 17 is Met, Phe, Ile or Leu, and the residue corresponding
to BPTI position 19 is Pro, Ser, Lys or Gln.
14. The protein of claim 3 where the residue corresponding to BPTI
position 15 is Leu, Ile or Val, the residue corresponding to BPTI
position 16 is Ala or Gly, the residue corresponding to BPTI
position 17 is Met, Phe, Ile or Leu, and the residue corresponding
to BPTI position 19 is Pro, Ser, Lys or Gln.
15. The protein of claim 5 where the residue corresponding to BPTI
position 15 is Leu, Ile or Val, the residue corresponding to BPTI
position 16 is Ala or Gly, the residue corresponding to BPTI
position 17 is Met, Phe, Ile or Leu, and the residue corresponding
to BPTI position 19 is Pro, Ser, Lys or Gln.
16. The protein of claim 1 where the residue corresponding to BPTI
position 16 is Ala.
17. The protein of claim 1 where the residue corresponding to BPTI
position 17 is Phe.
18. The protein of claim 1 where the residue corresponding to BPTI
position 19 is Pro.
19. The protein of claim 1 where the residues corresponding to BPTI
position 39 is selected from the group consisting of Gly, Gln, Leu,
Pro and Met.
20. The protein of claim 1 where the residue corresponding to BPTI
position 39 is Met.
21. The protein of claim 1 where the residue corresponding to BPTI
position 40 is Gly or Ala.
22. The protein of claim 1 in which the residue corresponding to
BPTI position 40 is Gly.
23. The protein of claim 1 where the residue corresponding to BPTI
position 41 is selected from the group consisting of Asn, Gin, Ser,
Thr, Tyr and Trp.
24. The protein of claim 1 in which the residue corresponding to
BPTI position 41 is Asn.
25. The protein of claim 1 in which residue corresponding to BPTI
position 42 is selected from the group consisting of Ala, Ser, Gly,
Gin and Asn.
26. The protein of claim 1 in which the residue corresponding to
BPTI position 42 is Gly.
27. The protein of claim 1 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Gly, Gin, Leu,
Pro and Met, where the residue corresponding to BPTI position 40 is
Gly or Ala, where the residue corresponding to BPTI position 41 is
selected from the group consisting of Asn, Gin, Ser, Thr, Tyr and
Trp, and where the residue corresponding to BPTI position 42 is
selected from the group consisting of Ala, Ser, Gly, Gin and
Asn.
28. The protein of claim 2 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Gly, Gin, Leu,
Pro and Met, where the residue corresponding to BPTI position 40 is
Gly or Ala, where the residue corresponding to BPTI position 41 is
selected from the group consisting of Asn, Gin, Ser, Thr, Tyr and
Trp, and where the residue corresponding to BPTI position 42 is
selected from the group consisting of Ala, Ser, Gly, Gin and
Asn.
29. The protein of claim 3 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Gly, Gln, Leu,
Pro and Met, where the residue corresponding to BPTI position 40 is
Gly or Ala, where the residue corresponding to BPTI position 41 is
selected from the group consisting of Asn, Gln, Ser, Thr, Tyr and
Trp, and where the residue corresponding to BPTI position 42 is
selected from the group consisting of Ala, Ser, Gly, Gln and
Asn.
30. The protein of claim 5 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Gly, Gln, Leu,
Pro and Met, where the residue corresponding to BPTI position 40 is
Gly or Ala, where the residue corresponding to BPTI position 41 is
selected from the group consisting of Asn, Gln, Ser, Thr, Tyr and
Trp, and where the residue corresponding to BPTI position 42 is
selected from the group consisting of Ala, Ser, Gly, Gln and
Asn.
31. The protein of claim 13 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Gly, Gln, Leu,
Pro and Met, where the residue corresponding to BPTI position 40 is
Gly or Ala, where the residue corresponding to BPTI position 41 is
selected from the group consisting of Asn, Gln, Ser, Thr, Tyr and
Trp, and where the residue corresponding to BPTI position 42 is
selected from the group consisting of Ala, Ser, Gly, Gln and
Asn.
32. The protein of claim 14 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Gly, Gln, Leu,
Pro and Met, where the residue corresponding to BPTI position 40 is
Gly or Ala, where the residue corresponding to BPTI position 41 is
selected from the group consisting of Asn, Gln, Ser, Thr, Tyr and
Trp, and where the residue corresponding to BPTI position 42 is
selected from the group consisting of Ala, Ser, Gly, Gln and
Asn.
33. The protein of claim 15 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Gly, Gln, Leu,
Pro and Met, where the residue corresponding to BPTI position 40 is
Gly or Ala, where the residue corresponding to BPTI position 41 is
selected from the group consisting of Asn, Gln, Ser, Thr, Tyr and
Trp, and where the residue corresponding to BPTI position 42 is
selected from the group consisting of Ala, Ser, Gly, Gln and
Asn.
34. The protein of claim 1 where the residues corresponding to BPTI
positions 39-42 are identical to the corresponding residues of a
human Kunitz domain.
35. The protein of claim 2 where the residues corresponding to BPTI
positions 39-42 are identical to the corresponding residues of a
human Kunitz domain.
36. The protein of claim 3 where the residues corresponding to BPTI
positions 39-42 are identical to the corresponding residues of a
human Kunitz domain.
37. The protein of claim 5 where the residues corresponding to BPTI
positions 39-42 are identical to the corresponding residues of a
human Kunitz domain.
38. The protein of claim 13 where the residues corresponding to
BPTI positions 39-42 are identical to the corresponding residues of
a human Kunitz domain.
39. The protein of claim 14 where the residues corresponding to
BPTI positions 39-42 are identical to the corresponding residues of
a human Kunitz domain.
40. The protein of claim 15 where the residues corresponding to
BPTI positions 39-42 are identical to the corresponding residues of
a human Kunitz domain.
41. The protein of claim 27 where the residues corresponding to
BPTI positions 39-42 are identical to the corresponding residues of
a human Kunitz domain.
42. The protein of claim 33 where the residues corresponding to
BPTI positions 39-42 are identical to the corresponding residues of
a human Kunitz domain.
43. The protein of claim 1 where the residues corresponding to BPTI
positions 40-42 are Gly-Asn-Gly.
44. The protein of claim 3 where the residues corresponding to BPTI
positions 40-42 are Gly-Asn-Gly.
45. The protein of claim 5 where the residues corresponding to BPTI
positions 40-42 are Gly-Asn-Gly.
46. The protein of claim 13 where the residues corresponding to
BPTI positions 40-42 are Gly-Asn-Gly.
47. The protein of claim 14 where the residues corresponding to
BPTI positions 40-42 are Gly-Asn-Gly.
48. The protein of claim 15 where the residues corresponding to
BPTI positions 40-42 are Gly-Asn-Gly.
49. The protein of claim 13 where the residue corresponding to BPTI
position 39 is Met.
50. The protein of claim 13 in which the residue corresponding to
BPTI position 40 is Gly.
51. The protein of claim 13 in which the residue corresponding to
BPTI position 41 is Asn.
52. The protein of claim 13 in which the residue corresponding to
BPTI position 42 is Gly.
53. The protein of claim 1 in which the residue corresponding to
BPTI position 31 is selected from the group consisting of Gln, Glu,
Leu, Lys, Val, Tyr and Asn.
54. The protein of claim 53 in which the residue corresponding to
BPTI position 31 is Gin.
55. The protein of claim 1 in which the residue corresponding to
BPTI position 34 is selected from the group consisting of Val, Ile,
Thr, Asp, Asn, Gin, Phe, His, Pro, Arg and Lys.
56. The protein of claim 55 in which the residue corresponding to
BPTI position 34 is Pro.
57. The protein of claim 1 in which the residue corresponding to
BPTI position 31 is selected from the group consisting of Gin, Glu,
Leu, Lys, Val, Tyr and Asn and the residue corresponding to BPTI
position 34 is selected from the group consisting of Val, Ile, Thr,
Asp, Asn, Gin, Phe, His, Pro, Arg and Lys.
58. The protein of claim 33 in which the residue corresponding to
BPTI position 31 is selected from the group consisting of Gin, Glu,
Leu, Lys, Val, Tyr and Asn and the residue corresponding to BPTI
position 34 is selected from the group consisting of Val, Ile, Thr,
Asp, Asn, Gin, Phe, His, Pro, Arg and Lys.
59. The protein of claim 40 in which the residue corresponding to
BPTI position 31 is selected from the group consisting of Gin, Glu,
Leu, Lys, Val, Tyr and Asn and the residue corresponding to BPTI
position 34 is selected from the group consisting of Val, Ile, Thr,
Asp, Asn, Gln, Phe, His, Pro, Arg and Lys.
60. The protein of claim 1 in which the residue corresponding to
BPTI position 31 is Gin and which the residue corresponding to BPTI
position 34 is Pro.
61. The protein of claim 33 in which the residue corresponding to
BPTI position 31 is Gln and the residue corresponding to BPTI
position 34 is Pro.
62. The protein of claim 40 in which the residue corresponding to
BPTI position 31 is Gln and the residue corresponding to BPTI
position 34 is Pro.
63. The protein of claim 1 where, for each residue corresponding to
a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
64. The protein of claim 3 where, for each residue corresponding to
a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
65. The protein of claim 5 where, for each residue corresponding to
a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
66. The protein of claim 13 where, for each residue corresponding
to a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
67. The protein of claim 14 where, for each residue corresponding
to a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
68. The protein of claim 15 where, for each residue corresponding
to a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
69. The protein of claim 27 where, for each residue corresponding
to a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
70. The protein of claim 33 where, for each residue corresponding
to a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
71. The protein of claim 34 where, for each residue corresponding
to a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
72. The protein of claim 42 where, for each residue corresponding
to a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
73. The protein of claim 57 where, for each residue corresponding
to a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
74. The protein of claim 59 where, for each residue corresponding
to a previously unspecified position in BPTI, the residue is one
listed in Tables 15 or 34 as an amino acid found in the
corresponding BPTI position in at least one of the BPTI homologues
set forth in Table 13A.
75. The protein of claim 63 where, each such amino acid is one
found in the corresponding BPTI position in an ITI Kunitz domain
listed in Table 13A.
76. The protein of claim 63 where each amino acid is one found in
the corresponding BPTI position in BPTI or human ITI-D1.
77. The protein of claim 1 where said domain has a higher
percentage identity to a naturally-occurring human Kunitz domain of
Table 13A than to any naturally-occurring nonhuman Kunitz domain of
Table 13A.
78. The protein of claim 1 where said domain consists of an amino
acid sequence identical to that of a protein selected from the
group consisting of EpiNe.alpha., EpiNE1, EpiNE2, EpiNE3, EpiNE4,
EpiNE5, EpiNE6, EpiNE7, EpiNE8.
79. The protein of claim 1 where the amino acid sequence of said
mutant Kunitz domain is of higher percentage identity to that of
ITI-D1 than to that of BPTI.
80. The protein of claim 1 where the amino acid sequence of said
mutant Kunitz domain is of higher percentage identity to that of
BPTI than to that of ITI-D1.
81. The protein of claim 1 which has a binding affinity for human
neutrophil elastase in the range of 100 pm to 1 pm.
82. The protein of claim 1 which has a binding affinity for human
neutrophil elastase which is greater than that of BPTI (K1SV,
R17L).
83. A non-naturally occurring or purified protein which inhibits
human neutrophil elastase, and comprises a Kunitz domain whose
amino acid sequence which at least 30% identical to that of BPTI,
where, in said sequence, the residue corresponding to BPTI position
18 is Phe, and where the residues corresponding to BPTI positions
39-42 are not charged residues.
84. The protein of claim 2 where the residue corresponding to BPTI
position 15 is Leu, Ile or Val, the residue corresponding to BPTI
position 16 is Ala or Gly, the residue corresponding to BPTI
position 17 is Met, Phe, Ile or Leu, and the residue corresponding
to BPTI position 19 is Pro, Ser, Lys or Gln.
85. The protein of claim 4 where the residue corresponding to BPTI
position 15 is Leu, Ile or Val, the residue corresponding to BPTI
position 16 is Ala or Gly, the residue corresponding to BPTI
position 17 is Met, Phe, Ile or Leu, and the residue corresponding
to BPTI position 19 is Pro, Ser, Lys or Gln.
86. The protein of claim 3 where the residue corresponds to BPTI
position 15 is Val, Ile or Leu, the residue corresponding to BPTI
position 16 is Ala, the residue corresponding to BPTI position 17
is Phe, and the residue corresponding to BPTI position 19 is
Pro.
87. The protein of claim 4 where the residue corresponds to BPTI
position 15 is Val, Ile or Leu, the residue corresponding to BPTI
position 16 is Ala, the residue corresponding to BPTI position 17
is Phe, and the residue corresponding to BPTI position 19 is
Pro.
88. The protein of claim 5 where the residue corresponds to BPTI
position 15 is Val, Ile or Leu, the residue corresponding to BPTI
position 16 is Ala, the residue corresponding to BPTI position 17
is Phe, and where the residue corresponding to BPTI position 19 is
Pro.
89. The protein of claim 2 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Arg, Gly, Lys,
Gln, Asp, Glu, Leu, Pro and Met, the residue corresponding to BPTI
position 40 is Gly or Ala, the residue corresponding to BPTI
position 41 is selected from the group consisting of Asn, Gln, Ser,
Thr, Tyr and Trp, and the residue corresponding to BPTI position 42
is selected from the group consisting of Ala, Ser, Gly, Gln and
Asn.
90. The protein of claim 4 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Arg, Gly, Lys,
Gln, Asp, Glu, Leu, Pro and Met, the residue corresponding to BPTI
position 40 is Gly or Ala, the residue corresponding to BPTI
position 41 is selected from the group consisting of Asn, Gln, Ser,
Thr, Tyr and Trp, and the residue corresponding to BPTI position 42
is selected from the group consisting of Ala, Ser, Gly, Gln and
Asn.
91. The protein of claim 6 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Arg, Gly, Lys,
Gln, Asp, Glu, Leu, Pro and Met, the residue corresponding to BPTI
position 40 is Gly or Ala, the residue corresponding to BPTI
position 41 is selected from the group consisting of Asn, Gln, Ser,
Thr, Tyr and Trp, and the residue corresponding to BPTI position 42
is selected from the group consisting of Ala, Ser, Gly, Gln and
Asn.
92. The protein of claim 7 where the residue corresponding to BPTI
position 39 is selected from the group consisting of Arg, Gly, Lys,
Gln, Asp, Glu, Leu, Pro and Met, the residue corresponding to BPTI
position 40 is Gly or Ala, the residue corresponding to BPTI
position 41 is selected from the group consisting of Asn, Gln, Ser,
Thr, Tyr and Trp, and the residue corresponding to BPTI position 42
is selected from the group consisting of Ala, Ser, Gly, Gln and
Asn.
93. The protein of claim 33 where the residue corresponds to BPTI
position 15 is Val, Ile or Leu the residue corresponding to BPTI
position 16 is Ala, the residue corresponding to BPTI position 17
is Phe, and the residue corresponding to BPTI position 19 is
Pro.
94. The protein of claim 93 where the residue corresponding to BPTI
position 31 is selected from the group consisting of Gln, Glu, Leu,
Lys, Val, Tyr and Asn, and the residue corresponding to BPTI
position 34 is selected from the group consisting of Val, Ile, Thr,
Asp, Asn, Gln, Phe, His, Pro, Arg and Lys.
95. The protein of claim 94 where the residue corresponding to BPTI
position 31 is Gln and the residue corresponding to BPTI position
34 is Pro.
96. A method of inhibiting human neutrophil elastase which
comprises contacting the elastase with the protein of claims 1, 2,
3, 4, 5, 13, 27, 29, 30, 83, or 94.
97. A method of inhibiting harmful human neutrophil elastase
activity in a subject which comprises administering to the subject
an inhibitorily effective amount of the protein of any of claims
1-95.
98. A method of treating emphysema which comprising administering,
to a subject suffering from emphysema, a therapeutically effective
amount of the protein of claims 1, 2, 3, 4, 5, 13, 27, 29, 30, 83
or 94.
99. A method of treating cystic fibrosis which comprising
administering, to a subject suffering from cystic fibrosis, a
therapeutically effective amount of the protein of claims 1, 2, 3,
4, 5, 13, 27, 29, 30, 83 or 94.
100. A method of inhibiting harmful human neutrophil elastase
activity in a subject which comprises administering to the subject
an inhibitorily effective amount of the protein of any of claims 1,
2, 3, 4, 5, 13, 27, 29, 30, 83 or 94, where the Kd of said protein
for trypsin, chymotrypsin and plasma is greater than 10.sup.-6M.
Description
[0001] This application is a continuation-in-part of Ladner,
Guterman, Roberts, and Markland, Ser. No. 07/487,063, filed Mar. 2,
1990, now pending, which is a continuation-in-part of Ladner and
Guterman, Ser. No. 07/240,160, filed Sep. 2, 1988, now pending.
Ser. No. 07/487,063 claimed priority under 35 U.S.C. 119 from PCT
Application No. PCT/US89/03731, filed Sep. 1, 1989. All of the
foregoing applications are hereby incorporated by reference.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] The following related and commonly-owned applications are
also incorporated by reference:
[0003] Robert Charles Ladner, Sonia Kosow Guterman, Rachael
Baribault Kent, and Arthur Charles Ley are named as joint inventors
on U.S. Ser. No. 07/293,980, filed Jan. 8, 1989, and entitled
GENERATION AND SELECTION OF NOVEL DNA-BINDING PROTEINS AND
POLYPEPTIDES. This application has been assigned to Protein
Engineering Corporation.
[0004] Robert Charles Ladner, Sonia Kosow Guterman, and Bruce
Lindsay Roberts are named as a joint inventors on a U.S. Ser. No.
07/470,651 filed January 26, 1990, entitled "PRODUCTION OF NOVEL
SEQUENCE-SPECIFIC DNA-ALTERING ENZYMES", likewise assigned to
Protein Engineering Corp.
[0005] Ladner, Guterman, Kent, Ley, and Markland, Ser. No.
07/558,011 is also assigned to Protein Engineering Corporation.
BACKGROUND OF THE INVENTION
[0006] Field of the Invention
[0007] This invention relates to development of novel binding
proteins (including mini-proteins) by an iterative process of
mutagenesis, expression, chromatographic selection, and
amplification. In this process, a gene encoding a potential binding
domain, said gene being obtained by random mutagenesis of a limited
number of predetermined codons, is fused to a genetic element which
causes the resulting chimeric expression product to be displayed on
the outer surface of a virus (especially a filamentous phage) or a
cell. Chromatographic selection is then used to identify viruses or
cells whose genome includes such a fused gene which coded for the
protein which bound to the chromatographic target.
Information Disclosure Statement
[0008] A. Protein Structure
[0009] The amino acid sequence of a protein determines its
three-dimensional (3D) structure, which in turn determines protein
function (EPST63, ANFI73). Shortle (SHOR85), Sauer and colleagues
(PAKU86, REID88a), and Caruthers and colleagues (EISE85) have shown
that some residues on the polypeptide chain are more important than
others in determining the 3D structure of a protein. The 3D
structure is essentially unaffected by the identity of the amino
acids at some loci; at other loci only one or a few types of amino
acid is allowed. In most cases, loci where wide variety is allowed
have the amino acid side group directed toward the solvent. Loci
where limited variety is allowed frequently have the side group
directed toward other parts of the protein. Thus substitutions of
amino acids that are exposed to solvent are less likely to affect
the 3D structure than are substitutions at internal loci. (See also
SCHU79, p169-171 and CREI84, p239-245, 314-315).
[0010] The secondary structure (helices, sheets, turns, loops) of a
protein is determined mostly by local sequence. Certain amino acids
have a propensity to appear in certain "secondary structures," they
will be found from time to time in other structures, and studies of
pentapeptide sequences found in different proteins have shown that
their conformation varies considerably from one occurrence to the
next (KABS84, ARGO87). As a result, a priori design of proteins to
have a particular 3D structure is difficult.
[0011] Several researchers have designed and synthesized proteins
de novo (MOSE83, MOSE87, ERIC86). These designed proteins are small
and most have been synthesized in vitro as polypeptides rather than
genetically. Hecht et al. (HECH90) have produced a designed protein
genetically. Moser, et al. state that design of biologically active
proteins is currently impossible.
[0012] B. Protein Binding Activity
[0013] Many proteins bind non-covalently but very tightly and
specifically to some other characteristic molecules (SCHU79,
CREI84). In each case the binding results from complementarity of
the surfaces that come into contact: bumps fit into holes, unlike
charges come together, dipoles align, and hydrophobic atoms contact
other hydrophobic atoms. Although bulk water is excluded,
individual water molecules are frequently found filling space in
intermolecular interfaces; these waters usually form hydrogen bonds
to one or more atoms of the protein or to other bound water. Thus
proteins found in nature have not attained, nor do they require,
perfect complementarity to bind tightly and specifically to their
substrates. only in rare cases is there essentially perfect
complementarity; then the binding is extremely tight (as for
example, avidin binding to biotin).
[0014] C. Protein Engineering
[0015] "Protein engineering" is the art of manipulating the
sequence of a protein in order to alter its binding
characteristics. The factors affecting protein binding are known,
(CHOT75, CHOT76, SCHU79, p98-107, and CREI84, Ch8), but designing
new complementary surfaces has proved difficult. Although some
rules have been developed for substituting side groups (SUTC87b),
the side groups of proteins are floppy and it is difficult to
predict what conformation a new side group will take. Further, the
forces that bind proteins to other molecules are all relatively
weak and it is difficult to predict the effects of these
forces.
[0016] Recently, Quiocho and collaborators (QUIO87) elucidated the
structures of several periplasmic binding proteins from
Gram-negative bacteria. They found that the proteins, despite
having low sequence homology and differences in structural detail,
have certain important structural similarities. Based on their
investigations of these binding proteins, Quiocho et al. suggest it
is unlikely that, using current protein engineering methods,
proteins can be constructed with binding properties superior to
those of proteins that occur naturally.
[0017] Nonetheless, there have been some isolated successes.
Wilkinson et al. (WILK84) reported that a mutant of the tyrosyl
tRNA synthetase of Bacillus stearothermophilus with the mutation
Thr.sub.51.fwdarw.Pro exhibits a 100-fold increase in affinity for
ATP. Tan and Kaiser (TANK77) and Tschesche et al. (TSCH87) showed
that changing a single amino acid in mini-protein greatly reduces
its binding to trypsin, but that some of the mutants retained the
parental characteristic of binding to an inhibiting chymotrypsin,
while others exhibited new binding to elastase. Caruthers and
others (EISE85) have shown that changes of single amino acids on
the surface of the lambda Cro repressor greatly reduce its affinity
for the natural operator O.sub.R3, but greatly increase the binding
of the mutant protein to a mutant operator. Changing three residues
in subtilisin from Bacillus amyloliguefaciens to be the same as the
corresponding residues in subtilisin from B. licheniformis produced
a protease having nearly the same activity as the latter
subtilisin, even though 82 amino acid sequence differences remained
(WELL87a). Insertion of DNA encoding 18 amino acids (corresponding
to Pro-Glu-Dynorphin-Gly) into the E. coli phoA gene so that the
additional amino acids appeared within a loop of the alkaline
phosphatase protein resulted in a chimeric protein having both phoA
and dynorphin activity (FREI90). Thus, changing the surface of a
binding protein may alter its specificity without abolishing
binding activity.
[0018] D. Techniques of Mutagenesis
[0019] Early techniques of mutating proteins involved manipulations
at the amino acid sequence level. In the semi-synthetic method
(TSCH87), the protein was cleaved into two fragments, a residue
removed from the new end of one fragment, the substitute residue
added on in its place, and the modified fragment joined with the
other, original fragment. Alternatively, the mutant protein could
be synthesized in its entirety (TANK77).
[0020] Erickson et al. suggested that mixed amino acid reagents
could be used to produce a family of sequence-related proteins
which could then be screened by affinity chromatography (ERIC86).
They envision successive rounds of mixed synthesis of variant
proteins and purification by specific binding. They do not discuss
how residues should be chosen for variation. Because proteins
cannot be amplified, the researchers must sequence the recovered
protein to learn which substitutions improve binding. The
researchers must limit the level of diversity so that each variety
of protein will be present in sufficient quantity for the isolated
fraction to be sequenced.
[0021] With the development of recombinant DNA techniques, it
became possible to obtain a mutant protein by mutating the gene
encoding the native protein and then expressing the mutated gene.
Several mutagenesis strategies are known. One, "protein surgery"
(DILL87), involves the introduction of one or more predetermined
mutations within the gene of choice. A single polypeptide of
completely predetermined sequence is expressed, and its binding
characteristics are evaluated.
[0022] At the other extreme is random mutagenesis by means of
relatively nonspecific mutagens such as radiation and various
chemical agents. See Ho et al. (HOCJ85) and Lehtovaara, E.P. Appln.
285,123.
[0023] It is possible to randomly vary predetermined nucleotides
using a mixture of bases in the appropriate cycles of a nucleic
acid synthesis procedure. The proportion of bases in the mixture,
for each position of a codon, will determine the frequency at which
each amino acid will occur in the polypeptides expressed from the
degenerate DNA population. Oliphant et al. (OLIP86) and oliphant
and Struhl (OLIP87) have demonstrated ligation and cloning of
highly degenerate oligonucleotides, which were used in the mutation
of promoters. They suggested that similar methods could be used in
the variation of protein coding regions. They do not say how one
should: a) choose protein residues to vary, or b) select or screen
mutants with desirable properties. Reidhaar-Olson and Sauer
(REID88a) have used synthetic degenerate oligo-nts to vary
simultaneously two or three residues through all twenty amino
acids. See also Vershon et al. (VERS86a; VERS86b). Reidhaar-Olson
and Sauer do not discuss the limits on how many residues could be
varied at once nor do they mention the problem of unequal abundance
of DNA encoding different amino acids. They looked for proteins
that either had wild-type dimerization or that did not dimerize.
They did not seek proteins having novel binding properties and did
not find any. This approach is likewise limited by the number of
colonies that can be examined (ROBE86).
[0024] To the extent that this prior work assumes that it is
desirable to adjust the level of mutation so that there is one
mutation per protein, it should be noted that many desirable
protein alterations require multiple amino acid substitutions and
thus are not accessible through single base changes or even through
all possible amino acid substitutions at any one residue.
[0025] D. Affinity Chromatography of Cells
[0026] Ferenci and coloborators have published a series of papers
on the chromatographic isolation of mutants of the
maltose-transport protein LamB of E. coli (FERE82a, FERE82b,
FERE83, FERE84, CLUN84, HEIN87 and papers cited therein) The
mutants were either spontaneous or induced with nonspecific
chemical mutagens. Levels of mutagenesis were picked to provide
single point mutations or single insertions of two residues. No
multiple mutations were sought or found.
[0027] While variation was seen in the degree of affinity for the
conventional LamB substrates maltose and starch, there was no
selection for affinity to a target molecule not bound at all by
native LamB, and no multiple mutations were sought or found. FERE84
speculated that the affinity chromatographic selection technique
could be adapted to development of similar mutants of other
"important bacterial surface-located enzymes", and to selecting for
mutations which result in the relocation of an intracellular
bacterial protein to the cell surface. Ferenci's mutant surface
proteins would not, however, have been chimeras of a bacterial
surface protein and an exogenous or heterologous binding
domain.
[0028] Ferenci also taught that there was no need to clone the
structural gene, or to know the protein structure, active site, or
sequence. The method of the present invention, however,
specifically utilizes a cloned structural gene. It is not possible
to construct and express a chimeric, outer surface-directed
potential binding protein-encoding gene without cloning.
[0029] Ferenci did not limit the mutations to particular loci or
particular substitutions. In the present invention, knowledge of
the protein structure, active site and/or sequence is used as
appropriate to predict which residues are most likely to affect
binding activity without unduly destabilizing the protein, and the
mutagenesis is focused upon those sites. Ferenci does not suggest
that surface residues should be preferentially varied. In
consequence, Ferenci's selection system is much less efficient than
that disclosed herein.
[0030] E. Bacterial and Viral Expression of Chimeric Surface
Proteins
[0031] A number of researchers have directed unmutated foreign
antigenic epitopes to the surface of bacteria or phage, fused to a
native bacterial or phage surface protein, and demonstrated that
the epitopes were recognized by antibodies. Thus, Charbit, et al.
(CHAR86) genetically inserted the C3 epitope of the VP1 coat
protein of poliovirus into the LamB outer membrane protein of E.
coli, and determined immunologically that the C3 epitope was
exposed on the bacterial cell surface. Charbit, et al. (CHAR87)
likewise produced chimeras of LamB and the A (or B) epitopes of the
preS2 region of hepatitis B virus.
[0032] A chimeric LacZ/ompB protein has been expressed in E. coli
and is, depending on the fusion, directed to either the outer
membrane or the periplasm (SILH77). A chimeric LacZ/OmpA surface
protein has also been expressed and displayed on the surface of E.
coli cells (Weinstock et al., WEIN83). Others have expressed and
displayed on the surface of a cell chimeras of other bacterial
surface proteins, such as E. coli type 1 fimbriae (Hedegaard and
Klemm (HEDE89)) and Bacterioides nodusus type 1 fimbriae (Jennings
et al., JENN89). In none of the recited cases was the inserted
genetic material mutagenized.
[0033] Dulbecco (DULB86) suggests a procedure for incorporating a
foreign antigenic epitope into a viral surface protein so that the
expressed chimeric protein is displayed on the surface of the virus
in a manner such that the foreign epitope is accessible to
antibody. In 1985 Smith (SMIT85) reported inserting a nonfunctional
segment of the EcoRI endonuclease gene into gene III of
bacteriophage f1, "Tin phase". The gene III protein is a minor coat
protein necessary for infectivity. Smith demonstrated that the
recombinant phage were adsorbed by immobilized antibody raised
against the EcoRI endonuclease, and could be eluted with acid. De
la Cruz et al. (DELA88) have expressed a fragment of the repeat
region of the circumsporozoite protein from Plasmodium falciparum
on the surface of M13 as an insert in the gene III protein. They
showed that the recombinant phage were both antigenic and
immunogenic in rabbits, and that such recombinant phage could be
used for B epitope mapping. The researchers suggest that similar
recombinant phage could be used for T epitope mapping and for
vaccine development.
[0034] None of these researchers suggested mutagenesis of the
inserted material, nor is the inserted material a complete binding
domain conferring on the chimeric protein the ability to bind
specifically to a receptor other than the antigen combining site of
an antibody.
[0035] McCafferty et al. (MCCA90) expressed a fusion of an Fv
fragment of an antibody to the N-terminal of the pIII protein. The
Fv fragment was not mutated.
[0036] F. Epitope Libraries on Fusion Phage
[0037] Parmley and Smith (PARM88) suggested that an epitope library
that exhibits all possible hexapeptides could be constructed and
used to isolate epitopes that bind to antibodies. In discussing the
epitope library, the authors did not suggest that it was desirable
to balance the representation of different amino acids. Nor did
they teach that the insert should encode a complete domain of the
exogenous protein. Epitopes are considered to be unstructured
peptides as opposed to structured proteins.
[0038] After the filing of the parent application whose benefit is
claimed herein under 35 U.S.C. 120, certain groups reported the
construction of "epitope libraries." Scott and Smith (SCOT90) and
Cwirla et al. (CWIR90) prepared "epitope libraries" in which
potential hexapeptide epitopes for a target antibody were randomly
mutated by fusing degenerate oligonucleotides, encoding the
epitopes, with gene III of fd phage, and expressing the fused gene
in phage-infected cells. The cells manufactured fusion phage which
displayed the epitopes on their surface; the phage which bound to
immobilized antibody were eluted with acid and studied. In both
cases, the fused gene featured a segment encoding a spacer region
to separate the variable region from the wild type pIII sequence so
that the varied amino acids would not be constrained by the nearby
pIII sequence. Devlin et al. (DEVL90) similarly screened, using M13
phage, for random 15 residue epitopes recognized by streptavidin.
Again, a spacer was used to move the random peptides away from the
rest of the chimeric phage protein. These references therefore
taught away from constraining the conformational repertoire of the
mutated residues.
[0039] Another problem with the Scott and Smith, Cwirla et al., and
Devlin et al., libraries was that they provided a highly biased
sampling of the possible amino acids at each position. Their
primary concern in designing the degenerate oligonucleotide
encoding their variable region was to ensure that all twenty amino
acids were encodible at each position; a secondary consideration
was minimizing the frequency of occurrence of stop signals.
Consequently, Scott and Smith and Cwirla et al. employed NNK
(N=equal mixture of G, A, T, C; K=equal mixture of G and T) while
Devlin et al. used NNS (S=equal mixture of G and C). There was no
attempt to minimize the frequency ratio of most favored-to-least
favored amino acid, or to equalize the rate of occurrence of acidic
and basic amino acids.
[0040] Devlin et al. characterized several affinity-selected
streptavidin-binding peptides, but did not measure the affinity
constants for these peptides. Cwirla et al. did determine the
affinity constant for his peptides, but were disappointed to find
that his best hexapeptides had affinities (350-300nM), "orders of
magnitude" weaker than that of the native Met-enkephalin epitope
(7nM) recognized by the target antibody. Cwirla et al. speculated
that phage bearing peptides with higher affinities remained bound
under acidic elution, possibly because of multivalent interactions
between phage (carrying about 4 copies of pIII) and the divalent
target IgG. Scott and Smith were able to find peptides whose
affinity for the target antibody (A2) was comparable to that of the
reference myohemerythrin epitope (50nM). However, Scott and Smith
likewise expressed concern that some high-affinity peptides were
lost, possibly through irreversible binding of fusion phage to
target.
[0041] G. Non-Commonly Owned Patents and Applications Naming Robert
Ladner as an Inventor
[0042] Ladner, U.S. Pat. No. 4,704,692, "Computer Based System and
Method for Determining and Displaying Possible Chemical Structures
for Converting Double- or Multiple-Chain Polypeptides to
Single-Chain Polypeptides" describes a design method for converting
proteins composed of two or more chains into proteins of fewer
polypeptide chains, but with essentially the same 3D structure.
There is no mention of variegated DNA and no genetic selection.
Ladner and Bird, WO88/01649 (Publ. Mar. 10, 1988) disclose the
specific application of computerized design of linker peptides to
the preparation of single chain antibodies.
[0043] Ladner, Glick, and Bird, WO88/06630 (publ. Sep. 7, 1988 and
having priority from U.S. application Ser. No. 07/021,046, assigned
to Genex Corp.) (LGB) speculate that diverse single chain antibody
domains (SCAD) may be screened for binding to a particular antigen
by varying the DNA encoding the combining determining regions of a
single chain antibody, subcloning the SCAD gene into the gpV gene
of phage lambda so that a SCAD/gpV chimera is displayed on the
outer surface of phage lambda, and selecting phage which bind to
the antigen through affinity chromatography. The only antigen
mentioned is bovine growth hormone. No other binding molecules,
targets, carrier organisms, or outer surface proteins are
discussed. Nor is there any mention of the method or degree of
mutagenesis. Furthermore, there is no teaching as to the exact
structure of the fusion nor of how to identify a successful fusion
or how to proceed if the SCAD is not displayed.
[0044] Ladner and Bird, WO88/06601 (publ. Sep. 7, 1988) suggest
that single chain "pseudodimeric" repressors (DNA-binding proteins)
may be prepared by mutating a putative linker peptide followed by
in vivo selection that mutation and selection may be used to create
a dictionary of recognition elements for use in the design of
asymmetric repressors. The repressors are not displayed on the
outer surface of an organism.
[0045] Methods of identifying residues in protein which can be
replaced with a cysteine in order to promote the formation of a
protein-stabilizing disulfide bond are given in Pantoliano and
Ladner, U.S. Pat. No. 4,903,773 (PANT90), Pantoliano and Ladner
(PANT87), Pabo and Suchenek (PABO86), MATS89, and SAUE86.
[0046] No admission is made that any cited reference is prior art
or pertinent prior art, and the dates given are those appearing on
the reference and may not be identical to the actual publication
date. All references cited in this specification are hereby
incorporated by reference.
SUMMARY OF THE INVENTION
[0047] The present invention is intended to overcome the
deficiencies discussed above. It relates to the construction,
expression, and selection of mutated genes that specify novel
proteins with desirable binding properties, as well as these
proteins themselves. The substances bound by these proteins,
hereinafter referred to as "targets", may be, but need not be,
proteins. Targets may include other biological or synthetic
macromolecules as well as other organic and inorganic
substances.
[0048] The fundamental principle of the invention is one of forced
evolution. In nature, evolution results from the combination of
genetic variation, selection for advantageous traits, and
reproduction of the selected individuals, thereby enriching the
population for the trait. The present invention achieves genetic
variation through controlled random mutagenesis ("variegation") of
DNA, yielding a mixture of DNA molecules encoding different but
related potential binding proteins. It selects for mutated genes
that specify novel proteins with desirable binding properties by 1)
arranging that the product of each mutated gene be displayed on the
outer surface of a replicable genetic package (GP) (a cell, spore
or virus) that contains the gene, and 2) using affinity
selection--selection for binding to the target material--to enrich
the population of packages for those packages containing genes
specifying proteins with improved binding to that target material.
Finally, enrichment is achieved by allowing only the genetic
packages which, by virtue of the displayed protein, bound to the
target, to reproduce. The evolution is "forced" in that selection
is for the target material provided.
[0049] The display strategy is first perfected by modifying a
genetic package to display a stable, structured domain (the
"initial potential binding domain", IPBD) for which an affinity
molecule (which may be an antibody) is obtainable. The success of
the modifications is readily measured by, e.g., determining whether
the modified genetic package binds to the affinity molecule.
[0050] The IPBD is chosen with a view to its tolerance for
extensive mutagenesis. Once it is known that the IPBD can be
displayed on a surface of a package and subjected to affinity
selection, the gene encoding the IPBD is subjected to a special
pattern of multiple mutagenesis, here termed "variegation", which
after appropriate cloning and amplification steps leads to the
production of a population of genetic packages each of which
displays a single potential binding domain (a mutant of the IPBD),
but which collectively display a multitude of different though
structurally related potential binding domains (PBDs). Each genetic
package carries the version of the pbd gene that encodes the PBD
displayed on the surface of that particular package. Affinity
selection is then used to identify the genetic packages bearing the
PBDs with the desired binding characteristics, and these genetic
packages may then be amplified. After one or more cycles of
enrichment by affinity selection and amplification, the DNA
encoding the successful binding domains (SBDs) may then be
recovered from selected packages.
[0051] If need be, the DNA from the SBD-bearing packages may then
be further "variegated", using an SBD of the last round of
variegation as the "parental potential binding domain" (PPBD) to
the next generation of PBDs, and the process continued until the
worker in the art is satisfied with the result. At that point, the
SBD may be produced by any conventional means, including chemical
synthesis.
[0052] When the number of different amino acid sequences obtainable
by mutation of the domain is large when compared to the number of
different domains which are displayable in detectable amounts, the
efficiency of the forced evolution is greatly enhanced by careful
choice of which residues are to be varied. First, residues of a
known protein which are likely to affect its binding activity
(e.g., surface residues) and not likely to unduly degrade its
stability are identified. Then all or some of the codons encoding
these residues are varied simultaneously to produce a variegated
population of DNA. The variegated population of DNA is used to
express a variety of potential binding domains, whose ability to
bind the target of interest may then be evaluated.
[0053] The method of the present invention is thus further
distinguished from other methods in the nature of the highly
variegated population that is produced and from which novel,
binding proteins are selected. We force the displayed potential
binding domain to sample the nearby "sequence space" of related
amino-acid sequences in an efficient, organized manner. Four goals
guide the various variegation plans used herein, preferably: 1) a
very large number (e.g., 10.sup.7) of variants is available, 2) a
very high percentage of the possible variants actually appears in
detectable amounts, 3) the frequency of appearance of the desired
variants is relatively uniform, and 4) variation occurs only at a
limited number of amino-acid residues, most preferably at residues
having side groups directed toward a common region on the surface
of the potential binding domain.
[0054] This is to be distinguished from the simple use of
indiscriminate mutagenic agents such as radiation and hydroxylamine
to modify a gene, where there is no (or very oblique) control over
the site of mutation. Many of the mutations will affect residues
that are not a part of the binding domain. Moreover, since at a
reasonable level of mutagenesis, any modified codon is likely to be
characterized by a single base change, only a limited and biased
range of possibilities will be explored. Equally remote is the use
of site-specific mutagenesis techniques employing mutagenic
oligonucleotides of nonrandomized sequence, since these techniques
do not lend themselves to the production and testing of a large
number of variants. While focused random mutagenesis techniques are
known, the importance of controlling the distribution of variation
has been largely overlooked.
[0055] In order to obtain the display of a multitude of different
though related potential binding domains, applicants generate a
heterogeneous population of replicable genetic packages each of
which comprises a hybrid gene including a first DNA sequence which
encodes a potential binding domain for the target of interest and a
second DNA sequence which encodes a display means, such as an outer
surface protein native to the genetic package but not natively
associated with the potential binding domain (or the parental
binding domain to which it is related) which causes the genetic
package to display the corresponding chimeric protein (or a
processed form thereof) on its outer surface.
[0056] It should be recognized that by expressing a hybrid protein
which comprises an outer surface transport signal not natively
associated with the binding domain, the utility of the present
invention is greatly extended. The binding domain need not be that
of a surface protein of the genetic package (or, in the case of a
viral package, of its host cell), since the provided outer surface
transport signal is responsible for achieving the desired display.
Thus, it is possible to display on the surface of a phage,
bacterial cell or bacterial spore a binding domain related to the
binding domain of a normally cytoplasmic binding protein, or the
binding domain of eukaryotic protein which is not found on the
surface of prokaryotic cells or viruses.
[0057] Another important aspect of the invention is that each
potential binding domain remains physically associated with the
particular DNA molecule which encodes it. Thus, once successful
binding domains are identified, one may readily recover the gene
and either express additional quantities of the novel binding
protein or further mutate the gene. The form that this association
takes is a "replicable-genetic package", a virus, cell or spore
which replicates and expresses the binding domain-encoding gene,
and transports the binding domain to its outer surface.
[0058] It is also possible chemically or enzymatically to modify
the PBDs before selection. The selection then identifies the best
modified amino acid sequence. For example, we could treat the
variegated population of genetic packages that display a variegated
population of binding domains with a protein tyrosine kinase and
then select for binding the target. Any tyrosines on the BD surface
will be phosphorylated and this could affect the binding
properties. Other chemical or enzymatic modifications are
possible.
[0059] By virtue of the present invention, proteins are obtained
which can bind specifically to targets other than the
antigen-combining sites of antibodies. A protein is not to be
considered a "binding protein" merely because it can be bound by an
antibody (see definition of "binding protein" which follows). While
almost any amino acid sequence of more than about 6-8 amino acids
is likely, when linked to an immunogenic carrier, to elicit an
immune response, any given random polypeptide is unlikely to
satisfy the stringent definition of "binding protein" with respect
to minimum affinity and specificity for its substrate. It is only
by testing numerous random polypeptides simultaneously (and, in the
usual case, controlling the extent and character of the sequence
variation, i.e., limiting it to residues of a potential binding
domain having a stable structure, the residues being chosen as more
likely to affect binding than stability) that this obstacle is
overcome.
[0060] In one embodiment, the invention relates to:
[0061] a) preparing a variegated population of replicable genetic
packages, each package including a nucleic acid construct coding
for an outer-surface-displayed potential binding protein other than
an antibody, comprising (i) a structural signal directing the
display of the protein (or a processed form thereof) on the outer
surface of the package and (ii) a potential binding domain for
binding said target, where the population collectively displays a
multitude of different potential binding domains having a
substantially predetermined range of variation in sequence,
[0062] b) causing the expression of said protein and the display of
said protein on the outer surface of such packages,
[0063] c) contacting the packages with target material, other than
an antibody with an exposed antigen-combining site, so that the
potential binding domains of the proteins and the target material
may interact, and separating packages bearing a potential binding
domain that succeeds in binding the target material from packages
that do not so bind,
[0064] d) recovering and replicating at least one package bearing a
successful binding domain,
[0065] e) determining the amino acid sequence of the successful
binding domain of a genetic package which bound to the target
material,
[0066] f) preparing a new variegated population of replicable
genetic packages according to step (a), the parental potential
binding domain for the potential binding domains of said new
packages being a successful binding domain whose sequence was
determined in step (e), and repeating steps (b)-(e) with said new
population, and, when a package bearing a binding domain of desired
binding characteristics is obtained,
[0067] g) abstracting the DNA encoding the desired binding domain
from the genetic package and placing it into a suitable expression
system. (The binding domain may then be expressed as a unitary
protein, or as a domain of a larger protein).
[0068] The invention is not, however, limited to proteins with a
single BD since the method may be applied to any or all of the BDs
of the protein, sequentially or simultaneously. The invention is
not, however, limited to biological synthesis of the binding
domains; peptides having an amino-acid sequence determined by the
isolated DNA can be chemically synthesized.
[0069] The invention further relates to a variegated population of
genetic packages. Said population may be used by one user to select
for binding to a first target, by a second user to select for
binding to a second target, and so on, as the present invention
does not require that the initial potential binding domain actually
bind to the target of interest, and the variegation is at residues
likely to affect binding. The invention also relates to the
variegated DNA used in preparing such genetic packages.
[0070] The invention likewise encompasses the procedure by which
the display strategy is verified. The genetic packages are
engineered to display a single IPBD sequence. (Variability may be
introduced into DNA subsequences adjacent to the ipbd subsequence
and within the osp-ipbd gene so that the IPBD will appear on the GP
surface.) A molecule, such as an antibody, having high affinity for
correctly folded IPBD is used to: a) detect IPBD on the GP surface,
b) screen colonies for display of IPBD on the GP surface, or c)
select GPs that display IPBD from a population, some members of
which might display IPBD on the GP surface. In one preferred
embodiment, this verification process (part I) involves:
[0071] 1) choosing a GP such as a bacterial cell, bacterial spore,
or phage, having a suitable outer surface protein (OSP),
[0072] 2) choosing a stable IPBD,
[0073] 3) designing an amino acid sequence that: a) includes the
IPBD as a subsequence and b) will cause the IPBD to appear on the
GP surface,
[0074] 4) engineering a gene, denoted osp-ipbd, that: a) codes for
the designed animo acid sequence, b) provides the necessary genetic
regulation, and c) introduces convenient sites for genetic
manipulation,
[0075] 5) cloning the osp-ipbd gene into the GP, and
[0076] 6) harvesting the transformed GPs and testing them for
presence of IPBD on the GP surface; this test is performed with an
affinity molecule having high affinity for IPBD, denoted
AfM(IPBD).
[0077] Once a GP(IPBD) is produced, it can be used many times as
the starting point for developing different novel proteins that
bind to a variety of different targets. The knowledge of how we
engineer the appearance of one IPBD on the surface of a GP can be
used to design and produce other GP(IPBD)s that display different
IPBDs.
[0078] Knowing that a particular genetic package and osp-ipbd
fusion are suitable for the practice of the invention, we may
variegate the genetic packages and select for binding to a target
of interest. Using IPBD as the PPBD to the first cycle of
variegation, we prepare a wide variety of osp-pbd genes that encode
a wide variety of PBDs. We use an affinity separation to enrich the
population of GP(vgPBD)s for GPs that display PBDs with binding
properties relative to the target that are superior to the binding
properties of the PPBD. An SBD selected from one variegation cycle
becomes the PPBD to the next variegation cycle. In a preferred
embodiment, Part II of the process of the present invention
involves:
[0079] 1) picking a target molecule, and an affinity separation
system which selects for proteins having an affinity for that
target molecule,
[0080] 2) picking a GP(IPBD),
[0081] 3) picking a set of several residues in the PPBD to vary;
the principal indicators of which residues to vary include: a) the
3D structure of the IPBD, b) sequences of homologous proteins, and
c) computer or theoretical modeling that indicates which residues
can tolerate different amino acids without disrupting the
underlying structure,
[0082] 4) picking a subset of the residues picked in Part II.3, to
be varied simultaneously; the principal considerations are the
number of different variants and which variants are within the
detection capabilities of the affinity separation system, and
setting the range of variation;
[0083] 5) implementing the variegation by:
[0084] a) synthesizing the part of the osp-pbd gene that encodes
the residues to be varied using a specific mixture of nucleotide
substrates for some or all of the bases encoding residues slated
for variation, thereby creating a population of DNA molecules,
denoted vgDNA,
[0085] b) ligating this vgDNA, by standard methods, into the
operative cloning vector (OCV) (e.g. a plasmid or
bacteriophage),
[0086] c) using the ligated DNA to transform cells, thereby
producing a population of transformed cells,
[0087] d) culturing (i.e. increasing in number) the population of
transformed cells and harvesting the population of GP(PBD)s, said
population being denoted as GP(vgPBD),
[0088] e) enriching the population for GPs that bind the target by
using affinity separation, with the chosen target molecule as
affinity molecule,
[0089] f) repeating steps II.5.d and II.5.e until a GP(SBD) having
improved binding to the target is isolated, and
[0090] g) testing the isolated SBD or SBDs for affinity and
specificity for the chosen target,
[0091] 6) repeating steps II.3, II.4, and II.5 until the desired
degree of binding is obtained.
[0092] Part II is repeated for each new target material. Part I
need be repeated only if no GP(IPBD) suitable to a chosen target is
available.
[0093] For each target, there are a large number of SBDs that may
be found by the method of the present invention. The process relies
on a combination of protein structural considerations,
probabilities, and targeted mutations with accumulation of
information. To increase the probability that some PBD in the
population will bind to the target, we generate as large a
population as we can conveniently subject to
selection-through-binding in one experiment. Key questions in
management of the method are "How many transformants can we
produce?", and "How small a component can we find through
selection-through-binding?". The optimum level of variegation is
determined by the maximum number of transformants and the selection
sensitivity, so that for any reasonable sensitivity we may use a
progressive process to obtain a series of proteins with higher and
higher affinity for the chosen target material.
[0094] The appended claims are hereby incorporated by reference
into this specification as an enumeration of the preferred
embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0095] FIG. 1 shows how a phage may be used as a genetic package.
At (a) we have a wild-type precoat protein lodged in the lipid
bilayer. The signal peptide is in the periplasmic space. At (b), a
chimeric precoat protein, with a potential binding domain
interposed between the signal peptide and the mature coat protein
sequence, is similarly trapped. At (c) and (d), the signal peptide
has been cleaved off the wild-type and chimeric proteins,
respectively, but certain residues of the coat protein sequence
interact with the lipid bilayer to prevent the mature protein from
passing entirely into the periplasm. At (e) and (f), mature
wild-type and chimeric protein are assembled into the coat of a
single stranded DNA phage as it emerges into the periplasmic space.
The phage will pass through the outer membrane into the medium
where it can be recovered and chromatographically evaluated.
[0096] FIG. 2 depicts (a) the optimal stereochemistry of a
disulfide bond, based on Creighton, "Disulfide Bonds and Protein
Stability" (CREI88) (the two possible torsion angles about the
disulfide bond of +90.degree. and -90.degree. are equally likely),
and (b) the standard geometric parameters for the disulfide bond,
following Katz and Kossiakoff (KATZ86). The average
C.alpha.-C.alpha. distance is 5-6 .ANG., and the typical S--S bond
length is .apprxeq.2.0 .ANG.. Many left-hand disulfides adopt as a
preferred geometry X1=-60.degree., X2=-60.degree., X3=-85.degree.,
X2=-60.degree., X1'=-60.degree., C.alpha.-C.alpha.=5.88 .ANG.;
right-hand disulfides are more variable.
[0097] FIG. 3 shows a mini-protein comprising eight residues,
numbered 4 through 11 and in which residues 5 and 10 are joined by
a disulfide. The .beta. carbons are labeled for residues 4, 6, 7,
8, 9, and 11; these residues are preferred sites of
variegation.
[0098] FIG. 4 shows the C.sub..alpha. of the coat protein of phage
f1.
[0099] FIG. 5 shows the construction of M13-MB51.
[0100] FIG. 6 shows construction of MR-BPTI, also known as BPTI-III
MK.
[0101] FIG. 7 illustrates fractionation of the Mini PEPI library on
HNE beads. The abscissae shows pH of buffer. The ordinants show
amount of phage (as fraction of input phage) obtained at given pH.
ordinants scaled by 10.sup.3.
[0102] FIG. 8 illustrates fractionation of the MYMUT PEPI library
on HNE beads. The abscissae shows pH of buffer. The ordinants show
amount of phage (as fraction of input phage) obtained at given pH.
Ordinants scaled by 10.sup.3.
[0103] FIG. 9 shows the elution profiles for EpiNE clones 1, 3, and
7. Each profile is scaled so that the peak is 1.0 to emphasize the
shape of the curve.
[0104] FIG. 10 shows pH profile for the binding of BPTI-III MK and
EpiNE1 on cathepsin G beads. The abscissae shows pH of buffer. The
ordinants show amount of phage (as fraction of input phage)
obtained at given pH. Ordinants scaled by 10.sup.3.
[0105] FIG. 11 shows pH profile for the fraxctionation of the MYMUT
Library on cathepsin G beads. The abscissae shows pH of buffer. The
ordinants show amount of phage (as fraction of input phage)
obtained at given pH. Ordinants scaled by 10.sup.3.
[0106] FIG. 12 shows a second fractionation of MYMUT library over
cathepsin G.
[0107] FIG. 13 shows elution profiles on immobilized cathepsin G
for phage selected for binding to cathepsin G.
[0108] FIG. 14 shows the C.sub..alpha.s of BPTI and interaction set
#2.
[0109] FIG. 15 shows the main chain of scorpion toxin (Brookhaven
Protein Data Bank entry 1SN3) residues 20 through 42. CYS.sub.25
and CYS.sub.41 are shown forming a disulfide. In the native protein
these groups form disulfides to other cysteines, but no main-chain
motion is required to bring the gamma sulphurs into acceptable
geometry. Residues, other than GLY, are labeled at the .beta.
carbon with the one-letter code.
[0110] FIG. 16 shows profiles of the elustion of phage that display
EpiNE7 and EpiNE7.23 froia HNE beads.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0111] OVERVIEW
[0112] I. DEFINITIONS AND ABBREVIATIONS
[0113] II. THE INITIAL POTENTIAL BINDING DOMAIN
[0114] A. Generally
[0115] B. Influence of Target Size on Choice of IPBD
[0116] C. Influence of Target Charge on Choice of IPBD
[0117] D. Other Considerations in the choice of IPBD
[0118] E. Bovine Pancreatic Trypsin Inhibitor (BPTI) as an IPBD
[0119] F. Mini-Proteins as IPBDs
[0120] G. Modified PBDs
[0121] III. VARIEGATION STRATEGY--MUTAGENESIS TO OBTAIN POTENTIAL
BINDING DOMAINS WITH DESIRED DIVERSITY
[0122] A. Generally
[0123] B. Identification of Residues to be Varied
[0124] C. Determining the Substitution Set for Each Parental
Residue
[0125] D. Special Considerations Relating to Variegation of
Mini-Proteins with Essential Cysteines
[0126] E. Planning the Second and Later Rounds of Variegation
[0127] IV. DISPLAY STRATEGY--DISPLAYING FOREIGN BINDING DOMAINS ON
THE SURFACE OF A "GENETIC PACKAGE"
[0128] A. General Requirements for Genetic Package
[0129] B. Phages for Use as Genetic Packages
[0130] C. Bacterial Cells as Genetic Packages
[0131] D. Bacterial Spores as Genetic Packages
[0132] E. Artificial Outer Surface Protein
[0133] F. Designing the osp::ipbd Gene Insert
[0134] G. Synthesis of Gene Inserts
[0135] H. Operative Cloning Vector
[0136] I. Transformation of Cells
[0137] J. Verification of Display Strategy
[0138] K. Analysis and Correction of Display Problems
[0139] V. AFFINITY SELECTION OF TARGET-BINDING MUTANTS
[0140] A. Affinity Separation Technology, Generally
[0141] B. Affinity Chromatography, Generally
[0142] C. Fluorescent-Activated Cell Sorting, Generally
[0143] D. Affinity Electrophoresis, Generally
[0144] E. Target Materials
[0145] F. Immobilization or Labeling of Target Material
[0146] G. Elution of Lower Affinity PBD-Bearing Packages
[0147] H. Optimization of Affinity Separation
[0148] I. Measuring the Sensitivity of Affinity Separation
[0149] J. Measuring the Efficiency of Separation
[0150] K. Reducing Selection due to Non-Specific Binding
[0151] L. Isolation of Genetic Package PBDs with Binding-to-Target
Phenotypes
[0152] M. Recovery of Packages
[0153] N. Amplifying the Enriched Packages
[0154] O. Determining Whether Further Enrichment is Needed
[0155] P. Characterizing the Putative SBDs
[0156] Q. Joint Selections
[0157] R. Selection for Non-Binding
[0158] S. Selection of Potential Binding Domains for Retention of
Structure
[0159] T. Engineering of Antagonists
[0160] VI. EXPLOITATION OF SUCCESSFUL BINDING DOMAINS AND
CORRESPONDING DNAS
[0161] A. Generally
[0162] B. Production of Novel Binding Proteins
[0163] C. Mini-Protein Production
[0164] D. Uses of Novel Binding Proteins
[0165] VII. EXAMPLES
[0166] I. Definitions and Abbreviations 1 K d ( x , y ) = [ x ] [ y
] [ x : y ] .
[0167] For the purposes of the appended claims, a protein P is a
binding protein if (1) For one molecular, ionic or atomic species
A, other than the variable domain of an antibody, the dissociation
constant K.sub.D (P,A) <10.sup.-6 moles/liter (preferably,
<10.sup.-7 moles/liter), and (2) for a different molecular,
ionic or atomic species B, K.sub.D (P,B) >10.sup.-4 moles/liter
(preferably, >10.sup.-1 moles/liter). As a result of these two
conditions, the protein P exhibits specificity for A over B, and a
minimum degree of affinity (or avidity) for A.
[0168] The exclusion of "variable domain of an antibody" in (1)
above is intended to make clear that for the purposes herein a
protein is not to be considered a "binding protein" merely because
it is antigenic. However, an antigen may nonetheless qualify as a
binding protein because it specifically binds to a substance other
than an antibody, e.g., an enzyme for its substrate, or a hormone
for its cellular receptor. Additionally, it should be pointed out
that "binding protein" may include a protein which binds
specifically to the Fc of an antibody, e.g., staphylococcal protein
A.
[0169] Normally, the binding protein will not be an antibody or a
antigen-binding derivative thereof. An antibody is a crosslinked
complex of four polypeptides (two heavy and two light chains). The
light chains of IgG have a molecular weight of .apprxeq.23,000
daltons and the heavy chains of .apprxeq.53,000 daltons. A single
binding unit is composed of the variable region of a heavy chain
(V.sub.H) and the variable region of a light chain (V.sub.L), each
about 110 amino-acid residues. The V.sub.H and V.sub.L regions are
held in proximity by a disulfide bond between the adjoining C.sub.L
and C.sub.H1 regions; altogether, these total 440 residues and
correspond to an Fab fragment. Derivatives of antibodies include
Fab fragments and the individual variable light and heavy domains.
A special case of antibody derivative is a "single chain antibody."
A "single-chain antibody" is a single chain polypeptide comprising
at least 200 amino acids, said amino acids forming two
antigen-binding regions connected by a peptide linker that allows
the two regions to fold together to bind the antigen in a manner
akin to that of an Fab fragment. Either the two antigen-binding
regions must be variable domains of known antibodies, or they must
(1) each fold into a .beta. barrel of nine strands that are
spatially related in the same way as are the nine strands of known
antibody variable light or heavy domains, and (2) fit together in
the same way as do the variable domains of said known antibody.
Generally speaking, this will require that, with the exception of
the amino acids corresponding to the hypervariable region, there is
at least 88% homology with the amino acids of the variable domain
of a known antibody.
[0170] While the present invention may be used to develop novel
antibodies through variegation of codons corresponding to the
hypervariable region of an antibody's variable domain, its primary
utility resides in the development of binding proteins which are
not antibodies or even variable domains of antibodies. Novel
antibodies can be obtained by immunological techniques; novel
enzymes, hormones, etc. cannot.
[0171] It will be appreciated that, as a result of evolution, the
antigen-binding domains of antibodies have acquired a structure
which tolerates great variability of sequence in the hypervariable
regions. The remainder of the variable domain is made up of
constant regions forming a distinctive structure, a nine strand
.beta. barrel, which hold the hypervariable regions (inter-strand
loops) in a fixed relationship with each other. Most other binding
proteins lack this molecular design which facilitates
diversification of binding characteristics. consequently, the
successful development of novel antibodies by modification of
sequences encoding known hypervariable regions--which, in nature,
vary from antibody to antibody--does not provide any guidance or
assurance of success in the development of novel,
non-immunoglobulin binding proteins.
[0172] It should further be noted that the affinity of antibodies
for their target epitopes is typically on the order of 10.sup.6 to
10.sup.10 liters/mole; many enzymes exhibit much greater affinities
(10.sup.9 to 10.sup.15 liters/mole) for their preferred substrates.
Thus, if the goal is to develop a binding protein with a very high
affinity for a target of interest, e.g., greater than 10.sup.10,
the antibody design may in fact be unduly limiting. Furthermore,
the complementarity-determining residues of an antibody comprises
many residues, 30 to 50. In most cases, it is not known which of
these residues participates directly in binding antigen. Thus,
picking an antibody as PPBD does not allow us to focus variegation
to a small number of residues.
[0173] Most larger proteins fold into distinguishable globules
called domains (ROSS81). Protein domains have been defined various
ways, but all definitions fall into one of three classes: a) those
that define a domain in terms of 3D atomic coordinates, b) those
that define a domain as an isolable, stable fragment of a larger
protein, and c) those that define a domain based on protein
sequence homology plus a method from class a) or b). Frequently,
different methods of defining domains applied to a single protein
yield identical or very similar domain boundaries. The diversity of
definitions for domains stems from the many ways that protein
domains are perceived to be important, including the concept of
domains in predicting the boundaries of stable fragments, and the
relationship of domains to protein folding, function, stability,
and evolution. The present invention emphasizes the retention of
the structured character of a domain even though its surface
residues are mutated. Consequently, definitions of "domain" which
emphasize stability--retention of the overall structure in the face
of perturbing forces such as elevated temperatures or chaotropic
agents--are favored, though atomic coordinates and protein sequence
homology are not completely ignored.
[0174] When a domain of a protein is primarily responsible for the
protein's ability to specifically bind a chosen target, it is
referred to herein as a "binding domain" (BD). A preliminary
operation is to engineer the appearance of a stable protein domain,
denoted as an "initial potential binding domain" (IPBD), on the
surface of a genetic package.
[0175] The term "variegated DNA" (vgDNA) refers to a mixture of DNA
molecules of the same or similar length which, when aligned, vary
at some codons so as to encode at each such codon a plurality of
different amino acids, but which encode only a single amino acid at
other codon positions. It is further understood that in variegated
DNA, the codons which are variable, and the range and frequency of
occurrence of the different amino acids which a given variable
codon encodes, are determined in advance by the synthesizer of the
DNA, even though the synthetic method does not allow one to know, a
priori, the sequence of ary individual DNA molecule in the mixture.
The number of designated variable codons in the variegated DNA is
preferably no more than 20 codons, and more preferably no more than
5-10 codons. The mix of amino acids encoded at each variable codon
may differ from codon to codon.
[0176] A population of genetic packages into which variegated DNA
has been introduced is likewise said to be "variegated".
[0177] For the purposes of this invention, the term "potential
binding protein" refers to a protein encoded by one species of DNA
molecule in a population of variegated DNA wherein the region of
variation appears in one or more subsequences encoding one or more
segments of the polypeptide having the potential of serving as a
binding domain for the target substance.
[0178] From time to time, it may be helpful to speak of the "parent
sequence" of the variegated DNA. When the novel binding domain
sought is an analogue of a known binding domain, the parent
sequence is the sequence that encodes the known binding domain. The
variegated DNA will be identical with this parent sequence at one
or more loci, but will diverge from it at chosen loci. When a
potential binding domain is designed from first principles, the
parent sequence is a sequence which encodes the amino acid sequence
that has been predicted to form the desired binding domain, and the
variegated DNA is a population of "daughter DNAS" that are related
to that parent by a recognizable sequence similarity.
[0179] A "chimeric protein" is a protein composed of a first amino
acid sequence substantially corresponding to the sequence of a
protein or to a large fragment of a protein (20 or more residues)
expressed by the species in which the chimeric protein is expressed
and a second amino acid sequence that does not substantially
correspond to an amino acid sequence of a protein expressed by the
first species but that does substantially correspond to the
sequence of a protein expressed by a second and different species
of organism. The second sequence is said to be foreign to the first
sequence.
[0180] One amino acid sequence of the chimeric proteins of the
present invention is typically derived from an outer surface
protein of a "genetic package" as hereafter defined. The second
amino acid sequence is one which, if expressed alone, would have
the characteristics of a protein (or a domain thereof) but is
incorporated into the chimeric protein as a recognizable domain
thereof. It may appear at the amino or carboxy terminal of the
first amino acid sequence (with or without an intervening spacer),
or it may interrupt the first amino acid sequence. The first amino
acid sequence may correspond exactly to a surface protein of the
genetic package, or it may be modified, e.g., to facilitate the
display of the binding domain.
[0181] In the present invention, the words "select" and "selection"
are used in the genetic sense; i.e. a biological process whereby a
phenotypic characteristic is used to enrich a population for those
organisms displaying the desired phenotype.
[0182] One affinity separation is called a "separation cycle"; one
pass of variegation followed by as many separation cycles as are
needed to isolate an SBD, is called a "variegation cycle". The
amino acid sequence of one SBD from one round becomes the PPBD to
the next variegation cycle. We perform variegation cycles
iteratively until the desired affinity and specificity of binding
between an SBD and chosen target are achieved.
[0183] The following abbreviations will be used throughout the
present specification:
1 Abbreviation Meaning GP Genetic Package, e.g. a bacteriophage
wtGP Wild-type GP X Any protein x Thegene for protein X BD Binding
Domain BPTI Bovine pancreatic trypsin inhibitor, identical to
aprotinin (Merck Index, entry 784, p. 119) IPBD Initial Potential
Binding Domain, e.g. BPTI PBD Potential Binding Domain, e.g. a
derivative of BPTI SBD Successful Binding Domain, e.g. a derivative
of BPTI selected for binding to a target PPBD Parental Potential
Binding Domain, i.e. an IPBD or an SBD from a previous selection
OSP Outer Surface Protein, e.g. coat protein of a phage or LamB
from E. coli OSP-PBD Fusion of an OSP and a PBD, order of fusion
not specified OSTS Outer Surface Transport Signal GP(x) A genetic
package containing the x gene GP(X) A genetic package that displays
X on its outer surface GP(osp-pbd) GP containing an osp-pbd gene
GP(OSP-PBD) A genetic package that displays PBD on its outside as a
fusion to OSP GP(pbd) GP containing a pbd gene, osp implicit
GP(PBD) A genetic package displaying PBD on its outside, OSP
unspecified {Q} An affinity matrix supporting "Q", e.g. {T4
lysozyme} is T4 lysozyme attached to an affinity matrix AfM(W) A
molecule having affinity for "W", e.g. trypsin is an AfM(BPTI)
AfM(W)* AfM(W) carrying a label, e.g. 125.sub.I XINDUCE A chemical
that can induce expression of a gene, e.g. IPTG for the lacUV5
promoter OCV Operative Cloning Vector K.sub.d A bimolecular
dissociation constant, K.sub.d = [A][B]/[A:B] K.sub.T K.sub.t = [T]
[SBD]/[T:SBD] (T is a target) K.sub.N K.sub.N = [N] [SBD]/[N:SBD]
(N is a non-target) DoAMoM Density of AfM(W) on affinity matrix
mfaa Most-Favored amino acid lfaa Least-Favored amino acid Abun(x)
Abundance of DNA molecules encoding amino acid x OMP Outer membrane
protein nt nucleotide SP-I Signal-sequence Peptidase I Y.sub.DQ
Yield of ssDNA up to Q bases long M.sub.DNA Maximum length of ssDNA
that can be synthesized in accep- table yield Y.sub.Pl Yield of
plasmid DNA per volume of culture L.sub.eff DNA ligation efficiency
M.sub.ntv Maximum number of transformants produced from Y.sub.D100
DNA of Insert .sup.ceff Efficiency of chromatographic enrichment,
enrichment per pass .sup.csensi Sensitivity of chromatographic
separation, can find 1 in N, .sup.Nchrom Maximum number of
enrichment cycles per variegation cycle .sup.Serr Error level in
synthesizing vgDNA :: in-frame genetic fusion or protein produced
from in-frame fused gene
[0184] II. The Initial Potential Binding Domain (IPBD):
[0185] II.A. Generally
[0186] The initial potential binding domain may be: 1) a domain of
a naturally occurring protein, 2) a non-naturally occurring domain
which substantially corresponds in sequence to a naturally
occurring domain, but which differs from it in sequence by one or
more substitutions, insertions or deletions, 3) a domain
substantially corresponding in sequence to a hybrid of subsequences
of two or more naturally occurring proteins, or 4) an artificial
domain designed entirely on theoretical grounds based on knowledge
of amino acid geometries and statistical evidence of secondary
structure preferences of amino acids. (However, the limitations of
a priori protein design prompted the present invention.) Usually,
the domain will be a known binding domain, or at least a homologue
thereof, but it may be derived from a protein which, while not
possessing a known binding activity, possesses a secondary or
higher structure that lends itself to binding activity (clefts,
grooves, etc.). The protein to which the IPBD is related need not
have any specific affinity for the target material.
[0187] In determining whether sequences should be deemed to
"substantially correspond", one should consider the following
issues: the degree of sequence similarity when the sequences are
aligned for best fit according to standard algorithms, the
similarity in the connectivity patterns of any crosslinks (e.g.,
disulfide bonds), the degree to which the proteins have similar
three-dimensional structures, as indicated by, e.g., X-ray
diffraction analysis or NMR, and the degree to which the sequenced
proteins have similar biological activity. In this context, it
should be noted that among the serine protease inhibitors, there
are families of proteins recognized to be homologous in which there
are pairs of members with as little as 30% sequence homology.
[0188] A candidate IPBD should meet the following criteria:
[0189] 1) a domain exists that will remain stable under the
conditions of its intended use (the domain may comprise the entire
protein that will be inserted, e.g. BPTI, .alpha.-conotoxin GI, or
CMTI-III),
[0190] 2) knowledge of the amino acid sequence is obtainable,
[0191] 3) a molecule is obtainable having specific and high
affinity for the IPBD, AfM(IPBD).
[0192] Preferably, in order to guide the variegation strategy,
knowledge of the identity of the residues on the domain's outer
surface, and their spatial relationships, is obtainable; however,
this consideration is less important if the binding domain is
small, e.g., under 40 residues.
[0193] Preferably, the IPBD is no larger than necessary because
small SBDs (for example, less than 30 amino acids) can be
chemically synthesized and because it is easier to arrange
restriction sites in smaller amino-acid sequences. For PBDs smaller
than about 40 residues, an added advantage is that the entire
variegated pbd gene can be synthesized in one piece. In that case,
we need arrange only suitable restriction sites in the osp gene. A
smaller protein minimizes the metabolic strain on the GP or the
host of the GP. The IPBD is preferably smaller than about 200
residues. The IPBD must also be large enough to have acceptable
binding affinity and specificity. For an IPBD lacking covalent
crosslinks, such as disulfide bonds, the IPBD is preferably at
least 40 residues; it may be as small as six residues if it
contains a crosslink. These small, crosslinked IPBDs, known as
"mini-proteins", are discussed in more detail later in this
section.
[0194] Some candidate IPBDs, which meet the conditions set forth
above, will be more suitable than others. Information about
candidate IPBDs that will be used to judge the suitability of the
IPBD includes: 1) a 3D structure (knowledge strongly preferred), 2)
one or more sequences homologous to the IPBD (the more homologous
sequences known, the better), 3) the pI of the IPBD (knowledge
desirable when target is highly charged), 4) the stability and
solubility as a function of temperature, pH and ionic strength
(preferably known to be stable over a wide range and soluble in
conditions of intended use), 5) ability to bind metal ions such as
Ca.sup.++ or Mg.sup.++ (knowledge preferred; binding per se, no
preference), 6) enzymatic activities, if any (knowledge preferred,
activity per se has uses but may cause problems), 7) binding
properties, if any (knowledge preferred, specific binding also
preferred), 8) availability of a molecule having specific and
strong affinity (K.sub.d<10.sup.-11 M) for the IPBD (preferred),
9) availability of a molecule having specific and medium affinity
(10.sup.-8 M<K.sub.d<10.sup.-6 M) for the IPBD (preferred),
10) the sequence of a mutant of IPBD that does not bind to the
affinity molecule(s) (preferred), and 11) absorption spectrum in
visible, UV, NMR, etc. (characteristic absorption preferred).
[0195] If only one species of molecule having affinity for IPBD
(AfM(IPBD)) is available, it will be used to: a) detect the IPBD on
the GP surface, b) optimize expression level and density of the
affinity molecule on the matrix, and c) determine the efficiency
and sensitivity of the affinity separation. As noted above,
however, one would prefer to have available two species of
AfM(IPBD), one with high and one with moderate affinity for the
IPBD. The species with high affinity would be used in initial
detection and in determining efficiency and sensitivity, and the
species with moderate affinity would be used in optimization.
[0196] If the IPBD is not itself a binding domain of a known
binding protein, or if its native target has not been purified, an
antibody raised against the IPBD may be used as the affinity
molecule. Use of an antibody for this purpose should not be taken
to mean that the antibody is the ultimate target.
[0197] There are many candidate IPBDs for which all of the above
information is available or is reasonably practical to obtain, for
example, bovine pancreatic trypsin inhibitor (BPTI, 58 residues),
CMTI-III (29 residues), crambin (46 residues), third domain of
ovomucoid (56 residues), heat-stable enterotoxin (ST-Ia of E. coli)
(18 residues), .alpha.-Conotoxin GI (13 residues), .mu.-Conotoxin
GIII (22 residues), Conus King Kong mini-protein (27 residues), T4
lysozyme (164 residues), and azurin (128 residues). Structural
information can be obtained from X-ray or neutron diffraction
studies, NMR, chemical cross linking or labeling, modeling from
known structures of related proteins, or from theoretical
calculations. 3D structural information obtained by X-ray
diffraction, neutron diffraction or NMR is preferred because these
methods allow localization of almost all of the atoms to within
defined limits. Table 50 lists several preferred IPBDs. Works
related to determination of 3D structure of small proteins via NMR
inculde: CHAZ85, PEAS90, PEAS88, CLOR86, CLOR87a, HEIT89, LECO87,
WAGN79, and PARD89.
[0198] In some cases, a protein having some affinity for the target
may be a preferred IPBD even though some other criteria are not
optimally met. For example, the V1 domain of CD4 is a good choice
as IPBD for a protein that binds to gp120 of HIV. It is known that
mutations in the region 42 to 55 of V1 greatly affect gp120 binding
and that other mutations either have much less effect or completely
disrupt the structure of V1. Similarly, tumor necrosis factor (TNF)
would be a good initial choice if one wants a TNF-like molecule
having higher affinity for the TNF receptor.
[0199] Membrane-bound proteins are not preferred IPBPs, though they
may serve as a source of outer surface transport signals. One
should distinguish between membrane-bound proteins, such as LamB or
OmpF, that cross the membrane several times forming a structure
that is embedded in the lipid bilayer and in which the exposed
regions are the loops that join trans-membrane segments, from
non-embedded proteins, such as the soluble domains of CD4, that are
simply anchored to the membrane. This is an important distinction
because it is quite difficult to create a soluble derivative of a
membrane-bound protein. Soluble binding proteins are in general
more useful since purification is simpler and they are more
tractable and more versatile assay reagents.
[0200] Most of the PBDs derived from a PPBD according to the
process of the present invention will have been derived by
variegation at residues having side groups directed toward the
solvent. Reidhaar-Olson and Sauer (REID88a) found that exposed
residues can accept a wide range of amino acids, while buried
residues are more limited in this regard. Surface mutations
typically have only small effects on melting temperature of the
PBD, but may reduce the stability of the PBD. Hence the chosen IPBD
should have a high melting temperature (50.degree. C. acceptable,
the higher the better; BPTI melts at 95.degree. C.) and be stable
over a wide pH range (8.0 to 3.0 acceptable; 11.0 to 2.0
preferred), so that the SBDs derived from the chosen IPBD by
mutation and selection-through-binding will retain sufficient
stability. Preferably, the substitutions in the IPBD yielding the
various PBDs do not reduce the melting point of the domain below
.apprxeq.40.degree. C. Mutations may arise that increase the
stability of SBDs relative to the IPBD, but the process of the
present invention does not depend upon this occurring. Proteins
containing covalent crosslinks, such as multiple disulfides, are
usually sufficient stable. A protein having at least two disulfides
and having at least 1 disulfide per every twenty residues may be
presumed to be sufficiently stable.
[0201] Two general characteristics of the target molecule, size and
charge, make certain classes of IPBDs more likely than other
classes to yield derivatives that will bind specifically to the
target. Because these are very general characteristics, one can
divide all targets into six classes: a) large positive, b) large
neutral, c) large negative, d) small positive, e) small neutral,
and f) small negative. A small collection of IPBDs, one or a few
corresponding to each class of target, will contain a preferred
candidate IPBD for any chosen target.
[0202] Alternatively, the user may elect to engineer a GP(IPBD) for
a particular target; criteria are given below that relate target
size and charge to the choice of IPBD.
[0203] II.B. Influence of Target Size on Choice of IPBD:
[0204] If the target is a protein or other macromolecule a
preferred embodiment of the IPBD is a small protein such as the
Cucurbita maxima trypsin inhibitor III (29 residues), BPTI from Bos
Taurus (58 residues), crambin from rape seed (46 residues), or the
third domain of ovomucoid from Coturnix coturnix Japonica (Japanese
quail) (56 residues), because targets from this class have clefts
and grooves that can accommodate small proteins in highly specific
ways. If the target is a macromolecule lacking a compact structure,
such as starch, it should be treated as if it were a small
molecule. Extended macromolecules with defined 3D structure, such
as collagen, should be treated as large molecules.
[0205] If the target is a small molecule, such as a steroid, a
preferred embodiment of the IPBD is a protein of about 80-200
residues, such as ribonuclease from Bos taurus (124 residues),
ribonuclease from Aspergillus oruzae (104 residues), hen egg white
lysozyme from Gallus callus (129 residues), azurin from Pseudomonas
aerugenosa (128 residues), or T4 lysozyme (164 residues), because
such proteins have clefts and grooves into which the small target
molecules can fit. The Brookhaven Protein Data Bank contains 3D
structures for all of the proteins listed. Genes encoding proteins
as large as T4 lysozyme can be manipulated by standard techniques
for the purposes of this invention.
[0206] If the target is a mineral, insoluble in water, one
considers the nature of the molecular surface of the mineral.
Minerals that have smooth surfaces, such as crystalline silicon,
are best addressed with medium to large proteins, such as
ribonuclease, as IPBD in order to have sufficient contact area and
specificity. Minerals with rough, grooved surfaces, such as
zeolites, could be bound either by small proteins, such as BPTI, or
larger proteins, such as T4 lysozyme.
[0207] II.C. Influence of Target Charge on Choice of IPBD:
[0208] Electrostatic repulsion between molecules of like charge can
prevent molecules with highly complementary surfaces from binding.
Therefore, it is preferred that, under the conditions of intended
use, the IPBD and the target molecule either have opposite charge
or that one of them is neutral. In some cases it has been observed
that protein molecules bind in such a way that like charged groups
are juxtaposed by including oppositely charged counter ions in the
molecular interface. Thus, inclusion of counter ions can reduce or
eliminate electrostatic repulsion and the user may elect to include
ions in the eluants used in the affinity separation step.
Polyvalent ions are more effective at reducing repulsion than
monovalent ions.
[0209] II.D. Other Considerations in the Choice of IPBD:
[0210] If the chosen IPBD is an enzyme, it may be necessary to
change one or more residues in the active site to inactivate enzyme
function. For example, if the IPBD were T4 lysozyme and the GP were
E. coli cells or M13, we would need to inactivate the lysozyme
because otherwise it would lyse the cells. If, on the other hand,
the GP were .PHI.X174, then inactivation of lysozyme may not be
needed because T4 lysozyme can be overproduced inside E. coli cells
without detrimental effects and .PHI.X174 forms intracellularly. It
is preferred to inactivate enzyme IPBDs that might be harmful to
the GP or its host by substituting mutant amino acids at one or
more residues of the active site. It is permitted to vary one or
more of the residues that were changed to abolish the original
enzymatic activity of the IPBD. Those GPs that receive osp-pbd
genes encoding an active enzyme may die, but the majority of
sequences will not be deleterious.
[0211] If the binding protein is intended for therapeutic use in
humans or animals, the IPBD may be chosen from proteins native to
the designated recipient to minimize the possibility of antigenic
reactions.
[0212] II.E. Bovine Pancreatic Trypsin Inhibitor (BPTI) as an
IPBD:
[0213] BPTI is an especially preferred IPBD because it meets or
exceeds all the criteria: it is a small, very stable protein with a
well known 3D structure. Marks et al. (MARK86) have shown that a
fusion of the phoA signal peptide gene fragment and DNA coding for
the mature form of BPTI caused native BPTI to appear in the
periplasm of E. coli, demonstrating that there is nothing in the
structure of BPTI to prevent its being secreted.
[0214] The structure of BPTI is maintained even when one or another
of the disulfides is removed, either by chemical blocking or by
genetic alteration of the amino-acid sequence. The stabilizing
influence of the disulfides in BPTI is not equally distributed.
Goldenberg (GOLD85) reports that blocking CYS14 and CYS38 lowers
the Tm of BPTI to .apprxeq.75.degree. C. while chemical blocking of
either of the other disulfides lowers Tm to below 40.degree. C.
Chemically blocking a disulfide may lower Tm more than mutating the
cysteines to other amino-acid types because the bulky blocking
groups are more destabilizing than removal of the disulfide. Marks
et al. (MARK87) replaced both CYS14 and CYS38 with either two
alanines or two threonines. The CYS14/CYS38 cystine bridge that
Marks et al. removed is the one very close to the scissile bond in
BPTI; surprisingly, both mutant molecules functioned as trypsin
inhibitors. Schnabel et al. (SCHN86) report preparation of
aprotinin(C14A,C38A) by use of Raney nickel. Eigenbrot et al.
(EIGE90) report the X-ray structure of BPTI(C30A/C51A) which is
stable to at least 50.degree. C. The backbone of this mutant is as
similar to BPTI as are the backbones of BPTI molecules that sit in
different crystal lattices. This indicates that BPTI is redundantly
stable and so is likely to fold into approximately the same
structure despite numerous surface mutations. Using the knowledge
of homologues, vide infra, we can infer which residues should not
be varied if the basic BPTI structure is to be maintained.
[0215] The 3D structure of BPTI has been determined at high
resolution by X-ray diffraction (HUBE77, MARQ83, WLOD84, WLOD87a,
WLOD87b), neutron diffraction (WLOD84), and by NMR (WAGN87). In one
of the X-ray structures deposited in the Brookhaven Protein Data
Bank, entry 6PTI, there was no electron density for A58, indicating
that A58 has no uniquely defined conformation. Thus we know that
the carboxy group does not make any essential interaction in the
folded structure. The amino terminus of BPTI is very near to the
carboxy terminus. Goldenberg and Creighton reported on circularized
BPTI and circularly permuted BPTI (GOLD83). Some proteins
homologous to BPTI have more or fewer residues at either
terminus.
[0216] BPTI has been called "the hydrogen atom of protein folding"
and has been the subject of numerous experimental and theoretical
studies (STAT87, SCHW87, GOLD83, CHAZ83, CREI74, CREI77a, CREI77b,
CREI80, SIEK87, SINH90, RUEH73, HUBE74, HUBE75, HUBE77 and
others).
[0217] BPTI has the added advantage that at least 59 homologous
proteins are known. Table 13 shows the sequences of 39 homologues.
A tally of ionizable groups in 59 homologues is shown in Table 14
and the composite of amino acid types occurring at each residue is
shown in Table 15.
[0218] BPTI is freely soluble and is not known to bind metal ions.
BPTI has no known enzymatic activity. BPTI is not toxic.
[0219] All of the conserved residues are buried; of the six fully
conserved residues only G37 has noticeable exposure. The solvent
accessibility of each residue in BPTI is given in Table 16 which
was calculated from the entry "6PTI" in the Brookhaven Protein Data
Bank with a solvent radius of 1.4 .ANG., the atomic radii given in
Table 7, and the method of Lee and Richards (LEEB71). Each of the
52 non-conserved residues can accommodate two or more kinds of
amino acids. By independently substituting at each residue only
those amino acids already observed at that residue, we could obtain
approximately 1.6.multidot.10.sup.43 different amino acid
sequences, most of which will fold into structures very similar to
BPTI.
[0220] BPTI will be especially useful as a IPBD for macromolecular
targets. BPTI and BPTI homologues bind tightly and with high
specificity to a number of enzyme macromolecules.
[0221] BPTI is strongly positively charged except at very high pH,
thus BPTI is useful as IPBD for targets that are not also strongly
positive under the conditions of intended use. There exist
homologues of BPTI, however, having quite different charges (viz.
SCI-III from Bombyx mori at -7 and the trypsin inhibitor from
bovine colostrum at -1). Once a genetic package is found that
displays BPTI on its surface, the sequence of the BPTI domain can
be replaced by one of the homologous sequences to produce acidic or
neutral IPBDS.
[0222] BPTI is quite small; if this should cause a pharmacological
problem, two or more BPTI-derived domains may be joined as in
humans BPTI homologues, one of which has two domains (BALD85,
ALBR83b) and another has three (WUNT88).
[0223] Another possible pharmacological problem is immunigenicity.
BPTI has been used in humans with very few adverse effects.
Siekmann et al. (SIEK89) have studied immunological characteristics
of BPTI and some homologues. It is an advantage of the method of
the present invention that a variety of SBDs can be obtained so
that, if one derivative proves to be antigenic, a different SBD may
be used. Furthermore, one can reduce the probability of immune
response by starting with a human protein, such as LACI (a BPTI
homologue) (WUNT88, GIRA89) or Inter-.alpha.-Trypsin Inhibitor
(ALBR83a, ALBR83b, DIAR90, ENGH89, TRIB86, GEBH86, GEBH90, KAUM86,
ODOM90, SALI90).
[0224] Further, a BPTI-derived gene fragment, coding for a novel
binding domain, could be fused in frame to a gene fragment coding
for other proteins, such as serum albumin or the constant parts of
IgG.
[0225] Tschesche et al. (TSCH87) reported on the binding of several
BPTI derivatives to various proteases:
2 Dissociation constants for BPTI derivatives, Molar. Trypsin
Chymotrypsin Elastase Elastase (bovine (bovine (porcine (human
Residue #15 pancreas) pancreas) pancreas) leukocytes) lysine 6.0
.multidot. 10.sup.-14 9.0 .multidot. 10.sup.-9 -- 3.5 .multidot.
10.sup.-6 glycine -- -- + 7.0 .multidot. 10.sup.-9 alanine + -- 2.8
.multidot. 10.sup.-8 2.5 .multidot. 10.sup.-9 valine -- -- 5.7
.multidot. 10.sup.-8 .sup. 1.1 .multidot. 10.sup.-10 leucine -- --
1.9 .multidot. 10.sup.-8 2.9 .multidot. 10.sup.-9
[0226] From the report of Tschesche et al. we infer that molecular
pairs marked "+" have K.sub.ds>3.5.multidot.10.sup.-6 M and that
molecular pairs marked "-" have
K.sub.ds>>3.5.multidot.10.sup.-6 M. Because of the wealth of
data about the binding of BPTI and various mutants to trypsin and
other proteases (TSCH87), we can proceed in various ways in
optimizing the affinity separation conditions. (For other PBDs, we
can obtain two different monoclonal antibodies, one with a high
affinity having K.sub.d of order 10.sup.-11 M, and one with a
moderate affinity having K.sub.d on the order of 10.sup.-6 M.)
[0227] Works concerning BPTI and its homologues include: KIDO88,
PONT88, KIDO90, AUER87, AUER90, SCOT87b, AUER88, AUER89, BECK88b,
WACH79, WACH80, BECK89a, DUFT85, FIOR88, GIRA89, GOLD84, GOLD88,
HOCH84, RITO83, NORR89a, NORR89b, OLTE89, SWAI88, and WAGN79.
[0228] II.F Mini-proteins as IPBDs:
[0229] A polypeptide is a polymer composed of a single chain of the
same or different amino acids joined by peptide bonds. Linear
peptides can take up a very large number of different conformations
through internal rotations about the main chain single bonds of
each .alpha. carbon. These rotations are hindered to varying
degrees by side groups, with glycine interfering the least, and
valine, isoleucine and, especially, proline, the most. A
polypeptide of 20 residues may have 10.sup.20 different
conformations which it may assume by various internal
rotations.
[0230] Proteins are polypeptides which, as a result of stabilizing
interactions between amino acids that are not in adjacent positions
in the chain, have folded into a well-defined conformation. This
folding is usually essential to their biological activity.
[0231] For polypeptides of 40-60 residues or longer, noncovalent
forces such as hydrogen bonds, salt bridges, and hydrophobic
"interactions" are sufficient to stabilize a particular folding or
conformation. The polypeptide's constituent segments are held to
more or less that conformation unless it is perturbed by a
denaturant such as rising temperature or decreasing pH, whereupon
the polypeptide unfolds or "melts". The smaller the peptide, the
more likely it is that its conformation will be determined by the
environment. If a small unconstrained peptide has biological
activity, the peptide ligand will be in essence a random coil until
it comes into proximity with its receptor. The receptor accepts the
peptide only in one or a few conformations because alternative
conformations are disfavored by unfavorable van der Waals and other
non-covalent interactions.
[0232] Small polypeptides have potential advantages over larger
polypeptides when used as therapeutic or diagnostic agents,
including (but not limited to):
[0233] a) better penetration into tissues,
[0234] b) faster elimination from the circulation (important for
imaging agents),
[0235] c) lower antigenicity, and
[0236] d) higher activity per mass.
[0237] Moreover, polypeptides of under about 50 residues have the
advantage of accessibility via chemical synthesis; polypeptides of
under about 30 residues are more easily synthesized than are larger
polypeptides. Thus, it would be desirable to be able to employ the
combination of variegation and affinity selection to identify small
polypeptides which bind a target of choice.
[0238] Polypeptides of this size, however, have disadvantages as
binding molecules. According to Olivera et al. (OLIV90a): "Peptides
in this size range normally equilibrate among many conformations
(in order to have a fixed conformation, proteins generally have to
be much larger)." Specific binding of a peptide to a target
molecule requires the peptide to take up one conformation that is
complementary to the binding site. For a decapeptide with three
isoenergetic conformations (e.g., .beta. strand, .alpha. helix, and
reverse turn) at each residue, there are about 6..multidot.10.sup.4
possible overall conformations. Assuming these conformations to be
equi-probable for the unconstrained decapeptide, if only one of the
possible conformations bound to the binding site, then the affinity
of the peptide for the target would expected to be about
6.multidot.10.sup.4 higher if it could be constrained to that
single effective conformation. Thus, the unconstrained decapeptide,
relative to a decapeptide constrained to the correct conformation,
would be expected to exhibit lower affinity. It would also exhibit
lower specificity, since one of the other conformations of the
unconstrained decapeptide might be one which bound tightly to a
material other than the intended target. By way of corollary, it
could have less resistance to degradation by proteases, since it
would be more likely to provide a binding site for the
protease.
[0239] In one embodiment, the present invention overcomes these
problems, while retaining the advantages of smaller polypeptides,
by fostering the biosynthesis of novel mini-proteins having the
desired binding characteristics. Mini-Proteins are small
polypeptides (usually less than about 60 residues) which, while too
small to have a stable conformation as a result of noncovalent
forces alone, are covalently crosslinked (e.g., by disulfide bonds)
into a stable conformation and hence have biological activities
more typical of larger protein molecules than of unconstrained
polypeptides of comparable size.
[0240] When mini-proteins are variegated, the residues which are
covalently crosslinked in the parental molecule are left unchanged,
thereby stabilizing the conformation. For example, in the
variegation of a disulfide bonded mini-protein, certain cysteines
are invariant so that under the conditions of expression and
display, covalent crosslinks (e.g., disulfide bonds between one or
more pairs of cysteines) form, and substantially constrain the
conformation which may be adopted by the hypervariable linearly
intermediate amino acids. In other words, a constraining
scaffolding is engineered into polypeptides which are otherwise
extensively randomized.
[0241] Once a mini-protein of desired binding characteristics is
characterized, it may be produced, not only by recombinant DNA
techniques, but also by nonbiological synthetic methods.
[0242] In vitro, disulfide bridges can form spontaneously in
polypeptides as a result of air oxidation. Matters are more
complicated in vivo. Very few intracellular proteins have disulfide
bridges, probably because a strong reducing environment is
maintained by the glutathione system. Disulfide bridges are common
in proteins that travel or operate in intracellular spaces, such as
snake venoms and other toxins (e.g., conotoxins, charybdotoxin,
bacterial enterotoxins), peptide hormones, digestive enzymes,
complement proteins, immunoglobulins, lysozymes, protease
inhibitors (BPTI and its homologues, CMTI-III (Cucurbita maxima
trypsin inhibitor III) and its homologues, hirudin, etc.) and milk
proteins.
[0243] Disulfide bonds that close tight intrachain loops have been
found in pepsin, thioredoxin, insulin A-chain, silk fibroin, and
lipoamide dehydrogenase. The bridged cysteine residues are
separated by one to four residues along the polypeptide chain.
Model building, X-ray diffraction analysis, and NMR studies have
shown that the .alpha. carbon path of such loops is usually flat
and rigid.
[0244] There are two types of disulfide bridges in immunoglobulins.
One is the conserved intrachain bridge, spanning about 60 to 70
amino acid residues and found, repeatedly, in almost every
immunoglobulin domain. Buried deep between the opposing .beta.
sheets, these bridges are shielded from solvent and ordinarily can
be reduced only in the presence of denaturing agents. The remaining
disulfide bridges are mainly interchain bonds and are located on
the surface of the molecule; they are accessible to solvent and
relatively easily reduced (STEI85). The disulfide bridges of the
mini-proteins of the present invention are intrachain linkages
between cysteines having much smaller chain spacings.
[0245] For the purpose of the appended claims, a mini-protein has
between about eight and about sixty residues. However, it will be
understood that a chimeric surface protein presenting a
mini-protein as a domain will normally have more than sixty
residues. Polypeptides containing intrachain disulfide bonds may be
characterized as cyclic in nature, since a closed circle of
covalently bonded atoms is defined by the two cysteines, the
intermediate amino acid residues, their peptidyl bonds, and the
disulfide bond. The terms "cycle", "span" and "segment" will be
used to define certain structural features of the polypeptides. An
intrachain disulfide bridge connecting amino acids 3 and 8 of a 16
residue polypeptide will be said herein to have a cycle of 6 and a
span of 4. If amino acids 4 and 12 are also disulfide bonded, then
they form a second cycle of 9 with a span of 7. Together, the four
cysteines divide the polypeptide into four intercysteine segments
(1-2, 5-7, 9-11, and 13-16). (Note that there is no segment between
Cys3 and Cys4.)
[0246] The connectivity pattern of a crosslinked mini-protein is a
simple description of the relative location of the termini of the
crosslinks. For example, for a mini-protein with two disulfide
bonds, the connectivity pattern "1-3, 2-4" means that the first
crosslinked cysteine is disulfide bonded to the third crosslinked
cysteine (in the primary sequence), and the second to the
fourth.
[0247] The degree to which the crosslink constrains the
conformational freedom of the mini-protein, and the degree to which
it stabilizes the mini-protein, may be assessed by a number of
means. These include absorption spectroscopy (which can reveal
whether an amino acid is buried or exposed), circular dichroism
studies (which provides a general picture of the helical content of
the protein), nuclear magnetic resonance imaging (which reveals the
number of nuclei in a particular chemical environment as well as
the mobility of nuclei), and X-ray or neutron diffraction analysis
of protein crystals. The stability of the mini-protein may be
ascertained by monitoring the changes in absorption at various
wavelengths as a function of temperature, pH, etc.; buried residues
become exposed as the protein unfolds. Similarly, the unfolding of
the mini-protein as a result of denaturing conditions results in
changes in NMR line positions and widths. Circular dichroism (CD)
spectra are extremely sensitive to conformation.
[0248] The variegated disulfide-bonded mini-proteins of the present
invention fall into several classes.
[0249] Class I mini-proteins are those featuring a single pair of
cysteines capable of interacting to form a disulfide bond, said
bond having a span of no more than nine residues. This disulfide
bridge preferably has a span of at least two residues; this is a
function of the geometry of the disulfide bond. When the spacing is
two or three residues, one residue is preferably glycine in order
to reduce the strain on the bridged residues. The upper limit on
spacing is less precise, however, in general, the greater the
spacing, the less the constraint on conformation imposed on the
linearly intermediate amino acid residues by the disulfide
bond.
[0250] The main chain of such a peptide has very little freedom,
but is not stressed. The free energy released when the disulfide
forms exceeds the free energy lost by the main-chain when locked
into a conformation that brings the cysteines together. Having lost
the free energy of disulfide formation, the proximal ends of the
side groups are held in more or less fixed relation to each other.
When binding to a target, the domain does not need to expend free
energy getting into the correct conformation. The domain can not
jump into some other conformation and bind a non-target.
[0251] A disulfide bridge with a span of 4 or 5 is especially
preferred. If the span is increased to 6, the constraining
influence is reduced. In this case, we prefer that at least one of
the enclosed residues be an amino acid that imposes restrictions on
the main-chain geometry. Proline imposes the most restriction.
Valine and isoleucine restrict the main chain to a lesser extent.
The preferred position for this constraining non-cysteine residue
is adjacent to one of the invariant cysteines, however, it may be
one of the other bridged residues. If the span is seven, we prefer
to include two amino acids that limit main-chain conformation.
These amino acids could be at any of the seven positions, but are
preferably the two bridged residues that are immediately adjacent
to the cysteines. If the span is eight or nine, additional
constraining amino acids may be provided.
[0252] The disulfide bond of a class I mini-proteins is exposed to
solvent. Thus, one should avoid exposing the variegated population
of GPs that display class I mini-proteins to reagents that rupture
disulfides; Creighton names several such reagents (CREI88).
[0253] Class II mini-proteins are those featuring a single
disulfide bond having a span of greater than nine amino acids. The
bridged amino acids form secondary structures which help to
stabilize their conformation. Preferably, these intermediate amino
acids form hairpin supersecondary structures such as those
schematized below: 1
[0254] Secondary structures are stabilized by hydrogen bonds
between amide nitrogen and carbonyl groups, by interactions between
charged side groups and helix dipoles, and by van der Waals
contacts. One abundant secondary structure in proteins is the
.alpha.-helix. The a helix has 3.6 residues per turn, a 1.5 .ANG.
rise per residue, and .alpha. helical radius of 2.3 .ANG.. All
observed .alpha.-helices are right-handed. The torsion angles .phi.
(-57.degree.) and .psi. (-47.degree.) are favorable for most
residues, and the hydrogen bond between the backbone carbonyl
oxygen of each residue and the backbone NH of the fourth residue
along the chain is 2.86 .ANG. long (nearly the optimal distance)
and virtually straight. Since the hydrogen bonds all point in the
same direction, the .alpha. helix has a considerable dipole moment
(carboxy terminus negative).
[0255] The .beta. strand may be considered an elongated helix with
2.3 residues per turn, a translation of 3.3 .ANG. per residue, and
.alpha. helical radius of 1.0 .ANG.. Alone, a .beta. strand forms
no main-chain hydrogen bonds. Most commonly, .beta. strands are
found in twisted (rather than planar) parallel, antiparallel, or
mixed parallel/antiparallel sheets.
[0256] A peptide chain can form a sharp reverse turn. A reverse
turn may be accomplished with as few as four amino acids. Reverse
turns are very abundant, comprising a quarter of all residues in
globular proteins. In proteins, reverse turns commonly connect
.beta. strands to form .beta. sheets, but may also form other
connections. A peptide can also form other turns that are less
sharp.
[0257] Based on studies of known proteins, one may calculate the
propensity of a particular residue, or of a particular dipeptide or
tripeptide, to be found in an a helix, .beta. strand or reverse
turn. The normalized frequencies of occurrence of the amino acid
residues in these secondary structures is given in Table 6-4 of
CREI84. For a more detailed treatment on the prediction of
secondary structure from the amino acid sequence, see Chapter 6 of
SCHU79.
[0258] In designing a suitable hairpin structure, one may copy an
actual structure from a protein whose three-dimensional
conformation is known, design the structure using frequency data,
or combine the two approaches. Preferably, one or more actual
structures are used as a model, and the frequency data is used to
determine which mutations can be made without disrupting the
structure.
[0259] Preferably, no more than three amino acids lie between the
cysteine and the beginning or end of the .alpha. helix or .beta.
strand.
[0260] More complex structures (such as a double hairpin) are also
possible.
[0261] Class III mini-proteins are those featuring a plurality of
disulfide bonds. They optionally may also feature secondary
structures such as those discussed above with regard to Class II
mini-proteins. Since the number of possible disulfide bond
topologies increases rapidly with the number of bonds (two bonds,
three topologies; three bonds, 15 topologies; four bonds, 105
topologies) the number of disulfide bonds preferably does not
exceed four. With two or more disulfide bonds, the disulfide bridge
spans preferably do not exceed 50, and the largest intercysteine
chain segment preferably does not exceed 20.
[0262] Naturally occurring class III mini-proteins, such as
heat-stable enterotoxin ST-Ia frequently have pairs of cysteines
that are adjacent in the amino-acid sequence. Adjacent cysteines
are very unlikely to form an intramolecular disulfide and cysteines
separated by a single amino acids form an intramolecular disulfide
with difficulty and only for certain intervening amino acids. Thus,
clustering cysteines within the amino-acid sequence reduces the
number of realizable disulfide bonding schemes. We utilize such
clustering in the class III mini-protein disclosed herein.
[0263] Metal Finger Mini-Proteins. The mini-proteins of the present
invention are not limited to those crosslinked by disulfide bonds.
Another important class of mini-proteins are analogues of finger
proteins. Finger proteins are characterized by finger structures in
which a metal ion is coordinated by two Cys and two His residues,
forming a tetrahedral arrangement around it. The metal ion is most
often zinc(II), but may be iron, copper, cobalt, etc. The "finger"
has the consensus sequence (Phe or Tyr)-(1 AA)-Cys-(2-4 AAs)-Cys-(3
AAs)-Phe-(5 AAs)-Leu-(2 AAs)-His-(3 AAs)-His-(5 AAs)(BERG88;
GIBS88). While finger proteins typically contain many repeats of
the finger motif, it is known that a single finger will fold in the
presence of zinc ions (FRAN87; PARR88). There is some dispute as to
whether two fingers are necessary for binding to DNA. The present
invention encompasses mini-proteins with either one or two fingers.
It is to be understood that the target need not be a nucleic
acid.
[0264] G. Modified PBSs
[0265] There exist a number of enzymes and chemical reagents that
can selectively modify certain side groups of proteins, including:
a) protein-tyrosine kinase, Ellmans reagent, methyl transferases
(that methylate GLU side groups), serine kinases, proline
hydroxyases, vitamin-K dependent enzymes that convert GLU to GLA,
maleic anhydride, and alkylating agents. Treatment of the
variegated population of GP(PBD)s with one of these enzymes or
reagents will modify the side groups affected by the chosen enzyme
or reagent. Enzymes and reagents that do not kill the GP are much
preferred. Such modification of side groups can directly affect the
binding properties of the displayed PBDs. Using affinity separation
methods, we enrich for the modified GPs that bind the predetermined
target. Since the active binding domain is not entirely genetically
specified, we must repeat the post-morphogenesis modification at
each enrichment round. This approach is particularly appropriate
with mini-protein IPBDs because we envision chemical synthesis of
these SBDs.
[0266] III. Variegation Strategy--Mutagenesis to Obtain Potential
Binding Domains with Desired Diversity
[0267] III.A. Generally
[0268] Using standard genetic engineering techniques, a molecule of
variegated DNA can be introduced into a vector so that it
constitutes part of a gene (OLIP86, OLIP87, AUSU87, REID88a). When
vector containing variegated DNA are used to transform bacteria,
each cell makes a version of the original protein. Each colony of
bacteria may produce a different version from any other colony. If
the variegations of the DNA are concentrated at loci known to be on
the surface of the protein or in a loop, a population of proteins
will be generated, many members of which will fold into roughly the
same 3D structure as the parent protein. The specific binding
properties of each member, however, may be different from each
other member.
[0269] We now consider the manner in which we generate a diverse
population of potential binding domains in order to facilitate
selection of a PBD-bearing GP which binds with the requisite
affinity to the target of choice. The potential binding domains are
first designed at the amino acid level. Once we have identified
which residues are to be mutagenized, and which mutations to allow
at those positions, we may then design the variegated DNA which is
to encode the various PBDs so as to assure that there is a
reasonable probability that if a PBD has an affinity for the
target, it will be detected. Of course, the number of independent
transformants obtained and the sensitivity of the affinity
separation technology will impose limits on the extent of
variegation possible within any single round of variegation.
[0270] There are many ways to generate diversity in a protein. (See
RICH86, CARU85, and OLIP86.) At one extreme, we vary a few residues
of the protein as much as possible (inter alia see CARU85, CARU87,
RICH86, and WHAR86). We will call this approach "Focused
Mutagenesis". A typical "Focused Mutagenesis" strategy is to pick a
set of five to seven residues and vary each through 13-20
possibilities. An alternative plan of mutagenesis ("Diffuse
Mutagenesis") is to vary many more residues through a more limited
set of choices (See VERS86a and PAKU86). The variegation pattern
adopted may fall between these extremes, e.g., two residues varied
through all twenty amino acids, two more through only two
possibilities, and a fifth into ten of the twenty amino acids.
[0271] There is no fixed limit on the number of codons which can be
mutated simultaneously. However, it is desirable to adopt a
mutagenesis strategy which results in a reasonable probability that
a possible PBD sequence is in fact displayed by at least one
genetic package. When the size of the set of amino acids
potentially encoded by each variable codon is the same for all
variable codons and within the set all amino acids are
equiprobable, this probability may be calculated as follows: Let
.GAMMA.(k,q) be the probability that amino acid number k will occur
at variegated codon q; these codons need not be contiguous. The
probability that a particular vgDNA molecule will encode a PBD
containing n variegated amino acids k.sub.1, . . . , k.sub.n
is:
p(k.sub.1, . . . , k.sub.n)=.GAMMA.(k.sub.1,1).multidot. . . .
.multidot..GAMMA.(k.sub.n,n)
[0272] Consider a library of N.sub.it independent transformants
prepared with said vgDNA; the probability that the sequence
k.sub.1, . . . , k.sub.n is absent is:
P(missing k.sub.1, . . . ,
k.sub.n)=exp{-N.sub.it.multidot.p(k.sub.1, . . . , k.sub.n)}.
P(k.sub.1, . . . , k.sub.n in
lib)=1-exp{-N.sub.it.multidot.p(k.sub.1, . . . , k.sub.n)}.
[0273] Preferably, the probability that a mutein encoded by the
vgDNA and composed of the least favored amino acids at each
variegated position will be displayed by at least one independent
transformant in the library is at least 0.50, and more preferably
at least 0.90. (Muteins composed of more favored amino acids would
of course be more likely to occur in the same library.)
[0274] Preferably, the variegation is such as will cause a typical
transformant population to display 10.sup.6-10.sup.7 different
amino acid sequences by means of preferably not more than 10-fold
more (more preferably not more than 3-fold) different DNA
sequences.
[0275] For a mini-protein that lacks .alpha. helices and .beta.
strands, one will, in any given round of mutation, preferably
variegate each of 4-6 non-cysteine codons so that they each encode
at least eight of the 20 possible amino acids. The variegation at
each codon could be customized to that position. Preferably,
cysteine is not one of the potential substitutions, though it is
not excluded.
[0276] When the mini-protein is a metal finger protein, in a
typical variegation strategy, the two Cys and two His residues, and
optionally also the aforementioned Phe/Tyr, Phe and Leu residues,
are held invariant and a plurality (usually 5-10) of the other
residues are varied.
[0277] When the mini-protein is of the type featuring one or more
.alpha. helices and .beta. strands, the set of potential amino acid
modifications at any given position is picked to favor those which
are less likely to disrupt the secondary structure at that
position. Since the number of possibilities at each variable amino
acid is more limited, the total number of variable amino acids may
be greater without altering the sampling efficiency of the
selection process.
[0278] For the last-mentioned class of mini-proteins, as well as
domains other than mini-proteins, preferably not more than 20 and
more preferably 5-10 codons will be variegated. However, if diffuse
mutagenesis is employed, the number of codons which are variegated
can be higher.
[0279] The decision as to which residues to modify is eased by
knowledge of which residues lie on the surface of the domain and
which are buried in the interior.
[0280] We choose residues in the IPBD to vary through consideration
of several factors, including: a) the 3D structure of the IPBD, b)
sequences homologous to IPBD, and c) modeling of the IPBD and
mutants of the IPBD. When the number of residues that could
strongly influence binding is greater than the number that should
be varied simultaneously, the user should pick a subset of those
residues to vary at one time. The user picks trial levels of
variegation and calculate the abundances of various sequences. The
list of varied residues and the level of variegation at each varied
residue are adjusted until the composite variegation is
commensurate with the sensitivity of the affinity separation and
the number of independent transformants that can be made.
[0281] Preferably, the abundance of PPBD-encoding DNA is 3 to 10
times higher than both 1/M.sub.ntv and 1/C.sub.sensi to provide a
margin of redundancy. M.sub.ntv is the number of transformants that
can be made from Y.sub.D100 DNA. With current technology M.sub.ntv
is approximately 5.multidot.10.sup.8, but the exact value depends
on the details of the procedures adapted by the user. Improvements
in technology that allow more efficient: a) synthesis of DNA, b)
ligation of DNA, or c) transformation of cells will raise the value
of M.sub.ntv. C.sub.sensi is the sensitivity of the affinity
separation; improvements in affinity separation will raise
C.sub.sensi. If the smaller of M.sub.ntv and C.sub.sensi is
increased, higher levels of variegation may be used. For example,
if C.sub.sensi is 1 in 10.sup.9 and M.sub.ntv is 10.sup.8, then
improvements in C.sub.sensi are less valuable than improvements in
M.sub.ntv.
[0282] While variegation normally will involve the substitution of
one amino acid for another at a designated variable codon, it may
involve the insertion or deletion of amino acids as well.
[0283] III.B. Identification of Residues to be Varied
[0284] We now consider the principles that guide our choice of
residues of the IPBD to vary. A key concept is that only structured
proteins exhibit specific binding, i.e. can bind to a particular
chemical entity to the exclusion of most others. Thus the residues
to be varied are chosen with an eye to preserving the underlying
IPBD structure. Substitutions that prevent the PBD from folding
will cause GPs carrying those genes to bind indiscriminately so
that they can easily be removed from the population.
[0285] Sauer and colleagues (PAKU86, REID88), and Caruthers and
colleagues (EISE85) have shown that some residues on the
polypeptide chain are more important than others in determining the
3D structure of a protein. The 3D structure is essentially
unaffected by the identity of the amino acids at some loci; at
other loci only one or a few types of amino acid is allowed. In
most cases, loci where wide variety is allowed have the amino acid
side group directed toward the solvent. Loci where limited variety
is allowed frequently have the side group directed toward other
parts of the protein. Thus substitutions of amino acids that are
exposed to solvent are less likely to affect the 3D structure than
are substitutions at internal loci. (See also SCHU79, p169-171 and
CREI84, p239-245, 314-315).
[0286] The residues that join helices to helices, helices to
sheets, and sheets to sheets are called turns and loops and have
been classified by Richardson (RICH81), Thornton (THOR88),
Sutcliffe et al. (SUTC87a) and others. Insertions and deletions are
more readily tolerated in loops than elsewhere. Thornton et al.
(THOR88) have summarized many observations indicating that related
proteins usually differ most at the loops which join the more
regular elements of secondary structure. (These observations are
relevant not only to the variegation of potential binding domains
but also to the insertion of binding domains into an outer surface
protein of a genetic package, as discussed in a later section.)
[0287] Burial of hydrophobic surfaces so that bulk water is
excluded is one of the strongest forces driving the binding of
proteins to other molecules. Bulk water can be excluded from the
region between two molecules only if the surfaces are
complementary. We should test as many surface variations as
possible to find one that is complementary to the target. The
selection-through-binding isolates those proteins that are more
nearly complementary to some surface on the target.
[0288] Proteins do not have distinct, countable faces. Therefore we
define an "interaction set" to be a set of residues such that all
members of the set can simultaneously touch one molecule of the
target material without any atom of the target coming closer than
van der Waals distance to any main-chain atom of the IPBD. The
concept of a residue "touching" a molecule of the target is
discussed below. From a picture of BPTI (such as FIG. 6-10, p. 225
of CREI84) we can see that residues 3, 7, 8, 10, 13, 39, 41, and 42
can all simultaneously contact a molecule the size and shape of
myoglobin. We also see that residue 49 can not touch a single
myoglobin molecule simultaneously with any of the first set even
though all are on the surface of BPTI. (It is not the intent of the
present invention, however, to suggest that use of models is
required to determine which part of the target molecule will
actually be the site of binding by PBD.)
[0289] Variations in the position, orientation and nature of the
side chains of the residues of the interaction set will alter the
shape of the potential binding surface defined by that set. Any
individual combination of such variations may result in a surface
shape which is a better or a worse fit for the target surface. The
effective diversity of a variegated population is measured by the
number of distinct shapes the potentially complementary surfaces of
the PBD can adopt, rather than the number of protein sequences.
Thus, it is preferable to maximize the former number, when our
knowledge of the IPBD permits us to do so.
[0290] To maximize the number of surface shapes generated for when
N residues are varied, all residues varied in a given round of
variegation should be in the same interaction set because variation
of several residues in one interaction set generates an exponential
number of different shapes of the potential binding surface.
[0291] If cassette mutagenesis is to be used to introduce the
variegated DNA into the ipbd gene, the protein residues to be
varied are, preferably, close enough together in sequence that the
variegated DNA (vgDNA) encoding all of them can be made in one
piece. The present invention is not limited to a particular length
of vgDNA that can be synthesized. With current technology, a
stretch of 60 amino acids (180 DNA bases) can be spanned.
[0292] Further, when there is reason to mutate residues further
than sixty residues apart, one can use other mutational means, such
as single-stranded-oligonucleotide-directed mutagenesis (BOTS85)
using two or more mutating primers.
[0293] Alternatively, to vary residues separated by more than sixty
residues, two cassettes may be mutated as follows: 1) vg DNA having
a low level of variegation (for example, 20 to 400 fold
variegation) is introduced into one cassette in the OCV, 2) cells
are transformed and cultured, 3) vg OCV DNA is obtained, 4) a
second segment of vgDNA is inserted into a second cassette in the
OCV, and 5) cells are transformed and cultured, GPs are harvested
and subjected to selection-through-bindin- g.
[0294] The composite level of variation preferably does not exceed
the prevailing capabilities to a) produce very large numbers of
independently transformed cells or b) detect small components in a
highly varied population. The limits on the level of variegation
are discussed later.
[0295] Data about the IPBD and the target that are useful in
deciding which residues to vary in the variegation cycle include:
1) 3D structure, or at least a list of residues on the surface of
the IPBD, 2) list of sequences homologous to IPBD, and 3) model of
the target molecule or a stand-in for the target.
[0296] These data and an understanding of the behavior of different
amino acids in proteins will be used to answer two questions:
[0297] 1) which residues of the IPBD are on the outside and close
enough together in space to touch the target simultaneously?
[0298] 2) which residues of the IPBD can be varied with high
probability of retaining the underlying IPBD structure?
[0299] Although an atomic model of the target material (obtained
through X-ray crystallography, NMR, or other means) is preferred in
such examination, it is not necessary. For example, if the target
were a protein of unknown 3D structure, it would be sufficient to
know the molecular weight of the protein and whether it were a
soluble globular protein, a fibrous protein, or a membrane protein.
Physical measurements, such as low-angle neutron diffraction, can
determine the overall molecular shape, viz. the ratios of the
principal moments of inertia. One can then choose a protein of
known structure of the same class and similar size and shape to use
as a molecular stand-in and yardstick. It is not essential to
measure the moments of inertia of the target because, at low
resolution, all proteins of a given size and class look much the
same. The specific volumes are the same, all are more or less
spherical and therefore all proteins of the same size and class
have about the same radius of curvature. The radii of curvature of
the two molecules determine how much of the two molecules can come
into contact.
[0300] The most appropriate method of picking the residues of the
protein chain at which the amino acids should be varied is by
viewing, with interactive computer graphics, a model of the IPBD. A
stick-figure representation of molecules is preferred. A suitable
set of hardware is an Evans & Sutherland PS390 graphics
terminal (Evans & Sutherland Corporation, Salt Lake City, Ut.)
and a MicroVAX II supermicro computer (Digital Equipment Corp.,
Maynard, Mass.). The computer should, preferably, have at least 150
megabytes of disk storage, so that the Brookhaven Protein Data Bank
can be kept on line. A FORTRAN compiler, or some equally good
higher-level language processor is preferred for program
development. Suitable programs for viewing and manipulating protein
models include: a) PS-FRODO, written by T. A. Jones (JONE85) and
distributed by the Biochemistry Department of Rice University,
Houston, Tex.; and b) PROTEUS, developed by Dayringer, Tramantano,
and Fletterick (DAYR86). Important features of PS-FRODO and PROTEUS
that are needed to view and manipulate protein models for the
purposes of the present invention are the abilities to: 1) display
molecular stick figures of proteins and other molecules, 2) zoom
and clip images in real time, 3) prepare various abstract
representations of the molecules, such as a line joining
C.sub..alpha.s and side group atoms, 4) compute and display
solvent-accessible surfaces reasonably quickly, 5) point to and
identify atoms, and 6) measure distance between atoms.
[0301] In addition, one could use theoretical calculations, such as
dynamic simulations of proteins, to estimate whether a substitution
at a particular residue of a particular amino-acid type might
produce a protein of approximately the same 3D structure as the
parent protein. Such calculations might also indicate whether a
particular substitution will greatly affect the flexibility of the
protein; calculations of this sort may be useful but are not
required.
[0302] Residues whose mutagenesis is most likely to affect binding
to a target molecule, without destabilizing the protein, are called
the "principal set". Using the knowledge of which residues are on
the surface of the IPBD (as noted above), we pick residues that are
close enough together on the surface of the IPBD to touch a
molecule of the target simultaneously without having any IPBD
main-chain atom come closer than van der Waals distance (viz. 4.0
to 5.0 .ANG.) from any target atom. For the purposes of the present
invention, a residue of the IPBD "touches" the target if: a) a
main-chain atom is within van der Waals distance, viz. 4.0 to 5.0
.ANG. of any atom of the target molecule, or b) the C.sub..beta. is
within D.sub.cutoff of any atom of the target molecule so that a
side group atom could make contact with that atom.
[0303] Because side groups differ in size (cf. Table 35), some
judgment is required in picking D.sub.cutoff. In the preferred
embodiment, we will use D.sub.cutoff=8.0 .ANG., but other values in
the range 6.0 .ANG. to 10.0 .ANG. could be used. If IPBD has G at a
residue, we construct a pseudo C.sub..beta. with the correct bond
distance and angles and judge the ability of the residue to touch
the target from this pseudo C.sub..beta..
[0304] Alternatively, we choose a set of residues on the surface of
the IPBD such that the curvature of the surface defined by the
residues in the set is not so great that it would prevent contact
between all residues in the set and a molecule of the target. This
method is appropriate if the target is a macromolecule, such as a
protein, because the PBDs derived from the IPBD will contact only a
part of the macromolecular surface. The surfaces of macromolecules
are irregular with varying curvatures. If we pick residues that
define a surface that is not too convex, then there will be a
region on a macromolecular target with a compatible curvature.
[0305] In addition to the geometrical criteria, we prefer that
there be some indication that the underlying IPBD structure will
tolerate substitutions at each residue in the principal set of
residues. Indications could come from various sources, including:
a) homologous sequences, b) static computer modeling, or c) dynamic
computer simulations.
[0306] The residues in the principal set need not be contiguous in
the protein sequence and usually are not. The exposed surfaces of
the residues to be varied do not need to be connected. We desire
only that the amino acids in the residues to be varied all be
capable of touching a molecule of the target material
simultaneously without having atoms overlap. If the target were,
for example, horse heart myoglobin, and if the IPBD were BPTI, any
set of residues in one interaction set of BPTI defined in Table 34
could be picked.
[0307] The secondary set comprises those residues not in the
primary set that touch residues in the primary set. These residues
might be excluded from the primary set because: a) the residue is
internal, b) the residue is highly conserved, or c) the residue is
on the surface, but the curvature of the IPBD surface prevents the
residue from being in contact with the target at the same time as
one or more residues in the primary set.
[0308] Internal residues are frequently conserved and the amino
acid type can not be changed to a significantly different type
without substantial risk that the protein structure will be
disrupted. Nevertheless, some conservative changes of internal
residues, such as I to L or F to Y, are tolerated. Such
conservative changes subtly affect the placement and dynamics of
adjacent protein residues and such "fine tuning" may be useful once
an SBD is found.
[0309] Surface residues in the secondary set are most often located
on the periphery of the principal set. Such peripheral residues can
not make direct contact with the target simultaneously with all the
other residues of the principal set. The charge on the amino acid
in one of these residues could, however, have a strong effect on
binding. Once an SBD is found, it is appropriate to vary the charge
of some or all of these residues. For example, the variegated codon
containing equimolar A and G at base 1, equimolar C and A at base
2, and A at base 3 yields amino acids T, A, K, and E with equal
probability.
[0310] The assignment of residues to the primary and secondary sets
may be based on: a) geometry of the IPBD and the geometrical
relationship between the IPBD and the target (or a stand-in for the
target) in a hypothetical complex, and b) sequences of proteins
homologous to the IPBD. However, it should be noted that the
distinction between the principal set and the secondary set is one
more of convenience than of substance; we could just as easily have
assigned each amino acid residue in the domain a preference score
that weighed together the different considerations affecting
whether they are suitable for variegation, and then ranked the
residues in order, from most preferred to least.
[0311] For any given round of variegation, it may be necessary to
limit the variegation to a subset of the residues in the primary
and secondary sets, based on geometry and on the maximum allowed
level of variegation that assures progressivity. The allowed level
of variegation determines how many residues can be varied at once;
geometry determines which ones.
[0312] The user may pick residues to vary in many ways. For
example, pairs of residues are picked that are diametrically
opposed across the face of the principal set. Two such pairs are
used to delimit the surface, up/down and right/left. Alternatively,
three residues that form an inscribed triangle, having as large an
area as possible, on the surface are picked. One to three other
residues are picked in a checkerboard fashion across the
interaction surface. Choice of widely spaced residues to vary
creates the possibility for high specificity because all the
intervening residues must have acceptable complementarity before
favorable interactions can occur at widely-separated residues.
[0313] The number of residues picked is coupled to the range
through which each can be varied by the restrictions discussed
below. In the first round, we do not assume any binding between
IPBD and the target and so progressivity is not an issue. At the
first round, the user may elect to produce a level of variegation
such that each molecule of vgDNA is potentially different through,
for example, unlimited variegation of 10 codons (20.sup.10
approx.=10.sup.13). One run of the DNA synthesizer produces
approximately 10.sup.13 molecules of length 100 nts. Inefficiencies
in ligation and transformation will reduce the number of proteins
actually tested to between 10.sup.7 and 5.multidot.10.sup.8.
Multiple replications of the process with such very high levels of
variegation will not yield repeatable results; the user decides
whether this is important.
[0314] III.C. Determining the Substitution Set for each Parental
Residue
[0315] Having picked which residues to vary, we now decide the
range of amino acids to allow at each variable residue. The total
level of variegation is the product of the number of variants at
each varied residue. Each varied residue can have a different
scheme of variegation, producing 2 to 20 different possibilities.
The set of amino acids which are potentially encoded by a given
variegated codon are called its "substitution set".
[0316] The computer that controls a DNA synthesizer, such as the
Milligen 7500, can be programmed to synthesize any base of an
oligo-nt with any distribution of nts by taking some nt substrates
(e.g. nt phosphoramidites) from each of two or more reservoirs.
Alternatively, nt substrates can be mixed in any ratios and placed
in one of the extra reservoir for so called "dirty bottle"
synthesis. Each codon could be programmed differently. The "mix" of
bases at each nucleotide position of the codon determines the
relative frequency of occurrence of the different amino acids
encoded by that codon.
[0317] Simply variegated codons are those in which those nucleotide
positions which are degenerate are obtained from a mixture of two
or more bases mixed in equimolar proportions. These mixtures are
described in this specification by means of the standardized
"ambiguous nucleotide" code (Table 1 and 37 CFR .sctn.1.822). In
this code, for example, in the degenerate codon "SNT", "S" denotes
an equimolar mixture of bases G and C, "N", an equimolar mixture of
all four bases, and "T", the single invariant base thymidine.
[0318] Complexly variegated codons are those in which at least one
of the three positions is filled by a base from an other than
equimolar mixture of two of more bases.
[0319] Either simply or complexly variegated codons may be used to
achieve the desired substitution set.
[0320] If we have no information indicating that a particular amino
acid or class of amino acid is appropriate, we strive to substitute
all amino acids with equal probability because representation of
one mini-protein above the detectable level is wasteful. Equal
amounts of all four nts at each position in a codon (NNN) yields
the amino acid distribution in which each amino acid is present in
proportion to the number of codons that code for it. This
distribution has the disadvantage of giving two basic residues for
every acidic residue. In addition, six times as much R, S, and L as
W or M occur. If five codons are synthesized with this
distribution, each of the 243 sequences encoding some combination
of L, R, and S are 7776-times more abundant than each of the 32
sequences encoding some combination of W and M. To have five Ws
present at detectable levels, we must have each of the (L,R,S)
sequences present in 7776-fold excess.
[0321] Preferably, we also consider the interactions between the
sites of variegation and the surrounding DNA. If the method of
mutagenesis to be used is replacement of a cassette, we consider
whether the variegation will generate gratuitous restriction sites
and whether they seriously interfere with the intended introduction
of diversity. We reduce or eliminate gratuitous restriction sites
by appropriate choice of variegation pattern and silent alteration
of codons neighboring the sites of variegation.
[0322] It is generally accepted that the sequence of amino acids in
a protein or polypeptide determine the three-dimensional structure
of the molecule, including the possibility of no definite
structure. Among polypeptides of definite length and sequence, some
have a defined tertiary structure and most do not.
[0323] Particular amino acid residues can influence the tertiary
structure of a defined polypeptide in several ways, including
by:
[0324] a) affecting the flexibility of the polypeptide main
chain,
[0325] b) adding hydrophobic groups,
[0326] c) adding charged groups,
[0327] d) allowing hydrogen bonds, and
[0328] e) forming cross-links, such as disulfides, chelation to
metal ions, or bonding to prosthetic groups.
[0329] Most works on proteins classify the twenty amino acids into
categories such as hydrophobic/hydrophilic,
positive/negative/neutral, or large/small. These classifications
are useful rules of thumb, but one must be careful not to
oversimplify. Proteins contain a variety of identifiable secondary
structural features, including: a) .alpha. helices, b) 3-10
helices, c) anti-parallel .beta. sheets, d) parallel .beta. sheets,
e) .OMEGA. loops, f) reverse turns, and g) various cross links.
Many people have analyzed proteins of known structures and assigned
each amino-acid to one category or another. Using the frequency at
which particular amino acids occur in various types of secondary
structures, people have a) tried to predict the secondary
structures of proteins for which only the amino-acid sequence is
known (CHOU74, CHOU78a, CHOU78b), and b) designed proteins de novo
that have a particular set of secondary structural elements
(DEGR87, HECH90). Although some amino acids show definite
predilection for one secondary form (e.g. VAL for .beta. structure
and ALA for .alpha. helices), these preferences are not very
strong; Creighton has tabulated the preferences (CREI84). In only
seven cases does the tendency exceed 2.0:
3 Amino acid distinction ratio MET .alpha./turn 3.7 PRO
turn/.alpha. 3.7 VAL .beta./turn 3.2 GLY turn/.alpha. 2.9 ILE
.beta./turn 2.8 PHE .beta./turn 2.3 LEU .alpha./turn 2.2
[0330] Every amino-acid type has been observed in every identified
secondary structural motif. ARG is particularly indiscriminate.
[0331] PRO is generally taken to be a helix breaker. Nevertheless,
proline often occurs at the beginning of helices or even in the
middle of a helix, where it introduces a slight bend in the helix.
Matthews and coworkers replaced a PRO that occurs near the middle
of an a helix in T4 lysozyme. To their surprise, the "improved"
protein is less stable than the wild-type. The rest of the
structure had been adapted to fit the bent helix.
[0332] Lundeen (LUND86) has tabulated the frequencies of amino
acids in helices, .beta. strands, turns, and coil in proteins of
known 3D structure and has distinguished between CYSs having free
thiol groups and half cystines. He reports that free CYS is found
most often in helixes while half cystines are found more often in p
sheets. Half cystines are, however, regularly found in helices.
Pease et al. (PEAS90) constructed a peptide having two cystines;
one end of each is in a very stable a helix. Apamin has a similar
structure (WEMM83, PEAS88).
[0333] Flexibility:
[0334] GLY is the smallest amino acid, having two hydrogens
attached to the C.sub..alpha.. Because GLY has no C.sub..beta., it
confers the most flexibility on the main chain. Thus GLY occurs
very frequently in reverse turns, particularly in conjunction with
PRO, ASP, ASN, SER, and THR.
[0335] The amino acids ALA, SER, CYS, ASP, ASN, LEU, MET, PHE, TYR,
TRP, ARG, HIS, GLU, GLN, and LYS have unbranched .beta. carbons. Of
these, the side groups of SER, ASP, and ASN frequently make
hydrogen bonds to the main chain and so can take on main-chain
conformations that are energetically unfavorable for the others.
VAL, ILE, and THR have branched .beta. carbons which makes the
extended main-chain conformation more favorable. Thus VAL and ILE
are most often seen in p sheets. Because the side group of THR can
easily form hydrogen bonds to the main chain, it has less tendency
to exist in a .beta. sheet.
[0336] The main chain of proline is particularly constrained by the
cyclic side group. The .phi. angle is always close to -60.degree..
Most prolines are found near the surface of the protein.
[0337] Charge:
[0338] LYS and ARG carry a single positive charge at any pH below
10.4 or 12.0, respectively. Nevertheless, the methylene groups,
four and three respectively, of these amino acids are capable of
hydrophobic interactions. The guanidinium group of ARG is capable
of donating five hydrogens simultaneously, while the amino group of
LYS can donate only three. Furthermore, the geometries of these
groups is quite different, so that these groups are often not
interchangeable.
[0339] ASP and GLU carry a single negative charge at any pH above
.apprxeq.4.5 and 4.6, respectively. Because ASP has but one
methylene group, few hydrophobic interactions are possible. The
geometry of ASP lends itself to forming hydrogen bonds to
main-chain nitrogens which is consistent with ASP being found very
often in reverse turns and at the beginning of helices. GLU is more
often found in a helices and particularly in the amino-terminal
portion of these helices because the negative charge of the side
group has a stabilizing interaction with the helix dipole (NICH88,
SALI88).
[0340] HIS has an ionization pK in the physiological range, viz.
6.2. This pK can be altered by the proximity of charged groups or
of hydrogen donators or acceptors. HIS is capable of forming bonds
to metal ions such as zinc, copper, and iron.
[0341] Hydrogen Bonds:
[0342] Aside from the charged amino acids, SER, THR, ASN, GLN, TYR,
and TRP can participate in hydrogen bonds.
[0343] Cross Links:
[0344] The most important form of cross link is the disulfide bond
formed between two thiols, especially the thiols of CYS residues.
In a suitably oxidizing environment, these bonds form
spontaneously. These bonds can greatly stabilize a particular
conformation of a protein or mini-protein. When a mixture of
oxidized and reduced thiol reagents are present, exchange reactions
take place that allow the most stable conformation to predominate.
Concerning disulfides in proteins and peptides, see also KATZ90,
MATS89, PERR84, PERR86, SAUE86, WELL86, JANA89, HORV89, KISH85, and
SCHN86.
[0345] Other cross links that form without need of specific enzymes
include:
4 1) (CYS).sub.4:Fe Rubredoxin (in CREI84, P. 376) 2)
(CYS).sub.4:Zn Aspartate Transcarbamylase (in CREI84, P. 376) and
Zn-fingers (HARD90) 3) (HIS).sub.2(MET)(CYS):Cu Azurin (in CREI84,
P. 376) and Basic "Blue" Cu Cucumber protein (GUSS88) 4)
(HIS).sub.4:Cu CuZn superoxide dismutase 5)
(CYS).sub.4:(Fe.sub.4S.sub.4) Ferredoxin (in CREI84, P. 376) 6)
(CYS).sub.2(HIS).sub.2:Zn Zinc-fingers (GIBS88) 7)
(CYS).sub.3(HIS):Zn Zinc-fingers (GAUS87, GIBS88)
[0346] Simply Variegated Codons
[0347] The following simply variegated codons are useful because
they encode a relatively balanced set of amino acids:
[0348] 1) SNT which encodes the set [L,P,H,R,V,A,D,G]: a) one
acidic (D) and one basic (R), b) both aliphatic (L,V) and aromatic
hydrophobics (H), c) large (L,R,H) and small (G,A) side groups, d)
ridged (P) and flexible (G) amino acids, e) each amino acid encoded
once.
[0349] 2) RNG which encodes the set [M,T,K,R,V,A,E,G]: a) one
acidic and two basic (not optimal, but acceptable), b) hydrophilics
and hydrophobics, c) each amino acid encoded once.
[0350] 3) RMG which encodes the set [T,K,A,E]: a) one acidic, one
basic, one neutral hydrophilic, b) three favor a helices, c) each
amino acid encoded once.
[0351] 4) VNT which encodes the set [L,P,H,R,I,T,N,S,V,A,D,G]: a)
one acidic, one basic, b) all classes: charged, neutral
hydrophilic, hydrophobic, ridged and flexible, etc., c) each 3mino
acid encoded once.
[0352] 5) RRS which encodes the set [N,S,K,R,D,E,G.sup.2]: a) two
acidics, two basics, b) two neutral hydrophilics, c) only glycine
encoded twice.
[0353] 6) NNT which encodes the set
[F,S,Y,C,L,P,H,R,I,T,N,V,A,D,G]: a) sixteen DNA sequences provide
fifteen different amino acids; only serine is repeated, all others
are present in equal amounts (This allows very efficient sampling
of the library.), b) there are equal numbers of acidic and basic
amino acids (D and R, once each), c) all major classes of amino
acids are present: acidic, basic, aliphatic hydrophobic, aromatic
hydrophobic, and neutral hydrophilic.
[0354] 7) NNG, which encodes the set
[L.sup.2,R.sup.2,S,W,P,Q,M,T,K,V,A,E,- G, stop]: a) fair
preponderance of residues that favor formation of .alpha.-helices
[L,M,A,Q,K,E; and, to a lesser extent, S,R,T]; b) encodes 13
different amino acids. (VHG encodes a subset of the set encoded by
NNG which encodes 9 amino acids in nine different DNA sequences,
with equal acids and bases, and 5/9 being .alpha.
helix-favoring.)
[0355] For the initial variegation, NNT is preferred, in most
cases. However, when the codon is encoding an amino acid to be
incorporated into an .alpha. helix, NNG is preferred.
[0356] Below, we analyze several simple variegations as to the
efficiency with which the libraries can be sampled.
[0357] Libraries of random hexapeptides encoded by (NNK).sup.6 have
been reported (SCOT90, CWIR90). Table 130 shows the expected
behavior of such libraries. NNK produces single codons for PHE,
TYR, CYS, TRP, HIS, GLN, ILE, MET, ASN, LYS, ASP, and GLU (.alpha.
set); two codons for each of VAL, ALA, PRO, THR, and GLY (.PHI.
set); and three codons for each of LEU, ARG, and SER (.OMEGA. set).
We have separated the 64,000,000 possible sequences into 28
classes, shown in Table 130A, based on the number of amino acids
from each of these sets. The largest class is
.PHI..OMEGA..alpha..alpha..alpha..alpha. with .apprxeq.14.6% of the
possible sequences. Aside from any selection, all the sequences in
one class have the same probability of being produced. Table 130B
shows the probability that a given DNA sequence taken from the
(NNK).sup.6 library will encode a hexapeptide belonging to one of
the defined classes; note that only .apprxeq.6.3% of DNA sequences
belong to the .PHI..OMEGA..alpha..alpha..alpha..alpha. class.
[0358] Table 130C shows the expected numbers of sequences in each
class for libraries containing various numbers of independent
transformants (viz. 10.sup.6, 3.multidot.10.sup.6, 10.sup.7,
3.multidot.10.sup.7, 10.sup.8, 3.multidot.10.sup.8, 10.sup.9, and
3.multidot.10.sup.9). At 10.sup.6 independent transformants (ITs),
we expect to see 56% of the
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA. class, but only 0.1% of
the .alpha..alpha..alpha..alpha..alpha..alpha. class. The vast
majority of sequences seen come from classes for which less than
10% of the class is sampled. Suppose a peptide from, for example,
class .PHI..PHI..OMEGA..OMEGA..alpha..alpha. is isolated by
fractionating the library for binding to a target. Consider how
much we know about peptides that are related to the isolated
sequence. Because only 4% of the
.PHI..PHI..OMEGA..OMEGA..alpha..alpha. class was sampled, we can
not conclude that the amino acids from the .OMEGA. set are in fact
the best from the .OMEGA. set. We might have LEU at position 2, but
ARG or SER could be better. Even if we isolate a peptide of the
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA. class, there is a
noticeable chance that better members of the class were not present
in the library.
[0359] With a library of 10.sup.7 ITs, we see that several classes
have been completely sampled, but that the
.alpha..alpha..alpha..alpha..alpha.- .alpha. class is only 1.1%
sampled. At 7.6.multidot.10.sup.7 ITs, we expect display of 50% of
all amino-acid sequences, but the classes containing three or more
amino acids of the .alpha. set are still poorly sampled. To achieve
complete sampling of the (NNK).sup.6 library requires about
3.multidot.10.sup.9 ITs, 10-fold larger than the largest
(NNK).sup.6 library so far reported.
[0360] Table 131 shows expectations for a library encoded by
(NNT).sup.4(NNG).sup.2. The expectations of abundance are
independent of the order of the codons or of interspersed unvaried
codons. This library encodes 0.133 times as many amino-acid
sequences, but there are only 0.0165 times as many DNA sequences.
Thus 5.0.multidot.10.sup.7 ITs (i.e. 60-fold fewer than required
for (NNK).sup.6) gives almost complete sampling of the library. The
results would be slightly better for (NNT).sup.6 and slightly, but
not much, worse for (NNG).sup.6. The controlling factor is the
ratio of DNA sequences to amino-acid sequences.
[0361] Table 132 shows the ratio of #DNA sequences/#AA sequences
for codons NNK, NNT, and NNG. For NNK and NNG, we have assumed that
the PBD is displayed as part of an essential gene, such as gene III
in Ff phage, as is indicated by the phrase "assuming stops vanish".
It is not in any way required that such an essential gene be used.
If a non-essential gene is used, the analysis would be slightly
different; sampling of NNK and NNG would be slightly less
efficient. Note that (NNT).sup.6 gives 3.6-fold more amino-acid
sequences than (NNK).sup.5 but requires 1.7-fold fewer DNA
sequences. Note also that (NNT).sup.7 gives twice as many
amino-acid sequences as (NNK).sup.6, but 3.3-fold fewer DNA
sequences.
[0362] Thus, while it is possible to use a simple mixture (NNS, NNK
or NNN) to obtain at a particular position all twenty amino acids,
these simple mixtures lead to a highly biased set of encoded amino
acids. This problem can be overcome by use of complexly variegated
codons.
[0363] Complexly Variegated Codons
[0364] Let Abun(x) be the abundance of DNA sequences coding for
amino acid x, defined by the distribution of nts at each base of
the codon. For any distribution, there will be a most-favored amino
acid (mfaa) with abundance Abun(mfaa) and a least-favored amino
acid (lfaa) with abundance Abun(lfaa). We seek the nt distribution
that allows all twenty amino acids and that yields the largest
ratio Abun(lfaa)/Abun(mfaa) subject, if desirable to further
constraints.
[0365] We first will present the mixture calculated to be optimal
when the nt distribution is subject to two constraints: equal
abundances of acidic and basic amino acids and the least possible
number of stop codons. Thus only nt distributions that yield
Abun(E)+Abun(D)=Abun(R)+Abun(K) are considered, and the function
maximized is:
{(1-Abun(stop))(Abun(lfaa)/Abun(mfaa))}.
[0366] We have simplified the search for an optimal nt distribution
by limiting the third base to T or G (C or G is equivalent). All
amino acids are possible and the number of accessible stop codons
is reduced because TGA and TAA codons are eliminated. The amino
acids F, Y, C, H, N, I, and D require T at the third base while W,
M, Q, K, and E require G. Thus we use an equimolar mixture of T and
G at the third base. However, it should be noted that the present
invention embraces use of complexly variegated codons in which the
third base is not limited to T or G (or to C or G).
[0367] A computer program, written as part of the present invention
and named "Find Optimum vgcodon" (See Table 9), varies the
composition at bases 1 and 2, in steps of 0.05, and reports the
composition that gives the largest value of the quantity
{(Abun(lfaa)/Abun(mfaa)(1-Abun(stop)))}- . A vg codon is
symbolically defined by the nucleotide distribution at each
base:
5 T C A G base #1 = t1 c1 a1 g1 base #2 = t2 c2 a2 g2 base #3 = t3
c3 a3 g3 t1 + c1 + a1 + g1 = 1.0 t2 + c2 + a2 + g2 = 1.0 t3 = g3 =
0.5, c3 = a3 = 0.
[0368] The variation of the quantities t1, c1, a1, g1, t2, c2, a2,
and g2 is subject to the constraint that:
Abun(E)+Abun(D)=Abun(K)+Abun(R)
Abun(E)+Abun(D)=g1*a2
Abun(K)+Abun(R)=a1*a2/2+c1*g2+a1*g2/2
g1*a2=a1*a2/2+c1*g2+a1*g2/2
[0369] Solving for g2, we obtain
g2=(g1*a2-0.5*a1*a2)/(c1+0.5*a1)
[0370] In addition,
t1=1-a1-c1-g1
t2=1-a2-c2-g2
[0371] We vary a1, c1, g1, a2, and c2 and then calculate t1, g2,
and t2. Initially, variation is in steps of 5%. Once an
approximately optimum distribution of nucleotides is determined,
the region is further explored with steps of 1%. The logic of this
program is shown in Table 9. The optimum distribution (the "fxS"
codon) is shown in Table 10A and yields DNA molecules encoding each
type amino acid with the abundances shown.
[0372] Note that this chemistry encodes all twenty amino acids,
with acidic and basic amino acids being equiprobable, and the most
favored amino acid (serine) is encoded only 2.454 times as often as
the least favored amino acid (tryptophan). The "fxS" vg codon
improves sampling most for peptides containing several of the amino
acids [F,Y,C,W,H,Q,I,M,N,K,D,E] for which NNK or NNS provide only
one codon. Its sampling advantages are most pronounced when the
library is relatively small.
[0373] A modification of "Fino Optimum vgcodon" varies the
composition at bases 1 and 2, in steps of 0.01, and reports the
composition that gives the largest value of the quantity
{(Abun(lfaa)/Abun(mfaa))} without any restraint on the relative
abundance of any amino acids. The results of this optimization is
shown in Table 10B. The changes are small, indicating that
insisting on equality of acids and bases and minimizing stop codons
costs us little. Also note that, without restraining the
optimization, the prevalence of acidic and basic amino acids comes
out fairly close. On the other hand, relaxing the restriction
leaves a distribution in which the least favored amino acid is only
0.412 times as prevalent as SER.
[0374] The advantages of an NNT codon are discussed elsewhere in
the present application. Unoptimized NNT provides 15 amino acids
encoded by only 16 DNA sequences. It is possible to improve on NNT
as follows. First note that the SER codons occur in the T and A
rows of the genetic-code table and in the C and G columns.
[SER]=T.sub.1.times.C.sub.2+A.sub.1.times.G.sub.2
[0375] If we reduce the prevalence of SER by reducing T.sub.1,
C.sub.2, A.sub.1, and G.sub.2 relative to other bases, then we will
also reduce the prevalence of PHE, TYR, CYS, PRO, THR, ALA, ARG,
GLY, ILE, and ASN. The prevalence of LEU, HIS, VAL, and ASP will
rise. If we assume that T.sub.1, C.sub.2, A.sub.1, and G.sub.2 are
all lowered to the same extent and that C.sub.1, G.sub.1, T.sub.2,
and A.sub.2 are increased by the same amount, we can compute a
shift that makes the prevalence of SER equal the prevalences of
LEU, HIS, VAL, and ASP. The decrease in PHE, TYR, CYS, PRO, THR,
ALA, ARG, GLY, ILE, and ASN is not equal; CYS and THR are reduced
more than the others.
[0376] Let the distribution be
6 T C A G base #1 = .25 - q .25 + q .25 - q .25 + q base #2 = .25 +
q .25 - q .25 + q .25 - q base #3 = 1.00 0.0 0.0 0.0 Setting [SER]
= [LEU] = [HIS] = [VAL] = [ASP] gives: (.25 - q) .multidot. (.25 -
q) + ( .25 - q) .multidot. ( .25 - q) = (.25 + q) .multidot. (.25 +
q) 2 .multidot. (.25 - q).sup.2 = (.25 + q).sup.2 q.sup.2 - 1.5 q +
.0625 = 0 q = (3/4) - {square root}2/2 = .0428
[0377] This distribution (shown in Table 10C) gives five amino
acids (SER, LEU, HIS, VAL, ASP) in very nearly equal amounts. A
further eight amino acids (PHE, TYR, ILE, ASN, PRO, ALA, ARG, GLY)
are present at 78% the abundance of SER. THR and CYS remain at half
the abundance of SER. When variegating DNA for disulfide-bonded
mini-proteins, it is often desirable to reduce the prevalence of
CYS. This distribution allows 13 amino acids to be seen at high
level and gives no stops; the optimized fxS distribution allows
only 11 amino acids at high prevalence.
[0378] The NNG codon can also be optimized. Table 10D shows an
approximately optimized NNG codon. When equimolar T,C,A,G are used
in NNG, one obtains double doses of LEU and ARG. To improve the
distribution, we increase G.sub.1 by 4.delta., decrease T.sub.1 and
A.sub.1 by .delta. each and C.sub.1 by 2.delta.. We adopt this
pattern because C.sub.1 affects both LEU and ARG while T.sub.1 and
A.sub.1 each affect either LEU or ARG, but not both. Similarly, we
decrease T.sub.2 and G.sub.2 by .tau. while we increase C.sub.2 and
A.sub.2 by .tau.. We adjusted .delta. and .tau. until
[ALA].apprxeq.[ARG]. There are, under this variegation, four
equally most favored amino acids: LEU, ARG, ALA, and GLU. Note that
there is one acidic and one basic amino acid in this set. There are
two equally least favored amino acids: TRP and MET. The ratio of
lfaa/mfaa is 0.5258. If this codon is repeated six times, peptides
composed entirely of TRP and MET are 2% as common as peptides
composed entirely of the most favored amino acids. We refer to this
as "the prevalence of (TRP/MET).sup.6 in optimized NNG.sup.6
vgDNA".
[0379] When synthesizing vgDNA by the "dirty bottle" method, it is
sometimes desirable to use only a limited number of mixes. One very
useful mixture is called the "optimized NNS mixture" in which we
average the first two positions of the fxS mixture: T.sub.1=0.24,
C.sub.1=0.17, A.sub.1=0.33, G.sub.1=0.26, the second position is
identical to the first, C.sub.3=G.sub.3=0.5. This distribution
provides the amino acids ARG, SER, LEU, GLY, VAL, THR, ASN, and LYS
at greater than 5% plus ALA, ASP, GLU, ILE, MET, and TYR at greater
than 4%.
[0380] An additional complexly variegated codon is of interest.
This codon is identical to the optimized NNT codon at the first two
positions and has T:G::90:10 at the third position. This codon
provides thirteen amino acids (ALA, ILE, ARG, SER, ASP, LEU, VAL,
PHE, ASN, GLY, PRO, TYR, and HIS) at more than 5.5%. THR at 4.3%
and CYS at 3.9% are more common than the LFAAs of NNK (3.125%). The
remaining five amino acids are present at less than 1%. This codon
has the feature that all amino acids are present; sequences having
more than two of the low-abundance amino acids are rare. When we
isolate an SBD using this codon, we can be reasonably sure that the
first 13 amino acids were tested at each position. A similar codon,
based on optimized NNG, could be used.
[0381] Table 10E shows some properties of an unoptimized NNS (or
NNK) codon. Note that there are three equally most-favored amino
acids: ARG, LEU, and SER. There are also twelve equally least
favored amino acids: PHE, ILE, MET, TYR, HIS, GLN, ASN, LYS, ASP,
GLU, CYS, and TRP. Five amino acids (PRO, THR, ALA, VAL, GLY) fall
in between. Note that a six-fold repetition of NNS gives sequences
composed of the amino acids [PHE, ILE, MET, TYR, HIS, GLN, ASN,
LYS, ASP, GLU, CYS, and TRP] at only .apprxeq.0.1% of the sequences
composed of [ARG, LEU, and SER]. Not only is this .apprxeq.20-fold
lower than the prevalence of (TRP/MET).sup.6 in optimized NNG.sup.6
vgDNA, but this low prevalence applies to twelve amino acids.
[0382] Diffuse Mutagenesis
[0383] Diffuse Mutagenesis can be applied to any part of the
protein at any time, but is most appropriate when some binding to
the target has been established. Diffuse Mutagenesis can be
accomplished by spiking each of the pure nts activated for DNA
synthesis (e.g. nt-phosphoramidites) with a small amount of one or
more of the other activated nts.
[0384] Contrary to general practice, the present invention sets the
level of spiking so that only a small percentage (1% to 0.00001%,
for example) of the final product will contain the initial DNA
sequence. This will insure that many single, double, triple, and
higher mutations occur, but that recovery of the basic sequence
will be a possible outcome. Let N.sub.b be the number of bases to
be varied, and let Q be the fraction of all sequences that should
have the parental sequence, then M, the fraction of the mixture
that is the majority component, is
M=exp{log.sub.e(Q)/N.sub.b}=10(log.sub.10(Q)/N.sub.b).
[0385] If, for example, thirty base pairs on the DNA chain were to
be varied and 1% of the product is to have the parental sequence,
then each mixed nt substrate should contain 86% of the parental nt
and 14% of other nts. Table 8 shows the fraction (fn) of DNA
molecules having n non-parental bases when 30 bases are synthesized
with reagents that contain fraction M of the majority component.
When M=0.63096, f24 and higher are less than 10.sup.8. The entry
"most" in Table 8 is the number of changes that has the highest
probability. Note that substantial probability for multiple
substitutions only occurs if the fraction of parental sequence (fo)
is allowed to drop to around 10.sup.-6. The N.sub.b base pairs of
the DNA chain that are synthesized with mixed reagents need not be
contiguous. They are picked so that between N.sub.b/3 and N.sub.b
codons are affected to various degrees. The residues picked for
mutation are picked with reference to the 3D structure of the IPBD,
if known. For example, one might pick all or most of the residues
in the principal and secondary set. We may impose restrictions on
the extent of variation at each of these residues based on
homologous sequences or other data. The mixture of non-parental nts
need not be random, rather mixtures can be biased to give
particular amino acid types specific probabilities of appearance at
each codon. For example, one residue may contain a hydrophobic
amino acid in all known homologous sequences; in such a case, the
first and third base of that codon would be varied, but the second
would be set to T. Other examples of how this might be done are
given in the horse heart myoglobin example. This diffuse
structure-directed mutagenesis will reveal the subtle changes
possible in protein backbone associated with conservative interior
changes, such as V to I, as well as some not so subtle changes that
require concomitant changes at two or more residues of the
protein.
[0386] III.D. Special Considerations Relating to Variegation of
Mini-proteins with Essential Cysteines
[0387] Several of the preferred simple or complex variegated codons
encode a set of amino acids which includes cysteine. This means
that some of the encoded binding domains will feature one or more
cysteines in addition to the invariant disulfide-bonded cysteines.
For example, at each NNT-encoded position, there is a one in
sixteen chance of obtaining cysteine. If six codons are so varied,
the fraction of domains containing additional cysteines is 0.33.
Odd numbers of cysteines can lead to complications, see Perry and
Wetzel (PERR84). On the other hand, many disulfide-containing
proteins contain cysteines that do not form disulfides, e.g.
trypsin. The possibility of unpaired cysteines can be dealt with in
several ways:
[0388] First, the variegated phage population can be passed over an
immobilized reagent that strongly binds free thiols, such as
SulfoLink (catalogue number 44895 H from Pierce Chemical Company,
Rockford, Illinois, 61105). Another product from Pierce is
TNB-Thiol Agarose (Catalogue Code 20409 H). BioRad sells Affi-Gel
401 (catalogue 153-4599) for this purpose.
[0389] Second, one can use a variegation that excludes cysteines,
such as:
[0390] NHT that gives [F,S,Y,L,P,H,I,T,N,V,A,D],
[0391] VNS that gives
[0392]
[L.sup.2,P.sup.2,H,Q,R.sup.3,I,M,T.sup.2,N,K,S,V.sup.2,A.sup.2,E,D,-
G.sup.2],
[0393] NNG that gives
[L.sup.2,S,W,P,Q,R.sup.2,M,T,K,R,V,A,E,G,stop],
[0394] SNT that gives [L,P,H,R,V,A,D,G],
[0395] RNG that gives [M,T,K,R,V,A,E,G],
[0396] RMG that gives [T,K,A,E],
[0397] VNT that gives [L,P,H,R,I,T,N,S,V,A,D,G], or
[0398] RRS that gives [N,S,K,R,D,E,G.sup.2].
[0399] However, each of these schemes has one or more of the
disadvantages, relative to NNT: a) fewer amino acids are allowed,
b) amino acids are not evenly provided, c) acidic and basic amino
acids are not equally likely), or d) stop codons occur.
Nonetheless, NNG, NHT, and VNT are almost as useful as NNT. NNG
encodes 13 different amino acids and one stop signal. Only two
amino acids appear twice in the 16-fold mix.
[0400] Thirdly, one can enrich the population for binding to the
preselected target, and evaluate selected sequences post hoc for
extra cysteines. Those that contain more cysteines than the
cysteines provided for conformational constraint may be perfectly
usable. It is possible that a disulfide linkage other than the
designed one will occur. This does not mean that the binding domain
defined by the isolated DNA sequence is in any way unsuitable. The
suitability of the isolated domains is best determined by chemical
and biochemical evaluation of chemically synthesized peptides.
[0401] Lastly, one can block free thiols with reagents, such as
Ellman's reagent, iodoacetate, or methyl iodide, that specifically
bind free thiols and that do not react with disulfides, and then
leave the modified phage in the population. It is to be understood
that the blocking agent may alter the binding properties of the
mini-protein; thus, one might use a variety of blocking reagent in
expectation that different binding domains will be found. The
variegated population of thiol-blocked genetic packages are
fractionated for binding. If the DNA sequence of the isolated
binding mini-protein contains an odd number of cysteines, then
synthetic means are used to prepare mini-proteins having each
possible linkage and in which the odd thiol is appropriately
blocked. Nishiuchi (NISH82, NISH86, and works cited therein)
disclose methods of synthesizing peptides that contain a plurality
of cysteines so that each thiol is protected with a different type
of blocking group. These groups can be selectively removed so that
the disulfide pairing can be controlled. We envision using such a
scheme with the alteration that one thiol either remains blocked,
or is unblocked and then reblocked with a different reagent.
[0402] III.E Planning the Second and Later Rounds of
Variegation
[0403] The method of the present invention allows efficient
accumulation of information concerning the amino-acid sequence of a
binding domain having high affinity for a predetermined target.
Although one may obtain a highly useful binding domain from a
single round of variegation and affinity enrichment, we expect that
multiple rounds will be needed to achieve the highest possible
affinity and specificity.
[0404] If the first round of variegation results in some binding to
the target, but the affinity for the target is still too low,
further improvement may be achieved by variegation of the SBDs.
Preferably, the process is progressive, i.e. each variegation cycle
produces a better starting point for the next variegation cycle
than the previous cycle produced. Setting the level of variegation
such that the ppbd and many sequences related to the ppbd sequence
are present in detectable amounts ensures that the process is
progressive. If the level of variegation is so high that the ppbd
sequence is present at such low levels that there is an appreciable
chance that no transformant will display the PPBD, then the best
SBD of the next round could be worse than the PPBD. At excessively
high level of variegation, each round of mutagenesis is independent
of previous rounds and there is no assurance of progressivity. This
approach can lead to valuable binding proteins, but repetition of
experiments with this level of variegation will not yield
progressive results. Excessive variation is not preferred.
[0405] Progressivity is not an all-or-nothing property. So long as
most of the information obtained from previous variegation cycles
is retained and many different surfaces that are related to the
PPBD surface are produced, the process is progressive. If the level
of variegation is so high that the ppbd gene may not be detected,
the assurance of progressivity diminishes. If the probability of
recovering PPBD is negligible, then the probability of progressive
behavior is also negligible.
[0406] A level of variegation that allows recovery of the PPBD has
two properties:
[0407] 1) we can not regress because the PPBD is available,
[0408] 2) an enormous number of multiple changes related to the
PPBD are available for selection and we are able to detect and
benefit from these changes.
[0409] It is very unlikely that all of the variants will be worse
than the PPBD; we desire the presence of PPBD at detectable levels
to insure that all the sequences present are indeed related to
PPBD.
[0410] An opposing force in our design considerations is that PBDs
are useful in the population only up to the amount that can be
detected; any excess above the detectable amount is wasted. Thus we
produce as many surfaces related to PPBD as possible within the
constraint that the PPBD be detectable.
[0411] If the level of variegation in the previous variegation
cycle was correctly chosen, then the amino acids selected to be in
the residues just varied are the ones best determined. The
environment of other residues has changed, so that it is
appropriate to vary them again. Because there are often more
residues in the principal and secondary sets than can be varied
simultaneously, we start by picking residues that either have never
been varied (highest priority) or that have not been varied for one
or more cycles. If we find that varying all the residues except
those varied in the previous cycle does not allow a high enough
level of diversity, then residues varied in he previous cycle might
be varied again. For example, if M.sub.ntv (the number of
independent transformants that car be produced from Y.sub.D100 of
DNA) and C.sub.sensi (the sensitivity of the affinity separation)
were such that seven residues could be varied, and if the principal
and secondary sets contained 13 residues, we would always vary
seven residues, even though that implies varying some residue twice
in a row. In such cases, we would pick the residues just varied
that contain the amino acids of highest abundance in the variegated
codons used.
[0412] It is the accumulation of information that allows the
process to select those protein sequences that produce binding
between the SBD and the target. Some interfaces between proteins
and other molecules involve twenty or more residues. Complete
variation of twenty residues would generate 10.sup.26 different
proteins. By dividing the residues that lie close together in space
into overlapping groups of five to seven residues, we can vary a
large surface but never need to test more than 10.sup.7 to 10.sup.9
candidates at once, a savings of 10.sup.19 to 10.sup.17 fold. The
power of selection with accumulation of information is well
illustrated in Chapter 3 of DAWK86.
[0413] Use of NNT or NNG variegated codons leads to very efficient
sampling of variegated libraries because the ratio of (different
amino-acid sequences)/(different DNA sequences) is much closer to
unity than it is for NNK or even the optimized vg codon (fxS).
Nevertheless, a few amino acids are omitted in each case. Both NNT
and NNG allow members of all important classes of amino acids:
hydrophobic, hydrophilic, acidic, basic, neutral hydrophilic,
small, and large. After selecting a binding domain, a subsequent
variegation and selection may be desirable to achieve a higher
affinity or specificity. During this second variegation, amino acid
possibilities overlooked by the preceding variegation may be
investigated
[0414] In the first round, we assume that the parental protein has
no known affinity for the target material. For example, consider
the parental mini-protein similar to that discussed in Example 11,
having the structure
X.sub.1--C.sub.2--X.sub.3--X.sub.4--X.sub.5--X.sub.6--C.sub.7---
X.sub.8 in which C.sub.2 and C.sub.7 form a disulfide bond.
Introduction of extra cysteines may cause alternative structures to
form which might be disadvantageous. Accidental cysteines at
positions 4 or 5 are thought to be potentially more troublesome
than at the other positions. We adopt the pattern of variegation:
X.sub.1:NNT, X.sub.3:NNT, X.sub.4:NNG, X.sub.5:NNG, X.sub.6:NNT,
and X.sub.8:NNT, so that cysteine can not occur at positions 4 and
5. (Table 131 shows the number of different amino acids expected in
libraries prepared with DNA variegated in this way and comprising
different numbers of independent transformants.)
[0415] In the second round of variegation, a preferred strategy is
to vary each position through a new set of residues which includes
the amino acid(s) which were found at that position in the
successful binding domains, and which include as many as possible
of the residues which were excluded in the first round of
variegation.
[0416] A few examples may be helpful. Suppose we obtained PRO using
NNT. This amino acid is available with either NNT or NNG. We can be
reasonably sure that PRO is the best amino acid from the set [PRO,
LEU, VAL, THR, ALA, ARG, GLY, PHE, TYR, CYS, HIS, ILE, ASN, ASP,
SER]. Thus we need to try a set that includes [PRO, TRP, GLN, MET,
LYS, GLU]. The set allowed by NNG is the preferred set.
[0417] What if we obtained HIS instead? Histidine is aromatic and
fairly hydrophobic and can form hydrogen bonds to and from the
imidazole ring. Tryptophan is hydrophobic and aromatic and can
donate a hydrogen to a suitable acceptor and was excluded by the
NNT codon. Methionine was also excluded and is hydrophobic. Thus,
one preferred course is to use the variegated codon HDS that allows
[HIS, GLN, ASN, LYS, TYR, CYS, TRP, ARG, SER, GLY,
<stop>].
[0418] GLN can be encoded by the NNG codon. If GLN is selected, at
the next round we might use the vg codon VAS that encodes three of
the seven excluded possibilities, viz. HIS, ASN, and ASP. The codon
VAS encodes 6 amino acid sequences in six DNA sequences. This
leaves PHE, CYS, TYR, and ILE untested, but these are all very
hydrophobic. Switching to NNT would be undesirable because that
would exclude GLN. One could use NAS that includes TYR and
<stop>. Suppose the successful amino acid encoded by an NNG
codon was ARG. Here we switch to NNT because this allows ARG plus
all the excluded possibilities.
[0419] THR is another possibility with the NNT codon. If THR is
selected, we switch to NNG because that includes the previously
excluded possibilities and includes THR. Suppose the successful
amino acid encoded by the NNT codon was ASP. We use RRS at the next
variegation because this includes both acidic amino acids plus LYS
and ARG. One could also use VRS to allow GLN.
[0420] Thus, later rounds of variegation test both amino acid
positions not previously mutated, and amino acid substitutions at a
previously mutated position which were not within the previous
substitution set.
[0421] If the first round of variegation is entirely unsuccessful,
a different pattern of variegation should be used. For example, if
more than one interaction set can be defined within a domain, the
residues varied in the next round of variegation should be from a
different set than that probed in the initial variegation. If
repeated failures are encountered, one may switch to a different
IPBD.
[0422] IV. Display Strategy: Displaying Foreign Binding Domains on
the Surface of a "Genetic Package"
[0423] IV.A. General Requirements for Genetic Packages
[0424] It is emphasized that the GP on which
selection-through-binding will be practiced must be capable, after
the selection, either of growth in some suitable environment or of
in vitro amplification and recovery of the encapsulated genetic
message. During at least part of the growth, the increase in number
is preferably approximately exponential with respect to time. The
component of a population that exhibits the desired binding
properties may be quite small, for example, one in 10.sup.6 or
less. Once this component of the population is separated from the
non-binding components, it must be possible to amplify it.
Culturing viable cells is the most powerful amplification of
genetic material known and is preferred. Genetic messages can also
be amplified in vitro, e.g. by PCR, but this is not the most
preferred method.
[0425] Preferred GPs are vegetative bacterial cells, bacterial
spores and bacterial DNA viruses. Eukaryotic cells could be used as
genetic packages but have longer dividing times and more stringent
nutritional requirements than do bacteria and it is much more
difficult to produce a large number of independent transformants.
They are also more fragile than bacterial cells and therefore more
difficult to chromatograph without damage. Eukaryotic viruses could
be used instead of bacteriophage but must be propagated in
eukaryotic cells and therefore suffer from some of the
amplification problems mentioned above.
[0426] Nonetheless, a strain of any living cell or virus is
potentially useful if the strain can be: 1) genetically altered
with reasonable facility to encode a potential binding domain, 2)
maintained and amplified in culture, 3) manipulated to display the
potential binding protein domain where it can interact with the
target material during affinity separation, and 4) affinity
separated while retaining the genetic information encoding the
displayed binding domain in recoverable form. Preferably, the GP
remains viable after affinity separation.
[0427] When the genetic package is a bacterial cell, or a phage
which is assembled periplasmically, the display means has two
components. The first component is a secretion signal which directs
the initial expression product to the inner membrane of the cell (a
host cell when the package is a phage). This secretion signal is
cleaved off by a signal peptidase to yield a processed, mature,
potential binding protein. The second component is an outer surface
transport signal which directs the package to assemble the
processed protein into its outer surface. Preferably, this outer
surface transport signal is derived from a surface protein native
to the genetic package.
[0428] For example, in a preferred embodiment, the hybrid gene
comprises a DNA encoding a potential binding domain operably linked
to a signal sequence (e.g., the signal sequences of the bacterial
phoA or bla genes or the signal sequence of M13 phage geneIII) and
to DNA encoding a coat protein (e.g., the M13 gene III or gene VIII
proteins) of a filamentous phage (e.g., M13). The expression
product is transported to the inner membrane (lipid bilayer) of the
host cell, whereupon the signal peptide is cleaved off to leave a
processed hybrid protein. The C-terminus of the coat protein-like
component of this hybrid protein is trapped in the lipid bilayer,
so that the hybrid protein does not escape into the periplasmic
space. (This is typical of the wild-type coat protein.) As the
single-stranded DNA of the nascent phage particle passes into the
periplasmic space, it collects both wild-type coat protein and the
hybrid protein from the lipid bilayer. The hybrid protein is thus
packaged into the surface sheath of the filamentous phage, leaving
the potential binding domain exposed on its outer surface. (Thus,
the filamentous phage, not the host bacterial cell, is the
"replicable genetic package" in this embodiment.)
[0429] If a secretion signal is necessary for the display of the
potential binding domain, in an especially preferred embodiment the
bacterial cell in which the hybrid gene is expressed is of a
"secretion-permissive" strain.
[0430] When the genetic package is a bacterial spore, or a phage
whose coat is assembled intracellularly, a secretion signal
directing the expression product to the inner membrane of the host
bacterial cell is unnecessary. In these cases, the display means is
merely the outer surface transport signal, typically a derivative
of a spore or phage coat protein.
[0431] There are several methods of arranging that the ipbd gene is
expressed in such a manner that the IPBD is displayed on the outer
surface of the GP. If one or more fusions of fragments of x genes
to fragments of a natural osp gene are known to cause X protein
domains to appear on the GP surface, then we pick the DNA sequence
in which an ipbd gene fragment replaces the x gene fragment in one
of the successful osp-x fusions as a preferred gene to be tested
for the display-of-IPBD phenotype. (The gene may be constructed in
any manner.) If no fusion data are available, then we fuse an ipbd
fragment to various fragments, such as fragments that end at known
or predicted domain boundaries, of the osp gene and obtain GPs that
display the osp-ipbd fusion on the GP outer surface by screening or
selection for the display-of-IPBD phenotype. The OSP may be
modified so as to increase the flexibility and/or length of the
linkage between the OSP and the IPBD and thereby reduce
interference between the two.
[0432] The fusion of ipbd and osp fragments may also include
fragments of random or pseudorandom DNA to produce a population,
members of which may display IPBD on the GP surface. The members
displaying IPBD are isolated by screening or selection for the
display-of-binding phenotype.
[0433] The replicable genetic entity (phage or plasmid) that
carries the osp-pbd genes (derived from the osp-ipbd gene) through
the selection-through-binding process, is referred to hereinafter
as the operative cloning vector (OCV). When the OCV is a phage, it
may also serve as the genetic package. The choice of a GP is
dependent in part on the availability of a suitable OCV and
suitable OSP.
[0434] Preferably, the GP is readily stored, for example, by
freezing. If the GP is a cell, it should have a short doubling
time, such as 20-40 minutes. If the GP is a virus, it should be
prolific, e.g., a burst size of at least 100/infected cell. GPs
which are finicky or expensive to culture are disfavored. The GP
should be easy to harvest, preferably by centrifugation. The GP is
preferably stable for a temperature range of -70 to 42.degree. C.
(stable at 4.degree. C. for several days or weeks); resistant to
shear forces found in HPLC; insensitive to UV; tolerant of
desiccation; and resistant to a pH of 2.0 to 10.0, surface active
agents such as SDS or Triton, chaotropes such as 4M urea or 2M
guanidinium HCl, common ions such as K.sup.+, Na.sup.+, and
SO.sub.4.sup.--, common organic solvents such as ether and acetone,
and degradative enzymes. Finally, there must be a suitable OCV.
[0435] Although knowledge of specific OSPs may not be required for
vegetative bacterial cells and endospores, the user of the present
invention, preferably, will know: is the sequence of any osp known?
(preferably yes, at least one required for phage). How does the OSP
arrive at the surface of GP? (knowledge of route necessary,
different routes have different uses, no route preferred per se).
Is the OSP post-translationally processed? (no processing most
preferred, predictable processing preferred over unpredictable
processing). What rules are known governing this processing, if
there is any processing? (no processing most preferred, predictable
processing acceptable). What function does the OSP serve in the
outer surface? (preferably not essential). Is the 3D structure of
an OSP known? (highly preferred). Are fusions between fragments of
osp and a fragment of x known? Does expression of these fusions
lead to X appearing on the surface of the GP? (fusion data is as
preferred as knowledge of a 3D structure). Is a "2D" structure of
an OSP available? (in this context, a "2D" structure indicates
which residues are exposed on the cell surface) (2D structure less
preferred than 3D structure). Where are the domain boundaries in
the OSP? (not as preferred as a 2D structure, but acceptable).
Could IPBD go through the same process as OSP and fold correctly?
(IPBD might need prosthetic groups) (preferably IPBD will fold
after same process). Is the sequence of an osp promoter known?
(preferably yes). Is osp gene controlled by regulatable promoter
available? (preferably yes). What activates this promoter?
(preferably a diffusible chemical, such as IPTG). How many
different OSPs do we know? (the more the better). How many copies
of each OSP are present on each package? (more is better).
[0436] The user will want knowledge of the physical attributes of
the GP: How large is the GP? (knowledge useful in deciding how to
isolate GPs) (preferably easy to separate from soluble proteins
such as IgGs). What is the charge on the "GP? (neutral preferred).
What is the sedimentation rate of the GP? (knowledge preferred, no
particular value preferred).
[0437] The preferred GP, OCV and OSP are those for which the fewest
serious obstacles can be seen, rather than the one that scores
highest on any one criterion.
[0438] Viruses are preferred over bacterial cells and spores (cp.
LUIT85 and references cited therein). The virus is preferably a DNA
virus with a genome size of 2 kb to 10 kb base pairs, such as (but
not limited to) the filamentous (Ff) phage M13, fd, and f1 (inter
alia see RASC86, BOEK80, BOEK82, DAYL88, GRAY81b, KUHN88, LOPE85,
WEBS85, MARV75, MARV80, MOSE82, CRIS84, SMIT88a, SMIT88b); the IncN
specific phage Ike and If1 (NAKA81, PEET85, PEET87, THOM83,
THOM88a); IncP-specific Pseudomonas aeruginosa phage Pf1 (THOM83,
THOM88a) and Pf3 (LUIT83, LUIT85, LUTI87, THOM88a); and the
Xanthomonas oryzae phage Xf (THOM83, THOM88a). Filamentous phage
are especially preferred.
[0439] Preferred OSPs for several GPs are given in Table 2.
References to osp-ipbd fusions in this section should be taken to
apply, mutatis mutandis, to osp-pbd and osp-sbd fusions as
well.
[0440] The species chosen as a GP should have a well-characterized
genetic system and strains defective in genetic recombination
should be available. The chosen strain may need to be manipulated
to prevent changes of its physiological state that would alter the
number or type of proteins or other molecules on the cell surface
during the affinity separation procedure.
[0441] IV.B. Phages for Use as GPs:
[0442] Unlike bacterial cells and spores, choice of a phage depends
strongly on knowledge of the 3D structure of an OSP and how it
interacts with other proteins in the capsid. This does not mean
that we need atomic resolution of the OSP, but that we need to know
which segments of the OSP interact to make the viral coat and which
segments are not constrained by structural or functional roles. The
size of the phage genome and the packaging mechanism are also
important because the phage genome itself is the cloning vector.
The osp-ipbd gene is inserted into the phage genome; therefore: 1)
the genome of the phage must allow introduction of the osp-ipbd
gene either by tolerating additional genetic material or by having
replaceable genetic material; 2) the virion must be capable of
packaging the genome after accepting the insertion or substitution
of genetic material, and 3) the display of the OSP-IPBD protein on
the phage surface must not disrupt virion structure sufficiently to
interfere with phage propagation.
[0443] The morphogenetic pathway of the phage determines the
environment in which the IPBD will have opportunity to fold.
Periplasmically assembled phage are preferred when IPBDs contain
essential disulfides, as such IPBDs may not fold within a cell
(these proteins may fold after the phage is released from the
cell). Intracellularly assembled phage are preferred when the IPBD
needs large or insoluble prosthetic groups (such as Fe.sub.4S.sub.4
clusters), since the IPBD may not fold if secreted because the
prosthetic group is lacking.
[0444] When variegation is introduced in Part II, multiple
infections could generate hybrid GPs that carry the gene for one
PBD but have at least some copies of a different PBD on their
surfaces; it is preferable to minimize this possibility by
infecting cells with phage under conditions resulting in a low
multiple-of-infection (MOI).
[0445] Bacteriophages are excellent candidates for GPs because
there is little or no enzymatic activity associated with intact
mature phage, and because the genes are inactive outside a
bacterial host, rendering the mature phage particles metabolically
inert.
[0446] The filamentous phages (e.g., M13) are of particular
interest.
[0447] For a given bacteriophage, the preferred OSP is usually one
that is present on the phage surface in the largest number of
copies, as this allows the greatest flexibility in varying the
ratio of OSP-IPBD to wild type OSP and also gives the highest
likelihood of obtaining satisfactory affinity separation. Moreover,
a protein present in only one or a few copies usually performs an
essential function in morphogenesis or infection; mutating such a
protein by addition or insertion is likely to result in reduction
in viability of the GP. Nevertheless, an OSP such as M13 gIII
protein may be an excellent choice as OSP to cause display of the
PBD.
[0448] It is preferred that the wild-type osp gene be preserved.
The ipbd gene fragment may be inserted either into a second copy of
the recipient osp gene or into a novel engineered osp gene. It is
preferred that the osp-ipbd gene be placed under control of a
regulated promoter. Our process forces the evolution of the PBDs
derived from IPBD so that some of them develop a novel function,
viz. binding to a chosen target. Placing the gene that is subject
to evolution on a duplicate gene is an imitation of the
widely-accepted scenario for the evolution of protein families. It
is now generally accepted that gene duplication is the first step
in the evolution of a protein family from an ancestral protein. By
having two copies of a gene, the affected physiological process can
tolerate mutations in one of the genes. This process is well
understood and documented for the globin family (cf. DICK83, p65ff,
and CREI84, p117-125).
[0449] The user must choose a site in the candidate OSP gene for
inserting a ipbd gene fragment. The coats of most bacteriophage are
highly ordered. Filamentous phage can be described by .alpha.
helical lattice; isometric phage, by an icosahedral lattice. Each
monomer of each major coat protein sits on a lattice point and
makes defined interactions with each of its neighbors. Proteins
that fit into the lattice by making some, but not all, of the
normal lattice contacts are likely to destabilize the virion by: a)
aborting formation of the virion, b) making the virion unstable, or
c) leaving gaps in the virion so that the nucleic acid is not
protected. Thus in bacteriophage, unlike the cases of bacteria and
spores, it is important to retain in engineered OSP-IPBD fusion
proteins those residues of the parental OSP that interact with
other proteins in the virion. For M13 gVIII, we retain the entire
mature protein, while for M13 gIII, it might suffice to retain the
last 100 residues (or even fewer). Such a truncated gIII protein
would be expressed in parallel with the complete gIII protein, as
gIII protein is required for phage infectivity.
[0450] Il'ichev et al. (ILIC89) have reported viable phage having
alterations in gene VIII. In one case, a point mutation changed one
amino acid near the amino terminus of the mature gVIII protein from
GLU to ASP. In the other case, five amino acids were inserted at
the site of the first mutation. They suggested that similar
constructions could be used for vaccines. They did not report on
any binding properties of the modified phage, nor did they suggest
mutagenizing the inserted material. Furthermore, they did not
insert a binding domain, nor did they suggest inserting such a
domain.
[0451] Further considerations on the design of the ipbd::osp gene
is discussed in section IV.F.
[0452] Filamentous Phage:
[0453] Compared to other bacteriophage, filamentous phage in
general are attractive and M13 in particular is especially
attractive because: 1) the 3D structure of the virion is known; 2)
the processing of the coat protein is well understood; 3) the
genome is expandable; 4) the genome is small; 5) the sequence of
the genome is known; 6) the virion is physically resistant to
shear, heat, cold, urea, guanidinium Cl, low pH, and high salt; 7)
the phage is a sequencing vector so that sequencing is especially
easy; 8) antibiotic-resistance genes have been cloned into the
genome with predictable results (HINESO); 9) It is easily cultured
and stored (FRIT85), with no unusual or expensive media
requirements for the infected cells, 10) it has a high burst size,
each infected cell yielding 100 to 1000 M13 progeny after
infection; and 11) it is easily harvested and concentrated (SALI64,
FRIT85).
[0454] The filamentous phage include M13, f1, fd, If1, Ike, Xf,
Pf1, and Pf3.
[0455] The entire life cycle of the filamentous phage M13, a common
cloning and sequencing vector, is well understood. M13 and f1 are
so closely related that we consider the properties of each relevant
to both (RASC86); any differentiation is for historical accuracy.
The genetic structure (the complete sequence (SCHA78), the identity
and function of the ten genes, and the order of transcription and
location of the promoters) of M13 is well known as is the physical
structure of the virion (BANN81, BOEK80, CHAN79, ITOK79, KAPL78,
KUHN85b, KUHN87, MAKO80, MARV78, MESS78, OHKA81, RASC86, RUSS81,
SCHA78, SMIT85, WEBS78, and ZIMM82); see RASC86 for a recent review
of the structure and function of the coat proteins. Because the
genome is small (6423 bp), cassette mutagenesis; is practical on RF
M13 (AUSU87), as is single-stranded oligo-nt directed mutagenesis
(FRITS5). M13 is a plasmid and transformation system in itself, and
an ideal sequencing vector. M13 can be grown on Rec.sup.- strains
of E. coli. The M13 genome is expandable (MESS78, FRIT85) and M13
does not lyse cells. Because the M13 genome is extruded through the
membrane and coated by a large number of identical protein
molecules, it can be used as a cloning vector (WATS87 p278, and
MESS77). Thus we can insert extra genes into M13 and they will be
carried along in a stable manner.
[0456] Marvin and collaborators (MARV78, MAKO80, BANN81) have
determined an approximate 3D virion structure of f1 by a
combination of genetics, biochemistry, and X-ray diffraction from
fibers of the virus. FIG. 4 is drawn after the model of Banner et
al. (BANN81) and shows only the C.sub..alpha.s of the protein. The
apparent holes in the cylindrical sheath are actually filled by
protein side groups so that the DNA within is protected. The amino
terminus of each protein monomer is to the outside of the cylinder,
while the carboxy terminus is at smaller radius, near the DNA.
Although other filamentous phages (e.g. Pf1 or Ike) have different
helical symmetry, all have coats composed of many short
.alpha.-helical monomers with the amino terminus of each monomer on
the virion surface.
[0457] The major coat protein is encoded by gene VIII. The 50 amino
acid mature gene VIII coat protein is synthesized as a 73 amino
acid precoat (ITOK79). The first 23 amino acids constitute a
typical signal-sequence which causes the nascent polypeptide to be
inserted into the inner cell membrane. Whether the precoat inserts
into the membrane by itself or through the action of host secretion
components, such as SecA and SecY, remains controversial, but has
no effect on the operation of the present invention.
[0458] An E. coli signal peptidase (SP-I) recognizes amino acids
18, 21, and 23, and, to a lesser extent, residue 22, and cuts
between residues 23 and 24 of the precoat (KUHN85a, KUHN85b,
OLIV87). After removal of the signal sequence, the amino terminus
of the mature coat is located on the periplasmic side of the inner
membrane; the carboxy terminus is on the cytoplasmic side. About
3000 copies of the mature 50 amino acid coat protein associate
side-by-side in the inner membrane.
[0459] The sequence of gene VIII is known, and the amino acid
sequence can be encoded on a synthetic gene, using lacUV5 promoter
and used in conjunction with the LacI.sup.q repressor. The lacUV5
promoter is induced by IPTG. Mature gene VIII protein makes up the
sheath around the circular ssDNA. The 3D structure of f1 virion is
known at medium resolution; the amino terminus of gene VIII protein
is on surface of the virion. A few modifications of gene VIII have
been made and are discussed below. The 2D structure of M13 coat
protein is implicit in the 3D structure. Mature M13 gene VIII
protein has only one domain.
[0460] When the GP is M13 the gene III and the gene VIII proteins
are highly preferred as OSP (see Examples I through IV). The
proteins from genes VI, VII, and IX may also be used.
[0461] As discussed in the Examples, we have constructed a
tripartite gene comprising:
[0462] 1) DNA encoding a signal sequence directing secretion of
parts (2) and (3) through the inner membrane,
[0463] 2) DNA encoding the mature BPTI sequence, and
[0464] 3) DNA encoding the mature M13 gVIII protein.
[0465] This gene causes BPTI to appear in active form on the
surface of M13 phage.
[0466] The gene VIII protein is a preferred OSP because it is
present in many copies and because its location and orientation in
the virion are known (BANN81). Preferably, the PBD is attached to
the amino terminus of the mature M13 coat protein. Had direct
fusion of PBD to M13 CP failed to cause PBD to be displayed on the
surface of M13, we would have varied part of the mini-protein
sequence and/or insert short random or nonrandom spacer sequences
between mini-protein and M13 CP. The 3D model of f1 indicates
strongly that fusing IPBD to the amino terminus of M13 CP is more
likely to yield a functional chimeric protein than any other fusion
site.
[0467] Similar constructions could be made with other filamentous
phage. Pf3 is a well known filamentous phage that infects
Pseudomonas aerugenosa cells that harbor an IncP-1 plasmid. The
entire genome has been sequenced (LUIT85) and the genetic signals
involved in replication and assembly are known (LUIT87). The major
coat protein of PF3 is unusual in having no signal peptide to
direct its secretion. The sequence has charged residues ASP.sub.7,
ARG.sub.37, LYS.sub.40, and PHE.sub.44-COO.sup.- which is
consistent with the amino terminus being exposed. Thus, to cause an
IPBD to appear on the surface of Pf3, we construct a tripartite
gene comprising:
[0468] 1) a signal sequence known to cause secretion in P.
aerugenosa (preferably known to cause secretion of IPBD) fused
in-frame to,
[0469] 2) a gene fragment encoding the IPBD sequence, fused
in-frame to,
[0470] 3) DNA encoding the mature Pf3 coat protein.
[0471] Optionally, DNA encoding a flexible linker of one to 10
amino acids is introduced between the ipbd gene fragment and the
Pf3 coat-protein gene. Optionally, DNA encoding the recognition
site for a specific protease, such as tissue plasminogen activator
or blood clotting Factor Xa, is introduced between the ipbd gene
fragment and the Pf3 coat-protein gene. Amino acids that form the
recognition site for a specific protease may also serve the
function of a flexible linker. This tripartite gene is introduced
into Pf3 so that it does not interfere with expression of any Pf3
genes. To reduce the possibility of genetic recombination, part (3)
is designed to have numerous silent mutations relative to the
wild-type gene. Once the signal sequence is cleaved off, the IPBD
is in the periplasm and the mature coat protein acts as an anchor
and phage-assembly signal. It matters not that this fusion protein
comes to rest in the lipid bilayer by a route different from the
route followed by the wild-type coat protein.
[0472] The amino-acid sequence of M13 pre-coat (SCHA78), called
AA_seq1, is
7 2
[0473] The single-letter codes for amino acids and the codes for
ambiguous DNA are given in Table 1. The best site for inserting a
novel protein domain into M13 CP is after A23 because SP-I cleaves
the precoat protein after A23, as indicated by the arrow. Proteins
that can be secreted will appear connected to mature M13 CP at its
amino terminus. Because the amino terminus of mature M13 CP is
located on the outer surface of the virion, the introduced domain
will be displayed on the outside of the virion. The uncertainty of
the mechanism by which M13CP appears in the lipid bilayer raises
the possibility that direct insertion of bpti into gene VIII may
not yield a functional fusion protein. It may be necessary to
change the signal sequence of the fusion to, for example, the phoA
signal sequence (MKQSTIALALLPLLFTPVTKA . . . ). Marks et al.
(MARK86) showed that the phoA signal peptide could direct mature
BPTI to the E. coli periplasm.
[0474] Another vehicle for displaying the IPBD is by expressing it
as a domain of a chimeric gene containing part or all of gene III.
This gene encodes one of the minor coat proteins of M13. Genes VI,
VII, and IX also encode minor coat proteins. Each of these minor
proteins is present in about 5 copies per virion and is related to
morphogenesis or infection. In contrast, the major coat protein is
present in more than 2500 copies per virion. The gene VI, VII, and
IX proteins are present at the ends of the virion; these three
proteins are not post-translationally processed (RASC86).
[0475] The single-stranded circular phage DNA associates with about
five copies of the gene III protein and is then extruded through
the patch of membrane-associated coat protein in such a way that
the DNA is encased in .alpha. helical sheath of protein (WEBS78).
The DNA does not base pair (that would impose severe restrictions
on the virus genome); rather the bases intercalate with each other
independent of sequence.
[0476] Smith (SMIT85) and de la Cruz et al. (DELA88) have shown
that insertions into gene III cause novel protein domains to appear
on the virion outer surface. The mini-protein's gene may be fused
to gene III at the site used by Smith and by de la Cruz et al., at
a codon corresponding to another domain boundary or to a surface
loop of the protein, or to the amino terminus of the mature
protein.
[0477] All published works use a vector containing a single
modified gene III of fd. Thus, all five copies of gIII are
identically modified. Gene III is quite large (1272 b.p. or about
20% of the phage genome) and it is uncertain whether a duplicate of
the whole gene can be stably inserted into the phage. Furthermore,
all five copies of gIII protein are at one end of the virion. When
bivalent target molecules (such as antibodies) bind a pentavalent
phage, the resulting complex may be irreversible. Irreversible
binding of the GP to the target greatly interferes with affinity
enrichment of the GPs that carry the genetic sequences encoding the
novel polypeptide having the highest affinity for the target.
[0478] To reduce the likelihood of formation of irreversible
complexes, we may use a second, synthetic gene that encodes
carboxy-terminal parts of III. We might, for example, engineer a
gene that consists of (from 5' to 3'):
[0479] 1) a promoter (preferably regulated),
[0480] 2) a ribosome-binding site,
[0481] 3) an initiation codon,
[0482] 4) a functional signal peptide directing secretion of parts
(5) and (6) through the inner membrane,
[0483] 5) DNA encoding an IPBD,
[0484] 6) DNA encoding residues 275 through 424 of M13 gIII
protein,
[0485] 7) a translation stop codon, and
[0486] 8) (optionally) a transcription stop signal.
[0487] We leave the wild-type gene III so that some unaltered gene
III protein will be present. Alternatively, we may use gene VIII
protein as the OSP and regulate the osp::ipbd fusion so that only
one or a few copies of the fusion protein appear on the phage.
[0488] M13 gene VI, VII, and IX proteins are not processed after
translation. The route by which these proteins are assembled into
the phase have rot been reported. These proteins are necessary for
normal morphogenesis and infectivity of the phage. Whether these
molecules (gene VI protein, gene VII protein, and gene IX protein)
attach themselves to the phage: a) from the cytoplasm, b) from the
periplasm, or c) from within the lipid bilayer, is not known. One
could use any of these proteins to introduce an IPBD onto the phage
surface by one of the constructions:
[0489] 1) ipbd::pmcp,
[0490] 2) Pmcp::ipbd,
[0491] 3) signal::ipbd::pmcp, and
[0492] 4) signal::pmcp::ipbd.
[0493] where ipbd represents DNA coding on expression for the
initial potential binding domain; pmcp represents DNA coding for
one of the phage minor coat proteins, VI, VII, and IX; signal
represents a functional secretion signal peptide, such as the phoA
signal (MKQSTIALALLPLLFTPVTKA)- ; and "::" represents in-frame
genetic fusion. The indicated fusions are placed downstream of a
known promoter, preferably a regulated promoter such as lacUV5,
tac, or trp. Fusions (1) and (2) are appropriate when the minor
coat protein attaches to the phage from the cytoplasm or by
autonomous insertion into the lipid bilayer. Fusion (1) is
appropriate if the amino terminus of the minor coat protein is free
and (2) is appropriate if the carboxy terminus is free. Fusions (3)
and (4) are appropriate if the minor coat protein attaches to the
phage from the periplasm or from within the lipid bilayer. Fusion
(3) is appropriate if the amino terminus of the minor coat protein
is free and (4) is appropriate if the carboxy terminus is free.
[0494] Bacteriophage .PHI.X174:
[0495] The bacteriophage .PHI.174 is a very small icosahedral virus
which has been thoroughly studied by genetics, biochemistry, and
electron microscopy (See The Single-Stranded DNA Phases (DENH78)).
To date, no proteins from .PHI.X174 have been studied by X-ray
diffraction. .PHI.X174 is not used as a cloning vector because
.PHI.X174 can accept very little additional DNA; the virus is so
tightly constrained that several of its genes overlap. Chambers et
al. (CHAM82) showed that mutants in gene G are rescued by the
wild-type G gene carried on a plasmid so that the host supplies
this protein.
[0496] Three gene products of .PHI.X174 are present on the outside
of the mature virion: F (capsid), G (major spike protein, 60 copies
per virion), and H (minor spike protein, 12 copies per virion). The
G protein comprises 175 amino acids, while H comprises 328 amino
acids. The F protein interacts with the single-stranded DNA of the
virus. The proteins F, G, and H are translated from a single mRNA
in the viral infected cells. If the G protein is supplied from a
plasmid in the host, then the viral g gene is no longer essential.
We introduce one or more stop codons into g so that no G is
produced from the viral gene. We fuse a pbd gene fragment to h,
either at the 3' or 5' terminus. We eliminate an amount of the
viral g gene equal to the size of pbd so that the size of the
genome is unchanged.
[0497] Large DNA Phages
[0498] Phage such as .lambda. or T4 have much larger genomes than
do M13 or .PHI.X174. Large genomes are less conveniently
manipulated than small genomes. Phage .lambda. has such a large
genome that cassette mutagenesis is not practicable. One can not
use annealing of a mutagenic oligonucleotide either, because there
is no ready supply of single-stranded .lambda. DNA. (.lambda. DNA
is packaged as double-stranded DNA.) Phage such as .lambda. and T4
have more complicated 3D capsid structures than M13 or .PHI.X174,
with more OSPs to choose from. Intracellular morphogenesis of phage
.lambda. could cause protein domains that contain disulfide bonds
in their folded forms not to fold.
[0499] Phage .lambda. virions and phage T4 virions form
intracellularly, so that IPBDs requiring large or insoluble
prosthetic groups might fold on the surfaces of these phage.
[0500] RNA Phages
[0501] RNA phage are not preferred because manipulation of RNA is
much less convenient than is the manipulation of DNA. If the RNA
phage MS2 were modified to make room for an osp-ipbd gene and if a
message containing the A protein binding site and the gene for a
chimera of coat protein and a PBD were produced in a cell that also
contained A protein and wild-type coat protein (both produced from
regulated genes on a plasmid), then the RNA coding for the chimeric
protein would get packaged. A package comprising RNA encapsulated
by proteins encoded by that RNA satisfies the major criterion that
the genetic message inside the package specifies something on the
outside. The particles by themselves are not viable unless the
modified A protein is functional. After isolating the packages that
carry an SBD, we would need to: 1) separate the RNA from the
protein capsid; 2) reverse transcribe the RNA into DNA, using AMV
or MMTV reverse transcriptase, and 3 use Thermus acuaticus DNA
polymerase for 25 or more cycles of Polymerase Chain Reaction.TM.
to amplify the osp-sbd DNA until there is enough to subclone the
recovered genetic message into a plasmid for sequencing and further
work.
[0502] Alternatively, helper phage could be used to rescue the
isolated phage. In one of these ways we can recover a sequence that
codes for an SBD having desirable binding properties.
[0503] IV.C. Bacterial Cells as Genetic Packages:
[0504] One may choose any well-characterized bacterial strain which
(1) may be grown in culture (2) may be engineered to display PBDs
on its surface, and (3) is compatible with affinity selection.
[0505] Among bacterial cells, the preferred genetic packages are
Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa,
Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae,
Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and
especially Escherichia coli. The potential binding mini-protein may
be expressed as an insert in a chimeric bacterial outer surface
protein (OSP). All bacteria exhibit proteins on their outer
surfaces. Works on the localization of OSPs and the methods of
determining their structure include: CALA90, HEIJ90, EHRM90,
BENZ88a, BENZ88b, MANO088, BAKE87, RAND87, HANC87, HENR87, NAKA86b,
MANO86, SILH85, TOMM85, NIKA84, LUGT83, and BECK83.
[0506] In E. coli, LamB is a preferred OSP. As discussed below,
there are a number of very good alternatives in E. coli and there
are very good alternatives in other bacterial species. There are
also methods for determining the topology of OSPs so that it is
possible to systematically determine where to insert an ipbd into
an osp gene to obtain display of an IPBD on the surface of any
bacterial species.
[0507] In view of the extensive knowledge of E. coli, a strain of
E. coli, defective in recombination, is the strongest candidate as
a bacterial GP.
[0508] Oliver has reviewed mechanisms of protein secretion in
bacteria (OLIV85a and OLIV87). Nikaido and Vaara (NIKA87), Benz
(BENZ88b), and Baker et al. (BAKE87) have reviewed mechanisms by
which proteins become localized to the outer membrane of
gram-negative bacteria. While moss bacterial proteins remain in the
cytoplasm, others are transported to the periplasmic space (which
lies between the plasma membrane and the cell wall of gram-negative
bacteria), or are conveyed and anchored to the outer surface of the
cell. Still others are exported (secreted) into the medium
surrounding the cell. Those characteristics of a protein that are
recognized by a cell and that cause it to be transported out of the
cytoplasm and displayed on the cell surface will be termed
"outer-surface transport signals".
[0509] Gram-negative bacteria have outer-membrane proteins (OMP),
that form a subset of OSPs. Many OMPs span the membrane one or more
times. The signals that cause OMPs to localize in the outer
membrane are encoded in the amino acid sequence of the mature
protein. Outer membrane proteins of bacteria are initially
expressed in a precursor form including a so-called signal peptide.
The precursor protein is transported to the inner membrane, and the
signal peptide moiety is extruded into the periplasmic space.
There, it is cleaved off by a "signal peptidase", and the remaining
"mature" protein can now enter the periplasm. Once there, other
cellular mechanisms recognize structures in the mature protein
which indicate that its proper place is on the outer membrane, and
transport it to that location.
[0510] It is well known that the DNA coding for the leader or
signal peptide from one protein may be attached to the DNA sequence
coding for another protein, protein X, to form a chimeric gene
whose expression causes protein X to appear free in the periplasm
(BECK83, INOU86 Ch10, LEEC86, MARK86, and BOQU87). That is, the
leader causes the chimeric protein to be secreted through the lipid
bilayer; once in the periplasm, it is cleaved off by the signal
peptidase SP-I.
[0511] The use of export-permissive bacterial strains (LISS85,
STAD89) increases the probability that a signal-sequence-fusion
will direct the desired protein to the cell surface. Liss et al.
(LISS85) showed that the mutation prlA4 makes E. coli more
permissive with respect to signal sequences. Similarly, Stader et
al. (STAD89) found a strain that bears a prlG mutation and that
permits export of a protein that is blocked from export in
wild-type cells. Such export-permissive strains are preferred.
[0512] OSP-IPBD fusion proteins need not fill a structural role in
the outer membranes of Gram-negative bacteria because parts of the
outer membranes are not highly ordered. For large OSPs there is
likely to be one or more sites at which osp can be truncated and
fused to ipbd such that cells expressing the fusion will display
IPBDs on the cell surface. Fusions of fragments of omp genes with
fragments of an x gene have led to X appearing on the outer
membrane (CHAR88b, BENS84, CLEM81). When such fusions have been
made, we can design an osp-ipbd gene by substituting ipbd for x in
the DNA sequence. Otherwise, a successful OMP-IPBD fusion is
preferably sought by fusing fragments of the best omp to an ipbd,
expressing the fused gene, and testing the resultant GPs for
display-of-IPBD phenotype. We use the available data about the OMP
to pick the point or points of fusion between omp and ipbd to
maximize the likelihood that IPBD will be displayed. (Spacer DNA
encoding flexible linkers, made, e.g., of GLY, SER, and ASN, may be
placed between the osp- and ipbd-derived fragments to facilitate
display.) Alternatively, we truncate osp at several sites or in a
manner that produces osp fragments of variable length and fuse the
osp fragments to ipbd; cells expressing the fusion are screened or
selected which display IPBDs on the cell surface. Freudl et al.
(FREU89) have shown that fragments of OSPs (such as OmpA) above a
certain size are incorporated into the outer membrane. An
additional alternative is to include short segments of random DNA
in the fusion of omp fragments to idbd and then screen or select
the resulting variegated population for members exhibiting the
display-of-IPBD phenotype.
[0513] In E. coli, the LamB protein is a well understood OSP and
can be used (BENS84, CHAR90, RONC90, VAND90, CHAP90, MOLL90,
CHAR88b, CHAR88c, CLEM81, DARG88, FERE82a, FERE82b, FERE83, FERE84,
FERE86a, FERE86b, FERE89a, FERE89b, GEHR87, HALL82, NAKA86a,
STAD86, HEIN88, BENS87b, BENS87c, BOUG84, BOUL86a, CHAR84). The E.
coli LamB has been expressed in functional form in S. typhimurium
(DEVR84, BARB85, HARK87), V. cholerae (HARK86), and K. pneumonia
(DEVR84, WEHM89), so that one could display a population of PBDs in
any of these species as a fusion to E. coli LamB. K. pneumonia
expresses a maltoporin similar to LamB (WEHM89) which could also be
used. In P. aeruginosa, the D1 protein (a homologue of LamB) can be
used (TRIA88).
[0514] LamB of E. coli is a porin for maltose and maltodextrin
transport, and serves as the receptor for adsorption of
bacteriophages .lambda. and K10. LamB is transported to the outer
membrane if a functional N-terminal sequence is present; further,
the first 49 amino acids of the mature sequence are required for
successful transport (BENS84). As with other OSPs, LamB of E. coli
is synthesized with a typical signal-sequence which is subsequently
removed. Homology between parts of LamB protein and other outer
membrane proteins OmpC, OmpF, and PhoE has been detected (NIKA84),
including homology between LamB amino acids 39-49 and sequences of
the other proteins. These subsequences may label the proteins for
transport to the outer membrane.
[0515] The amino acid sequence of LamB is known (CLEM81), and a
model has been developed of how it anchors itself to the outer
membrane (Reviewed by, among others, BENZ88b). The location of its
maltose and phage binding domains are also known (HEIN88). Using
this information, one may identify several strategies by which a
PBD insert may be incorporated into LamB to provide a chimeric OSP
which displays the PBD on the bacterial outer membrane.
[0516] When the PBDs are to be displayed by a chimeric
transmembrane protein like LamB, the PBD could be inserted into a
loop normally found on the surface of the cell (cp. BECK83,
MANO86). Alternatively, we may fuse a 5' segment of the osp gene to
the ipbd gene fragment; the point of fusion is picked to correspond
to a surface-exposed loop of the OSP and the carboxy terminal
portions of the OSP are omitted. In LamB, it has been found that up
to 60 amino acids may be inserted (CHAR88b) with display of the
foreign epitope resulting; the structural features of OmpC, OmpA,
OmpF, and PhoE are so similar that one expects similar behavior
from these proteins.
[0517] It should be noted that while LamB may be characterized as a
binding protein, it is used in the present invention to provide an
OSTS; its binding domains are not variegated.
[0518] Other bacterial outer surface proteins, such as OmpA, OmpC,
OmpF, PhoE, and pilin, may be used in place of LamB and its
homologues. OmpA is of particular interest because it is very
abundant and because homologues are known in a wide variety of
gram-negative bacterial species. Baker et al. (BAKE87) review
assembly of proteins into the outer membrane of E. coli and cite a
topological model of OmpA (VOGE86) that predicts that residues
19-32, 62-73, 105-118, and 147-158 are exposed on the cell surface.
Insertion of a ipbd encoding fragment at about codon 111 or at
about codon 152 is likely to cause the IPBD to be displayed on the
cell surface. Concerning OmpA, see also MACI88 and MANO88. Porin
Protein F of Pseudomonas aeruginosa has been cloned and has
sequence homology to OmpA of E. coli (DUCH88). Although this
homology is not sufficient to allow prediction of surface-exposed
residues on Porin Protein F, the methods used to determine the
topological model of OmpA may be applied to Porin Protein F. Works
related to use of OmpA as an OSP include BECK80 and MACI88.
[0519] Misra and Benson (MISR88a, MISR88b) disclose a topological
model of E. coli OmpC that predicts that, among others, residues
GLY.sub.164 and IEU.sub.250 are exposed on the cell surface. Thus
insertion of an ipbd gene fragment at about codon 164 or at about
codon 250 of the E. coli ompC gene or at corresponding codons of
the S. typhimurium ompC gene is likely to cause IPBD to appear on
the cell surface. The OmpC genes of other bacterial species may be
used. Other works related to OmpC include CATR87 and CLIC88.
[0520] OmpF of E. coli is a very abundant OSP, .gtoreq.10.sup.4
copies/cell. Pages et al. (PAGE90) have published a model of OmpF
indicating seven surface-exposed segments. Fusion of an ipbd gene
fragment, either as an insert or to replace the 3' part of ompF, in
one of the indicated regions is likely to produce a functional
ompF::ipbd gene the expression of which leads to display of IPBD on
the cell surface. In particular, fusion at about codon 111, 177,
217, or 245 should lead to a functional ompF::ipbd gene. Concerning
OmpF, see also REID88b, PAGE88, BENS88, TOMM82, and SODE85.
[0521] Pilus proteins are of particular interest because piliated
cells express many copies of these proteins and because several
species (N. gonorrhoeae, P. aeruginosa, Moraxella bovis,
Bacteroides nodosus, and E. coli) express related pilins. Getzoff
and coworkers (GETZ88, PARG87, SOME85) have constructed a model of
the gonococcal pilus that predicts that the protein forms a
four-helix bundle having structural similarities to tobacco mosaic
virus protein and myohemerythrin. On this model, both the amino and
carboxy termini of the protein are exposed. The amino terminus is
methylated. Elleman (ELLE88) has reviewed pilins of Bacteroides
nodosus and other species and serotype differences can be related
to differences in the pilin protein and that most variation occurs
in the C-terminal region. The amino-terminal portions of the pilin
protein are highly conserved. Jennings et al. (JENN89) have grafted
a fragment of foot-and-mouth disease virus (residues 144-159) into
the B. nodosus type 4 fimbrial protein which is highly homologous
to gonococcal pilin. They found that expression of the 3'-terminal
fusion in P. aeruginosa led to a viable strain that makes
detectable amounts of the fusion protein. Jennings et al. did not
vary the foreign epitope nor did they suggest any variation. They
inserted a GLY-GLY linker between the last pilin residue and the
first residue of the foreign epitope to provide a "flexible
linker". Thus a preferred place to attach an IPBD is the carboxy
terminus. The exposed loops of the bundle could also be used,
although the particular internal fusions tested by Jennings et al.
(JENN89) appeared to be lethal in P. aeruginosa. Concerning pilin,
see also MCKE85 and ORND85.
[0522] Judd (JUDD86, JUDD85) has investigated Protein IA of N.
gonorrhoeae and found that the amino terminus is exposed; thus, one
could attach an IPBD at or near the amino terminus of the mature
P.IA as a means to display the IPBD on the N. gonorrhoeae
surface.
[0523] A model of the topology of PhoE of E. coli has been
disclosed by van der Ley et al. (VAND86). This model predicts eight
loops that are exposed; insertion of an IPBD into one of these
loops is likely to lead to display of the IPBD on the surface of
the cell. Residues 158, 201, 238, and 275 are preferred locations
for insertion of and IPBD.
[0524] Other OSPs that could be used include E. coli BtuB, FepA,
FhuA, IutA, FecA, and FhuE (GUDM89) which are receptors for
nutrients usually found in low abundance. The genes of all these
proteins have been sequenced, but topological models are not yet
available. Gudmunsdottir et al. (GUDM89) have begun the
construction of such a model for BtuB and FepA by showing that
certain residues of BtuB face the periplasm and by determining the
functionality of various BtuB::FepA fusions. Carmel et al. (CARM90)
have reported work of a similar nature for FhuA. All Neisseria
species express outer surface proteins for iron transport that have
been identified and, in many cases, cloned. See also MORS87 and
MORS88.
[0525] Many gram-negative bacteria express one or more
phospholipases. E. coli phospholipase A, product of the pldA gene,
has been cloned and sequenced by de Geus et al. (DEGE84). They
found that the protein appears at the cell surface without any
posttranslational processing. A ipbd gene fragment can be attached
at either terminus or inserted at positions predicted to encode
loops in the protein. That phospholipase A arrives on the outer
surface without removal of a signal sequence does not prove that a
PldA::IPBD fusion protein will also follow this route. Thus we
might cause a PldA::IPBD or IPBD::PldA fusion to be secreted into
the periplasm by addition of an appropriate signal sequence. Thus,
in addition to simple binary fusion of an ipbd fragment to one
terminus of pldA, the constructions:
[0526] 1) sss::ipbd::pldA
[0527] 2) ss::pldA::ipbd
[0528] should be tested. Once the PldA::IPBD protein is free in the
periplasm it does not remember how it got there and the structural
features of PldA that cause it to localize on the outer surface
will direct the fusion to the same destination.
[0529] IV.D. Bacterial Spores as Genetic Packages:
[0530] Bacterial spores have desirable properties as GP candidates.
Spores are much more resistant than vegetative bacterial cells or
phage to chemical and physical agents, and hence permit the use of
a great variety of affinity selection conditions. Also, Bacillus
spores neither actively metabolize nor alter the proteins on their
surface. Spores have the disadvantage that the molecular mechanisms
that trigger sporulation are less well worked out than is the
formation of M13 or the export of protein to the outer membrane of
E. coli.
[0531] Bacteria of the genus Bacillus form endospores that are
extremely resistant to damage by heat, radiation, desiccation, and
toxic chemicals (reviewed by Losick et al. (LOSI86)). This
phenomenon is attributed to extensive intermolecular crosslinking
of the coat proteins. Endospores from the genus Bacillus are more
stable than are exospores from Streptomyces. Bacillus subtilis
forms spores in 4 to 6 hours, but Streptomyces species may require
days or weeks to sporulate. In addition, genetic knowledge and
manipulation is much more developed for B. subtilis than for other
spore-forming bacteria. Thus Bacillus spores are preferred over
Streptomyces spores. Bacteria of the genus Clostridium also form
very durable endospores, but clostridia, being strict anaerobes,
are not convenient to culture.
[0532] Viable spores that differ only slightly from wild-type are
produced in B. subtilis even if any one of four coat proteins is
missing (DONO87). Moreover, plasmid DNA is commonly included in
spores, and plasmid encoded proteins have been observed on the
surface of Bacillus spores (DEBR86). For these reasons, we expect
that it will be possible to express during sporulation a gene
encoding a chimeric coat protein, without interfering materially
with spore formation.
[0533] Donovan et al. have identified several polypeptide
components of B. subtilis spore coat (DONO87); the sequences of two
complete coat proteins and amino-terminal fragments of two others
have been determined. Some, but not all, of the coat proteins are
synthesized as precursors and are then processed by specific
proteases before deposition in the spore coat (DONO87). The 12 kd
coat protein, CotD, contains 5 cysteines. CotD also contains an
unusually high number of histidines (16) and prolines (7). The llkd
coat protein, CotC, contains only one cysteine and one methionine.
CotC has a very unusual amino-acid sequence with 19 lysines (K)
appearing as 9 K-K dipeptides and one isolated K. There are also 20
tyrosines (Y) of which 10 appear as 5 Y-Y dipeptides. Peptides rich
in Y and K are known to become crosslinked in oxidizing
environments (DEVO78, WAIT83, WAIT85, WAIT86). CotC contains 16 D
and E amino acids that nearly equals the 19 Ks. There are no A, F,
R, I, L, N, P, Q, S, or W amino acids in CotC. Neither CotC nor
CotD is post-translationally cleaved, but the proteins CotA and
CotB are.
[0534] Since, in B. subtilis, some of the spore coat proteins are
post-translationally processed by specific proteases, it is
valuable to know the sequences of precursors and mature coat
proteins so that we can avoid incorporating the recognition
sequence of the specific protease into our construction of an
OSP-IPBD fusion. The sequence of a mature spore coat protein
contains information that causes the protein to be deposited in the
spore coat; thus gene fusions that include some or all of a mature
coat protein sequence are preferred for screening or selection for
the display-of-IPBD phenotype.
[0535] Fusions of ipbd fragments to cotC or cotD fragments are
likely to cause IPBD to appear on the spore surface. The genes cotC
and cotD are preferred osp genes because CotC and CotD are not
post-translationally cleaved. Subsequences from cotA or cotB could
also be used to cause an IPBD to appear on the surface of B.
subtilis spores, but we must take the post-translational cleavage
of these proteins into account. DNA encoding IPBD could be fused to
a fragment of cotA or cotB at either end of the coding region or at
sites interior to the coding region. Spores could then be screened
or selected for the display-of-IPBD phenotype.
[0536] The promoter of a spore coat protein is most active: a) when
spore coat protein is being synthesized and deposited onto the
spore and b) in the specific place that spore coat proteins are
being made. The sequences of several sporulation promoters are
known; coding sequences operatively linked to such promoters are
expressed only during sporulation. Ray et al. (RAYC87) have shown
that the G4 promoter of B. subtilis is directly controlled by RNA
polymerase bound to .sigma..sup.E. To date, no Bacillus sporulation
promoter has been shown to be inducible by an exogenous chemical
inducer as the lac promoter of E. coli. Nevertheless, the quantity
of protein produced from a sporulation promoter can be controlled
by other factors, such as the DNA sequence around the
Shine-Dalgarno sequence or codon usage. Chemically inducible
sporulation promoters can be developed if necessary.
[0537] IV.E. Artificial OSPs
[0538] It is generally preferable to use as the genetic package a
cell, spore or virus for which an outer surface protein which can
be engineered to display a IPBD has already been identified.
However, the present invention is not limited to such genetic
packages.
[0539] It is believed that the conditions for an outer surface
transport signal in a bacterial cell or spore are not particularly
stringent, i.e., a random polypeptide of appropriate length
(preferably 30-100 amino acids) has a reasonable chance of
providing such a signal. Thus, by constructing a chimeric gene
comprising a segment encoding the IPBD linked to a segment of
random or pseudorandom DNA (the potential OSTS), and placing this
gene under control of a suitable promoter, there is a possibility
that the chimeric protein so encoded will function as an
OSP-IPBD.
[0540] This possibility is greatly enhanced by constructing
numerous such genes, each having a different potential OSTS,
cloning them into a suitable host, and selecting for transformants
bearing the IPBD (or other marker) on their outer surface. Use of
secretion-permissive mutants, such as PrlA4 (LISS85) or PrlG
(STAD89), can increase the probability of obtaining a working
OSP-IPBD.
[0541] When seeking to display a IPBD on the surface of a bacterial
cell, as an alternative to choosing a natural OSP and an insertion
site in the OSP, we can construct a gene (the "display probe")
comprising: a) a regulatable promoter (e.g. lacUV5), b) a
Shine-Dalgarno sequence, c) a periplasmic transport signal
sequence, d) a fusion of the ipbd gene with a segment of random DNA
(as in Kaiser et al. (KAIS87)), e) a stop codon, and f) a
transcriptional terminator.
[0542] When the genetic package is a spore, we can use the approach
described above for attaching a IPBD to an E. coli cell, except
that: a) a sporulation promoter is used, and b) no periplasmic
signal sequence should be present
[0543] For phage, because the OSP-IPBD fulfills a structural role
in the phage coat, it is unlikely that any particular random DNA
sequence coupled to the ipbd gene will produce a fusion protein
that fits into the coat in a functional way. Nevertheless, random
DNA inserted between large fragments of a coat protein gene and the
pbd gene will produce a population that is likely to contain one or
more members that display the IPBD on the outside of a viable
phage.
[0544] As previously stated, the purpose of the random DNA is to
encode an OSTS, like that embodied in known OSPs. The fusion of
ipbd and the random DNA could be in either order, but ipbd upstream
is slightly preferred. Isolates from the population generated in
this way can be screened for display of the IPBD. Preferably, a
version of selection-through-binding is used to select GPs that
display IPBD on the GP surface. Alternatively, clonal isolates of
GPs may be screened for the display-of-IPBD phenotype.
[0545] The preference for ipbd upstream of the random DNA arises
from consideration of the manner in which the successful GP(IPBD)
will be used. The present invention contemplates introducing
numerous mutations into the pbd region of the osp-pbd gene, which,
depending on the variegation scheme, might include gratuitous stop
codons. If pbd precedes the random DNA, then gratuitous stop codons
in pbd lead to no OSP-PBD protein appearing on the cell surface. If
pbd follows the random DNA, then gratuitous stop codons in pbd
might lead to incomplete OSP-PBD proteins appearing on the cell
surface. Incomplete proteins often are non-specifically sticky so
that GPs displaying incomplete PBDs are easily removed from the
population.
[0546] The random DNA may be obtained in a variety of ways.
Degenerate synthetic DNA is one possibility. Alternatively,
pseudorandom DNA can be generated from any DNA having high sequence
diversity, e.g., the genome of the organism, by partially digesting
with an enzyme that cuts very often, e.g., Sau3AI. Alternatively,
one could shear DNA having high sequence diversity, blunt the
sheared DNA with the large fragment of E. coli DNA polymerase I
(hereinafter referred to as Klenow fragment), and clone the sheared
and blunted DNA into blunt sites of the vector (MANI82, p295,
AUSU87).
[0547] If random DNA and phenotypic selection or screening are used
to obtain a GP(IPBD), then we clone random DNA into one of the
restriction sites that was designed into the display probe. A
plasmid carrying the display probe is digested with the appropriate
restriction enzyme and the fragmented, random DNA is annealed and
ligated by standard methods. The ligated plasmids are used to
transform cells that are grown and selected for expression of the
antibiotic-resistance gene. Plasmid-bearing GPs are then selected
for the display-of-IPBD phenotype by the affinity selection methods
described hereafter, using AfM(IPBD) as if it were the target.
[0548] As an alternative to selecting GP(IPBD)s through binding to
an affinity column, we can isolate colonies or plaques and screen
for successful artificial OSPs through use of one of the methods
listed below for verification of the display strategy.
[0549] IV.F Designing the osp-ipbd Gene Insert:
[0550] Genetic Construction and Expression Considerations
[0551] The (i)pbd-osp gene may be: a) completely synthetic, b) a
composite of natural and synthetic DNA, or c) a composite of
natural DNA fragments. The important point is that the pbd segment
be easily variegated so as to encode a multitudinous and diverse
family of PBDs as previously described. A synthetic ipbd segment is
preferred because it allows greatest control over placement of
restriction sites. Primers complementary to regions abutting the
osp-ipbd gene on its 31 flank and to parts of the osp-ipbd gene
that are not to be varied are needed for sequencing.
[0552] The sequences of regulatory parts of the gene are taken from
the sequences of natural regulatory elements: a) promoters, b)
Shine-Dalgarno sequences, and c) transcriptional b) terminators.
Regulatory elements could also be designed from knowledge of
consensus sequences of natural regulatory regions. The sequences of
these regulatory elements are connected to the coding regions;
restriction sites are also inserted in or adjacent to the
regulatory regions to allow convenient manipulation.
[0553] The essential function of the affinity separation is to
separate GPs that bear PBDs (derived from IPBD) having high
affinity for the target from GPs bearing PBDs having low affinity
for the target. If the elution volume of a GP depends on the number
of PBDs on the GP surface, then a GP bearing many PBDs with low
affinity, GP(PBDw), might co-elute with a GP bearing fewer PBDs
with high affinity, GP(PBDs). Regulation of the osp-pbd gene
preferably is such that most packages display sufficient PBD to
effect a good separation according to affinity. Use of a
regulatable promoter to control the level of expression of the
osp-pbd allows fine adjustment of the chromatographic behavior of
the variegated population.
[0554] Induction of synthesis of engineered genes in vegetative
bacterial cells has been exercised through the use of regulated
promoters such as lacUV5, trpP, or tac (MANI82). The factors that
regulate the quantity of protein synthesized include: a) promoter
strength (cf. HOOP87), b) rate of initiation of translation (cf.
GOLD87), c) codon usage, d) secondary structure of mRNA, including
atternators (cf. LAND87) and terminators (cf. YAGE87), e)
interaction of proteins with mRNA (cf. MCPH86, MILL87b, WINT87), f)
degradation rates of mRNA (cf. BRAW87, KING86), g) proteolysis (cf.
GOTT87). These factors are sufficiently well understood that a wide
variety of heterologous proteins can now be produced in E. coli, B.
subtilis and other host cells in at least moderate quantities
(SKER88, BETT88). Preferably, the promoter for the osp-ipbd gene is
subject to regulation by a small chemical inducer. For example, the
lac promoter and the hybrid trp-lac (tac) promoter are regulatable
with isopropyl thiogalactoside (IPTG). Hereinafter, we use
"XINDUCE" as a generic term for a chemical that induces expression
of a gene. The promoter for the constructed gene need not come from
a natural osp gene; any regulatable bacterial promoter can be
used.
[0555] Transcriptional regulation of gene expression is best
understood and most effective, so we focus our attention on the
promoter. If transcription of the osp-ipbd gene is controlled by
the chemical XINDUCE, then the number of OSP-IPBDs per GP increases
for increasing concentrations of XINDUCE until a fall-off in the
number of viable packages is observed or until sufficient IPBD is
observed on the surface of harvested GP(IPBD)s. The attributes that
affect the maximum number of OSP-IPBDs per GP are primarily
structural in nature. There may be steric hindrance or other
unwanted interactions between IPBDs if OSP-IPBD is substituted for
every wild-type OSP. Excessive levels of OSP-IPBD may also
adversely affect the solubility or morphogenesis of the GP. For
cellular and viral GPs, as few as five copies of a protein having
affinity for another immobilized molecule have resulted in
successful affinity separations (FERE82a, FERE82b, and SMIT85).
[0556] A non-leaky promoter is preferred. Non-leakiness is useful:
a) to show that affinity of GP(osp-ipbd)s for AfM(IPBD) is due to
the osp-ipbd gene, and b) to allow growth of GP(osp-ipbd) in the
absence of XINDUCE if the expression of osp-ipbd is
disadvantageous. The lacUV5 promoter in conjunction with the
LacI.sup.q repressor is a preferred example.
[0557] An exemplary osp-ipbd gene has the DNA sequence shown in
Table 25 and there annotated to explain the useful restriction
sites and biologically important features, viz. the lacUV5
promoter, the lacO operator, the Shine-Dalgarno sequence, the amino
acid sequence, the stop codons, and the trp attenuator
transcriptional terminator.
[0558] The present invention is not limited to a single method of
gene design. The osp-ipbd gene need not be synthesized in toto;
parts of the gene may be obtained from nature. One may use any
genetic engineering method to produce the correct gene fusion, so
long as one can easily and accurately direct mutations to specific
sites in the pbd DNA subsequence. In all of the methods of
mutagenesis considered in the present invention, however, it is
necessary that the coding sequence for the osp-ipbd gene be
different from any other DNA in the OCV. The degree and nature of
difference needed is determined by the method of mutagenesis to be
used. If the method of mutagenesis is to be replacement of
subsequences coding for the PBD with vgDNA, then the subsequences
to be mutagenized are preferably bounded by restriction sites that
are unique with respect to the rest of the OCV. Use of non-unique
sites involves partial digestion which is less efficient than
complete digestion of a unique site and is not preferred. If
single-stranded-oligonucleotide-directed mutagenesis is to be used,
then the DNA sequence of the subsequence coding for the IPBD must
be unique with respect to the rest of the OCV.
[0559] The coding portions of genes to be synthesized are designed
at the protein level and then encoded in DNA. The amino acid
sequences are chosen to achieve various goals, including: a)
display of a IPBD on the surface of a GP, b) change of charge on a
IPBD, and c) generation of a population of PBDs from which to
select an SBD. These issues are discuss in more detail below. The
ambiguity in the genetic code is exploited to allow optimal
placement of restriction sites and to create various distributions
of amino acids at variegated codons.
[0560] While the invention does not require any particular number
or placement of restriction sites, it is generally preferable to
engineer restriction sites into the gene to facilitate subsequent
manipulations. Preferably, the gene provides a series of fairly
uniformly spaced unique restriction sites with no more than a
preset maximum number of bases, for example 100, between sites.
Preferably, the gene is designed so that its insertion into the OCV
does not destroy the uniqueness of unique restriction sites of the
OCV. Preferred recognition sites are those for restriction enzymes
which a) generate cohesive ends, b) have unambiguous recognition,
or c) have higher specific activity.
[0561] The ambiguity of the DNA between the restriction sites is
resolved from the following considerations. If the given amino acid
sequence occurs in the recipient organism, and if the DNA sequence
of the gene in the organism is known, then, preferably, we maximize
the differences between the engineered and natural genes to
minimize the potential for recombination. In addition, the
following codons are poorly translated in E. coli and, therefore,
are avoided if possible: cta(L), cga (R), cgg (R), and agg (R). For
other host species, different codon restrictions would be
appropriate. Finally, long repeats of any one base are prone to
mutation and thus are avoided. Balancing these considerations, we
can design a DNA sequence.
[0562] Structural Considerations
[0563] The design of the amino-acid sequence for the ipbd-osp gene
to encode involves a number of structural considerations. The
design is somewhat different for each type of GP. In bacteria, OSPs
are not essential, so there is no requirement that the OSP domain
of a fusion have any of its parental functions beyond lodging in
the outer membrane.
[0564] Relationship between PBD and OSP
[0565] It is not required that the PBD and OSP domains have any
particular spatial relationship; hence the process of this
invention does not require use of the method of U.S. patent
'692.
[0566] It is, in fact, desirable that the OSP not constrain the
orientation of the PBD domain; this is not to be confused with lack
of constraint within the PBD. Cwirla et al. (CWIR90), Scott and
Smith (SCOT90), and Devlin et al. (DEVL90), have taught that
variable residues in phage-displayed random peptides should be free
of influence from the phage OSP. We teach that binding domains
having a moderate to high degree of conformational constraint will
exhibit higher specificity and that higher affinity is also
possible. Thus, we prescribe picking codons for variegation that
specify amino acids that will appear in a well-defined framework.
The nature of the side groups is varied through a very wide range
due to the combinatorial replacement of multiple amino acids. The
main chain conformations of most PBDs of a given class is very
similar. The movement of the PBD relative to the OSP should not,
however, be restricted. Thus it is often appropriate to include a
flexible linker between the PBD and the OSP. Such flexible linkers
can be taken from naturally occurring proteins known to have
flexible regions. For example, the gIII protein of M13 contains
glycine-rich regions thought to allow the amino-terminal domains a
high degree of freedom. Such flexible linkers may also be designed.
Segments of polypeptides that are rich in the amino acids GLY, ASN,
SER, and ASP are likely to give rise to flexibility. Multiple
glycines are particularly preferred.
[0567] Constraints Imposed by OSP
[0568] When we choose to insert the PBD into a surface loop of an
OSP such as LamB, OmpA, or M13 gIII protein, there are a few
considerations that do not arise when PBD is joined to the end of
an OSP. In these cases, the OSP exerts some constraining influence
on the PBD; the ends of the PBD are held in more or less fixed
positions. We could insert a highly varied DNA sequence into the
osp gene at codons that encode a surface-exposed loop and select
for cells that have a specific-binding phenotype. When the
identified amino-acid sequence is synthesized (by any means), the
constraint of the OSP is lost and the peptide is likely to have a
much lower affinity for the target and a much lower specificity.
Tan and Kaiser (TANN77) found that a synthetic model of BPTI
containing all the amino acids of BPTI that contact trypsin has a
Kd for trypsin .apprxeq.10.sup.7 higher than BPTI. Thus, it is
strongly preferred that the varied amino acids be part of a PBD in
which the structural constrains are supplied by the PBD.
[0569] It is known that the amino acids adjoining foreign epitopes
inserted into LamB influence the immunological properties of these
epitopes (VAND90). We expect that PBDs inserted into loops of LamB,
OmpA, or similar OSPs will be influenced by the amino acids of the
loop and by the OSP in general. To obtain appropriate display of
the PBD, it may be necessary to add one or more linker amino acids
between the OSP and the PBD. Such linkers may be taken from natural
proteins or designed on the basis of our knowledge of the
structural behavior of amino acids. Sequences rich in GLY, SER,
ASN, ASP, ARG, and THR are appropriate. One to five amino acids at
either junction are likely to impart the desired degree of
flexibility between the OSP and the PBD.
[0570] Phage OSP
[0571] A preferred site for insertion of the ipbd gene into the
phage osp gene is one in which: a) the IPBD folds into its original
shape, b) the OSP domains fold into their original shapes, and c)
there is no interference between the two domains.
[0572] If there is a model of the phage that indicates that either
the amino or carboxy terminus of an OSP is exposed to solvent, then
the exposed terminus of that mature OSP becomes the prime candidate
for insertion of the ipbd gene. A low resolution 3D model
suffices.
[0573] In the absence of a 3D structure, the amino and carboxy
termini of the mature OSP are the best candidates for insertion of
the ipbd gene. A functional fusion may require additional residues
between the IPBD and OSP domains to avoid unwanted interactions
between the domains. Random-sequence DNA or DNA coding for a
specific sequence of a protein homologous to the IPBD or OSP, can
be inserted between the osp fragment and the ipbd fragment if
needed.
[0574] Fusion at a domain boundary within the OSP is also a good
approach for obtaining a functional fusion. Smith exploited such a
boundary when subcloning heterologous DNA into gene III of f1
(SMIT85).
[0575] The criteria for identifying OSP domains suitable for
causing display of an IPBD are somewhat different from those used
to identify and IPBD. When identifying an OSP, minimal size is not
so important because the OSP domain will not appear in the final
binding molecule nor will we need to synthesize the gene repeatedly
in each variegation round. The major design concerns are that: a)
the OSP::IPBD fusion causes display of IPBD, b) the initial genetic
construction be reasonably convenient, and c) the osp::ipbd gene be
genetically stable and easily manipulated. There are several
methods of identifying domains. Methods that rely on atomic
coordinates have been reviewed by Janin and Chothia (JANI85). These
methods use matrices of distances between .alpha. carbons
(C.sub..alpha.), dividing planes (cf. ROSE85), or buried surface
(RASH84). Chothia and collaborators have correlated the behavior of
many natural proteins with domain structure (according to their
definition). Rashin correctly predicted the stability of a domain
comprising residues 206-316 of thermolysin (VITA84, RASH84).
[0576] Many researchers have used partial proteolysis and protein
sequence analysis to isolate and identify stable domains. (See, for
example, VITA84, POTE83, SCOT87a, and PABO79.) Pabo et al. used
calorimetry as an indicator that the cI repressor from the
coliphage .lambda. contains two domains; they then used partial
proteolysis to determine the location of the domain boundary.
[0577] If the only structural information available is the amino
acid sequence of the candidate OSP, we can use the sequence to
predict turns and loops. There is a high probability that some of
the loops and turns will be correctly predicted (cf. Chou and
Fasman, (CHOU74)); these locations are also candidates for
insertion of the ipbd gene fragment.
[0578] Bacterial OSPs
[0579] In bacterial OSPs, the major considerations are: a) that the
PBD is displayed, and b) that the chimeric protein not be
toxic.
[0580] From topological models of OSPs, we can determine whether
the amino or carboxy termini of the OSP is exposed. If so, then
these are excellent choices for fusion of the osp fragment to the
ipbd fragment.
[0581] The lamB gene has been sequenced and is available on a
variety of plasmids (CLEM81, CHAR88). Numerous fusions of fragments
of lamB with a variety of other genes have been used to study
export of proteins in E. coli. From various studies, Charbit et al.
(CHAR88) have proposed a model that specifies which residues of
LamB are: a) embedded in the membrane, b) facing the periplasm, and
c) facing the cell surface; we adopt the numbering of this model
for amino acids in the mature protein. According to this model,
several loops on the outer surface are defined, including: 1)
residues 88 through 111, 2) residues 145 through 165, and 3) 236
through 251.
[0582] Consider a mini-protein embedded in LamB. For example,
insertion of DNA encoding G.sub.1NXCX.sub.5XXXCX.sub.10SG.sub.12
between codons 153 and 154 of lamB is likely to lead to a wide
variety of LamB derivatives being expressed on the surface of E.
coli cells. G.sub.1, N.sub.2, S.sub.11, and G.sub.12 are supplied
to allow the mini-protein sufficient orientational freedom that is
can interact optimally with the target. Using affinity enrichment
(involving, for example, FACS via a fluorescently labeled target,
perhaps through several rounds of enrichment), we might obtain a
strain (named, for example, BEST) that expresses a particular LamB
derivative that shows high affinity for the predetermined target.
An octapeptide having the sequence of the inserted residues 3
through 10 from BEST is likely to have an affinity and specificity
similar to that observed in BEST because the octapeptide has an
internal structure that keeps the amino acids in a conformation
that is quite similar in the LamB derivative and in the isolated
mini-protein.
[0583] Consideration of the Signal Peptide
[0584] Fusing one or more new domains to a protein may make the
ability of the new protein to be exported from the cell different
from the ability of the parental protein. The signal peptide of the
wild-type coat protein may function for authentic polypeptide but
be unable to direct export of a fusion. To utilize the
Sec-dependent pathway, one may need a different signal peptide.
Thus, to express and display a chimeric BPTI/M13 gene VIII protein,
we found it necessary to utilize a heterologous signal peptide
(that of phoA).
[0585] Provision of a Means to Remove PBD from the GP
[0586] GPs that display peptides having high affinity for the
target may be quite difficult to elute from the target,
particularly a multivalent target. (Bacteria that are bound very
tightly can simply multiply in situ.) For phage, one can introduce
a cleavage site for a specific protease, such as blood-clotting
Factor Xa, into the fusion OSP protein so that the binding domain
can be cleaved from the genetic package. Such cleavage has the
advantage that all resulting phage have identical OSPs and
therefore are equally infective, even if polypeptide-displaying
phage can be eluted from the affinity matrix without cleavage. This
step allows recovery of valuable genes which might otherwise be
lost. To our knowledge, no one has disclosed or suggested using a
specific protease as a means to recover an information-containing
genetic package or of converting a population of phage that vary in
infectivity into phage having identical infectivity.
[0587] IV.G. Synthesis of Gene Inserts
[0588] The present invention is not limited as to how a designed
DNA sequence is divided for easy synthesis. An established method
is to synthesize both strands of the entire gene in overlapping
segments of 20 to 50 nucleotides (nts) (THER88). An alternative
method that is more suitable for synthesis of vgDNA is an
adaptation of methods published by Oliphant et al. (OLIP86 and
OLIP87) and Ausubel et al. (AUSU87). It differs from previous
methods in that it: a) uses two synthetic strands, and b) does not
cut the extended DNA in the middle. Our goals are: a) to produce
longer pieces of dsDNA than can be synthesized as ssDNA on
commercial DNA synthesizers, and b) to produce strands
complementary to single-stranded vgDNA. By using two synthetic
strands, we remove the requirement for a palindromic sequence at
the 3' end.
[0589] DNA synthesizers can currently produce oligo-nts of lengths
up to 200 nts in reasonable yield, M.sub.DNA=200. The parameters
N.sub.w (the length of overlap needed to obtain efficient
annealing) and N.sub.s (the number of spacer bases needed so that a
restriction enzyme can cut near the end of blunt-ended dsDNA) are
determined by DNA and enzyme chemistry. N.sub.w=10 and N.sub.s=5
are reasonable values. Larger values of N.sub.w and N.sub.s are
allowed but add to the length of ssDNA that is to be synthesized
and reduce the net length of dsDNA that can be produced.
[0590] Let A.sub.L be the actual length of dsDNA to be synthesized,
including any spacers. A.sub.L must be no greater than (2
M.sub.DNA-N.sub.w). Let Q.sub.w be the number of nts that the
overlap window can deviate from center,
Q.sub.w(2M.sub.DNA-N.sub.w-A.sub.L)/2.
[0591] Q.sub.w is never negative. It is preferred that the two
fragments be approximately the same length so that the amounts
synthesized will be approximately equal. This preference may be
overridden by other considerations. The overall yield of dsDNA is
usually dominated by the synthetic yield of the longer
oligo-nt.
[0592] We use the following procedure to generate dsDNA of lengths
up to (2 M.sub.DNA-N.sub.w) nts through the use of Klenow fragment
to extend synthetic ss DNA fragments that are not more than
M.sub.DNA nts long. When a pair of long oligo-nts, complementary
for N.sub.w nts at their 3' ends, are annealed there will be a free
3' hydroxyl and a long ssDNA chain continuing in the 5' direction
on either side. We will refer to this situation as a 5'
superoverhang. The procedure comprises:
[0593] 1) picking a non-palindromic subsequence of N.sub.w to
N.sub.w+4 nts near the center of the dsDNA to be synthesized; this
region is called the overlap (typically, N.sub.w is 10),
[0594] 2) synthesizing a ss DNA molecule that comprises that part
of the anti-sense strand from its 5' end up to and including the
overlap,
[0595] 3) synthesizing a ss DNA molecule that comprises that part
of the sense strand from its 5' end up to and including the
overlap,
[0596] 4) annealing the two synthetic strands that are
complementary throughout the overlap region, and
[0597] 5) extending both superoverhangs with Klenow fragment and
all four deoxynucleotide triphosphates.
[0598] Because M.sub.DNA is not rigidly fixed at 200, the current
limits of 390 (=2 M.sub.DNA-N.sub.w) nts overall and 200 in each
fragment are not rigid, but can be exceeded by 5 or 10 nts. Going
beyond the limits of 390 and 200 will lead to lower yields, but
these may be acceptable in certain cases.
[0599] Restriction enzymes do not cut well at sites closer than
about five base pairs from the end of blunt ds DNA fragments
(OLIP87 and p.132 New England BioLabs 1990-1991 Catalogue).
Therefore N.sub.s nts (with N.sub.s typically set to 5) of spacer
are added to ends that we intend to cut with a restriction enzyme.
If the plasmid is to be cut with a blunt-cutting enzyme, then we do
not add any spacer to the corresponding end of the ds DNA
fragment.
[0600] To choose the optimum site of overlap for the oligo-nt
fragments, first consider the anti-sense strand of the DNA to be
synthesized, including any spacers at the ends, written (in upper
case) from 5' to 3' and left-to-right. N.B.: The N.sub.w nt long
overlap window can never include bases that are to be variegated.
N.B.: The N.sub.w nt long overlap should not be palindromic lest
single DNA molecules prime themselves. Place a Nw nt long window as
close to the center of the anti-sense sequence as possible. Check
to see whether one or more codons within the window can be changed
to increase the GC content without: a) destroying a needed
restriction site, b) changing amino acid sequence, or c) making the
overlap region palindromic. If possible, change some AT base pairs
to GC pairs. If the GC content of the window is less than 50%,
slide the window right or left as much as Qw nts to maximize the
number of C's and G's inside the window, but without including any
variegated bases. For each trial setting of the overlap window,
maximize the GC content by silent codon changes, but do not destroy
wanted restriction sites or make the overlap palindromic. If the
best setting still has less than 50% GC, enlarge the window to
N.sub.w+2 nts and place it within five nts of the center to obtain
the maximum GC content. If enlarging the window one or two nts will
increase the GC content, do so, but do not include variegated
bases.
[0601] Underscore the anti-sense strand from the 5' end up to the
right edge of the window. Write the complementary sense sequence
3'-to-5' and left-to-right and in lower case letters, under the
anti-sense strand starting at the left edge of the window and
continuing all the way to the right end of the anti-sense
strand.
[0602] We will synthesize the underscored anti-sense strand and the
part of the sense strand that we wrote. These two fragments,
complementary over the length of the window of high GC content, are
mixed in equimolar quantities and annealed. These fragments are
extended with Klenow fragment and all four deoxynucleotide
triphosphates to produce ds blunt-ended DNA. This DNA can be cut
with appropriate restriction enzymes to produce the cohesive ends
needed to ligate the fragment to other DNA.
[0603] The present invention is not limited to any particular
method of DNA synthesis or construction. Conventional DNA
synthesizers may be used, with appropriate reagent modifications
for production of variegated DNA (similar to that now used for
production of mixed probes). For example, the Milligen 7500 DNA
synthesizer has seven vials from which phosphoramidites may be
taken. Normally, the first four contain A, C, T, and G. The other
three vials may contain unusual bases such as inosine or mixtures
of bases, the so-called "dirty bottle". The standard software
allows programmed mixing of two, three, or four bases in equimolar
quantities.
[0604] The synthesized DNA may be purified by any art recognized
technique, e.g., by high-pressure liquid chromatography (HPLC) or
PAGE.
[0605] The osp-pbd genes may be created by inserting vgDNA into an
existing parental gene, such as the osp-ipbd shown to be
displayable by a suitably transformed GP. The present invention is
not limited to any particular method of introducing the vgDNA,
however, two techniques are discussed below.
[0606] In the case of cassette mutagenesis, the restriction sites
that were introduced when the gene for the inserted domain was
synthesized are used to introduce the synthetic vgDNA into a
plasmid or other OCV. Restriction digestions and ligations are
performed by standard methods (AUSU87).
[0607] In the case of single-stranded-oligonucleotide-directed
mutagenesis, synthetic vgDNA is used to create diversity in the
vector (BOTS85).
[0608] The modes of creating diversity in the population of GPs
discussed herein are not the only modes possible. Any method of
mutagenesis that preserves at least a large fraction of the
information obtained from one selection and then introduces other
mutations in the same domain will work. The limiting factors are
the number of independent transformants that can be produced and
the amount of enrichment one can achieve through affinity
separation. Therefore the preferred embodiment uses a method of
mutagenesis that focuses mutations into those residues that are
most likely to affect the binding properties of the PBD and are
least likely to destroy the underlying structure of the IPBD.
[0609] Other modes of mutagenesis might allow other GPs to be
considered. For example, the bacteriophage .lambda. is not a useful
cloning vehicle for cassette mutagenesis because of the plethora of
restriction sites. One can, however, use
single-stranded-oligo-nt-directed mutagenesis on .lambda. without
the need for unique restriction sites. No one has used
single-stranded-oligo-nt-directed mutagenesis to introduce the high
level of diversity called for in the present invention, but if it
is possible, such a method would allow use of phage with large
genomes.
[0610] IV.H. Operative Cloning Vector
[0611] The operative cloning vector (OCV) is a replicable nucleic
acid used to introduce the chimeric ipbd-osp or ipbd-osp gene into
the genetic package. When the genetic package is a virus, it may
serve as its own OCV. For cells and spores, the OCV may be a
plasmid, a virus, a phagemid, or a chromosome.
[0612] The OCV is preferably small (less than 10 KB), stable (even
after insertion of at least 1 kb DNA), present in multiple copies
within the host cell, and selectable with appropriate media. It is
desirable that cassette mutagenesis be practical in the OCV;
preferably, at least 25 restriction enzymes are available that do
not cut the OCV. It is likewise desirable that single-stranded
mutagenesis be practical. If a suitable OCV does not already exist,
it may be engineered by manipulation of available vectors.
[0613] When the GP is a bacterial cell or spore, the OCV is
preferably a plasmid because genes on plasmids are much more easily
constructed and mutated than are genes in the bacterial chromosome.
When bacteriophage are to be used, the osp-ipbd gene is inserted
into the phage genome. The synthetic osp-ipbd genes can be
constructed in small vectors and transferred to the GP genome when
complete.
[0614] Phage such as M13 do not confer antibiotic resistance on the
host so that one can not select for cells infected with M13. An
antibiotic resistance gene can be engineered into the M13 genome
(HINE80). More virulent phage, such as .PHI.X174, make discernable
plaques that can be picked, in which case a resistance gene is not
essential; furthermore, there is no room in the .PHI.X174 virion to
add any new genetic material. Inability to include an antibiotic
resistance gene is a disadvantage because it limits the number of
GPs that can be screened.
[0615] It is preferred that GP(IPBD) carry a selectable marker not
carried by wtGP. It is also preferred that wtGP carry a selectable
marker not carried by GP(IPBD).
[0616] A derivative of M13 is the most preferred OCV when the phage
also serves as the GP. Wild-type M13 does not confer any
resistances on infected cells; M13 is a pure parasite. A "phagemid"
is a hybrid between a phage and a plasmid, and is used in this
invention. Double-stranded plasmid DNA isolated from
phagemid-bearing cells is denoted by the standard convention, e.g.
pXY24. Phage prepared from these cells would be designated XY24.
Phagemids such as Bluescript K/S (sold by Stratagene) are not
preferred for our purposes because Bluescript does not contain the
full genome of M13 and must be rescued by coinfection with
competent wild-type M13. Such coinfections could lead to genetic
recombination yielding heterogeneous phage unsuitable for the
purposes of the present invention. Phagemids may be entirely
suitable for developing a gene that clauses an IPBD to appear on
the surface of phage-like genetic packages.
[0617] It is also well known that plasmids containing the ColE1
origin of replication can be greatly amplified if protein synthesis
is halted in a log-phase culture. Protein synthesis can be halted
by addition of chloramphenicol or other agents (MANI82).
[0618] The bacteriophage M13 bla 61 (ATCC 37039) is derived from
wild-type M13 through the insertion of the .beta. lactamase gene
(HINE80). This phage contains 8.13 kb of DNA. M13 bla cat 1 (ATCC
37040) is derived from M13 bla 61 through the additional insertion
of the chloramphenicol resistance gene (HINE80); M13 bla cat 1
contains 9.88 kb of DNA. Although neither of these variants of M13
contains the ColE1 origin of replication, either could be used as a
starting point to construct a cloning vector with this feature.
[0619] IV.I. Transformation of Cells:
[0620] When the GP is a cell, the population of GPs is created by
transforming the cells with suitable OCVs. When the GP is a phage,
the phage are genetically engineered and then transfected into host
cells suitable for amplification. When the GP is a spore, cells
capable of sporulation are transformed with the OCV while in a
normal metabolic state, and then sporulation is induced so as to
cause the OSP-PBDs to be displayed. The present invention is not
limited to any one method of transforming cells with DNA. The
procedure given in the examples is a modification of that of
Maniatis (p250, MANI82). One preferably obtains at least 10.sup.7
and more preferably at least 10.sup.8 transformants/.mu.g of CCC
DNA.
[0621] The transformed cells are grown first under non-selective
conditions that allow expression of plasmid genes and then selected
to kill untransformed cells. Transformed cells are then induced to
express the osp-pbd gene at the appropriate level of induction. The
GPs carrying the IPBD or PBDs are then harvested by methods
appropriate to the GP at hand, generally, centrifugation to
pelletize GPs and resuspension of the pellets in sterile medium
(cells) or buffer (spores or phage). They are then ready for
verification that the display strategy was successful (where the
GPs all display a "test" IPBD) or for affinity selection (where the
GPs display a variety of different PBDs).
[0622] IV.J. Verification of Display Strategy:
[0623] The harvested packages are tested to determine whether the
IPBD is present on the surface. In any tests of GPs for the
presence of IPBD on the GP surface, any ions or cofactors known to
be essential for the stability of IPBD or AM(IPBD) are included at
appropriate levels. The tests can be done: a) by affinity labeling,
b) enzymatically, c) spectrophotometrically, d) by affinity
separation, or e) by affinity precipitation. The AfM(IPBD) in this
step is one picked to have strong affinity (preferably,
K.sub.d<10.sup.-11 M) for the IPBD molecule and little or no
affinity for the wtGP. For example, if BPTI were the IPBD, trypsin,
anhydrotrypsin, or antibodies to BPTI could be used as the
AfM(BPTI) to test for the presence of BPTI. Anhydrotrypsin, a
trypsin derivative with serine 195 converted to dehydroalanine, has
no proteolytic activity but retains its affinity for BPTI (AKOH72
and HUBE77).
[0624] Preferably, the presence of the IPBD on the surface of the
GP is demonstrated through the use of a soluble, labeled derivative
of a AfM(IPBD) with high affinity for IPBD. The label could be: a)
a radioactive atom such as .sup.125I, b) a chemical entity such as
biotin, or 3) a fluorescent entity such as rhodamine or
fluorescein. The labeled derivative of AfM(IPBD) is denoted as
AfM(IPBD)*. The preferred procedure is:
[0625] 1) mix AfM(IPBD)* with GPs that are to be tested for the
presence of IPBD; conditions of mixing should favor binding of IPBD
to AfM(IPBD)*,
[0626] 2) separate GPs from unbound AfM(IPBD)* by use of:
[0627] a) a molecular sizing filter that will pass AfM(IPBD)* but
not GPs,
[0628] b) centrifugation, or
[0629] c) a molecular sizing column (such as Sepharose or Sephadex)
that retains free AfM(IPBD)* but not GPs,
[0630] 3) quantitate the AfM(IPBD)* bound by GPs.
[0631] Alternatively, if the iPBD has a known biochemical activity
(enzymatic or inhibitory), its presence on the GP can be verified
through this activity. For example, if the IPBD were BPTI, then one
could use the stoichiometric inactivation of trypsin not only to
demonstrate the presence of BPTI, but also to quantitate the
amount.
[0632] If the IPBD has strong, characteristic absorption bands in
the visible or UV that are distinct from absorption by the wtGP,
then another alternative for measuring the IPBD displayed on the GP
is a spectrophotometric measurement. For example, if IPBD were
azurin, the visible absorption could be used to identify GPs that
display azurin.
[0633] Another alternative is to label the GPs and measure the
amount of label retained by immobilized AfM(IPBD). For example, the
GPs could be grown with a radioactive precursor, such as .sup.32P
or .sup.3H-thymidine, and the radioactivity retained by immobilized
AfM(IPBD) measured.
[0634] Another alternative is to use affinity chromatography; the
ability of a GP bearing the IPBD to bind a matrix that supports a
AfM(IPBD) is measured by reference to the wtGP.
[0635] Another alternative for detecting the presence of IPBD on
the GP surface is affinity precipitation.
[0636] If random DNA has been used, then affinity selection
procedures are used to obtain a clonal isolate that has the
display-of-IPBD phenotype. Alternatively, clonal isolates may be
screened for the display-of-IPBD phenotype. The tests of this step
are applied to one or more of these clonal isolates.
[0637] If no isolates that bind to the affinity molecule are
obtained we take corrective action as disclosed below.
[0638] If one or more of the tests above indicates that the IPBD is
displayed on the GP surface, we verify that the binding of
molecules having known affinity for IPBD is due to the chimeric
osp-ipbd gene through the use of standard genetic and biochemical
techniques, such as:
[0639] 1) transferring the osp-ipbd gene into the parent GP to
verify that osp-ipbd confers binding,
[0640] 2) deleting the osp-ipbd gene from the isolated GP to verify
that loss of osp-ipbd causes loss of binding,
[0641] 3) showing that binding of GPs to AfM(IPBD) correlates with
[XINDUCE] (in those cases that expression of osp-ipbd is controlled
by [XINDUCE]), and
[0642] 4) showing that binding of GPs to AfM(IPBD) is specific to
the immobilized AfM(IPBD) and not to the support matrix.
[0643] Variation of: a) binding of GPs by soluble AfM(IPBD)*, b)
absorption caused by IPBD, and c) biochemical reactions of IPBD are
linear in the amount of IPBD displayed. Presence of IPBD on the GP
surface is indicated by a strong correlation between [XINDUCE] and
the reactions that are linear in the amount of IPBD. Leakiness of
the promoter is not likely to present problems of high background
with assays that are linear in the amount of IPBD. These
experiments may be quicker and easier than the genetic tests.
Interpreting the effect of [XINDUCE) on binding to a {AfM(IPBD)}
column, however, may be problematic unless the regulated promoter
is completely repressed in the absence of [XINDUCE]. The affinity
retention of GP(IPBD)s is not linear in the number of IPBDs/GP and
there may be, for example, little phenotypic difference between GPs
bearing 5 IPBDs and GPs bearing 50 IPBDs. The demonstration that
binding is to AfM(IPBD) and the genetic tests are essential; the
tests with XINDUCE are optional.
[0644] We sequence the relevant ipbd gene fragment from each of
several clonal isolates to determine the construction. We also
establish the maximum salt concentration and pH range for which the
GP(IPBD) binds the chosen AfM(IPBD). This is preferably done by
measuring, as a function of salt concentration and pH, the
retention of AfM(IPBD)* on molecular sizing filters that pass
AfM(IPBD)* but not GP. This information will be used in refining
the affinity selection scheme.
[0645] IV.K. Analysis and Correction of Display Problems
[0646] If the IPBD is displayed on the outside of the GP, and if
that display is clearly caused by the introduced osp-ipbd gene, we
proceed with variegation, otherwise we analyze the result and adopt
appropriate corrective measures. If we have unsuccessfully
attempted to fuse an ipbd fragment to a natural osp fragment, our
options are :1) pick a different fusion to the same osp by a) using
opposite end of osp, b) keeping more or fewer residues from osp in
the fusion; for example, in increments of 3 or 4 residues, c)
trying a known or predicted domain boundary, d) trying a predicted
loop or turn position, 2) pick a different osp, or 3) switch to
random DNA method. If we have just tried the random DNA method
unsuccessfully, our options are: 1) choose a different relationship
between ipbd fragment and random DNA (ipbd first, random DNA second
or vice versa), 2) try a different degree of partial digestion, a
different enzyme for partial digestion, a different degree of
shearing or a different source of natural DNA, or 3) switch to the
natural OSP method. If all reasonable OSPs of the current GP have
been tried and the random DNA method has been tried, both without
success, we pick a new GP.
[0647] We may illustrate the ways in which problems may be attacked
by using the example of BPTI as the IPBD, the M13 phage as the GP,
and the major coat (gene VIII) protein as the OSP. The following
amino-acid sequence, called AA_seq2, illustrates how the sequence
for mature BPTI (shown underscored) may be inserted immediately
after the signal sequence of M13 precoat protein (indicated by the
arrow) and before the sequence for the M13 CP.
8 3
[0648] We adopt the convention that sequence numbers of fusion
proteins refer to the fusion, as coded, unless otherwise noted.
Thus the alanine that begins M13 CP is referred to as "number 82",
"number 1 of M13 CP", or "number 59 of the mature BPTI-M13 CP
fusion".
[0649] It is desirable to determine where, exactly, the BPTI
binding domain is being transported: is it remaining in the
cytoplasm? Is it free within the periplasm? Is it attached to the
inner membrane? Proteins in the periplasm can be freed through
spheroplast formation using lysozyme and EDTA in a concentrated
sucrose solution (BIRD67, MALA64). If BPTI were free in the
periplasm, it would be found in the supernatant. Trypsin labeled
with .sup.125I would be mixed with supernatant and passed over a
non-denaturing molecular sizing column and the radioactive
fractions collected. The radioactive fractions would then be
analyzed by SDS-PAGE and examined for BPTI-sized bands by silver
staining.
[0650] Spheroplast formation exposes proteins anchored in the inner
membrane. Spheroplasts would be mixed with AHTrp* and then either
filtered or centrifuged to separate them from unbound AHTrp*. After
washing with hypertonic buffer, the spheroplasts would be analyzed
for extent of AHTrp* binding.
[0651] If BPTI were found free in the periplasm, then we would
expect that the chimeric protein was being cleaved both between
BPTI and the M13 mature coat sequence and between BPTI and the
signal sequence. In that case, we should alter the BPTI/M13 CP
junction by inserting vgDNA at codons for residues 78-82 of
AA_seq2.
[0652] If BPTI were found attached to the inner membrane, then two
hypotheses can be formed. The first is that the chimeric protein is
being cut after the signal sequence, but is not being incorporated
into LG7 virion; the treatment would also be to insert vgDNA
between residues 78 and 82 of AA_seq2. The alternative hypothesis
is that BPTI could fold and react with trypsin even if signal
Sequence is not cleaved. N-terminal amino acid sequencing of
trypsin-binding material isolated from cell homogenate determines
what processing is occurring. If signal sequence were being
cleaved, we would use the procedure above to vary residues between
C78 and A82; subsequent passes would add residues after residue 81.
If signal sequence were not being cleaved, we would vary residues
between 23 and 27 of AA_seq2. Subsequent passes through that
process would add residues after 23.
[0653] If BPTI were found neither in the periplasm nor on the inner
membrane, then we would expect that the fault was in the signal
sequence or the signal-sequence-to-BPTI junction. The treatment in
this case would be to vary residues between 23 and 27.
[0654] Analytical experiments to determine what has gone wrong take
time and effort and, for the foreseen outcomes, indicate variations
in only two regions. Therefore, we believe it prudent to try the
synthetic experiments described below without doing the analysis.
For example, these six experiments that introduce variegation into
the bpti-gene VIII fusion could be tried:
[0655] 1) 3 variegated codons between residues 78 and 82 using
olig#12 and olig#13,
[0656] 2) 3 variegated codons between residues 23 and 27 using
olig#14 and olig#15,
[0657] 3) 5 variegated codons between residues 78 and 82 using
olig#13 and olig#12a,
[0658] 4) 5 variegated codons between residues 23 and 27 using
olig#15 and olig#14a,
[0659] 5) 7 variegated codons between residues 78 and 82 using
olig#13 and oligt12b, and
[0660] 6) 7 variegated codons between residues 23 and 27 using
olig#15 and olig#14b.
[0661] To alter the BPTI-M13 CP junction, we introduce DNA
variegated at codons for residues between 78 and 82 into the SphI
and SfiI sites of pLG7. The residues after the last cysteine are
highly variable in amino acid sequences homologous to BPTI, both in
composition and length; in Table 25 these residues are denoted as
G79, G80, and A81. The first part of the M13 CP is denoted as A82,
E83, and G84. One of the oligo-nts olig#12, olig#12a, or olig#12b
and the primer olig#13 are synthesized by standard methods. The
oligo-nts are:
9 residue 75 76 77 78 79 80 81 82 83 5'
gc.vertline.gag.vertline.cGC.vertline.ATG.vertline.CGT.vertline.ACC.vertl-
ine.TGC.vertline.qfk.vertline.qfk.vertline.qfk.vertline.GCT.vertline.GAA.v-
ertline.- 84 85 86 87 88 89 90 91
GGT.vertline.GAT.vertline.GAT.vertline.CCG.vertline.GCC.vertline.AAA.ve-
rtline.GCG.vertline.GCC.vertline.gcg.vertline.cc 3' olig#12 residue
75 76 77 78 79 80 81 81a 81b 5'
gc.vertline.gag.vertline.cGC.vertline.ATG.vertline.CGT.vertline.ACC.vertl-
ine.TGC.vertline.qfk.vertline.qfk.vertline.qfk.vertline.qfk.vertline.qfk.v-
ertline.- 82 83 84 85 86 87
GCT.vertline.GAA.vertline.GGT.vertline.GAT.vertline.GAT.vertline.CCG.ve-
rtline.- 88 89 90 91
GCC.vertline.AAA.vertline.GCG.vertline.GCC.vertline.gcg.ve-
rtline.cc 3' olig#12a residue 75 76 77 78 79 80 81 81a 81b 5'
gc.vertline.gag.vertline.cGC.vertline.ATG.vertli-
ne.CGT.vertline.ACC.vertline.TGC.vertline.qfk.vertline.qfk.vertline.qfk.ve-
rtline.qfk.vertline.qfk.vertline.- 81c 81d 82 83 84 85 86 87
qfj.vertline.qfk.vertline.GCT.vertlin-
e.GAA.vertline.GGT.vertline.GAT.vertline.GAT.vertline.CCG.vertline.-
88 89 90 91
GCC.vertline.AAA.vertline.GCG.vertline.GCC.vertline.gcg.vertline.cc
3' olig#12b residue 91 90 89 88 87 86 5'
gg.vertline.cgc.vertline.GGC.vertline.CGC.vertline.TTT.vertline.GGC.vertl-
ine.CGG.vertline.ATC 3' olig#13
[0662] where q is a mixture of (0.26 T, 0.18C, 0.26 A, and 0.30 G),
f is a mixture of (0.22 T, 0.16 C, 0.40 A, and 0.22 G), and k is a
mixture of equal parts of T and G. The bases shown in lower case at
either end are spacers and are not incorporated into the cloned
gene. The primer is complementary to the 31 end of each of the
longer oligo-nts. One of the variegated oligo-nts and the primer
olig#13 are combined in equimolar amounts and annealed. The dsDNA
is completed with all four (nt)TPs and Klenow fragment. The
resulting dsDNA and RF pLG7 are cut with both SfiI and SphI,
purified, mixed, and ligated. We then select a transformed clone
that, when induced with IPTG, binds AHTrp.
[0663] To vary the junction between M13 signal sequence and BPTI,
we introduce DNA variegated at codons for residues between 23 and
27 into the KpnI and XhoI sites of pLG7. The first three residues
are highly variable in amino acid sequences homologous to BPTI.
Homologous sequences also vary in length at the amino terminus. One
of the oligo-nts olig#14, olig#14a, or olig#14b and the primer
olig#15 are synthesized by standard methods. The oligo-nts are:
10 residue : 17 18 19 20 21 22 23 24 25 5'
g.vertline.gcc.vertline.gcG.vertline.GTA.vertline.CCG.vertline.ATG.vertli-
ne.CTG.vertline.TCT.vertline.TTT.vertline.GCT.vertline.fxk.vertline.fxk.ve-
rtline.- 26 27 28 29 30
fxk.vertline.TTC.vertline.TGT.vertline.CTC.vertline.GAG.vertline.cgc.vert-
line.ccg.vertline.cga.vertline. 3' olig#14 residue 17 18 19 20 21
22 23 24 25 26 5'g.vertline.gcc.vertline.gcG.-
vertline.GTA.vertline.CCG.vertline.ATG.vertline.CTG.vertline.TCT.vertline.-
TTT.vertline.GCT.vertline.fxk.vertline.fxk.vertline.fxk.vertline.-
26a 26b 27 28 29 30 .vertline.fxk.vertline.fxk.vert-
line.TTC.vertline.TGT.vertline.CTC.vertline.GAG.vertline.cgc.vertline.ccg.-
vertline.cga.vertline. 3' olig#14a, residue 17 18 19 20 21 22 23 24
25 26 5'g.vertline.gcc.vertline.gcG.vertl-
ine.GTA.vertline.CCG.vertline.ATG.vertline.CTG.vertline.TCT.vertline.TTT.v-
ertline.GCT.vertline.fxk.vertline.fxk.vertline.fxk.vertline.- 26a
26b 26c 26d 27 28 29 30 .vertline.fxk.vertline.fxk.ver-
tline.fxk.vertline.fxk.vertline.TTC.vertline.TGT.vertline.CTC.vertline.GAG-
.vertline.cgc.vertline.ccg.vertline.cga.vertline.3'olig#14b 5'
tcg.vertline.cgg.vertline.gcg.vertline.CTC.vertline.GAG.vertline.A-
CA.vertline.GAA.vertline. 3' olig#15
[0664] where f is a mixture of (0.26 T, 0.18 C, 0.26 A, and 0.30
G), x is a mixture of (0.22 T, 0.16 C, 0.40 A, and 0.22 G), and k
is a mixture of equal parts of T and G. The bases shown in lower
case at either end are spacers and are not incorporated into the
cloned gene. One of the variegated oligo-nts and the primer are
combined in equimolar amounts and annealed. The ds DNA is completed
with all four (nt)TPs and Klenow fragment. The resulting dsDNA and
RF pLG7 are cut with both KpnI and XhoI, purified, mixed, and
ligated. We select a transformed clone that, when induced with
IPTG, binds AHTrp or trp.
[0665] Other numbers of variegated codons could be used.
[0666] If none of these approaches produces a working chimeric
protein, we may try a different signal sequence. If that doesn't
work, we may try a different OSP.
[0667] V. Affinity Selection of Target-binding Mutants
[0668] V.A. Affinity Separation Technology, Generally
[0669] Affinity separation is used initially in the present
invention to verify that the display system is working, i.e., that
a chimeric outer surface protean has been expressed and transported
to the surface of the genetic package and is oriented so that the
inserted binding domain is accessible to target material. When used
for this purpose, the binding domain is a known binding domain for
a particular target and that target is the affinity molecule used
in the affinity separation process. For example, a display system
may be validated by using inserting DNA encoding BPTI into a gene
encoding an outer surface protein of the genetic package of
interest, and testing for binding to anhydrotrypsin, which is
normally bound by BPTI.
[0670] If the genetic packages bind to the target, then we have
confirmation that the corresponding binding domain is indeed
displayed by the genetic package. Packages which display the
binding domain (and thereby bind the target) are separated from
those which do not.
[0671] Once the display system is validated, it is possible to use
a variegated population of genetic packages which display a variety
of different potential binding domains, and use affinity separation
technology to determine how well they bind to one or more targets.
This target need not be one bound by a known binding domain which
is parental to the displayed binding domains, i.e., one may select
for binding to a new target.
[0672] For example, one may variegate a BPTI binding domain and
test for binding, not to trypsin, but to another serine protease,
such as human neutrophil elastase or cathepsin G, or even to a
wholly unrelated target, such as horse heart myoglobin.
[0673] The term "affinity separation means" includes, but is not
limited to: a) affinity column chromatography, b) batch elution
from an affinity matrix material, c) batch elution from an affinity
material attached to a plate, d) fluorescence activated cell
sorting, and e) electrophoresis in the presence of target material.
"Affinity material" is used to mean a material with affinity for
the material to be purified, called the "analyte". In most cases,
the association of the affinity material and the analyte is
reversible so that the analyte can be freed from the affinity
material once the impurities are washed away.
[0674] The procedures described in sections V.H, V.I and V.J are
not required for practicing the present invention, but may
facilitate the development of novel binding proteins thereby.
[0675] V.B. Affinity Chromatography, Generally
[0676] Affinity column chromatography, batch elution from an
affinity matrix material held in some container, and batch elution
from a plate are very similar and hereinafter will be treated under
"affinity chromatography."
[0677] If affinity chromatography is to be used, then:
[0678] 1) the molecules of the target material must be of
sufficient size and chemical reactivity to be applied to a solid
support suitable for affinity separation,
[0679] 2) after application to a matrix, the target material
preferably does not react with water,
[0680] 3) after application to a matrix, the target material
preferably does not bind or degrade proteins in a non-specific way,
and
[0681] 4) the molecules of the target material must be sufficiently
large that attaching the material to a matrix allows enough
unaltered surface area (generally at least 500 .ANG..sup.2,
excluding the atom that is connected to the linker) for protein
binding.
[0682] Affinity chromatography is the preferred separation means,
but FACS, electrophoresis, or other means may also be used.
[0683] V.C. Fluorescent-activated Cell Sorting, Generally
[0684] Fluorescent-activated cell sorting involves use of an
affinity material that is fluorescent per se or is labeled with a
fluorescent molecule. Current commercially available cell sorters
require 800 to 1000 molecules of fluorescent dye, such as Texas
red, bound to each cell. FACS can sort 10.sup.3 cells or
viruses/sec.
[0685] FACS (e.g. FACStar from Beckton-Dickinson, Mountain View,
Calif.) is most appropriate for bacterial cells and spores because
the sensitivity of the machines requires approximately 1000
molecules of fluorescent label bound to each GP to accomplish a
separation. OSPs such as OmpA, OmpF, OmpC are present at
.gtoreq.10.sup.4/cell, often as much as 10.sup.5/cell. Thus use of
FACS with PBDs displayed on one of the OSPs of a bacterial cell is
attractive. This is particularly true if the target is quite small
so that attachment to a matrix has a much greater effect than would
attachment to a dye. To optimize FACS separation or GPs, we use a
derivative of Afm(IPBD) that is labeled with a fluorescent
molecule, denoted Afm(IPBD)*. The variables to be optimized
include: a) amount of IPBD/GP, b) concentration of Afm(IPBD)*, c)
ionic strength, d) concentration of GPs, and e) parameters
pertaining to operation of the FACS machine. Because Afm(IPBD)* and
GPs interact in solution, the binding will be linear in both
[Afm(IPBD)*] and [displayed IPBD]. Preferably, these two parameters
are varied together. The other parameters can be optimized
independently.
[0686] If FACS is to be used as the affinity separation means,
then:
[0687] 1) the molecules of the target material must be of
sufficient size and chemical reactivity to be conjugated to a
suitable fluorescent dye or the target must itself be
fluorescent,
[0688] 2) after any necessary fluorescent labeling, the target
preferably does not react with water,
[0689] 3) after any necessary fluorescent labeling, the target
material preferably does not bind or degrade proteins in a
non-specific way, and
[0690] 4) the molecules of the target material must be sufficiently
large that attaching the material to a suitable dye allows enough
unaltered surface area (generally at least 500 .ANG..sup.2,
excluding the atom that is connected to the linker) for protein
binding.
[0691] V.D. Affinity Electrophoresis, Generally
[0692] Electrophoretic affinity separation involves electrophoresis
of viruses or cells in the presence of target material, wherein the
binding of said target material changes the net charge of the virus
particles or cells. It has been used to separate bacteriophages on
the basis of charge. (SERW87).
[0693] Electrophoresis is most appropriate to bacteriophage.
because of their small size (SERW87). Electrophoresis is a
preferred separation means if the target is so small that
chemically attaching it to a column or to a fluorescent label would
essentially change the entire target. For example, chloroacetate
ions contain only seven atoms and would be essentially altered by
any linkage. GPs that bind chloroacetate would become more
negatively charged than GPs that do not bind the ion and so these
classes of GPs could be separated.
[0694] If affinity electrophoresis is to be used, then:
[0695] 1) the target must either be charged or of such a nature
that its binding to a protein will change the charge of the
protein,
[0696] 2) the target material preferably does not react with
water,
[0697] 3) the target material preferably does not bind or degrade
proteins in a non-specific way, and
[0698] 4) the target must be compatible with a suitable gel
material.
[0699] The present invention makes use of affinity separation of
bacterial cells, or bacterial viruses (or other genetic packages)
to enrich a population for those cells or viruses carrying genes
that code for proteins with desirable binding properties.
[0700] V.E. Target Materials
[0701] The present invention may be used to select for binding
domains which bind to one or more target materials, and/or fail to
bind to one or more target materials. Specificity, of course, is
the ability of a binding molecule to bind strongly to a limited set
of target materials, while binding more weakly or not at all to
another set of target materials from which the first set must be
distinguished.
[0702] The target materials may be organic macromolecules, such as
polypeptides, lipids, polynucleic acids, and polysaccharides, but
are not so limited. Almost any molecule that is stable in aqueous
solvent may be used as a target. The following list of possible
targets is given as illustration and not as limitation. The
categories are not strictly mutually exclusive. The omission of any
category is not to be construed to imply that said category is
unsuitable as a target.
[0703] A. Peptides
[0704] 1) human .beta. endorphin (Merck Index 3528)
[0705] 2) dynorphin (MI 3458)
[0706] 3) Substance P (MI 8834)
[0707] 4) Porcine somatostatin (MI 8671)
[0708] 5) human atrial natriuretic factor (MI 887)
[0709] 6) human calcitonin
[0710] 7) glucagon
[0711] B. Proteins
[0712] I. Soluble Proteins
[0713] a. Hormones
[0714] 1) human TNF (MI 9411)
[0715] 2) Interleukin-1 (MI 4895)
[0716] 3) Interferon-.gamma. (MI 4894)
[0717] 4) Thyrotropin (MI 9709)
[0718] 5) Interferon-.alpha. (MI 4892)
[0719] 6) Insulin (MI 4887, p.789)
[0720] b. Enzymes
[0721] 1) human neutrophil elastase
[0722] 2) Human thrombin
[0723] 3) human Cathepsin G
[0724] 4) human tryptase
[0725] 5) human chymase
[0726] 6) human blood clotting Factor Xa
[0727] 7) any retro-viral Pol protease
[0728] 8) any retrd-viral Gag protease
[0729] 9) dihydrofolate reductase
[0730] 10) Pseudomonas putida cytochrome P450CAM
[0731] 11) human pyruvate kinase
[0732] 12) E. coli pyruvate kinase
[0733] 13) jack bean urease
[0734] 14) aspartate transcarbamylase (E. coli)
[0735] 15) ras protein
[0736] 16) any protein-tyrosine kinase
[0737] c. Inhibitors
[0738] 1) aprotinin (MI 784)
[0739] 2) human .alpha.1-anti-trypsin
[0740] 3) phage .lambda. cI (inhibits DNA transcription)
[0741] d. Receptors
[0742] 1) TNF receptor
[0743] 2) IgE receptor
[0744] 3) LamB
[0745] 4) CD4
[0746] 5) IL-1 receptor
[0747] e. Toxins
[0748] 1) ricin (also an enzyme)
[0749] 2) .alpha. Conotoxin GI
[0750] 3) mellitin
[0751] 4) Bordetella pertussis adenylate cyclase (also an
enzyme)
[0752] 5) Pseudomonas aeruginosa hemolysin
[0753] f. Other proteins
[0754] 1) horse heart myoglobin
[0755] 2) human sickle-cell haemoglobin
[0756] 3) human deoxy haemoglobin
[0757] 4) human CO haemoglobin
[0758] 5) human low-density lipoprotein (a lipoprotein)
[0759] 6) human IgG (combining site removed or blocked) (a
glycoprotein)
[0760] 7) influenza haemagglutinin
[0761] 8) phage .lambda. capsid
[0762] 9) fibrinogen
[0763] 10) HIV-1 gp120
[0764] 11) Neisseria gonorrhoeae pilin
[0765] 12) fibril or flagellar protein from spirochaete bacterial
species such as those that cause syphilis, Lyme disease, or
relapsing fever
[0766] 13) pro-enzymes such as prothrombin and trypsinogen
[0767] II. Insoluble Proteins
[0768] 1) silk
[0769] 2) human elastin
[0770] 3) keratin
[0771] 4) collagen
[0772] 5) fibrin
[0773] C. Nucleic acids
[0774] a. DNA
11 4
[0775] b. RNA
[0776] 1) yeast Phe tRNA
[0777] 2) ribosomal RNA
[0778] 3) segment of mRNA
[0779] D. Organic molecules (not peptide, protein, or nucleic
acid)
[0780] I. Small and monomeric
[0781] 1) cholesterol
[0782] 2) aspartame
[0783] 3) bilirubin
[0784] 4) morphine
[0785] 5) codeine
[0786] 6) heroine
[0787] 7) dichlorodiphenyltrichlorethane (DDT)
[0788] 8) prostaglandin PGE2
[0789] 9) actinomycin
[0790] 10) 2,2,3 trimethyldecane
[0791] 11) Buckminsterfullerene
[0792] 12) cortavazol (MI 2536, p.397)
[0793] II. Polymers
[0794] 1) cellulose
[0795] 2) chitin
[0796] III. Others
[0797] 1) O-antigen of Salmonella enteritidis (a
lipopolysaccharide)
[0798] E. Inorganic compounds
[0799] 1) asbestos
[0800] 2) zeolites
[0801] 3) hydroxylapatite
[0802] 4) 111 face of crystalline silicon
[0803] 5) paulingite
[0804] 6) U(IV) (uranium ions)
[0805] 7) Au(III) (gold ions)
[0806] F. Organometallic compounds
[0807] 1) iron(III) haem
[0808] 2) cobalt haem
[0809] 3) cobalamine
[0810] 4) (isopropylamino).sub.6Cr(III)
[0811] Serine proteases are an especially interesting class of
potential target materials. Serine proteases are ubiquitous in
living organisms and play vital roles in processes such as:
digestion, blood clotting, fibrinolysis, immune response,
fertilization, and post-translational processing of peptide
hormones. Although the role these enzymes play is vital,
uncontrolled or inappropriate proteolytic activity can be very
damaging. Several serine proteases are directly involved in serious
disease states. Uncontrolled neutrophil elastase (NE) (also known
as leukocyte elastase) is thought to be the major cause of
emphysema (BEIT86, HUBB86, HUBB89, HUTC87, SOMM90, WEWE87) whether
caused by congenital lack of .alpha.-1-antitrypsin or by smoking.
NE is also implicated as an essential ingredient in the pernicious
cycle of: 5
[0812] observed in cystic fibrosis (CF) (NADE90). Inappropriate NE
activity is very harmful and to stop the progression of emphysema
or to alleviate the symptoms of CF, an inhibitor of very high
affinity is needed. The inhibitor must be very specific to NE lest
it inhibit other vital serine proteases or esterases. Nadel
(NADE90) has suggested that onset of excess secretion is initiated
by 10.sup.-10 M NE; thus, the inhibitor must reduce the
concentration of free NE to well below this level. Thus human
neutrophil elastase is a preferred target and a highly stable
protein is a preferred IPBD. In particular, BPTI, ITI-D1, or
another BPTI homologue is a preferred IPBD for development of an
inhibitor to HNE. Other preferred IPBDs for making an inhibitor to
HNE include CMTI-III, SLPI, Eglin, .alpha.-conotoxin GI, and
.OMEGA. Conotoxins.
[0813] HNE is not the only serine protease for which an inhibitor
would be valuable. Works concerning uses of protease inhibitors and
diseases thought to result from inappropriate protease activity
include: NADE87, REST88, SOMM90, and SOMM89. Tryptase and chymase
may be involved in asthma, see FRAN89 and VAND89. There are reports
that suggest that Proteinase 3 (also known as p29) is as important
or even more important than HNE; see NILE89, ARNA90, KAOR88,
CAMP90, and GUPT90. Cathepsin G is another protease that may cause
disease when present in excess; see FERR90, PETE89, SALV87, and
SOMM90. These works indicate that a problem exists and that
blocking one or another protease might well alleviate a disease
state. Some of the cited works report inhibitors having measurable
affinity for a target protease, but none report truly excellent
inhibitors that have K.sub.d in the range of 10.sup.-12 M as may be
obtained by the method of the present invention. The same IPBDs
used for HNE can be used for any serine protease.
[0814] The present invention is not, however, limited to any of the
above-identified target materials. The only limitation is that the
target material be suitable for affinity separation.
[0815] A supply of several milligrams of pure target material is
desired. With HNE (as discussed in Examples II and III), 400 .mu.g
of enzyme is used to prepare 200 .mu.l of ReactiGel beads. This
amount of beads is sufficient for as many as 40 fractionations.
Impure target material could be used, but one might obtain a
protein that binds to a contaminant instead of to the target.
[0816] The following information about the target material is
highly desirable: 1) stability as a function of temperature, pH,
and ionic strength, 2) stability with respect to chaotropes such as
urea or guanidinium Cl, 3) pI, 4) molecular weight, 5) requirements
for prosthetic groups or ions, such as haem or Ca.sup.+2, and 6)
proteolytic activity, if any. It is also potentially useful to
know: 1) the target's sequence, if the target is a macromolecule,
2) the 3D structure of the target, 3) enzymatic activity, if any,
and 4) toxicity, if any.
[0817] The user of the present invention specifies certain
parameters of the intended use of the binding protein: 1) the
acceptable temperature range, 2) the acceptable pH range, 3) the
acceptable concentrations of ions and neutral solutes, and 4) the
maximum acceptable dissociation constant for the target and the
bBD:
K.sub.T=[Target][SBD]/[Target:SBD].
[0818] In some cases, the user may require discrimination between
T, the target, and N, some non-target. Let
K.sub.T=[T][SBD]/[T:SBD] , and
K.sub.N=[N][SBD]/[N:SBD],
then K.sub.T/K.sub.N=([T][N:SBD])/([N][T:SBD]).
[0819] The user then specifies a maximum acceptable value for the
ratio K.sub.T/K.sub.N.
[0820] The target material preferably is stable under the specified
conditions of pH, temperature, and solution conditions.
[0821] If the target material is a protease, one considers the
following points:
[0822] 1) a highly specific protease can be treated like any other
target,
[0823] 2) a general protease, such as subtilisin, may degrade the
OSPs of the GP including OSP-PBDs; there are several alternative
Ways of dealing with general proteases, including: a) use a
protease inhibitor as PPBD so that the SBD is an inhibitor of the
protease, b) a chemical inhibitor may be used to prevent
proteolysis (e.g. phenylmethylfluorosulfate (PMFS) that inhibits
serine proteases), c) one or more active-site residues may be
mutated to create an inactive protein (e.g. a serine protease in
which the active serine is mutated to alanine), or d) one or more
active-site amino-acids of the protein may be chemically modified
to destroy the catalytic activity (e.g. a serine protease in which
the active serine is converted to anhydroserine),
[0824] 3) SBDs selected for binding to a protease need not be
inhibitors; SBDs that happen to inhibit the protease target are a
fairly small subset of SBDs that bind to the protease target,
[0825] 4) the more we modify the target protease, the less like we
are to obtain an SBD that inhibits the target protease, and
[0826] 5) if the user requires that the SBD inhibit the target
protease, then the active site of the target protease must not be
modified any more than necessary; inactivation by mutation or
chemical modification are preferred methods of inactivation and a
protein protease inhibitor becomes a prime candidate for IPBD. For
example, BPTI has been mutated, by the methods of the present
invention, to bind to proteases other than trypsin.
[0827] Example III-VI disclose that uninhibited serine proteases
may be used as targets quite successfully and that protein protease
inhibitors derived from BPTI and selected for binding to these
immobilized proteases are excellent inhibitors.
[0828] V.F. Immobilization or Labeling of Target Material
[0829] For chromatography, FACS, or electrophoresis there may be a
need to covalently link the target material to a second chemical
entity. For chromatography the second entity is a matrix, for FACS
the second entity is a fluorescent dye, and for electrophoresis the
second entity is a strongly charged molecule. In many cases, no
coupling is required because the target material already as the
desired property of: a) immobility, b) fluorescence, or c) charge.
In other cases, chemical or physical coupling is required.
[0830] Various means may be used to immobilize or label the target
materials. The means of immobilization or labeling is, in part,
determined by the nature of the target. In particular, the physical
and chemical nature of the target and its functional groups of the
target material determine which types of immobilization reagents
may be most easily used.
[0831] For the purpose of selecting an immobilization method, it
may be more helpful to classify target materials as follows: (a)
solid, whether crystalline or amorphous, and insoluble in an
aqueous solvent (e.g., many minerals, and fibrous organics such as
cellulose and silk); (b) solid, whether crystalline or amorphous,
and soluble in an aqueous solvent; (c) liquid, but insoluble in
aqueous phase (e.g., 2,3,3-trimethyldecane); or (d) liquid, and
soluble in aqueous media.
[0832] It is not necessary that the actual target material be used
in preparing the immobilized or labeled analogue that is to be used
in affinity separation; rather, suitable reactive analogues of the
target material may be more convenient. If 2,3,3-trimethyldecane
were the target material, for example, then
2,3,3-trimethyl-10-aminodecane would be far easier to immobilize
than the parental compound. Because the latter compound is modified
at one end of the chain, it retains almost all of the shape and
charge attributes that differentiate the former compound from other
alkanes.
[0833] Target materials that do not have reactive functional groups
may be immobilized by first creating a reactive functional group
through the use of some powerful reagent, such as a halogen. For
example, an alkane can be immobilized for affinity by first
halogenating it and then reacting the halogenated derivative with
an immobilized or immobilizable amine.
[0834] In some cases, the reactive groups of the actual target
material may occupy a part on the target molecule that is to be
left undisturbed. In that case, additional functional groups may be
introduced by synthetic chemistry. For example, the most reactive
groups in cholesterol are on the steroid ring system, viz, --OH and
>C.dbd.C. We may wish to leave this ring system as it is so that
it binds to the novel binding protein. In this case, we prepare an
analogue having a reactive group attached to the aliphatic chain
(such as 26-aminocholesterol) and immobilize this derivative in a
manner appropriate to the reactive group so attached.
[0835] Two very general methods of immobilization are widely used.
The first is to biotinylate the compound of interest and then bind
the biotinylated derivative to immobilized avidin. The second
method is to generate antibodies to the target material, immobilize
the antibodies by any of numerous methods, and then bind the target
material to the immobilized antibodies. Use of antibodies is more
appropriate for larger target materials; small targets (those
comprising, for example, ten or fewer non-hydrogen atoms) may be so
completely engulfed by an antibody that very little of the target
is exposed in the target-antibody complex.
[0836] Non-covalent immobilization of hydrophobic molecules without
resort to antibodies may also be used. A compound, such as
2,3,3-trimethyldecane is blended with a matrix precursor, such as
sodium alginate, and the mixture is extruded into a hardening
solution. The resulting beads will have 2,3,3-trimethyldecane
dispersed throughout and exposed on the surface.
[0837] Other immobilization methods depend on the presence of
particular chemical functionalities. A polypeptide will present
--NH.sub.2 (N-terminal; Lysines), --COOH (C-terminal; Aspartic
Acids; Glutamic Acids), --OH (Serines; Threonines; Tyrosines), and
--SH (Cysteines). A polysaccharide has free --OH groups, as does
DNA, which has a sugar backbone.
[0838] The following table is a nonexhaustive review of reactive
functional groups and potential immobilization reagents:
12 Group Reagent R--NH.sub.2 Derivatives of 2,4,6-trinitro benzene
sulfonates (TNBS), (CREI84, p. 11) R--NH.sub.2 Carboxylic acid
anhydrides, e.g. derivatives of succinic anhydride, maleic
anhydride, citraconic anhydride (CREI84, p. 11) R--NH.sub.2
Aldehydes that form reducible Schiff bases (CREI84, p. 12) guanido
cyclohexanedione derivatives (CREI84, p. 14) R--CO.sub.2H Diazo
cmpds (CREI84, p. 10) R--CO.sub.2-- Epoxides (CREI84, p. 10) R--OH
Carboxylic acid anhydrides Aryl-OH Carboxylic acid anhydrides
Indole ring Benzyl halide and sulfenyl halides (CREI84, p. 19)
R--SH N-alkylmaleimides (CREI84, p. 21) R--SH ethyleneimine
derivatives (CREI84, p. 21) R--SH Aryl mercury compounds, (CREI84,
P. 21) R--SH Disulfide reagents, (CREI84, p. 23) Thiol ethers Alkyl
iodides, (CREI84, p. 20) Ketones Make Schiff's base and reduce with
NaBH.sub.4. (CREI84, p. 12-13) Aldehydes Oxidize to COOH, vide
supra. R--SO.sub.3H Convert to R--SO.sub.2Cl and react with
immobilized alcohol or amine. R--PO.sub.3H Convert to R--PO.sub.2Cl
and react with immobilized alcohol or amine. CC double bonds Add
HBr and then make amine or thiol.
[0839] The next table identifies the reactive groups of a number of
potential targets.
13 Reactive groups or Compound (Item#, page)* [derivatives]
prostaglandin E2 (2893,1251) --OH, keto, --COOH, C.dbd.C aspartame
(861,132) --NH.sub.2, --COOH, --COOCH.sub.3 haem (4558, 732) vinyl,
--COOH, Fe bilirubin (1235,189) vinyl, --COOH, keto, --NH--
morphine (6186,988) --OH, --C.dbd.C--, reactive phenyl ring codeine
(2459,384) --OH, --C.dbd.C--, reactive phenyl ring
dichlorodiphenyltrichlorethane aromatic chlorine, aliphatic
(2832,446) chlorine benzo(a)pyrene (1113,172)
[Chlorinate.fwdarw.amine, or make sulfonate.fwdarw.Aryl-SO.sub.2Cl]
actinomycin D (2804,441) aryl-NH.sub.2, --OH cellulose self
immobilized hydroxylapatite self immobilized cholesterol (2204,341)
--OH, >C.dbd.C-- *Note: Item# and page refer to The Merck Index,
llth Edition.
[0840] The extensive literature on affinity chromatography and
related techniques will provide further examples.
[0841] Matrices suitable for use as support materials include
polystyrene, glass, agarose and other chromatographic supports, and
may be fabricated into beads, sheets, columns, wells, and other
forms as desired. Suppliers of support material for affinity
chromatography include: Applied Protein Technologies Cambridge,
Mass.; BioRad Laboratories, Rockville Center, N.Y.; Pierce Chemical
Company, Rockford, Ill. Target materials are attached to the matrix
in accord with the directions of the manufacturer of each matrix
preparation with consideration of good presentation of the
target.
[0842] Early in the selection process, relatively high
concentrations of target materials may be applied to the matrix to
facilitate binding; target concentrations may subsequently be
reduced to select for higher affinity SBDs.
[0843] V.G. Elution of Lower Affinity PBD-bearing Genetic
Packacres
[0844] The population of GPs is applied to an affinity matrix under
conditions compatible with the intended use of the binding protein
and the population is fractionated by passage of a gradient of some
solute over the column. The process enriches for PBDs having
affinity for the target and for which the affinity for the target
is least affected by the eluants used. The enriched fractions are
those containing viable GPs that elute from the column at greater
concentration of the eluant.
[0845] The eluants preferably are capable of weakening noncovalent
interactions between the displayed PBDs and the immobilized target
material. Preferably, the eluants do not kill the genetic package;
the genetic message corresponding to successful mini-proteins is
most conveniently amplified by reproducing the genetic package
rather than by in vitro procedures such as PCR. The list of
potential eluants includes salts (including Na+, NH.sub.4+, Rb+,
SO.sub.4--, H.sub.2PO.sub.4-, citrate, K+, Li+, Cs+, HSO.sub.4-,
CO.sub.3--, Ca++, Sr++, Cl-, PO.sub.4---, HCO.sub.3-, Mg++, Ba++,
Br-, HPO.sub.4-- and acetate), acid, heat, compounds known to bind
the target, and soluble target material (or analogues thereof).
[0846] Because bacteria continue to metabolize during affinity
separation, the choice of buffer components is more restricted for
bacteria than for bacteriophage or spores. Neutral solutes, such as
ethanol, acetone, ether, or urea, are frequently used in protein
purification and are known to weaken non-covalent interactions
between proteins and other molecules. Many of these species are,
however, very harmful to bacteria and bacteriophage. Urea is known
not to harm M13 up to 8 M. Bacterial spores, on the other hand, are
impervious to most neutral solutes. Several affinity separation
passes may be made within a single round of variegation. Different
solutes may be used in different analyses, salt in one, pH in the
next, etc.
[0847] Any ions or cofactors needed for stability of PBDs (derived
from IPBD) or target are included in initial and elution buffers at
appropriate levels. We first remove GP(PBD)s that do not bind the
target by washing the matrix with the initial buffer. We determine
that this phase of washing is complete by plating aliquots of the
washes or by measuring the optical density (at 260 nm or 280 nm).
The matrix is then eluted with a gradient of increasing: a) salt,
b) [H+] (decreasing pH), c) neutral solutes, d) temperature
(increasing or decreasing), or e) some combination of these
factors. The solutes in each of the first three gradients have been
found generally to weaken non-covalent interactions between
proteins and bound molecules. Salt is a preferred solute for
gradient formation in most cases. Decreasing pH is also a highly
preferred eluant. In some cases, the preferred matrix is not stable
to low pH so that salt and urea are the most preferred reagents.
Other solutes that generally weaken non-covalent interaction
between proteins and the target material of interest may also be
used.
[0848] The uneluted genetic packages contain DNA encoding binding
domains which have a sufficiently high affinity for the target
material to resist the elution conditions. The DNA encoding such
successful binding domains may be recovered in a variety of ways.
Preferably, the bound genetic packages are simply eluted by means
of a change in the elution conditions. Alternatively, one may
culture the genetic package in situ, or extract the
target-containing matrix with phenol (or other suitable solvent)
and amplify the DNA by PCR or by recombinant DNA techniques.
Additionally, if a site for a specific protease has been engineered
into the display vector, the specific protease is used to cleave
the binding domain from the GP.
[0849] V.H. Optimization of Affinity Chromatography Separation:
[0850] For linear gradients, elution volume and eluant
concentration are directly related. Changes in eluant concentration
cause GPs to elute from the column. Elution volume, however, is
more easily measured and specified. It is to be understood that the
eluant concentration is the agent causing GP release and that an
eluant concentration can be calculated from an elution volume and
the specified gradient.
[0851] Using a specified elution regime, we compare the elution
volumes of GP(IPBD)s with the elution volumes of wtGP on affinity
columns supporting AfM(IPBD). Comparisons are made at various: a)
amounts of IPBD/GP, b) densities of AfM(IPBD)/(volume of matrix)
(DoAMoM), c) initial ionic strengths, d) elution rates, e) amounts
of GP/(volume of support), f) pHs, and g) temperatures, because
these are the parameters most likely to affect the sensitivity and
efficiency of the separation. We then pick those conditions giving
the best separation.
[0852] We do not optimize pH or temperature; rather we record
optimal values for the other parameters for one or more values of
pH and temperature. The pH used must be within the range of pH for
which GP(IPBD) binds the AfM(IPBD) that is being used in this step.
The conditions of intended use specified by the user may include a
specification of pH or temperature. If pH is specified, then pH
will not be varied in eluting the column. Decreasing pH may,
however, be used to liberate sound GPs from the matrix. Similarly,
if the intended use specifies a temperature, we will hold the
affinity column at the specified temperature during elution, but we
might vary the temperature during recovery. If the intended use
specifies the pH or temperature, then we prefer that the affinity
separation be optimized for all other parameters at the specified
pH and temperature.
[0853] In the optimization devised in this step, we preferably use
a molecule known to have moderate affinity for the IPBD (K.sub.d in
the range 10.sup.-6 M to 10.sup.-8 M), for the following reason.
When populations of GP(vgPBD)s are fractionated, there will be
roughly three subpopulations: a) those with no binding, b) those
that have some binding but can be washed off with high salt or low
pH, and c) those that bind very tightly and are most easily rescued
in situ. We optimize the parameters to separate (a) from (b) rather
than (b) from (c). Let PBD.sub.w be a PBD having weak binding to
the target and PBD.sub.s be a PBD having strong binding. Higher
DoAMoM might, for example, favor retention of GP(PBDW) but also
make it very difficult to elute viable GP(PBD.sub.w). We will
optimize the affinity separation to retain GP(PBD.sub.w) rather
than to allow release of GP(PBD.sub.s) because a tightly bound
GP(PBD.sub.s) can be rescued by in situ growth. If we find that
DoAMoM strongly affects the elution volume, then in part III we may
reduce the amount of target on the affinity column when an SBD has
been found with moderately strong affinity (Kd on the order of
10.sup.-7 M) for the target.
[0854] In case the promoter of the osp-ipbd gene is not regulated
by a chemical inducer, we optimize DoAMoM, the elution rate, and
the amount of GP/volume of matrix. If the optimized affinity
separation is acceptable, we proceed. If not, we develop a means to
alter the amount of IPBD per GP. Among GPs considered in the
present invention, this case could arise only for spores because
regulatable promoters are available for all other systems.
[0855] If the amount of IPBD/spore is too high, we could engineer
an operator site into the osp-ipbd gene. We choose the operator
sequence such that a repressor sensitive to a small diffusible
inducer recognizes the operator. Alternatively, we could alter the
Shine-Dalgarno sequence to produce a lower homology with consensus
Shine-Dalgarno sequences. If the amount of IPBD/spore is too low,
we can introduce variability into the promoter or Shine-Dalgarno
sequences and screen colonies for higher amounts of IPBD/spore.
[0856] In this step, we measure elution volumes of genetically pure
GPs that elute from the affinity matrix as sharp bands that can be
detected by UV absorption. Alternatively, samples from effluent
fractions can be plated on suitable medium (cells or spores) or on
sensitive cells (phage) and colonies or plaques counted.
[0857] Several values of IPBD/GP, DoAMoM, elution rates, initial
ionic strengths, and loadings should be examined. The following is
only one of many ways in which the affinity separation could be
optimized. We anticipate that optimal values of IPBD/GP and DoAMoM
will be correlated and therefore should be optimized together. The
effects of initial ionic strength, elution rate, and amount of
GP/(matrix volume) are unlikely to be strongly correlated, and so
they can be optimized independently.
[0858] For each set of parameters to be tested, the column is
eluted in a specified manner. For example, we may use a regime
called Elution Regime 1: a KCl gradient runs from 10 mM to maximum
allowed for the GP(IPBD) viability in 100 fractions of 0.05
V.sub.V, followed by 20 fractions of 0.05 vV at maximum allowed
KCl; pH of the buffer is maintained at the specified value with a
convenient buffer such as phosphate, Tris, or MOPS. Other elution
regimes can be used; what is important is that the conditions of
this optimization be similar to the conditions that are used in
Part III for selection for binding to target and recovery of GPs
from the chromatographic system.
[0859] When the osp-ipbd gene is regulated by [XINDUCE], IPBD/GP
can be controlled by varying [XINDUCE]. Appropriate values of
[XINDUCE] depend on the identity of [XINDUCE] and the promoter; if,
for example, XINDUCE is isopropylthiogalactoside (IPTG) and the
promoter is lacUV5, then [IPTG]=0, 0.1 uM, 1.0 uM, 10.0 uM, 100.0
uM, and 1.0 mM would be appropriate levels to test. The range of
variation of [XINDUCE] is extended until an optimum is found or an
acceptable level of expression is obtained.
[0860] DoAMoM is varied from the maximum that the matrix material
can bind to 1% or 0.1% of this level in appropriate steps. We
anticipate that the efficiency of separation will be a smooth
function of DoAMoM so that it is appropriate to cover a wide range
of values for DoAMoM with a coarse grid and then explore the
neighborhood of the approximate optimum with a finer grid.
[0861] Several values of initial ionic strength are tested, such as
1.0 mM, 5.0 mM, 10.0 mM and 20.0 mM. Low ionic strength favors
binding between oppositely charged groups, but could also cause GP
to precipitate.
[0862] The elution rate is varied, by successive factors of 1/2,
from the maximum attainable rate to {fraction (1/16)} of this
value. If the lowest elution rate tested gives the best separation,
we test lower elution rates until we find an optimum or adequate
separation.
[0863] The goal of the optimization is to obtain a sharp transition
between bound and unbound GPs, triggered by increasing salt or
decreasing pH or a combination of both. This optimization need be
performed only: a) for each temperature to be used, b) for each pH
to be used, and c) when a new GP(IPBD) is created.
[0864] V.I. Measuring the Sensitivity of Affinity Separation:
[0865] Once the values of IPBD/GP, DoAMoM, initial ionic strength,
elution rate, and amount of GP/(volume of affinity support) have
been optimized, we determine the sensitivity of the affinity
separation (C.sub.sensi) by the following procedure that measures
the minimum quantity of GP(IPBD) that can be detected in the
presence of a large excess of wtGP. The user chooses a number of
separation cycles, denoted N.sub.chrom, that will be performed
before an enrichment is abandoned; preferably, N.sub.chrom is in
the range 6 to 10 and N.sub.chrom must be greater than 4.
Enrichment can be terminated by isolation of a desired GP(SBD)
before N.sub.chrom passes.
[0866] The measurement of sensitivity is significantly expedited if
GP(IPBD) and wtGP carry different selectable markers because such
markers allow easy identification of colonies obtained by plating
fractions obtained from the chromatography column. For example, if
wtGP carries kanamycin resistance and GP(IPBD) carries ampicillin
resistance, we can plate fractions from a column on non-selective
media suitable for the GP. Transfer of colonies onto ampicillin or
kanamycin-containing media will determine the identity of each
colony.
[0867] Mixtures of GP(IPBD) and wtGP are prepared in the ratios of
l:V.sub.lim, where V.sub.lim ranges by an appropriate factor (e.g.
{fraction (1/10)}) over an appropriate range, typically 101l
through 104. Large values of V.sub.lim are tested first; once a
positive result is obtained for one value of V.sub.lim, no smaller
values of V.sub.lim need be tested. Each mixture is applied to a
column supporting, at the optimal DoAMoM, an AfM(IPBD) having high
affinity for IPED and the column is eluted by the specified elution
regime, such as Elution Regime 1. The last fraction that contains
viable GPs and an inoculum of the column matrix material are
cultured. If GP(IPBD) and wtGP have different selectable markers,
then transfer onto selection plates identifies each colony. If
GP(IPBD) and wtGP have no selectable markers or the same selectable
markers, then a number (e.g. 32) of GP clonal isolates are tested
for presence of IPBD. If IPBD is not detected on the surface of any
of the isolated GPs, then GPs are pooled from: a) the last few
(e.g. 3 to 5) fractions that contain viable GPs, and b) an inoculum
taken from the column matrix. The pooled GPs are cultured and
passed over the same column and enriched for GP(IPBD) in the manner
described. This process is repeated until N.sub.chrom passes have
been performed, or until the IPBD has been detected on the GPs. If
GP(IPBD) is not detected after N.sub.chrom passes, V.sub.lim is
decreased and the process is repeated.
[0868] Once a value for V.sub.lim is found that allows recovery of
GP(IPBD)s, the factor by which V.sub.lim is varied is reduced and
additional values are tested until V.sub.lim is known to within a
factor of two.
[0869] C.sub.sensi equals the highest value of V.sub.lim for which
the user can recover GP(IPBD) within N.sub.chrom passes. The number
of chromatographic cycles (K.sub.cyc) that were needed to isolate
GP(IPBD) gives a rough estimate of C.sub.eff; C.sub.eff is
approximately the K.sub.cycth root of V.sub.lim:
C.sub.eff.apprxeq.exp{log.sub.e(V.sub.lim)/K.sub.cyc}
[0870] For example, if V.sub.lim were 4.0.times.10.sup.8 and three
separation cycles were needed to isolate GP(IPBD), then
C.sub.eff.apprxeq.736.
[0871] V.J. Measuring the Efficiency of Separation
[0872] To determine C.sub.eff more accurately, we determine the
ratio of GP(IPBD)/wtGP loaded onto an AfM(IPBD) column that yields
approximately equal amounts of GP(IPBD) and wtGP after elution. We
prepare mixtures of GP(IPBD) and wtGP in ratios GP(IPBD):wtGP::1:Q;
we start Q at twenty times the approximate C.sub.eff found above. A
1:Q mixture of GP(IPBD) and wtGP is applied to a AfM(IPBD) column
and eluted by the specified elution regime, such as Elution Regime
1. A sample of the last fraction that contains viable GPs is plated
at a dilution that gives well separated colonies or plaques. The
presence of IPBD or the osp-ipbd gene in each colony or plaque can
be determined by a number of standard methods, including: a) use of
different selectable markers, b) nitrocellulose filter lift of GPs
and detection with AfM(IPBD)* (AUSU87), or c) nitrocellulose filter
lift of GPs and detection with radiolabeled DNA that is
complementary to the osp-ipbd gene (AUSU87). Let F be the fraction
of GP(IPBD) colonies found in the last fraction containing viable
GPs. When a Q is found such that 0.20<F<0.80, then
C.sub.eff=Q * F.
[0873] If F<0.2, then we reduce Q by an appropriate factor (e.g.
{fraction (1/10)}) and repeat the procedure. If F>0.8, then we
increase Q by an appropriate factor (e.g. 2) and repeat the
procedure.
[0874] V.K. Reducing Selection Due to Non-specific Binding:
[0875] When affinity chromatography is used for separating bound
and unbound GPs, we may reduce non-specific binding of GP(PBD)s to
the matrix that bears the target in the following ways:
[0876] 1) we treat the column with blocking agents such as
genetically defective GPs or a solution of protein before the
population of GP(vgPBD)s is chromatographed, and
[0877] 2) we pass the population of GP(vgPBD)s over a matrix
containing no target or a different target from the same class as
the actual target prior to affinity chromatography.
[0878] Step (1) above saturates any non-specific binding that the
affinity matrix might show toward wild-type GPs or proteins in
general; step (2) removes components of our population that exhibit
non-specific binding to the matrix or to molecules of the same
class as the target. If the target were horse heart myoglobin, for
example, a column supporting bovine serum albumin could be used to
trap GPs exhibiting PBDs with strong non-specific binding to
proteins. If cholesterol were the target, then a hydrophobic
compound, such as p-tertiarybutylbenzyl alcohol, could be used to
remove GPs displaying PBDs having strong non-specific binding to
hydrophobic compounds. It is anticipated that PBDs that fail to
fold or that are prematurely terminated will be non-specifically
sticky. These sequences could outnumber the PBDs having desirable
binding properties. Thus, the capacity of the initial column that
removes indiscriminately adhesive PBDs should be greater (e.g. 5
fold greater) than the column that supports the target
molecule.
[0879] Variation in the support material (polystyrene, glass,
agarose, cellulose, etc.) in analysis of clones carrying SBDs is
used to eliminate enrichment for packages that bind to the support
material rather than the target.
[0880] FACs may be used to separate GPs that bind fluorescent
labeled target. We discriminate against artifactual binding to the
fluorescent label by using two or more different dyes, chosen to be
structurally different. GPs isolated using target labeled with a
first dye are cultured. These GPs are then tested with target
labeled with a second dye.
[0881] Electrophoretic affinity separation uses unaltered target so
that only other ions in the buffer can give rise to artifactual
binding. Artifactual binding to the gel material gives rise to
retardation independent of field direction and so is easily
eliminated.
[0882] A variegated population of GPs will have a variety of
charges. The following 2D electrophoretic procedure accommodates
this variation in the population. First the variegated population
of GPs is electrophoresed in a gel that contains no target
material. The electrophoresis continues until the GP s are
distributed along the length of the lane. The gels described by
Sewer for phage are very low in agarose and lack mechanical
stability. The target-free lane in which the initial
electrophoresis is conducted is separate from a square of gel that
contains target material by a removable baffle. After the first
pass, the baffle is removed and a second electrophoresis is
conducted at right angles to the first. GPs that do not bind target
migrate with unaltered mobility while GP s that do bind target
will, separate from the majority that do not bind target. A
diagonal line of non-binding GPs will form. This line is excised
and discarded. Other parts of the gel are dissolved and the GPs
cultured.
[0883] V.L. Isolation of GP(PBD)s with Binding-to-target
Pheno-types:
[0884] The harvested packages are now enriched for the
binding-to-target phenotype by use of affinity separation involving
the target material immobilized on an affinity matrix. Packages
that fail to bind to the target material are washed away. If the
packages are bacteriophage or endospores, it may be desirable to
include a bacteriocidal agent, such as azide, in the buffer to
prevent bacterial growth. The buffers used in chromatography
include: a) any ions or other solutes needed to stabilize the
target, and b) any ions or other solutes needed to stabilize the
PBDs derived from the IPBD.
[0885] V.M. Recovery of Packages:
[0886] Recovery of packages that display binding to an affinity
column may be achieved in several ways, including:
[0887] 1) collect fractions eluted from the column with a gradient
as described above; fractions eluting later in the gradient contain
GPs more enriched for genes encoding PBDs with high affinity for
the column,
[0888] 2) elute the column with the target material in soluble
form,
[0889] 3) flood the matrix with a nutritive medium and grow the
desired packages in situ,
[0890] 4) remove parts of the matrix and use them to inoculate
growth medium,
[0891] 5) chemically or enzymatically degrade the linkage holding
the target to the matrix so that GPs still bound to target are
eluted, or
[0892] 6) degrade the packages and recover DNA with phenol or other
suitable solvent; the recovered DNA is used to transform cells that
regenerate GPs.
[0893] It is possible to utilize combinations of these methods. It
should be remembered that what we want to recover from the affinity
matrix is not the GPs per se, but the information in them. Recovery
of viable GPs is very strongly preferred, but recovery of genetic
material is essential. If cells, spores, or virions bind
irreversibly to the matrix but are not killed, we can recover the
information through in situ cell division, germination, or
infection respectively. Proteolytic degradation of the packages and
recovery of DNA is not preferred.
[0894] Although degradation of the bound GPs and recovery of
genetic material is a possible mode of operation, inadvertent
inactivation of the GPs is very deleterious. It is preferred that
maximum limits for solutes that do not inactivate the GPs or
denature the target or the column are determined. If the affinity
matrices are expendable, one may use conditions that denature the
column to elute GPs; before the target is denatured, a portion of
the affinity matrix should be removed for possible use as an
inoculum. As the GPs are held together by protein-protein
interactions and other non-covalent molecular interactions, there
will be cases in which the molecular package will bind so tightly
to the target molecules on the affinity matrix that the GPs can not
be washed off in viable form. This will only occur when very tight
binding has been obtained. In these cases, methods (3) through (5)
above can be used to obtain the bound packages or the genetic
messages from the affinity matrix.
[0895] It is possible, by manipulation of the elution conditions,
to isolate SBDs that bind to the target at one pH (pH.sub.b) but
not at another pH (pH.sub.o) The population is applied at pHb and
the column is washed thoroughly at pH.sub.b. The column is then
eluted with buffer at pH.sub.o and GPs that come off at the new pH
are collected and cultured. Similar procedures may be used for
other solution parameters, such as temperature. For example,
GP(vgPBD)s could be applied to a column supporting insulin. After
eluting with salt to remove GPs with little or no binding to
insulin, we elute with salt and glucose to liberate GPs that
display PBDs that bind insulin or glucose in a competitive
manner.
[0896] V.N. Amplifying the Enriched Packages
[0897] Viable GPs having the selected binding trait are amplified
by culture in a suitable medium, or, in the case of phage,
infection into a host so cultivated. If the GPs have been
inactivated by the chromatography, the OCV carrying the osp-ipbd
gene are recovered from the GP, and introduced into a new, viable
host.
[0898] V.O. Determining Whether Further Enrichment is Needed:
[0899] The probability of isolating a GP with improved binding
increases by C.sub.eff with each separation cycle. Let N be the
number of distinct amino-acid sequences produced by the
variegation. We want to perform K separation cycles before
attempting to isolate an SBD, where K is such that the probability
of isolating a single SBD is 0.10 or higher.
K=the smallest integer>=log.sub.10(0.10
N)/log.sub.10(C.sub.eff)
[0900] For example, if N were 1.0.multidot.10.sup.7 and
C.sub.eff=6.31.multidot.10.sup.2, then
log.sub.10(1.0.multidot.10.sup.6)/-
log.sub.10(6.31.multidot.10.sup.2)=6.0000/2.8000=2.14. Therefore we
would attempt to isolate SBDs after the third separation cycle.
After only two separation cycles, the probability of finding an SBD
is
(6.31.times.10.sup.2).sup.2/(1.0.multidot.10.sup.7)=0.04
[0901] and attempting to isolate SBDs might be profitable.
[0902] Clonal isolates from the last fraction eluted which
contained any viable GPs, as well as clonal isolates obtained by
culturing an inoculum taken from the affinity matrix, are cultured
in a growth step that is similar to that described previously.
Other fractions may be cultured too. If K separation cycles have
been completed, samples from a number, e.g. 32, of these clonal
isolates are tested for elution properties on the (target) column.
If none of the isolated, genetically pure GPs show improved binding
to target, or if K cycles have not yet been completed, then we pool
and culture, in a manner similar to the manner set forth
previously, the GPs from the last few fractions eluted that
contained viable GPs and from the GPs obtained by culturing an
inoculum taken from the column matrix. We then repeat the
enrichment procedure described above. This cyclic enrichment may
continue N.sub.chrom passes or until an SBD is isolated.
[0903] If one or more of the isolated GPs has improved retention on
the (target) column, we determine whether the retention of the
candidate SBDs is due to affinity for the target material as
follows. A second column is prepared using a different support
matrix with the target material bound at the optimal density. The
elution volumes, under the same elution conditions as used
previously, of candidate GP(SBD)s are compared to each other and to
GP(PPBD of this round). If one or more candidate GP(SBD)s has a
larger elution volume than GP(PPBD of this round), then we pick the
GP(SBD) having the highest elution volume and proceed to
characterize the population. If none of the candidate GP(SBD)s has
higher elution volume than GP(PPBD of this round), then we pool and
culture, in a manner similar to the manner used previously, the GPs
from the last few fractions that contained viable GPs and the GPs
obtained by culturing an inoculum taken from the column matrix. We
then repeat the enrichment procedure.
[0904] If all of the SBDs show binding that is superior to PPBD of
this round, we pool and culture the GPs from the last fraction that
contains viable GPs and from the inoculum taken from the column.
This population is re-chromatographed at least one pass to
fractionate further the GPs based on K.sub.d.
[0905] If an RNA phage were used as GP, the RNA would either be
cultured with the assistance of a helper phage or be reverse
transcribed and the DNA amplified. The amplified DNA could then be
sequenced or subcloned into suitable plasmids.
[0906] V.P. Characterizing the Putative SBDS:
[0907] We characterize members of the population showing desired
binding properties Dy genetic and biochemical methods. We obtain
clonal isolates and test these strains by genetic and affinity
methods to determine genotype and phenotype with respect to binding
to target. For several genetically pure isolates that show binding,
we demonstrate that the binding is caused by the artificial
chimeric gene by excising the osp-sbd gene and crossing it into the
parental GP. We also ligate the deleted backbone of each GP from
which the osp-sbd is removed and demonstrate that each backbone
alone cannot confer binding to the target on the GP. We sequence
the osp-sbd gene from several clonal isolates. Primers for
sequencing are chosen from the DNA flanking the osp-ppbd gene or
from parts of the osp-ppbd gene that are not variegated.
[0908] The present invention is not limited to a single method of
determining protein sequences, and reference in the appended claims
to determining the amino acid sequence of a domain is intended to
include any practical method or combination of methods, whether
direct or indirect. The preferred method, in most cases, is to
determine the sequence of the DNA that encodes the protein and then
to infer the amino acid sequence. In some cases, standard methods
of protein-sequence determination may be needed to detect
post-translational processing.
[0909] The present invention is not limited to a single method of
determining the sequence of nucleotides (nts) in DNA subsequences.
In the preferred embodiment, plasmids are isolated and denatured in
the presence of a sequencing primer, about 20 nts long, that
anneals to a region adjacent, on the 5' side, to the region of
interest. This plasmid is then used as the template in the four
sequencing reactions with one dideoxy substrate in each. Sequencing
reactions, agarose gel electrophoresis, and polyacrylamide gel
electrophoresis (PAGE) are performed by standard procedures
(AUSU87).
[0910] For one or more clonal isolates, we may subclone the sbd
gene fragment, without the osp fragment, into an expression vector
such that each SBD can be produced as a free protein. Because
numerous unique restriction sites were built into the inserted
domain, it is easy to subclone the gene at any time. Each SBD
protein is purified by normal means, including affinity
chromatography. Physical measurements of the strength of binding
are then made on each free SBD protein by one of the following
methods: 1) alteration of the Stokes radius as a function of
binding of the target material, measured by characteristics of
elution from a molecular sizing column such as agarose, 2)
retention of radiolabeled binding protein on a spun affinity column
to which has been affixed the target material, or 3) retention of
radiolabeled target material on a spun affinity column to which has
been affixed the binding protein. The measurements of binding for
each free SBD are compared to the corresponding measurements of
binding for the PPBD.
[0911] In each assay, we measure the extent of binding as a
function of concentration of each protein, and other relevant
physical and chemical parameters such as salt concentration,
temperature, pH, and prosthetic group concentrations (if any).
[0912] In addition, the SBD with highest affinity for the target
from each round is compared to the best SBD of the previous round
(IPBD for the first round) and to the IPBD (second and later
rounds) with respect to affinity for the target material.
Successive rounds of mutagenesis and selection-through-binding
yield increasing affinity until desired levels are achieved.
[0913] If we find that the binding is not yet sufficient, we decide
which residues to vary next. If the binding is sufficient, then we
now have a expression vector bearing a gene encoding the desired
novel binding protein.
[0914] V.O. Joint Selections:
[0915] One may modify the affinity separation of the method
described to select a molecule that binds to material A but not to
material B. One needs to prepare two selection columns, one with
material A and the other with material B. The population of genetic
packages is prepared in the manner described, but before applying
the population to A, one passes the population over the B column so
as to remove those members of the population that have high
affinity for B ("reverse affinity chromatography"). In the
preceding specification, the initial column supported some other
molecule simply to remove GP(PBD)s that displayed PBDs having
indiscriminate affinity for surfaces.
[0916] It may be necessary to amplify the population that does not
bind to B before passing it over A. Amplification would most likely
be needed if A and B were in some ways similar and the PPBD has
been selected for having affinity for A. The optimum order of
interactions might be determined empirically. For example, to
obtain an SBD that binds A but not B, three columns could be
connected in series: a) a column supporting some compound, neither
A nor B, or only the matrix material, b) a column supporting B, and
c) a column supporting A. A population of GP(vgPBD)s is applied to
the series of columns and the columns are washed with the buffer of
constant ionic strength that is used in the application. The
columns are uncoupled, and the third column is eluted with a
gradient to isolate GP(PBD)s that bind A but not B.
[0917] One can also generate molecules that bind to both A and B.
In this case we can use a 3D model and mutate one face of the
molecule in question to get binding to A. One can then mutate a
different face to produce binding to B. When an SBD binds at least
somewhat to both A and B, one can mutate the chain by Diffuse
Mutagenesis to refine the binding and use a sequential joint
selection for binding to both A and B.
[0918] The materials A and B could be proteins that differ at only
one or a few residues. For example, A could be a natural protein
for which the gene has been cloned and B could be a mutant of A
that retains the overall 3D structure of A. SBDs selected to bind A
but not B probably bind to A near the residues that are mutated in
B. If the mutations were picked to be in the active site of A
(assuming A has an active site), then an SBD that binds A but not B
will bind to the active site of A and is likely to be an inhibitor
of A.
[0919] To obtain a protein that will bind to both A and B, we can,
alternatively, first obtain an SBD that binds A and a different SBD
that binds B. We can then combine the genes encoding these domains
so that a two-domain single-polypeptide protein is produced. The
fusion protein will have affinity for both A and B because one of
its domains binds A and the other binds B.
[0920] One can also generate binding proteins with affinity for
both A and B, such that these materials will compete for the same
site on the binding protein. We guarantee competition by
overlapping the sites for A and B. Using the procedures of the
present invention, we first create a molecule that binds to target
material A. We then vary a set of residues defined as: a) those
residues that were varied to obtain binding to A, plus b) those
residues close in 3D space to the residues of set (a) but that are
internal and so are unlikely to bind directly to either A or B.
Residues in set (b) are likely to make small changes in the
positioning of the residues in set (a) such that the affinities for
A and B will be changed by small amounts. Members of these
populations are selected for affinity to both A and B.
[0921] V.R. Selection for Non-binding:
[0922] The method of the present invention can be used to select
proteins that do not bind to selected targets. Consider a protein
of pharmacological importance, such as streptokinase, that is
antigenic to an undesirable extent. We can take the
pharmacologically important protein as IPBD and antibodies against
it as target. Residues on the surface of the pharmacologically
important protein would be variegated and GP(PBD)s that do not bind
to an antibody column would be collected and cultured. Surface
residues may be identified in several ways, including: a) from a 3D
structure, b) from hydrophobicity considerations, or c) chemical
labeling. The 3D structure of the pharmacologically important
protein remains the preferred guide to picking residues to vary,
except now we pick residues that are widely spaced so that we leave
as little as possible of the original surface unaltered.
[0923] Destroying binding frequently requires only that a single
amino acid in the binding interface be changed. If polyclonal
antibodies are used, we face the problem that all or most of the
strong epitopes must be altered in a single molecule. Preferably,
one would have a set of monoclonal antibodies, or a narrow range of
antibody species. If we had a series of monoclonal antibody
columns, we could obtain one or more mutations that abolish binding
to each monoclonal antibody. We could then combine some or all of
these mutations in one molecule to produce a pharmacologically
important protein recognized by none of the monoclonal antibodies.
Such mutants are tested to verify that the pharmacologically
interesting properties have not be altered to an unacceptable
degree by the mutations.
[0924] Typically, polyclonal antibodies display a range of binding
constants for antigen. Even if we have only polyclonal antibodies
that bind to the pharmacologically important protein, we may
proceed as follows. We engineer the pharmacologically important
protein to appear on the surface of a replicable GP. We introduce
mutations into residues that are on the surface of the
pharmacologically important protein or into residues thought to be
on the surface of the pharmacologically important protein so that a
population of GPs is obtained. Polyclonal antibodies are attached
to a column and the population of GPs is applied to the column at
low salt. The column is eluted with a salt gradient. The GPs that
elute at the lowest concentration of salt are those which bear
pharmacologically important proteins that have been mutated in a
way that eliminates binding to the antibodies having maximum
affinity for the pharmacologically important protein. The GPs
eluting at the lowest salt are isolated and cultured. The isolated
SBD becomes the PPBD to further rounds of variegation so that the
antigenic determinants are successively eliminated.
[0925] V.S. Selection of PBDs for Retention of Structure:
[0926] Let us take an SBD with known affinity for a target as PPBD
to a variegation of a region of the PBD that is far from the
residues that were varied to create the SBD. We can use the target
as an affinity molecule to select the PBDs that retain binding for
the target, and that presumably retain the underlying structure of
the IPBD. The variegations in this case could include insertions
and deletions that are likely to disrupt the IPBD structure. We
could also use the IPBD and AfM(IPBD) in the same way.
[0927] For example, if IPBD were BPTI and AfM(BPTI) were trypsin,
we could introduce four or five additional residue after residue 26
and select GPs that display PBDs having specific affinity for
AfM(BPTI). Residue 26 is chosen because it is in a turn and because
it is about 25 A from K15, a key amino acid in binding to
trypsin.
[0928] The underlying structure is most likely to be retained if
insertions or deletions are made at loops or turns.
[0929] V.T. Engineering of Antagonists
[0930] It may be desirable to provide an antagonist to an enzyme or
receptor. This may be achieved by making a molecule that prevents
the natural substrate or agonist from reaching the active site.
Molecules that bind directly to the active site may be either
agonists or antagonists. Thus we adopt the following strategy. We
consider enzymes and receptors together under the designation TER
(Target Enzyme or Receptor).
[0931] For most TERs, there exist chemical inhibitors that block
the active site. Usually, these chemicals are useful only as
research tools due to highly toxicity. We make two affinity
matrices: one with active TER and one with blocked TER. We make a
variegated population of GP(PBD)s and select for SBPs that bind to
both forms of the enzyme, thereby obtaining SDPs that do not bind
to the active site. We expect that SBDs will be found that bind
different places on the enzyme surface. Pairs of the sbd genes are
fused with an intervening peptide segment. For example, if SBD-1
and SBD-2 are binding domains that show high affinity for the
target enzyme and for which the binding is non-competitive, then
the gene sbd-1::linker::sbd-2 encodes a two-domain protein that
will show high affinity for the target. We make several fusions
having a variety of SBDs and various linkers. Such compounds have a
reasonable probability of being an antagonist to the target
enzyme.
[0932] VI. Exploitation of Successful Binding Domains and
Corresponding DNAS
[0933] VI.A. Generally
[0934] Using the method of the present invention, we can obtain a
replicable genetic package that displays a novel protein domain
having high affinity and specificity for a target material of
interest. Such a package carries both amino-acid embodiments of the
binding protein domain and a DNA embodiment of the gene encoding
the novel binding domain. The presence of the DNA facilitates
expression of a protein comprising the novel binding protein domain
within a high-level expression system, which need not be the same
system used during the developmental process.
[0935] VI.B. Production of Novel Binding Proteins
[0936] We can proceed to production of the novel binding protein in
several ways; including: a) altering of the gene encoding the
binding domain so that the binding domain is expressed as a soluble
protein, not attached to a genetic package (either by deleting
codons 5' of those encoding the binding domain or by inserting stop
codons 3' of those encoding the binding domain), b) moving the DNA
encoding the binding domain into a known expression system, and c)
utilizing the genetic package as a purification system. (If the
domain is small enough, it may be feasible to prepare it by
conventional peptide synthesis methods.)
[0937] Option (c) may be illustrated as follows. Assume that a
novel BPTI derivative has been obtained by selection of M13
derivatives in which a population of BPTI-derived domains are
displayed as fusions to mature coat protein. Assume that a specific
protease cleavage site (e.g. that of activated clotting factor X)
is engineered into the amino-acid sequence between the carboxy
terminus of the BPTI-derived domain and the mature coat domain.
Furthermore, we alter the display system to maximize the number of
fusion proteins displayed on each phage. The desired phage can be
produced and purified, for example by centrifugation, so that no
bacterial products remain. Treatment of the purified phage with a
catalytic amount of factor X cleaves the binding domains from the
phage particles. A second centrifugation step separates the cleaved
protein from the phage, leaving a very pure protein
preparation.
[0938] VI.C. Mini-protein Production
[0939] As previously mentioned, an advantage inhering from the use
of a mini-protein as an IPBD is that it is likely that the derived
SBD will also behave like a mini-protein and will be obtainable by
means of chemical synthesis. (The term "chemical synthesis", as
used herein, includes the use of enzymatic agents in a cell-free
environment.)
[0940] It is also to be understood that mini-proteins obtained by
the method of the present invention may be taken as lead compounds
for a series of homologues that contain non-naturally occurring
amino acids and groups other than amino acids. For example, one
could synthesize a series of homologues in which each member of the
series has one amino acid replaced by its D enantiomer. One could
also make homologues containing constituents such as .beta.
alanine, aminobutyric acid, 3-hydroxyproline, 2-Aminoadipic acid,
N-ethylasperagine, norvaline, etc.; these would be tested for
binding and other properties of interest, such as stability and
toxicity.
[0941] Peptides may be chemically synthesized either in solution or
on supports. Various combinations of stepwise synthesis and
fragment condensation may be employed.
[0942] During synthesis, the amino acid side chains are protected
to prevent branching. Several different protective groups are
useful for the protection of the thiol groups of cysteines:
[0943] 1) 4-methoxybenzyl (MBzl; Mob) (NISH82; ZAFA88), removable
with HF;
[0944] 2) acetamidomethyl (Acm)(NISH82; NISH86; BECK89c), removable
with iodine; mercury ions (e.g., mercuric acetate); silver nitrate;
and
[0945] 3) S-para-methoxybenzyl (HOUG84).
[0946] Other thiol protective groups may be found in standard
reference works such as Greene, PROTECTIVE GROUPS IN ORGANIC
SYNTHESIS (1981).
[0947] Once the polypeptide chain has been synthesized, disulfide
bonds must be formed. Possible oxidizing agents include air
(HOUG84; NISH86), ferricyanide (NISH82; HOUG84), iodine (NISH82),
and performic acid (HOUG84). Temperature, pH, solvent, and
chaotropic chemicals may affect the course of the oxidation.
biologically active form: conotoxin G1 (13AA, 4 Cys)(NISH-82);
heat-stable enterotoxin ST (18AA, 6 Cys) (HOUG84); analogues of ST
(BHAT86); .OMEGA.-conotoxin GVIA (27AA, 6Cys) (NISH86; RIVI87b);
106 -conotoxin MVIIA (27 AA, 6 Cys) (OLIV87b); .alpha.-conotoxin SI
(13 AA, 4 Cys) (ZAFA88); .mu.-conotoxin IIIa (22AA, 6 Cys)
(BECK89c, CRUZ89, HATA90). Sometimes, the polypeptide naturally
folds so that the correct disulfide bonds are formed. Other times,
it must be helped along by use of a differently removable
protective group for each pair of cysteines.
[0948] VI.D. Uses of Novel Binding Proteins
[0949] The successful binding domains of the present invention may,
alone or as part of a larger protein, be used for any purpose for
which binding proteins are suited, including isolation or detection
of target materials. In furtherance of this purpose, the novel
binding proteins may be coupled directly or indirectly, covalently
or noncovalently, to a label, carrier or support.
[0950] When used as a pharmaceutical, the novel binding proteins
may be contained with suitable carriers or adjuvanants.
[0951] All references cited anywhere in this specification are
incorporated by reference to the extent which they may be
pertinent.
EXAMPLE I
[0952] Display of BPTI as a Fusibn to M13 Gene VIII Protein:
[0953] Example I involves display of BPTI on M13 as a fusion to the
mature gene VIII coat protein. Each of the DNA constructions was
confirmed by restriction digestion analysis and DNA sequencing.
[0954] 1. Construction of the
viii-signal-sequence::bpti::mature-viii-coat- -protein Display
Vector.
[0955] A. Operative Cloning Vectors (OCV).
[0956] The operative cloning vectors are M13 and phagemids derived
from M13 or f1. The initial construction was in the f1-based
phagemid pGEM-3Zf(-).TM. (Promega Corp., Madison, Wis.).
[0957] A gene comprising, in order,: i) a modified lacUV5 promoter,
ii) a Shine-Dalgarno sequence, iii) DNA encoding the M13 gene VIII
signal sequence, iv) a sequence encoding mature BPTI, v) a sequence
encoding the mature-M13-gene-VIII coat protein, vi) multiple stop
codons, and vii) a transcription terminator, was constructed. This
gene is illustrated in Tables 101-105; each table shows the same
DNA sequence with different features annotated. There are a number
of differences between this gene and the one proposed in the
hypothetical example in the generic specification of the parent
application. Because the actual construction was made in
pGEM-3Zf(-), the ends of the synthetic DNA were made compatible
with SalI and BamHI. The lacO operator of lacUV5 was changed to the
symmetrical lacO with the intention of achieving tighter repression
in the absence of IPTG. Several silent codon changes were made so
that the longest segment that is identical to wild-type gene VIII
is minimized so that genetic recombination with the co-existing
gene VIII is unlikely.
[0958] i) OCV Based upon pGEM-3Zf.
[0959] pGEM-3Zf.TM. (Promega Corp., Madison, Wis.) is a
plasmid-based vector containing the amp gene, bacterial origin of
replication, bacteriophage f1 origin of replication, a lacZ operon
containing a multiple cloning site sequence, and the T7 and SP6
polymerase binding sequences.
[0960] Two restriction enzyme recognition sites were introduced, by
site-directed oligonucleotide mutagenesis, at the boundaries of the
lacZ operon. This allowed for the removal of the lacZ operon and
its replacement with the synthetic gene. A BamHI recognition site
(GGATCC) was introduced at the 5' end of the lacZ operon by the
mutation of bases C.sub.331 and T.sub.332 to G and A respectively
(numbering of Promega). A SalI recognition site (GTCGAC) was
introduced at the 3' end of the operon by the mutation of bases
C.sub.3021 and T.sub.3023 to G and C respectively. A construct
combining these variants of pGEM-3Zf was designated pGEM-MB3/4.
[0961] ii) OCV Based Upon M13mp18.
[0962] M13mpl8 (YANI85) is an M13 bacteriophage-based vector
(available from, inter alia, New England Biolabs, Beverly, Mass.)
consisting of the whole of the phage genome into which has been
inserted a lacZ operon containing a multiple cloning site sequence
(MESS77). Two restriction enzyme sites were introduced into M13mp18
using standard methods. A BamHI recognition site (GGATCC) was
introduced at the 5' end of the lacZ operon by the mutation of
bases C.sub.6003 and G.sub.6004 to A and T respectively (numbering
of Messing). This mutation also destroyed a unique NarI site. A
SalI recognition site (GTCGAC) was introduced at the 3' end of the
operon by the mutation of bases A.sub.6430 and C.sub.6432 to C and
A respectively. A construct combining these variants of M13mp18 was
designated M13-MB1/2.
[0963] B) Synthetic Gene.
[0964] A synthetic gene
(VIII-signal-sequence::mature-bpti::mature-VIII-co- at-protein) was
constructed from 16 synthetic oligonucleotides (Table 105), custom
synthesized by Genetic Designs Inc. of Houston, Tex., using methods
detailed in KIMH89 and ASHM89. Table 101 shows the DNA sequence;
Table 102 contains an annotated version of this sequence. Table 103
shows the overlaps of the synthetic oligonucleotides in
relationship to the restriction sites and coding sequence. Table
104 shows the synthetic DNA in double-stranded form. Table 105
shows each of the 16 synthetic oligonucleotides from 5'-to-3'. The
oligonucleotides were phosphorylated, with the exception of the 5'
most molecules, using standard methods, annealed and ligated in
stages such that a final synthetic duplex was generated. The
overhanging ends of this duplex was filled in with T4 DNA
polymerase and it was cloned into the HincII site of pGEM-3Zf(-);
the initial construct is called pGEM-MB1 (Table 101a).
Double-stranded DNA of PGEM-MB1 was cut with PstI, filled in with
T4 DNA polymerase and ligated to a SalI linker (New England
BioLabs) so that the synthetic gene is bounded by BamHI and SalI
sites (Table 101b and Table 102b). The synthetic gene was obtained
on a BamHI-SalI cassette and cloned into pGEM-MB3/4 and M13-MB1/2
utilizing the BamHI and SalI sites previously introduced, to
generate the constructs designated pGEM-MB16 and M13-MB15,
respectively. The full length of the synthetic insert was sequenced
and found to be unambiguously correct except for: 1) a missing G in
the Shine-Dalgarno sequence; and 2) a few silent errors in the
third bases of some codons (shown as upper case in Table 101).
Table 102 shows the Ribosome-binding site A.sub.104GGAGG but the
actual sequence is A.sub.104GAGG. Efforts to express protein from
this construction, in vivo and in vitro, were unavailing.
[0965] C) Alterations to the Synthetic Gene.
[0966] i) Ribosome Binding Site (RBS).
[0967] Starting with the construct pGEM-MB16, a fragment of DNA
bounded by the restriction enzyme sites SacI and NheI (containing
the original RBS) was replaced with a synthetic oligonucleotide
duplex (with compatible SacI and NheI overhangs) containing the
sequence for a new RBS that is very similar to the RBS of E. coli
phoA and that has been shown to be functional.
14 Original putative RBS (5'-to-3') GAGCTCagaggCTTACTATGAAGA-
AATCTCTGGTTCTTAAGGCTAGC .vertline.SacI.vertline. .vertline. Nhe I
.vertline. New RBS (5'-to-3')
GAGCTCTggaggaAATAAAATGAAGAAATCTCTGGTTCTTAAGGCTAGC
.vertline.SacI.vertline. .vertline. Nhe I .vertline.
[0968] The putative RBSs above are lower case and the initiating
methionine codon is underscored and bold. The resulting construct
was designated pGEM-MB20. In vitro expression of the gene carried
by pGEM-MB20 produced a novel protein species of the expected size,
about 14.5 kd.
[0969] ii) tac promoter.
[0970] In order to obtain higher expression levels of the fusion
protein, the lacUV5 promoter was changed to a tac promoter.
Starting with the construct PGEM-MB16, which contains the lacUV5
promoter, a fragment of DNA bounded by the restriction enzyme sites
BamHI and HpaI was excised and replaced with a compatible synthetic
oligonucleotide duplex containing the -35 sequence of the trp
promoter, Cf RUSS82. This converted the lacUV5 promoter to a tac
promoter in a construct designated pGEM-MB22, Table 112.
15 MB16 5'- GATCC tctagagtcggc TTTACA ctttatgcttc(cg-gctcg..-3' 3'-
G agatctcagccg aaatgt gaaatacgaag gc(cgagc..-5' .vertline.
.vertline. .vertline. -35.vertline. .vertline. .vertline. BamHI
HpaII MB22 insert 5'- GATCC actccccatccccctg TTGACA attaatcat -3'
3'- G tgaggggtagggggac AACTGT taattagtagc-5' .vertline. .vertline.
.vertline. -35.vertline. .vertline. BamHI (HpaII)
[0971] Promoter and RBS variants of the fusion protein gene were
constructed by basic DNA manipulation techniques to generate the
following:
16 Promoter RBS Encoded Protein. pGEM-MB16 lac old
VIIIs.p.-BPTI-matureVIII pGEM-MB20 lac new " PGEM-MB22 tac old "
pGEM-MB26 tac new "
[0972] The synthetic gene from variants pGEM-MB20 and pGEM-MB26
were recloned into the altered phage vector M13-MB1/2 to generate
the phage constructs designated M13-MB27 and M13-MB28
respectively.
[0973] iii. Signal Peptide Sequence.
[0974] In vitro expression of the synthetic gene regulated by tac
and the "new" RBS produced a novel protein of the expected size for
the unprocessed protein (about 16 kd). In vivo expression also
produced novel protein of full size; no processed protein could be
seen on phage or in cell extracts by silver staining or by Western
analysis with anti-BPTI antibody.
[0975] Thus we analyzed the signal sequence of the fusion. Table
106 shows a number of typical signal sequences. Charged residues
are generally thought to be of great importance and are shown bold
and underscored. Each signal sequence contains a long stretch of
uncharged residues that are mostly hydrophobic; these are shown in
lower case. At the right, in parentheses, is the length of the
stretch of uncharged residues. We note that the fusions of gene
VIII signal to BPTI and gene III signal to BPTI have rather short
uncharged segments. These short uncharged segments may reduce or
prevent processing of the fusion peptides. We know that the gene
III signal sequence is capable of directing: a) insertion of the
peptide comprising (mature-BPTI)::(mature-gene-III-protein) into
the lipid bilayer, and b) translocation of BPTI and most of the
mature gene III protein across the lipid bilayer (vide infra). That
the gene III remains anchored in the lipid bilayer until the phage
is assembled is directed by the uncharged anchor region near the
carboxy terminus of the mature gene III protein (see Table 116) and
not by the secretion signal sequence. The phoA signal sequence can
direct secretion of mature BPTI into the periplasm of E. coli
(MARK86). Furthermore, there is controversy over the mechanism by
which mature authentic gene VIII protein comes to be in the lipid
bilayer prior to phage assembly.
[0976] Thus we decided to replace the DNA coding on expression for
the gene-VIII-putative-signal-sequence by each of: 1) DNA coding on
expression for the phoA signal sequence, 2) DNA coding on
expression for the bla signal sequence, or 3) DNA coding on
expression for the M13 gene III signal. Each of these replacements
produces a tripartite gene encoding a fusion protein that
comprises, in order: (a) a signal peptide that directs secretion
into the periplasm of parts (b) and (c), derived from a first gene;
(b) an initial potential binding domain (BPTI in this case),
derived from a second gene (in this case, the second gene is an
animal gene); and (c) a structural packaging signal (the mature
gene VIII coat protein), derived from a third gene.
[0977] The process by which the IPBD::packaging-signal fusion
arrives on the phage surface is illustrated in FIG. 1. In FIG. 1a,
we see that authentic gene VIII protein appears (by whatever
process) in the lipid bilayer so that both the amino and carboxy
termini are in the cytoplasm. Signal peptidase-I cleaves the gene
VIII protein liberating the signal peptide (that is absorbed by the
cell) and mature gene VIII coat protein that spans the lipid
bilayer. Many copies of mature gene VIII coat protein accumulate in
the lipid bilayer awaiting phage assembly (FIG. 1c). Some signal
sequences are able to direct the translocation of quite large
proteins across the lipid bilayer. If additional codons are
inserted after the codons that encode the cleavage site of the
signal peptidase-I of such a potent signal sequence, the encoded
amino acids will be translocated across the lipid bilayer as shown
in FIG. 1b. After cleavage by signal peptidase-I, the amino acids
encoded by the added codons will be in the periplasm but anchored
to the lipid bilayer by the mature gene VIII coat protein, Figure
Id. The circular single-stranded phage DNA is extruded through a
part of the lipid bilayer containing a high concentration of mature
gene VIII coat protein; the carboxy terminus of each coat protein
molecule packs near the DNA while the amino terminus packs on the
outside. Because the fusion protein is identical to mature gene
VIII coat protein within the trans-bilayer domain, the fusion
protein will co-assemble with authentic mature gene VIII coat
protein as shown in FIG. 1e.
[0978] In each case, the mature VIII coat protein moiety is
intended to co-assemble with authentic mature VIII coat protein to
produce phage particle having BPTI domains displayed on the
surface. The source and character of the secretion signal sequence
is not important because the signal sequence is cut away and
degraded. The structural packaging signal, however, is quite
important because it must co-assemble with the authentic coat
protein to make a working virus sheath.
[0979] a) Bacterial Alkaline Phosphatase (phoA) Signal Peptide.
[0980] Construct pGEM-MB26 contains a fragment of DNA bounded by
restriction enzyme sites SacI and AccIII which contains the new RBS
and sequences encoding the initiating methionine and the signal
peptide of M13 gene VIII pro-protein. This fragment was replaced
with a synthetic duplex (constructed from four annealed
oligonucleotides) containing the RBS and DNA coding for the
initiating methionine and signal peptide of PhoA (INOU82). The
resulting construct was designated pGEM-MB42; the sequence of the
fusion gene is shown in Table 113. M13MB48 is a derivative of
GemMB42. A BamHI-SalI DNA fragment from GenMB42, containing the
gene construct, was ligated into a similarly cleaved vector
M13MB1/2 giving rise to M13MB48.
[0981] PhoA RBS and signal peptide sequence
17 PhoA RBS and signal peptide sequence
5'-GAGCTCCATGGGAGAAAATAAA.ATG.AAA.CAA.AGC.ACG.-
.vertline.SacI.vertline. met lys gln ser thr
.ATC.GCA.CTC.TTA.CCG.TTA.CTG.TTT.ACC.CCT.GTG.- ile ala leu leu pro
leu leu phe thr pro val ACA.AAA.GCC.CGT.CCG.GAT.-3' thr lys ala arg
pro asp . . . .vertline.AccIII.vertline.
[0982] b) Beta-lactamase Signal Peptide.
[0983] To enable the introduction of the beta-lactamase (amp)
promoter and DNA coding for the signal peptide into the gene
encoding (mature-BPTI)::(mature-VIII-coat-protein) an initial
manipulation of the amp gene (encoding beta-lactamase) was
required. Starting with pGEM-3Zf an AccIII recognition site
(TCCGGA) was introduced into the amp gene adjacent to the DNA
sequence encoding the amino acids at the beta-lactamase signal
peptide cleavage site. Using standard methods of in vitro
site-directed oligo-nucleotide mutagenesis bases C.sub.2504 and
A.sub.2501 were converted to T and G respectively to generate the
construct designated pGEM-MB40. Further manipulation of pGEM-MB40
entailed the insertion of a synthetic oligonucleotide linker
(CGGATCCG) containing the BamHI recognition sequence (GGATCC) into
the AatII site (GACGTC starting at nucleotide number 2260) to
generate the construct designated pGEM-MB45. The DNA bounded by the
restriction enzyme sites of BamHI and AccIII contains the amp
promoter, amp RBS, initiating methionine and beta-lactamase signal
peptide. This fragment was used to replace the corresponding
fragment from pGEM-MB26 to generate construct pGEM-MB46.
[0984] amp gene promoter and signal peptide sequences
18 amp gene promoter and signal peptide sequences
5'-GGATCCGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTT-
GTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAAT-
AACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT-
ATG.AGT.ATT.CAA.CAT.TTC.CGT.GTC.GCC.CTT.ATT.- met ser ile gln his
phe arg val ala leu ile CCC.TTT.TTT.GCG.GCA.TTT.TG-
C.CTT.CCT.GTT.TTT.- pro phe phe ala ala phe cys leu pro val phe
GCT.CAT.CCG.-3' ala his pro . . .
[0985] c) M13-gene-III-signal::bpti::mature-VIII-coat-protein
[0986] We may also construct, as depicted in FIG. 5, M13-MB51 which
would carry a gene encoding a fusion of M13-gene-III-signal-peptide
to the previously described BPTI::mature VIII coat protein. First
the BstEII site that follows the stop codons of the synthetic gene
VIII is changed to an AlwNI site as follows. DNA of pGEM-MB26 is
cut with BstEII and the ends filled in by use of Klenow enzyme; a
blunt AlwNI linker is ligated to this DNA. This construction is
called pGEM-MB26Alw. The XhoI to AlwNI fragment (approximately 300
bp) of pGEM-MB26Alw is purified. RF DNA from phage MK-BPTI (vide
infra) is cut with AlwNI and XhoI and the large fragment purified.
These two fragments are ligated together; the resulting
construction is named M13-MB51. Because M13-MB51 contains no gene
III, the phage can not form plaques. M13-MB51 can, however, render
cells Km.sup.R. Infectious phage particles can be obtained by use
of helper phage. As explained below, the gene III signal sequence
is capable of directing (BPTI)::(mature-gene-III-protein) to the
surface of phage. In M13-MB51, we have inserted DNA encoding gene
VIII coat protein (50 amino acids) and three stop codons 5' to the
DNA encoding the mature gene III protein. Summary of signal peptide
fusion protein variants.
19 Signal Fusion Promoter RBS sequence protein pGEM-MB26 tac new
VIII BPTI/VIII-coat pGEM-MB42 tac new phoA BPTI/VIII-coat pGEM-MB46
amp amp amp BPTI/VIII-coat pGEM-MB51 III III III BPTI/VIII-coat M13
MB48 tac new phoA BPTI/VIII-coat
[0987] 2. Analysis of the Protein Products Encoded by the Synthetic
(signal-peptide::mature-bpti::viii-coat-protein) Genes
[0988] i) In vitro Analysis
[0989] A coupled transcription/translation prokaryotic system
(Amersham Corp., Arlington Heights, Ill.) was utilized for the in
vitro analysis of the protein products encoded by the BPTI/VIII
synthetic gene and the variants derived from this.
[0990] Table 107 lists the protein products encoded by the listed
vectors which are visualized by the standard method of fluorography
following in vitro synthesis in the presence of .sup.35S-methionine
and separation of the products using SDS polyacrylamide gel
electrophoresis. In each sample a pre-beta-lactamase product
(approximately 31 kd) can be seen. This is derived from the amp
gene which is the common selection gene for each of the vectors. In
addition, a (pre-BPTI/VIII) product encoded by the synthetic gene
and variants can be seen as indicated. The migration of these
species (approximately 14.5 kd) is consistent with the expected
size of the encoded proteins.
[0991] ii) In vivo Analysis.
[0992] The vectors detailed in sections (B) and (C) were freshly
transfected into the E. coli strain XL1-blue.TM. (Stratagene, La
Jolla, Calif.) and in strain SEF'. E. coli strain SE6004 (LISS85)
carries the prlA4 mutation and is more permissive in secretion than
strains that carry the wild-type prlA allele. SE6004 is F.sup.- and
is deleted for lacI; thus the cells can not be infected by M13 and
lacUV5 and tac promoters can not be regulated with IPTG. Strain
SEF' is derived from strain SE6004 (LISS85) by crossing with
XL1-Blue.TM.; the F' in XL1-Blue.TM. carries Tc.sup.R and
lacI.sup.q. SE6004 is streptomycin.sup.R, Tc.sup.S while
XL1-Blue.TM. is streptomycin.sup.S, Tc.sup.R so that both parental
strains can be killed with the combination of Tc and streptomycin.
SEF' retains the secretion-permissive phenotype of the parental
strain, SE6004(prlA4).
[0993] The fresh transfectants were grown in NZYCM medium (SAMB89)
for 1 hour after which IPTG was added over the range of
concentrations 1.0 .mu.M to 0.5 mM (to derepress the lacUV5 and tac
promoters) and grown for an additional 1.5 hours.
[0994] Aliquots of the bacterial cells expressing the synthetic
insert encoded proteins together with the appropriate controls (no
vector, vector with no insert and zero IPTG) were lysed in SDS gel
loading buffer and electrophoresed in 20% polyacrylamide gels
containing SDS and urea. Duplicate gels were either silver stained
(Daiichi, Tokyo, Japan) or electrotransferred to a nylon matrix
(Immobilon from Millipore, Bedford, Mass.) for western analysis by
standard means using rabbit anti-BPTI pqlyclonal antibodies.
[0995] Table 108 lists the interesting proteins visualized on a
silver stained gel and by western analysis of an identical gel. We
can see clearly in the western analysis that protein species
containing BPTI epitopes are present in the test strains which are
absent from the control strains and which are also IPTG inducible.
In XL1-Blue.TM., the migration of this species is predominantly
that of the unprocessed form of the pro-protein although a small
proportion of the encoded proteins appear to migrate at a size
consistent with that of a fully processed form. In SEF', the
processed form predominates, there being only a faint band
corresponding to the unprocessed species.
[0996] Thus in strain SEF', we have produced a tripartite fusion
protein that is specifically cleaved after the secretion signal
sequence. We believe that the mature protein comprises BPTI
followed by the gene VIII coat protein and that the coat protein
moiety spans the membrane. We believe that it is highly likely that
one or more copies, perhaps hundreds of copies, of this protein
will co-assemble into M13 derived phage or M13-like phagemids. This
construction will allow us to a) mutagenize the BPTI domain, b)
display each of the variants on the coat of one or more phage (one
type per phage), and c) recover those phage that display variants
having novel binding properties with respect to target materials of
our choice.
[0997] Rasched and Oberer (RASC86) report that phage produced in
cells that express two alleles of gene VIII, that have differences
within the first 11 residues of the mature coat protein, contain
some of each protein. Thus, because we have achieved in vivo
processing of, the phoA(signal)::bpti::matureVIII fusion gene, it
is highly likely that co-expression of this gene with wild-type
VIII will lead to production of phage bearing BPTI domains on their
surface. Mutagenesis of the bpti domain of these genes will provide
a population of phage, each phage carrying a gene that codes for
the variant of BPTI displayed on the phage surface.
[0998] VIII Display Phage: Production, Preparation and
Analysis.
[0999] i. Phage Production.
[1000] The OCV can be grown in XL1-Blue.TM. in the absence of the
inducing agent, IPTG. Typically, a plaque plug is taken from a
plate and grown in 2 ml of medium, containing freshly diluted
bacterial cells, for 6 to 8 hours. Following centrifugation of this
culture the supernatant is taken and the phage titer determined.
This is kept as a phage stock for further infection, phage
production and display of the gene product of interest.
[1001] A 100 fold dilution of a fresh overnight culture of SEF'
bacterial cells in 500 ml of NZCYM medium is allowed to grow to a
cell density of 0.4 (Ab 600 nm) in a shaker incubator at 37.degree.
C. To this culture is added a sufficient amount of the phage stock
to give a MOI of 10 together with IPTG to give a final
concentration of 0.5 mM. The culture is allowed to grow for a
further 2 hrs.
[1002] ii. Phage Preparation and Purification.
[1003] The phage producing bacterial culture is centrifuged to
separate the phage in the supernatant from the bacterial pellet. To
the supernatant is added one quarter by volume of phage
precipitation solution (20% PEG, 3.75 M ammonium acetate) and PMSF
to a final concentration of lM. It is left on ice for 2 hours after
which the precipitated phage is retrieved by centrifugation. The
phage pellet is redissolved in TrisEDTA containing .0.1% Sarkosyl
and left at 4.degree. C. for 1 hour after which any bacteria and
bacterial debris is removed by centrifugation. The phage in the
supernatant is reprecipitated with PEG overnight at 4.degree. C.
The phage pellet is resuspended in LB medium and repreciptated
another two times to remove the detergent. The phage is stored in
LB medium at 4.degree. C., titered and used for analysis and
binding studies.
[1004] A more stringent phage purification scheme involves
centrifugation in a CsCl gradient. 3.86 g of CsCl is dissolved in
NET buffer (0.1 M NaCl, 1 mM EDTA, 0.1M Tris pH 7.7) upto a volure
of 10 ml. 10.sup.12 to 10.sup.13 phage in TE Sarkosyl buffer
.sup.are mixed with 5 ml of CsCl NET buffer and transferred to a
sealable ultracentrifuge tube. Centrifugation is performed
overnight at 34K rpm in a Sorvall OTD-65B Ultracentrifuge. The
tubes are opened and 400 .mu.l aliqouts are carefully removed. 5
.mu.l aliqouts are removed from the fractions and analysed by
agarose gel electrophoresis after heating at 65.degree. C. for 15
minutes together with the gel loading buffer containing 0.1% SDS.
Fractions containing phage are pooled, the phage reprecipitated and
finally redissolved in LB medium to a concentration of 10.sup.12 to
10.sup.13 phage per ml.
[1005] iii. Phage Analysis.
[1006] The display phage, together with appropriate controls are
analyzed using standard methods of polyacrylamide gel
electrophoresis and either silver staining of the gel or
electrotransfer to a nylon matrix followed by analysis with
anti-BPTI antiserum (Western analysis). Quantitation of the display
of heterologous proteins is achieved by running a serial dilution
of the starting protein, for example BPTI, together with the
display phage samples in the electrophoresis and Western analyses
described above. An alternative method involves running a 2 fold
serial dilution of a phage in which both the major coat protein and
the fusion protein are visualized by silver staining. A comparison
of the relative ratios of the two protein species allows one to
estimate the number of fusion proteins per phage since the number
of VIII gene encoded proteins per phage (approximately 3000) is
known.
[1007] Incorporation of Fusion Protein into Bacteriophage.
[1008] In vivo expression of the processed BPTI:VIII fusion
protein, encoded by vectors GemMB42 (above and Table 113) and
M13MB48 (above), implied that the processed fusion product was
likely to be correctly located within the bacterial cell membrane.
This localization made it possible that it could be incorporated
into the phage and that the BPTI moiety would be displayed at the
bacteriophage surface.
[1009] SEF' cells were infected with either M13MB48 (consisting of
the starting phage vector M13mp18, altered as described above,
containing the synthetic gene consisting of a tac promoter,
functional ribosome binding site, phoA signal peptide, mature BPTI
and mature major coat protein) or M13mp18, as a control. Phage
infections, preparation and purification was performed as described
in Example VIII.
[1010] The resulting phage were electrophoresed (approximately
10.sup.11 phage per lane) in a 20% polyacrylamide gel containing
urea followed by electrotransfer to a nylon matrix and western
analysis using anti-BPTI rabbit serum. A single species of protein
was observed in phage derived from infection with the M13MB48 stock
phage which was not observed in the control infection. This protein
had a migration of about 12 kd, consistent with that of the fully
processed fusion protein.
[1011] Western analysis of SEF' bacterial lysate with or without
phage infection demonstrated another species of protein of about 20
kd. This species was also present, to a lesser degree, in phage
preparations which were simply PEG precipitated without further
purification (for example, using nonionic detergent or by CsCl
gradient centrifugation). A comparison of M13MB48 phage progoff
eparations made in the presence or absence of detergent
aldemonstrated that sarkosyl treatment and CsCl gradient
purification did remove the bacterial contaminant while having no
effect on the presence of the BPTI:VIII fusion protein. This
indicates that the fusion protein has been incorporated and is a
constituent of the phage body.
[1012] The time course of phage production and BPTI:VIII
incorporation was followed post-infection and after IPTG induction.
Phage production and fusion protein incorporation appeared to be
maximal after two hours. This time course was utilized in further
phage productions and analyses.
[1013] Polyacrylamide electrophoresis of the phage preparations,
followed by silver staining, demonstrated that the preparations
were essentially free of contaminating protein species and that an
extra protein band was present in M13MB48 derived phage which was
not present in the control phage. The size of the new protein was
consistent with that seen by western analysis. A similar analysis
of a serially diluted BPTI:VIII incorporated phage demonstrated
that the ratio of fusion protein to major coat protein was
typically in the range of 1:150. Since the phage is known to
contain in the order of 3000 copies of the gene VIII product, this
means that the phage population contains, on average, 10's of
copies of the fusion protein per phage.
[1014] Altering the Initiating Methionine of the Natural Gene
VIII.
[1015] The OCV M13MB48 contains the synthetic gene encoding the
BPTI:VIII fusion protein in the intergenic region of the modified
M13mp18 phage vector. The remainder of the vector consists of the
M13 genome which contains the genes necessary for various
bacteriophage functions, such as DNA replication and phage
formation etc. In an attempt to increase the phage incorporation of
the fusion protein, we decided to try to diminish the production of
the natural gene VIII product, the major coat protein, by altering
the codon for the initiating methionine of this gene to one
encoding leucine. In such cases, methionine is actually
incorporated, but the rate of initiation is reduced. The change was
achieved by standard methods of site-specific oligonucleotide
mutagenesis as follows.
20 M K K S -rest of VIII ACT.TCC.TC.ATG.AAA.AAG.TCT. rest of XI - T
S S stop
[1016] Site-specific Mutagenesis.
21 (L) K K S -rest of VIII ACT.TCC.AG.CTG.AAA.AAG.TCT. rest of XI -
T S S stop
[1017] Note that the 3' end of the XI gene overlaps with the 5' end
of the VIII gene. Changes in DNA sequence were designed such that
the desired change in the VIII gene product could be achieved
without alterations to the predicted amino acid sequence of the
gene XI product. A diagnostic pvuII recognition site was introduced
at this site.
[1018] It was anticipated that initiation of the natural gene VIII
product would be hindered, enabling a higher proportion of the
fusion protein to be incorporated into the resulting phage.
[1019] Analyses of the phage derived from this modified vector
indicated that there was a significant increase in the ratio of
fusion protein to major coat protein. Quantitative estimates
indicated that within a phage population as much as 100 copies of
the BPTI:VIII fusion were incorporated per phage.
[1020] Incorporation of Interdomain Extension Fusion Proteins into
Phage.
[1021] A phage pool containing a variegated pentapeptide extension
at the BPTI:coat protein interface (see Example VII) was used to
infect SEF' cells. IPTG induction, phage production and preparation
were as described in Example VIII. Using the criteria detailed in
the previous section, it was determined that extended fusion
proteins were incorporated into phage. Gel electrophoresis of the
generated phage, followed by either silver staining or western
analysis with anti-BPTI rabbit serum, demonstrated fusion proteins
that migrated similarly to but discernably slower that of the
starting fusion protein.
[1022] With regard to the `EGGGS linker` extensions of the domain
interface, individual phage stocks predicted to contain one or more
5-amino-acid unit extensions were analyzed in a similar fashion.
The migration of the extended fusion proteins were readily
distinguishable from the parent fusion protein when viewed by
western analysis or silver staining. Those clones analyzed in more
detail included M13.3X4 (which contains a single inverted EGGGS
linker with a predicted amino acid sequence of GGGSL), M13.3X7
(which contains a correctly orientated linker with a predicted
amino acid sequence of EGGGS), M13.3X11 (which contains 3 linkers
with an inversion and a predicted amino acid sequence for the
extension of EGGGSGSSSLGSSSL) and M13.3Xd which contains an
extension consisting of at least 5 linkers or 25 amino acids.
[1023] The extended fusion proteins were all incorporated into
phage at high levels (on average lOs of copies per phage were
present and when analyzed by gel electrophoresis migrated rates
consistent with the predicted size of the extension. Clones M13.3X4
and M13.3X7 migrated at a position very similar to but discernably
different from the parent fusion protein, while M13.3X11 and
M13.3Xd were markedly larger.
[1024] Display of BPTI:VIII Fusion Protein by Bacteriophage.
[1025] The BPTI:VIII fusion protein had been shown to be
incorporated into the body of the phage. This phage was analyzed
further to demonstrate that the BPTI moiety was accessible to
specific antibodies and hence displayed at the phage surface.
[1026] The assay is detailed in section EE, but principally
involves the addition of purified anti-BPTI IgG (from the serum of
BPTI injected rabbits) to a known titer of phage. Following
incubation, protein A-agarose beads are added to bind the IgG and
left to incubate overnight. The IgG-protein A beads and any bound
phage are removed by centrifugation followed by a retitering of the
supernatant to determine any loss of phage. The phage bound to the
beads can be acid eluted and titered also. Appropriate controls are
included in the assay, such as a wild type phage stock (M13mp18)
and IgG purified from normal rabbit pre-immune serum.
[1027] Table 140 shows that while the titer of the wild type phage
is unaltered by the presence of anti-BPTI IgG, BPTI-IIIMK (the
positive control for the assay), demonstrated a significant drop in
titer with or without the extra addition of protein A beads. (Note
that since the BPTI moiety is part of the III gene product which is
involved in the binding of phage to bacterial pili, such a
phenomenon is entirely expected.) Two batches of M13MB48 phage
(containing the BPTI:VIII fusion protein) demonstrated a
significant reduction in titer, as judged by plaque forming units,
when anti-BPTI antibodies and protein A beads were added to the
phage. The initial drop in titer with the antibody alone, differs
somewhat between the two batches of phage. This may be a result of
experimental or batch variation. Retrieval of the
immunoprecipitated phage, while not quantitative, was significant
when compared to the wild type phage control.
[1028] Further control experiments relating to this section are
shown in Table 141 lad Table 142. The data demonstrated that the
loss in titer observed for the BPTI:VIII containing phage is a
result of the display of BPTI epitopes by these phage and the
specific interaction with anti-BPTI antibodies. No significant
interaction with either protein A agarose beads or IgG purified
from normal rabbit serum could be demonstrated. The larger drop in
titer for M13MB48 batch five reflects the higher level
incorporation of the fusion protein in this preparation.
[1029] Functionality of the BPTI Moiety in the BPTI-VIII Display
Phage.
[1030] The previous two sections demonstrated that the BPTI:VIII
fusion protein has been incorporated into the phage body and that
the BPTI moiety is displayed at the phage surface. To demonstrate
that the displayed molecule is functional, binding experiments were
performed in a manner almost identical to that described in the
previous section except that proteases were used in place of
antibodies. The display phage, together with appropriate controls,
are allowed to interact with immobilized proteases or immobilized
inactivated proteases. Binding can be assessed by monitoring the
loss in titer of the display phage or by determining the number of
phage bound to the respective beads.
[1031] Table 143 shows the results of an experiment in which
BPTI.VIII display phage, M13MB48, were allowed to bind to
anhydrotrypsin-agarose beads. There was a significant drop in titer
when compared to wild type phage, which do not display BPTI. A pool
of phage (5AA Pool), each contain a variegated 5 amino acid
extension at the BPTI:major coat protein interface, demonstrated a
similar decline in titer. In a control experiment (table 143) very
little non-specific binding of the above display phage was observed
with agarose beads to which an unrelated protein (streptavidin) is
attached.
[1032] Actual binding of the display phage is demonstrated by the
data shown for two experiments in Table 144. The negative control
is wild type M13mp18 and the positive control is BPTI-IIIMK, a
phage in which the BPTI moiety, attached to the gene III protein,
has been shown to be displayed and functional. M13MB48 and M13MB56
both bind to anhydrotrypsin beads in a manner comparable to that of
the positive control, being 40 to 60 times better than the negative
control (non-display phage). Hence functionality of the BPTI
moiety, in the major coat fusion protein, was established.
[1033] To take this analysis one step further, a comparison of
phage binding to active and inactivated trypsin is shown in Table
145. The control phage, M13mp18 and BPTI-III MK, demonstrated
binding similar to that detailed in Example III. Note that the
relative binding is enhanced with trypsin due to the apparent
marked reduction in the non-specific binding of the wild type phage
to the active protease. M13.3X7 and M13.3X11, which both contain
`EGGGS` linker extensions at the domain interface, bound to
anhydrotrypsin and trypsin in a manner similar to BPTI-IIIMK phage.
The binding, relative to non-display phage, was approximately 100
fold higher in the anhydrotrypsin binding assay and at least 1000
fold higher in the trypsin binding assay. The binding of another
`EGGGS` linker variant (M13.3Xd) was similar to that of
M13.3X7.
[1034] To demonstrate the specificity of binding the assays were
repeated with human neutrophil elastase (HNE) beads and compared to
that seen with trypsin beads Table 146. BPTI has a very high
affinity for trypsin and a low affinity for HNE, hence the BPTI
display phage should reflect these affinities when used in binding
assays with these beads. The negative and positive controls for
trypsin binding were as already described above while an additional
positive control for the HNE beads, BPTI(K15L,MGNG)-III MA (see
Example III) was included. The results, shown in Table 146,
confirmed this prediction. M13MB48, M13.3X7 and M13.3X11 phage
demonstrated good binding to trypsin, relative to wild type phage
and the HNE control (BPTI(K15L,MGNG)-III MA), being comparable to
BPTI-IIIMK phage. conversely poor binding occurred when HNE beads
were used, with the exception of the HNE positive control
phage.
[1035] Taken together the accumulated data demonstrated that when
BPTI is part of a fusion protein with the major coat protein of M13
phage, the molecule is both displayed at the surface of the phage
and a significant proportion of it is functional in a specific
protease binding manner.
EXAMPLE II
Construction of BPTI/Gene-III Display Vector
[1036] DNA manipulations were conducted according to standard
procedures as described in Maniatis et al. (MANI82). First the
unwanted lacZ gene of M13-MB1/2 was removed. M13-MB1/2 RF was cut
with BamHI and SalI and the large fragment was isolated by agarose
gel electrophoresis. The recovered 6819 bp fragment was filled in
with Klenow fragment of E. coli DNA polymerase and ligated to a
synthetic HindIII 8mer linker (CAAGCTTG). The ligation sample was
used to transfect competent XL1-Blue.sup.(TM) (Stratagene, La
Jolla, Calif.) cells which were subsequently plated for plaque
formation. RF DNA was prepared from chosen plaques and a clone,
M13-MB1/2-delta, containing regenerated BamHI and SalI sites as
well as a new HindIII site, all 500 bp upstream of the BglII site
(6935) was picked.
[1037] A unique NarI site was introduced into codons 17 and 18 of
gene III (changing the amino acids from H-S to G-A, Cf. Table 110).
10.sup.6 phage produced from bacterial cells harboring the
M13-MB1/2-delta RF DNA were used to infect a culture of CJ236 cells
(relevant genotype: F', dut1, ung1, Cm.sup.R) (OD595=0.35).
Following overnight incubation at 37.degree. C., phage were
recovered and uracil-containing ss DNA was extracted from phage in
accord with the instructions for the MUTA-GENE.RTM. M13 in vitro
Mutagenesis Kit (Catalogue Number 170-3571, Bio-Rad, Richmond,
Calif.). Two hundred nanograms of the purified single stranded DNA
was annealed to 3 picomoles of a phosphorylated 25mer mutagenic
oligonucleotide,
[1038] 5'-gtttcagcggCgCCagaatagaaag-3',
[1039] where upper case indicates the changes). Following filling
in with T4 DNA polymerase and ligation with T4 DNA ligase, the
reaction sample was used to transfect competent XL1-Blue.sup.(TM)
cells which were subsequently plated to permit the formation of
plaques.
[1040] RF DNA, isolated from phage-infected cells which had been
allowed to propagate in liquid culture for 8 hours, was denatured,
spotted on a Nytran membrane, baked and hybridized to the 25mer
mutagenic oligonucleotide which had previously been phosphorylated
with .sup.32P-ATP. Clones exhibiting strong hybridization signals
at 70.degree. C. (6.degree. C. less than the theoretical Tm of the
mutagenic oligonucleotide) were chosen for large scale RF
preparation. The presence of a unique NarI site at nucleotide 1630
was confirmed by restriction enzyme analysis. The resultant RF DNA,
M13-MB1/2-delta-NarI was cut with BamHI, dephosphorylated with calf
intestinal phosphatase, and ligated to a 1.3 Kb BamHI fragment,
encoding the kanamycin-resistance gene (kan), derived from plasmid
pUC4K (Pharmacia, Piscataway, N.J.). The ligation sample was used
to transfect compatent XL1-Blue.sup.(TM) cells which were
subsequently plated onto LB plates containing kanamycin (Km). RF
DNA prepared from KmR colonies was prepared and subjected to
restriction enzyme analysis to confirm the insertion of kan into
M13-MB1/2-delta-NarI DNA thereby creating the phage MK. Phage MK
grows as well as wild-type M13, indicating that the changes at the
cleavage site of gene III protein are not detectably deleterious to
the phage.
[1041] Insertion of Synthetic BPTI Gene
[1042] The construction of the BPTI-III expression vector is shown
in FIG. 6. The synthetic bpti-VIII fusion contains a NarI site that
comprises the last two codons of the BPTI-encoding region. A second
NarI site was introduced upstream of the BPTI-encoding region as
follows. RF DNA of phage M13-MB26 was cut with AccIII and ligated
to the dsDNA adaptor:
22 5'-TATTCTGGCGCCCGT -3' 3'-ATAAGACCGCGGGCAGGCC-5'
.vertline.NarI.vertline. .vertline.AccIII
[1043] The ligation sample was subsequently restricted with NarI
and a 180 bp DNA fragment encoding BPTI was isolated by agarose gel
electrophoresis. RF DNA of phage MK was digested with NarI,
dephosphorylated with calf intestinal phosphatase and ligated to
the 180 bp fragment. Ligation samples were used to transfect
competent XL1-Blue.sup.(TM) cells which were plated to enable the
formation of plaques. DNA, isolated from phage derived from
plaques, was denatured, applied to a Nytran membrane, baked and
hybridized to a .sup.32P-phosphorylated double stranded DNA probe
corresponding to the BPTI gene. Large scale RF preparations were
made for clones exhibiting a strong hybridization signal.
Restriction enzyme digestion analysis confirmed the insertion of a
single copy of the synthetic BPTI gene iron gene III of MK to
generate phage MK-BPTI. Subsequent DNA sequencing confirmed that
the sequence of the bpti-III fusion gene is correct and that the
correct reading frame is maintained (Table 111). Table 116 shows
the entire coding region, the translation into protein sequence,
and the functional parts of the polypeptide chain.
[1044] Expression of the BPTI-III Fusion Gene in vitro
[1045] MK-BPTI RF DNA was added to a coupled prokaryotic
transcription-translation extract (Amersham). Newly synthesized
radiolabelled proteins were produced and subsequently separated by
electrophoresis on a 15% SDS-polyacrylamide gel subjected to
fluorography. The MK-BPTI DNA directs the synthesis of an
unprocessed gene III fusion protein which is 7 Xd larger than the
gene III product encoded by MK. This is consistent with the
insertion of 58 amino acids of BPTI into the gene III protein.
Immunoprecipitation of radiolabelled proteins generated by the
cell-free prokaryotic extract was conducted. Neither rabbit
anti(M13-gene-VIII-protein) IgG nor normal rabbit IgG were able to
immunoprecipitate the gene III protein encoded by either MK or
MK-BPTI. However, rabbit anti-BPTI IgG is able to immunoprecipitate
the gene III protein encoded by MK-BPTI but not by MK. This
confirms that the increase in size of the III protein encoded by
MK-BPTI is attributable to the insertion of the BPTI protein.
[1046] Western Analysis
[1047] Phage were recovered from bacterial cultures by PEG
precipitation. To remove residual bacterial cells, recovered phage
were resuspended in a high salt buffer and subjected to
centrifugation, in accord with the instructions for the
MUTA-GENE.RTM. M13 in vitro Mutagenesis Kit (Catalogue Number
170-3571, Bio-Rad, Richmond, Calif.). Aliquots of phage (containing
up to 40 .mu.l of protein) were subjected to electrophoresis on a
12.5% SDS-urea-polyacrylamide gel and proteins were transferred to
a sheet of Immobilon by electro-transfer. Western blots were
developed using rabbit anti-BPTI serum, which had previously been
incubated with an E. coli extract, followed by goat ant-rabbit
antibody conjugated to alkaline phosphatase. An immunoreactive
protein of 67 Kd is detected in preparations of the MK-BPTI but not
the MK phage. The size of the immunoreactive protein is consistent
with the predicted size of a processed BPTI-III fusion protein (6.4
Kd plus 60 Kd). These data indicate that BPTI-specific epitopes are
presented on the surface of the MK-BPTI phage but not the MK
phage.
[1048] Neutralization of Phage Titer with Agarose-immobilized
Anhydro-trypsin
[1049] Anhydro-trypsin is a derivative of trypsin in which the
active site serine has been converted to dehydroalanine.
Anhydro-trypsin retains the specific binding of trypsin but not the
protease activity. Unlike polyclonal-antibodies, anhydro-trypsin is
not expected to bind unfolded BPTI or incomplete fragments.
[1050] Phage MK-BPTI and MK were diluted to a concentration
1.4.multidot.10.sup.12 particles per ml. in TBS buffer (PARM88)
containing 1.0 mg/ml BSA. Thirty microliters of diluted phage were
added to 2, 5, or 10 microliters of a 50% slurry of
agarose-immobilized anhydro-trypsin (Pierce Chemical Co., Rockford,
Ill.) in TBS/BSA buffer. Following incubation at 25.degree. C.,
aliquots were removed, diluted in ice cold LB broth and titered for
plaque-forming units on a lawn of XL1-Blue.sup.(TM) cells. Table
114 illustrates that incubation of the MK-BPTI phage with
immobilized anhydro-trypsin results in a very significant loss in
titer over a four hour period while no such effect is observed with
the MK (control) phage The reduction in phage titer is also
proportional to the amount of immobilized anhydro-trypsin added to
the MK-BPTI phage. Incubation with five microliters of a 50% slurry
of agarose-immobilized streptavidin (Sigma, St. Louis, Mo.) in
TBS/BSA buffer does not reduce the titer of either the MK-BPTI or
MK phage. These data are consistent with the presentation of a
correctly-folded, functional BPTI protein on the surface of the
MK-BPTI phage but not on the MK phage. Unfolded or incomplete BPTI
domains are not expected to bind anhydro-trypsin. Furthermore,
unfolded BPTI domains are expected to be non-specifically
sticky.
[1051] Neutralization of Phage Titer with Anti-BPTI Antibody
[1052] MK-BPTI and MK phage were diluted to a concentration of
4.multidot.10.sup.8 plaque-forming units per ml in LB broth.
Fifteen microliters of diluted phage were added to an equivalent
volume of either rabbit anti-BPTI serum or normal rabbit serum
(both diluted 10 fold in LB broth). Following incubation at
37.degree. C., aliquots were removed, diluted by 104 in ice-cold LB
broth and titered for plaque-forming units on a lawn of
XL1-Blue.sup.(TM) cells. Incubation of the MK-BPTI phage with
anti-BPTI serum results in a steady loss in titer over a two hour
period while no such effect is observed with the MK phage. As
expected, normal rabbit serum does not reduce the titer of either
the MK-BPTI or the MK phage. Prior incubation of the anti-BPTI
serum with authentic BPTI protein but not with an equivalent amount
of E. coli protein, blocks the ability of the serum to reduce the
titer of the MK-BPTI phage. This data is consistent with the
presentation of BPTI-specific epitopes on the surface of the
MK-BPTI phage but not the MK phage. More specifically, the data
indicates that these BPTI epitopes are associated with the gene III
protein and that association of this fusion protein with an
anti-BPTI antibody blocks its ability to mediate the infection of
bacterial cells.
[1053] Neutralization of Phage Titer with Trypsin
[1054] MK-BPTI and MK phage were diluted to a concentration of
4.multidot.10.sup.8 plaque-forming units per ml in LB broth.
Diluted phage were added to an equivalent volume of trypsin diluted
to various concentrations in LB broth. Following incubation at
37.degree. C., aliquots were removed, diluted by 10.sup.4 in ice
cold LB broth and titered for plaque-forming units on a lawn of
XL1-Blue.sup.(TM) cells. Incubation of the MK-BPTI phage with 0.15
.mu.g of trypsin results in a 70% loss in titer after a two hour
period while only a 15% loss in titer is observed for the MK phage.
A reduction in the amount of trypsin added to phage results in a
reduction in the loss of titer. However, at all trypsin
concentrations investigated, the MK-BPTI phage are more sensitive
to incubation with trypsin than the MK phage. An interpretation of
this data is that association of the BPTI-III fusion protein
displayed on the surface of the MK-BPTI phage with trypsin blocks
its ability to mediate the infection of bacterial cells.
[1055] The reduction in titer of phage MK by trypsin is an example
of a phenomenon that is likely to be general: proteases, if present
in sufficient quantity, will degrade proteins on the phage and
reduce infectivity. The present application lists several means
that can be used to overcome this problem.
[1056] Affinity Selection System
[1057] Affinity Selection with Immobilized Anhydro-trypsin
[1058] MK-BPTI and MK phage were diluted to a concentration of
1.4.multidot.10.sup.12 particles per ml in TBS buffer (PARM88
containing 1.0 mg/ml BSA. We added 4.0.multidot.10.sup.10 phage to
5 microliters of a 50% slurry of either agarose-immobilized
anhydro-trypsin beads (Pierce Chemical Co.) or agarose-immobilized
streptavidin beads (Sigma) in TBS/BSA. Following a 3 hour
incubation at room temperature, the beads were pelleted by
centrifugation for 30 seconds at 5000 rpm in a microfuge and the
supernatant fraction was collected. The beads were washed 5 times
with TBS/Tween buffer (PARM88) and after each wash the beads were
pelleted by centrifugation and the supernatant was removed.
Finally, beads were resuspended in elution buffer (0.1 N HCl
containing 1.0 mg/ml BSA adjusted to pH 2.2 with glycine) and
following a 5 minute incubation at room temperature, the beads were
pelleted by centrifugation. The supernatant was removed and
neutralized by the addition of 1.0 M Tris-HCl buffer, pH 8.0.
[1059] Aliquots of phage samples were applied to a Nytran membrane
using a Schleicher and Schuell (Keene, N.H.) filtration minifold
and phage DNA was immobilized onto the Nytran by baking at
80.degree. C. for 2 hours. The baked filter was incubated at
42.degree. C. for 1 hour in pre-wash solution (MANI82) and
pre-hybridization solution (5Prime-3Prime, West Chester, Pa.). The
1.0 Kb NarI (base 1630)/XmnI (base 2646) DNA fragment from MK RF
was radioactively labelled with .sup.32P-dCTP using an
oligolabelling kit (Pharmacia, Piscataway, N.J.). The radioactive
probe was added to the Nytran filter in hybridization solution
(5Prime-3Prime) and, following overnight incubation at 42.degree.
C., the filter was washed and subjected to autoradiography.
[1060] The efficiency of this affinity selection system can be
semi-quantitatively determined using the dot-blot procedure
described elsewhere in the present application. Exposure of
MK-BPTI-phage-treated anhydro-trypsin beads to elution buffer
releases bound MK-BPTI phage. Streptavidin beads do not retain
phage MK-BPTI. Anhydro-trypsin beads do not retain phage MK. In the
experiment depicted in Table 115, we estimate that 20% of the total
MK-BPTI phage were bound to 5 microliters of the immobilized
anhydrotrypsin and were subsequently recovered by washing the beads
with elution buffer (pH 2.2 HCl/glycine). Under the same
conditions, no detectable MK-BPTI phage were bound and subsequently
recovered from the streptavidin beads. The amount of MK-BPTI phage
recovered in the elution fraction is proportional to the amount of
immobilized anhydro-trypsin added to the phage. No detectable MIK
phage were bound to either the immobilized anhydro-trypsin or
streptavidin beads and no phage were recovered with elution buffer.
These data indicate that the affinity selection system described
above can be utilized to select for phage displaying a specific
folded protein (in this case, BPTI). Unfolded or incomplete BPTI
domains are not expected to bind anhydro-trypsin.
[1061] Affinity Selection with Anti-BPTI Antibodies
[1062] MK-BPTI and MK phage were diluted to a concentration of
1-1010 particles per ml in Tris buffered saline solution (PARM88)
containing 1.0 mg/ml BSA. Two.multidot.10.sup.8 phage were added to
2.5 .mu.g of either biotinylated rabbit anti-BPTI IgG in TBS/BSA or
biotinylated rabbit anti-mouse antibody IgG (Sigma) in TBS/BSA, and
incubated overnight at 4.degree. C. A 50% slurry of
streptavidin-agarose (Sigma), washed three times with TBS buffer
prior to incubation with 30 mg/ml BSA in TBS buffer for 60 minutes
at room temperature, was washed three times with TBS/Tween buffer
(PARM88) and resuspended to a final concentration of 50% in this
buffer. Samples containing phage and biotinylated IgG were diluted
with TBS/Tween prior to the addition of streptavidin-agarose in
TBS/Tween buffer. Following a 60 minute incubation at room
temperature, streptavidin-agarose beads were pelleted by
centrifugation for 30 seconds and the supernatant fraction was
collected. The beads were washed 5 times with TBS/Tween buffer and
after each wash, the beads were pelleted by centrifugation and the
supernatant was removed. Finally, the streptavidin-agarose beads
were resuspended in elution buffer (0.1 N HCl containing 1.0 mg/ml
BSA adjusted to pH 2.2 with glycine), incubated 5 minute at room
temperature, and pelleted by centrifugation. The supernatant was
removed and neutralized by the addition of 1.0 M Tris-HCl buffer,
pH 8.0.
[1063] Aliquots of phage samples were applied to a Nytran membrane
using a Schleicker and Schuell minifold apparatus. Phage DNA was
immobilized onto the Nytran by baking at 80.degree. C. for 2 hours.
Filters were washed for 60 minutes in pre-wash solution (MANI82) at
42.degree. C. then incubated at 42.degree. C. for 60 minutes in
Southern pre-hybridization solution (5Prime-3Prime). The 1.0 Kb
NarI (1630bp)/XmnI (2646 bp) DNA fragment from MK RF was
radioactively labelled with .sup.32P-adCTP using an oligolabelling
kit (Pharmacia, Piscataway, N.J.). Nytran membranes were
transferred from pre-hybridization solution to Southern
hybridization solution (5Prime-3Prime) at 42.degree. C. The
radioactive probe was added to the hybridization solution and
following overnight incubation at 42.degree. C., the filter was
washed 3 times with 2.times. SSC, 0.1% SDS at room temperature and
once at 65.degree. C. in 2.times. SSC, 0.1% SDS. Nytran membranes
were subjected to autoradiography. The efficiency of the affinity
selection system can be semi-quantitatively determined using the
above dot blot procedure. Comparison of dots A1 and B1 or C1 and D1
indicates that the majority of phage did not stick to the
streptavidin-agarose beads. Washing with TBS/Tween buffer removes
the majority of phage which are non-specifically associated with
streptavidin beads. Exposure of the streptavidin beads to elution
buffer releases bound phage only in the case of MK-BPTI phage which
have previously been incubated with biotinylated rabbit anti-BPTI
IgG. This data indicates that the affinity selection system
described above can be utilized to select for phage displaying a
specific antigen (in this case BPTI). We estimate an enrichment
factor of at least 40 fold based on the calculation 2 Enrichment
Factor = Percent MK-BPTI phage recovered Percent MK phage
recovered
EXAMPLE III
[1064] Characterization and Fractionation of Clonally Pure
Populations of Phage, Each Displaying a Single Chimeric Aprotinin
Homologue/M13 Gene III Protein:
[1065] This Example demonstrates that chimeric phage proteins
displaying a target-binding domain can be eluted from immobilized
target by decreasing pH, and the pH at which the protein is eluted
is dependent on the binding affinity of the domain for the
target.
[1066] Standard Procedures:
[1067] Unless otherwise noted, all manipulations were carried out
at room temperature. Unless otherwise noted, all cells are
XL1-Blue.sup.(TM) (Stratagene, La Jolla, Calif.).
[1068] 1) Demonstration of the Binding of BPTI-III MK Phaae to
Active Trypsin Beads
[1069] Previous experiments designed to verify that BPTI displayed
by fusion phage is functional relied on the use of immobilized
anhydro-trypsin, a catalytically inactive form of trypsin. Although
anhydro-trypsin is essentially identical to trypsin structurally
(HUBE75, YOK077) and in binding properties (VINC74, AKOH72), we
demonstrated that BPTI-III fusion phage also bind immobilized
active trypsin. Demonstration of the binding of fusion phage to
immobilized active protease and subsequent recovery of infectious
phage facilitates subsequent experiments where the preparation of
inactive forms of serine proteases by protein modification is
laborious or not feasible.
[1070] Fifty .mu.l of BPTI-III MK phage (identified as MK-BPTI in
U.S. Ser. No. 07/487,063) (3.7.multidot.10.sup.11 pfu/ml) in either
50 mM Tris, pH 7.5, 150 mM NaCl, 1.0 mg/ml BSA (TBS/BSA) buffer or
50 mM sodium citrate, pH 6.5, 150 mM NaCl, 1.0 mg/ml BSA (CBS/BSA)
buffer were added to 10 .mu.l of a 25% slurry of immobilized
trypsin (Pierce Chemical Co., Rockford, Ill.) also in TBS/BSA or
CBS/BSA. As a control, 50 .mu.l MK phage (9.3.multidot.10.sup.12
pfu/ml) were added to 10 .mu.l of a 25% slurry of immobilized
trypsin in either TBS/BSA or CBS/BSA buffer. The infectivity of
BPTI-III MK phage is 25-fold lower than that of MK phage; thus the
conditions chosen above ensure that an approximately equivalent
number of phage particles are added to the trypsin beads. After 3
hours of mixing on a Labquake shaker (Labindustries Inc., Berkeley,
Calif.) 0.5 ml of either TBS/BSA or CBS/BSA was added where
appropriate to the samples. Beads were washed for 5 min and
recovered by centrifugation for 30 sec. The supernatant was removed
and 0.5 ml of TBS/0.1% Tween-20 was added. The beads were mixed for
5 minutes on the shaker and recovered by centrifugation as above.
The supernatant was removed and the beads were washed an additional
five times with TBS/0.1% Tween-20 as described above. Finally, the
beads were resuspended in 0.5 ml of elution buffer (0.1 M HCl
containing 1.0 mg/ml BSA adjusted to pH 2.2 with glycine), mixed
for 5 minutes and recovered by centrifugation. The supernatant
fraction was removed and neutralized by the addition of 130 .mu.l
of 1 M Tris, pH 8.0. Aliquots of the neutralized elution sample
were diluted in LB broth and titered for plaque-forming units on a
lawn of cells.
[1071] Table 201 illustrates that a significant percentage of the
input BPTI-III MK phage bound to immobilized trypsin and was
recovered by washing with elution buffer. The amount of fusion
phage which bound to the beads was greater in TBS buffer (pH 7.5)
than in CBS buffer (pH 6.5). This is consistent with the
observation that the affinity of BPTI for trypsin is greater at pH
7.5 than at pH 6.5 (VINC72, VINC74). A much lower percentage of the
MK control phage (which do not display BPTI) bound to immobilized
trypsin and this binding was independent of the pH conditions. At
pH 6.5, 1675 times more of the BPTI-III MK phage than of the MK
phage bound to trypsin beads while at pH 7.5, a 2103-fold
difference was observed. Hence fusion phage displaying BPTI adhere
not only to anhydro-trypsin beads but also to active trypsin beads
and can be recovered as infectious phage. These data, in
conjunction with earlier findings, strongly suggest that BPTI
displayed on the surface of fusion phage is appropriately folded
and functional.
[1072] 2) Generation of P1 Mutants of BPTI
[1073] To demonstrate the specificity of interaction of BPTI-III
fusion phage with immobilized serine proteases, single amino acid
substitutions were introduced at the P1 position (residue 15 of
mature BPTI) of the BPTI-III fusion protein by site-directed
mutagenesis. A 25mer mutagenic oligonucleotide (PI) was designed to
substitute a LEU codon for the LYS.sub.15 codon. This alteration is
desired because BPTI(K15L) is a moderately good inhibitor of human
neutrophil elastase (HNE) (K.sub.d=2.9.multidot.10.sup.-9 M)
(BECK88b) and a poor inhibitor of trypsin. A fusion phage
displaying BPTI(K15L) should bind to immobilized HNE but not to
immobilized trypsin. BPTI-III MK fusion phage would be expected to
display the opposite phenotype (bind to trypsin, fail to bind to
HNE). These observations would illustrate the binding specificity
of BPTI-III fusion phage for immobilized serine proteases.
[1074] Mutagenesis of the P1 region of the BPTI-VIII gene contained
within the intergenic region of recombinant phage MB46 was carried
out using the Muta-Gene M13 In Vitro Mutagenesis Kit (Bio-Rad,
Richmond, Calif.). MB46 phage (7.5.multidot.10.sup.6 pfu) were used
to infect a 50 ml culture of CJ236 cells (O.D.600=0.5). Following
overnight incubation at 37.degree. C., phage were recovered and
uracil-containing single-stranded DNA was extracted from the phage.
The single-stranded DNA was further purified by NACS chromatography
as recommended by the manufacturer (B.R.L., Gaithersburg, Md.).
[1075] Two hundred nanograms of the purified single-stranded DNA
were annealed to 3 picomoles of the phosphorylated 25mer mutagenic
oligonucleotide (P1). Following filling in with T4 DNA polymerase
and ligation with T4 DNA ligase, the sample was used to transfect
competent cells which were subsequently plated on LB plates to
permit the formation of plaques. Phage derived from picked plaques
were applied to a Nytran membrane using a Schleicher and Schuell
(Keene, NH) minifold I apparatus (Dot Blot Procedure). Phage DNA
was immobilized onto the filter by baking at 80.degree. C. for 2
hours. The filter was bathed in 1.times. Southern pre-hybridization
buffer (5Prime-3Prime, West Chester, Pa.) for 2 hours.
Subsequently, the filter was incubated in 1.times. Southern
hybridization solution (5Prime-3Prime) containing a 21mer probing
oligonucleotide (LEUl) which had been radioactively labelled with
gamma-.sup.32P-ATP (N.E.N./DuPont, Boston, Mass.) by T4
polynucleotide kinase (New England BioLabs (NEB), Beverly, Mass.).
Following overnight hybridization, the filter was washed 3 times
with 6.times. SSC at room temperature and once at 60.degree. C. in
6.times. SSC prior to autoradiography. Clones exhibiting strong
hybridization signals were chosen for large scale Rf preparation
using the PZ523 spin column protocol (5Prime-3Prime). Restriction
enzyme analysis confirmed that the structure of the Rf was correct
and DNA sequencing confirmed the substitution of a LEU codon (TTG)
for the LYS.sub.15 codon (AAA). This Rf DNA was designated
MB46(K15L).
[1076] 3) Generation of the BPTI-III MA Vector
[1077] The original gene III fusion phage MK can be detected on the
basis of its ability to transduce cells to kanamycin resistance
(Km.sup.R). It was deemed advantageous to generate a second gene
III fusion vector which can confer resistance to a different
antibiotic, namely ampicillin (Ap). One could then mix a fusion
phage conferring Ap.sup.R while displaying engineered protease
inhibitor A (EPI-A) with a second fusion phage conferring Km.sup.R
while displaying EPI-B. The mixture could be added to an
immobilized serine protease and, following elution of bound fusion
phage, one could evaluate the relative affinity of the two EPIs for
the immobilized protease from the relative abundance of phage that
transduce cells to Km.sup.R or Ap.sup.R.
[1078] The ap.sup.R gene is contained in the vector pGem3Zf
(Promega Corp., Madison, Wis.) which can be packaged as single
stranded DNA contained in bacteriophage when helper phage are added
to bacteria containing this vector. The recognition sites for
restriction enzymes SmaI and SnaBI were engineered into the 3'
non-coding region of the Ap.sup.R (.beta.-lactamase) gene using the
technique of synthetic oligonucleotide directed site specific
mutagenesis. The single stranded DNA was used as the template for
in vitro mutagenesis leading to the following DNA sequence
alterations (numbering as supplied by Promega): a) to create a SmaI
(or XmaI) site, bases T.sub.1115.fwdarw.C and A.sub.1116.fwdarw.C,
and b) to create a SnaBI site, G.sub.1125.fwdarw.T,
C.sub.1129.fwdarw.T, and T.sub.1130.fwdarw.A. The alterations were
confirmed by radiolabelled probe analysis with the mutating
oligonucleotide and restriction enzyme analysis; this plasmid is
named pSGK3.
[1079] Plasmid SGK3 was cut with AatII and SmaI and treated with T4
DNA polymerase (NEB) to remove overhanging 3' ends (MANI82,
SAMB89). Phosphorylated HindIII linkers (NEB) were ligated to the
blunt ends of the DNA and following HindIII digestion, the 1.1 kb
fragment was isolated by agarose gel electrophoresis followed by
purification on an Ultrafree-MC filter unit as recommended by the
manufacturer (Millipore, Bedford, Mass.). M13-MB1/2-delta Rf DNA
was cut with HindIII and the linearized Rf was purified and ligated
to the 1.1 kb fragment derived from pSGK3. Ligation samples were
used to transfect competent cells which were plated on LB plates
containing Ap. Colonies were picked and grown in LB broth
containing Ap overnight at 37.degree. C. Aliquots of the culture
supernatants were assayed for the presence of infectious phage. Rf
DNA was prepared from cultures which were both Ap.sup.R and
contained infectious phage. Restriction enzyme analysis confirmed
that the Rf contained a single copy of the Ap.sup.R gene inserted
into the intergenic region of the M13 genome in the same
transcriptional orientation as the phage genes. This Rf DNA was
designated MA.
[1080] The 5.9 kb BalII/BsmI fragment from MA Rf DNA and the 2.2 kb
BglII/BsmI fragment from BPTI-III MK Rf DNA were ligated together
and a portion of the ligation mixture was used to transfect
competent cells which were subsequently plated to permit plaque
formation on a lawn of cells. Large and small size plaques were
observed on the plates. Small size plaques were picked for further
analysis since BPTI-III fusion phage give rise to small plaques due
to impairment of gene III protein function. Small plaques were
added to LB broth containing Ap and cultures were incubated
overnight at 37.degree. C. An Ap.sup.R culture which contained
phage which gave rise to small plaques when plated on a lawn of
cells was used as a source of Rf DNA. Restriction enzyme analysis
confirmed that the BPTI-III fusion gene had been inserted into the
MA vector. This Rf was designated BPTI-III MA.
[1081] 4) Construction of BPTI(K15L)-III MA
[1082] MB46(Kl5L) Rf DNA was digested with XhoI and EagI and the
125 bp DNA fragment was isolated by electrophoresis on a 2% agarose
gel followed by extraction from an agarose slice by centrifugation
through an Ultrafree-MC filter unit. The 8.0 kb XhoI/EagI fragment
derived from BPTI-III MA Rf was also prepared. The above two
fragments were ligated and the ligation sample was used to
transfect competent cells which were plated on LB plates containing
Ap. Colonies were picked and used to inoculate LB broth containing
Ap. Cultures were incubated overnight at 37.degree. C. and phage
within the culture supernatants was probed using the Dot Blot
Procedure. Filters were hybridized to a radioactively labelled
oligonucleotide (LEU1). Positive clones were identified by
autoradiography after washing filters under high stringency
conditions. Rf DNA was prepared from Ap.sup.R cultures which
contained phage carrying the K15L mutation. Restriction enzyme
analysis and DNA sequencing confirmed that the K15L mutation had
been introduced into the BPTI-III MA Rf. This Rf was designated
BPTI(K15L)-III MA. Interestingly, BPTI(K15L)-III MA phage gave rise
to extremely small plaques on a lawn of cells and the infectivity
of the phage is 4 to 5 fold less than that of BPTI-III MK phage.
This suggests that the substitution of LEU for LYS.sub.15 impairs
the ability of the BPTI:gene III fusion protein to mediate phage
infection of bacterial cells.
[1083] 5) Preparation of Immobilized Human Neutrophil Elastase
[1084] One ml of Reacti-Gel 6.times. CDI activated agarose (Pierce
Chemical Co.) in acetone (200 .mu.l packed beads) was introduced
into an empty Select-D spin column (5Prime-3Prime). The acetone was
drained out and the beads were washed twice rapidly with 1.0 ml of
ice cold water and 1.0 ml of ice cold 100 mM boric acid, pH 8.5,
0.9% NaCl. Two hundred .mu.l of 2.0 mg/ml human neutrophil elastase
(HNE) (CalBiochem, San Diego, Calif.) in borate buffer were added
to the beads. The column was sealed and mixed end over end on a
Labquake Shaker at 4.degree. C. for 36 hours. The HNE solution was
drained off and the beads were washed with ice cold 2.0 M Tris, pH
8.0 over a 2 hour period at 4.degree. C. to block remaining
reactive groups. A 50% slurry of the beads in TBS/BSA was prepared.
To this was added an equal volume of sterile 100% glycerol and the
beads were stored as a 25% slurry at -20.degree. C. Prior to use,
the beads were washed 3 times with TBS/BSA and a 50% slurry in
TBS/BSA was prepared.
[1085] 6) Characterization of the Affinity of BPTI-III MK and
BPTI(K15L)-III MA Phage for Immobilized Trypsin and Human
Neutrophil Elastase
[1086] Thirty .mu.l of BPTI-III MX phage in TBS/BSA
(1.7.multidot.10.sup.11 pfu/ml) was added to 5 .mu.l of a 50%
slurry of either immobilized human neutrophil elastase or
immobilized trypsin (Pierce Chemical Co.) also in TBS/BSA.
Similarly 30 .mu.l of BPTI(K15L)-III MA phage in TBS/BSA (3.2-1010
pfu/ml) was added to either immobilized HNE or trypsin. Samples
were mixed on a Labquake shaker for 3 hours. The beads were washed
with 0.5 ml of TBS/BSA for 5 minutes and recovered by
centrifugation. The supernatant was removed and the beads were
washed 5 times with 0.5 ml of TBS/0.1% Tween-20. Finally, the beads
were resuspended in 0.5 ml of elution buffer (0.1 M HCl containing
1.0 mg/ml BSA adjusted to pH 2.2 with glycine), mixed for 5 minutes
and recovered by centrifugation. The supernatant fraction was
removed, neutralized with 130 .mu.l of 1 M Tris, pH 8.0, diluted in
LB broth, and titered for plaque-forming units on a lawn of
cells.
[1087] Table 202 illustrates that 82 times more of the BPTI-III MX
input phage bound to the trypsin beads than to the HNE beads. By
contrast, the BPTI(K15L)-III MA phage bound preferentially to HNE
beads by a factor of 36. These results are consistent with the
known affinities of wild type and the Kl5L variant of BPTI for
trypsin and HNE. Hence BPTI-III fusion phage bind selectively to
immobilized proteases and the nature of the BPTI variant displayed
on the surface of the fusion phage dictates which particular
protease is the optimum receptor for the fusion phage.
[1088] 7) Effect of pH on the Dissociation of Bound BPTI-III MK and
BPTI(K15L)-III MA Phage from Immobilized Neutrophil Elastase
[1089] The affinity of a given fusion phage for an immobilized
serine protease can be characterized on the basis of the amount of
bound fusion phage which elutes from the beads by washing with a pH
2.2 buffer. This represents rather extreme conditions for the
dissociation of fusion phage from beads. Since the affinity of the
BPTI variants described above for HNE is not high
(K.sub.d>1.multidot.10.sup.-9 M) it was anticipated that fusion
phage displaying these variants might dissociate from HNE beads
under less severe pH conditions. Furthermore fusion phage might
dissociate from HNE beads under specific pH conditions
characteristic of the particular BPTI variant displayed by the
phage. Low pH buffers providing stringent wash conditions might be
required to dissociate fusion phage displaying a BPTI variant with
a high affinity for HNE whereas neutral pH conditions might be
sufficient to dislodge a fusion phage displaying a BPTI variant
with a weak affinity for HNE.
[1090] Thirty .mu.l of BPTI(K15L)-III MA phage
(1.7.multidot.10.sup.10 pfu/ml in TBS/BSA) were added to 5 .mu.l of
a 50% slurry of immobilized HNE also in TBS/BSA. Similarly, 30
.mu.l of BPTI-III MA phage (8.6.multidot.10.sup.10 pfu/ml in
TBS/BSA) were added to 5 .mu.l of immobilized HNE. The above
conditions were chosen to ensure that an approximately equivalent
number of phage particles were added to the beads. The samples were
incubated for 3 hours on a Labquake shaker. The beads were washed
with 0.5 ml of TBS/BSA for 5 min on the shaker, recovered by
centrifugation and the supernatant was removed. The beads were
washed with 0.5 ml of TBS/0.1% Tween-20 for 5 minutes and recovered
by centrifugation. Four additional washes with TBS/0.1% Tween-20
were performed as described above. The beads were washed as above
with 0.5 ml of 100 mM sodium citrate, pH 7.0 containing 1.0 mg/ml
BSA. The beads were recovered by centrifugation and the supernatant
was removed. Subsequently, the HNE beads were washed sequentially
with a series of 100 mM sodium citrate, 1.0 mg/ml BSA buffers of pH
6.0, 5.0, 4.0 and 3.0 and finally with the 2.2 elution buffer
described above. The pH washes were neutralized by the addition of
1 M Tris, pH 8.0, diluted in LB broth and titered for
plaque-forming units on a lawn of cells.
[1091] Table 203 illustrates that a low percentage of the input
BPTI-III MK fusion phage adhered to the HNE beads and was recovered
in the pH 7.0 and 6.0 washes predominantly. By contrast, a
significantly higher percentage of the BPTI(K15L)-III MA phage
bound to the HNE beads and was recovered predominantly in the pH
5.0 and 4.0 washes. Hence lower pH conditions (i.e. more stringent)
are required to dissociate BPTI(K15L)-III MA than BPTI-MK phage
from immobilized HNE. The affinity of BPTI(K15L) is over 1000 times
greater than that of BPTI for HNE (based on reported K.sub.d values
(BECK88b)). Hence this suggests that lower pH conditions are indeed
required to dissociate fusion phage displaying a BPTI variant with
a higher affinity for HNE.
[1092] 8) Construction of BPTI(MGNG)-III MA Phase
[1093] The light chain of bovine inter-.alpha.-trypsin inhibitor
contains 2 domains highly homologous to BPTI. The amino terminal
proximal domain (called BI-8e) has been generated by proteolysis
and shown to be a potent inhibitor of HNE
(K.sub.d=4.4.multidot.10.sup.-11 M) (ALBR83). By contrast a BPTI
variant with the single substitution of LEU for LYS.sub.15 exhibits
a moderate affinity for HNE (K.sub.d=2.9.multidot.10.- sup.-9 M)
(BECK88b). It has been proposed that the Pi residue is the primary
determinant of the specificity and potency of BPTI-like molecules
(BECK88b, LASK80 and works cited therein). Although both BI-8e and
BPTI(K15L) feature LEU at their respective P1 positions, there is a
66 fold difference in the affinities of these molecules for HNE.
Structural features, other than the P1 residue, must contribute to
the affinity of BPTI-like molecules for HNE.
[1094] A comparison of the structures of BI-8e and BPTI-(K15L)
reveals the presence of three positively charged residues at
positions 39, 41, and 42 of BPTI which are absent in BI-8e. These
hydrophilic and highly charged residues of BPTI are displayed on a
loop which underlies the loop containing the P1 residue and is
connected to it via a disulfide bridge. Residues within the
underlying loop (in particular residue 39) participate in the
interaction of BPTI with the surface of trypsin near the catalytic
pocket (BLOW72) and may contribute significantly to the tenacious
binding of BPTI to trypsin. However, these hydrophilic residues
might hamper the docking of BPTI variants with HNE. In support of
this hypothesis, BI-8e displays a high affinity for HNE and
contains no charged residues in the region spanning residues 39-42.
Hence residues 39 through 42 of wild type BPTI were replaced with
the corresponding residues of the human homologue of BI-8e. We
anticipated that a BPTI derivative containing the MET-GLY-ASN-GLY
(MGNG) sequence would exhibit a higher affinity for HNE than
corresponding derivatives which retain the sequence of wild type
BPTI at residues 39-42.
[1095] A double stranded oligonucleotide with AccI and EaqI
compatible ends was designed to introduce the desired alteration of
residues 39 to 42 via cassette mutagenesis. Codon 45 was altered to
create a new XmnI site, unique in the structure of the BPTI gene,
which could be used to screen for mutants. This alteration at codon
45 does not alter the encoded amino-acid sequence. BPTI-III MA Rf
DNA was digested with AccI. Two oligonucleotides (CYSB and CYST)
corresponding to the bottom and top strands of the mutagenic DNA
were annealed and ligated to the AccI digested BPTI-III MA Rf DNA.
The sample was digested with BglII and the 2.1 kb BalII/EagI
fragment was purified. BPTI-III MA Rf was also digested with BglII
and EagI and the 6.0 kb fragment was isolated and ligated to the
2.1 kb BglII/EaaI fragment described above. Ligation samples were
used to transfect competent cells which were plated to permit the
formation of plaques on a lawn of cells. Phage derived from plaques
were probed with a radioactively labelled oligonucleotide (CYSB)
using the Dot Blot Procedure. Positive clones were identified by
autoradiography of the Nytran membrane after washing at high
stringency conditions. Rf DNA was prepared from Ap.sup.R cultures
containing fusion phage which hybridized to the CYSB probe.
Restriction enzyme analysis and DNA sequencing confirmed that
codons 39-42 of BPTI had been altered. The Rf DNA was designated
BPTI(MGNG)-III MA.
[1096] 9) Construction of BPTI(K15L,MGNG)-III MA
[1097] BPTI(MGNG)-III MA Rf DNA was digested with AccI and the 5.6
kb fragment was purified. BPTI(K15L)-III MA was digested with AccI
and the 2.5 kb DNA fragment was purified. The two fragments above
were ligated together and ligation samples were used to transfect
competent cells which were plated for plaque production. Large and
small plaques were observed on the plate. Representative plaques of
each type were picked and phage were probed with the LEU1
oligonucleotide via the Dot Blot Procedure. After the Nytran filter
had been washed under high stringency conditions, positive clones
were identified by autoradiography. only the phage which hybridized
to the LEU1 oligonucleotide gave rise to the small plaques
confirming an earlier observation that substitution of LEU for
LYS.sub.15 substantially reduces phage infectivity. Appropriate
cultures containing phage which hybridized to the LEU1
oligonucleotide were used to prepare Rf DNA. Restriction enzyme
analysis and DNA sequencing confirmed that the K15L mutation had
been introduced into BPTI(MGNG)-III MA. This Rf DNA was designated
BPTI (K5L,MGNG)-III MA.
[1098] 10) Effect of Mutation of Residues 39-42 of BPTI(K15L) on
its Affinity for Immobilized HNE
[1099] Thirty .mu.l of BPTI(K15L,MGNG)-III MA phage
(9.2.multidot.10.sup.9 pfu/ml in TBS/BSA) were added to 5 .mu.l of
a 50% slurry of immobilized HNE also in TBS/BSA. Similarly 30 .mu.l
of BPTI(K15L)-III MA phage (1.2.multidot.10.sup.10 pfu/ml in
TBS/BSA) were added to immobilized HNE. The samples were incubated
for 3 hours on a Labquake shaker. The beads were washed for 5 min
with 0.5 ml of TBS/BSA and recovered by centrifugation. The beads
were washed 5 times with 0.5 ml of TBS/0.1% Tween-20 as described
above. Finally, the beads were washed sequentially with a series of
100 mM sodium citrate buffers of pH 7.0, 6.0, 5.5, 5.0, 4.75, 4.5,
4.25, 4.0 and 3.5 as described above. pH washes were neutralized,
diluted in LB broth and titered for plaque-forming units on a lawn
of cells.
[1100] Table 204 illustrates that almost twice as much of the
BPTI(K15L,MGNG)-III MA as BPTI(K15L)-III MA phage bound to HNE
beads. In both cases the pH 4.75 fraction contained the largest
proportion of the recovered phage. This confirms that replacement
of residues 39-42 of wild type BPTI with the corresponding residues
of BI-8e enhances the binding of the BPTI(KL5L) variant to HNE.
[1101] 11) Fractionation of a Mixture of BPTI-III MK and
BPTI(K15L,MGNG)-III MA Fusion Phage
[1102] The observations described above indicate that
BPTI(K15L,MGNG)-III MA and BPTI-III MK phage exhibit different pH
elution profiles from immobilized HNE. It seemed plausible that
this property could be exploited to fractionate a mixture of
different fusion phage.
[1103] Fifteen .mu.l of BPTI-III MK phage (3.92-1010 pfu/ml in
TBS/BSA), equivalent to 8.91.multidot.10.sup.7 KmR transducing
units, were added to 15 .mu.l of BPTI(K15L,MGNG)-III MA phage
(9.85.multidot.10.sup.9 pfu/ml in TBS/BSA), equivalent to
4.44.multidot.10.sup.7 Ap.sup.R transducing units. Five Al of a 50%
slurry of immobilized HNE in TBS/BSA was added to the phage and the
sample was incubated for 3 hours on a Labquake mixer. The beads
were washed for 5 minutes with 0.5 ml of TBS/BSA prior to being
washed 5 times with 0.5 ml of TBS/2.0% Tween-20 as described above.
Beads were washed for 5 minutes with 0.5 ml of 100 mM sodium
citrate, pH 7.0 containing 1.0 mg/ml BSA. The beads were recovered
by centrifugation and the supernatant was removed. Subsequently,
the HNE beads were washed sequentially with a series of 100 mM
citrate buffers of pH 6.0, 5.0 and 4.0. The pH washes were
neutralized by the addition of 130 .mu.l of 1 M Tris, pH 8.0.
[1104] The relative proportion of BPTI-III MK and
BPTI(K15L,MGNG)-III MA phage in each pH fraction was evaluated by
determining the number of phage able to transduce cells to Km.sup.R
as opposed to Ap.sup.R. Fusion phage diluted in 1.times. Minimal A
salts were added to 100 .mu.l of cells (O.D.600=0.8 concentrated to
{fraction (1/20)} original culture volume) also in Minimal salts in
a final volume of 200 .mu.l. The sample was incubated for 15 min at
37.degree. C. prior to the addition of 200 .mu.l of 2.times. LB
broth. After an additional 15 min incubation at 37.degree. C.,
duplicate aliquots of cells were plated on LB plates containing
either Ap or Km to permit the formation of colonies. Bacterial
colonies on each type of plate were counted and the data was used
to calculate the number of Ap.sup.R and Km.sup.R transducing units
in each pH fraction. The number of Ap.sup.R transducing units is
indicative of the amount of BPTI(K15L,MGNG)-III MA phage in each pH
fraction while the total number of Km.sup.R transducing units is
indicative of the amount of BPTI-III MK phage.
[1105] Table 205 illustrates that a low percentage of the BPTI-III
MK input phage (as judged by Km.sup.R transducing units) adhered to
the HNE beads and was recovered predominantly in the pH 7.0
fraction. By contrast, a significantly higher percentage of the
BPTI(K15L,MGNG)-III MA phage (as judged by Ap.sup.R transducing
units) adhered to the HNE beads and was recovered predominantly in
the pH 4.0 fraction. A comparison of the total number of Ap.sup.R
and Km.sup.R transducing units in the pH 4.0 fraction shows that a
984-fold enrichment of BPTI(K15L,MGNG)-III MA phage over BPTI-III
MK phage was achieved. Hence, the above procedure can be utilized
to fractionate mixtures of fusion phage on the basis of their
relative affinities for immobilized HNE.
[1106] 12) Construction of BPTI(K15V,R17L)-III MA
[1107] A BPTI variant containing the alterations K15V and R17L
demonstrates the highest affinity for HNE of any BPTI variant
described to date (K.sub.d=6.multidot.10.sup.-11 M) (AUER89). As a
means of testing the selection system described herein, a fusion
phage displaying this variant of BPTI was generated and used as a
"reference" phage to characterize the affinity for immobilized HNE
of fusion phage displaying a BPTI variant with a known affinity for
free HNE. A 76 bp mutagenic oligonucleotide (VALL) was designed to
convert the LYS.sub.15 codon (AAA) to a VAL codon (GTT) and the
ARG.sub.17 codon (CGA) to a LEU codon (CTG). At the same time
codons 11, 12 and 13 were altered to destroy the ApaI site resident
in the wild type BPTI gene while creating a new RsrII site, which
could be used to screen for correct clones.
[1108] The single stranded VAL1 oligonucleotide was converted to
the double stranded form following the procedure described in
Current Protocols in Molecular Biology (AUSU87). One .mu.g of the
VALl oligonucleotide was annealed to one .mu.g of a 20 bp primer
(MB8). The sample was heated to 80.degree. C., cooled to 62.degree.
C. and incubated at this temperature for 30 minutes before being
allowed to cool to 37.degree. C. Two .mu.l of a 2.5 mM mixture of
dNTPs and 10 units of Sequenase (U.S.B., Cleveland, Ohio) were
added to the sample and second strand synthesis was allowed to
proceed for 45 minutes at 37.degree. C. One hundred units of XhoI
was added to the sample and digestion was allowed to proceed for 2
hours at 37.degree. C. in 100 .mu.l of 1.times. XhoI digestion
buffer. The digested DNA was subjected to electrophoreses on a 4%
GTG NuSieve agarose (FMC Bioproducts, Rockland, Me.) gel and the 65
bp fragment was excised and purified from melted agarose by phenol
extraction and ethanol precipitation. A portion of the recovered 65
bp fragment was subjected to electrophoresis on a 4% GTG NuSieve
agarose gel for quantitation. One hundred nanograms of the
recovered fragment was dephosphorylated with 1.9 .mu.l of HK(TM)
phosphatase (Epicentre Technologies, Madison, Wis.) at 37.degree.
C. for 60 minutes. The reaction was stopped by heating at
65.degree. C. for 15 minutes. BPTI-MA Rf DNA was digested with XhoI
and StuI and the 8.0 kb fragment was isolated. one Al of the
dephosphorylation reaction (5 ng of double-stranded VAL1
oligonucleotide) was ligated to 50 ng of the 8.0 kb XhoI/StuI
fragment derived from BPTI-III MA Rf. Ligation samples were
subjected to phenol extraction and DNA was recovered by ethanol
precipitation. Portions of the recovered ligation DNA were added to
40 .mu.l of electro-competent cells which were shocked using a
Bio-Rad Gene Pulser device set at 1.7 kv, 25 .mu.F and 800 .OMEGA..
One ml of SOC media was immediately added to the cells which were
allowed to recover at 37.degree. C. for one hour. Aliquots of the
electroporated cells were plated onto LB plates containing Ap to
permit the formation of colonies.
[1109] Phage contained within cultures derived from picked Ap.sup.R
colonies were probed with two radiolabelled oligonucleotides (PRP1
and ESP1) via the Dot Blot Procedure. Rf DNA was prepared from
cultures containing phage which exhibited a strong hybridization
signal with the ESP1 oligonucleotide but not with the PRP1
oligonucleotide. Restriction enzyme analysis verified loss of the
ApaI site and acquisition of a new RsrII site diagnostic for the
changes in the P1 region. Fusion phage were also probed with a
radiolabelled oligonucleotide (VLPl) via the Dot Blot Procedure.
Autoradiography confirmed that fusion phage which previously failed
to hybridize to the PRP1 probe, hybridized to the VLP1 probe. DNA
sequencing confirmed that the LYS.sub.15 and ARG.sub.17 codons had
been converted to VAL and LEU codons respectively. The Rf DNA was
designated BPTI(K15V,R17L)-III MA.
[1110] 13) Affinity of BPTI(K15V,R17L)-III MA Phaae for Immobilized
HNE
[1111] Forty .mu.l of BPTI(K15,Rl7L)-III MA phage
(9.8.multidot.10.sup.10 pfu/ml) in TBS/BSA were added to 10 .mu.l
of a 50% slurry of immobilized HNE also in TBS/BSA. Similarly, 40
.mu.l of BPTI(K15L,MGNG)-III MA phage (5.13.multidot.10.sup.9
pfu/ml) in TBS/BSA were added to immobilized HNE. The samples were
mixed for 1.5 hours on a Labquake shaker. Beads were washed once
for 5 min with 0.5 ml of TBS/BSA and then 5 times with 0.5 ml of
TBS/1.0% Tween-20 as described previously. Subsequently the beads
were washed sequentially with a series of 50 mM sodium citrate
buffers containing 150 mM NaCl, 1.0 mg/ml BSA of pH 7.0, 6.0, 5.0,
4.5, 4.0, 3.75, 3.5 and 3.0. In the case of the BPTI(K15L,MGNG)-III
MA phage, the pH 3.75 and 3.0 washes were omitted. Two washes were
performed at each pH and the supernatants were pooled, neutralized
with 1 M Tris pH 8.0, diluted in LB broth and titered for
plaque-forming units on a lawn of cells.
[1112] Table 206 illustrates that the pH 4.5 and 4.0 fractions
contained the largest proportion of the recovered
BPTI(K15V,R17L)-III MA phage. By contrast, the BPTI(K15L,MGNG)-III
MA phage, like BPTI(K15L)-III MA phage, were recovered
predominantly in the pH 5.0 and 4.5 fractions, as shown above. The
affinity of BPTI(K15V,R17L) is 48 times greater than that of
BPTI(K15L) for HNE (based on reported Kd values, AUER89 for
BPTI(K15V,R17L) and BECK88b for BPTI(K15L)). That the pH elution
profile for BPTI(K15V,R17L)-III MA phage exhibits a peak at pH 4.0
while the profile for BPTI(K15L)-III MA phage displays a peak at pH
4.5 supports the contention that lower pH conditions are required
to dissociate, from immobilized HNE, fusion phage displaying a BPTI
variant with a higher affinity for free HNE.
EXAMPLE IV
[1113] Construction of a Variegated Population of Phage Displaying
BPTI Derivates and Fractionation for Members that Display Binding
Domains having High Affinity for Human Neutrophil Elastase:
[1114] We here describe generation of a library of 1000 different
potential engineered protease inhibitiors (PEPIs) and the
fractionation with immobilized HNE to obtain an engineered protease
inhibitor (Epi) having high affinity for HNE. Successful Epis that
bind HNE are designated EpiNEs.
[1115] 1) Design of a Mutagenic Oligonucleotide to Create a Library
of Fusion Phage
[1116] A 76 bp variegated oligonucleotide (MYMUT) was designed to
construct a library of fusion phage displaying 1000 different PEPIs
derived from BPTI. The oligonucleotide contains 1728 different DNA
sequences but due to the degeneracy of the genetic code, it encodes
1000 different protein sequences. The oligonucleotide was designed
so as to destroy an ApaI site (shown in Table 113) encompassing
codons 12 and 13. ApaI digestion could be used to select against
the parental Rf DNA used to construct the library.
[1117] The MYMUT oligonucleotide permits the substitution of 5
hydrophobic residues (PHE, LEU, ILE, VAL, and MET via a DTS codon
(D=approximately equimolar A, T, and G; S=approximately equimolar C
and G)) for LYS.sub.15. Replacement of LYS.sub.15 in BPTI with
aliphatic hydrophobic residues via semi-synthesis has provided
proteins having higher affinity for HNE than BPTI (TANN77,
JERI74a,b, WENZ80, TSCH86, BECK88b). At position 16, either GLY or
ALA are permitted (GST codon). This is in keeping with the
predominance of these two residues at the corresponding positions
in a variety of BPTI homologues (CREI87). The variegation scheme at
position 17 is identical to that at 15. Limited data is available
on the relative contribution of this residue to the interaction of
BPTI homologues with HNE. A variety of hydrophobic residues at
position 17 was included with the anticipation that they would
enhance the docking of a BPTI variant with HNE. Finally at
positions 18 and 19, 4 (PHE, SER, THR, and ILE via a WYC codon
(W=approximately equimolar A and T; Y=approximately equimolar T and
C)) and 5 (SER, PRO, THR, LYS, GLN, and stop via an HMA codon
(H=approximately equimolar A, C, and T; M=approximately equimolar A
and C)) different amino acids respectively are encoded. These
different amino acid residues are found in the corresponding
positions of BPTI homologues that are known to bind to HNE
(CREI87). Although the amino acids included in the PEPI library
were chosen because there was some indication that they might
facilitate binding to HNE, it was not and is not possible to
predict which combination of these amino acids will lead to high
affinity for HNE. The mutagenic oligonucleotide MYMUT was
synthesized by Genetic Design Inc. (Houston, Tex.).
[1118] 2) Construction of Library of Fusion Phage Displaying
Potential Engineered Protease Inhibitors
[1119] The single-stranded mutagenic MYMUT DNA was converted to the
double stranded form with compatible XhoI and StuI ends and
dephosphorylated with HK.sup.(TM) phosphatase as described above
for the VALl oligonucleotide. BPTI(MGNG)-III MA Rf DNA was digested
with XhoI and StuI for 3 hours at 37.degree. C. to ensure complete
digestion. The 8.0 kb DNA fragment was purified by agarose gel
electrophoresis and Ultrafree-MC unit filtration. One .mu.l of the
dephosphorylated MYMUT DNA (5 ng) was ligated to 50 ng of the 8.0
kb fragment derived from BPTI(MGNG)-III MA Rf DNA. Under these
conditions, the 10:1 molar ratio of insert to vector was found to
be optimal for the generation of transformants. Ligation samples
were extracted with phenol, phenol/chloroform/IAA (25:24:1, v:v:v)
and chloroform/IAA (24:1, v:v) and DNA was ethanol precipitated
prior to electroporation. One .mu.l of the recovered ligation DNA
was added to 40 .mu.l of electro-competent cells. Cells were
shocked using a Bio-Rad Gene Pulser device as described above.
Immediately following electroshock, 1.0 ml of SOC media was added
to the cells which were allowed to recover at 37.degree. C. for 60
minutes with shaking. The electroporated cells were plated onto LB
plates containing Ap to permit the formation of colonies.
[1120] To assess the efficiency of the cassette mutagenesis
procedure, 39 transformants were picked at random and phage present
in culture supernatants were applied to a Nytran membrane and
probed using the Dot Blot Procedure. Two Nytran membranes were
prepared in this manner. The first filter was allowed to hybridize
to the CYSB oligo-nucleotide which had previously been
radiolabelled. The second membrane was allowed to hybridize to the
PRP1 oligonucleotide which had also been radiolabelled. Filters
were subjected to autoradiography following washing under high
stringency conditions. Of the 39 phage samples applied to the
membrane, all 39 hybridized to the CYSB probe. This indicated that
there was fusion phage in the culture supernatants and that at
least the DNA encoding residues 35-47 appeared to be present in the
phage genomes. Only 11 of the 39 samples hybridized to the PRP1
oligonucleotide indicating that 28% of the transformants were
probably the parental phage BPTI(MGNG)-III MA used to generate the
library. The remaining 28 clones failed to hybridize to the PRP1
probe indicating that substantial alterations were introduced into
the P1 region by cassette mutagenesis using the MYMUT
oligonucleotide. Of these 28 samples, all were found to contain
infectious phage indicating that mutagenesis did not result in
frame shift mutations which would lead to the generation of
defective gene III products and non-infectious phage. (These 28
PEPI-displaying phage constitute a mini-library, the fractionation
of which is discussed below.) Hence the overall efficiency of
mutagenesis was estimated to be 72% in those cases where ligation
DNA was not subjected to ApaI digestion prior to
electroporation.
[1121] Bacterial colonies were harvested by overlaying chilled LB
plates containing Ap with 5 ml of ice cold LB broth and scraping
off cells using a sterile glass rod. A total of 4899 transformants
were harvested in this manner of which 3299 were obtained by
electroporation of ligation samples which were not digested with
ApaI. Hence we estimate that 72% of these transformants (i.e. 2375)
represent mutants of the parental BPTI(MGNG)-III MA phage derived
by cassette mutagenesis of the P1 position. An additional 1600
transformants were obtained by electroporation of ligation samples
which had been digested with ApaI. If we assume that all of these
clones contain new sequences at the P1 position then the total
number of mutants in the pool of 4899 transformants is estimated to
be 2375+1600=3975. The total number of potentially different DNA
sequences in the MYMUT library is 1728. We calculate that the
library should display about 90% of the potential engineered
protease inhibitor sequences as follows: 3 N displayed = N possible
( 1 - exp { - Libsize / N ( DNA ) } ) = 1000 ( 1 - exp { - 3975 /
1728 } ) = 900 % ofpossible sequences displayed = 100 ( 900 1000 )
= 90 %
[1122] 3) Fractionation of a Mini-library of Fusion Phage
[1123] We studied the fractionation of the mini library of 28 PEPIs
to establish the appropriate parameters for fractionation of the
entire MYMUT PEPI library. We anticipated that fractionation could
be easier when the library of fusion phage was much less diverse
than the entire MYMUT library. Fewer cycles of fractionation might
be required to affinity purify a fusion phage exhibiting a high
affinity for HNE. Secondly, since the sequences of all the fusion
phage in the mini-library can be determined, one can determine the
probability of selecting a given fusion phage from the initial
population.
[1124] Two ml of the culture supernatants of the 28 PEPIs described
above were pooled. Fusion phage were recovered, resuspended in 300
mM NaCl, 100 mM Tris, pH 8.0, 1 mM EDTA and stored on ice for 15
minutes. Insoluble material was removed by centrifugation for 3
minutes in a microfuge at 4.degree. C. The supernatant fraction was
collected and PEPI phage were precipitated with PEG-8000. The final
phage pellet was resuspended in TBS/BSA. Aliquots of the recovered
phage were titered for plaque-forming units on a lawn of cells. The
final stock solution consisted of 200 .mu.l of fusion phage at a
concentration of 5.6.multidot.10.sup.12 pfu/ml.
[1125] a) First Enrichment Cycle
[1126] Forty .mu.l of the above phage stock was added to 10 .mu.l
of a 50% slurry of HNE beads in TBS/BSA. The sample was allowed to
mix on a Labquake shaker for 1.5 hours. Five hundred .mu.l of
TBS/BSA was added to the sample and after an additional 5 minutes
of mixing, the HNE beads were collected by centrifugation. The
supernatant fraction was removed and the beads were resuspended in
0.5 ml of TBS/0.5% Tween-20. Beads were washed for 5 minutes on the
shaker and recovered by centrifugation as above. The supernatant
fraction was removed and the beads were subjected to 4 additional
washes with TBS/Tween-20 as described above to reduce non-specific
binding of fusion phage to HNE beads. Beads were washed twice as
above with 0.5 ml of 50 mM sodium citrate pH 7.0, 150 mM NaCl
containing 1.0 mg/ml BSA. The supernatants from the two washes were
pooled. Subsequently, the HNE beads were washed sequentially with a
series of 50 mM sodium citrate, 150 mM NaCl, 1.0 mg/ml BSA buffers
of pH 6.0, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5 and 2.0. Two washes were
performed at each pH and the supernatants were pooled and
neutralized by the addition of 260 .mu.l of 1 M Tris, pH 8.0.
Aliquots of each pH fraction were diluted in LB broth and titered
for plaque-forming units on a lawn of cells. The total amount of
fusion phage (as judged by pfu) appearing in each pH wash fraction
was determined.
[1127] FIG. 7 illustrates that the largest percentage of input
phage which bound to the HNE beads was recovered in the pH 5.0
fraction. The elution peak exhibits a trailing edge on the low pH
side suggesting that a small proportion of the total bound fusion
phage might elute from the HNE beads at a pH<5. BPTI(K15L)-III
phage display a BPTI variant with a moderate affinity for HNE
(K.sub.d=2.9.multidot.10.sup.-9 M) (BECK88b). Since BPTI(K15L)-III
phage elute from HNE beads as a peak centered on pH 4.75 and the
highest peak in the first passage of the mini-library over HNE
beads is centered on pH 5.0, we infer that many members of the
MYMUT PEPI mini-library display PEPIs having moderate to high
affinity for HNE.
[1128] To enrich for fusion phage displaying the highest affinity
for HNE, phage contained in the lowest pH fraction (pH 2.0) from
the first enrichment cycle were amplified and subjected to a second
round of fractionation. Amplification involved the Transduction
Procedure described above. Fusion phage (2000 pfu) were incubated
with 100 .mu.l of cells for 15 minutes at 37.degree. C. in 200
.mu.l of 1.times. Minimal A salts. Two hundred .mu.l of 2.times. LB
broth was added to the sample and cells were allowed to recover for
15 minutes at 37.degree. C. with shaking. One hundred .mu.l
portions of the above sample were plated onto LB plates containing
Ap. Five such transduction reactions were performed yielding a
total of 20 plates, each containing approximately 350 colonies
(7000 transformants in total). Bacterial cells were harvested as
described for the preparation of the MYMUT library and fusion phage
were collected as described for the preparation of the
mini-library. A total of 200 .mu.l of fusion phage
(4.3.multidot.10.sup.12 pfu/ml in TBS/BSA) derived from the pH 2.0
fraction from the first passage of the mini-library was obtained in
this manner.
[1129] b) Second Enrichment Cycle
[1130] Forty .mu.l of the above phage stock was added to 10 .mu.l
of a 50% slurry of HNE beads in TBS/BSA. The sample was allowed to
mix for 1.5 hours and the HNE beads were washed with TBS/BSA,
TBS/0.5% Tween and sodium citrate buffers as described above.
Aliqouts of neutralized pH fractions were diluted and titered as
described above.
[1131] The elution profile for the second passage of the
mini-library over HNE beads is shown in FIG. 7. The largest
percentage of the input phage which bound to the HNE beads was
recovered in the pH 3.5 wash. A smaller peak centered on pH 4.5 may
represent residual fusion phage from the first passage of the
mini-library which eluted at pH 5.0. The percentage of total input
phage which eluted at pH 3.5 in the second cycle exceeds the
percentage of input phage which eluted at pH 5.0 in the first
cycle. This is indicative of more avid binding of fusion phage to
the HNE matrix. Taken together, the significant shift in the pH
elution profile suggests that selection for fusion phage displaying
BPTI variants with higher affinity for HNE occurred.
[1132] c) Third Cycle
[1133] Phage obtained in the pH 2.0 fraction from the second
passage of the mini-library were amplified as above and subjected
to a third round of fractionation. The pH elution profile is shown
in FIG. 7. The largest percentage of input phage was recovered in
the pH 3.5 wash as is the case with the second passage of the
mini-library. However, the minor peak centered on pH 4.5 is
diminished in the third passage relative to the second passage.
Furthermore, the percentage of input phage which eluted at pH 3.5
is greater in the third passage than in the second passage. In
comparison, the BPTI(K15V,R17L)-III fusion phage elute from HNE
beads as a peak centered on pH 4.25. Taken together, the data
suggests that a significant selection for fusion phage displaying
PEPIs with high affinity for HNE occurred. Furthermore, since more
extreme pH conditions are required to elute fusion phage in the
third passage of the MYMUT library relative to those conditions
needed to elute BPTI(K15V,R17L)-III MA phage, this suggests that
those fusion phage which appear in the pH 3.5 fraction may display
a PEPI with a higher affinity for HNE than the BPTI(K15V,R17L)
variant (i.e. K.sub.d<6.multidot.10.sup.-11 M).
[1134] d) Characterization of Selected Fusion Phage
[1135] The pH 2.0 fraction from the third passage of the
mini-library was titered and plaques were obtained on a lawn of
cells. Twenty plaques were picked at random and phage derived from
plaques were probed with the CYSB oligonucleotide via the Dot Blot
Procedure. Autoradiography of the filter revealed that all 20
samples gave a positive hybridization signal indicating that fusion
phage were present and the DNA encoding residues 35 to 47 of
BPTI(MGNG) is contained within the recombinant M13 genomes. Rf DNA
was prepared for the 20 clones and initial dideoxy sequencing
revealed that 12 clones were identical. This sequence was
designated EpiNE.alpha. (Table 207). No DNA sequence changes were
observed apart from the planned variegation. Hence the cassette
mutagenesis procedure preserved the context of the planned
variegation of the pepi gene. The Dot Blot Procedure was employed
to probe all 20 selected clones from the pH 2.0 fraction from the
third passage of the mini-library with an oligonucleotide
homologous to the sequence of EpiNE.alpha.. Following high
stringency washing, autoradiography revealed that all 20 selected
clones were identical in the P1 region. Furthermore dot blot
analysis revealed that of the 28 different phage samples pooled to
create the mini-library, only one contained the EpiNE.alpha.
sequence. Hence in just three passes of the mini-library over HNE
beads, 1 out of 28 input fusion phage was selected for and appears
as a pure population in the lowest pH fraction from the third
passage of the library. That the EpiNE.alpha. phage elute at pH 3.5
while BPTI(K15V,R17L)-III MA phage elute at a higher pH strongly
suggests that the EpiNE.alpha. protein has a significantly higher
affinity than BPTI(K15V,R17L) for HNE.
[1136] 4) Fractionation of the MYMUT Library
[1137] a) Three Cycles of Enrichment
[1138] The same procedure used above to fractionation the
mini-library was used to fractionate the entire MYMUT PEPI library
consisting of fusion phage displaying 1000 different proteins. The
phage inputs for the first, second and third rounds of
fractionation were 4.0.multidot.10.sup.11, 5.8.multidot.10.sup.10,
and 1.1.multidot.10.sup.11 pfu respectively. FIG. 8 illustrates
that the largest percentage of input phage which bound to the HNE
matrix was recovered in the pH 5.0 wash in the first enrichment
cycle. The pH elution profile is very similar to that seen for the
first passage of the mini-library over HNE beads. A trailing edge
is also observed on the low pH side of the pH 5.0 peak however this
is not as prominent as that observed for the mini-library. The
percentage of input phage which eluted in the pH 7.0 wash was
greater than that eluted in the pH 6.0 wash. This is in contrast to
the result obtained for the first passage of the mini library and
may reflect the presence of .apprxeq.20% parental BPTI(MGNG)-III MA
phage in the MYMUT library pool. These phage adhere to the HNE
beads weakly (if at all) and elute in the pH 7.0 fraction. That no
parent phage were present in the mini-library is consistent with
the absence of a peak at pH 7.0 in the first passage of the
mini-library.
[1139] Phage present in the pH 2.0 fraction from the first passage
of the MYMUT library were amplified as described previously and
subjected to a second round of fractionation. The largest
percentage of input phage which bound to the HNE beads was
recovered in the pH 3.5 wash (FIG. 8). A minor peak centered on pH
4.5 was also evident. The fact that more extreme pH conditions were
required to elute the majority of bound fusion phage suggested that
selection of fusion phage displaying PEPIs with higher affinity for
HNE had occurred. This was also indicated by the fact that the
total percentage of input phage which appeared in the pH 3.5 wash
in the second enrichment cycle was 10 times greater than the
percentage of input which appeared in the pH 5.0 wash in the first
cycle.
[1140] Fusion phage from the pH 2.0 fraction of the second pass of
the NYMUT library were amplified and subjected to a third passage
over HNE beads. The proportion of fusion phage appearing in the pH
3.5 fraction relative to that in the 4.5 fraction was greater in
the third passage than in the second passage (FIG. 8). Also the
amount of fusion phage appearing in the pH 3.5 fraction was higher
in the third passage than in the second passage. The fact that wash
conditions less than pH 4.25 were required to elute bound fusion
phage derived from the MYMUT library suggests that the EpiNEs
displayed by these phage possess a higher affinity for HNE than the
BPTI(K15V,R17L) variant.
[1141] b) Characterization of Selected Clones
[1142] The pH 2.0 fraction from the third enrichment cycle of the
MYMUT library was titered on a lawn of cells. Twenty plaques were
picked at random. Rf DNA was prepared for each of the clones and
fusion phage were collected by PEG precipitation. Clonally pure
populations of fusion phage in TBS/BSA were prepared and
characterized with respect to their affinity for immobilized HNE.
pH elution profiles were obtained to determine the stringency of
the conditions required to elute bound fusion phage from the HNE
matrix. FIG. 9 illustrates the pH profiles obtained for EpiNE
clones 1, 3, and 7. The pH profiles for all 3 clones exhibit a peak
centered on pH 3.5. Unlike the pH profile obtained for the third
passage of the MYMUT library, no minor peak centered on pH 4.5 is
evident. This is consistent with the clonal purity of the selected
EpiNE phage utilized to generate the profiles. The elution peaks
are not symmetrical and a prominent trailing edge on the low pH
side. In all probability, the 10 minute elution period employed is
inadequate to remove bound fusion phage at the low pH conditions.
EpiNE clones 1 through 8 have the following characteristics: five
clones (identified as EpiNE1, EpiNE3, EpiNE5, EpiNE6, and EpiNE7)
display very similar pH profiles centered on pH 3.5. The remaining
3 clones elute in the pH 3.5 to 4.0 range. There remains some
diversity amongst the 20 randomly chosen clones obtained from the
pH 2.0 fraction of the third passage of the MYMUT library and these
clones might exhibit different affinities for HNE.
[1143] c) Sequences of the EpiNE Clones
[1144] The DNA sequences encoding the P1 regions of the different
EpiNE clones were determined by dideoxy sequencing of Rf DNA. The
sequences are shown in Table 208. Essentially, only the codons
targeted for mutagenesis (i.e. 15 to 19) were altered as a
consequence of cassette mutagenesis using the MYMUT
oligonucleotide. only 1 codon outside the target region was found
to contain an unexpected alteration. In this case, codon 21 of
EpiNE8 was altered from a tyrosine codon (TAT) to a SER codon (TCT)
by a single nucleotide substitution. This error could have been
introduced into the MYMUT oligonucleotide during its synthesis.
Alternatively, an error could have been introduced when the
single-stranded MYMUT oligonucleotide was converted to the
double-stranded form by Sequenase. Regardless of the reason, the
error rate is extremely low considering only 1 unexpected
alteration was observed after sequencing 20 codons in 19 different
clones. Furthermore, the value of such a mutation is not diminished
by its accidental nature.
[1145] Some of the EpiNE clones are identical. The sequences of
EpiNE1, EpiNE3, and EpiNE7 appear a total of 4, 6 and 5 times
respectively. Assuming the 1745 potentially different DNA sequences
encoded by the T oligonucleotide were present at equal frequency in
the fusion phage library, the frequent appearance of the sequences
for clones EpiNE1, EpiNE3, and EpiNE7 may have important
implications. EpiNE1, EpiNE3, and EpiNE7 fusion phage may display
BPTI variants with the highest affinity for HNE of all the 1000
potentially different BPTI variants in the MYMUT library.
[1146] An examination of the sequences of the EpiNE clones is
illuminating. A strong preference for either VAL or ILE at the P1
position (residue 15) is indicated with VAL being favored over ILE
by 14 to 6. In the MYMUT library, VAL at position 15 is
approximately twice as prevalent as ILE. No examples of LEU, PHE,
or MET at the Pi position were observed although the MYMUT
oligonucleotide has the potential to encode these residues at Pi.
This is consistent with the observation that BPTI variants with
single amino acid substitutions of LEU, PHE, or MET for LYS.sub.15
exhibit a significantly lower affinity for HNE than their
counterparts containing either VAL or ILE (BECK88b).
[1147] PHE is strongly favored at position 17, appearing in 12 of
20 codons. MET is the second most prominent residue at this
position but it only appears when VAL is present at position 15. At
position 18 PHE was observed in all 20 clones sequenced even though
the MYMUT oligonucleotide is capable of encoding other residues at
this position. This result is quite surprising and could not be
predicted from previous mutational analysis of BPTI, model
building, or on any theoretical grounds. We infer that the presence
of PHE at position 18 significantly enhances the ability each of
the EpiNEs to bind to HNE. Finally at position 19, PRO appears in
10 of 20 codons while SER, the second most prominent residue,
appears at 6 of 20 codons. Of the residues targeted for mutagenesis
in the present study, residue 19 is the nearest to the edge of the
interaction surface of a PEPI with HNE. Nevertheless, a
preponderance of PRO is observed and may indicate that PRO at 19,
like PHE at 18, enhances the binding of these proteins to HNE.
Interestingly, EpiNEs appears only once and differs from EpiNE1
only at position 19; similarly, EpiNE6 differs from EpiNE3 only at
position 19. These alterations may have only a minor effect on the
ability of these proteins to interact with HNE. This is supported
by the fact that the pH elution profiles for EpiNE5 and EpiNE6 are
very similar to those of EpiNE1 and EpiNE3 respectively.
[1148] Only EpiNE2 and EpiNE8 exhibit pH profiles which differ from
those of the other selected clones. Both clones contain LYS at
position 19 which may restrict the interaction of BPTI with HNE.
However, we can not exclude the possibility that other alterations
within EpiNE2 and EpiNE8 (R15L and Y21S respectively) influence
their affinity for HNE.
[1149] EpiNE7 was expressed as a soluble protein and analyzed for
HNE inhibition activity by the fluorometric assay of Castillo et
al. (CAST79); the data were analyzed by the method of Green and
Work (GREE53). Preliminary results indicate that
K.sub.d(HNE,EpiNE7).ltoreq.8.- .multidot.10.sup.-12 M, i.e. at
least 7.5-fold lower than the lowest K.sub.d reported for a BPTI
derivative with restect to HNE.
[1150] C. Summary
[1151] Taken together, these data show that the alterations which
appear in the P1 region of the EPI mutants confer the ability to
bind to HNE and hence be selected through the fractionation
process. That the sequences of EpiNE1, EpiNE3, and EpiNE7 appear
frequently in the population of selected clones suggests that these
clones display BPTI variants with the highest affinity for HNE of
any of the 1000 potentially different variants in the MYMUT
library. Furthermore, that pH conditions less than 4.0 are required
to elute these fusion phage from immobilized HNE suggests that they
display BPTI variants having a higher affinity for HNE than
BPTI(K15V,R17L). EpiNE7 exhibits a lower Kd toward HNE than does
BPTI(K15V,R17L); EpiNE1 and EpiNE3 should are also expected to
exhibit lower Kds for HNE than BPTI(K15V,R17L). It is possible that
all of the listed EpiNEs have lower K.sub.ds than
BPRI(K15V,R17L).
[1152] Position 18 has not previously been identified as a key
position in determining specificity or affinity of aprotinin
homologues or derivatives for particular serine proteases. None
have reported or suggested that phenylalanine at position 18 will
confer specificity and high affinity for HNE. One of the powerful
advantages of the present invention is that many diverse amino-acid
sequences may be tested simultaneously.
EXAMPLE V
[1153] Screening of the MYMUT Library for Binding to Cathepsin G
Beads.
[1154] We fractionated the MYMUT library over immobilized human
Cathepsin G to find an engineered protease inhibitor having high
affinity for Cathepsin G, hereafter designated as an EpiC. The
details of phage binding, elution of bound phage with buffers of
decreasing pH (pH profile), titering of the phage contained in
these fractions, composition of the MYMUT library, and the
preparation of cathepsin G (Cat G) beads are essentially the same
as detailed in Example IV.
[1155] A pH profile for the binding of two starting controls,
BPTI-III MK and EpiNE1, are shown in FIG. 10. BPTI-III MK phage,
which contains wild type BPTI fused to the III gene product, shows
no apparent binding to Cat G beads in this assay. EpiNE1 phage was
obtained by enrichment with HNE beads (Example IV and Table 208).
EpiNE1-III MK demonstrated little binding to Cat G beads in the
assay, although a small peak or shoulder is visible in the pH 5
eluted fraction.
[1156] FIG. 11 shows the pH profiles of the MYMUT library phage
when bound to Cat G beads. Library-Cat G interaction was monitored
using three cycles of binding, pH elution, transduction of the pH 2
eluted phage, growth of the transduced phage and rebinding of any
selected phage to Cat G beads, in an exact copy of that used to
find variants of BPTI which bound to HNE. In contrast to the pH
profiles elicited with HNE beads, little enhancement of binding was
observed for the same phage library when cycled with Cat G beads
(with the exception of a possible `shoulder` developing in the pH5
elutions).
[1157] To investigate the elution profile around the pH 5 point in
more detail, the binding of phage taken from the pH 4 eluted
fraction (bound to Cat G beads) rather than the previously used pH
2 fraction was examined. FIG. 12 demonstrates a marked enhancement
of phage binding to the Cat G beads with an apparent elution peak
of pH 5. The binding, as a fraction of the input phage population,
increased with subsequent binding and elution cycles.
[1158] Individual phage clones were picked, grown and analyzed for
binding to Cat G beads. FIG. 13 shows the binding and pH profiles
for the individual Cat G binding clones (designated EpiC variants).
All clones exhibited minor peaks, superimposed upon a gradual fall
in bound phage, at pH elutions of 5 (clones 1, 8, 10 and 11) or pH
4.5 (clone 7).
[1159] DNA sequencing of the EpiC clones, shown in Table 209,
demonstrated that the clones selected for binding to Cat G beads
represented a distinct subset of the available sequences in the
MYMUT library and a cluster of sequences different from that
obtained when enriched with HNE beads. The P1 residue in the EpiC
mutants is predominantly MET, with one example of PHE, while in
BPTI it is LYS and in the EpiNE variants it is either VAL or LEU.
In the EpiC mutants residue 16 is predominantly ALA with one
example of GLY and residue 17 is PHE, ILE or LEU. Interestingly
residues 16 and 17 appear to pair off by complementary size, at
least in this small sample. The small GLY residue pairs with the
bulky PHE while the relatively larger ALA residue pairs with the
less bulky LEU and ILE. The majority of the available residues in
the MYMUT library for positions 18 and 19 are represented in the
EpiC variants.
[1160] Hence, a distinct subset of related sequences from the MYMUT
library have been selected for and demonstrated to bind to Cat G. A
comparison of the pH profiles elicited for the EpiC variants with
Cat C and the EpiNE variants for HNE indicates that the EpiNE
variants have a high affinity for HNE while the EpiC variants have
a moderate affinity for Cat G. Nonetheless, the starting molecule,
BPTI, has virtually no detectable affinity for Cat G and the
selection of clones with a moderate affinity is a significant
finding.
EXAMPLE VI
[1161] Second Round of Variegation of EpiNE7 to Enhance Binding to
HNE
[1162] A. Mutagenesis of EpiNE7 Protein in the Loop Comprising
Residues 34-41
[1163] In Example IV, we described engineered protease inhibitors
EpiNE1 through EpiNE8 that were obtained by affinity selection.
Modeling of the structure of the BPTI-Trypsin complex (Brookhaven
Protein Data Bank entry 1TPA) indicates that the EpiNE protein
surface that interacts with HNE is formed not only by residues
15-19 but also by residues 34-40 that are brought close to this
primary loop when the protein folds (HUBE74, HUBE75, OAST88).
Acting upon this assumption, we changed amino acid residues in a
second loop of the EpiNE7 protein to find EpiNE7 derivatives having
higher affinity for HNE.
[1164] In the complex of BPTI and trypsin found in Brookhaven
Protein Data Bank entry 1TPA ("1TPA complex"), VAL.sub.34 contacts
TYR.sub.151 and GLN192. (Residues in trypsin or HNE are underscored
to distinguish them from the inhibitor.) In HNE, the corresponding
residues are ILE.sub.151 and PHE.sub.192, ILE is smaller and more
hydrophobic than TYR. PHE is larger and more hydrophobic than GLN.
Neither of the HNE side groups have the possibility to form
hydrogen bonds. When side groups larger than that of VAL are
substituted at position 34, interactions with residues other than
151 and 192 may be possible. In particular, an acidic residue at 34
might interact with ARG.sub.147 of HNE that corresponds to
SER.sub.147 of trypsin in lTPA. Table 15 shows that, in 59
homologues of BPTI, 13 different amino acids have been seen at
position 34. Thus we allow all twenty amino acids at 34.
[1165] Position 36 is not highly varied; only GLY, SER, and ARG
have been observed with GLY by far the most prevalent. In the 1TPA
complex, GLY.sub.36 contacts HIS.sub.57 and GLN.sub.192. HIS.sub.57
is conserved and GLN.sub.192 corresponds to PHE.sub.192 of HNE.
Adding a methyl group to GLY.sub.36 could increase hydrophobic
interactions with PHE.sub.192 of HNE. GLY.sub.36 is in a
conformation that most amino acids can achieve: .phi.=-79.degree.
and .psi.=-9.degree. (Deisenhoffer cited in CREI84, p.222.).
[1166] In the lTPA complex, ARG.sub.39 contacts SER.sub.96,
ASN.sub.97, THR.sub.98, LEU.sub.99, GLN.sub.175, and TRP.sub.215.
In HNE, all of the corresponding residues are different! SER96 is
deleted; ASN.sub.97 corresponds to ASP.sub.97 (bearing a negative
charge); THR.sub.98 corresponds to PRO.sub.98; LEU.sub.99
corresponds to the residues VAL.sub.99, ASN.sub.99a, and
LEU.sub.99b; GLN.sub.175 is deleted; and TRP.sub.215 corresponds to
PHE.sub.215. Position 39 shows a moderately high degree of
variability with 7 different amino acids observed, viz. ARG, GLY,
LYS, GLN, ASP, PRO, and MET. Having seen PRO (the most rigid amino
acid), GLY (the most flexible amino acid), LYS and ASP (basic and
acidic amino acids), we assume that all amino acids are
structurally compatible with the aprotinin backbone. Because the
context of residue 39 has changed so much, we allow all 20 amino
acids.
[1167] Position 40 is not highly variable; only GLY and ALA have
been observed (with similar frequency, 24:16). Position 41 is
moderately varied, showing ASN, LYS, ASP, GLN, HIS, GLU, and TYR.
The side groups of residues 40 and 41 are not thought to contact
trypsin in the 1TPA complex. Nevertheless, these residues can exert
electrostatic effects and can influence the dynamic properties of
residues 39, 38, and others. The choice of residues 34, 36, 39, 40,
and 41 to be varied simultaneously illustrates the rule that the
varied residues should be able to touch one molecule of the target
material at one time or be able to influence residues that touch
the target. These residues are not contiguous in sequence, nor are
they contiguous on the surface of EpiNE7. They can, nonetheless,
all influence the contacts between the EpiNE and HNE.
[1168] Amino acid residues VAL.sub.34, GLY.sub.36, MET.sub.39,
GLY.sub.40, and ASN.sub.41 were variegated as follows: any of 20
genetically encodable amino acids at positions 34 and 39 (NNS
codons in which N is approximately equimolar A,C,T,G and S is
approximately equimolar C and G), GLY or ALA at position 36 and 40
(GST codon), and [ASP, GLU, HIS, LYS, ASN, GLN, TYR, or stop] at
position 41 (NAS codon). Because the PEPIs are displayed fused to
gIII protein, DNA containing stop codons will not give rise to
infectuous phage in non-suppressor hosts.
[1169] For cassette mutagenesis, a 61 base long oligonucleotide DNA
population was synthesized that contained 32,768 different DNA
sequences coding on expression for a total of 11,200 amino acid
sequences. This oligonucleotide extends from the third base of
codon 51 in Table 113 (the middle of the StuI site) to base 2 of
codon 70 (the EagI site (identified as XmaIII in Table 113)).
[1170] We used a mutagenesis method similar to that described by
Cwirla et al. (CWIR90) and other standard DNA manipulations
described in Maniatis et al. (MANI82) and Sambrook et al. (SAMB89).
EpiNE7 RF DNA was restricted with EaqI and StuI, agarose gel
purified, and dephosphorylated using HK.sup.(TM) phosphatase
(Epicentre Technologies). We prepared insert by annealing two
small, 16 base and 17 base, phosphorylated synthetic DNA primers to
the phosphorylated 61 base long oligonucleotide population
described above. The resulting insert DNA population had the
following features: double stranded DNA ends capable of
regenerating upon ligation the EagI (5' overhang) and StuI (blunt)
restricted sites of the EpiNE7 RF DNA, and single stranded DNA in
the central mutagenic region. Insert and EpiNE7 vector DNA were
ligated. Ligation samples were used to transfect competent
XL1-Blue.sup.(TM) cells which were subsequently plated for
formation of ampicillin resistant (Ap.sup.R) colonies. The
resulting phage-producing, Ap.sup.R colonies were harvested and
recombinant phage was isolated. By following these procedures, a
phage library of 1.2.multidot.10.sup.5 independent transformants
was assembled. We estimated that 97.4% of the approximately
3.3.multidot.10.sup.4 possible DNA sequences were represented:
0.974=(1-exp{-1.2.multidot.10.sup.5/32768})
[1171] The probability of observing the parental sequence is higher
than 0.974 because VAL occurs twice in the NNS codon: 4 Probability
of seeing ( V 34 , G 36 , M 39 , G 40 , N 41 ) = ( 1 - exp { - 1.2
10 5 .times. 2 / 32768 ) } = ( 1 - exp { - 7.32 } ) = ( 1 - 6.5 10
- 4 ) = 0.99934
[1172] Furthermore, we expect that a small amount (for example, 1
part in 1000) of uncut or once-cut and religated parental vector
would come through the procedures used. Thus the parental sequence
is almost certainly present in the library. This library is
designated the KLMUT library.
[1173] B. Affinity Selection with Immobilized Human Neutrophil
Elastase
[1174] 1) First Fractionation
[1175] We added 1.1.multidot.10.sup.8 plaque forming units of the
KLMUT library to 10 .mu.l of a 50% slurry of agarose-immobilized
human neutrophil elastase beads (HNE from Calbiochem cross-linked
to Reacti-Gel.sup.(TM) agarose beads from Pierce Chemical Co.
following manufacturer's directions) in TBS/BSA. Following 3 hours
incubation at room temperature, the beads were washed and phage was
eluted as done in the selection of EpiNE phage isolates (Example
IV). The progression in lowering pH during the elution was: pH 7.0,
6.0, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, and 2.0. Beads carrying phage
remaining after pH 2.0 elution were used to infect
XL1-Blue.sup.(TM) cells that were plated to allow plaque formation.
The 348 resulting plaques were pooled to form a phage population
for further affinity selection. A population of phage particles
containing 6.0.multidot.10.sup.8 plaque forming units was added to
10 .mu.l of a 50% slurry of agarose-immobilized HNE beads in
TBS/BSA and the above selection procedure was repeated.
[1176] Following this second round of affinity selection, a portion
of the beads was mixed with XL1-Blue.sup.(TM) cells and plated to
allow plaque formation. of the resulting plaques, 480 were pooled
to form a phage population for a third affinity selection. We
repeated the selection procedure described above using a population
of phage particles containing 3.0.multidot.10.sup.9 plaque forming
units. Portions of the pH 2.0 eluate and of the beads were plated
with XL1-Blue.sup.(TM) cells to allow formation of plaques.
Individual plaques were picked for preparation of RF DNA. From DNA
sequencing, we determined the amino acid sequence in the mutated
secondary loop of 15 EpiNE7-homolog clones. The sequences are given
in Table 210 as EpiNE7.1 through EpiNE7.20. Three sequences were
observed twice: EpiNE7.4 and EpiNE7.14; EpiNE7.8 and EpiNE7.9; and
EpiNE7.10 and EpiNE7.20. EpiNE7.4 was eluted at pH 2 while
EpiNE7.14 was obtained by culturing HNE beads that had been washed
with pH 2 buffer. Similarly, EpiNE7.10 came from pH 2 elution but
EpiNE7.20 came from beads. EpiNE7.8 and EpiNE7.9 both came from pH
2 elution. Interestingly, EpiNE7.8 is found in both the first and
second fractionations (EpiNE7.31 (vide infra)).
[1177] b 2) Second Fractionation
[1178] The purpose of affinity fractionation is to reduce diversity
on the basis of affinity for the target. The first enrichment step
of the first fractionation reduced the population from
3.multidot.10.sup.4 possible DNA sequences to no more than 348.
This might be too severe and some of the loss of diversity might
not be related to affinity. Thus we carried out a second
fractionation of the entire KLMUT library seeking to reduce the
diversity more gradually.
[1179] We added 2.0.multidot.10.sup.11 plaque forming units of the
KLMUT library to 10 .mu.l of a 50% slurry of agarose-immobilized
HNE beads in TBS/BSA. Following 3 hours incubation at room
temperature, phage were eluted as described above. We then
transduced XL1-Blue.sup.(TM) cells with portions of the pH 2.0
eluate and plated for Ap.sup.R colonies.
[1180] The resulting phage-producing colonies were harvested to
obtain amplified phage for further affinity selection. A population
of these phage particles containing 2.0.multidot.10.sup.10 plaque
forming units was added to 10 .mu.l of a 50% slurry of
agarose-immobilized HNE beads in TBS/BSA and incubated for 90
minutes at room temperature. Phage were eluted as described above
and portions of the pH 2.0 eluate were used to transduce
XL1-Blue.sup.(TM) cells. We plated the transductants for Ap.sup.R
colonies and obtained amplified phage from the harvested
colonies.
[1181] In a third round of affinity selection, a population of
phage particles containing 3.0.multidot.10.sup.10 plaque forming
units was added to 20 .mu.l of 50% slurry of agarose-immobilized
HNE beads and incubated for 2 hours at room temperature. We eluted
the phage with the following pH washes: pH 7.0, 6.0, 5.0, 4.5, 4.0,
3.5, 3.25, 3.0, 2.75, 2.5, 2.25, and 2.0. After plating a portion
of the pH 2.0 eluate fraction for plaque formation, we picked
individual plaques for preparation of RF DNA. DNA sequencing
yielded the amino acid sequence in the mutated secondary loop for
20 EpiNE7 homolog clones. These sequences, together with EpiNE7,
are given in Table 210 as EpiNE7.21 through EpiNE7.40. The plaques
observed when EpiNEs are plated display a variety of sizes.
EpiNE7.21 through EpiNE7.30 were picked with attention to plaque
size: 7.21, 7.22, and 7.23 from small plaques, 7.24 through 7.30
from plaques of increasing size, with 7.30 coming from a large
plaque. TRP occurs at position 39 in EpiNE7.21, 7.22, 7.23, 7.25,
and 7.30. Thus plaque size does not correlate with the appearance
of TRP at 39. One sequence, EpiNE7.31, from this fractionation is
identical to sequences EpiNE7.8 and EpiNE7.9 obtained in the first
fractionation. EpiNE7.30, EpiNE7.34, and EpiNE7.35 are identical,
indicating that the diversity of the library has been greatly
reduced. It is believed that these sequences have an affinity for
HNE that is at least comparable to that of EpiNE7 and probably
higher. Because the parental EpiNE7 sequence did not recur, it is
quite likely that some or all of the EpiNE7.nn derivatives have
higher affinity for HNE than does EpiNE7.
[1182] 3) Conclusions
[1183] One can draw some conclusions. First, because some sequences
have been isolated repeatedly, the fractionation is nearly
complete. The diversity has been reduced from .gtoreq.10.sup.4 to a
few tens of sequences.
[1184] Second, the parental sequence has not recurred. At 39, MET
did not occur! At position 34 VAL occurred only once in 35
sequences. At 41, ASN occurred only 4 of 35 times. At 40, GLY
occurred 17 of 35 times. At position 36, GLY occurred 34 of 35
times, indicating that ALA is undesirable here. EpiNE7.24 and
EpiNE7.36 are most like EpiNE7, having three of the varied residues
identical to EpiNE7.
[1185] Third, the results of the first and second fractionation are
similar. In the second fractionation, the prevalence of TRP at
position 39 is more marked ({fraction (5/15)} in fractionation #1,
{fraction (14/20)} in #2). It is possible that the first
fractionation lost some high-affinity EPIs through under-sampling.
Nevertheless, the first fractionation was clearly quite
successful.
[1186] Fourth, there are strong preferences at positions 39 and 36
and lesser but significant preferences at positions 34 and 41 with
little preference at 40.
[1187] Heretofore, no homologues of aprotinin have been reported
having ALA at 36. In the selected EpiNE7.nn sequences, the
preference for GLY over ALA at position 36 is 34:1. This preference
is probably not due to differences in protein stability. The
process of the present invention, as applied in the present
example, does not select against proteins on the basis of stability
so long as the protein does fold and function at the temperature
used in the procedure. ALA is probably tolerated at position 36
well enough to allow those proteins having ALA.sub.36 to fold and
function; one example was found having ALA.sub.36. It may be
relevant that the sole sequence having ALA.sub.36 also has
GLY.sub.34. The flexibility of GLY at 34 may allow the methyl of
ALA at 36 to fit into HNE in a way that is not possible when other
amino acids occupy position 34.
[1188] At position 39, all 20 amino acids were allowed, but only
seven were seen. TRP is strongly preferred with 19 occurrences, HIS
second with six occurences, and LEU third with 5 occurrences. No
homologues of aprotinin have been reported having either TRP or HIS
at position 39 as are now disclosed. Although LEU is represented in
the NNS codon thrice, TRP and HIS have but one codon each and their
prevalence is surprising. We constructed a model having HNE
(Brookhaven Protein Data Bank entry 1HNE) and EpiNE7.9 spatially
related as in the 1TPA complex. (The a carbons of HNE of conserved
internal residues were superimposed on the corresponding .alpha.
carbons of trypsin, rms deviation .apprxeq.0.5 .ANG..) Inspection
of this model indicates that TRP.sub.39 could interact with the
loop of HNE that comprises VAL.sub.99, ASN.sub.99a, and
LEU.sub.99b. HIS is observed in six cases; HIS is hydrophobic,
aromatic, and in some ways similar to TRP. LEU.sub.39 in EpiNE7.5
could also interact with these residues if the loop moves a short
distance. GLU occurred twice while LYS, ARG, and GLN occurred once
each. In BPTI, the C.sub..alpha. of residue 39 is .apprxeq.10 .ANG.
from the C.sub..alpha. of residue 15 so that TRP.sub.39 interacts
with different features of HNE than do the amino acids substituted
at position 15. Residue 34 is well separated from each of the
residues 15, 18, and 39; thus it contacts different features on the
HNE surface from these residues. Although serine proteases are
highly similar near the catalytic site, the similarity diminishes
rapidly outside this conserved region. The specificity of serine
proteases is in fact determined by more interactions than the P1
residue. To make an inhibitor that is highly specific to HNE, we
must go beyond matching the requirement at P1. Thus, the
substitutions at 18 (determined in Example IV), 39, 34, and other
non-P1 positions are invaluable in customizing the EpiNE to HNE.
When making an inhibitor customized to a different serine protease,
it is likely that many, if not all, of these positions will be
changed to obtain high affinity and specificity. It is a major
advantage of the present method that many such derivatives may be
tested rapidly.
[1189] At position 34, all 20 amino acids were allowed. Fourteen
have been seen. LYS appeared seven times, GLU five times, THR four
times, LEU three times, GLY, ASP, GLN, MET, ASN, and HIS twice
each, and ARG, PRO, VAL, and TYR once each. There were no instances
of ALA, CYS, PHE, ILE, SER, or TRP. No homologue of aprotinin with
GLU, GLY, or MET at 34 has been reported heretofore. Here, as at
position 39, the library contains an excess of LEU over LYS and
GLU. Thus, we infer that the prevalence of LYS, GLU, THR, and LEU
is related to tighter binding of EpiNEs having these amino acids at
position 34. The prevalence of LYS is surprising, as there are no
acidic groups on HNE in the neighborhood. The N.sub.zeta of
LYS.sub.34 could interact with a main-chain carbonyl oxygen while
the methylene groups interact with ILE.sub.151 and/or PHE.sub.192.
LEU.sub.34 could interact with ILE.sub.151 and/or PHE.sub.192 while
GLU.sub.34 could interact with ARG.sub.147.
[1190] There has been little if any enrichment at positions 40 and
41. Alanine is somewhat preferred at 40; ALA:GLY::19:16. Both ALA
and GLY have been reported in aprotinin homologues.
[1191] Position 41 shows a preponderance of LYS (12 occurrences)
and GLU (7), but all eight possibilities have been seen. The
overall distribution is LYS.sup.12, GLU.sup.7, ASP.sup.4,
ASN.sup.4, GLN.sup.3, HIS.sup.3, and TYR.sup.2. Heretofore, no
homologues of aprotinin having GLU, GLN, HIS, or TYR at position 41
have been reported.
[1192] One sequence, EpiNE7.25 contains an unexpected change at
position 47, SER to LEU. Heretofore, all homologues of aprotinin
reported have had either SER or THR at position 47. The side groups
of SER and THR can form hydrogen bonds to main-chain atoms at the
beginning of the short .alpha. helix.
[1193] The consensus sequence, LYS.sub.34, GLY.sub.36, TRP.sub.39,
ALA.sub.40, LYS.sub.41 was not observed. EpiNE7.23 is quite close,
differing only at position 40 where the preference for ALA is very,
very weak.
[1194] We tested EpiNE7.23 (the sequence closest to consensus)
against EpiNE7 on HNE beads. FIG. 16 shows the fractionation of
strains of phage that display these two EpiNEs. Phage that display
EpiNE7 are eluted at higher pH than are phage that display
EpiNE7.23. Furthermore, more of the EpiNE7.23 phage are retained
than of the EpiNE7 phage. Note the peak at pH 2.25 in the EpiNE7.23
elution. This suggests that EpiNE7.23 has a higher affinity for HNE
than does EpiNE7. In a similar way, we tested EpiNE7.4 and found
that it is not retained on HNE so well as EpiNE7. This is
consistent with the fractionation not being complete.
[1195] Further fractionation, characterization of clonally pure
EpiNE7.nn strains, and biochemical characterization of soluble
EpiNE7.nn derivatives will reveal which sequences in this
collection have the highest affinity for HNE.
[1196] Fractionation of the library involves a number of factors.
Differential binding allows phage that display PBDs having the
desired binding properties to be enriched. Differences in
infectivity, plaque size, and phage yield are related to
differences in the sequence of the PBDs, but are not directly
correlated to affinity for the target. These factors may reduce the
effectiveness of the desired fractionation. An additional factor
that may be present is differential abundance of PBD sequences in
the initial library. One step we employ to reduce the effect of
differential infectivity is to transduce cells with isolated phage
rather than to infect them. In the first fractionation, we did not
obtain sufficient material for transduction and so infected cells;
this fractionation was successful. Because the parental sequence,
EpiNE7, was selected for a sequence at residues 15 through 19 that
confer high affinity for HNE, we believe that many, if not most,
members of the KLMUT population have significant affinity for HNE.
Thus the present fractionations must separate variants having very
high affinity for HNE from those merely having high affinity for
HNE. It is perhaps relevant that BPTI-III MK phage are only
partially eluted from immobilized trypsin at pH 2.2.;
K.sub.d(trypsin,BPTI)=6.0.multidot.10.sup.-14 M. Elution of
EpiNE7-III MA phage from immobilized HNE gives a peak at about pH
3.5 with some phage appearing at lower pH;
K.sub.d(HNE,EpiNE7).ltoreq.1..mult- idot.10.sup.-11 M. We recycled
phage that either were eluted at pH 2.0 or that were retained after
elution with pH 2.0 buffer. A large percentage of EpiNE7-III MA
phage would have been washed away with the fractions at pHs less
acid than 2.0. This, together with the marked preferences at
positons 39, 36, and 34, strongly sugestes that we have
successfully fractionated the KLMUT library on the basis of
affinity for HNE and that the EpiNE7.nn proteins have higher
affinity for HNE than does EpiNE7 or any other reported aprotinin
derivative.
[1197] Fractionation in a few stringent steps emphasizes the
affinity of the PBD and allows isolation of variants that confer a
small-plaque phenotype on cells (through low infectivity or by
slowing cell growth). More gradual fractionation allows observation
of a wider variety of variants that show high affinity and favors
sequences that start at low abundance. Gradual fractionation also
favors selection of variants that do not confer a small-plague
phenotype; such variants may be easier to work with and are
preferred for some purposes. In either case, it is preferred to
fractionate until there is a manageable number of distinct isolates
and to characterize these isolates as pure clones. Thus, it is
desirable, in most cases, to fractionate a library in more than one
way.
[1198] None have identified positions 39 and 34 as key in
determining the affinity and specificity of aprotinin homologues
and derivatives for particular serine proteases. None have
suggested the tryptophan at 39 or charged amino acids (LYS or GLU)
at 34 will enhance binding of an aprotinin homologue to HNE.
Different substitutions at these positions is likely to confer
different specificity on those derivatives. One of the major
advantages of the present invention is that many substitutions at
several locations may be tested with an amount of effort not much
greater than is required to test a single derivative by previously
used methods.
[1199] There exist a number of proteases produced by lymphocytes.
Neutrophil elastase is not the only lymphocytic protease that
degrades elastin. The protease p29 is related to HNE. Screening the
MYMUT and KLMUT libraries against immobilized p29 is likely to
allow isolation of an aprotinin derivative having high affinity for
p29.
EXAMPLE VII
[1200] BPTI:VIII Boundary Extensions.
[1201] The aim of this work was to introduce peptide extensions
between the C-terminus of the BPTI domain and the N-terminus of the
M13 major coat protein within the fusion protein. The reasons for
this were two fold; firstly to alter potential protease cleavage
sites at the interdomain boundary (as evidenced by an apparent
instability of the fusion protein) and secondly to increase
interdomain flexibility.
[1202] 1) Insertion of a Variegated Pentapentide at the BPTI:VIII
Interface.
[1203] The gene shown in Table 113 was modified by insertion of
five RVT codons between codon 81 and 82. Two synthetic
oligonucleotides were designed and custom synthesized. The first
consisted of, from 5' to 3': a) from base 2 of codon 77 to the end
of codon 81, b) five copies of RVT, and c) from codon 82 to the
second base of codon 94. The second comprised 20 bases
complementary to the 3' end of the first oligonucleotide. Each RVT
codon allows one of the amino acids [T, N, S, A, D, and G] to be
encoded. This variegation codon was picked because: a) each amino
acid occurs once, and b) all these amino acids are thought to
foster a flexible linker. When annealed, the primed variegated
oligonucleotide was converted to double-stranded DNA using standard
methods.
[1204] The duplex was digested with restriction enzymes SfiI and
NarI and the resulting 45 base-pair fragment was ligated into a
similarly cleaved OCV, M13MB48 (Example I.1.iii.a). The ligated
material was transfected into competent E. coli cells (strain
XL1-Blue.sup.(TM)) and plated onto a lawn of the same cells on
normal bacterial growth plates to form plaques. The bacteriophage
contained within the plaques were analyzed using standard methods
of nitrocellulose lifts and probing using a .sup.32P-labeled
oligonucleotide complementary to the DNA sequence encoding the
fusion protein interface. Approximately 80% of the plaques probed
poorly with this oligonucleotide and hence contained new sequences
at this position.
[1205] A pool of phages, containing the novel interface
pentapeptide extensions, was collected by combining the phage
extracted from the plated plaques.
[1206] 2. Adding Multiple Unit Extensions to the Fusion Protein
Interface.
[1207] The M13 gene III product contains `stalk-like` regions as
implied by electron micrographic visualization of the bacteriophage
(LOPE85). The predicted amino acid sequence of this protein
contains repeating motifs, which include:
[1208] glu.gly.gly.gly.ser (EGGGS) seven times
[1209] gly.gly.gly.ser (GGGS) three times
[1210] glu.gly.gly.gly.thr (EGGGT) once.
[1211] The aim of this section was to insert, at the domain
interface, multiple unit extensions which would mirror the
repeating motifs observed in the III gene product.
[1212] Two synthetic oligonucleotides were designed and custom
synthesized. GLY is encoded by four codons (GGN); when translated
in the opposite direction, these codons give rise to THR, PRO, ALA,
and SER. The third base of these codons was picked so that
translation of the oligonucleotide in the opposite direction would
encode SER. When annealed the synthetic oligonucleotides give the
following unit duplex sequence (an EGGGS linker):
23 E G G G S 5' C.GAG.GGA.GGA.GGA.TC 3' 3' TC.CCT.CCT.CCT.AGG.C 5'
(L) (S) (S) (S) (G)
[1213] The duplex has a common two base pair 5' overhang (GC) at
either end of the linker which allows for both the ligation of
multiple units and the ability to clone into the unique NarI
recognition sequence present in OCV's M13MB48 and Gem MB42. This
site is positioned within 1 codon of the DNA encoding the
interface. The cloning of an EGGGS linker (or multiple linker) into
the vector NarI site destroys this recognition sequence. Insertion
of the EGGGS linker in reverse orientation leads to insertion of
GSSSL into the fusion protein.
[1214] Addition of a single EGGGS linker at the NarI site of the
gene shown in Table 113 leads to the following gene:
24 79 80 80a 80b 80c 80d 80e 81 82 83 84 G G E G G G S A A E G
GGT.GGC.GAG.GGA.GGA.GGA.- TCC.GCC.GCT.GAA.GGT
[1215] Note that there is no preselection for the orientation of
the linker(s) inserted into the OCV and that multiple linkers of
either orientation (with the predicted EGGGS or GSSSL amino acid
sequence) or a mixture of orientations (inverted repeats of DNA)
could occur.
[1216] A ladder of increasingly large multiple linkers was
established by annealing and ligating the two starting
oligonucleotides containing different proportions of 5'
phosphorylated and non-phosphorylated ends. The logic behind this
is that ligation proceeds from the 3' unphosphorylated end of an
oligonucleotide to the 5' phosphorylated end of another. The use of
a mixture of phosphorylated and non-phosphorylated oligonucleotides
allows for an element of control over the extent of multiple linker
formation. A ladder showing a range of insert sizes was readily
detected by agarose gel electrophoresis spanning 15 bp (1 unit
duplex-5 amino acids) to greater than 600 base pairs (40 ligated
linkers-200 amino acids).
[1217] Large inverted repeats can lead to genetic instability. Thus
we chose to remove them, prior to ligation into the OCV, by
digesting the population of multiple linkers with the restriction
enzymes AccIII or XhoI, since the linkers, when ligated
`head-to-head` or `tail-to-tail`, generate these recognition
sequences. Such a digestion significantly reduces the range in
sizes of the multiple linkers to between 1 and 8 linker units (i.e.
between 5 and 40 amino acids in steps of 5), as assessed by agarose
gel electrophoresis.
[1218] The linkers were ligated (as a pool of different insert
sizes or as gel-purified discrete fragments) into NarI cleaved OCVs
M13MB48 or GemMB42 using standard methods. Following ligation the
restriction enzyme NarI was added to remove the self-ligating
starting OCV (since linker insertion destroys the NarI recognition
sequence). This mixture was used to transform competent XL-1 blue
cells and appropriately plated for plaques (OCV M13MB48) or
ampicillin resistant colonies (OCV GemMB42).
[1219] The transformants were screened using dot blot DNA analysis
with one of two .sup.32P labeled oligonucleotide probes. One probe
consisted of a sequence complementary to the DNA encoding the P1
loop of BPTI while the second had a sequence complementary to the
DNA encoding the domain interface region. Suitable linker
candidates would probe positively with the first probe and
negatively or poorly with the second. Plaque purified clones were
used to generate phage stocks for binding analyses and BPTI display
while the Rf DNA derived from phage infected bacterial cells was
used for restriction enzyme analysis and sequencing. Representative
insert sequences of selected clones analyzed are as follows:
25 M13.3X4 (GG)C.GGA.TCC.TCC.TCC.CT(C.GCC) gly ser ser ser leu
M13.3X7 (G C.GAG.GGA.GGA.GGA.TC(C.GCC) glu gly gly gly ser M13.3X11
(GG)C.GAG.GGA.GGA.GGA.TCC.GGA.TCC.TCC. glu gly gly gly ser gly ser
ser TCC.CTC.GGA.TCC.TCC.TCC.CT(C.GCCC) ser leu gly ser ser ser
leu
[1220] These highly flexible oligomeric linkers are believed to be
useful in joining a binding domain to the major coat (gene VIII)
protein of filamentous phage to facilitate the display of the
binding domain on the phage surface. They may also be useful in the
construction of chimeric OSPs for other genetic packages as
well.
EXAMPLE VIII
[1221] Bacterial Expression Vectors.
[1222] The expression vectors were designed for the bacterial
production of BPTI analogues resulting from the mutagenesis and
screening for variants with specific binding properties. The
expression vectors used are derivatives of the OCV's M13MB48 and
GemMB42. The conversion was achieved by replacing the first codon
of the mature VIII gene (codon 82 as shown in Table 113) with a
translational stop codon by site specific mutagenesis.
[1223] The salient points of the expression vector composition are
identical to that of the parent OCV's, namely a lacUV5 promoter
(hence IPTG induction), ribosome binding site, initiating
methionine, pho A signal peptide and transcriptional termination
signal (see Table 113). The placement of the stop codon allows for
the expression of only the first half the fusion protein. The
Gem-based expression system, containing the genes encoding BPTI
analogues, is stored as plasmid DNA, being freshly transfected into
cells for expression of the analogue protein. The M13-based
expression system is stored as both RF DNA and as phage stocks. The
phage stocks are used to infect fresh bacterial cells for
expression of the protein of interest.
[1224] Bacterial Expression of BPTI and Analogues.
[1225] i. Gem-based Expression Vector and Protocol.
[1226] The gem-based expression vector is a derivative of the OCV
GemMB42 (Eample I and Table 113). This vector, at least when it
contains the BPTI or analogue genes, has demonstrated a degree of
insert instability on prolonged growth in liquid culture. To reduce
the risk of this the following protocol is used.
[1227] Expression vector DNA (containing the BPTI or analogue gene)
is transfected into the E. coli strain, XL1-Blue.sup.(TM), which is
plated on bacterial plates containing ampicillin and allowed to
incubate overnight at 37.degree. C. to give a dense population of
colonies. The colonies are scraped from the plate with a glass
spreader in lml of NZCYM medium and combined with the scraped cells
from other duplicate plates. This stock of cells is diluted
approximately one hundred fold into NZCYM liquid medium containing
ampicillin (100 .mu.g per ml) and allowed to grow in a shaking
incubator to a cell density of approximately half log (absorbance
of 0.3 at 600 nm). IPTG is added to a final concentration of 0.5 mM
and the induced culture allowed to grow for a further two hours
when it is processed as described below.
[1228] ii. M13-based Expression Vector and Protocol.
[1229] The M13-based expression vector is derived from OCV M13MB48
(Example I). The BPTI gene (or analogue) is contained within the
intergenic region and its transcription is under the control of a
lacUV5 promoter, hence IPTG inducible. The expression vector,
containing the gene of interest, is maintained and utilized as a
phage stock. This method enables a potentially lethal or
deleterious gene to be supplied to a bacterial culture and gene
induction to occur only when the bacterial culture has achieved
sufficient mass. Poor growth and insert instability can be
circumvented to a large extent, giving this system an advantage
over the Gem-based vector described above.
[1230] An overnight bacterial culture of XL1-Blue.sup.(TM) or SEF'
is grown in LB medium containing tetracycline (50 .mu.g per ml) to
ensure the presence of pili as sites for bacteriophage binding and
infection. This culture is diluted 100-fold into NZCYM medium
containing tetracycline and bacterial growth allowed to proceed in
an incubator shaker until a cell density of 1.0 (Ab 600 nm) has
been achieved. Phage, containing the expression vector and gene of
interest, are added to the bacterial culture at a multiplicity of
infection (MOI) of 10 and allowed to infect the cells for 30
minutes. Gene expression is then induced by the addition of IPTG to
a final concentration of 0.5 mM and the culture allowed to grow
overnight. Media collection and cell fractionation is as described
elsewhere.
[1231] Bacterial Cell Fractionation.
[1232] After heterologous gene expression the bacterial cell
culture can be separated into the following fractions: conditioned
medium, periplasmic fraction and post-periplasmic cell lysate. This
is achieved using the following procedures.
[1233] The culture is centrifuged to pellet the bacteria, allowing
the supernatant to be stored as conditioned medium. This fraction
contains any exported proteins. The pellet is taken up in 20%
sucrose, 30 mM Tris pH 8 and 1 mM EDTA (80 ml of buffer per gram of
fresh weight pellet) and allowed to sit at room temperature for 10
minutes. The cells are repelleted and taken up in the same volume
of ice cold 5 mM MgSO.sub.4 and left on ice for 10 minutes.
Following centrifugation, to pellet the cells, the supernatant
(periplasmic fraction) is stored. A second round of osmotic shock
fractionation can be undertaken if desired.
[1234] The post-periplasmic pellet can be further lysed as follows.
The pellet is resuspended in 1.5 ml of 20% sucrose, 40 mM Tris pH
8, 50 mM EDTA and 2.5 mg of lysozyme (per gram fresh weight of
starting pellet). After 15 minutes at room temperature 1.15 ml of
0.1% Triton X is added together with 300 .mu.l of 5M NaCl and
incubated for a further 15 minutes. 2.5 ml of 0.2 M triethanolamine
(pH 7.8), 150 .mu.l of 1M CaCl.sub.2, 100 .mu.l of 1M MgCl.sub.2
and 5 .mu.g of DNA'se are added and allowed to incubate, with
end-over-end mixing, for 20 minutes to reduce viscosity. This is
followed by centrifugation with the supernatant being retained as
the post-periplasmic lysate.
[1235] The present invention is not, of course, limited to any
particular expression system, whether bacterial or not.
EXAMPLE IX
[1236] Construction of an ITI-domain I/Gene III Display Vector
[1237] 1. ITI domain I as an IPBD
[1238] Inter-.alpha.-trypsin inhibitor (ITI) is a large (M.sub.r ca
240,000) circulating protease inhibitor found in the plasma of many
mammalian species (for recent reviews see ODOM90, SALI90, GEBH90,
GEBH86). The intact inhibitor is a glycoprotein and is currently
believed to consist of three glycosylated subunits that interact
through a strong glycosaminoglycan linkage (ODOM90, SALI90, ENGH89,
SELL87). The anti-trypsin activity of ITI is located on the
smallest subunit (ITI light chain, unglycosylated M.sub.r ca
15,000) which is identical in amino acid sequence to an acid stable
inhibitor found in urine (UTI) and serum (STI) (GEBH86, GEBH90).
The mature light chain consists of a 21 residue N-terminal
sequence, glycosylated at SER.sub.10, followed by two tandem
Kunitz-type domains the first of which is glycosylated at
ASN.sub.45 (ODOM90). In the human protein, the second Kunitz-type
domain has been shown to inhibit trypsin, chymotrypsin, and plasmin
(ALBR83a, ALBR83b, SELL87, SWAI88). The first domain lacks these
activities but has been reported to inhibit leukocyte elastase
(10.sup.-6>K.sub.i>10.s- up.-9) (ALBR83a,b, ODOM90). CDNA
encoding the ITI light chain also codes for .alpha.-1-microglobulin
(TRAB86, KAUM86, DIAR90); the proteins are separated
post-translationally by proteolysis.
[1239] The N-terminal Kunitz-type of the ITI light chain (ITI-D1,
comprising residues 22 to 76 of the UTI sequence shown in FIG. 1 of
GEBH86) possesses a number of characteristics that make it useful
as an IPBD. The domain is highly homologous to both BPTI and the
EpiNE series of proteins described elsewhere in the present
application. Although an x-ray structure of the isolated domain is
not available, crystallographic studies of the related Kunitz-type
domain isolated from the Alzheimer's amyloid .beta.-protein
(AA.beta.P) precursor show that this polypeptide assumes a crystal
structure almost identical to that of BPTI (HYNE90). Thus, it is
likely that the solution structure of the isolated ITI-D1
polypeptide will be highly similar to the structures of BPTI and
AA.beta.P. In this case, the advantages described previously for
use of BPTI as an IPBD apply to ITI-D1. ITI-D1 provides additional
advantages as an IDBP for the development of specific anti-elastase
inhibitory activity. First, this domain has been reported to
inhibit both leukocyte elastase (ALBR83a,b, ODOM90) and Cathepsin-G
(SWAI88, ODOM90), activities which BPTI lacks. Second, ITI-D1 lacks
affinity for the related serine proteases trypsin, chymotrypsin,
and plasmin (ALBR83a,b, SWAI88), an advantage for the development
of specificity in inhibition. Finally, ITI-D1 is a human-derived
polypeptide so derivatives are anticipated to show minimal
antigenicity in clinical applications.
[1240] 2. Construction of the Display Vector.
[1241] For purposes of this discussion, numbering of the nucleic
acid sequence for the ITI light chain gene is that of TRAB86 and of
the amino acid sequence is that shown for UTI in FIG. 1 of GEBH86.
DNA manipulations were conducted according to standard methods as
described in SAMB89 and AUSU87.
[1242] The protein sequence of human ITI-D1 consists of 56 amino
acid residues extending from LYS.sub.22 to ARG.sub.77 of the
complete ITI light chain sequence. This sequence is encoded by the
168 bases between positions 750 and 917 in the cDNA sequence
presented in TRAB86. The majority of the domain is contained
between a BglI site spanning bases 663 to 773 and a PstI site
spanning bases 903 to 908. The insertion of the ITI-D1 sequence
into M13 gene III was conducted in two steps. First a linker
containing the appropriate ITI sequences outside the central BglI
to PstI region was ligated into the NarI site of phage MA RF DNA.
In the second step, the remainder of the ITI-D1 sequence was
incorporated into the linker-bearing phage RF DNA.
[1243] The linker DNA consisted of two synthetic oligonucleotides
(top and bottom strands) which, when annealed, produced a 54 bp
double-stranded fragment with the following structure (5' to
3'):
[1244] NARI OVERHANG/ITI-5'/BGLI/STUFFER/PSTI/ITI-3'/NARI
OVERHANG
[1245] The NarI OVERHANG sequences provide compatible ends for
ligation into a cut NarI site. The ITI-5' sequence consists of ds
DNA corresponding to the thirteen positions from A750 to T662
immediately 5' adjacent to the BglI site in the ITI-D1 sequence.
Two changes, both silent, are introduced in this sequence: T to C
at position 658 (changes codon for ASP.sub.24 from GAT to GAC) and
G to T at position 661 (changes codon for SER.sub.25 from TCG to
TCT). The sequences BGLI and PSTI are identical to the BglI and
PstI sites, respectively, in the ITI-D1 sequence. The ITI-3'
sequence consists of dsDNA corresponding to the nine positions from
A909 to T917 immediately 3' adjacent to the PstI site in the ITI-D1
sequence. The one base change included in this sequence, A to T at
position 917, is silent and changes the codon for ARG.sub.77 from
CGA to CGT. The STUFFER sequence consists of dsDNA encoding three
residues (5' to 3'): LEU (TTA), TRP(TGG), and SER(TCA). The reverse
complement of the STUFFER sequence encodes two translation
termination codons (TGA and TAA). Phage expressing gene III
containing the linker in opposite orientation to that shown above
will not produce a functional gene III product.
[1246] Phage MA RF DNA was digested with NarI and the linear ca.
8.2 kb fragment was gel purified and subsequently dephosphorylated
using HK phosphatase (Epicentre). The linker oligonucleotides were
annealed to form the linker fragment described above, which was
then kinased using T4 Polynucleotide Kinase. The kinased linker was
ligated to the NarI-digested MA RF DNA in a 10:1 (linker:RF) molar
ratio. After 18 hrs at 16.degree. C., the ligation was stopped by
incubation at 65.degree. C. for 10 min and the ligation products
were ethanol precipitated in the presence of 10 .mu.g of yeast
tRNA. The dried precipitate was dissolved in 5 .mu.l of water and
used to transform D1210 cells by electroporation. After 60 min of
growth in SOC at 37.degree. C., transformed cells were plated onto
LB plates supplemented with ampicillin (Ap, 200 .mu.g/ml). RF DNA
prepared from Ap.sup.r isolates was subjected to restriction enzyme
analysis. The DNA sequences of the linker insert and the
immediately surrounding regions were confirmed by DNA sequencing.
Phage strains containing the ITI Linker sequence inserted into the
NarI site in gene III are called MA-IL.
[1247] Phage MA-IL RF DNA was partially digested with BalI and the
ca. 8.2 kb linear fragment was gel purified. This fragment was
digested with PstI and the large linear fragment was gel purified.
The BglI to PstI fragment of ITI-D1 was isolated from pMGlA (a
plasmid carrying the sequence shown in TRAB86). pMGlA was digested
to completion with BglI and the ca. 1.6 kb fragment was isolated by
agarose gel electrophoresis and subsequent Geneclean (Bio101, La
Jolla, Calif.) purification. The purified BglI fragment was
digested to completion with PstI and EcoRI and the resulting
mixture of fragments was used in a ligation with the BglI and PstI
cut MA-IL RF DNA described above. Ligation, transformation, and
plating were as described above. After 18 hr. of growth on LB Ap
plates at 37.degree. C., Ap.sup.r colonies were harvested with LB
broth supplemented with Ap (200 .mu.g/ml) and the resulting cell
suspension was grown for two hours at 37.degree. C. Cells were
pelleted by centrifugation (10 min at 5000.times. g, 4.degree. C.).
The supernatant fluid was transferred to sterile centrifugation
tubes and recentrifuged as above. The supernatant fluid from the
second centrifugation step was retained as the phage stock
POP1.
[1248] PCR was used to demonstrate the presence of phage containing
the complete ITI-D1-III fusion gene. Upstream PCR primers, 1UP and
2UP, are located spanning nucleotides 1470 to 1494 and 1593 to 1618
of the phage M13 DNA sequence, respectively. A downstream PCR
primer 3DN spans nucleotides 1779 to 1804. Two ITI-D1-specific
primers, IAI-1 and IAI-2, are located spanning positions 789 to 810
and 894 to 914, respectively, in the ITI light chain sequence of
TRAB86. IAI-1 and IAI-2 are used as downstream primers in PCR
reactions with 1UP or 2UP. IAI-1 is entirely contained within the
BglI to PstI region of the ITI-D1 sequence, while IAI-2 spans the
PstI site in the ITI-D1 sequence. When aliquots of POP1 phage were
used as substrates for PCR, template-specific products of
characteristic size were produced in reactions containing lUP or
2UP plus IAI-1 or IAI-2 primer pairs. No such products are obtained
using MA-IL phage as template. No PCR products with sizes
corresponding to complete ITI-D1-gene III templates were obtained
using POP1 phage and the 1UP or 2UP plus 3DN primer pairs. This
last result reflects the low abundance (<1%) of phage containing
the complete ITI-D1 sequence in POP1.
[1249] Preparative PCR was used to generate substrate amounts of
the 330 bp PCR product of a reaction using the 1UP and IAI-2 primer
pair to amplify the POP1 template. The 330 bp PCR product was gel
purified and then cut to completion with BglI and PstI. The 138 bp
BglI to PstI fragment from ITI-D1 was isolated by agarose gel
electrophoresis followed by Qiaex extraction (Qiagen, Studio City,
Calif.). MA-IL phage RF DNA was digested to completion with PstI.
The ca. 8.2 kb linear fragment was gel purified and subsequently
digested to completion with BglI. The BglI digest was extracted
once with phenol:chloroform (1:1), the aqueous phase was ethanol
precipitated, and the pellet was dissolved in TE (pH8.0). An
aliquot of this solution was used in a ligation reaction with the
138 bp BglI to PstI fragment as described above. The ethanol
precipitated ligation products were used to transform
XL1-Blue.sup.(TM) cells by electroporation and after 1 hr growth in
SOC at 37.degree. C., cells were plated on LB Ap plates. A phage
population, POP2, was prepared from Ap.sup.r colonies as described
previously.
[1250] Phage stocks obtained from individual plaques produced on
titration of POP2 were tested by PCR for the presence of the
complete ITI-D1-III gene fusion. PCR results indicate the entire
fusion gene was present in seven of nine isolates tested. RF DNA
from the seven isolates testing positive was subjected to
restriction enzyme analysis. The complete sequence of the ITI-D1
insertion into gene III was confirmed in four of the seven isolates
by DNA sequence analysis. Phage isolates containing the ITI-D1-III
fusion gene are called MA-ITI.
[1251] 3. Expression and display of ITI-DI.
[1252] Expression of the ITI domain I-Gene III fusion protein and
its display on the surface of phage were demonstrated by Western
analysis and phage titer neutralization experiments.
[1253] For Western analysis, aliquots of PEG-purified phage
preparations containing up to 4.multidot.10.sup.10 infective
particles were subjected to electrophoresis on a 12.5%
SDS-urea-polyacrylamide gel. Proteins were transferred to a sheet
of Immobilon-P transfer membrane (Millipore, Bedford, MA) by
electrotransfer. Western blots were developed using a rabbit
anti-ITI serum (SALI87) which had previously been incubated with an
E. coli extract, followed by goat anti-rabbit IgG conjugated to
horse radish peroxidase (#401315, Calbiochem, La Jolla, Calif.). An
immunoreactive protein with an apparent size of ca. 65-69 kD is
detected in preparations of MA-ITI phage but not with preparations
of the parental MA phage. The size of the immunoreactive protein is
consistent with the expected size of the processed ITI-D1-III
fusion protein (ca. 67 kD, as previously observed for the BPTI-III
fusion protein).
[1254] Rabbit anti-BPTI serum has been shown to block the ability
of MK-BPTI phage to infect E. coli cells (Example II). To test for
a similar effect of rabbit anti-ITI serum on the infectivity of
MA-ITI phage, 10 .mu.l aliquots of MA or MA-ITI phage were
incubated in 100 .mu.l reactions containing 10 .mu.l aliquots of
PBS, normal rabbit serum (NRS), or anti-ITI serum. After a three
hour incubation at 37.degree. C., phage suspensions were titered to
determine residual plaque-forming activity. These data are
summarized in Table 211. Incubation of MA-ITI phage with rabbit
anti-ITI serum reduces titers 10- to 100-fold, depending on initial
phage titer. A much smaller decrease in phage titer (10 to 40%) is
observed when MA-ITI phage are incubated with NRS. In contrast, the
titer of the parental MA phage is unaffected by either NRS or
anti-ITI serum.
[1255] Taken together, the results of the Western analysis and the
phage-titer neutralization experiments are consistent with the
expression of an ITI-DI-III fusion protein in MA-ITI phage, but not
in the parental MA phage, such that ITI-specific epitopes are
present on the phage surface. The ITI-specific epitopes are located
with respect to III such that antibody binding to these epitopes
prevents phage from infecting E. coli cells.
[1256] 4. Fractionation of MA-ITI Phage Bound to
Agarose-immobilized Protease Beads.
[1257] To test if phage displaying the ITI-DI-III fusion protein
interact strongly with the proteases human neutrophil elastase
(HNE) or cathepsin-G, aliquots of display phage were incubated with
agarose-immobilized HNE or cathepsin-G beads (HNE beads or Cat-G
beads, respectively). The beads were washed and bound phage eluted
by pH fractionation as described in Examples II and III. The
procession in lowering pH during the elution was: pH 7.0, 6.0, 5.5,
5.0, 4.5, 4.0, 3.5, 3.0, 2.5, and 2.0. Following elution and
neutralization, the various input, wash, and pH elution fractions
were titered.
[1258] The results of several fractionations are summarized in
Table 212 (EpiNE-7 or MA-ITI phage bound to HNE beads) and Table
213 (EpiC-10 or MA-ITI phage bound to Cat-G beads). For the two
types of beads (HNE or Cat-G), the pH elution profiles obtained
using the control display phage (EpiNE-7 or EpiC-10, respectively)
were similar to those seen previously (Examples II and III). About
0.3% of the EpiNE-7 display phage applied to the HNE beads were
eluted during the fractionation procedure and the elution profile
had a maximum for elution at about pH 4.0. A smaller fraction,
0.02%, of the EpiC-10 phage applied to the Cat-G beads were eluted
and the elution profile displayed a maximum near pH 5.5.
[1259] The MA-ITI phage show no evidence of great affinity for
either HNE or cathepsin-G immobilized on agarose beads. The pH
elution profiles for MA-ITI phage bound to HNE or Cat-G beads show
essentially monotonic decreases in phage recovered with decreasing
pH. Further, the total fractions of the phage applied to the beads
that were recovered during the fractionation procedures were quite
low: 0.002% from HNE beads and 0.003% from Cat-G beads.
[1260] Published values of K.sub.i for inhibition neutrophil
elastase by the intact, large (M.sub.r=240,000) ITI protein range
between 60 and 150 nM and values between 20 and 6000 nM have been
reported for the inhibition of Cathepsin G by ITI (SWAI88, ODOM90).
our own measurements of pH fraction of display phage bound to HNE
beads show that phage displaying proteins with low affinity
(>.mu.M) for HNE are not bound by the beads while phage
displaying proteins with greater affinity (nM) bind to the beads
and are eluted at about pH 5. If the first Kunitz-type domain ot
the ITI light chain is entirely responsible for the inhibitory
activity of ITI against HNE, and if this domain is correctly
displayed on the MA-ITI phage, then it appears that the minimum
affinity of an inhibitor for HNE that allows binding and
fractionation of display phage on HNE beads is 50 to 100 nM.
[1261] 5. Alteration of the P1 region of ITI-DI.
[1262] If ITI-DI and EpiNE-7 assume the same configuration in
solution as BPTI, then these two polypeptides have identical amino
acid sequences in both the primary and secondary binding loops with
the exception of four residues about the P1 position. For ITI-DI
the sequence for positions 15 to 20 is (position 15 in ITI-DI
corresponds to position 36 in the UTI sequence of GEBH86): MET15,
GLY16, MET17, THR18, SER19, ARG20. In EpiNE-7 the equivalent
sequence is: VAL15, ALA16, MET17, PHE18, PRO19, ARG20. These two
proteins appear to differ greatly in their affinities for HNE. To
improve the affinity of ITI-DI for HNE, the EpiNE-7 sequence shown
above was incorporated into the ITI-DI sequence at positions 15
through 20.
[1263] The EpiNE-7 sequence was incorporated into the ITI-DI
sequence in MA-ITI by cassette mutagenesis. The mutagenic cassette
consisted of two synthetic 51 base oligonucleotides (top and bottom
stands) which were annealed to make double stranded DNA containing
an Eag I overhang at the 5' end and a Stv I overhang at the 3' end.
The DNA sequence between the Eag I and Stv I overhangs is identical
to the ITI-DI sequence between these sites except at four codons:
the codon for position 15, AT (MET), was changed to GTC (VAL), the
codon for position 16, GGA (GLY), was changed to GCT (ALA), the
codon for position 18, ACC (THR) was changed to TTC (PHE), and the
codon for position 19, AGC (SER), was changed to CCA (PRO). MA-ITI
RF DNA was digested with Eag I and Stv I. The large, linear
fragment was gel purified and used in a ligation with the mutagenic
cassette described above. Ligation products were used to transform
XL1-Blue.sup.tm cells as described previously. Phage stocks
obtained from overnight cultures of Ap.sup.r transductants were
screened by. PCR for incorporation of the altered sequence and the
changes in the codons for positions 15, 16, 18, and 19 were
confirmed by DNA sequencing. Phage isolates containing the
ITI-DI-III fusion gene with the EpiNE-7 changes around the P1
position are called MA-ITI-E7.
[1264] 6. Fractionation of MA-ITI-E7 Phage.
[1265] To test if the changes at positions 15, 16, 18, and 19 of
the ITI-DI-III fusion protein influence binding of display phage to
HNE beads, abbreviated pH elution profiles were measured. Aliquots
of EpiNE-7, MA-ITI, and MA-ITI-E7 display phage were incubated with
HNE beads for three hours at room temperature. The beads were
washed and phage were eluted as described (Example III), except
that only three pH elutions were performed: pH 7.0, 3.5, and 2.0.
The results of these elutions are shown in Table 214.
[1266] Binding and elution of the EpiNE-7 and MA-ITI display phage
were found to be as previously described. The total fraction of
input phages was high (0.4%) for EpiNE-7 phage and low (0.001%) for
MA-ITI phage. Further, the EpiNE-7 phage showed maximum phage
elution in the pH 3.5 fraction while the MA-ITI phage showed only a
monotonic decrease in phage yields with decreasing pH, as seen
above.
[1267] The two strains of MA-ITI-E7 phage show increased levels of
binding to HNE beads relative to MA-ITI phage. The total fraction
of the input phage eluted from the beads is 10-fold greater for
both MA-ITI-E7 phage strains than for MA-ITI phage (although still
40-fold lower that EpiNE-7 phage). Further, the pH elution profiles
of the MA-ITI-E7 phage strains show maximum elutions in the pH 3.5
fractions, similar to EpiNE-7 phage.
[1268] To further define the binding properties of MA-ITI-E7 phage,
the extended pH fractionation procedure described previously was
performed using phage bound to HNE beads. These data are summarized
in Table 215. The pH elution profile of EpiNE-7 display phage is as
previously described. In this more resolved, pH elution profile,
MA-ITI-E7 phage show a broad elution maximum centered around pH 5.
Once again, the total fraction of MA-ITI-E7 phage obtained on pH
elution from HNE beads was about 40-fold less than that obtained
using EpiNE-7 display phage.
[1269] The pH elution behavior of MA-ITI-E7 phage bound to HNE
beads is qualitatively similar to that seen using BPTI[K15L]-III-MA
phage. BPTI with the K15L mutation has an affinity for HNE of
.apprxeq.3..multidot.10- .sup.-9 M. Assuming all else remains the
same, the pH elution profile for MA-ITI-E7 suggests that the
affinity of the free ITI-DI-E7 domain for HNE might be in the nM
range. If this is the case, the substitution of the EpiNE-7
sequence in place of the ITI-DI sequence around the P1 region has
produced a 20- to 50-fold increase in affinity for HNE (assuming
K.sub.i=60 to 150 nM for the unaltered ITI-DI).
[1270] If EpiNE-7 and ITI-DI-E7 have the same solution structure,
these proteins present the identical amino acid sequences to HNE
over the interaction surface. Despite this similarity, EpiNE-7
exhibits a roughly 1000-fold greater affinity for HNE than does
ITI-DI-E7. Again assuming similar structure, this observation
highlights the importance of non-contacting secondary residues in
modulating interaction strengths.
[1271] Native ITI light chain is glycosylated at two positions,
SER10 and ASN45 (GEBH86). Removal of the glycosaminoglycan chains
has been shown to decrease the affinity of the inhibitor for HNE
about 5-fold (SELL87). Another potentially important difference
between EpiNE-7 and ITI-DI-E7 is that of net charge. The changes in
BPTI that produce EpiNE-7 reduce the total charge on the molecule
from +6 to +1. Sequence differences between EpiNE-7 and ITI-DI-E7
further reduce the charge on the latter to -1. Furthermore, the
change in net charge between these two molecules arises from
sequence differences occurring in the central portions of the
molecules. Position 26 is LYS in EpiNE-7 and is THR in ITI-DI-E7,
while at position 31 these residues are GLN and GLU, respectively.
These changes in sequence not only alter the net charge on the
molecules but also position negatively charged residue close to the
interaction surface in ITI-DI-E7. It may be that the occurrence of
a negative charge at position 31 (which is not found in any other
of the HNE inhibitors described here) destabilized the
inhibitor-protease interaction.
EXAMPLE X
Generation of a Variegated ITI-DI Population
[1272] The following is a hypothetical example demonstating how to
obtain a derivative of ITI having high affinity for HNE.
[1273] The results of Example IX demonstrate that the nature of the
protein sequence around the P1 position in ITI-DI can significantly
influence the strength of the interaction between ITI-DI and HNE.
While incorporation of the EpiNE-7 sequence increases the affinity
of ITI-DI for HNE, it is unlikely that this particular sequence is
optimal for binding.
[1274] We generate a large population of potential binding proteins
having differing sequences in the P1 region of ITI-DI using the
oligonucleotide ITIMUT. ITIMUT is designed to incorporate
variegation in ITI-DI at the six positions about and including the
P1 residue: 13, 15, 16, 17, 18, and 19. ITIMUT is synthesized as
one long (top strand) 73 base oligonucleotide and one shorter (24
base) bottom strand oligonucleotide. The top strand sequence
extends from position 770 (G) to position 842 (G) in the sequence
of TREB86. This sequence includes the codons for the positions of
variegation as well as the recognition sequences for the flanking
restriction enzymes Eag I (778 to 783) and Sty I (829 to 834). The
bottom strand oligonucleotide comprises the complement of the
sequence from positions 819 to 842.
[1275] To generate the mutagenic cassette, the top and bottom
strand oligonucleotides are annealed and the resulting duplex is
completed in an extension reaction using DNA polymerase. Following
digestion of the 73 bp dsDNA with Eag I and Stv I, the purified 51
bp mutagenic cassette is ligated with the large linear fragment
obtained from a similar digestion of MA-ITI RF DNA. Ligation
products are used to transform competent cells by electroporation
and phage stocks produced from Ap.sup.r transductants are analyzed
for the presence and nature of novel sequences as described
previously.
[1276] The variegation in the ITIMUT cassette is confined to the
codons for the six positions in ITI-DI (13, 15, 16, 17, 18, and
19), and employs three different nucleotide mixes: N, R, and S. For
this mutagenesis, the composition of the N-mix is 36%A, 17%C, 23%G,
and 24%T, and corresponds to the N-mix composition in the optimized
NNS codon described elsewhere. The R-mix composition is 50%A, 50%G,
and the S-mix composition is 50%C, 50%G.
[1277] The codon for ITI-DI position 13 (CCC, PRO) is changed to
SNG in ITIMUT. This codon encodes the eight residues PRO, VAL, GLU,
ALA, GLY, LEU, GLN, and ARG. The encoded group includes the
parental residue (PRO) as well as the more commonly observed
variants at the position, ARG and LEU (see Table 15), and also
provides for the occurrence of acidic (GLU), large polar (GLN) and
nonpolar (VAL), and small (ALA, GLY) residues.
[1278] The codons for positions 15 and 17 (ATG, MET) are changed to
the optimized NNS codon. All 20 natural amino acid residues and a
translation termination are allowed.
[1279] The codon for position 16 (CGA, GLY) is changed to RNS in
ITIMUT. This codon encodes the twelve amino acids GLY, ALA, ASP,
GLU, VAL, MET, ILE, THR, SER, ARG, ASN, and LYS. The encoded group
includes the most commonly observed residues at this position, ALA
and GLY, and provides for the occurrence of both positively (ARG,
LYS) and negatively (GLU, ASP) charged amino acids. Large nonpolar
residues are also included (ILE, MET, VAL).
[1280] Finally, at positions 18 and 19, the ITI-DI sequence is
changed from ACC.AGC (THR.SER) to NNT.NNT. The NNT codon encodes
the fifteen amino acid residues PHE, SER, TYR, CYS, LEU, PRO, HIS,
ARG, ILE, THR, ASN, VAL, ALA, ASP, and GLY. This group includes the
parental residues and the further advantages of the NNT codon have
been discussed elsewhere.
[1281] The ITIMUT DNA sequence encodes a total of:
8*20*12*20*15*15=8,640,000
[1282] different protein sequences in a total of:
2.sup.25=33,554,422
[1283] different DNA sequences. The total number of protein
sequences encoded by ITIMUT is only 7.4-fold fewer than the total
possible number of natural sequences obtained from variation at six
positions (=20.sup.6=6.4.multidot.10.sup.7). However, this degree
of variation in protein sequence is obtained from a minimum of
1.07>10.sup.9 (NNS.sup.6=2.sup.30) DNA sequences, a 32-fold
greater number than that comprising ITIMUT. Thus, ITIMUT is an
efficient vehicle for the generation of a large and diverse
population of potential binding proteins.
EXAMPLE XI
[1284] Development and Selection of BPTI Mutants for Binding to
Horse Heart Nyogiobin (HHMB)
[1285] The following example is hypothetical and illustrates
alternative embodiments of the invention not given in other
examples.
[1286] HHMb is chosen as a typical protein target; any other
protein could be used. HHMb satisfies all of the criteria for a
target: 1) it is large enough to be applied to an affinity matrix,
2) after attachment it is not reactive, and 3) after attachment
there is sufficient unaltered surface to allow specific binding by
PBDs.
[1287] The essential information for HHMb is known: 1) HHMb is
stable at least up to 70.degree. C., between pH 4.4 and 9.3, 2)
HHMb is stable up to 1.6 M Guanidinium Cl, 3) the pI of HHMb is
7.0, 4) for HHMb, M.sub.r=16,000, 5) HHMb requires haem, 6) HHMb
has no proteolytic activity.
[1288] In addition, the following information about HHMb and other
myoglobins is available: 1) the sequence of HHMB is known, 2) the
3D structure of sperm whale myoglobin is known; HHMb has 19 amino
acid differences and it is generally assumed that the 3D structures
are almost identical, 3) HHMb has no enzymatic activity, 4) HHMB is
not toxic.
[1289] We set the specifications of an SBD as 1) T=25.degree. C.;
2) pH=8.0; 3) Acceptable solutes ((A) for binding: i) phosphate, as
buffer, 0 to 20 mM, and ii) KCl, 10 mM; (B) for column elution: i)
phosphate, as buffer, 0 to 30 mM, ii) KCl, up to 5 M, and iii)
Guanidinium Cl, up to 0.8 M.); 4) Acceptable Kd
<1.0.multidot.10.sup.-8 M.
[1290] As stated in Sec. III.B, the residues to be varied are
picked, in part, through the use of interactive computer graphics
to visualize the structures. In this example, all residue numbers
refer to BPTI. We pick a set of residues that forms a surface such
that all residues can contact one target molecule. Information that
we refer to during the process of choosing residues to vary
includes: 1) the 3D structure of BPTI, 2) solvent accessibility of
each residue as computed by the method of Lee and Richards
(LEEB71), 3) a compilation of sequences of other proteins
homologous to BPTI, and 4) knowledge of the structural nature of
different amino acid types.
[1291] Tables 16 and 34 indicate which residues of BPTI: a) have
substantial surface exposure, and b) are known to tolerate other
amino acids in other closely related proteins. We use interactive
computer graphics to pick sets of eight to twenty residues that are
exposed and variable and such that all members of one set can touch
a molecule of the target material at one time. If BPTI has a small
amino acid at a given residue, that amino acid may not be able to
contact the target simultaneously with all the other residues in
the interaction set, but a larger amino acid might well make
contact. A charged amino acid might affect binding without making
direct contact. In such cases, the residue should be included in
the interaction set, with a notation that larger residues might be
useful. In a similar way, large amino acids near the geometric
center of the interaction set may prevent residues on either side
of the large central residue from making simultaneous contact. If a
small amino acid, however, were substituted for the large amino
acid, then the surface would become flatter and residues on either
side could make simultaneous contact. Such a residue should be
included in the interaction set with a notation that small amino
acids may be useful.
[1292] Table 35 was prepared from standard model parts and shows
the maximum span between C.sub..beta. and the tip of each type of
side group. C.sub..beta. is used because it is rigidly attached to
the protein main-chain; rotation about the
C.sub..alpha.-C.sub..beta. bond is the most important degree of
freedom for determining the location of the side group.
[1293] Table 34 indicates five surfaces that meet the given
criteria. The first surface comprises the set of residues that
actually contacts trypsin in the complex of trypsin with BPTI as
reported in the Brookhaven Protein Data Bank entry "1TPA". This set
is indicated by the number "1". The exposed surface of the residues
in this set (taken from Table 16) totals 1148 .ANG..sup.2. Although
this is not strictly the area of contact between BPTI and trypsin,
it is approximately the same.
[1294] Other surfaces, numbered 2 to 5, were picked by first
picking one exposed, variable residue and then picking neighboring
residues until a surface was defined. The choice of sets of
residues shown in Table 34 is in no way exhaustive or unique; other
sets of variable, surface residues can be picked. Set #2 is shown
in stereo view, FIG. 14, including the .alpha. carbons of BPTI, the
disulfide linkages, and the side groups of the set. We take the
orientation of BPTI in FIG. 14 as a standard orientation and
hereinafter refer to K15 as being at the top of the molecule, while
the carboxy and amino termini are at the bottom.
[1295] Solvent accessibilities are useful, easily tabulated
indicators of a residue's exposure. Solvent accessibilities must be
used with some caution; small amino acids are under-represented and
large amino acids overrepresented. The user must consider what the
solvent accessibility of a different amino acid would be when
substituted into the structure of BPTI.
[1296] To create specific binding between a derivative of BPTI and
HHMb, we will vary the residues in set #2. This set includes the
twelve principal residues 17(R), 19(I), 21(Y), 27(A), 28(G), 29(L),
31(Q), 32(T), 34(V), 48(A), 49(E), and 52(M) (Sec. III.B). None of
the residues in set #2 is completely conserved in the sample of
sequences reported in Table 34; thus we can vary them with a high
probability of retaining the underlying structure. Independent
substitution at each of these twelve residues of the amino acid
types observed at that residue would produce approximately
4.4.multidot.10.sup.9 amino acid sequences and the same number of
surfaces.
[1297] BPTI is a very basic protein. This property has been used in
isolating and purifying BPTI and its homologues so that the high
frequency of arginine and lysine residues may reflect bias in
isolation and is not necessarily required by the structure. Indeed,
SCI-III from Bombyx mori contains seven more acidic than basic
groups (SASA84).
[1298] Residue 17 is highly variable and fully exposed and can
contain R, K, A, Y, H, F, L, M, T, G, Y, P, or S. All types of
amino acids are seen: large, small, charged, neutral, and
hydrophobic. That no acidic groups are observed may be due to bias
in the sample.
[1299] Residue 19 is also variable and fully exposed, containing P,
R, I, S, K, Q, and L.
[1300] Residue 21 is not very variable, containing F or Y in 31 of
33 cases and I and W in the remaining cases. The side group of Y21
fills the space between T32 and the main chain of residues 47 and
48. The OH at the tip of the Y side group projects into the
solvent. Clearly one can vary the surface by substituting Y or F so
that the surface is either hydrophobic or hydrophilic in that
region. It is also possible that the other aromatic amino acid
(viz. H) or the other hydrophobics (L, M, or V) might be
tolerated.
[1301] Residue 27 most often contains A, but S, K, L, and T are
also observed. On structural grounds, this residue will probably
tolerate any hydrophilic amino acid and perhaps any amino acid.
[1302] Residue 28 is G in BPTI. This residue is in a turn, but is
not in a conformation peculiar to glycine. Six other types of amino
acids have been observed at this residue: K, N, Q, R, H, and N.
Small side groups at this residue might not contact HHMb
simultaneously with residues 17 and 34. Large side groups could
interact with HHMb at the same time as residues 17 and 34. Charged
side groups at this residue could affect binding of HHMb on the
surface defined by the other residues of the principal set. Any
amino acid, except perhaps P, should be tolerated.
[1303] Residue 29 is highly variable, most often containing L. This
fully exposed position will probably tolerate almost any amino acid
except, perhaps, P.
[1304] Residues 31, 32, and 34 are highly variable, exposed, and in
extended conformations; any amino acid should be tolerated.
[1305] Residues 48 and 49 are also highly variable and fully
exposed, any amino acid should be tolerated.
[1306] Residue 52 is in an a helix. Any amino acid, except perhaps
P, might be tolerated.
[1307] Now we consider possible variation of the secondary set
(Sec. 13.1.2) of residues that are in the neighborhood of the
principal set. Neighboring residues that might be varied at later
stages include 9(P), 11(T), 15(K), 16(A), 18(I), 20(R), 22(F),
24(N), 26(K), 35(Y), 47(S), 50(D), and 53(R).
[1308] Residue 9 is highly variable, extended, and exposed. Residue
9 and residues 48 and 49 are separated by a bulge caused by the
ascending chain from residue 31 to 34. For residue 9 and residues
48 and 49 to contribute simultaneously to binding, either the
target must have a groove into which the chain from 31 to 34 can
fit, or all three residues (9, 48, and 49) must have large amino
acids that effectively reduce the radius of curvature of the BPTI
derivative.
[1309] Residue 11 is highly variable, extended, and exposed.
Residue 11, like residue 9, is slightly far from the surface
defined by the principal residues and will contribute to binding in
the same circumstances.
[1310] Residue 15 is highly varied. The side group of residue 15
points away form the face defined by set #2. Changes of charge at
residue 15 could affect binding on the surface defined by residue
set #2.
[1311] Residue 16 is varied but points away from the surface
defined by the principal set. Changes in charge at this residue
could affect binding on the face defined by set #2.
[1312] Residue 18 is I in BPTI. This residue is in an extended
conformation and is exposed. Five other amino acids have been
observed at this residue: M, F, L, V, and T. Only T is hydrophilic.
The side group points directly away from the surface defined by
residue set #2. Substitution of charged amino acids at this residue
could affect binding at surface defined by residue set #2.
[1313] Residue 20 is R in BPTI. This residue is in an extended
conformation and is exposed. Four other amino acids have been
observed at this residue: A, S, L, and Q. The side group points
directly away from the surface defined by residue set #2.
Alteration of the charge at this residue could affect binding at
surface defined by residue set #2.
[1314] Residue 22 is only slightly varied, being Y, F, or H in 30
of 33 cases. Nevertheless, A, N, and S have been observed at this
residue. Amino acids such as L, M, I, or Q could be tried here.
Alterations at residue 22 may affect the mobility of residue 21;
changes in charge at residue 22 could affect binding at the surface
defined by residue set #2.
[1315] Residue 24 shows some variation, but probably can not
interact with one molecule of the target simultaneously with all
the residues in the principal set. Variation in charge at this
residue might have an effect on binding at the surface defined by
the principal set.
[1316] Residue 26 is highly varied and exposed. Changes in charge
may affect binding at the surface defined by residue set #2;
substitutions may affect the mobility of residue 27 that is in the
principal set.
[1317] Residue 35 is most often Y, W has been observed. The side
group of 35 is buried, but substitution of F or W could affect the
mobility of residue 34.
[1318] Residue 47 is always T or S in the sequence sample used. The
O.sub.gamma probably accepts a hydrogen bond from the NH of residue
50 in the alpha helix. Nevertheless, there is no overwhelming
steric reason to preclude other amino acid types at this residue.
In particular, other amino acids the side groups of which can
accept hydrogen bonds, viz. N, D, Q, and E, may be acceptable
here.
[1319] Residue 50 is often an acidic amino acid, but other amino
acids are possible.
[1320] Residue 53 is often R, but other amino acids have been
observed at this residue. Changes of charge may affect binding to
the amino acids in interaction set #2.
[1321] Stereo FIG. 14 shows the residues in set #2, plus R39. From
FIG. 14, one can see that R39 is on the opposite side of BPTI form
the surface defined by the residues in set #2. Therefore, variation
at residue 39 at the same time as variation of some residues in set
#2 is much less likely to improve binding that occurs along surface
#2 than is variation of the other residues in set #2.
[1322] In addition to the twelve principal residues and 13
secondary residues, there are two other residues, 30(C) and 33(F),
involved in surface #2 that we will probably not vary, at least not
until late in the procedure. These residues have their side groups
buried inside BPTI and are conserved. Changing these residues does
not change the surface nearly so much as does changing residues in
the principal set. These buried, conserved residues do, however,
contribute to the surface area of surface #2. The surface of
residue set #2 is comparable to the area of the trypsin-binding
surface. Principal residues 17, 19, 21, 27, 28, 29, 31, 32, 34, 48,
49, and 52 have a combined solvent-accessible area of 946.9
A.sup.2. Secondary residues 9, 11, 15, 16, 18, 20, 22, 24, 26, 35,
47, 50, and 53 have combined surface of 1041.7 A.sup.2. Residues 30
and 33 have exposed surface totaling 38.2 A.sup.2. Thus the three
groups' combined surface is 2026.8 A.sup.2.
[1323] Residue 30 is C in BPTI and is conserved in all homologous
sequences. It should be noted, however, that C14/C38 is conserved
in all natural sequences, yet Marks et al. (MARK87) showed that
changing both C14 and C38 to A,A or T,T yields a functional trypsin
inhibitor. Thus it is possible that BPTI-like molecules will fold
if C30 is replaced.
[1324] Residue 33 is F in BPTI and in all homologous sequences.
Visual inspection of the BPTI structure suggests that substitution
of Y, M, H, or L might be tolerated.
[1325] Having identified twenty residues that define a possible
binding surface, we must choose some to vary first. Assuming a
hypothetical affinity separation sensitivity, C.sub.sensi, of 1 in
4.multidot.10.sup.8, we decide to vary six residues (leaving some
margin for error in the actual base composition of variegated
bases). To obtain maximal recognition, we choose residues from the
principal set that are as far apart as possible. Table 36 shows the
distances between the p carbons of residues in the principal and
peripheral set. R17 and V34 are at one end of the principal
surface. Residues A27, G28, L29, A48, E49, and M52 are at the other
end, about twenty Angstroms away; of these, we will vary residues
17, 27, 29, 34, and 48. Residues 28, 49, and 52 will be varied at
later rounds.
[1326] Of the remaining principal residues, 21 is left to later
variations. Among residues 19, 31, and 32, we arbitrarily pick 19
to vary.
[1327] Unlimited variation of six residues produces
6.4.multidot.10.sup.7 amino acid sequences. By hypothesis,
C.sub.sensi is 1 in 4.multidot.10.sup.8. Table 37 shows the
programmed variegation at the chosen residues. The parental
sequence is present as 1 part in 5.5.multidot.10.sup.7, but the
least favored sequences are present at only 1 part in
4.2.multidot.10.sup.9. Among single-amino-acid substitutions from
the PPBD, the least favored is F17-I19-A27-L29-V34-A48 and has a
calculated abundance of 1 part in 1.6.multidot.10.sup.8. Using the
optimal qfk codon, we can recover the parental sequence and all
one-amino-acid substitutions to the PPBD if actual nt compositions
come within 5% of programmed compositions. The number of
transformants is M.sub.ntv=1.0.multidot.10.sup.9 (also by
hypothesis), thus we will produce most of the programmed
sequences.
[1328] The residue numbers of the preceding section are referred to
mature BPTI (R1-P2- . . . -A58). Table 25 has residue numbers
referring to the pre-M13CP-BPTI protein; all mature BPTI sequence
numbers have been increased by the length of the signal sequence,
i.e. 23. Thus in terms of the pre-OSP-PBD residue numbers, we wish
to vary residues 40, 42, 50, 52, 57, and 71. A DNA subsequence
containing all these codons is found between the (ADaI/DraII/PssI)
sites at base 191 and the Sph I site at base 309 of the osp-pbd
gene. Among ApaI, DraI, and PssI, ApaI is preferred because it
recognizes six bases without any ambiguity. DraII and PssI, on the
other hand, recognize six bases with two-fold ambiguity at two of
the bases. The vgDNA will contain more DraII and PssI recognition
sites at the varied locations than it will contain ApaI recognition
sites. The unwanted extraneous cutting of the vgDNA by ApaI and
SphI will eliminate a few sequences from our population. This is a
minor problem, but by using the more specific enzyme (ApaI), we
minimize the unwanted effects. The sequence shown in Table 37
illustrates an additional way in which gratuitous restriction sites
can be avoided in some cases. The osp-ipbd gene had the codon GGC
for g51; because we are varying both residue 50 and 52, it is
possible to obtain an ApaI site. If we change the glycine codon to
GGT, the ApaI site can no longer arise. ApaI recognizes the DNA
sequence (GGGCC/C).
[1329] Each piece of dsDNA to be synthesized needs six to eight
bases added at either end to allow cutting with restriction enzymes
and is shown in Table 37. The first synthetic base (before cutting
with ApaI and SphI) is 184 and the last is 322. There are 142 bases
to be synthesized. The center of the piece to the synthesized lies
between Q54 and V57. The overlap can not include varied bases, so
we choose bases 245 to 256 as the overlap that is 12 bases long.
Note that the codon for F56 has been changed to TTC to increase the
GC content of the overlap. The amino acids that are being varied
are marked as X with a plus over them. Codons 57 and 71 are
synthesized on the sense (bottom) strand. The design calls for
"qfk" in the antisense strand, so that the sense strand contains
(from 5' to 3') a) equal part C and A (i.e. the complement of k),
b) (0.40 T, 0.22 A, 0.22 C, and 0.16 G) (i.e. the complement of f),
and c) (0.26 T, 0.26 A, 0.30 C, and 0.18 G).
[1330] Each residue that is encoded by "qfk" has 21 possible
outcomes, each of the amino acids plus stop. Table 12 gives the
distribution of amino acids encoded by "qfk", assuming 5% errors.
The abundance of the parental sequence is the product of the
abundances of R.times.I.times.A.times.L.times.V.times.A. The
abundance of the least-favored sequence is 1 in
4.2.multidot.10.sup.9.
[1331] Olig#27 and olig#28 are annealed and extended with Klenow
fragment and all four (nt)TPs. Both the ds synthetic DNA and RF
pLG7 DNA are cut with both ApaI and SphI. The cut DNA is purified
and the appropriate pieces ligated (See Sec. 14.1) and used to
transform competent PE383. (Sec. 14.2). In order to generate a
sufficient number of transformants, V.sub.c is set to 5000 ml.
[1332] 1) culture E. coli in 5.0 1 of LB broth at 37.degree. C.
until cell density reaches 5.multidot.10.sup.7 to
7.multidot.10.sup.7 cells/ml,
[1333] 2) chill on ice for 65 minutes, centrifuge the cell
suspension at 4000g for 5 minutes at 4.degree. C.,
[1334] 3) discard supernatant; resuspend the cells in 1667 ml of an
ice-cold, sterile solution of 60 mM Cacl.sub.2,
[1335] 4) chill on ice for 15 minutes, and then centrifuge at 4000g
for 5 minutes at 4.degree. C.,
[1336] 5) discard supernatant; resuspend cells in 2.times.400 ml of
ice-cold, sterile 60 mM CaCl.sub.2; store cells at 4.degree. C. for
24 hours,
[1337] 6) add DNA in ligation or TE buffer; mix and store on ice
for 30 minutes; 20 ml of solution containing 5 .mu.g/ml of DNA is
used,
[1338] 7) heat shock cells at 42.degree. C. for 90 seconds,
[1339] 8) add 200 ml LB broth and incubate at 37.degree. C. for 1
hour,
[1340] 9) add the culture to 2.0 1 of LB broth containing
ampicillin at 35-100 .mu.g/ml and culture for 2 hours at 37.degree.
C.,
[1341] 10) centrifuge at 8000 g for 20 minutes at 4.degree. C.,
[1342] 11) discard supernatant, resuspend cells in 50 ml of LB
broth plus ampicillin and incubate 1 hour at 37.degree. C.,
[1343] 12) plate cells on LB agar containing ampicillin,
[1344] 13) harvest virions by method of Salivar et al.
(SALI64).
[1345] The heat shock of step (7) can be done by dividing the 200
ml into 100 200 .mu.l aliquots in 1.5 ml plastic Eppendorf tubes.
It is possible to optimize the heat shock for other volumes and
kinds of container. It is important to: a) use all or nearly all
the vgDNA synthesized in ligation, this will require large amounts
of pLG7 backbone, b) use all or nearly all the ligation mixture to
transform cells, and c) culture all or nearly all the transformants
at high density. These measures are directed at maintaining
diversity.
[1346] IPTG is added to the growth medium at 2.0 mM (the optimal
level) and virions are harvested in the usual way. It is important
to collect virions in a way that samples all or nearly all the
transformants. Because F cells are used in the transformation,
multiple infections do not pose a problem.
[1347] HHMb has a pI of 7.0 and we carry out chromatography at pH
8.0 so that HHMb is slightly negative while BPTI and most of its
mutants are positive. HHMb is fixed (Sec. V.F) to a 2.0 ml column
on Affi-Gel 10.sup.(TM) or Affi-Gel 15.sup.(TM) at 4.0 mg/ml
support matrix, the same density that is optimal for a column
supporting trp.
[1348] We note that charge repulsion between BPTI and HHMb should
not be a serious problem and does not impose any constraints on
ions or solutes allowed as eluants. Neither BPTI nor HHMb have
special requirements that constrain choice of eluants. The eluant
of choice is KCl in varying concentrations.
[1349] To remove variants of BPTI with strong, indiscriminate
binding for any protein or for the support matrix, we pass the
variegated population of virions over a column that supports bovine
serum albumin (BSA) before loading the population onto the {HHMb)
column. Affi-Gel 10.sup.(TM) or Affi-Gel 15.sup.(TM) is used to
immobilize BSA at the highest level the matrix will support. A 10.0
ml column is loaded with 5.0 ml of Affi-Gel-linked-BSA; this
column, called (BSA}, has V.sub.V=5.0 ml. The variegated population
of virions containing 10.sup.12 pfu in 1 ml (0.2 x V.sub.V) of 10
mM KC1, 1 mM phosphate, pH 8.0 buffer is applied to {BSA}. We wash
(BSA) with 4.5 ml (0.9.times.V.sub.V) of 50 mM KCl, 1 mM phosphate,
pH 8.0 buffer. The wash with 50 mM salt will elute virions that
adhere slightly to BSA but not virions with strong binding. The
pooled effluent of the {BSA} column is 5.5 ml of approximately 13
mM KC1.
[1350] The column {HHMb} is first blocked by treatment with
10.sup.11 virions of M13(am429) in 100 ul of 10 mM KC1 buffered to
pH 8.0 with phosphate; the column is washed with the same buffer
until OD.sub.260 returns to base line or 2.times.V.sub.V have
passed through the column, whichever comes first. The pooled
effluent from {BSA} is added to tHHMb) in 5.5 ml of 13 mM KCl, mM
phosphate, pH 8.0 buffer. The column is eluted in the following
way:
[1351] 1) 10 mM KCl buffered to pH 8.0 with phosphate, until
optical density at 280 nm falls to base line or 2.times.V.sub.V,
whichever is first, (effluent discarded),
[1352] 2) a gradient of 10 mM to 2 M KCl in 3.times.V.sub.V, pH
held at 8.0 with phosphate, (30.multidot.100 .mu.l fractions),
[1353] 3) a gradient of 2 M to 5 M KCl in 3.times.V.sub.V,
phosphate buffer to pH 8.0 (30.multidot.100 .mu.l fractions),
[1354] 4) constant 5 M KCl plus 0 to 0.8 M guanidinium Cl in
2.times.V.sub.V, with phosphate buffer to pH 8.0, (20.multidot.100
.mu.l fractions), and
[1355] 5) constant 5 M KCl plus 0.8 M guanidinium Cl in
1.times.V.sub.V, with phosphate buffer to pH 8.0, (10.multidot.100
.mu.l fractions).
[1356] In addition to the elution fractions, a sample is removed
from the column and used as an inoculum for phage-sensitive
Sup.sup.- cells (Sec. V). A sample of 4 .mu.l from each fraction is
plated on phage-sensitive Sup.sup.- cells. Fractions that yield too
many colonies to count are replated at lower dilution. An
approximate titre of each fraction is calculated. Starting with the
last fraction and working toward the first fraction that was
titered, we pool fractions until approximately 10.sup.9 phage are
in the pool, i.e. about 1 part in 1000 of the phage applied to the
column. This population is infected into 3.multidot.10.sup.11
phage-sensitive PE384 in 300 ml of LB broth. The very low
multiplicity of infection (moi) is chosen to reduce the possibility
of multiple infection. After thirty minutes, viable phage have
entered recipient cells but have not yet begun to produce new
phage. Phage-born genes are expressed at this phase, and we can add
ampicillin that will kill uninfected cells. These cells still carry
F-pili and will absorb phage helping to prevent multiple
infections.
[1357] If multiple infection should pose a problem that cannot be
solved by growth at low multiple-of-infection on F.sup.+ cells, the
following procedure can be employed to obviate the problem. Virions
obtained from the affinity separation are infected into F.sup.+ E.
coli and cultured to amplify the genetic messages (Sec. V). CCC DNA
is obtained either by harvesting RF DNA or by in vitro extension of
primers annealed to ss phage DNA. The CCC DNA is used to transform
F.sup.- cells at a high ratio of cells to DNA. Individual virions
obtained in this way should bear only proteins encoded by the DNA
within.
[1358] The phagemid population is grown and chromatographed three
times and then examined for SBDs (Sec. V). In each separation
cycle, phage from the last three fractions that contain viable
phage are pooled with phage obtained by removing some of the
support matrix as an inoculum. At each cycle, about 10.sup.12 phage
are loaded onto the column and about 10.sup.9 phage are cultured
for the next separation cycle. After the third separation cycle,
SBD colonies are picked from the last fraction that contained
viable phage.
[1359] Each of the SBDs is cultured and tested for retention on a
Pep-Tie column supporting HHMb. The phage showing the greatest
retention on the Pep-Tie {HHMb} column. This SBD! becomes the
parental amino-acid sequence to the second variegation cycle.
[1360] Assume for the sake of argument that, in SBD!, R40 changed
to D, I42 changed to Q, A50 changed to E, L52 remained L, and A71
changed to W (see Table 38). If so, a rational plan for the second
round of variegation would be that which is set forth in Table 39.
The residues to be varied are chosen by: a) choosing some of the
residues in the principal set that were not varied in the first
round (viz. residues 42, 44, 51, 54, 55, 72, or 75 of the fusion),
and b) choosing some residues in the secondary set. Residues 51,
54, 55, and 72 are varied through all twenty amino acids and,
unavoidably, stop. Residue 44 is only varied between Y and F. Some
residues in the secondary set are varied through a restricted
range; primarily to allow different charges (+, 0, -) to appear.
Residue 38 is varied through K, R, E, or G. Residue 41 is varied
through I, V, K, or E. Residue 43 is varied through R, S, G, N, K,
D, E, T, or A.
[1361] Now assume that in the most successful SBD of the second
round of variegation (SBD-2!), residue 38 (K15 of BPTI) changed to
E, 41 becomes V, 43 goes to N, 44 goes to F, 51 goes to F, 54 goes
to S, 55 goes to A, and 72 goes to Q (see Table 40). A third round
of variation is illustrated in Table 41; eight amino acids are
varied. Those in the principal set, residues 40, 55, and 57, are
varied through all twenty amino acids. Residue 32 is varied through
P, Q, T, K, A, or E. Residue 34 is varied through T, P, Q, K, A, or
E. Residue 44 is varied through F, L, Y, C, W, or stop. Residue 50
is varied through E, K, or Q. Residue 52 is varied through L, F, I,
M, or V. The result of this variation is shown in Table 42.
[1362] This example is hypothetical. It is anticipated that more
variegation cycles will be needed to achieve dissociation constants
of 10.sup.-8 M. It is also possible that more than three separation
cycles will be needed in some variegation cycles. Real DNA
chemistry and DNA synthesizers may have larger errors than our
hypothetical 5%. If S.sub.err>0.05, then we may not be able to
vary six residues at once. Variation of 5 residues at once is
certainly possible.
EXAMPLE XII
[1363] Design and Mutagenesis of a Class 1 Mini-protein
[1364] To obtain a library of binding domains that are
conformationally constrained by a single disulfide, we insert DNA
coding for the following family of mini-proteins into the gene
coding for a suitable OSP. 6
[1365] Where 7
[1366] indicates disulfide bonding; this mini-protein is depicted
in FIG. 3. Disulfides normally do not form between cysteines that
are consecutive on the polypeptide chain. One or more of the
residues indicated above as X.sub.n will be varied extensively to
obtain novel binding. There may be one or more amino acids that
precede X.sub.1 or follow X8, however, these additional residues
will not be significantly constrained by the diagrammed disulfide
bridge, and it is less advantageous to vary these remote, unbridged
residues. The last X residue is connected to the OSP of the genetic
package.
[1367] X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6,
X.sub.7, and X.sub.8 can be varied independently; i.e. a different
scheme of variegation could be used at each position. X.sub.1 and
X.sub.8 are the least constrained residues and may be varied less
than other positions.
[1368] X.sub.1 and X.sub.8 can be, for example, one of the amino
acids [E, K, T, and A]; this set of amino acids is preferred
because: a) the possibility of positively charged, negatively
charged, and neutral amino acids is provided, b) these amino acids
can be provided in 1:1:1:1 ratio via the codon RMG (R=equimolar A
and G, M=equimolar A and C), and c) these amino acids allow proper
processing by signal peptidases.
[1369] One option for variegation of X.sub.2, X.sub.3, X.sub.4,
X.sub.5, X.sub.6, and X.sub.7 is to vary all of these in the same
way. For example, each of X.sub.2, X.sub.3, X.sub.4, X.sub.5,
X.sub.6, and X.sub.7 can be chosen from the set [F, S, Y, C, L, P,
H, R, I, T, N, V, A, D, and G] which is encoded by the mixed codon
NNT. Tables 10 and 130 compares libraries in which six codons have
been varied either by NNT or NNK codons. NNT encodes 15 different
amino acids and only 16 DNA sequences. Thus, there are
1.139.multidot.10.sup.7 amino-acid sequences, no stops, and only
1.678.multidot.10.sup.7 DNA sequences. A library of 10.sup.8
independent transformants will contain 99% of all possible
sequences. The NNK library contains 6.4.multidot.10.sup.7
sequences, but complete sampling requires a much larger number of
independent transformants.
EXAMPLE XIII
[1370] A CYS::HELIX::TURN::STRAND::CYS Unit
[1371] The parental Class 2 mini-proteins may be a
naturally-occurring Class 2 mini-protein. It may also be a domain
of a larger protein whose structure satisfies or may be modified so
as to satisfy the criteria of a class 2 mini-protein. The
modification may be a simple one, such as the introduction of a
cysteine (or a pair of cysteines) into the base of a hairpin
structure so that the hairpin may be closed off with a disulfide
bond, or a more elaborate one, so as the modification of
intermediate residues so as to achieve the hairpin structure. The
parental class 2 mini-protein may also be a composite of structures
from two or more naturally-occurring proteins, e.g., an .alpha.
helix of one protein and a .beta. strand of a second protein.
[1372] One mini-protein motif of potential use comprises a
disulfide loop enclosing a helix, a turn, and a return strand. Such
a structure could be designed or it could be obtained from a
protein of known 3D structure. Scorpion neurotoxin, variant 3,
(ALMA83a, AIMA83b) (hereafter ScorpTx) contains a structure
diagrammed in FIG. 15 that comprises a helix (residues N22 through
N33), a turn (residues 33 through 35), and a return strand
(residues 36 through 41). ScorpTx contains disulfides that join
residues 12-65, 16-41, 25-46, and 29-48. CYS.sub.25 and CYS.sub.41
are quite close and could be joined by a disulfide without
deranging the main chain. FIG. 15 shows CYS.sub.25 joined to
CYS.sub.41. In addition, CYS.sub.29 has been changed to GLN. It is
expected that a disulfide will form between 25 and 41 and that the
helix shown will form; we know that the amino-acid sequence shown
is highly compatible with this structure. The presence of
GLY.sub.35, GLY.sub.36, and GLY.sub.39 give the turn and extended
strand sufficient flexibility to accommodate any changes needed
around CYS.sub.41 to form the disulfide.
[1373] From examination of this structure (as found in entry 1SN3
of the Brookhaven Protein Data Bank), we see that the following
sets of residues would be preferred for variegation:
26 Residue Codon Allowed amino acids Naa/Ndna 1) T.sub.27 NNG
L.sup.2 R.sup.2 MVSPTAQKEWG. 13/15 2) E.sub.28 VHG LMVPTAGKE 9/9 3)
A.sub.31 VHG LMVPTAGKE 9/9 4) K.sub.32 VHG LMVPTAGKE 9/9 5) G24 NNG
L.sup.2 R.sup.2 MVSPTAQKEWG. 13/15 6) E23 VHG LMVPTAGKE 9/9 7) Q34
VAS HQNKED 6/6 Note: Exponents on amino acids indicate multiplicity
of codons.
[1374] Positions 27, 28, 31, 32, 24, and 23 comprise one face of
the helix. At each of these locations we have picked a variegating
codon that a) includes the parental amino acid, b) includes a set
of residues having a predominance of helix favoring residues, c)
provides for a wide variety of amino acids, and d) leads to as even
a distribution as possible. Position 34 is part of a turn. The side
group of residue 34 could interact with molecules that contact the
side groups of resideus 27, 28, 31, 32, 24, and 23. Thus we allow
variegation here and provide amino acids that are compatible with
turns. The variegation shown leads to 6.65.multidot.10.sup.6 amino
acid sequences encoded by 8.85.multidot.10.sup.6 DNA sequences.
27 SET 2 Residue Codon Allowed amino acids Naa/Ndna 1) D.sub.26 VHS
L.sup.2 IMV.sup.2 P.sup.2 T.sup.2 A.sup.2 HQNKDE 13/18 2) T.sub.27
NNG L.sup.2 R.sup.2 MVSPTAQKEWG. 13/15 3) K.sub.30 VHG KEQPTALMV
9/9 4) A.sub.31 VHG KEQPTALMV 9/9 5) K.sub.32 VHG LMVPTAGKE 9/9 6)
S.sub.37 RRT SNDG 4/4 7) Y.sub.38 NHT YSFHPLNTIDAV 9/9
[1375] Positions 26, 27, 30, 31, and 32 are variegated so as to
enhance helix-favoring amino acids in the population. Residues 37
and 38 are in the return strand so that we pick different
variegation codons. This variegation allows 4.43.multidot.10.sup.6
amino-acid sequences and 7.08.multidot.10.sup.6 DNA sequences. Thus
a library that embodies this scheme can be sampled very
efficiently.
EXAMPLE XIV
[1376] Design and Mutagenesis of Class 3 Mini-protein
[1377] Two Disulfide Bond Parental Mini-proteins
[1378] Mini-proteins with two disulfide bonds may be modelled after
the .alpha.-conotoxins, e.g., GI, GIA, GII, MI, and SI. These have
the following conserved structure: 8
[1379] Hashimoto et al. (HASH85) reported synthesis of twenty-four
analogues of .alpha. conotoxins GI, GII, and MI. Using the
numbering scheme for GI (CYS at positions 2, 3, 7, and 13),
Hashimoto et al. reported alterations at 4, 8, 10, and 12 that
allows the proteins to be toxic. Almquist et al. (ALMQ89)
synthesized [des-GLU.sub.1] .alpha. Conotoxin GI and twenty
analogues. They found that substituting GLY for PRO.sub.5 gave rise
to two isomers, perhaps related to different disulfide bonding.
They found a number of substitutions at residues 8 through 11 that
allowed the protein to be toxic. Zafaralla et al. (ZAFA88) found
that substituting PRO at position 9 gives an active protein. Each
of the groups cited used only in vivo toxicity as an assay for the
activity. From such studies, one can infer that an active protein
has the parental 3D structure, but one can not infer that an
inactive protein lacks the parental 3D structure.
[1380] Pardi et al. (PARD89) determined the 3D structure of .alpha.
Conotoxin GI obtained from venom by NMR. Kobayashi et al. (KOBA89)
have reported a 3D structure of synthetic a Conotoxin GI from NMR
data which agrees with that of PARD89. We refer to FIG. 5 of Pardi
et al.
[1381] Residue GLU.sub.1 is known to accomodate GLU, ARG, and ILE
in known analogues or homologues. A preferred variegation codon is
NNG that allows the set of amino acids
[L.sup.2R.sup.2MVSPTAQKEWG<stop>]. From FIG. 5 of Pardi et
al. we see that the side group of GLU.sub.1 projects into the same
region as the strand comprising residues 9 through 12. Residues 2
and 3 are cysteines and are not to be varied. The side group of
residue 4 points away from residues 9 through 12; thus we defer
varying this residue until a later round. PRO.sub.5 may be needed
to cause the correct disulfides to form; when GLY was substituted
here the peptide folded into two forms, neither of which is toxic.
It is allowed to vary PRO.sub.5, but not perferred in the first
round.
[1382] No substitutions at ALA.sub.6 have been reported. A
preferred variegation codon is RMG which gives rise to ALA, THR,
LYS, and GLU (small hydrophobic, small hydrophilic, positive, and
negative). CYS.sub.7 is not varied. We prefer to leave GLY.sub.8 as
is, although a homologous protein having ALA.sub.8 is toxic.
Homologous proteins having various amino acids at position 9 are
toxic; thus, we use an NNT variegation codon which allows
FS.sup.2YCLPHRITNVADG. We use NNT at positions 10, 11, and 12 as
well. At position 14, following the fourth CYS, we allow ALA, THR,
LYS, or GLU (via an RMG codon). This variegation allows
1.053.multidot.10.sup.7 anino-acid sequences, encoded by
1.68.multidot.10.sup.7 DNA sequences. Libraries having
2.0.multidot.10.sup.7, 3.0.multidot.10.sup.7, and
5.0.multidot.10.sup.7 independent transformants will, respectively,
display .apprxeq.70%, .apprxeq.83%, and .apprxeq.95% of the allowed
sequences. Other variegations are also appropriate. Concerning
.alpha. conotoxins, see, inter alia, ALMQ89, CRUZ85, GRAY83,
GRAY84, and PARD89.
[1383] The parental mini-protein may instead be one of the proteins
designated "Hybrid-I" and "Hybrid-II" by Pease et al. (PEAS90); cf.
FIG. 4 of PEAS90. One preferred set of residues to vary for either
protein consists of:
28 Parental Variegated Allowed AA seqs/ Amino acid Codon Amino
acids DNA seqs A5 RVT ADGTNS 6/6 P6 VYT PTALIV 6/6 E7 RRS
EDNKSRG.sup.2 7/8 T8 VHG TPALMVQKE 9/9 A9 VHG ATPLMVQKE 9/9 A10 RMG
AEKT 4/4 K12 VHG KQETPALMV 9/9 Q16 NNG L.sup.2 R.sup.2 S.WPQMTKVAEG
13/15
[1384] This provides 9.55.multidot.10.sup.6 amino-acid sequences
encoded by 1.26.multidot.10.sup.7 DNA sequences. A library
comprising 5.0.multidot.10.sup.7 transformants allows expression of
98.2% of all possible sequences. At each position, the parental
amino acid is allowed.
[1385] At position 5 we provide amino acids that are compatible
with a turn. At position 6 we allow ILE and VAL because they have
branched .beta. carbons and make the chain ridged. At position 7 we
allow ASP, ASN, and SER that often appear at the amino termini of
helices. At positions 8 and 9 we allow several helix-favoring amino
acids (ALA, LEU, MET, GLN, GLU, and LYS) that have differing
charges and hydrophobicities because these are part of the helix
proper. Position 10 is further around the edge of the helix, so we
allow a smaller set (ALA, THR, LYS, and GLU). This set not only
includes 3 helix-favoring amino acids plus THR that is well
tolerated but also allows positive, negative, and neutral
hydrophilic. The side groups of 12 and 16 project into the same
region as the residues already recited. At these positions we allow
a wide variety of amino acids with a bias toward helix-favoring
amino acids.
[1386] The parental mini-protein may instead be a polypeptide
composed of residues 9-24 and 31-40 of aprotinin and possessing two
disulfides (Cys9-Cys22 and Cysl4-Cys38). Such a polypeptide would
have the same disulfide bond topology as .alpha.-conotoxin, and its
two bridges would have spans of 12 and 17, respectively.
[1387] Residues 23, 24 and 31 are variegated to encode the amino
acid residue set [G,S,R,D,N,H,P,T,A] so that a sequence that favors
a turn of the necessary geometry is found. We use trypsin or
anhydrotrypsin as the affinity molucule to enrich for GPs that
display a mini-protein that folds into a stable structure similar
to BPTI in the P1 region.
[1388] Three Disulfide Bond Parental Mini-proteins
[1389] The cone snails (Conus) produce venoms (conotoxins) which
are 10-30 amino acids in length and exceptionally rich in disulfide
bonds. They are therefore archetypal mini-proteins. Novel
mini-proteins with three disulfide bonds may be modelled after the
.mu.-(GIIIA, GIIIB, GIIIC) or .OMEGA.-(GVIA, GVIB, GVIC, GVIIA,
GVIIB, MVIIA, MVIIB, etc.) conotoxins. The .mu.-conotoxins have the
following conserved structure: 9
[1390] No 3D structure of a .mu.-conotoxin has been published.
Hidaka et al. (HIDA90) have established the connectivity of the
disulfides. The following diagram depicts geographutoxin I (also
known as .mu.-conotoxin GIIIA). 10
[1391] The connection from R19 to C20 could go over or under the
strand from Q14 to C15. One preferred form of variegation is to
vary the residues in one loop. Because the longest loop contains
only five amino acids, it is appropriate to also vary the residues
connected to the cysteines that form the loop. For example, we
might vary residues 5 through 9 plus 2, 11, 19, and 22. Another
useful variegation would be to vary residues 11-14 and 16-19, each
through eight amino acids. Concerning .mu. conotoxins, see BECK89b,
BECK89c, CRUZ89, and HIDA90.
[1392] The .OMEGA.-conotoxins may be represented as follows: 11
[1393] The King Kong peptide has the same disulfide arrangement as
the .OMEGA.-conotoxins but a different biological activity.
Woodward et al. (WOOD90) report the sequences of three homologuous
proteins from C. textile. Within the mature toxin domain, only the
cysteines are conserved. The spacing of the cysteines is exactly
conserved, but no other position has the same amino acid in all
three sequences and only a few positions show even pair-wise
matches. Thus we conclude that all positions (except the cysteines)
may be substituted freely with a high probability that a stable
disulfide structure will form. Concerning .OMEGA. conotoxins, see
HILL89 and SUNX87.
[1394] Another mini-protein which may be used as a parental binding
domain is the Cucurbita maxima trypsin inhibitor I (CMTI-I);
CMTI-III is also appropriate. They are members of the squash family
of serine protease inhibitors, which also includes inhibitors from
summer squash, zucchini, and cucumbers (WIEC85). McWherter et al.
(MCWH89) describe synthetic sequence-variants of the squash-seed
protease inhibitors that have affinity for human leukocyte elastase
and cathepsin G. Of course, any member of this family might be
used.
[1395] CMTI-I is one of the smallest proteins known, comprising
only 29 amino acids held in a fixed comformation by three disulfide
bonds. The structure has been studied by Bode and colleagues using
both X-ray diffraction (BODE89) and NMR (HOLA89a,b). CMTI-I is of
ellipsoidal shape; it lacks helices or .beta.-sheets, but consists
of turns and connecting short polypeptide stretches. The disulfide
pairing is Cys3-Cys20, Cys10-Cys22 and Cys16-Cys28. In the
CMTI-I:trypsin complex studied by Bode et al., 13 of the 29
inhibitor residues are in direct contact with trypsin; most of them
are in the primary binding segment Val2(P4)-Glu9 (P4') which
contains the reactive site bond Arg5(P1)-Ile6 and is in a
conformation observed also for other serine proteinase
inhibitors.
[1396] CMTI-I has a K.sub.i for trypsin of
.apprxeq.1.5.multidot.10.sup.-1- 2 M. McWherter et al. suggested
substitution of "moderately bulky hydrophobic groups" at P1 to
confer HLE specificity. They found that a wider set of residues
(VAL, ILE, LEU, ALA, PHE, MET, and GLY) gave detectable binding to
HLE. For cathepsin G, they expected bulky (especially aromatic)
side groups to be strongly preferred. They found that PHE, LEU,
MET, and ALA were functional by their criteria; they did not test
TRP, TYR, or HIS. (Note that ALA has the second smallest side group
available.)
[1397] A preferred initial variegation strategy would be to vary
some or all of the residues ARG.sub.1, VAL.sub.2, PRO.sub.4,
ARG.sub.5, ILE.sub.6, LEU.sub.7, MET.sub.8, GLU.sub.9, LYS.sub.11,
HIS.sub.25, GLY.sub.26, TYR.sub.27, and GLY.sub.29. If the target
were HNE, for example, one could synthesize DNA embodying the
following possibilities:
29 vg Allowed #AA seqs/ Parental Codon amino acids #DNA seqs
ARG.sub.1 VNT RSLPHITNVADG 12/12 VAL.sub.2 NWT VILFYHND 8/8
PRO.sub.4 VYT PLTIAV 6/6 ARG.sub.5 VNT RSLPHITNVADG 12/12 ILE.sub.6
NNK all 20 20/31 LEU.sub.7 VWG LQMKVE 6/6 TYR.sub.27 NAS YHONKDE.
7/8
[1398] This allows about 5.81.multidot.10.sup.6 amino-acid
sequences encoded by about 1.03.multidot.10.sup.7 DNA sequences. A
library comprising 5.0.multidot.10.sup.7 independent transformants
would give .apprxeq.99% of the possible sequences. Other
variegation schemes could also be used.
[1399] Other inhibitors of this family include: Trypsin inhibitor I
from Citrullus vulgaris (OTLE87), Trypsin inhibitor II from Bryonia
dioica (OTLE87), Trypsin inhibitor I from Cucurbita maxima (in
OTLE87), trypsin inhibitor III from Cucurbita maxima (in OTIE87),
trypsin inhibitor IV from Cucurbita maxima (in OTLE87), trypsin
inhibitor II from Cucurbita pepo (in OTLE87), trypsin inhibitor III
from Cucurbita pepo (in OTLE87), trypsin inhibitor IIb from Cucumis
sativus (in OTLE87), trypsin inhibitor IV from Cucumis sativus (in
OTLE87), trypsin inhibitor II from Ecballium elaterium (FAVE89),
and inhibitor CM-1 from Momordica repens (in OTLE87).
[1400] Another mini-protein that may be used as an initial
potential binding domain is the heat-stable enterotoxins derived
from some enterotoxogenic E. coli, Citrobacter freundii, and other
bacteria (GUAR89). These mini-proteins are known to be secreted
from E. coli and are extremely stable. Works related to synthesis,
cloning, expression and properties of these proteins include:
BHAT86, SEKI85, SHIM87, TAKA85, TAKE90, THOM85a,b, YOSH85, DALL90,
DWAR89, GARI87, GUZM89, GUZM90, HOUG84, KUBO89, KUPE90, OKAM87,
OKAM88, and OKAM90.
[1401] Another preferred IPBD is crambin or one of its homologues,
the phoratoxins and ligatoxins (LECO87). These proteins are
secreted in plants. The 3D structure of crambin has been
determined. NMR data on homologues indicate that the 3D structure
is conserved. Residues thought to be on the surface of crambin,
phoratoxin, or ligatoxin are preferred residues to vary.
EXAMPLE XV
[1402] A Mini-protein having a Cross-link Consisting of CU(II), One
Cysteine, Two Histidines, and One Methionine.
[1403] Sequences such as
HIS-ASN-GLY-MET-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-HIS-ASN-G- LY-CYS and
CYS-ASN-GLY-MET-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-HIS-ASN-GLY-HIS are likely
to combine with Cu(II) to form structures as shown in the diagram:
12
[1404] Other arrangements of HIS, MET, HIS, and CYS along the chain
are also likely to form similar structures. The amino acids ASN-GLY
at positions 2 and 3 and at positions 12 and 13 give the amino
acids that carry the metal-binding ligands enough flexibility for
them to come together and bind the metal. Other connecting
sequences may be used, e.g. GLY-ASN, SER-GLY, GLY-PRO, GLY-PRO-GLY,
or PRO-GLY-ASN could be used. It is also possible to vary one or
more residues in the loops that join the first and second or the
third and fourth metal-binding residues. For example, 13
[1405] is likely to form the diagrammed structure for a wide
variety of amino acids at Xaa4. It is expected that the side groups
of Xaa4 and Xaa6 will be close together and on the surface of the
mini-protein.
[1406] The variable amino acids are held so that they have limited
flexibility. This cross-linkage has some differences from the
disulfide linkage. The separation between C.sub..alpha.4 and
C.sub..alpha.11 is greater than the separation of the
C.sub..alpha.s of a cystine. In addition, the interaction of
residues 1 through 4 and 11 through 14 with the metal ion are
expected to limit the motion of residues 5 through 10 more than a
disulfide between rsidues 4 and 11. A single disulfide bond exerts
strong distance constrains on the .alpha. carbons of the joined
residues, but very little directional constraint on, for example,
the vector from N to C in the main-chain.
[1407] For the desired sequence, the side groups of residues 5
through 10 can form specific interactions with the target. Other
numbers of variable amino acids, for example, 4, 5, 7, or 3, are
appropriate. Larger spans may be used when the enclosed sequence
contains segments having a high potential to form .alpha. helices
or other secondary structure that limits the conformational freedom
of the polypeptide main chain. Whereas a mini-protein having four
CYSs could form three distinct pairings, a mini-protein having two
HISs, one MET, and one CYS can form only two distinct complexes
with Cu. These two structures are related by mirror symmetry
through the Cu. Because the two HISs are distinguishable, the
structures are different.
[1408] When such metal-containing mini-proteins are displayed on
filamentous phage, the cells that produce the phage can be grown in
the presence of the appropriate metal ion, or the phage can be
exposed to the metal only after they are separated from the
cells.
EXAMPLE XVI
[1409] A Mini-protein having a Cross-link Consisting of ZN(II) And
Four Cysteines
[1410] A cross link similar to the one shown in Example XV is
exemplified by the Zinc-finger proteins (GIBS88, GAUS87, PARR88,
FRAN87, CHOW87, HARD90). One family of Zinc-fingers has two CYS and
two HIS residues in conserved positions that bind Zn.sub.++
(PARR88, FRANS7, CHOW87, EVAN88, BERG88, CHAV88). Gibson et al.
(GIBS88) review a number of sequences thought to form zinc-fingers
and propose a three-dimensional model for these compounds. Most of
these sequences have two CYS and two HIS residues in conserved
positions, but some have three CYS and one HIS residue. Gauss et
al. (GAUS87) also report a zinc-finger protein having three CYS and
one HIS residues that bind zinc. Hard et al. (HARD90) report the 3D
structure of a protein that comprises two zinc-fingers, each of
which has four CYS residues. All of these zinc-binding proteins are
stable in the reducing intracellular environment.
[1411] One preferred example of a CYS::zinc cross linked
mini-protein comprises residues 440 to 461 of the sequence shown in
FIG. 1 of HARD90. The resiudes 444 through 456 may be variegated.
One such variegation is as follows:
30 Parental Allowed #AA/#DNA SER444 SER, ALA 2/2 ASP445 ASP, ASN,
GLU, LYS 4/4 GLU446 GLU, LYS, GLN 3/3 ALA447 ALA, THR, GLY, SER 4/4
SER448 SER, ALA 2/2 GLY449 GLY, SER, ASN, ASP 4/4 CYS450 CYS, PHE,
ARG, LEU 4/4 H15451 HIS, GLN, ASN, LYS, ASP, GLU 6/6 TYR452 TYR,
PHE, HIS, LEU 4/4 GLY453 GLY, SER, ASN, ASP 4/4 VAL454 VAL, ALA,
ASP, GLY, SER, ASN, THR, ILE 8/8 LEU455 LEU, HIS, ASP, VAL 4/4
THR456 THR, ILE, ASN, SER 4/4
[1412] This leads to 3.77.multidot.10.sup.7 DNA sequences that
encode the same number of amino-acid sequences. A library having
1.0.multidot.10.sup.8 indepentent transformants will display 93% of
the allowed sequences; 2.0.multidot.10.sup.8 independent
transformants will display 99.5% of allowed sequences.
31TABLE 1 Single-letter codes. Single-letter code is used for
proteins: a = ALA c = CYS d = ASP e = GLU f = PHE g = GLY h = HIS i
= ILE k = LYS l = LEU m = MET n = ASN p = PRO q = GLN r = ARG s =
SER t = THR v = VAL w = TRP y = TYR . = STOP * = any amino acid b =
n or d z = e or q x = any amino acid Single-letter IUB codes for
DNA: T, C, A, G stand for themselves M for A or C R for puRines A
or G W for A or T S for C or G Y for pYrimidines T or C K for G or
T V for A, C, or G (not T) H for A, C, or T (not G) D for A, G, or
T (not C) B for C, G, or T (not A) N for any base.
[1413]
32TABLE 2 Preferred Outer-Surface Proteins Preferred Genetic
Outer-Surface Package Protein Reason for preference M13 coat
protein a) exposed amino terminus, (gpVIII) b) predictable post-
translational processing, c) numerous copies in virion. d) fusion
data available gp III a) fusion data available. b) amino terminus
exposed. c) working example available. PhiXl74 G protein a) known
to be on virion exterior, b) small enough that the G-ipbd gene can
replace H gene. E. coli LamB a) fusion data available, b)
non-essential. Ompc a) topological model b) non-essential; abundant
OmpA a) topological model b) non-essential; abundant c) homologues
in other genera OmpF a) topological model b) non-essential;
abundant PhoE a) topological model b) non-essential; abundant c)
inducible B. subtilis CotC a) no post-translational spores
processing, b) distinctive sdequence that causes protein to
localize in spore coat, c) non-essential. CotD Same as for
CotC.
[1414]
33TABLE 3 Ambiguous DNA for AA_seq2 14 15 16 17 18 19 20 21 22 23
24 25 26 27 28 29 30
[1415]
34TABLE 4 Table of Restriction Enzyme Suppliers Suppliers: Sigma
Chemical Co. P.O. Box 14508 St. Louis, Mo. 63178 Bethesda Research
Laboratories P.O. Box 6009 Gaithersburg, Maryland, 20877 Boehringer
Mannheim Biochemicals 7941 Castleway Drive Indianapolis, Indiana,
46250 International Biochemicals, Inc. P.O. Box 9558 New Haven,
Connecticutt, 06535 New England BioLabs 32 Tozer Road Beverly,
Massachusetts, 01915 Promega 2800 S. Fish Hatchery Road Madison,
Wisconsin, 53711 Stratagene Cloning Systems 11099 North Torrey
Pines Road La Jolla, California, 92037
[1416]
35TABLE 5 Potential sites in ipbd gene. Summary of cuts. Enz = %
Acc I has 3 elective sites: 96 169 281 Enz = Afl II has 1 elective
sites: 19 Enz = Apa I has 2 elective sites: 102 103 Enz = Asu II
has 1 elective sites: 381 Enz = Ava III has 1 elective sites: 314
Enz = BspM II has 1 elective sites: 72 Enz = BssH II has 2 elective
sites: 67 115 Enz = % BstX I has 1 elective sites: 323 Enz = +Dra
II has 3 elective sites: 102 103 226 Enz = +EcoN I has 2 elective
sites: 62 94 Enz = +Esp I has 2 elective sites: 57 187 Enz = Hind
III has 6 elective sites: 9 23 60 287 361 386 Enz = Kpn I has 1
elective sites: 48 Enz = Mlu I has 1 elective sites: 314 Enz = Nar
I has 2 elective sites: 238 343 Enz = Nco I has 1 elective sites:
323 Enz = Nhe I has 3 elective sites: 25 289 388 Enz = Nru I has 2
elective sites: 38 65 Enz = +PflM I has 1 elective sites: 94 Enz =
PmaC I has 1 elective sites: 228 Enz = +PpuM I has 2 elective
sites: 102 226 Enz = +Rsr II has 1 elective sites: 102 Enz = +Sfi I
has 2 elective sites: 24 261 Enz = Spe I has 3 elective sites: 12
45 379 Enz = Sph I has 1 elective sites: 221 Enz = Stu I has 5
elective sites: 23 70 150 287 386 Enz = % Sty I has 6 elective
sites: 11 44 143 263 323 383 Enz = Xba I has 1 elective sites: 84
Enz = Xho I has 1 elective sites: 85 Enz = Xma III has 3 elective
sites: 70 209 242 Enzymes not cutting ipbd. Avr II BamH I Bcl I
BstE II EcoR I EcoR V Hpa I Not I Sac I Sal I Sau I Sma I Xma I
[1417]
36TABLE 6 Exposure of amino acid types in T4 lzm & HEWL. HEADER
HYDROLASE (O-GLYCOSYL) 18-AUG-86 2LZM COMPND LYSOZYME
(E.C.3.2.1.17) AUTHOR L.H.WEAVER,B.W.MATTHEWS Coordinates from
Brookhaven Protein Data Bank: 1LYM. Only Molecule A was considered.
HEADER HYDROLASE(O-GLYCOSYL) 29-JUL-82 1LYM COMPND LYSOZYME
(E.C.3.2.1.17) AUTHOR J.HOGLE,S.T.RAO,M.SUNDARALINGAM Solvent
radius = 1.40 Atomic radii in Table 7. Surface area measured in
.ANG..sup.2. Max Type N <area> sigma max min
exposed(fraction) ALA 27 211.0 1.47 214.3 207.1 85.1( 0.40) CYS 10
239.8 3.56 245.5 234.4 38.3( 0.16) ASP 17 271.1 5.36 281.4 262.5
127.1( 0.47) GLU 10 297.2 5.78 304.9 285.4 100.7( 0.34) PHE 8 316.6
5.92 325.4 307.5 99.8( 0.32) GLY 23 185.5 1.31 188.3 183.3 91.9(
0.50) HIS 2 297.7 3.23 301.0 294.5 32.9( 0.11) ILE 16 278.1 3.61
285.6 269.6 57.5( 0.21) LYS 19 309.2 5.38 321.9 300.1 147.1( 0.48)
LEU 24 282.6 6.75 304.0 269.8 109.9( 0.39) MET 7 293.0 5.70 299.5
283.1 88.2( 0.30) ASN 26 273.0 5.75 285.1 262.6 143.4( 0.53) PRO 5
239.9 2.75 242.1 234.6 128.7( 0.54) GLN 8 299.5 4.75 305.8 291.5
145.9( 0.49) ARG 24 344.7 8.66 355.8 326.7 240.7( 0.70) SER 16
228.6 3.59 236.6 223.3 98.2( 0.43) THR 18 250.3 3.89 257.2 244.2
139.9( 0.56) VAL 15 254.3 4.05 261.8 245.7 111.1( 0.44) TRP 9 359.4
3.38 366.4 355.1 102.0( 0.28) TYR 9 335.8 4.97 342.0 325.0 72.6(
0.22)
[1418]
37TABLE 7 Atomic radii .ANG. C.sub..alpha. 1.70 .sup.Ocarbonyl 1.52
.sup.Namide 1.55 Other atoms 1.80
[1419]
38TABLE 8 Fraction of DNA molecules having n non-parental bases
when reagents that have fraction M of parental nucleotode. M .9965
.97716 .92612 .8577 .79433 .63096 f0 .9000 .5000 .1000 .0100 .0010
.000001 f1 .09499 .35061 .2393 .04977 .00777 .0000175 f2 .00485
.1188 .2768 .1197 .0292 .000149 f3 .00016 .0259 .2061 .1854 .0705
.000812 f4 .000004 .00409 .1110 .2077 .1232 .003207 f8 0. 2
.multidot. 10.sup.-7 .00096 .0336 .1182 .080165 f16 0. 0. 0. 5
.multidot. 10.sup.-7 .00006 .027281 f23 0. 0. 0. 0. 0. .0000089
most 0 0 2 5 7 12 "most" is the value of n having the highest
probability.
[1420]
39TABLE 9 best vgCodon Program "Find Optimum vgCodon."
INITIALIZE-MEMORY-OF-ABUNDANCES DO ( t1 = 0.21 to 0.31 in steps of
0.01 ) DO ( c1 = 0.13 to 0.23 in steps of 0.01 ) DO ( a1 = 0.23 to
0.33 in steps of 0.01 ) Comment calculate g1 from other
concentrations g1 = 1.0 - t1 - c1 - a1 IF( g1 .ge. 0.15 ) DO ( a2 =
0.37 to 0.50 in steps of 0.01 ) DO ( c2 = 0.12 to 0.20 in steps of
0.01 ) Comment Force D+E = R + K g2 = (g1*a2 -.5*a1*a2)/(c1+0.5*a1)
Comment Calc t2 from other concentrations. t2 = 1. - a2 - c2 - g2
IF(g2.gt. 0.1.and. t2.gt.0.1) CALCULATE-ABUNDANCES
COMPARE-ABUNDANCES-TO-PREVIOUS-ONES end_IF_block end_DO_loop ! c2
end_DO_loop ! a2 end_IF_block ! if g1 big enough end_DO_loop ! a1
end_DO_loop ! c1 end_DO_loop ! t1 WRITE the best distribution and
the abundances.
[1421]
40TABLE 10 Abundances obtained from various vgCodons A. Optimized
fxS Codon, Restrained by [D] + [E] = [K] + [R] T C A G 1 .26 .18
.26 .30 f 2 .22 .16 .40 .22 x 3 .5 .0 .0 .5 S Amino Amino acid
Abundance acid Abundance A 4.80% C 2.86% D 6.00% E 6.00% F 2.86% G
6.60% H 3.60% I 2.86% K 5.20% L 6.82% M 2.86% N 5.20% P 2.88% Q
3.60% R 6.82% S 7.02% mfaa T 4.16% V 6.60% W 2.86% lfaa Y 5.20%
stop 5.20% [D] + [E] .ident. [K] + [R] = .12 ratio =
Abun(W)/Abun(S) = 0.4074 j (l/ratio).sup.j (ratio).sup.j stop-free
1 2.454 .4074 .9480 2 6.025 .1660 .8987 3 14.788 .0676 .8520 4
36.298 .0275 .8077 5 89.095 .0112 .7657 6 218.7 4.57 .multidot.
10.sup.-3 .7258 7 536.8 1.86 .multidot. 10.sup.-3 .6881 B.
Unrestrained, optimized T C A G 1 .27 .19 .27 .27 2 .21 .15 .43 .21
3 .5 .0 .0 .5 Amino Amino acid Abundance acid Abundance A 4.05% C
2.84% D 5.81% E 5.81% F 2.84% G 5.67% H 4.08% I 2.84% K 5.81% L
6.83% M 2.84% N 5.81% P 2.85% Q 4.08% R 6.83% S 6.89% mfaa T 4.05%
V 5.67% W 2.84% lfaa Y 5.81% stop 5.81% [D] + [E] = 0.1162 [K] +
[R] = 0.1264 ratio = Abun(W)/Abun(S) = 0.41176 j (l/ratio).sup.j
(ratio).sup.j stop-free 1 2.4286 .41176 .9419 2 5.8981 .16955 .8872
3 14.3241 .06981 .8356 4 34.7875 .02875 .7871 5 84.4849 .011836
.74135 6 205.180 .004874 .69828 7 498.3 2.007 .multidot. 10.sup.-3
.6577 C. Optimized NNT T C A G 1 .2071 .2929 .2071 .2929 2 .2929
.2071 .2929 .2071 3 1. .0 .0 .0 Amino Amino acid Abundance acid
Abundance A 6.06% C 4.29% lfaa D 8.58% E none F 6.06% G 6.06% H
8.58% I 6.06% K none L 8.58% M none N 6.06% P 6.06% Q none R 6.06%
S 8.58% mfaa T 4.29% lfaa V 8.58% W none Y 6.06% stop none j
(l/ratio).sup.j (ratio).sup.j stop-free 1 2.0 .5 1. 2 4.0 .25 1. 3
8.0 .125 1. 4 16.0 .0625 1. 5 32.0 .03125 1. 6 64.0 .015625 1. 7
128.0 .0078125 1. D. Optimized NNG T C A G 1 .23 .21 .23 .33 2 .215
.285 .285 .215 3 .0 .0 .0 1.0 Amino Amino acid Abundance acid
Abundance A 9.40% C none D none E 9.40% F none G 7.10% H none I
none K 6.60% L 9.50% mfaa M 4.90% N none P 6.00% Q 6.00% R 9.50% S
6.60% T 6.6% V 7.10% W 4.90% lfaa Y none stop 6.60% j
(l/ratio).sup.j (ratio).sup.j stop-free 1 1.9388 .51579 0.934 2
3.7588 .26604 0.8723 3 7.2876 .13722 0.8148 4 14.1289 .07078 0.7610
5 27.3929 3.65 .multidot. 10.sup.-2 0.7108 6 53.109 1.88 .multidot.
10.sup.-2 0.6639 7 102.96 9.72 .multidot. 10.sup.-3 0.6200 E.
Unoptimized NNS (NNK gives identical distribution) T C A G 1 .25
.25 .25 .25 2 .25 .25 .25 .25 3 .0 .5 .0 0.5 Amino Amino acid
Abundance acid Abundance A 6.25% C 3.125% D 3.125% E 3.125% F
3.125% G 6.25% H 3.125% I 3.125% K 3.125% L 9.375% M 3.125% N
3.125% P 6.25% Q 3.125% R 9.375% S 9.375% T 6.25% V 6.25% W 3.125%
Y 3.125% stop 3.125% j (l/ratio).sup.j (ratio).sup.j stop-free 1
3.0 .33333 .96875 2 9.0 .11111 .9385 3 27.0 .03704 .90915 4 81.0
.01234567 .8807 5 243.0 .0041152 .8532 6 729.0 1.37 .multidot.
10.sup.-3 .82655 7 2187.0 4.57 .multidot. 10.sup.-4 .8007
[1422]
41TABLE 11 Calculate worst codon. Program "Find worst vgCodon
within Serr of given distribution." INITIALIZE-MEMORY-OF-ABUNDANCES
Comment Serr is % error level. READ Serr Comment T1i,C1i,A1i,G1i,
T2i,C2i,A2i,G2i, T3i,G3i Comment are the intended nt-distribution.
READ T1i, C1i, A1i, G1i READ T2i, C2i, A2i, G2i READ T3i, G3i Fdwn
= 1.-Serr Fup = 1.+Serr DO ( t1 = T1i*Fdwn to T1i*Fup in 7 steps)
DO ( c1 = C1i*Fdwn to C1i*Fup in 7 steps) DO ( a1 = A1i*Fdwn to
A1i*Fup in 7 steps) g1 = 1. - t1 - c1 - a1 IF( (g1-G1i)/G1i .lt.
-Serr) Comment g1 too far below G1i, push it back g1 = G1i*Fdwn
factor = (1.-g1)/(t1 + c1 + a1) t1 = t1*factor c1 = c1*factor a1 =
a1*factor end_IF_block IF( (g1-G1i)/G1i .gt. Serr) Comment g1 too
far above G1i, push it back g1 = G1i*Fup factor = (1.-g1)/(t1 + c1
+ a1) t1 = t1*factor c1 = c1*factor a1 = a1*factor end_IF_block DO
( a2 = A2i*Fdwn to A2i*Fup in 7 steps) DO ( c2 = C2i*Fdwn to
C2i*Fup in 7 steps) DO (g2=G2i*Fdwn to G2i*Fup in 7 steps) Comment
Calc t2 from other concentrations. t2 = 1. - a2 - c2 - g2 IF(
(t2-T2i)/T2i .lt. -Serr) Comment t2 too far below T2i, push it back
t2 = T2i*Fdwn factor = (1.-t2)/(a2 + c2 + g2) a2 = a2*factor c2 =
c2*factor g2 = g2*factor end_IF_block IF( (t2-T2i)/T2i .gt. Serr)
Comment t2 too far above T2i, push it back t2 = T2i*Fup factor =
(1.-t2)/(a2 + c2 + g2) a2 = a2*factor c2 = c2*factor g2 = g2*factor
end_IF_block IF(g2.gt. 0.0 .and. t2.gt.0.0) t3 = 0.5*(1.-Serr) g3 =
1. - t3 CALCULATE-ABUNDANCES COMPARE-ABUNDANCES-TO-PREVIOUS-- ONES
t3 = 0.5 g3 = 1. - t3 CALCULATE-ABUNDANCES
COMPARE-ABUNDANCES-TO-PREVIOUS-ONES t3 = 0.5*(1.+Serr) g3 = 1. - t3
CALCULATE-ABUNDANCES COMPARE-ABUNDANCES-TO-PREVIOUS-ONES
end_IF_block end_DO_loop ! g2 end_DO_loop ! c2 end_DO_loop ! a2
end_DO_loop ! a1 end_DO_loop ! c1 end_DO_loop ! t1 WRITE the WORST
distribution and the abundances.
[1423]
42TABLE 12 Abundances obtained using optimum vgCodon assuming 5%
errors Amino Amino acid Abundance acid Abundance A 4.59% C 2.76% D
5.45% E 6.02% F 2.49% lfaa G 6.63% H 3.59% I 2.71% K 5.73% L 6.71%
M 3.00% N 5.19% P 3.02% Q 3.97% R 7.68% mfaa S 7.01% T 4.37% V
6.00% W 3.05% Y 4.77% stop 5.27% Ratio = Abun(F)/Abun(R) = 0.3248 j
(l/ratio).sup.j (ratio).sup.j stop-free 1 3.079 .3248 .9473 2 9.481
.1055 .8973 3 29.193 .03425 .8500 4 89.888 .01112 .8052 5 276.78
3.61 .multidot. 10.sup.-3 .7627 6 852.22 1.17 .multidot. 10.sup.-3
.7225 7 2624.1 3.81 .multidot. 10.sup.-4 .6844
[1424]
43TABLE 13 BPTI Homologues R # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
16 17 18 19 -3 -- -- -- F -- -- -- -- -- -- -- -- -- -- -- -- Z --
-- -2 -- -- -- Q T -- -- -- -- -- -- Q -- -- -- H G Z -- -1 -- --
-- T E -- -- -- -- -- -- P -- -- -- D D G -- 1 R R R P R R R R R R
R L A R R R K R A 2 P P P P P P P P P P P R A P P P R P A 3 D D D D
D D D D D D D K K D R T D S K 4 F F F L F F F F F F F L Y F F F I F
Y 5 C C C C C C C C C C C C C C C C C C 6 L L L Q L L L L L L L I K
E E N R N K 7 E E E L E E E E E E E L L L L L L L L 8 P P P P P P P
P P P P H P P P P P P P 9 P P P Q P P P P P P P R L A A P P A V 10
Y Y Y A Y Y Y Y Y Y Y N R E E E E E R 11 T T T R T T T T T T T P I
T T S Q T Y 12 G G G G G G G G G G G G G G G G G G G 13 P P P P P P
P P P P P R P L L R P P P 14 C T A C C C C C C C C C C C C C C C C
15 K K K K K V G A L I K Y K K K R K K K 16 A A A A A A A A A A A Q
R A A G G A K 17 R R R A A R R R R R R K K Y R H R S K 18 I I I L M
I I I I I I I I I I I L I F 19 I I I L I I I I I I I P P R R R P R
P 20 R R R R R R R R R R R A S S S R R Q S 21 Y Y Y Y Y Y Y Y Y Y Y
F F F F I Y Y F 22 F F F F F F F F F F F Y Y H H Y F Y Y 23 Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y 24 N N N N N N N N N N N N K N N N N
N N 25 A A A S A A A A A A A Q W L R L P S W 26 K K K T K K K K K K
K K K A A E A K K 27 A A A S A A A A A A A K A A A S S S A 28 G G G
N G G G G G G G K K Q Q N R G K 29 L L L A F L L L L L L Q Q Q Q K
N G Q 30 C C C C C C C C C C C C C C C C C C C 31 Q Q Q E E Q Q Q Q
Q Q E L L L K E Q L 32 T T T P T T T T T T T G P Q E V S Q P 33 F F
F F F F F F F F F F F F F F F F F 34 V V V T V V V V V V V T D I I
F I I N 35 Y Y Y Y Y Y Y Y Y Y W Y Y Y Y Y Y Y 36 G G G G G G G G G
G G S S G G G G G S 37 G G G G G G C G G C G G G G G G G G G 38 C T
A C C C C C C C C C C C C C C C C 39 R R R Q R R R R R R R G G G G
G K R G 40 A A A G A A A A A A A G G G G G G G G 41 K K K N K K K K
K K K N N N N N N N N 42 R R R N S R R R R R R S A A A A K Q A 43 N
N N N N N N N N N N N N N N N N N N 44 N N N N N N N N N N N R R R
R N N R R 45 F F F F F F F F F F F F F F F F F F F 46 K K K E K K K
K K K K K K K K E K D K 47 S S S T S S S S S S S T T T T T T T T 48
A A A T A A A A A A A I I I I R K T I 49 E E E E E E E E E E E E E
D D D A Q E 50 D D D M D D D D D D D E E E E E E Q E 51 C C C C C C
C C C C C C C C C C C C C 52 M M M L M M M M M M E R R R H R V Q R
53 R R R R R R R R R R R R R R R E R G R 54 T T T I T T T T T T T T
T T T T A V T 55 C C C C C C C C C C C C C C C C C C C 56 G G G E G
G G G G G G I V V V G R V V 57 G G G P G G G G G G G R G G G G P --
G 58 A A A P A A A A A A A K -- -- -- K P -- -- 59 -- -- -- Q -- --
-- -- -- -- -- -- -- -- -- -- E -- -- 60 -- -- -- Q -- -- -- -- --
-- -- -- -- -- -- -- R -- -- 61 -- -- -- T -- -- -- -- -- -- -- --
-- -- -- -- P -- -- 62 -- -- -- D -- -- -- -- -- -- -- -- -- -- --
-- -- -- -- 63 -- -- -- K -- -- -- -- -- -- -- -- -- -- -- -- -- --
-- 64 -- -- -- S -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- R #
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 -5 -- -- -- -- --
-- -- -- -- -- -- -- D -- -- -- -4 -- -- -- -- -- -- -- -- -- -- --
-- E -- -- -- -3 -- -- -- -- -- -- -- -- -- -- -- -- T P -- -- -2 Z
-- L Z R K -- -- -- R R -- E T -- -- -1 P -- Q D D N -- -- -- Q K
-- R T -- -- 1 R R H H R R I K T R R R G D K T 2 R P R P P P N E V
H H P F L A V 3 K Y T K K T G D A R P D L P D E 4 L A F F F F D S A
D D F D I S A 5 C C C C C C C C C C C C C C C C 6 I E K Y Y N E Q N
D D L T E Q N 7 L L L L L L L L L K K E S Q L L 8 H I P P P L P G P
P P P P A D P 9 R V A A A P K Y V P P P P FG Y I 10 N A E D D E V S
I D D Y V D S V 11 P A P P P T V A R K T T T A Q Q 12 G G G G G G G
G G G K G G G G G 13 R P P R R R P P P N I P P L P P 14 C C C C C C
C C C C C C C C C C 15 Y M K K L N R M R -- -- K R F L R 16 D F A A
A A A G A G Q A A G G A 17 K F S H Y L R M F P T K G Y L F 18 I I I
I M I F T I V V M F M F I 19 P S P P P P P S Q R R I K K K Q 20 A A
A R R A R R L A A R R L R L 21 F F F F F F Y Y W F F Y Y Y Y W 22 Y
Y Y Y Y Y Y F A Y Y F N S F A 23 Y Y Y Y Y Y Y Y F Y Y Y Y Y Y F 24
N S N D N N N N D D K N N N N D 25 Q K W S P S S G A T P A T Q G A
26 K G A A A H S T V R S K R E T V 27 K A A S S L S S K L A A T T S
K 28 K N K N N H K M G K K G K K M G 29 Q K K K K K R A K T R F Q N
A K 30 C C C C C C C C C C C C C C C C 31 E Y Q N E Q E E V K V E E
E E V 32 R P L K K K K T L A Q T P E T R 33 F F F F F F F F F F F F
F F F F 34 D T H I I N I Q P Q R V K I L S 35 W Y Y Y Y Y Y Y Y Y Y
Y Y Y Y Y 36 S S G G G G G G G R G G G G G G 37 G G G G G G G G G G
G G G G G G 38 C C C C C C C C C C C C C C C C 39 G R K P R G G M Q
D D K K Q M K 40 G G G G G G G G G G G A G G G G 41 N N N N N N N N
N D D K N N N N 42 S A A A A A A G G H H S G D L G 43 N N N N N N N
N N G G N N N N N 44 R R R N N N N N K N N N R R N K 45 F F F F F F
F F F F F F Y F F F 46 K K S K K K H V Y K K R K S L Y 47 T T T T T
T T T S T S S S T S S 48 I I I W W I L E E E D A E L Q Q 49 E E E D
D D E K K T H E Q A K K 50 E E K E E E E E E L L D D E E E 51 C C C
C C C C C C C C C C C C C 52 R R R R R Q E L R R R M L E L K 53 R R
H Q H R K Q E C C R D Q Q E 54 T T A T T T V T Y E E T A K T Y 55 C
C C C C C C C C C C C C C C C 56 I V V G V A G R G L E G S I R G 57
G V G A A A V -- V V L G G N -- I 58 -- -- -- S S K R -- P Y Y A F
-- -- P 59 -- -- -- A G Y S -- G P R -- -- -- -- G 60 -- -- -- -- I
G -- -- D -- -- -- -- -- -- E 61 -- -- -- -- -- -- -- -- E -- -- --
-- -- -- A R # 36 37 38 39 40 -5 -- -- -- -- -- -4 -- -- -- -- --
-3 -- -- -- -- -- -2 -- -- -- -- -- -1 -- Z -- -- -- 1 R R R R R 2
P P P P P 3 D D D D D 4 F F F F F 5 C C C C C 6 L L L L L 7 E E E E
E 8 P P P P P 9 P P P P P 10 Y Y Y Y Y 11 T T T T T 12 G G G G G 13
P P P P P 14 C C C C C 15 R K K K K 16 A A A A A 17 R R R R K 18 I
M I M M 19 I I I I I 20 R R R R R 21 Y Y Y Y Y 22 F F F F F 23 Y Y
Y Y Y 24 N N N N N 25 A A A A A 26 K K K K K 27 A A A A A 28 C G G
G G 29 L L L L F 30 C C C C C 31 Q Q Q Q E 32 T P P P T 33 F F F F
F 34 V V V V V 35 Y Y Y Y Y 36 G G G G G 37 G G G G G 38 C C C C C
39 R R R R K 40 A A A A A 41 K K K K K 42 R S R R S 43 N N N N N 44
N N N N N 45 F F F F F 46 K K K K R 47 S S S S S 48 A A S A A 49 E
E E E E 50 D D D D D 51 C C C C C 52 E N N M M 53 R R R R R 54 T T
T T T 55 C C C C C 56 G G G G G 57 G G G G G 58 A A A A A 59 -- --
-- -- -- 60 -- -- -- -- -- 61 -- -- -- -- -- 1 BPTI 2 Engineered
BPTI From MARK87 3 Engineered BPTI From MARK87 4 Bovine Colostrum
(DUFT85) 5 Bovine Serum (DUFT85) 6 Semisynthetic BPTI, TSCH87 7
Semisynthetic BPTI, TSCH87 8 Semisynthetic BPTI, TSCH87 9
Semisynthetic BPTI, TSCH87 10 Semisynthetic BPTI, TSCH87 11
Engineered BPTI, AUER87 12 Dendroaspis polylepis polylepis (Black
mamba) venom I (DUFT85) 13 Dendroaspis polylepis polylepis (Black
Mamba) venom K (DUFT85) 14 Hemachatus hemachates (Ringhals Cobra)
HHV II (DUFT85) 15 Naja nivea (Cape cobra) NNV II (DUFT85) 16
Vipera russelli (Russel's viper) RVV II (TAKA74) 17 Red sea turtle
egg white (DUFT85) 18 Snail mucus (Helix pomania) (WAGN78) 19
Dendroaspis angusticeps (Eastern green mamba) C13 S1 C3 toxin
(DUFT85) 20 Dendroaspis angusticeps (Eastern Green Mamba) C13 S2 C3
toxin (DUFT85) 21 Dendroaspis polylepis polylepes (Black mamba) B
toxin (DUFT85) 22 Dendroaspis polylepis polylepes (Black Mamba) E
toxin (DUFT85) 23 Vipera ammodytes TI toxin (DUFT85) 24 Vipera
ammodytes CTI toxin (DUFT85) 25 Bungarus fasciatus VIII B toxin
(DUFT85) 26 Anemonia sulcata (sea anemone) 5 II (DUFT85) 27 Homo
sapiens HI-14 "inactive" domain (DUFT85) 28 Homo sapiens HI-8
"active" domain (DUFT85) 29 beta bungarotoxin B1 (DUFT85) 30 beta
bungarotoxin B2 (DUFT85) 31 Bovine spleen TI II (FIOR85) 32
Tachypleus tridentatus (Horseshoe crab) hemocyte inhibitor (NAKA87)
33 Bombyx mori (silkworm) SCI-III (SASA84) 34 Bos taurus (inactive)
BI-14 35 Bos taurus (active) BI-8 36: Engineered BPTI (KR15, ME52):
Auerswald '88, Biol Chem Hoppe-Seyler, 369 Supplement, pp27-35. 37:
Isoaprotinin G-1: Siekmann, Wenzel, Schroder, and Tschesche '88,
Biol Chem Hoppe-Seyler, 369:157-163. 38: Isoaprotinin 2: Siekmann,
Wenzel, Schroder, and Tschesche '88, Biol Chem Hoppe-Seyler,
369:l57-163. 39: Isoaprotinin G-2: Siekmann, Wenzel, Schroder, and
Tschesche '88, Biol Chem Hoppe-Seyler, 369:157-163. 40:
Isoaprotinin 1: Siekmann, Wenzel, Schroder, and Tschesche '88, Biol
Chem Hoppe-Seyler, 369:157-163. Notes a) both beta bungarotoxins
have residue 15 deleted. b) B. mori has an extra residue between C5
and C14; we have assigned F and G to residue 9. c) all natural
proteins have C at 5, 14, 30, 38, 50, & 55. d) all homologues
have F33 and G37. e) extra C's in bungarotoxins form interchain
cystine bridges
[1425]
44TABLE 13A Identification codes for Tables 14 and 15 1 BPTI 2
synthetic BPTI, Tan & Kaiser, biochem. 16(8)1531-41 3
Semisynthetic BPTI, TSCH87 4 Semisynthetic BPTI, TSCH87 5
Semisynthetic BPTI, TSCH87 6 Semisynthetic BPTI, TSCH87 7
Semisynthetic BPTI, TSCH87 8 Engineered BPTI, AUER87 9 BPTI
Auerswald & al GB 2 208 511A 10 BPTI Auerswald & al GB 2
208 511A 11 Engineered BPTI From MARK87 12 Engineered BPTI From
MARK87 13 BPTI(KR15,ME52): Auerswald '88, Biol Chem Hoppe-Seyler,
369 Suppl, pp27-35. 14 BPTI CA30/CA51 Eigenbrot & al, Protein
Engineering 3(7)591-598 ('90) 15 Isoaprotinin 2 Siekmann et al '88,
Biol Chem Hoppe-Seyler, 369:157-163. 16 Isoaprotinin G-2: Siekmann
et al '88, Biol Chem Hoppe-Seyler, 369:157-163. 17 BPTI Engineered,
Auerswald & al GB 2 208 511A 18 BPTI Engineered, Auerswald
& al GB 2 208 511A 19 BPTI Engineered, Auerswald & al GB 2
208 511A 20 Isoaprotinin G-1 Siekmann & al '88, Biol Chem
Hoppe-Seyler, 369:157-163. 21 BPTI Engineered, Auerswald & al
GB 2 208 511A 22 BPTI Engineered, Auerswald & al GB 2 208 511A
23 Bovine Serum (in Dufton '85) 24 Bovine spleen TI II (FIOR85) 25
Snail mucus (Helix pomatia) (WAGN78) 26 Hemachatus hemachates
(Ringhals Cobra) HHV II (in Dufton '85) 27 Red sea turtle egg white
(in Dufton '85) 28 Bovine Colostrum (in Dufton '85) 29 Naja nivea
(Cape cobra) NNV II (in Dufton '85) 30 Bungarus fasciatus VIII B
toxin (in Dufton '85) 31 Vipera ammodytes TI toxin (in Dufton '85)
32 Porcine ITI domain 1, (in CREI87) 33 Human Alzheimer's beta APP
protease inhibitor, (SHIN90) 34 Equine ITI domain 1, in Creighton
& Charles 35 Bos taurus (inactive) BI-8e (ITI domain 1) 36
Anemonia sulcata (sea anemone) 5 II (in Dufton '85) 37 Dendroaspis
polylepis polylepes (Black Mamba) E toxin (in Dufton '85) 38 Vipera
russelli (Russel's viper) RVV II (TAKA74) 39 Tachypleus tridentatus
(Horseshoe crab) hemocyte inhibitor (NAKA87) 40 LACI 2 (Factor Xa)
(WUNT88) 41 Vipera ammodytes CTI toxin (in Dufton '85) 42
Dendroaspis polylepis polylepis (Black Mamba) venom K (in Dufton
'85) 43 Homo sapiens HI-8e "inactive" domain (in Dufton '85) 44
Green Mamba toxin K, (in CREI87) 45 Dendroaspis angusticeps
(Eastern green mamba) C13 SI C3 toxin (in Dufton '85) 46 LACI 3 47
Equine ITI domain 2, (CREI87) 48 LACI 1 (VIIa) 49 Dendroaspis
polylepis polylepes (Black mamba) B toxin (in Dufton '85) 50
Porcine ITI domain 2, Creighton and Charles 51 Homo sapiens HI-8t
"active" domain (in Dufton '85) 52 Bos taurus (active) BI-8t 53
Trypstatin Kito & al ('88) J Biol Chem 263(34)18104-07 54
Dendroaspis angusticeps (Eastern Green Mamba) C13 S2 C3 toxin (in
Dufton '85) 55 Green Mamba I venom Creighton & Charles '87
CSHSQB 52:511-519. 56 beta bungarotoxin B2 (in Dufton '85) 57
Dendroaspis polylepis polylepis (Black mamba) venom I (in Dufton
'85) 58 beta bungarotoxin Bl (in Dufton '85) 59 Bombyx mori
(silkworm) SCI-III (SASA84)
[1426]
45TABLE 14 Tally of Ionizable groups Identifier D E K R Y H NH CO2
+ ions 1 2 2 4 6 4 0 1 1 6 16 2 2 2 4 6 4 0 1 1 6 16 3 2 2 3 6 4 0
1 1 5 15 4 2 2 3 6 4 0 1 1 5 15 5 2 2 3 6 4 0 1 1 5 15 6 2 2 3 6 4
0 1 1 5 15 7 2 2 3 6 4 0 1 1 5 15 8 2 3 4 6 4 0 1 1 5 17 9 2 2 3 5
4 0 1 1 4 14 10 2 3 3 6 4 0 1 1 4 16 11 2 2 4 6 4 0 1 1 6 16 12 2 2
4 6 4 0 1 1 6 16 13 2 3 3 7 4 0 1 1 5 17 14 2 2 4 6 4 0 1 1 6 16 15
2 2 4 6 4 0 1 1 6 16 16 2 2 4 6 4 0 1 1 6 16 17 2 2 3 5 4 0 1 1 4
14 18 2 3 3 5 4 0 1 1 3 15 19 2 3 3 5 4 0 1 1 3 15 20 2 2 4 5 4 0 1
1 5 15 21 2 3 3 4 4 0 1 1 2 14 22 2 4 3 4 4 0 1 1 1 15 23 2 4 4 4 4
0 1 1 2 16 24 2 3 5 4 4 0 1 1 4 16 25 1 1 2 4 4 0 1 1 4 10 26 2 3 2
5 3 1 1 1 2 14 27 2 4 6 8 3 0 1 1 8 22 28 2 4 2 3 3 0 1 1 -1 13 29
1 4 2 7 2 2 1 1 4 16 30 1 2 5 3 4 2 1 1 5 13 31 4 1 5 3 4 2 1 1 3
15 32 1 4 3 2 4 1 1 1 0 12 33 2 6 1 5 3 0 1 1 -2 16 34 2 4 2 2 3 1
1 1 -2 12 35 2 2 3 2 4 0 1 1 1 11 36 1 5 4 5 4 1 1 1 3 17 37 0 2 6
3 3 3 1 1 7 13 38 2 5 3 7 3 2 1 1 3 19 39 3 3 5 5 4 0 1 1 4 18 40 3
7 4 3 4 0 1 1 3 19 41 3 2 4 6 5 1 1 1 5 17 42 1 2 8 5 4 0 1 1 10 18
43 1 4 2 2 4 0 1 1 -1 11 44 1 2 9 4 5 0 1 1 10 18 45 0 2 8 4 5 0 1
1 10 16 46 1 3 5 5 3 0 1 1 6 16 47 3 4 4 3 3 0 1 1 0 16 48 3 6 5 4
1 1 1 1 0 20 49 0 3 3 5 5 0 1 1 5 13 50 2 6 4 2 3 0 1 1 -2 16 51 2
4 4 3 3 0 1 1 1 15 52 1 4 6 2 3 0 1 1 3 15 53 2 2 5 1 4 0 1 1 2 12
54 2 3 6 8 3 1 1 1 9 21 55 1 3 6 7 3 1 1 1 9 19 56 6 2 6 7 4 3 1 1
5 23 57 0 3 7 7 3 1 1 1 11 19 58 6 2 5 7 4 2 1 1 4 22 59 4 7 3 1 4
0 1 1 -7 17
[1427]
46TABLE 15 Frequency of Amino Acids at Each Position in BPTI and 58
Homologues Res. Different Id. AAs Contents First -5 2 -58 D -- -4 2
-58 E -- -3 5 -55 P T Z F -- -2 10 -43 R3 Z3 Q3 T2 E G H K L -- -1
11 -41 D4 P3 R2 T2 Q2 G K N Z E -- 1 13 R35 K6 T4 A3 H2 G2 L M N P
I D -- R 2 10 P35 R6 A4 V4 H3 E3 N F I L P 3 11 D32 K8 S4 A3 T3 R2
E2 P2 G L Y D 4 9 F34 A6 D4 L4 S4 Y3 I2 W V F 5 1 C59 C 6 13 L25 N7
E6 K4 Q4 I3 D2 S2 Y2 R F T A L 7 7 L28 E25 K2 F Q S T E 8 10 P46 H3
D2 G2 E I K L A Q P 9 12 P30 A9 I4 V4 R3 Y3 L F Q H E K P 9a 2 -58G
-- 10 9 Y24 E8 D8 V6 R3 S3 A3 N3 I Y 11 11 T31 Q8 P7 R3 A3 Y2 K S D
V I T 12 2 G58 K G 13 5 P45 R7 L4 I2 N P 14 3 C57 A T C 15 12 K22
R12 L7 V6 Y3 M2 -2 N I A F G K 16 7 A41 G9 F2 D2 K2 Q2 R A 17 14
R19 L8 K7 F5 M4 Y4 H2 A2 S2 G2 I N T P R 18 8 I41 M7 F4 L2 V2 E T A
I 19 10 I24 P12 R8 K5 S4 Q2 L N E T I 20 5 R39 A8 L6 S5 Q R 21 5
Y35 F17 W51 L Y 22 6 F32 Y18 A5 H2 S N F 23 2 Y52 F7 Y 24 4 N47 D8
K3 S N 25 13 A29 S6 Q4 G4 W4 P3 T2 L2 R N K V I A 26 11 K31 A9 T5
S3 V3 R2 E2 G H F Q K 27 8 A32 S11 K5 T4 Q3 L2 I E A 28 7 G32 K13
N5 M4 Q2 R2 H G 29 10 L22 K13 Q11 A5 F2 R2 N G M T L 30 2 C58 A C
31 10 Q25 E17 L5 V5 K2 N A R I Y Q 32 11 T25 P11 K4 Q4 L4 R3 E3 G2
S A V T 33 1 F59 F 34 13 V24 I10 T5 N3 Q3 D3 K3 F2 H2 R S P L V 35
2 Y56 W3 Y 36 3 G50 S8 R G 37 1 G59 G 38 3 C57 A T C 39 9 R25 G13
K6 Q4 E3 M3 L2 D2 P R 40 2 G35 A24 A 41 3 N33 K24 D2 K 42 12 R22
A12 G8 S6 Q2 H2 N2 M D E K L R 43 2 N57 G2 N 44 3 N40 R14 K5 N 45 2
F58 Y F 46 11 K39 Y5 E4 S2 V2 D2 R H T A L K 47 2 S36 T23 S 48 11
A23 I11 E6 Q6 L4 K2 T2 W2 S D R A 49 8 E37 K8 D6 Q3 A2 P H T E 50 7
E27 D25 K2 L2 M Q Y D 51 2 C58 A C 52 9 M17 R15 E8 L7 K6 Q2 T2 H V
M 53 11 R37 E6 Q5 K2 C2 H2 A N G D W R 54 8 T41 Y5 A4 V3 I2 E2 M K
T 55 1 C59 C 56 10 G33 V9 R5 I4 E3 L A S T K G 57 12 G34 V6 -5 A3
R2 I2 P2 D K S L N G 58 10 A25 -15 P7 K3 S2 Y2 G2 F D R A
[1428]
47TABLE 16 Exposure in BPTI Coordinates taken from Brookhaven
Protein Data Bank entry 6PTI. HEADER PROTEINASE INHIBITOR (TRYPSIN)
13-MAY-87 6PTI COMPND BOVINE PANCREATIC TRYPSIN INHIBITOR COMPND
2(/BPTI$,CRYSTAL FORM /III$) AUTHOR A.WLODAWER Solvent radius =
1.40 Atomic radii given in Table 7 Areas in .ANG..sup.2. Not Not
Total Covered covered Residue area by M/C fraction at all fraction
ARG 1 342.45 205.09 0.5989 152.49 0.4453 PRO 2 239.12 92.65 0.3875
47.56 0.1989 ASP 3 272.39 158.77 0.5829 143.23 0.5258 PHE 4 311.33
137.82 0.4427 43.21 0.1388 CYS 5 241.06 48.36 0.2006 0.23 0.0010
LEU 6 280.98 151.45 0.5390 115.87 0.4124 GLU 7 291.39 128.91 0.4424
90.39 0.3102 PRO 8 236.12 128.71 0.5451 99.98 0.4234 PRO 9 236.09
109.82 0.4652 45.80 0.1940 TYR 10 330.97 153.63 0.4642 79.49 0.2402
THR 11 249.20 80.10 0.3214 64.99 0.2608 GLY 12 184.21 56.75 0.3081
23.05 0.1252 PRO 13 240.07 130.25 0.5426 75.27 0.3136 CYS 14 237.10
75.55 0.3186 53.52 0.2257 LYS 15 310.77 200.25 0.6444 192.00 0.6178
ALA 16 209.41 66.63 0.3182 45.59 0.2177 ARG 17 351.09 243.67 0.6940
201.48 0.5739 ILE 18 277.10 100.51 0.3627 58.95 0.2127 ILE 19
278.03 146.06 0.5254 96.05 0.3455 ARG 20 339.11 144.65 0.4266 43.81
0.1292 TYR 21 333.60 102.24 0.3065 69.67 0.2089 PHE 22 306.08 70.64
0.2308 23.01 0.0752 TYR 23 338.66 77.05 0.2275 17.34 0.0512 ASN 24
264.88 99.03 0.3739 38.69 0.1461 ALA 25 211.15 85.13 0.4032 48.20
0.2283 LYS 26 313.29 216.14 0.6899 202.84 0.6474 ALA 27 210.66
96.05 0.4560 54.78 0.2601 GLY 28 186.83 71.52 0.3828 32.09 0.1718
LEU 29 280.70 132.42 0.4718 93.61 0.3335 CYS 30 238.15 57.27 0.2405
19.33 0.0812 GLN 31 301.15 141.80 0.4709 82.64 0.2744 THR 32 251.26
138.17 0.5499 76.47 0.3043 PHE 33 304.27 59.79 0.1965 18.91 0.0622
VAL 34 251.56 109.78 0.4364 42.36 0.1684 TYR 35 332.64 80.52 0.2421
15.05 0.0452 GLY 36 187.06 11.90 0.0636 1.97 0.0105 GLY 37 185.28
84.26 0.4548 39.17 0.2114 CYS 38 234.56 73.64 0.3139 26.40 0.1125
ARG 39 417.13 304.62 0.7303 250.73 0.6011 ALA 40 209.53 94.01
0.4487 52.95 0.2527 LYS 41 314.60 166.23 0.5284 108.77 0.3457 ARG
42 349.06 232.83 0.6670 179.59 0.5145 ASN 43 266.47 38.53 0.1446
5.32 0.0200 ASN 44 269.65 91.08 0.3378 23.39 0.0867 PHE 45 313.22
69.73 0.2226 14.79 0.0472 LYS 46 309.83 217.18 0.7010 155.73 0.5026
SER 47 224.78 69.11 0.3075 24.80 0.1103 ALA 48 211.01 82.06 0.3889
31.07 0.1473 GLU 49 286.62 161.00 0.5617 100.01 0.3489 ASP 50
299.53 156.42 0.5222 95.96 0.3204 CYS 51 238.68 24.51 0.1027 0.00
0.0000 MET 52 293.05 89.48 0.3054 66.70 0.2276 ARG 53 356.20 224.61
0.6306 189.75 0.5327 THR 54 251.53 116.43 0.4629 51.64 0.2053 CYS
55 240.40 69.95 0.2910 0.00 0.0000 GLY 56 184.66 60.79 0.3292 32.78
0.1775 GLY 57 106.58 49.71 0.4664 38.28 0.3592 ALA 58 no position
given in Protein Data Bank "Total area" is the area measured by a
rolling sphere of radius 1.4 .ANG., where only the atoms within the
residue are considered. This takes account of conformation. "Not
covered is the area measured by a rolling sphere by N/C" of radius
1.4 .ANG. where all main-chain atoms are considered, fraction is
the exposed area divided by the total area. Surface buried by
main-chain atoms is more definitely covered than is surface covered
by side group atoms. "Not covered is the area measured by a rolling
sphere at all" of radius 1.4 .ANG. where all atoms of the protein
are considered.
[1429]
48TABLE 17 Plasmids used in Detailed Example I Phage Contents LG1
M13mpl8 with Ava II/Aat II/Acc I/Rsr II/Sau I adaptor pLG2 LG1 with
amp.sup.R and ColE1 of pBR322 cloned into Aat II/Acc I sites pLG3
pLG2 with Acc I site removed pLG4 pLG3 with first part of osp-pbd
gene cloned into Rsr II/Sau I sites, Avr II/Asu II sites created
pLG5 pLG4 with second part of osp-pbd gene cloned into Avr II/Asu
II sites, BssH I site created pLG6 pLG5 with third part of osp-pbd
gene cloned into Asu II/BssH I sites, Bbe I site created pLG7 pLG6
with last part of osp-pbd gene cloned into Bbe I/Asu II sites pLG8
pLG7 with disabled osp-pbd gene, same length DNA. pLG9 pLG7 mutated
to display BPTI(V15.sub.BPTI) pLGlO pLG8 + tet.sup.Rgene -
amp.sup.R gene pLGll pLG9 + tet.sup.Rgene - amp.sup.R gene
[1430]
49TABLE 18 Enzyme sites eliminated when M13mp18 is cut by AvaII and
Bsu36I AhaII NarI GdiII PvuI FspI BglI HgiEII Bsu36I EcoRI SacI
KpnI XmaI SmaI BamHI XbaI SalI HindIII AccI PstI SphI HindII
[1431]
50TABLE 19 Enzymes not cutting M13mp18 AatII AflI ApaI AvrII BbvII
BclI BspMI BssHI BstBI BstEII BstXI EagI Eco57I EcoNI EcoO109I
EcoRV EspI HpaI MluI NcoI NheI NotI NruI NsiI PflMI PmaCI PpaI
PpuMI RsrI SacI ScaI SfiI SpeI StuI StyI Tth111I XcaI XhoI
[1432]
51TABLE 20 Enzymes cutting AmpR gene and ori AatII BbvII Eco57I
PpaI ScaI Tth111I AhaII GdiII PvuI FspI BglI HgiEII HindII PstI
XbaI AflIII NdeI
[1433]
52TABLE 21 Enzymes tested on Ambig DNA Enzyme Recognition Symm cuts
Supply %AccI GTMKAC P 2 & 4 <B,M,I,N,P,T AflII CTTAAG P 1
& 5 <N ApaI GGGCCC P 5 & 1 <M,I,N,P,T AsuII TTCGAA P
2 & 4 <P,N(BstBI) AvaIII ATGCAT P 5 & 1 <T;
NsiI:M,N,P,T; EcoT22I:T AvrII CCTAGG P 1 & 5 <N BamHI GGATCC
P 1 & 5 <S,B,M,I,N,P,T BclI TGATCA P 1 & 5
<S,B,M,I,N,T BspMII TCCGGA P 1 & 5 <N BssHII GCGCGC P 1
& 5 <N,T +BstEII GGTNACC P 1 & 6 <S,B,M,N,T %BstXI
CCANNNNN P 8 & 4 <N,P,T +DraII RGGNCCY P 2 & 5 <M,T ;
Eco0109I:N +EcoNI CCTNNNNN P 5 & 6 <N(soon) EcoRI GAATTC P 1
& 5 <S,B,M,I,N,P,T EcoRV GATATC P 3 & 3
<S,B,M,I,N,P,T +EspI GCTNAGC P 2 & 5 <T HindIII AAGCTT P
1 & 5 <S,B,M,I,N,P,T HpaI GTTAAC P 3 & 3
<S,B,M,I,N,P,T KpnI GGTACC P 5 & 1 <S,B,M,I,N,P,T ;
Asp718:M MluI ACGCGT P 1 & 5 <M,N,P,T NarI GGCGCC P 2 &
4 <B,N,T NcoI CCATGG P 1 & 5 <B,M,N,P,T NheI GCTAGC P 1
& 5 <M,N,P,T NotI GCGGCCGC P 2 & 6 <M,N,P,T NruI
TCGCGA P 3 & 3 <B,M,N,T +PflMI CCANNNNN P 7 & 4 <N
PmaCI CACGTG P 3 & 3 <none +PpuMI RGGWCCY P 2 & 5 <N
+RsrII CGGWCCG P 2 & 5 <N,T SacI GAGCTC P 5 & 1
<B(SstI),M,I,N,P, T SalI GTCGAC P 1 & 5 <B,M,I,N,P,T
+SauI CCTNAGG P 2 & 5 <M; CvnI:B; +EE,UNS MstII :T;
Bsu36I:N; +EE,UNS AocI:T +SfiI GGCCNNNNNGGCC P 8 & 5 <N,P,T
SmaI CCCGGG P 3 & 3 <B,M,I,N,P,T SpeI ACTAGT P 1 & 5
<M,N,T SphI GCATGC P 5 & 1 <B,M,I,N,P,T StuI AGGCCT P 3
& 3 <M,N,I(AatI),P,T %StyI CCWWGG P 1 & 5 <N,P,T XcaI
GTATAC P 3 & 3 <N(soon) XhoI CTCGAG P 1 & 5
<B,M,I,P,T; CcrI: T ; PaeR7I:N XmaI CCCGGG P 1 & 5
<I,N,P,T XmaIII CCGCCG P 1 & 5 <B; EagI:N; Eco52I:T
N_restrct = 43
[1434]
53TABLE 22 ipbd gene pbd mod10 29III88: lacUV5
RsrII/AvrII/gene/TrpA attenuator/MstII; ! 5'- CGGaCCG TaT ! RsrII
site CCAGGC tttaca CTTTATGCTTCCGGCTCG tataat GTG ! lacUV5 TGG
aATTGTGAGCGGATAACAATT ! lacO operator CCT AGGAgg CtcaCT !
Shine-Dalgarno seq. atg aag aaa tct ctg gtt ctt aag gct agc ! 10,
M13 leader gtt gct gtc gcg acc ctg gta ccg atg ctg ! 20 tct ttt gct
cgt ccg gat ttc tgt ctc gag ! 30 ccg cca tat act ggg ccc tgc aaa
gcg cgc ! 40 atc atc cgt tat ttc tac aac gct aaa gca ! 50 ggc ctg
tgc cag acc ttt gta tac ggt ggt ! 60 tgc cgt gct aag cgt aac aac
ttt aaa tcg ! 70 gcc gaa gat tgc atg cgt acc tgc ggt ggc ! 80 gcc
gct gaa ggt gat gat ccg gcc aaa gcg ! 90 gcc ttt aac tct ctg caa
gct tct gct acc ! 100 gaa tat atc ggt tac gcg tgg gcc atg gtg ! 110
gtg gtt atc gtt ggt gct acc atc ggt atc ! 120 aaa ctg ttt aag aaa
ttt act tcg aaa gcg ! 130 tct taa tag tga ggttacc! BstEII agtcta
agcccgc ctaatga gcgggct tttttttt ! terminator CCTgAGG -3' !
MstII
[1435]
54TABLE 23 ipbd DNA sequence DNA Sequence file=INS_M13PTIM13.DNA;
17 DNA Sequence title= pbd mod10 29III88: lac-UV5
RsrII/AvrII/gene/TrpA attenuator/MstII; ! 1
A.vertline.GGA.vertline.CCG.vertline.TAT.vertline.CCA.vertli-
ne.GGC.vertline.TTT.vertline.ACA.vertline.CTT.vertline.TAT.vertline.GCT.ve-
rtline.TCC.vertline.GGC.vertline.TCG.vertline. 41
TAT.vertline.AAT.vertline.GTG.vertline.TGG.vertline.AAT.vertline.TGT.vert-
line.GAG.vertline.CGG.vertline.ATA.vertline.ACA.vertline.ATT.vertline.CCT.-
vertline.AGG.vertline.AGG.vertline. 83
CTC.vertline.ACT.vertline.ATG.vertline.AAG.vertline.AAA.vertline.TCT.vert-
line.CTG.vertline.GTT.vertline.CTT.vertline.AAG.vertline.GCT.vertline.AGC.-
vertline.GTT.vertline.GCT.vertline. 125
GTC.vertline.GCG.vertline.ACC.vertline.CTG.vertline.GTA.vertline.CCG.vert-
line.ATG.vertline.CTG.vertline.TCT.vertline.TTT.vertline.GCT.vertline.CGT.-
vertline.CCG.vertline.GAT.vertline. 167
TTC.vertline.TGT.vertline.CTC.vertline.GAG.vertline.CCG.vertline.CCA.vert-
line.TAT.vertline.ACT.vertline.GGG.vertline.CCC.vertline.TGC.vertline.AAA.-
vertline.GCG.vertline.CGC.vertline. 209
ATC.vertline.ATC.vertline.CGT.vertline.TAT.vertline.TTC.vertline.TAC.vert-
line.AAC.vertline.GCT.vertline.AAA.vertline.GCA.vertline.GGC.vertline.CTG.-
vertline.TGC.vertline.CAG.vertline. 251
ACC.vertline.TTT.vertline.GTA.vertline.TAC.vertline.GGT.vertline.GGT.vert-
line.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.CGT.vertline.AAC.-
vertline.AAC.vertline.TTT.vertline. 293
AAA.vertline.TCG.vertline.GCC.vertline.GAA.vertline.GAT.vertline.TGC.vert-
line.ATG.vertline.CGT.vertline.ACC.vertline.TGC.vertline.GGT.vertline.GGC.-
vertline.GCC.vertline.GCT.vertline. 335
GAA.vertline.GGT.vertline.GAT.vertline.GAT.vertline.CCG.vertline.GCC.vert-
line.AAA.vertline.GCG.vertline.GCC.vertline.TTT.vertline.AAC.vertline.TCT.-
vertline.CTG.vertline.CAA.vertline. 377
GTG.vertline.GTT.vertline.ATC.vertline.GTT.vertline.GGT.vertline.GCT.vert-
line.ACC.vertline.ATC.vertline.GGT.vertline.ATC.vertline.AAA.vertline.CTG.-
vertline.TTT.vertline.AAG.vertline. 419
GTG.vertline.GTT.vertline.ATC.vertline.GTT.vertline.GGT.vertline.GCT.vert-
line.ACC.vertline.ATC.vertline.GGT.vertline.ATC.vertline.AAA.vertline.CTG.-
vertline.TTT.vertline.AAG.vertline. 461
AAA.vertline.TTT.vertline.ACT.vertline.TCG.vertline.AAA.vertline.GCG.vert-
line.TCT.vertline.TAA.vertline.TAG.vertline.TGA.vertline.GGT.vertline.TAC.-
vertline.CAG.vertline.TCT.vertline. 503
AAG.vertline.CCC.vertline.GCC.vertline.TAA.vertline.TGA.vertline.GCG.vert-
line.GGC.vertline.TTT.vertline.TTT.vertline.TTT.vertline.CCT.vertline.GAG.-
vertline.G Total = 539 bases
[1436]
55TABLE 24 Summary of Restriction Cuts Enz = % Acc I has 1 observed
sites: 259 Enz = Acc III has 1 observed sites: 162 Enz = Acy I has
1 observed sites: 328 Enz = Afl II has 1 observed sites: 109 Enz =
% Afl III has 1 observed sites: 404 Enz = Aha III has 1 observed
sites: 292 Enz = Apa I has 1 observed sites: 193 Enz = Asp718 has 1
observed sites: 138 Enz = Asu II has 1 observed sites: 471 Enz = %
Ava I has 1 observed sites: 175 Enz = Avr II has 1 observed sites:
76 Enz = % Ban I has 3 observed sites: 138 328 540 Enz = Bbe I has
1 observed sites: 328 Enz = +Bgl I has 1 observed sites: 352 Enz =
+Bin I has 1 observed sites: 346 Enz = % BspM I has 1 observed
sites: 319 Enz = BssH II has 1 observed sites: 205 Enz = +BstE II
has 1 observed sites: 493 Enz = % BstX I has 1 observed sites: 413
Enz = Cfr I has 2 observed sites: 299 350 Enz = +Dra II has 1
observed sites: 193 Enz = +Esp I has 1 observed sites: 277 Enz = %
Fok I has 1 observed sites: 213 Enz = Gdi II has 2 observed sites:
299 350 Enz = Hae I has 1 observed sites: 240 Enz = Hae II has 1
observed sites: 328 Enz = +Hga I has 1 observed sites: 478 Enz = %
HgiC I has 3 observed sites: 138 328 540 Enz = % HgiJ II has 1
observed sites: 193 Enz = Hind III has 1 observed sites: 377 Enz =
+Hph I has 1 observed sites: 340 Enz = Kpn I has 1 observed sites:
138 Enz = +Mbo II has 2 observed sites: 93 304 Enz = Mlu I has 1
observed sites: 404 Enz = Nar I has 1 observed sites: 328 Enz = Nco
I has 1 observed sites: 413 Enz = Nhe I has 1 observed sites: 115
Enz = Nru I has 1 observed sites: 128 Enz = Nsp (7524) has 1
observed sites: 311 Enz = NspB II has 1 observed sites: 332 Enz =
+PflM I has 1 observed sites: 184 Enz = +Pss I has 1 observed
sites: 193 Enz = +Rsr II has 1 observed sites: 3 Enz = +Sau I has 1
observed sites: 535 Enz = % SfaN I has 2 observed sites: 144 209
Enz = +Sfi I has 1 observed sites: 351 Enz = Sph I has 1 observed
sites: 311 Enz = Stu I has 1 observed sites: 240 Enz = % Sty I has
2 observed sites: 76 413 Enz = Xca I has 1 observed sites: 259 Enz
= Xho I has 1 observed sites: 175 Enz = Xma III has 1 observed
sites: 299 Enzymes that do not cut Aat II AlwN I ApaL I Ase I Ava
III Bal I BamH I Bbv I Bbv II Bcl I Bgl II Bsm I BspH I Cla I Dra
III Eco47 III EcoN I EcoR I EcoR V HaiA I Hinc II Hpa I Mst I Nae I
Nde I Not I Ple I PmaC I PpuM I Pst I Pvu I Pvu II Sac I Sac II Sal
I Sca I Sma I SnaB I Spe I Ssp I Tag II Tth111 I Tth111 II Xho II
Xma I Xmn I
[1437]
56TABLE 25 Annotated Sequence of ipbd gene 31 28 32 52 33 73 34 88
35 118 36 148 37 178 38 208 39 235 40 268 41 295 42 325 43 346 44
361 45 388 46 409 47 424 48 448 49 478 50 502 51 532 52 539 53
[1438]
57TABLE 26 DNA_seq1 54 55 56 57 58 59 60 61
[1439]
58TABLE 27 DNA_synth1
5'.vertline.CCG.vertline.TCC.vertline.GTC.vertline.GGA.vertline.CCG.vertl-
ine.TAT.vertline.CCA.vertline.GGC.vertline.TTT.vertline.ACA.vertline.CTT.v-
ertline. .vertline.TAT .vertline.GCT.vertline.TCC.vertline-
.GGC.vertline.TCG.vertline.TAT.vertline.AAT.vertline.GTG.vertline.TGG.vert-
line. .vertline.AAT.vertline.TGT.vertline.GAG.vertline.-
CGG.vertline.ATA.vertline.ACA.vertline.ATT.vertline. olig#4=3'-gt
taa 10 .vertline.CTT.vertline.AGG.vertline. gga tcc /3'=olig#3
.vertline.GCG.vertline.GCT.vertline.CCT.vertline.TCG.v-
ertline.AAA.vertline.GCG.vertline. cgg cga gga agc ttt cgc
.vertline.TCT.vertline.TAA.vertline.TAG.vertline.TGA.-
vertline.GGT.vertline.TAC.vertline.CAG.vertline.TCT.vertline. aga
att atc act cca atg gtc aga .vertline.AAG.vertline.CCC.ve-
rtline.GCC.vertline.TAA.vertline.TGA.vertline.GCG.vertline.GGC.vertline.TT-
T.vertline.TTT.vertline.TTT.vertline. ttc ggg cgg att act cgc ccg
aaa aaa aaa .vertline.CCT.vertline.GAG.vertline.GCA.vertl-
ine.GGT.vertline.GAG.vertline.CG gga ctc cgt cca ctg gc-5' "Top"
strand 99 "Bottom" strand 100 Overlap 23 (14 c/g and 9 a/t) Net
length 158
[1440]
59TABLE 28 DNA_seq2
5'-.vertline.gcg.vertline.cca.vertline.acg.vertline.
.vertline.spacer .vertline.
.vertline.CCT.vertline.AGG.vertline.AGG.vertline.CTC.vertline.A-
CT.vertline. Avr II.vertline. S. D. .vertline. .vertline. m
.vertline. k .vertline. k .vertline. s .vertline. l .vertline. v
.vertline. l .vertline. k .vertline. a .vertline. s .vertline.
.vertline. 1 .vertline. 2 .vertline. 3 .vertline. 4 .vertline. 5
.vertline. 6 .vertline. 7 .vertline. 8 .vertline. 9 .vertline.
10.vertline.
.vertline.ATG.vertline.AAG.vertline.AAA.vertline.TCT.vertline.CTG.vertlin-
e.GTT.vertline.CTT.vertline.AAG.vertline.GCT.vertline.AGC.vertline.
.vertline.Afl II.vertline.Nhe I .vertline. .vertline. v .vertline.
a .vertline. v .vertline. a .vertline. t .vertline. l .vertline. v
.vertline. p .vertline. m .vertline. l .vertline. .vertline.
11.vertline. 12.vertline. 13.vertline. 14.vertline. 15.vertline.
16.vertline. 17.vertline. 18.vertline. 19.vertline. 20.vertline.
.vertline.GTT.vertline.GCT-
.vertline.GTC.vertline.GCG.vertline.ACC.vertline.CTG.vertline.GTA.vertline-
.CCG.vertline.ATG.vertline.CTG.vertline. .vertline.Nru I .vertline.
.vertline.Kpn I .vertline. .vertline. s .vertline. f .vertline. a
.vertline. r .vertline. p .vertline. d .vertline. f .vertline. c
.vertline. l .vertline. e .vertline. .vertline. 21.vertline.
22.vertline. 23.vertline. 24.vertline. 25.vertline. 26.vertline.
27.vertline. 28.vertline. 29.vertline. 30.vertline.
.vertline.TCT.vertline.TTT.vertline.GCT.vertline.CGT-
.vertline.CCG.vertline.GAT.vertline.TTC.vertline.TGT.vertline.CTC.vertline-
.GAG.vertline. .vertline.AccIII.vertline. .vertline.Ava I
.vertline. .vertline.Xho I .vertline. .vertline. p .vertline. p
.vertline. y .vertline. t .vertline. g .vertline. p .vertline. c
.vertline. k .vertline. a .vertline. r .vertline. .vertline.
31.vertline. 32.vertline. 33.vertline. 34.vertline. 35.vertline.
36.vertline. 37.vertline. 38.vertline. 39.vertline. 40.vertline.
.vertline.CCG.vertline.CCG.vertline.TAT.vertline.ACT.vertline.GGG.vertlin-
e.CCC.vertline.TGC.vertline.AAA.vertline.GCG.vertline.CGC.vertline.
.vertline. PflM I .vertline. .vertline.BssH II.vertline.
.vertline.Apa I .vertline. .vertline.Dra II .vertline.
.vertline.Pss I .vertline. .vertline. i .vertline. i .vertline. r
.vertline. .vertline. 41.vertline. 42.vertline. 43.vertline.
.vertline.atc.vertline.atc.vertline.cgt.vertline. .vertline.t
.vertline.s .vertline.k .vertline.
.vertline.127.vertline.128.vertline.12- 9.vertline.
.vertline.ACT.vertline.TCG.vertline.AA-
c.vertline.gcg.vertline.gct.vertline.gcg.vertline.-3' .vertline.Asu
II.vertline. spacer .vertline.
[1441]
60TABLE 29 DNA_synth2
5'-.vertline.GCA.vertline.CCA.vertline.ACG.vertline.
.vertline.CCT.vertline.AGG.vertline.AGG.vertline.CTC.vertline-
.ACT.vertline. .vertline.ATG.vertline.AAG.vertline.AAA.v-
ertline.TCT.vertline.CTG.vertline.GTT.vertline.CTT.vertline.AAG.vertline.G-
CT.vertline.AGC.vertline. .vertline.GGT.vertline.GCT.ver-
tline.GTC.vertline.GCG.vertline.ACC.vertline.CTG.vertline.GTA.vertline.CCG-
.vertline.ATG.vertline.CTG.vertline. olig.multidot.6=3'-ggc tac gac
/ 3'=olig#5
.vertline.TCT.vertline.TTT.vertline.GCT.vertline.CGT.ve-
rtline.CCG.vertline.GAT.vertline.TTC.vertline.TGT.vertline.CTC.vertline.GA-
G.vertline. aga aaa cga gca ggc cta aag aca gag ctc
.vertline.CCG.vertline.CCA.vertline.TAT.vertline.ACT.vertline.GGG.vert-
line.CCC.vertline.TGC.vertline.AAA.vertline.GCG.vertline.CGC.vertline.
ggc ggt ata tga ccc ggg acg ttt cgc gcg
.vertline.ATC.vertline.ATC.vertline.CGT.vertline. tag tag gca
.vertline.ACT.vertline.TCG.vertline.AAA.ver-
tline.CGC.vertline.GCT.vertline.GCG.vertline. tga agc ttt cgc cga
cgc-5' "Top" strand 99 "Bottom" strand 99 Overlap 24 (14 c/g and 10
a/t) Net length 155
[1442]
61TABLE 30 DNA_seq3 .vertline.a .vertline.r .vertline.
.vertline.39.vertline.40.vertline.
5'-.vertline.ccc.vertline.tgc.vertline.aca.vertline.GCG.vertli-
ne.CGC.vertline. .vertline. spacer .vertline.BssH II.vertline.
.vertline.i .vertline.i .vertline.r .vertline.y .vertline.f
.vertline.y .vertline.n .vertline.a .vertline.k .vertline.
.vertline.41 .vertline.42.vertline.43.vert-
line.44.vertline.45.vertline.46.vertline.47.vertline.48.vertline.49.vertli-
ne. .vertline.ATC.vertline.ATC.vertline.CGT.vertline.TAT.vertline.-
TTC.vertline.TAC.vertline.AAC.vertline.GCT.vertline.AAA.vertline.
.vertline.a .vertline.g .vertline.l .vertline.c .vertline.q
.vertline.t .vertline.f .vertline.v .vertline.y .vertline.g
.vertline.g .vertline.
.vertline.50.vertline.51.vertline.52.vertline.53.vertl-
ine.54.vertline.55.vertline.56.vertline.57.vertline.58.vertline.59.vertlin-
e.60.vertline. .vertline.GCA.vertline.GGC.vertline.CTG.vertline.TG-
C.vertline.CAG.vertline.ACC.vertline.TTT.vertline.GTA.vertline.TAC.vertlin-
e.GGT.vertline.GGT.vertline. .vertline.Stu I .vertline.
.vertline.Acc I .vertline. .vertline.Xca I .vertline. .vertline.c
.vertline.r .vertline.a .vertline.k .vertline.r .vertline.n
.vertline.n .vertline.f .vertline.k .vertline.
.vertline.61.vertline.62.vertline.63.vertl-
ine.64.vertline.65.vertline.66.vertline.67.vertline.68.vertline.69.vertlin-
e. .vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.C-
GT.vertline.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline.
.vertline.Esp I .vertline. .vertline.s .vertline.a .vertline.e
.vertline.d .vertline.c .vertline.m .vertline.r .vertline.t
.vertline.c .vertline.g .vertline.
.vertline.70.vertline.71.vertline.72.vertline.73.vertline.74.vertline.75.-
vertline.76.vertline.77.vertline.78.vertline.79.vertline.
.vertline.TCG.vertline.GCC.vertline.GAA.vertline.GAT.vertline.TGC.vertlin-
e.ATG.vertline.CGT.vertline.ACC.vertline.TGC.vertline.GGT.vertline.
.vertline.XmaIII.vertline. .vertline.Sph I.vertline. .vertline.g
.vertline.a .vertline. .vertline.80.vertline.81.vertline.
.vertline.GGC.vertline.GCC.ver- tline.gct.vertline.gaa.vertline.
.vertline.Bbe I .vertline.spacer.vertline. .vertline.Nar I
.vertline. .vertline.t .vertline.s .vertline.k .vertline.
.vertline.127.vertline.128.vertline.129.vertline.
.vertline.ttt.vertline.acT.vertline.TCG.vertline.AAa.vertline.gcg.vert-
line.tcg.vertline.ccg.vertline.-3' .vertline.Asu II.vertline.
[1443]
62TABLE 31 DNA_synth3
5'-.vertline.CCC.vertline.TGC.vertline.ACA.vertline.GCG.vertli-
ne.CGC.vertline. .vertline.ATC.vertline.ATC.vertline.CGT-
.vertline.TAT.vertline.TTC.vertline.TAC.vertline.AAC.vertline.GCT.vertline-
.AAA.vertline. .vertline.GCA.vertline.GGC.vertline.CTG.v-
ertline.TGC.vertline.CAG.vertline.ACC.vertline.TTT.vertline.GTA.vertline.T-
AC.vertline.GGT.vertline.GGT.vertline. olig#8=3'-g cca cca /
3'=olig#3 .vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.ve-
rtline.CGT.vertline.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline.
acg gca cga ttc gca ttg ttg aaa ttt
.vertline.TCG.vertline.GCC.vertline.GAA.vertline.GAT.vertline.TGC.vertlin-
e.ATG.vertline.CGT.vertline.ACC.vertline.TGC.vertline.GGT.vertline.
agc cgg ctt cta acg tac gca tgg acg cca
.vertline.GGC.vertline.GCC.vertline.GCT.vertline.GAA.vertline. ccg
ccg cgt ctt .vertline.TTT.vertline.ACT.vert-
line.TCG.vertline.AAA.vertline.GCG.vertline.TCG.vertline.CCG.vertline.
aaa tga agc ttt cgc agc ggc-5' "Top" strand 93 "Bottom" strand 97
Overlap 25 (15 g/c & 10 a/t) Net length 146
[1444]
63TABLE 32 DNA.sub.-seq4 .vertline.g .vertline.a .vertline.a
.vertline.e .vertline.g .vertline.d .vertline.d .vertline. 5'
.vertline.80.vertline.81.vertline.82.vertline.83.vertline.84.vertline.85.-
vertline.86.vertline.
.vertline.cct.vertline.cgc.vertline.ctt.vertl-
ine.GGC.vertline.GCC.vertline.GCT.vertline.GAA.vertline.GGT.vertline.GAT.v-
ertline.GAT.vertline. .vertline. spacer .vertline.Bbe I .vertline.
.vertline.Nar I .vertline. .vertline.p .vertline.a .vertline.k
.vertline.a .vertline.a .vertline.
.vertline.87.vertline.88.vertline.89.vertline.90.vertline.91.v-
ertline. .vertline.CCG.vertline.GCC.vertline.AAA.vertline.GCG.v-
ertline.GCC.vertline. .vertline. Sfi I .vertline. .vertline.f
.vertline.n .vertline.s .vertline.l .vertline.q .vertline.a
.vertline.s .vertline.a .vertline.t .vertline.
----.vertline.92.vertline.93.vertline.94.vertline.95.vertline.96.vert-
line.97.vertline.98.vertline.99.vertline.100.vertline.
.vertline.TTT.vertline.AAC.vertline.TCT.vertline.CTG.vertline.CAA.vertlin-
e.GCT.vertline.TCT.vertline.GCT.vertline.ACC.vertline.
.vertline.Hind 3.vertline. .vertline.e .vertline.y .vertline.i
.vertline.g .vertline.y .vertline.a .vertline.w .vertline.
.vertline.101.vertline.102.vertline.103.vertline.104.ve-
rtline.105.vertline.106.vertline.107.vertline.
.vertline.GAA.vertline.TAT.vertline.ATC.vertline.GGT.vertline.TAC.vertlin-
e.GCG.vertline.TGG.vertline. .vertline.Mlu I.vertline. .vertline.a
.vertline.m .vertline.v .vertline.v .vertline.v .vertline.
.vertline.108.vertline.109.vert-
line.110.vertline.111.vertline.112.vertline.
.vertline.GCC.vertline.ATG.vertline.GTG.vertline.GTG.vertline.GTT.vertlin-
e. .vertline. BstX I .vertline. .vertline.Nco I.vertline.
.vertline.i .vertline.v .vertline.g .vertline.a .vertline.t
.vertline.i .vertline.g .vertline.i .vertline.
.vertline.113.vertline.114.vertline.115.vertline.116.vertline.117.ver-
tline.118.vertline.119.vertline.120.vertline.
.vertline.ATC.vertline.GTT.vertline.GGT.vertline.GCT.vertline.ACC.vertlin-
e.ATC.vertline.GGT.vertline.ATC.vertline. .vertline.k .vertline.l
.vertline.f .vertline.k .vertline.k .vertline.f .vertline.t
.vertline.s .vertline.k .vertline. .vertline.121.vertline.122.vert-
line.123.vertline.124.vertline.125.vertline.126.vertline.127.vertline.128.-
vertline.129.vertline.
.vertline.AAA.vertline.CTG.vertline.TTT.vert-
line.AAG.vertline.AAA.vertline.TTT.vertline.ACT.vertline.TCG.vertline.AAa.-
vertline.gcg.vertline.tcg.vertline.ggc.vertline.-3' .vertline.Asu
II.vertline. spacer .vertline.
[1445]
64TABLE 33 DNA_synth4
5'.vertline.GCT.vertline.CGC.vertline.CCT.vertline.GGC.vertline.GCC.vertl-
ine.GCT.vertline.GAA.vertline.GGT.vertline.GAT.vertline.GAT.vertline.
.vertline.CCG.vertline.GCC.vertline.AAA.vertline.GCG.vertli-
ne.GCC.vertline. .vertline.TTT.vertline.AAC.vertline.TC-
T.vertline.CTG.vertline.CAA.vertline.GCT.vertline.TCT.vertline.GCT.vertlin-
e.ACC.vertline. .vertline.GAA.vertline.TAT.ver-
tline.ATC.vertline.GGT.vertline.TAC.vertline.GCG.vertline.TGG.vertline.
olig#10=3'- ata tag cca atg cgc aac / 3'=olig#9
.vertline.GCC.vertline.ATG.vertline.GTG.vertline.GTG.ver-
tline.GTT.vertline. ccg tac cac cac caa
.vertline.ATC.vertline.GTT.vertline.GGT.vertline.GCT.vertline.ACC.vertlin-
e.ATC.vertline.GGT.vertline.ATC.vertline. tag caa cca cga tgg tag
cca tag .vertline.AAA.vertline.CTG.vertline.TTT.vertline.-
AAG.vertline.AAA.vertline.TTT.vertline.ACT.vertline.TCG.vertline.AAA.vertl-
ine.GCG.vertline.TCT.vertline.TGA.vertline. tct gac aaa ttc ttt aaa
tga agc ttt cgc aga act-5' "Top" strand 100 "Bottom" strand 93
Overlap 25 (14 c/g and 11 alt) Net length 149
[1446]
65TABLE 34 Some interaction sets in BPTI Number Res. Diff. # AAs
Contents BPTI 1 2 3 4 5 -5 2 D -32 -- -4 2 E -32 -- -3 5 T P F Z
-29 -- -2 10 Z3 R3 Q2 T2 H G L K E -18 -- -1 10 D4 T2 P2 Q2 E G N K
R -18 -- 1 10 R21 A2 K2 H2 P L I T G D R 5 2 9 P20 R4 A2 H2 N E V F
L P s 5 3 10 D15 K6 T3 R2 P2 S Y G A L D 4 s 4 7 F19 D4 L3 Y2 I2 A2
S F s 5 5 1 C33 C x x 6 10 L11 E5 N4 K3 Q2 I2 Y2 D2 T R L 4 7 5 L18
E11 K2 S Q E s 4 8 7 P26 H2 A2 I L G F P 3 4 9 9 P17 A6 V3 R2 Q L K
Y F P s 3 4 10 10 Y11 E7 D4 A2 N2 R2 V2 S I D Y s s 4 11 10 T17 P5
A3 R2 I S Q Y V K T 1 s 3 4 12 2 G32 K C x x x 13 5 P22 R6 L3 N I P
1 s 4 s 14 3 C31 T A C 1 s s 5 15 12 K15 R4 Y2 M2 L2 -2 V G A I N F
K 1 s 3 4 s 16 7 A22 G5 Q2 R K D F A 1 s s s 5 17 12 R12 K5 A2 Y3
H2 S2 F2 L M T G P R 1 2 3 s 18 6 I21 M4 F3 L2 V2 T I 1 s s 5 19 7
I11 P10 R6 S2 K2 L Q I 1 2 3 s 20 5 R19 A7 S4 L2 Q R s s s 5 21 4
Y18 F13 W I Y 2 s s s 22 6 F14 Y14 H2 A N S F s 3 4 23 2 Y32 F Y s
s 24 4 N26 K3 D3 S N s 3 25 10 A12 S5 Q3 P3 W3 L2 T2 K G R A s s 26
9 K16 A6 T2 E2 S2 R2 G H V K s 3 4 27 5 A18 S8 K3 L2 T2 A 2 3 4 28
7 G13 K10 N5 Q2 R H M G 2 s s 29 10 L9 Q7 K7 A2 F2 R2 M G T N L 2 3
30 1 C33 C x x x 31 7 Q12 E11 L4 K2 V2 Y N Q 2 3 4 32 11 T12 P5 K4
Q3 E2 L2 G V S R A T 2 3 s 33 1 F33 F x x x x 34 11 V11 I8 T3 D2 N2
Q2 F H P R K V 1 2 3 s 35 2 Y31 W2 Y s s s 5 36 3 G27 S5 R G 1 37 1
G33 G x x 38 3 C31 TA C 1 s 5 39 7 R13 G9 K4 Q3 D2 P M R 1 4 s 40 2
G22 A11 A s s 5 41 3 N20 K11 D2 K 4 s 42 9 A11 R9 S4 G3 H2 D Q K N
R s 5 43 2 N31 G2 N s 44 3 N21 R11 K N s 45 2 F32 Y F s 46 8 K24 E2
S2 D H V Y R K 5 47 2 T19 S14 S s 5 48 9 A11 I9 E4 T2 W2 L2 R K D A
2 s s 49 7 E19 D6 A2 Q2 K2 T H E 2 s 50 6 E16 D12 L2 M Q K D s 5 51
1 C33 C x x 52 7 R13 M10 L3 E3 Q2 H V N 2 s 53 8 R21 Q3 E2 H2 C2 G
K D R s 5 54 7 T23 A3 V2 E2 I Y K T 5 55 1 C33 C x 56 8 G15 V8 I3
E2 R2 A L S G 57 8 G19 V4 A3 P2 -2 R L N G 58 8 A11 -10 P3 K3 S2 Y2
R F A 59 9 -24 G2 Q E A Y S P R -- 60 6 -28 Q R I G D -- 61 3 -31 T
P -- 62 2 -32 D -- 63 2 -32 K -- 64 2 -32 S -- s indicates
secondary set x indicates in or close to surface but buried and/or
highly conserved.
[1447]
66TABLE 35 Distances from C.sub..beta. to Tip of Side Group in A
Amino Acid type Distance A 0.0 C (reduced) 1.8 D 2.4 E 3.5 F 4.3 G
-- H 4.0 I 2.5 K 5.1 L 2.6 M 3.8 N 2.4 P 2.4 Q 3.5 R 6.0 S 1.5 T
1.5 V 1.5 W 5.3 Y 5.7 Notes: These distances were calculated for
standard model parts with all side groups fully extended.
[1448]
67TABLE 36 Distances, BPTI residue set #2 Distances in .ANG.
between C.sub..beta. Hypothetical C.sub..beta. was added to each
Glycine. R17 I19 Y21 A27 G28 L29 Q31 T32 V34 A48 E49 M52 P9 T11 X15
A16 I18 R20 F22 N24 K26 C30 F33 Y35 S47 D50 C51 R53 I19 7.7 Y21
15.1 8.4 A27 22.6 17.1 12.2 G28 26.6 20.4 13.8 5.3 L29 22.5 15.8
9.6 5.1 5.2 Q31 16.1 10.4 6.8 6.8 10.6 6.8 T32 11.7 5.2 6.1 12.0
15.5 10.9 5.4 V34 5.6 6.51 1.6 17.6 21.7 18.0 11.4 8.2 A48 18.5
11.0 5.4 12.6 13.3 8.4 8.8 8.3 15.7 E49 22.0 14.7 8.9 16.9 16.1
12.2 13.9 13.3 19.8 5.5 M52 23.6 16.3 8.6 12.2 10.3 7.6 11.3 13.2
20.0 6.2 6.1 P9 14.0 11.3 9.0 12.2 15.4 13.3 7.9 9.2 8.7 13.9 17.7
15.5 T11 9.5 11.2 13.5 18.8 22.5 19.8 13.5 12.1 5.7 18.5 22.1 21.5
7.2 K15 7.9 14.6 20.1 27.4 31.3 27.9 21.4 18.1 10.3 24.6 27.5 28.7
16.4 9.5 A16 5.5 10.1 15.9 25.2 28.5 24.6 18.6 14.5 8.6 19.8 22.2
24.2 14.9 9.8 6.2 I18 6.1 6.0 11.2 21.3 24.4 20.2 14.7 10.4 7.0
15.0 17.4 19.5 12.2 9.5 10.4 4.9 R20 10.6 5.9 5.4 16.0 18.5 14.6
9.8 6.9 7.8 10.2 13.0 13.8 8.0 9.4 14.9 10.6 6.2 F22 15.6 10.9 5.6
10.5 12.8 10.3 6.2 8.1 10.8 10.3 13.8 11.4 4.1 10.6 19.1 16.3 12.7
6.9 N24 19.9 14.7 9.4 4.1 7.3 6.1 4.8 10.0 14.7 11.4 15.6 11.2 8.4
15.3 24.1 21.9 18.2 12.7 6.6 K26 24.4 20.1 15.2 5.4 7.7 9.8 10.1
15.3 19.0 17.0 20.9 15.7 12.1 18.6 27.9 26.6 23.3 18.1 11.6 5.9 C30
18.9 12.1 4.6 8.8 9.5 5.3 5.9 8.2 14.9 4.9 8.7 5.6 10.6 16.6 24.1
20.2 15.7 9.8 6.8 6.9 12.4 F33 10.8 7.4 7.7 12.6 16.4 13.0 6.6 5.6
5.5 12.2 16.5 15.4 4.2 7.1 15.0 12.8 9.6 6.1 5.6 9.3 13.9 10.1 Y35
8.4 7.4 9.4 18.4 21.4 17.9 12.2 9.5 5.8 14.4 17.2 17.8 7.8 5.8 11.0
7.6 4.9 4.3 8.8 14.8 19.5 13.5 6.4 S47 17.6 10.6 6.6 17.3 17.9 13.4
12.6 10.4 15.9 5.3 4.7 9.1 15.3 18.5 23.1 17.6 12.8 9.1 12.0 15.3
21.0 8.8 13.5 13.2 D50 20.0 13.6 7.2 17.2 16.8 13.5 13.5 12.9 17.6
7.6 5.5 7.7 14.7 18.6 24.2 19.2 14.7 9.9 11.0 14.7 20.1 8.6 14.3
13.7 5.0 C51 18.9 12.2 4.0 12.1 12.2 8.8 8.8 9.7 15.3 5.4 7.1 5.4
11.0 16.4 23.5 19.2 14.6 8.7 6.9 9.6 15.0 3.7 10.9 12.5 6.9 5.2 R53
25.4 18.6 11.0 17.2 15.0 13.0 15.7 16.7 22.3 9.7 6.3 5.6 17.9 23.1
29.6 24.8 20.3 15.0 13.8 15.5 19.9 9.9 18.2 18.8 9.4 5.8 7.4 R39
15.4 16.9 17.1 24.9 27.2 24.9 20.1 18.7 13.8 22.3 23.9 24.0 13.0
9.5 12.0 11.8 12.5 12.8 14.7 20.8 24.3 20.6 14.4 9.6 20.4 19.0 18.8
23.4
[1449]
68TABLE 37 vgDNA to vary BPTI set #2.1 + .vertline.g .vertline.p
.vertline.c .vertline.k .vertline.a .vertline.X .vertline.
.vertline.35.vertline.36.vertline.37.ve-
rtline.38.vertline.39.vertline.40.vertline.
5'-.vertline.CAC.vertline.CCT.vertline.GGG.vertline.CCC.vertline.TGC.vert-
line.AAA.vertline.GCG.vertline.gfk.vertline. 208 .vertline.spacer
.vertline.Apa I .vertline. + .vertline.i .vertline.X .vertline.r
.vertline.y .vertline.f .vertline.y .vertline.n .vertline.a
.vertline.k .vertline.
.vertline.41.vertline.42.vertline.43.vertline.44.vertline.45.vertline.46.-
vertline.47.vertline.48.vertline.49.vertline.
.vertline.ACT.vertline.pfk.vertline.CGT.vertline.TAT.vertline.TTC.vertlin-
e.TAC.vertline.AAC.vertline.GCT.vertline.AAA.vertline. 235 /
3'=olig#27 72nts + ! + .vertline. + .vertline.X .vertline.g
.vertline.X .vertline.c .vertline.q .vertline.t .vertline.f
.vertline.X .vertline.y .vertline.g .vertline.g .vertline.
.vertline.50.vertline.51.vertli-
ne.52.vertline.53.vertline.54.vertline.55.vertline.56.vertline.57.vertline-
.58.vertline.59.vertline.60.vertline.
.vertline.qfk.vertline.GGt.ve-
rtline.qfk.vertline.TGC.vertline.CAG.vertline.ACC.vertline.TTc.vertline.qf-
k.vertline.TAC.vertline.GGT.vertline.GGT.vertline. 268 olig#28=3'-
acg gtc tgg aag **m atg cca cca 79 nts Overlap=12 (7 cg, 5 at)
.vertline.c .vertline.r .vertline.a .vertline.k .vertline.r
.vertline.n .vertline.n .vertline.f .vertline.k .vertline.
.vertline.61.vertline.62.vertline.63.vertline.64.vertli-
ne.65.vertline.66.vertline.67.vertline.68.vertline.69.vertline.
.vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.CGT.vertlin-
e.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline. 295 acg gca
cga ttc gca ttg ttg aaa ttt .vertline.Esp I .vertline. .vertline.s
.vertline.X .vertline.e .vertline.d .vertline.c .vertline.m
.vertline. .vertline.70.vertline.71.vertline.72.vertli-
ne.73.vertline.74.vertline.75.vertline.
.vertline.TCT.vertline.qfk.-
vertline.GAG.vertline.GAT.vertline.TGC.vertline.ATG.vertline.C 322
agc **m ctc cta acg tac gca ccc acc-5' .vertline.Sph I.vertline.
spacer .vertline. k = equal parts of T and G; m = equal parts of C
and A; q = (.26 T, .18 C, .26 A, and .30 G); f = (.22 T, .16 C, .40
A, and .22 G); * = complement ofsymbol above Residue 40 42 50 52 57
71 Possibilities
21.times.21.times.21.times.21.times.21.times.21.times.-
=8.6.times.10.sup.7 Abundance .times. 10: of PPBD .768 .271 .459
.671 .600 .459 Produce = 1.77.times.10.sup.-8 Parent =
1/(5.5.times.10.sup.7) least favored=1/(4.2.times.10.sup.9) Least
favored one-amino-acid substitution from PPBD present at 1 in
1.6.times.10.sup.7
[1450]
69TABLE 38 Result of varying set#2 of EPTI 2.1 .vertline.l
.vertline.e .vertline. .vertline.29.vertline.30.vertline.
.vertline.CTC.vertline.GAG.vertline. 178 .vertline.Ava I .vertline.
.vertline.Xho I .vertline. .vertline.p .vertline.p .vertline.y
.vertline.t .vertline.g .vertline.p .vertline.c .vertline.k
.vertline.a .vertline.D .vertline. .vertline.31.vertline.32.vertli-
ne.33.vertline.34.vertline.35.vertline.36.vertline.37.vertline.38.vertline-
.39.vertline.40.vertline.
.vertline.CCG.vertline.CCA.vertline.TAT.v-
ertline.ACT.vertline.GGG.vertline.CCC.vertline.TGC.vertline.AAA.vertline.G-
CG.vertline.GAT.vertline. 208 .vertline. PflM I .vertline.
.vertline.Apa I .vertline. .vertline.Dra II .vertline.
.vertline.Pss I .vertline. .vertline.i .vertline.Q .vertline.r
.vertline.y .vertline.f .vertline.y .vertline.n .vertline.a
.vertline.k .vertline.
.vertline.41.vertline.42.vertline.43.vertline.44.vertli-
ne.45.vertline.46.vertline.47.vertline.48.vertline.49.vertline.
.vertline.ATC.vertline.CAG.vertline.CGT.vertline.TAT.vertline.TTC.vertlin-
e.TAC.vertline.AAC.vertline.GCT.vertline.AAA.vertline. 235
.vertline.E .vertline.g .vertline.L .vertline.c .vertline.q
.vertline.t .vertline.f .vertline.s .vertline.y .vertline.g
.vertline.g .vertline.
.vertline.50.vertline.51.vertline.52.vertline.53.vertline.54.vertline-
.55.vertline.56.vertline.57.vertline.58.vertline.59.vertline.60.vertline.
.vertline.GAG.vertline.GGC.vertline.CTG.vertline.TGC.vertline.CAG.-
vertline.ACC.vertline.TTT.vertline.TCG.vertline.TAC.vertline.GGT.vertline.-
GGT.vertline. 268 .vertline.c .vertline.r .vertline.a .vertline.k
.vertline.r .vertline.n .vertline.n .vertline.f .vertline.k
.vertline. .vertline.61.vertline.62.vertline.63.vertline.64.vertli-
ne.65.vertline.66.vertline.67.vertline.68.vertline.69.vertline.
.vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.CGT.vertlin-
e.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline. 295
.vertline.Esp I .vertline. .vertline.s .vertline.W .vertline.e
.vertline.d .vertline.c .vertline.m .vertline.r .vertline.t
.vertline.c .vertline.g .vertline. .vertline.70.vertline.71.vertli-
ne.72.vertline.73.vertline.74.vertline.75.vertline.76.vertline.77.vertline-
.78.vertline.79.vertline.
.vertline.TCG.vertline.TGG.vertline.GAA.v-
ertline.GAT.vertline.TGC.vertline.ATG.vertline.CGT.vertline.ACC.vertline.T-
GC.vertline.GGT.vertline. 325 .vertline.Sph I.vertline. .vertline.g
.vertline.a .vertline. .vertline.80.vertline.81.vertline.
.vertline.GGC.vertline.GCC.vert- line. .vertline.Bbe I .vertline.
.vertline.Nar I .vertline.
[1451]
70TABLE 39 vgDNA to vary set#t2 BPTI 2.2 + .vertline. g .vertline.
p .vertline. c .vertline. X.vertline. a .vertline. D .vertline.
.vertline. 35.vertline. 36.vertline. 37.vertline. 38.vertline.
39.vertline. 40.vertline. 5'-cg gca
cgc.vertline.GGG.vertline.CCC.vertline.TGC.vertline.mrA.vertlin-
e.GCG.vertline.GAT.vertline. 208 .vertline. spacer .vertline. Apa I
.vertline. + + + .vertline. X.vertline. Q .vertline. X.vertline.
X.vertline. f .vertline. y .vertline. n .vertline. a .vertline. k
.vertline. .vertline. 41.vertline. 42.vertline. 43.vertline.
44.vertline. 45.vertline. 46.vertline. 47.vertline. 48.vertline.
49.vertline.
.vertline.rwA.vertline.CAG.vertline.rvk.vertline.TwT.vertline.TTC.vertlin-
e.TAC.vertline.AAC.vertline.GCT.vertline.AAA.vertline. 235 + + +
.vertline. E .vertline. X.vertline. L .vertline. c .vertline.
X.vertline. X.vertline. f .vertline. S .vertline. y .vertline. g
.vertline. g .vertline. .vertline. 50.vertline. 51.vertline.
52.vertline. 53.vertline. 54.vertline. 55.vertline. 56.vertline.
57.vertline. 58.vertline. 59.vertline. 60.vertline.
.vertline.GAG.vertline.gfk.vertline.CTG.vertline.TGC.vertline.gfk.vertlin-
e.gfk.vertline.TTT.vertline.TCG.vertline.TAC.vertline.GGT.vertline.GGT.ver-
tline. 268 91 nts olig#30 3'-g cca cca Overlap=15 (11 CG, 4AT) /-
3' olig#29 94 nts .vertline. c .vertline. r .vertline. a .vertline.
k .vertline. r .vertline. n .vertline. n .vertline. f .vertline. k
.vertline. .vertline. 61.vertline. 62.vertline. 63.vertline.
64.vertline. 65.vertline. 66.vertline. 67.vertline. 68.vertline.
69.vertline.
.vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.CGT.vertlin-
e.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline. 295 acg gca
cga ttc gca ttg ttg aaa ttt .vertline. Esp I .vertline. +
.vertline. s .vertline. W .vertline. X.vertline. d .vertline. c
.vertline. m .vertline. .vertline. 70.vertline. 71.vertline.
72.vertline. 73.vertline. 74.vertline. 75.vertline.
.vertline.TCG.vertline.TGG.vertline.qfk.vertline.GAT.-
vertline.TGC.vertline.ATG.vertline.C agc acc **m cta acg tac gcg
acc tgc -5' .vertline. Sph I.vertline. spacer .vertline. k = equal
parts of T and G; v equal parts of C, A, and G; m = equal parts of
C and A; r=equal parts of A and G; w = equal parts of A and T; q =
(.26 T, .18 C, .26 A, and .30 G); f = (.22 T, .16 C, .40 A, and .22
G); * = complement of symbol above Residue 38 41 43 44 51 54 55 72
Possibilities 4 .times. 4 .times. 9 .times. 2 .times. 21 .times. 21
.times. 21 .times. 21 = 6.2 .times. 10.sup.7 Abundance .times. 10
2.5 2.5 .833 5. .663 .397 .437 .602 Product = 2.3.times.10.sup.-8
Parent = 1/(4.4 .times. 10.sup.7) least favored = 1/(1.25 .times.
10.sup.9) Least favored one-amino-acid substitution from PPBD
present at 1 in 1.2 .times. 10.sup.7
[1452]
71TABLE 40 Result of varying set#2 of BPTI 2.2 .vertline. 1
.vertline. e .vertline. .vertline. 29.vertline. 30.vertline.
.vertline.CTC.vertline.GAG.vertline. 178 .vertline. Xho I
.vertline. .vertline. p .vertline. p .vertline. y .vertline. t
.vertline. g .vertline. p .vertline. c .vertline. E .vertline. a
.vertline. D .vertline. .vertline. 31.vertline. 32.vertline.
33.vertline. 34.vertline. 35.vertline. 36.vertline. 37.vertline.
38.vertline. 39.vertline. 40.vertline.
.vertline.CCG.vertline.CCA.vertline.TAT.vertline.ACT.vertline.GGG.vertlin-
e.CCC.vertline.TGC.vertline.GAG.vertline.GCG.vertline.GAT.vertline.
208 .vertline. PflM I .vertline. .vertline. Apa I .vertline.
.vertline. V .vertline. Q .vertline. N .vertline. F .vertline. f
.vertline. y .vertline. n .vertline. a .vertline. k .vertline.
.vertline. 41.vertline. 42.vertline. 43.vertline. 44.vertline.
45.vertline. 46.vertline. 47.vertline. 48.vertline. 49.vertline.
.vertline.GTT.vertline.CAG.-
vertline.AAT.vertline.TTT.vertline.TTC.vertline.TAC.vertline.AAC.vertline.-
GCT.vertline.AAA.vertline. 235 .vertline. E .vertline. F .vertline.
L .vertline. c .vertline. S .vertline. A .vertline. f .vertline. S
.vertline. y .vertline. g .vertline. g .vertline. .vertline.
50.vertline. 51.vertline. 52.vertline. 53.vertline. 54.vertline.
55.vertline. 56.vertline. 57.vertline. 58.vertline. 59.vertline.
60.vertline. .vertline.GAG.vertline.TTT.vertline.CTG.-
vertline.TGC.vertline.TCT.vertline.GCT.vertline.TTT.vertline.TCG.vertline.-
TAC.vertline.GGT.vertline.GGT.vertline. 268 .vertline. c .vertline.
r .vertline. a .vertline. k .vertline. r .vertline. n .vertline. n
.vertline. f .vertline. k .vertline. .vertline. 61.vertline.
62.vertline. 63.vertline. 64.vertline. 65.vertline. 66.vertline.
67.vertline. 68.vertline. 69.vertline.
.vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.CGT.vertlin-
e.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline. 295
.vertline. Esp I .vertline. .vertline. s .vertline. W .vertline. Q
.vertline. d .vertline. c .vertline. m .vertline. r .vertline. t
.vertline. c .vertline. g .vertline. .vertline. 70.vertline.
71.vertline. 72.vertline. 73.vertline. 74.vertline. 75.vertline.
76.vertline. 77.vertline. 78.vertline. 79.vertline.
.vertline.TGC.vertline.TGG.vertline.CAG.vertline.GAT.vertline.TGC.vertlin-
e.ATG.vertline.CGT.vertline.ACC.vertline.TGC.vertline.GGT.vertline.
325 .vertline. Sph I.vertline. .vertline. g .vertline. a .vertline.
.vertline. 80.vertline. 81.vertline.
.vertline.GGC.vertline.GCC.vertline. .vertline. Bbe I .vertline.
.vertline. Nar I .vertline.
[1453]
72TABLE 41 vg DNA set#2 of BPTI 2.3 .vertline. 1 .vertline. e
.vertline. .vertline. 29.vertline. 30.vertline. 5'-cg agc
ctg.vertline.CTC.vertline.CAG.vertline. 178 .vertline. spacer
.vertline. Xho I .vertline. .vertline. p .vertline. X.vertline. y
.vertline. X.vertline. g .vertline. p .vertline. c .vertline. E
.vertline. a .vertline. X.vertline. .vertline. 31.vertline.
32.vertline. 33.vertline. 34.vertline. 35.vertline. 36.vertline.
37.vertline. 38.vertline. 39.vertline. 40.vertline.
.vertline.CCG.vertline.vmg.vertline.TAT.vertline.vmg.-
vertline.GGG.vertline.CCC.vertline.TGC.vertline.GAG.vertline.GCG.vertline.-
qfk.vertline. 208 .vertline. V .vertline. Q .vertline. N .vertline.
X.vertline. f .vertline. y .vertline. n .vertline. a .vertline. k
.vertline. .vertline. 41.vertline. 42.vertline. 43.vertline.
44.vertline. 45.vertline. 46.vertline. 47.vertline. 48.vertline.
49.vertline. .vertline.GTT.vertline.CAG.vertline.AAT.-
vertline.Tdk.vertline.TTC.vertline.TAC.vertline.AAC.vertline.GCc.vertline.-
AAg.vertline.-3' olig#33 71 nts 67 nts olig#34 3'- g atg ttg cgg
ttc Overlap=13 (7 CG, 6 AT) + + + + .vertline. X.vertline. F
.vertline. X.vertline. c .vertline. S .vertline. X.vertline. f
.vertline. X.vertline. y .vertline. g .vertline. g .vertline.
.vertline. 50.vertline. 51.vertline. 52.vertline. 53.vertline.
54.vertline. 55.vertline. 56.vertline. 57.vertline. 58.vertline.
59.vertline. 60.vertline.
.vertline.vAG.vertline.TTT.vertline.nTk.vertline.TGC.-
vertline.TCT.vertline.qfk.vertline.TTT.vertline.qfk.vertline.TAC.vertline.-
GGT.vertline.GGT.vertline. 268 btc aaa nam acg aga **m aaa **m atg
cca cca .vertline. c .vertline. r .vertline. a .vertline. k
.vertline. .vertline. 61.vertline. 62.vertline. 63.vertline.
64.vertline. .vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.-
vertline.C acg gca cga ttc gcg acc ggc .vertline. Esp I .vertline.
spacer .vertline. k = equal parts of T and G; m = equal parts of C
and A; w = equal parts of A and T; n = equal parts of A,C,G,T; d =
equal parts A,G,T; V = equal parts A,C,G; q = (.26 T, .18 C, .26 A,
and .30 G); f = (.22 T, .16 C, .40 A, and .22 G); * = complement of
symbol above Residue 38 41 43 44 51 54 55 72 Possibilities 4
.times. 4 .times. 9 .times. 2 .times. 21 .times. 21 .times. 21
.times. 21 = 6.2 .times. 10.sup.7 Abundance .times. 10 2.5 2.5 .833
5. .663 .397 .437 .602 Product = 2.3.times.10.sup.-8 Parent =
1/(4.4 .times. 10.sup.7) least favored = 1/(1.25 .times. 10.sup.9)
Least favored one-amino-acid substitution from PPBD present at 1 in
1.2 .times. 10.sup.7
[1454]
73TABLE 42 Result of varying set#2 of BPTI 2.3 .vertline. 1
.vertline. e .vertline. .vertline. 29.vertline. 30.vertline.
.vertline.CTC.vertline.GAG.vertline. 178 .vertline. Ava I
.vertline. .vertline. Xho I .vertline. .vertline. p .vertline. e
.vertline. y .vertline. Q .vertline. g .vertline. p .vertline. c
.vertline. E .vertline. a .vertline. A .vertline. .vertline.
31.vertline. 32.vertline. 33.vertline. 34.vertline. 35.vertline.
36.vertline. 37.vertline. 38.vertline. 39.vertline. 40.vertline.
.vertline.CCG.vertline.GAG.vertline.TAT.vertline.CAG.vertline.GGG.vertlin-
e.CCC.vertline.TGC.vertline.GAG.vertline.GCG.vertline.GCT.vertline.
208 .vertline. Apa I .vertline. .vertline. V .vertline. Q
.vertline. N .vertline. W .vertline. f .vertline. y .vertline. n
.vertline. a .vertline. k .vertline. .vertline. 41.vertline.
42.vertline. 43.vertline. 44.vertline. 45.vertline. 46.vertline.
47.vertline. 48.vertline. 49.vertline.
.vertline.GTT.vertline.CAG.vertline.AAT.vertline.TGG.vertline.TTC.vertlin-
e.TAC.vertline.AAC.vertline.GCT.vertline.AAA.vertline. 235
.vertline. Q .vertline. F .vertline. M .vertline. c .vertline. S
.vertline. L .vertline. f .vertline. H .vertline. y .vertline. g
.vertline. g .vertline. .vertline. 50.vertline. 51.vertline.
52.vertline. 53.vertline. 54.vertline. 55.vertline. 56.vertline.
57.vertline. 58.vertline. 59.vertline. 60.vertline.
.vertline.CAG.vertline.TTT.vertline.ATG.vertline.TGC.vertline.TCT.vertlin-
e.CCT.vertline.TTT.vertline.CAT.vertline.TAC.vertline.GGT.vertline.GGT.ver-
tline. 268 .vertline. c .vertline. r .vertline. a .vertline. k
.vertline. r .vertline. n .vertline. n .vertline. f .vertline. k
.vertline. .vertline. 61.vertline. 62.vertline. 63.vertline.
64.vertline. 65.vertline. 66.vertline. 67.vertline. 68.vertline.
69.vertline. .vertline.TGC.vertline.CGT.vertline.GCT.-
vertline.AAG.vertline.CGT.vertline.AAC.vertline.AAC.vertline.TTT.vertline.-
AAA.vertline. 295 .vertline. Esp I .vertline. .vertline. s
.vertline. W .vertline. Q .vertline. d .vertline. c .vertline. m
.vertline. r .vertline. t .vertline. c .vertline. g .vertline.
.vertline. 70.vertline. 71.vertline. 72.vertline. 73.vertline.
74.vertline. 75.vertline. 76.vertline. 77.vertline. 78.vertline.
79.vertline. .vertline.TGC.vertline.TGG.vertline.CAG.-
vertline.GAT.vertline.TGC.vertline.ATG.vertline.CGT.vertline.ACC.vertline.-
TGC.vertline.GGT.vertline. 325 .vertline. Sph I.vertline.
.vertline. g .vertline. a .vertline. .vertline. 80.vertline.
81.vertline. .vertline.GGC.vertline.GCC.ve- rtline. .vertline. Bbe
I .vertline. .vertline. Nar I .vertline.
[1455]
74TABLE 50 Number Amino Cross Source IPBD Acids Structure Links
Secreted Organism AfM Preferred IPBDs Aprotinin 58 X-ray, NMR 3 SS
yes Bos taurus trypsin 5-55, 14-38 30-51 (1:6, 2:4, 3:5) Crambin 46
X-ray, NMR 3 SS yes rape seed ?, Mab CMTI-III 26 NMR 3 SS yes
cucumber trypsin ST-I.sub.A 13 NMR 3 SS yes E. coli MAbs &
guanylate cyclase Third domain, 56 X-ray, NMR 3 SS yes Coturnix
trypsin ovomucoid coturnix japonica Ribonuclease A 124 X-ray, NMR
yes Bos taurus RNA, DNA Ribonuclease 104 X-ray, NMR? yes A. oruzae
RNA, DNA Lysozyme 129 X-ray, NMR? 4 SS yes Gallus gallus
NAG-NAM-NAG Azurin 128 X-ray Cu: CYS, P. aerugenosa Mab
HIS.sup.2,MET Characteristics of Known IPBDs .alpha.-Conotoxins
13-15 NMR 2 SS yes Conus snails Receptor .mu.-Conotoxins 20-25 NMR
3 SS yes Conus snails Receptor .OMEGA.-Conotoxins 25-30 - 3 SS yes
Conus snails Receptor King-kong 25-30 - 3 SS yes Conus snails Mabs
peptides Nuclease 141 X-ray none yes S aurius RNA, DNA
(staphylococcal) Charybdotoxin 37 NMR 3 SS yes Leiurus Ca.sup.+2
(scorpion toxin) 7-28, 13-33 quinquestriatus -dependent 17-35
hebraeus K.sup.+channel (1:4, 2:5, 3:6) Apamin 12 NMR 2 SS yes Bees
Mabs, (bee venom) (1:3, 2:4) Receptor(?) Other suitable IPBDS
Ferredoxin Secretory trypsin inhibitor Soybean trypsin inhibitor
SLPI (Secretory Leukocyte Protease Inhibitor) (THOM86) and SPAI
(ARAK90) Cystatin and homologues (MACH89, STUB90) Eglin (MCPH85)
Barley inhibitor (CLOR87a, CLOR87b, SVEN82)
[1456]
75TABLE 101a VIIIsignal::bpti::VIII-coatgene pbd mod14: 9 V 89 :
Sequence cloned into pGEM-MB1 pGEM-3Zf(-) [HincII]::lacUV5 SacI
gene TrpA attenuator (SalI)::pGEM-3Zf(-) [HincII]! 5'-(GAATTC
GAGCTCGGTACCCGG GGATCC TCTAGAGTC)- !polylinker GGC tttaca
CTTTATGCTTCCGGCTCG tataat GTG ! lacUV5 TGG aATTGTGAGCGcTcACAATT !
lacO-symm operator gagctc AG(G)AGG CttaCT ! Sac I; Shine-Dalgarno
seq..sup.a atg aag aaa tct ctg gtt ctt aag gct agc ! 10, M13 leader
gtt gct gtc gcg acc ctg gta cct atg ttg ! 20 .rarw.codon # tcc ttc
gct cgt ccg gat ttc tgt ctc gag ! 30 cca cca tac act ggg ccc tgc
aaa gcg cgc ! 40 atc atc cgC tat ttc tac aat gct aaa gca ! 50 ggc
ctg tgc cag acc ttt gta tac ggt ggt ! 60 tgc cgt gct aag cgt aac
aac ttt aaa tcg ! 70 gcc gaa gat tgc atg cgt acc tgc ggt ggc ! 80
gcc gct gaa ggt gat gat ccg gcc aaG gcg ! 90 gcc ttc aat tct ctG
caa gct tct gct acc ! 100 gag tat att ggt tac gcg tgg gcc atg gtg !
110 gtg gtt atc gtt ggt gct acc atc ggg atc ! 120 aaa ctg ttc aag
aag ttt act tcg aag gcg ! 130 tct taa tga tag GGTTACC ! BstEII
AGTCTA AGCCCGC CTAATGA GCGGGCT TTTTTTTT ! terminator aTCGA- ! (SalI
ghost) (GACCTGCAGGCATGCAAGCTT . . . -3') ! pGEM polylinker Notes:
.sup.aDesigned sequence contained AGGAGG, but sequencing indicates
that actual DNA contains AGAGG.
[1457]
76TABLE 101b VIII-signal::bpti::VIII-coat gene BamHI-SalI cassette,
after insertion of Sall linker in PstI site of pGEM-MB1.
pGEM-3Zf(-) [HincII)::lacUV5 SacI gene TrpA attenuator (SalI):
:pGEM-3Zf(-) [HincII] 5'-GAATTC GAGCTC GGTACCCGG GGATCC TCTAGA GTC-
! BamHI GGC tttaca CTTTATGCTTCCGGCTCG tataat GTG ! lacUV5 TGG
aATTGTGAGCGcTcACAATT ! lacO-symm operator gagctc AGAGG CttaCT ! Sac
I; Shine-Dalgarno seq. atg aag aaa tct ctg gtt ctt aag gct agc !
10, M13 leader gtt got gto gcg acc ctg gta cct atg ttg ! 20 .rarw.-
codon # tcc ttc gct cgt ccg gat ttc tgt ctc gag ! 30 cca cca tac
act ggg ccc tgc aaa gcg cgc ! 40 atc atc cgC tat ttc tac aat gct
aaa gca ! 50 ggc ctg tgc cag acc ttt gta tac ggt ggt ! 60 tgc cgt
gct aag cgt aac aac ttt aaa tcg ! 70 gcc gaa gat tgc atg cgt acc
tgc ggt ggc ! 80 gcc gct gaa ggt gat gat ccg gcc aaG gcg ! 90 gcc
ttc aat tct ctG caa gct tct gct acc ! 100 gag tat att ggt tac gcg
tgg gcc atg gtg ! 110 gtg gtt atc gtt ggt gct acc atc ggg atc ! 120
aaa ctg ttc aag aag ttt act tcg aag gcg ! 130 tct taa tga tag
GGTTACC ' BstEII AGTCTA AGCCCGC CTAATGA GCGGGCT TTTTTTTT !
terminator aTCGA GACctgca GGTCGACC ggcatgc-3'
.vertline.SalI.vertline.
[1458]
77TABLE 102a Annotated Sequence of gene found in pGEM-MB1
nucleotide number 5'-(G GATCC TCTAGA GTC) GGC- 3 from pGEM
polylinker tttaca CTTTATGCTTCCGGCTCG tataat GTGTGG- 39 -35 lacUV5
-10 aATTGTGAGCGcTcACAATT- 59 lacO-symm operator gagctc AG(G)AGG
CttaCT- 77 SacI Shine-Dalgarno seq..sup.a .vertline.fM .vertline. K
.vertline. K .vertline. S .vertline. L .vertline. V .vertline. L
.vertline. K .vertline. A .vertline. S .vertline. .vertline. 1
.vertline. 2 .vertline. 3 .vertline. 4 .vertline. 5 .vertline. 6
.vertline. 7 .vertline. 8 .vertline. 9 .vertline. 10.vertline.
.vertline.ATG.vertline.AAG.vertline.AAA.ve-
rtline.TCT.vertline.CTG.vertline.GTT.vertline.CTT.vertline.AAG.vertline.GC-
T.vertline.AGC.vertline.- 107 .vertline. Afl II.vertline. Nhe I
.vertline. .vertline. V .vertline. A .vertline. V .vertline. A
.vertline. T .vertline. L .vertline. V .vertline. P .vertline. M
.vertline. L .vertline. .vertline. 11.vertline. 12.vertline.
13.vertline. 14.vertline. 15.vertline. 16.vertline. 17.vertline.
18.vertline. 19.vertline. 20.vertline.
.vertline.GTT.vertline.GCT.vertline.GTC.vertline.GCG.vertline.ACC.vertlin-
e.CTG.vertline.GTA.vertline.CCT.vertline.ATG.vertline.TTG.vertline.-
137 .vertline. Nru I.vertline. .vertline. Kpn I.vertline.
.vertline. S .vertline. F .vertline. A .vertline. R .vertline. P
.vertline. D .vertline. F .vertline. C .vertline. L .vertline. E
.vertline. .vertline. 21.vertline. 22.vertline. 23.vertline.
24.vertline. 25.vertline. 26.vertline. 27.vertline. 28.vertline.
29.vertline. 30.vertline.
.vertline.TCC.vertline.TTC.vertline.GCT.vertline.CGT.vertline.CCG.vertlin-
e.GAT.vertline.TTC.vertline.TGT.vertline.CTC.vertline.GAG.vertline.-
167 .Arrow-up bold. .vertline.AccIII.vertline. .vertline. Ava I
.vertline. M13/BPTI Jnct .vertline. Xho I .vertline. .vertline. P
.vertline. P .vertline. Y .vertline. T .vertline. G .vertline. P
.vertline. C .vertline. K .vertline. A .vertline. R .vertline.
.vertline. 31.vertline. 32.vertline. 33.vertline. 34.vertline.
35.vertline. 36.vertline. 37.vertline. 38.vertline. 39.vertline.
40.vertline.
.vertline.CCA.vertline.CCA.vertline.TAC.vertline.ACT.vertline.GGG.vertlin-
e.CCC.vertline.TGC.vertline.AAA.vertline.GCG.vertline.CGC.vertline.-
197 .vertline. PflM I .vertline. .vertline..vertline.
.vertline.BssH II.vertline. .vertline. Apa I .vertline..vertline.
.vertline. Dra II .vertline. .vertline. Pss I .vertline. .vertline.
I .vertline. I .vertline. R .vertline. Y .vertline. F .vertline. Y
.vertline. N .vertline. A .vertline. K .vertline. A .vertline.
.vertline. 41.vertline. 42.vertline. 43.vertline. 44.vertline.
45.vertline. 46.vertline. 47.vertline. 48.vertline. 49.vertline.
50.vertline. .vertline.ATC.vertline.ATC.vertline.CGC-
.vertline.TAT.vertline.TTC.vertline.TAC.vertline.AAT.vertline.GCT.vertline-
.AAA.vertline.GC .vertline.- 226 .vertline. G .vertline. L
.vertline. C .vertline. Q .vertline. T .vertline. F .vertline. V
.vertline. Y .vertline. G .vertline. C .vertline. .vertline.
51.vertline. 52.vertline. 53.vertline. 54.vertline. 55.vertline.
56.vertline. 57.vertline. 58.vertline. 59.vertline. 60.vertline.
A.vertline.GGC.vertline.CTG.vertline.TGC.vertline.CAG.vertline.ACC.vertli-
ne.TTT.vertline.GTA.vertline.TAC.vertline.GGT.vertline.GGT.vertline.-
257 .vertline. Stu I.vertline. .vertline. Acc I .vertline.
.vertline. Xca I .vertline. .vertline. C .vertline. R .vertline. A
.vertline. K .vertline. R .vertline. N .vertline. N .vertline. F
.vertline. K .vertline. .vertline. 61.vertline. 62.vertline.
63.vertline. 64.vertline. 65.vertline. 66.vertline. 67.vertline.
68.vertline. 69.vertline.
.vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG-
.vertline.CGT.vertline.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline-
.- 284 .vertline. Esp I .vertline. .vertline. S .vertline. A
.vertline. E .vertline. D .vertline. C .vertline. M .vertline. R
.vertline. T .vertline. C .vertline. G .vertline. .vertline.
70.vertline. 71.vertline. 72.vertline. 73.vertline. 74.vertline.
75.vertline. 76.vertline. 77.vertline. 78.vertline. 79.vertline.
.vertline.TCG.vertline.GCC.vertline.GAA-
.vertline.GAT.vertline.TGC.vertline.ATG.vertline.CGT.vertline.ACC.vertline-
.TGC.vertline.GGT.vertline.- 314 .vertline.XmaIII.vertline.
.vertline. Sph I.vertline. BPTI/M13 boundary .dwnarw. .vertline. G
.vertline. A .vertline. A .vertline. E .vertline. G .vertline. D
.vertline. D .vertline. P .vertline. A .vertline. K .vertline. A
.vertline. A .vertline. .vertline. 80.vertline. 81.vertline.
82.vertline. 83.vertline. 84.vertline. 85.vertline. 86.vertline.
87.vertline. 88.vertline. 89.vertline. 90.vertline. 91.vertline.
.vertline.GGC.vertline.GCC-
.vertline.GCT.vertline.GAA.vertline.GGT.vertline.GAT.vertline.GAT.vertline-
.CCG.vertline.GCC.vertline.AAG.vertline.GCG.vertline.GCC.vertline.-
350 .vertline. Bbe I .vertline. .vertline. Sfi I .vertline.
.vertline. Nar I .vertline. .vertline. F .vertline. N .vertline. S
.vertline. L .vertline. Q .vertline. A .vertline. S .vertline. A
.vertline. T .vertline. .vertline. 92.vertline. 93.vertline.
94.vertline. 95.vertline. 96.vertline. 97.vertline. 98.vertline.
99.vertline.100.vertline.
.vertline.TTC.vertline.AAT.vertline.TCT.vertline.CTG.vertline.CAA.vertlin-
e.GCT.vertline.TCT.vertline.GCT.vertline.ACC.vertline.- 377
.vertline.Hind 3.vertline. .vertline. E .vertline. Y .vertline. I
.vertline. C .vertline. Y .vertline. A .vertline. W .vertline.
.vertline.101.vertline.102.vertline.103.v-
ertline.104.vertline.105.vertline.106.vertline.107.vertline.
.vertline.GAG.vertline.TAT.vertline.ATT.vertline.GGT.vertline.TAC.vertlin-
e.GCG.vertline.TGG.vertline.- 398 .vertline. A .vertline. M
.vertline. V .vertline. V .vertline. V .vertline. I .vertline. V
.vertline. G .vertline. A .vertline.
.vertline.108.vertline.109.vertline.110.vertline.111.vertline.112.vertlin-
e.113.vertline.114.vertline.115.vertline.116.vertline.
.vertline.CCC.vertline.ATG.vertline.GTG.vertline.GTG.vertline.GTT.vertlin-
e.ATC.vertline.GTT.vertline.GGT.vertline.GCT.vertline.- 425
.vertline. BstX I .vertline. .vertline. Nco I.vertline. .vertline.
T .vertline. I .vertline. G .vertline. I .vertline.
.vertline.117.vertline.118.vertline.- 119.vertline.120.vertline.
.vertline.ACC.vertline.ATC.vertlin- e.GGG.vertline.ATC.vertline.-
437 .vertline. K .vertline. L .vertline. F .vertline. K .vertline.
K .vertline. F .vertline. T .vertline. S .vertline. K .vertline. A
.vertline.
.vertline.121.vertline.122.vertline.123.vertline.124.vertline.125.vertlin-
e.126.vertline.127.vertline.128.vertline.129.vertline.130.vertline.
.vertline.AAA.vertline.CTG.vertline.TTC.vertline.AAG.vertline.AAG.vertli-
ne.TTT.vertline.ACT.vertline.TCG.vertline.AAG.vertline.GCG.vertline.-
467 .vertline.Asu II.vertline. .vertline. S .vertline. . .vertline.
. .vertline. . .vertline.
.vertline.131.vertline.132.vertline.133.vertline.134.vertline.
.vertline.TCT.vertline.TAA.vertline.TGA.vertline.TAG.vertline.
GGTTACC- 486 BstE II AGTCTA AGCCCGC CTAATGA GCGGGCT TTTTTTTT- 521
terminator aTCGA (GACctgcaggcatgc)-3' (SalI ) from pGEM polylinker
Notes: .sup.aDesigned called for Shine-Dalgarno sequence, AGGAGG,
but sequencing shows that actual constructed gene contains AGAGG.
Note the following enzyme equivalences, Xma III = Eag I Acc III =
BspM II Dra II = EcoO109 I Asu II = BstB I
[1459]
78TABLE 102b Annotated Sequence of gene after insertion of SalI
linker nucleotide number 5'-(GGATCC TCTAGA GTC) GGC- 3 from pGEM
polylinker tttaca CTTTATGCTTCCGGCTCG tataat GTGTGG- 39 -35 lacUV5
-10 aATTGTGAGCGcTcACAATT- 59 lacO-symm operator gagctc AGAGG
CttaCT- 77 SacI Shine-Dalgarno seq. .vertline.fM .vertline. K
.vertline. K .vertline. S .vertline. L .vertline. V .vertline. L
.vertline. K .vertline. A .vertline. S .vertline. .vertline. 1
.vertline. 2 .vertline. 3 .vertline. 4 .vertline. 5 .vertline. 6
.vertline. 7 .vertline. 8 .vertline. 9 .vertline. 10.vertline.
.vertline.ATG.vertline.AAG.vertline.AAA.ve-
rtline.TCT.vertline.CTG.vertline.GTT.vertline.CTT.vertline.AAG.vertline.GC-
T.vertline.AGC.vertline.- 107 .vertline. Afl II.vertline. Nhe I
.vertline. .vertline. V .vertline. A .vertline. V .vertline. A
.vertline. T .vertline. L .vertline. V .vertline. P .vertline. M
.vertline. L .vertline. .vertline. 11.vertline. 12.vertline.
13.vertline. 14.vertline. 15.vertline. 16.vertline. 17.vertline.
18.vertline. 19.vertline. 20.vertline.
.vertline.GTT.vertline.GCT.vertline.GTC.vertline.GCG.vertline.ACC.vertlin-
e.CTG.vertline.GTA.vertline.CCT.vertline.ATG.vertline.TTG.vertline.-
137 .vertline. Nru I.vertline. .vertline. Kpn I.vertline.
.vertline. S .vertline. F .vertline. A .vertline. R .vertline. P
.vertline. D .vertline. F .vertline. C .vertline. L .vertline. E
.vertline. .vertline. 21.vertline. 22.vertline. 23.vertline.
24.vertline. 25.vertline. 26.vertline. 27.vertline. 28.vertline.
29.vertline. 30.vertline. .vertline.TCC.vertline.TTC.-
vertline.GCT.vertline.CGT.vertline.CCG.vertline.GAT.vertline.TTC.vertline.-
TGT.vertline.CTC.vertline.GAG.vertline.- 167 .vertline.
.vertline.AccIII.vertline. .vertline. Ava I .vertline. M13 BPTI
Jnct .vertline. Xho I .vertline. .vertline. P .vertline. P
.vertline. Y .vertline. T .vertline. G .vertline. P.vertline. C
.vertline. K .vertline. A .vertline. R .vertline. .vertline.
31.vertline. 32.vertline. 33.vertline. 34.vertline. 35.vertline.
36.vertline. 37.vertline. 38.vertline. 39.vertline. 40.vertline.
.vertline.CCA.vertline.CCA.vertline.TAC.-
vertline.ACT.vertline.GGG.vertline.CCC.vertline.TGC.vertline.AAA.vertline.-
GCG.vertline.CGC.vertline.- 197 .vertline. PflM I .vertline.
.vertline..vertline. .vertline.BssH II.vertline. .vertline. Apa I
.vertline..vertline. .vertline. Dra II .vertline. .vertline. Pss I
.vertline. .vertline. I .vertline. I .vertline. R .vertline. Y
.vertline. F .vertline. Y .vertline. N .vertline. A .vertline. K
.vertline. A .vertline. .vertline. 41.vertline. 42.vertline.
43.vertline. 44.vertline. 45.vertline. 46.vertline. 47.vertline.
48.vertline. 49.vertline. 50.vertline.
.vertline.ATC.vertline.ATC.vertline.CGC.vertline.TAT.vertline.TTC.vertlin-
e.TAC.vertline.AAT.vertline.GCT.vertline.AAA.vertline.GC
.vertline.- 226 .vertline. G .vertline. L .vertline. C .vertline. Q
.vertline. T .vertline. F .vertline. V .vertline. Y .vertline. G
.vertline. G .vertline. .vertline. 51.vertline. 52.vertline.
53.vertline. 54.vertline. 55.vertline. 56.vertline. 57.vertline.
58.vertline. 59.vertline. 60.vertline. A.vertline.GGC.vertline.CTG-
.vertline.TGC.vertline.CAG.vertline.ACC.vertline.TTT.vertline.GTA.vertline-
.TAC.vertline.GGT.vertline.GGT.vertline.- 257 .vertline. Stu
I.vertline. .vertline. Acc I .vertline. .vertline. Xca I .vertline.
.vertline. C .vertline. R .vertline. A .vertline. K .vertline. R
.vertline. N .vertline. N .vertline. F .vertline. K .vertline.
.vertline. 61.vertline. 62.vertline. 63.vertline. 64.vertline.
65.vertline. 66.vertline. 67.vertline. 68.vertline. 69.vertline.
.vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.CGT.vertlin-
e.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline. 284
.vertline. Esp I .vertline. .vertline. S .vertline. A .vertline. E
.vertline. D .vertline. C .vertline. M .vertline. R .vertline. T
.vertline. C .vertline. G .vertline. .vertline. 70.vertline.
71.vertline. 72.vertline. 73.vertline. 74.vertline. 75.vertline.
76.vertline. 77.vertline. 78.vertline. 79.vertline.
.vertline.TCG.vertline.GCC.vertline.GAA.vertline.GAT.vertline.TGC.vertlin-
e.ATG.vertline.CGT.vertline.ACC.vertline.TGC.vertline.GGT.vertline.-
314 .vertline.XmaIII.vertline. .vertline. Sph I.vertline. BPTI M13
boundary .dwnarw. .vertline. G .vertline. A .vertline. A .vertline.
E .vertline. G .vertline. D .vertline. D .vertline. P .vertline. A
.vertline. K .vertline. A .vertline. A .vertline. .vertline.
80.vertline. 81.vertline. 82.vertline. 83.vertline. 84.vertline.
85.vertline. 86.vertline. 87.vertline. 88.vertline. 89.vertline.
90.vertline. 91.vertline.
.vertline.GGC.vertline.GCC.vertline.GCT.vertline.GAA.-
vertline.GGT.vertline.GAT.vertline.GAT.vertline.CCG.vertline.GCC.vertline.-
AAG.vertline.GCG.vertline.GCC.vertline.- 350 .vertline. Bbe I
.vertline. .vertline. Sfi I .vertline. .vertline. Nar I .vertline.
.vertline. F .vertline. N .vertline. S .vertline. L .vertline. Q
.vertline. A .vertline. S .vertline. A .vertline. T .vertline.
.vertline. 92.vertline. 93.vertline. 94.vertline. 95.vertline.
96.vertline. 97.vertline. 98.vertline. 99.vertline.100.vertline.
.vertline.TTC.vertline.AAT.-
vertline.TCT.vertline.CTG.vertline.CAA.vertline.GCT.vertline.TCT.vertline.-
GCT.vertline.ACC.vertline. 377 .vertline.Hind 3.vertline.
.vertline. E .vertline. Y .vertline. I .vertline. G .vertline. Y
.vertline. A .vertline. W .vertline.
.vertline.101.vertline.102.vertline.103.vertline.104.vertline.105.vertlin-
e.106.vertline.107.vertline.
.vertline.GAG.vertline.TAT.vertline.AT-
T.vertline.GGT.vertline.TAC.vertline.GCG.vertline.TGG.vertline.-
398 .vertline. A .vertline. M .vertline. V .vertline. V .vertline.
V .vertline. I .vertline. V .vertline. G .vertline. A .vertline.
.vertline.108.vertline.109.vertline.110.vertline.111-
.vertline.112.vertline.113.vertline.114.vertline.115.vertline.116.vertline-
. .vertline.GCC.vertline.ATG.vertline.GTG.vertline.GTG.vertline.-
GTT.vertline.ATC.vertline.GTT.vertline.GGT.vertline.GCT.vertline.-
425 .vertline. BstX I .vertline. .vertline. Nco I.vertline.
.vertline. T .vertline. I .vertline. G .vertline. I .vertline.
.vertline.117.vertline.118.vertline.1- 19.vertline.120.vertline.
.vertline.ACC.vertline.ATC.vertline.- GGG.vertline.ATC.vertline.
437 .vertline. K .vertline. L .vertline. F .vertline. K .vertline.
K .vertline. F .vertline. T .vertline. S .vertline. K .vertline. A
.vertline.
.vertline.121.vertline.122.vertline.123.vertline.124.vertline.125.vertlin-
e.126.vertline.127.vertline.128.vertline.129.vertline.130.vertline.
.vertline.AAA.vertline.CTG.vertline.TTC.vertline.AAG.vertline.AAG.vertlin-
e.TTT.vertline.ACT.vertline.TCG.vertline.AAG.vertline.GCG.vertline.-
467 .vertline.Asu II.vertline. .vertline. S .vertline. . .vertline.
. .vertline. . .vertline.
.vertline.131.vertline.132.vertline.133.vertline.134.vertline.
.vertline.TCT.vertline.TAA.vertline.TGA.vertline.TAG.vertline.
GGTTACC- 437 BstE II AGTCTA AGCCCGC CTAATGA GCGGGCT TTTTTTTT- 521
terminator aTCGA GACctgca GGTCGACC ggcatgc-3'
.vertline.SalI.vertline. Note the following enzyme equivalences,
Xma III = Eag I Acc III = BspM II Dra II = EcoO109 I Asu II = BstB
I
[1460]
79TABLE 102c Calculated properties of Peptide For the apoprotein
Molecular weight of peptide = 16192 Charge on peptide = 9 [A + G +
P] = 36 [C + F + H + I + L + M + V + W + Y] = 48 [D + E + K + R + N
+ Q + S + T + .] = 48 For the mature protein Molecular weight of
peptide = 13339 Charge on peptide = 6 [A + G + P] 31 [C + F + H + I
+ L + M + V + W + Y] = 37 [D + E + K + R + N + Q + S + T + .] =
41
[1461]
80TABLE 102d Codon Usage First Second Base Base t c a g Third base
t 3 4 2 1 t 5 1 4 5 c 0 0 0 0 a 1 2 0 1 g c 1 1 0 4 t 1 1 0 2 c 0 2
1 0 a 5 2 1 0 g a 1 2 2 0 t 5 5 2 1 c 0 0 5 0 a 4 0 7 0 g g 4 9 4 6
t 1 5 0 2 c 2 1 2 0 a 2 5 2 2 g
[1462]
81 TABLE 102e Amino-acid frequency Encoded polypeptide AA # AA # AA
# AA # A 20 C 6 D 4 E 4 F 8 G 10 H 0 I 6 K 12 L 8 M 4 N 4 P 6 Q 2 R
6 S 8 T 7 V 9 W 1 Y 6 .multidot. 1 Mature protein A 16 C 6 D 4 E 4
F 7 G 10 H 0 I 6 K 9 L 4 M 2 N 4 P 5 Q 2 R 6 S 5 T 6 V 5 W 1 Y
6
[1463]
82TABLE 102f Enzymes used to manipulate BPTI-gp8 fusion SacI
GAGCT.vertline.C AflII C.vertline.TTAAG NheI G.vertline.CTAGC NruI
TCG.vertline.CGA KpnI GGTAC.vertline.C AccIII = BsPMII
T.vertline.CCGGA AvaI C.vertline.yCGrG XhoI C.vertline.TCGAG PflMI
CCAnnnn.vertline.nTGG BssHII G.vertline.CGCGC ApaI GGGCC.vertline.C
DraII = Eco109I rGGnC.vertline.Cy (Same as PssI) StuI
AGG.vertline.CCT AccI GT.vertline.mkAC XcaI GTA.vertline.TAC EspI
GC.vertline.TnAGC XmaIII C.vertline.GGCCG (Supplier ?) SphI
GCATG.vertline.C BbeI GGCGC.vertline.C (Supplier ?) NarI
GGCG.vertline.CC SfiI GGCCnnnn.vertline.nGGCC HindIII
A.vertline.AGCTT BstXI CCAnnnnn.vertline.nTGG NcoI C.vertline.CATGG
AsuII = BstBI TT.vertline.CGAA BstEII G.vertline.GTnACC SalI
G.vertline.TCGAC
[1464]
83TABLE 103 Annotated Sequence of osp-ipbd gene Underscored bases
indicate sites of overlap between annealed synthetic duplexes. 5'-
/GGC tttaca CTTTAT,GCTTCCGGCTCG tataat GTGTGG- lacUV5
aATTGTGAGCGcTcACAATT- lacO-symm operator gagctc AG(G)/AGG CttaCT-
Sac I Shine-Dalgarno seq. .vertline.fM .vertline. K .vertline. K
.vertline. S .vertline. L .vertline. V .vertline. L .vertline. K
.vertline. A .vertline. S .vertline. .vertline. 1 .vertline. 2
.vertline. 3 .vertline. 4 .vertline. 5 .vertline. 6 .vertline. 7
.vertline. 8 .vertline. 9 .vertline. 10.vertline.
.vertline.ATG.vertline.AAG,.vertline.AAA.vertline.TCT.vertline.CTG.vertli-
ne.GTT.vertline.CTT.vertline.AAG.vertline.GCT.vertline.AGC.vertline.-
.vertline. Afl II.vertline. Nhe I .vertline. .vertline. V
.vertline. A .vertline. V .vertline. A .vertline. T .vertline. L
.vertline. V .vertline. P .vertline. M .vertline. L .vertline.
.vertline. 11.vertline. 12.vertline. 13.vertline. 14.vertline.
15.vertline. 16.vertline. 17.vertline. 18.vertline. 19.vertline.
20.vertline. .vertline.GTT.vertline.-
GCT.vertline.GTC.vertline.GCG.vertline.ACC.vertline.CTG.vertline.GTA.vertl-
ine.CCT.vertline.ATG.vertline.T/TG.vertline.- .vertline. Nru
I.vertline. .vertline. Kpn I.vertline. .vertline. S .vertline. F
.vertline. A .vertline. R .vertline. P .vertline. D .vertline. F
.vertline. C .vertline. L .vertline. E .vertline. .vertline.
21.vertline. 22.vertline. 23.vertline. 24.vertline. 25.vertline.
26.vertline. 27.vertline. 28.vertline. 29.vertline. 30.vertline.
.vertline.TCC.vertline.TTC.vertline.G-
CT.vertline.CG,T.vertline.CCG.vertline.GAT.vertline.TTC.vertline.TGT.vertl-
ine.CTC.vertline.GAG.vertline.- .Arrow-up bold.
.vertline.AccIII.vertline. .vertline. Ava I .vertline. M13/BPTI
Jnct .vertline. Xho I .vertline. .vertline. P .vertline. P
.vertline. Y .vertline. T .vertline. G .vertline. P .vertline. C
.vertline. K .vertline. A .vertline. R .vertline. .vertline.
31.vertline. 32.vertline. 33.vertline. 34.vertline. 35.vertline.
36.vertline. 37.vertline. 38.vertline. 39.vertline. 40.vertline.
.vertline.CCA.vertline.CCA.vertline.T-
AC.vertline.ACT.vertline.GGG.vertline.CCC.vertline.TGC.vertline.AAA.vertli-
ne.GCG.vertline.CGC.vertline._ .vertline. PflM I .vertline.
.vertline..vertline. .vertline.BssH II.vertline. .vertline. Apa I
.vertline..vertline. .vertline. Dra II .vertline. .vertline. Pss I
.vertline. .vertline. I .vertline. I .vertline. R .vertline. Y
.vertline. F .vertline. Y .vertline. N .vertline. A .vertline. K
.vertline. A .vertline. .vertline. 41.vertline. 42.vertline.
43.vertline. 44.vertline. 45.vertline. 46.vertline. 47.vertline.
48.vertline. 49.vertline. 50.vertline.
.vertline.ATC.vertline.ATC.vertline.CG/C.vertline.TAT.vertline.TTC.vert-
line.TAC.vertline.AAT.vertline.GC,T.vertline.AAA.vertline.GC
.vertline.- .vertline. G .vertline. L .vertline. C .vertline. Q
.vertline. T .vertline. F .vertline. V .vertline. Y .vertline. G
.vertline. G .vertline. .vertline. 51.vertline. 52.vertline.
53.vertline. 54.vertline. 55.vertline. 56.vertline. 57.vertline.
58.vertline. 59.vertline. 60.vertline. A.vertline.GGC.vertline.CT-
G.vertline.TGC.vertline.CAG.vertline.ACC.vertline.TTT.vertline.GTA.vertlin-
e.TAC.vertline.GGT.vertline.GGT.vertline.- .vertline. Stu
I.vertline. .vertline. Acc I .vertline. .vertline. Xca I .vertline.
.vertline. C .vertline. R .vertline. A .vertline. K .vertline. R
.vertline. N .vertline. N .vertline. F .vertline. K .vertline.
.vertline. 61.vertline. 62.vertline. 63.vertline. 64.vertline.
65.vertline. 66.vertline. 67.vertline. 68.vertline. 69.vertline.
.vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.CGT.vertl-
ine./AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline.-
.vertline. Esp I .vertline. .vertline. S .vertline. A .vertline. E
.vertline. D .vertline. C .vertline. M .vertline. R .vertline. T
.vertline. C .vertline. G .vertline. .vertline. 70 .vertline.
71.vertline. 72.vertline. 73.vertline. 74.vertline. 75.vertline.
76.vertline. 77.vertline. 78.vertline. 79.vertline.
.vertline.TCG,.vertline.GCC.vertline.GAA.vertline.-
GAT.vertline.TGC.vertline.ATG.vertline.CGT.vertline.ACC.vertline.TGC.vertl-
ine.GGT.vertline.- .vertline.Xma III.vertline. .vertline. Sph
I.vertline. BPTI/M13 boundary .dwnarw. .vertline. G .vertline. A
.vertline. A .vertline. E .vertline. G .vertline. D .vertline. D
.vertline. P .vertline. A .vertline. K .vertline. A .vertline. A
.vertline. .vertline. 80.vertline. 81.vertline. 82.vertline.
83.vertline. 84.vertline. 85.vertline. 86.vertline. 87.vertline.
88.vertline. 89.vertline. 90.vertline. 91.vertline.
.vertline.GGC.vertline.-
GCC.vertline.GCT.vertline.GAA.vertline.GGT.vertline.GAT.vertline.GAT.vertl-
ine.CCG.vertline.GCC.vertline.AAG.vertline.GCG.vertline.G/CC.vertline.-
.vertline. Bbe I .vertline. .vertline. Sfi I .vertline. .vertline.
Nar I .vertline. .vertline. F .vertline. N .vertline. S .vertline.
L .vertline. Q .vertline. A .vertline. S .vertline. A .vertline. T
.vertline. .vertline. 92.vertline. 93.vertline. 94.vertline.
95.vertline. 96.vertline. 97.vertline. 98.vertline.
99.vertline.100.vertline.
.vertline.TTC.vertline.AAT.vertline.TCT.vertline.CTG.vertline.C,AA.vertl-
ine.GCT.vertline.TCT.vertline.GCT.vertline.ACC.vertline.-
.vertline.Hind 3.vertline. .vertline. E .vertline. Y .vertline. I
.vertline. G .vertline. Y .vertline. A .vertline. W .vertline.
.vertline.101.vertline.102.vertline.103-
.vertline.104.vertline.105.vertline.106.vertline.107.vertline.
.vertline.GAG.vertline.TAT.vertline.ATT.vertline.GGT.vertline.TAC.vertlin-
e.GCG.vertline.TGG.vertline.- .vertline. A .vertline. M .vertline.
V .vertline. V .vertline. V .vertline. I .vertline. V .vertline. G
.vertline. A .vertline.
.vertline.108.vertline.109.vertline.110.vertline.111.vertline.112.vertlin-
e.113.vertline.114.vertline.115.vertline.116.vertline.
.vertline.GCC.vertline.ATG.vertline.GTG.vertline.GTG.vertline.GTT.vertlin-
e.AT/C.vertline.GTT.vertline.GGT.vertline.GCT.vertline.- .vertline.
BstX I .vertline. .vertline. Nco I.vertline. .vertline. T
.vertline. I .vertline. G .vertline. I .vertline. .vertline.
117.vertline.118.vertline.119.vertline.120.vertline.
.vertline.ACC,.vertline.ATC.vertline.GGG.vertline.ATC.vertline.-
.vertline. K .vertline. L .vertline. F .vertline. K .vertline. K
.vertline. F .vertline. T .vertline. S .vertline. K .vertline. A
.vertline. .vertline.121.vertline.122.vertline.123.vertline.124-
.vertline.125.vertline.126.vertline.127.vertline.128.vertline.129.vertline-
.130.vertline. .vertline.AAA.vertline.CTG.vertline.TTC.vertline.-
AAG.vertline.AAG.vertline.TTT.vertline.ACT.vertline.TCG.vertline.AAG.vertl-
ine.GCG.vertline.- .vertline.Asu II.vertline. .vertline. S
.vertline. . .vertline. . .vertline. . .vertline.
.vertline.131.vertline.132.vertline.133- .vertline.134.vertline.
.vertline.TCT.vertline.TAA.vertline.TGA.- vertline.TAG.vertline.
GGTTA/CC- BstE II AGTCTA AGCCC,GC CTAATGA GCGGGCT TTTTTTTT-
terminator a/(TCGA),-3' (Sal I)
[1465]
84TABLE 104 Definition and alignment of oligonucleotides Lines
ending with "-" are continued on a following line. Blocks +TL,1/47
of ten bases are delimited by "-" within a line. When a break in
one strand does not correspond to a ten-base mark in the other
strand, "--" is inserted in the other strand. .dwnarw. Olig #801
(68 bases) 5'-GG-CTTTACACTT-TAT--GCTTCCG- 3'-cc-gaaatgtgaa-ata
cgaaggc- filled in .Arrow-up bold. .dwnarw. Olig #802 (67 bases)
GCTCGTATAA-TGTGTGGAAT-TGTGAGCGCT-CACAATTGAG-CTCAGG AGGC-TTACTATGAA-
cgagcatatt-acacacctta-acactcgcga-gtgttaactc-gagtc-
c--tccg-aatgatactt- Olig #803 (70 bases) .dwnarw.
G--AAATCTCTG-GTTCTTAAGG-CTAGCGTTGC- -TGTCGCGACC-CTGGTACCTA-TGT
TGTCCTT- c tttagagac-caagaattcc-gatcgc-
aacg-acagcgctgg-gaccatggat-aca--acagga- .Arrow-up bold. Olig #817
(68 bases) CGCTCG--TCCG-GATTTCTGTC-TCGAGCCACC-ATACACTGGG--
CCCTGCAAAG-CGCGCATCAT- gcgagc aggc-ctaaagacag-agctcggtgg-tatgtgacc-
c-gggacgtttc-gcgcgtagta- .Arrow-up bold. Olig #816 (65 bases)
.dwnarw.Olig #804 (67 bases) CCG
CTATTTC-TACAATGC--TA-AAGCAGGCCT-GTGCCAGACC-TTTGTATACG-GTGGTTGCCG-
ggc--gataaag-atgttacg
at-ttcgtccgga-cacggtctgg-aaacatatgc-caccaacggc- .Arrow-up bold.
Olig #815 (72 bases) .dwnarw. Olig #805 (76 bases) TGCTAAGCGT
AACAACTTTA-AATCG--GCCGA-AGATTGCATG-CGTACCTGCG-GTGGCGCCGC-
acgattcgca--ttgttgaaat-ttagc
cggct-tctaacgtac-gcatggacgc-caccgcggcg- .Arrow-up bold. Olig #814
(67 bases) .dwnarw. Olig #806 (67 bases)
TGAAGGTGAT-GATCCGGCCA-AGGCGG
CCTT-CAATTCTCTG-C--AAGCTTCTG-CTACCGAGTA-
acttccacta-ctaggccggt-tccgcc--ggaa-gttaagagac-g
ttcgaagac-gatggctcat- .Arrow-up bold. Olig #813 (76 bases) .dwnarw.
Olig #807 (69 bases) TATTGGTTAC-GCGTGGGCCA-TGGTGGTGGT-TAT
CGTTGGT-GCTACC--ATCG-GGATCAAACT-
ataaccaatg-cgcacccggt-accaccacca-ata--gcaacca-cgatgg
tagc-cctagtttga- .Arrow-up bold. Olig #812 (65 bases) .dwnarw. Olig
#808 (38 bases) GTTCAAGAAG-TTTACTTCGA-AGGCGTCTTA-ATGATAGGGT-TA
CCAGTCTA-AGCCC--GCCTA-
caagttcttc-aaatgaagct-tccgcagaat-tactatccca-at--ggtcagat-tcggg
cggat- Olig #811 (69 bases) .Arrow-up bold. .dwnarw. filled in
ATGAGCGGGC-TTTTTTTTTA TCGA-3' tactcgcccg-aaaaaaaaat-agct-5'
.Arrow-up bold. Olig #810 (29 bases) Overlap Sequences Junction Tm
AGGCTTACTATGAAG 802:817 42. TGTCCTTCGCTCG 803:816 42.
CTATTTCTACAATGC 804:815 40. AACAACTTTAAATCG 805:814 38.
CCTTCAATTCTCTGC 806:813 44. CGTTGGTGCTACC 807:812 42. CCAGTCTAAGCCC
808:811 42.
[1466]
85TABLE 105 Individual sequences of Oligonucleotides 801-817. Olig
#801 (68 bases) 5'-ggcTTTAcAc TTTATgcTTc cggcTcgTAT AATgTgTggA
ATTgTgAgcg cTcAcAATTg AgcTcAgg-3 Olig #802 (67 bases) 5'-AggcTTAcTA
TgAAgAAATc TcTggTTcTT AAggcTAgcg TTgcTgTcgc gAcccTggTA ccTATgT-3'
Olig #803 (70 bases) 5'-TgTccTTcgC TcgTccggAT TTcTgTcTcg AgccAccATA
cAcTgggccc TgcAAAgcgc gcATcATccg-3' Olig #804 (67 bases)
5'-cTATTTcTAc AATgcTAAAg cAggccTgTg ccAgAccTTT gTATAcggTg
gTTgccgTgc TAAgcgT-3' Olig #805 (76 bases) 5'-AAcAAcTTTA AATcggccgA
AgATTgcATg cgTAccTgcg gTggcgccgc TgAAggTgAT gATccggccA Aggcgg-3'
Olig #806 (67 bases) 5'-ccTTCAATTc TcTgcAAgcT TcTgcTAccg AgTATATTgg
TTAcgcgTgg gccATggTgg TggTTAT-3' Olig #807 (69 bases) 5'-cgTTggTgcT
AccATcgggA TcAAAcTgTT cAAgAAgTTT AcTTcgAAgg cgTcTTAATg ATAgggTTA-3'
Olig #808 (38 bases) 5'-ccAgTcTAAg cccgccTAAT gAgcgggcTT
TTTTTTTA-3' Olig #810 (29 bases) 5'-TcgATAAAAA AAAAgcccgc
TcATTAggc-3' Olig #811 (69 bases) 5'-gggcTTAgAc TggTAAcccT
ATcATTAAgA cgccTTcgAA gTAAAcTTcT TgAAcAgTTT gATcccgAT-3' Olig #812
(65 bases) 5'-ggTAgcAccA AcgATAAccA ccAccATggc ccAcgcgTAA
ccAATATAcT cggTAgcAgA AgcTT-3' Olig #813 (76 bases) 5'-gcAgAgAATT
gAAggccgcc TTggccggAT cATcAccTTc AgcggcgccA ccgcAggTAc gcATgcAATc
.vertline..vertline..vertline.TTcggc-3' Olig #814 (67 bases)
5'-cgATTTAAAg TTgTTAcgcT TAgcAcggcA AccAccgTAT AcAAAggTcT
ggcAcAggcc TgcTTTA-3' Olig #815 (72 bases) 5'-gcATTgTAgA AATAgcggAT
gATgcgcgcT TTgcAgggcc cAgTgTATgg TggcTcgAgA cAgAAATccg gA-3' Olig
#816 (65 bases) 5'-cgAgcgAAgg AcAAcATAgg TAccAgggTc gcgAcAgcAA
cgcTAgccTT AAgAAccAgA gaTTT-3' Olig #817 (68 bases) 5'-cTTcATAgTA
AgccTccTgA gcTcAATTgT gAgcgcTcAc AATTccAcAc ATTATAcgAg
ccggAAgc-3'
[1467]
86TABLE 106 Signal Peptides PhoA M K q s t i a l a l l p l l f t p
v t K A /R T . . . (17) MalE M K I K T G A R i l a l s a l t t m m
f s a s a l a /K I . . . (18) OmpF M M K R n i l a v i v p a l l v
a g t a n a /a E . . . (19) Bla M S I Q H F R v a l i p f f a a f c
l p v f a /h p . . . (>18) LamB M M I T L R K l p l a v a v a a
g v m s a q a m a /v D . . . (19) Lpp M K A T K l v l g a v i l g s
t l l a g /c s . . . (>17) gpIII M K K l l f a i p l v v p f y s
h s /a E T V E . . . (16) gpIII-BPTI M K K l l f a i p l v v p f y
s g a /R P D . . . (15) gpVIII M K K S L V L K a s v a v a t l v p
m l s f a /a E G D D . . . (16) gpVIII-BPTI M K K S L V L K a s v a
v a t l v p m l s f a /R P D . . . (15) gpVIII' M K K s l v l l a s
v a v a t l v p m l s f a /a E G D D . . . (21)
[1468]
87TABLE 107 In vitro transcription/translation analysis of
vector-encoded signal::BPTI::mature VIII protein species 31 kd
species.sup.a 14.5 kd species.sup.b No DNA (control) .sup. -.sup.c
- pGEN-3Zf (-) + - pGEM-MB16 + - pGEM-MB20 + + pGEM-MB26 + +
pGEM-MB42 + + pGEM-MB46 ND ND Notes: .sup.apre-beta-lactamase,
encoded by the amp (bla) gene. .sup.bpre-BPTI/VIII peptides encoded
by the synthetic gene and derived constructs. .sup.c- for absence
of product; + for presence of product; ND for Not Determined.
[1469]
88TABLE 108 Western analysis.sup.a of in vivo expressed
signal::BPTI::mature VIII protein species signal 14.5 kd
species.sup.b 12 kd species.sup.c A) expression in strain XL1-Blue
pGEM-3Zf(-) -- .sup. -.sup.d - PGEM-MB16 VIII - - PGEM-MB20 VIII ++
- PGEM-MB26 VIII +++ +/- PGEM-MB42 phoA ++ + B) expression in
strain SEF.sup.1 pGEM-MB42 phoA +/- +++ Notes: .sup.aAnalysis using
rabbit anti-BPTI polyclonal antibodies and
horse-radish-peroxidase-conjugated goat anti-rabbit IgG antibody.
.sup.bpro-BPTI/VIII peptides encoded by the synthetic gene and
derived constructs. .sup.cprocessed BPTI/VIII peptide encoded by
the synthetic gene. .sup.dnot present . . . - weakly present . . .
+/- present . . . + strong presence . . . ++ very strong presence
+++
[1470]
89TABLE 109 M13 gene III 1579 5'-GT GAAAAAATTA TTATTCGCAA
TTCCTTTAGT 1611 TGTTCCTTTC TATTCTCACT CCGCTGAAAC TGTTGAAAGT 1651
TGTTTAGCAA AACCCCATAC AGAAAATTCA TTTACTAACG 1691 TCTGGAAAGA
CGACAAAACT TTAGATCGTT ACGCTAACTA 1731 TGAGGGTTGT CTGTGGAATG
CTACAGGCGT TGTAGTTTGT 1771 ACTGGTGACG AAACTCAGTG TTACGGTACA
TGGGTTCCTA 1811 TTGGGCTTGC TATCCCTGAA AATGAGGGTG GTGGCTCTGA 1851
GGGTGGCGGT TCTGAGGGTG GCGGTTCTGA GGGTGGCGGT 1891 ACTAAACCTC
CTGAGTACGG TGATACACCT ATTCCGGGCT 1931 ATACTTATAT CAACCCTCTC
GACGGCACTT ATCCGCCTGG 1971 TACTGAGCAA AACCCCGCTA ATCCTAATCC
TTCTCTTGAG 2011 GAGTCTCAGC CTCTTAATAC TTTCATGTTT CAGAATAATA 2051
GGTTCCGAAA TAGGCAGGGG GCATTAACTG TTTATACGGG 2091 CACTGTTACT
CAAGGCACTG ACCCCGTTAA AACTTATTAC 2131 CAGTACACTC CTGTATCATC
AAAAGCCATG TATGACGCTT 2171 ACTGGAACGG TAAATTCAGA GACTGCGCTT
TCCATTCTGG 2211 CTTTAATGAG GATCCATTCG TTTGTGAATA TCAAGGCCAA 2251
TCGTCTGACC TGCCTCAACC TCCTGTCAAT GCTGGCGGCG 2291 GCTCTGGTGG
TGGTTCTGGT GGCGGCTCTG AGGGTGGTGG 2331 CTCTGAGGGT GGCGGTTCTG
AGGGTGGCGG CTCTGAGGGA 2371 GGCGGTTCCG GTGGTGGCTC TGGTTCCGGT
GATTTTGATT 2411 ATGAAAAGAT GGCAAACGCT AATAAGGGGG CTATGACCGA 2451
AAATGCCGAT GAAAACGCGC TACAGTCTGA CGCTAAAGGC 2491 AAACTTGATT
CTGTCGCTAC TGATTACGGT GCTGCTATCG 2531 ATGGTTTCAT TGGTGACGTT
TCCGGCCTTG CTAATGGTAA 2571 TGGTGCTACT GGTGATTTTG CTGGCTCTAA
TTCCCAAATG 2611 GCTCAAGTCG GTGACGGTGA TAATTCACCT TTAATGAATA 2651
ATTTCCGTCA ATATTTACCT TCCCTCCCTC AATCGGTTGA 2691 ATGTCGCCCT
TTTGTCTTTA GCGCTGGTAA ACCATATGAA 2731 TTTTCTATTG ATTGTGACAA
AATAAACTTA TTCCGTGGTG 2771 TCTTTGCGTT TCTTTTATAT GTTGCCACCT
TTATGTATGT 2811 ATTTTCTACG TTTGCTAACA TACTGCGTAA TAAGGAGTCT 2851
TAATCATGCC AGTTCTTTTG GGTATTCCGT
[1471]
90TABLE 110 Introduction of NarI into gene III A) Wild-type III,
portion encoding the signal peptide M K K L L F A I P L 1 2 3 4 5 6
7 8 9 10 1579 5'-GTG AAA AAA TTA TTA TTC GCA ATT CCT TTA / Cleavage
site .dwnarw. V V P F Y S H S A E T V 11 12 13 14 15 16 17 18 19 20
21 22 1609 GTT GTT CCT TTC TAT TCT CAC TCC GCT GAA ACT GTT-3' B)
III, portion encoding the signal peptide with NarI site m k k l l f
a I p l 1 2 3 4 5 6 7 8 9 10 1579 5'-gtg aaa aaa tta tta ttc gca
att cot tta / cleavage site .dwnarw. v v p f y s G A a e t V 11 12
13 14 15 16 17 18 19 20 21 22 1609 gtt gtt cct ttc tat tct GGc Gcc
gct gaa act gtt-3'
[1472]
91TABLE 111 IIIsp::bpti::mautreIII fusion gene. m k k l l f a I p l
1 2 3 4 5 6 7 8 9 10 5'-gtg aaa aaa tta tta ttc gca att cct tta
.vertline..rarw.--- gene III signal peptide -------- / cleavage
site .dwnarw. v v p f y s G A 11 12 13 14 15 16 17 18 gtt gtt cct
ttc tat tct GGc Gcc ----------------------------.fwdarw..vertlin-
e. .vertline. R .vertline. P .vertline. D .vertline. F .vertline. C
.vertline. L .vertline. E .vertline. .vertline. 19.vertline.
20.vertline. 21.vertline. 22.vertline. 23.vertline. 24.vertline.
25.vertline.
.vertline.CGT.vertline.CCG.vertline.GAT.vertline.TTC.vertline.TGT.vertlin-
e.CTC.vertline.GAG.vertline.- .Arrow-up bold.
.vertline.AccIII.vertline. .vertline. Ava I .vertline. M13/BPTI
Jnct .vertline. Xho I .vertline. .vertline. P .vertline. P
.vertline. Y .vertline. T .vertline. G .vertline. P .vertline. C
.vertline. K .vertline. A .vertline. R .vertline. .vertline.
26.vertline. 27.vertline. 28.vertline. 29.vertline. 30.vertline.
31.vertline. 32.vertline. 33.vertline. 34.vertline. 35.vertline.
.vertline.CCA.vertline.CCA.vertline.TAC-
.vertline.ACT.vertline.GGG.vertline.CCC.vertline.TGC.vertline.AAA.vertline-
.GCG.vertline.CGC.vertline.- .vertline. PflM I .vertline.
.vertline..vertline. .vertline.BssH II.vertline. .vertline. Apa I
.vertline..vertline. .vertline. Dra II .vertline. .vertline. Pss I
.vertline. .vertline. I .vertline. I .vertline. R .vertline. Y
.vertline. F .vertline. Y .vertline. N .vertline. A .vertline. K
.vertline. A .vertline. .vertline. 36.vertline. 37.vertline.
38.vertline. 39.vertline. 40.vertline. 41.vertline. 42.vertline.
43.vertline. 44.vertline. 45.vertline.
.vertline.ATC.vertline.ATC.vertline.CGC.vertline.TAT.vertline.TTC.vertlin-
e.TAC.vertline.AAT.vertline.GCT.vertline.AAA.vertline.GC
.vertline.- .vertline. G .vertline. L .vertline. C .vertline. Q
.vertline. T .vertline. F .vertline. V .vertline. Y .vertline. G
.vertline. G .vertline. .vertline. 46.vertline. 47.vertline.
48.vertline. 49.vertline. 50.vertline. 51.vertline. 52.vertline.
53.vertline. 54.vertline. 55.vertline. A.vertline.GGC.vertline.CT-
G.vertline.TGC.vertline.CAG.vertline.ACC.vertline.TTT.vertline.GTA.vertlin-
e.TAC.vertline.GGT.vertline.GGT.vertline.- .vertline. Stu
I.vertline. .vertline. Acc I .vertline. .vertline. Xca I .vertline.
.vertline. C .vertline. R .vertline. A .vertline. K .vertline. R
.vertline. N .vertline. N .vertline. F .vertline. K .vertline.
.vertline. 56.vertline. 57.vertline. 58.vertline. 59.vertline.
60.vertline. 61.vertline. 62.vertline. 63.vertline. 64.vertline.
.vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.CGT.vertlin-
e.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline.- .vertline.
Esp I .vertline. .vertline. S .vertline. A .vertline. E .vertline.
D .vertline. C .vertline. M .vertline. R .vertline. T .vertline. C
.vertline. G .vertline. .vertline. 65.vertline. 66.vertline.
67.vertline. 68.vertline. 69.vertline. 70.vertline. 71.vertline.
72.vertline. 73.vertline. 74.vertline.
.vertline.TCG.vertline.GCC.vertline.GAA.vertline.GAT.vertline.TGC.vertlin-
e.ATG.vertline.CGT.vertline.ACC.vertline.TGC.vertline.GGT.vertline.-
.vertline.XmaIII.vertline. .vertline. Sph I.vertline. BPTI/M13
boundary .dwnarw. .vertline. G .vertline. A .vertline. .vertline.
75.vertline. 76.vertline. .vertline.GGC.vertline.GCC.vertline.-
.vertline. Bbe I .vertline. .vertline. Nar I .vertline. G A a e t v
e s 77 78 79 80 81 82 83 84 GGc Gcc gct gaa act gtt GAA AGT 1651
TGTTTAGCAA AACCCCATAC AGAAAATTCA TTTACTAACG 1691 TCTGGAAAGA
CGACAAAACT TTAGATCGTT ACGCTAACTA 1731 TGAGGGTTGT CTGTGGAATG
CTACAGGCGT TGTAGTTTGT 1771 ACTGGTGACG AAACTCAGTG TTACGGTACA
TGGGTTCCTA 1811 TTGGGCTTGC TATCCCTGAA AATGAGGGTG GTGGCTCTGA 1851
GGGTGGCGGT TCTGAGGGTG GCGGTTCTGA GGGTGGCGGT 1891 ACTAAACCTC
CTGAGTACGG TGATACACCT ATTCCGGGCT 1931 ATACTTATAT CAACCCTCTC
GACGGCACTT ATCCGCCTGG 1971 TACTGAGCAA AACCCCGCTA ATCCTAATCC
TTCTCTTGAG 2011 GAGTCTCAGC CTCTTAATAC TTTCATGTTT CAGAATAATA 2051
GGTTCCGAAA TAGGCAGGGG GCATTAACTG TTTATACGGG 2091 CACTGTTACT
CAAGGCACTG ACCCCGTTAA AACTTATTAC 2131 CAGTACACTC CTGTATCATC
AAAAGCCATG TATGACGCTT 2171 ACTGGAACGG TAAATTCAGA GACTGCGCTT
TCCATTCTGG 2211 CTTTAATGAG GATCCATTCG TTTGTGAATA TCAAGGCCAA 2251
TCGTCTGACC TGCCTCAACC TCCTGTCAAT GCTGGCGGCG 2291 GCTCTGGTGG
TGGTTCTGGT GGCGGCTCTG AGGGTGGTGG 2331 CTCTGAGGGT GGCGGTTCTG
AGGGTGGCGG CTCTGAGGGA 2371 GGCGGTTCCG GTGGTGGCTC TGGTTCCGGT
GATTTTGATT 2411 ATGAAAAGAT GGCAAACGCT AATAAGGGGG CTATGACCGA 2451
AAATGCCGAT GAAAACGCGC TACAGTCTGA CGCTAAAGGC 2491 AAACTTGATT
CTGTCGCTAC TGATTACGGT GCTGCTATCG 2531 ATGGTTTCAT TGGTGACGTT
TCCGGCCTTG CTAATGGTAA 2571 TGGTGCTACT GGTGATTTTG CTGGCTCTAA
TTCCCAAATG 2611 GCTCAAGTCG GTGACGGTGA TAATTCACCT TTAATGAATA 2651
ATTTCCGTCA ATATTTACCT TCCCTCCCTC AATCGGTTGA 2691 ATGTCGCCCT
TTTGTCTTTA GCGCTGGTAA ACCATATGAA 2731 TTTTCTATTG ATTGTGACAA
AATAAACTTA TTCCGTGGTG 2771 TCTTTGCGTT TCTTTTATAT GTTGCCACCT
TTATGTATGT 2811 ATTTTCTACG TTTGCTAACA TACTGCGTAA TAAGGAGTCT 2851
TAATCATGCC AGTTCTTTTG GGTATTCCGT
[1473]
92TABLE 112 Annotated Sequence of Ptac::RBS(GGAGGAAATAAA)::
VIII-signal::mature-bpti::mature-VIII-co- at-protein gene 5'-GGATCC
actccccatcccc .vertline. .vertline. BamHI ctg TTGACA
attaatcatcgGCTCG tataat GTGTGG- -35 tac -10 aATTGTGAGCGcTcACAATT-
lacO-symm operator GAGCTC T ggagga AATAAA- SacI Shine-Dalgarno seq.
.vertline.fM .vertline. K .vertline. K .vertline. S .vertline. L
.vertline. V .vertline. L .vertline. K .vertline. A .vertline. S
.vertline. .vertline. 1 .vertline. 2 .vertline. 3 .vertline. 4
.vertline. 5 .vertline. 6 .vertline. 7 .vertline. 8 .vertline. 9
.vertline. 10.vertline.
.vertline.ATG.vertline.AAG.vertline.AAA.vertline.TCT.vertline.CTG.vertlin-
e.GTT.vertline.CTT.vertline.AAG.vertline.GCT.vertline.AGC.vertline.-
.vertline. Afl II .vertline. Nhe I .vertline. .vertline. V
.vertline. A .vertline. V .vertline. A .vertline. T .vertline. L
.vertline. V .vertline. P .vertline. M .vertline. L .vertline.
.vertline. 11.vertline. 12.vertline. 13.vertline. 14.vertline.
15.vertline. 16.vertline. 17.vertline. 18.vertline. 19.vertline.
20.vertline. .vertline.GTT.vertline.G-
CT.vertline.GTC.vertline.GCG.vertline.ACC.vertline.CTG.vertline.GTA.vertli-
ne.CCT.vertline.ATG.vertline.TTG.vertline.- .vertline. Nru
I.vertline. .vertline. Kpn I.vertline. .vertline. S .vertline. F
.vertline. A .vertline. R .vertline. P .vertline. D .vertline. F
.vertline. C .vertline. L .vertline. E .vertline. .vertline.
21.vertline. 22.vertline. 23.vertline. 24.vertline. 25.vertline.
26.vertline. 27.vertline. 28.vertline. 29.vertline. 30.vertline.
.vertline.TCC.vertline.TTC.vertline.G-
CT.vertline.CGT.vertline.CCG.vertline.GAT.vertline.TTC.vertline.TGT.vertli-
ne.CTC.vertline.GAG.vertline.- .Arrow-up bold.
.vertline.AccIII.vertline. .vertline. Ava I .vertline. M13/BPTI
Jnct .vertline. Xho I .vertline. .vertline. P .vertline. P
.vertline. Y .vertline. T .vertline. G .vertline. P .vertline. C
.vertline. K .vertline. A .vertline. R .vertline. .vertline.
31.vertline. 32.vertline. 33.vertline. 34.vertline. 35.vertline.
36.vertline. 37.vertline. 38.vertline. 39.vertline. 40.vertline.
.vertline.CCA.vertline.CCA.vertline.T-
AC.vertline.ACT.vertline.GGG.vertline.CCC.vertline.TGC.vertline.AAA.vertli-
ne.GCG.vertline.CGC.vertline.- .vertline. Pf1M I .vertline.
.vertline..vertline. .vertline.BSSH II.vertline. .vertline. Apa I
.vertline..vertline. .vertline. Dra II .vertline. .vertline. Pss I
.vertline. .vertline. I .vertline. I .vertline. R .vertline. Y
.vertline. F .vertline. Y .vertline. N .vertline. A .vertline. K
.vertline. A .vertline. .vertline. 41.vertline. 42.vertline.
43.vertline. 44.vertline. 45.vertline. 46.vertline. 47.vertline.
48.vertline. 49.vertline. 50.vertline.
.vertline.ATC.vertline.ATC.vertline.CGC.vertline.TAT.vertline.TTC.vertli-
ne.TAC.vertline.AAT.vertline.GCT.vertline.AAA.vertline.GC
.vertline.- .vertline. G .vertline. L .vertline. C .vertline. Q
.vertline. T .vertline. F .vertline. V .vertline. Y .vertline. G
.vertline. G .vertline. .vertline. 51.vertline. 52.vertline.
53.vertline. 54.vertline. 55.vertline. 56.vertline. 57.vertline.
58.vertline. 59.vertline. 60.vertline. A.vertline.GGC.vertline.CT-
G.vertline.TGC.vertline.CAG.vertline.ACC.vertline.TTT.vertline.GTA.vertlin-
e.TAC.vertline.GGT.vertline.GGT.vertline.- .vertline. Stu
I.vertline. .vertline. Acc I .vertline. .vertline. Xca I .vertline.
.vertline. C .vertline. R .vertline. A .vertline. K .vertline. R
.vertline. N .vertline. N .vertline. F .vertline. K .vertline.
.vertline. 61.vertline. 62.vertline. 63.vertline. 64.vertline.
65.vertline. 66.vertline. 67.vertline. 68.vertline. 69.vertline.
.vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.CGT.vertlin-
e.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline.- .vertline.
Esp I .vertline. .vertline. S .vertline. A .vertline. E .vertline.
D .vertline. C .vertline. M .vertline. R .vertline. T .vertline. C
.vertline. G .vertline. .vertline. 70.vertline. 71.vertline.
72.vertline. 73.vertline. 74.vertline. 75.vertline. 76.vertline.
77.vertline. 78.vertline. 79.vertline.
.vertline.TCG.vertline.GCC.vertline.GAA.vertline.GAT.vertline.TGC.vertli-
ne.ATG.vertline.CGT.vertline.ACC.vertline.TGC.vertline.GGT.vertline.-
.vertline.XmaIII.vertline. .vertline. Sph I.vertline. BPTI/M13
boundary .dwnarw. .vertline. G .vertline. A .vertline. A .vertline.
E .vertline. G .vertline. D .vertline. D .vertline. P .vertline. A
.vertline. K .vertline. A .vertline. A .vertline. .vertline.
80.vertline. 81.vertline. 82.vertline. 83.vertline. 84.vertline.
85.vertline. 86.vertline. 87.vertline. 88.vertline. 89.vertline.
90.vertline. 91.vertline.
.vertline.GGC.vertline.GCC.vertline.GCT.vertline.G-
AA.vertline.GGT.vertline.GAT.vertline.GAT.vertline.CCG.vertline.GCC.vertli-
ne.AAG.vertline.GCG.vertline.GCC.vertline.- .vertline. Bbe I
.vertline. .vertline. Sfi I .vertline. .vertline. Nar I .vertline.
.vertline. F .vertline. N .vertline. S .vertline. L .vertline. Q
.vertline. A .vertline. S .vertline. A .vertline. T .vertline.
.vertline. 92.vertline. 93.vertline. 94.vertline. 95.vertline.
96.vertline. 97.vertline. 98.vertline. 99.vertline.100.vertline.
.vertline.TTC.vertline.AAT.vertline.TCT.vertline.CTG.vertline.CAA.vertlin-
e.GCT.vertline.TCT.vertline.GCT.vertline.ACC.vertline.-
.vertline.Hind 3.vertline. .vertline. E .vertline. Y .vertline. I
.vertline. G .vertline. Y .vertline. A .vertline. W .vertline.
.vertline.101.vertline.102.vertline.103-
.vertline.104.vertline.105.vertline.106.vertline.107.vertline.
.vertline.GAG.vertline.TAT.vertline.ATT.vertline.GGT.vertline.TAC.vertlin-
e.GCG.vertline.TGG.vertline.- .vertline. A .vertline. M .vertline.
V .vertline. V .vertline. V .vertline. I .vertline. V .vertline. G
.vertline. A .vertline.
.vertline.108.vertline.109.vertline.110.vertline.111.vertline.112.vertlin-
e.113.vertline.114.vertline.115.vertline.116.vertline.
.vertline.GCC.vertline.ATG.vertline.GTG.vertline.GTG.vertline.GTT.vertlin-
e.ATC.vertline.GTT.vertline.GGT.vertline.GCT.vertline.- .vertline.
BstX I .vertline. .vertline. Nco I.vertline. .vertline. T
.vertline. I .vertline. G .vertline. I .vertline.
.vertline.117.vertline.118.vertl- ine.119.vertline.120.vertline.
.vertline.ACC.vertline.ATC.v- ertline.GGG.vertline.ATC.vertline.-
.vertline. K .vertline. L .vertline. F .vertline. K .vertline. K
.vertline. F .vertline. T .vertline. S .vertline. K .vertline. A
.vertline.
.vertline.121.vertline.122.vertline.123.vertline.124.vertline.125.vertlin-
e.126.vertline.127.vertline.128.vertline.129.vertline.130.vertline.
.vertline.AAA.vertline.CTG.vertline.TTC.vertline.AAG.vertline.AAG.vert-
line.TTT.vertline.ACT.vertline.TCG.vertline.AAG.vertline.GCG.vertline.-
.vertline.Asu II.vertline. .vertline. S .vertline. . .vertline. .
.vertline. . .vertline.
.vertline.131.vertline.132.vertline.133.vertline.134.vertline.
.vertline.TCT.vertline.TAA.vertline.TGA.vertline.TAG.vertline.
GGTTACC- BstE II AGTCTA AGCCCGC CTAATGA GCGGGCT TTTTTTTT-
terminator aTCGA GACctgca GGTCGACC ggcatgc-3'
.vertline.SalI.vertline.
[1474]
93TABLE 113 Annotated Sequence of pGEM-MB42 comprising
Ptac::RBS(GGAGGAAATAAA):: phoA-signal::mature-bpti::mat-
ure-VIII-coat-protein 5'-GGATCC actccccatcccc .vertline. .vertline.
BamHI ctg TTGACA attaatcatcgGCTCG tataat GTGTGG -35 tac -10
aATTGTGAGCGcTcACAATT- lacO-symm operator .vertline. M .vertline. K
.vertline. Q .vertline. S .vertline. T .vertline. .vertline. 1
.vertline. 2 .vertline. 3 .vertline. 4 .vertline. 5 .vertline.
GAGCTCCATGGGAGAAAATAAA.vertline.ATG.ve-
rtline.AAA.vertline.CAA.vertline.AGC.vertline.ACG.vertline.-
.vertline.SacI.vertline. .vertline..rarw.---- phoA signal peptide
.vertline. I .vertline. A .vertline. L .vertline. L .vertline. P
.vertline. L .vertline. L .vertline. F .vertline. T .vertline. P
.vertline. V .vertline. T .vertline. .vertline. 6 .vertline. 7
.vertline. 8 .vertline. 9 .vertline. 10.vertline. 11.vertline.
12.vertline. 13.vertline. 14.vertline. 15.vertline. 16.vertline.
17.vertline. .vertline.ATC.vertline.GCA-
.vertline.CTC.vertline.TTA.vertline.CCG.vertline.TTA.vertline.CTG.vertline-
.TTT.vertline.ACC.vertline.CCT.vertline.GTG.vertline.ACA.vertline.-
---------------- phoA signal continues ---------- (There are no
residues 20-23.) .vertline. K .vertline. A .vertline. R .vertline.
P .vertline. D .vertline. F .vertline. C .vertline. L .vertline. E
.vertline. .vertline. 18.vertline. 19.vertline. 24.vertline.
25.vertline. 26.vertline. 27.vertline. 28.vertline. 29.vertline.
30.vertline.
.vertline.AAA.vertline.GCC.vertline.CGT.vertline.CCG.vertline.GAT.vertlin-
e.TTC.vertline.TGT.vertline.CTC.vertline.GAG.vertline.- phoA
signal.fwdarw..Arrow-up bold. .vertline.AccIII.vertline. .vertline.
Ava I .vertline. phoA/BPTI Jnct .vertline. Xho I .vertline.
.vertline..rarw.---- BPTI insert --------- .vertline. P .vertline.
P .vertline. Y .vertline. T .vertline. G .vertline. P .vertline. C
.vertline. K .vertline. A .vertline. R .vertline. .vertline.
33.vertline. 34.vertline. 35.vertline. 36.vertline. 37.vertline.
38.vertline. 39.vertline. 40.vertline. .vertline.CCA.vertline.CCA-
.vertline.TAC.vertline.ACT.vertline.GGG.vertline.CCC.vertline.TGC.vertline-
.AAA.vertline.GCG.vertline.CGC.vertline._ .vertline. PflM I
.vertline. .vertline..vertline. .vertline.BssH II.vertline.
.vertline. Apa I.vertline. .vertline. .vertline. Dra II .vertline.
.vertline. Pss I .vertline. .vertline. I .vertline. I .vertline. R
.vertline. Y .vertline. F .vertline. Y .vertline. N .vertline. A
.vertline. K .vertline. A .vertline. .vertline. 41.vertline.
42.vertline. 43.vertline. 44.vertline. 45.vertline. 46.vertline.
47.vertline. 48.vertline. 49.vertline. 50.vertline.
.vertline.ATC.vertline.ATC.vertline.CGC.vertline.TAT.vertline.TTC.vertli-
ne.TAC.vertline.AAT.vertline.GCT.vertline.AAA.vertline.GC
.vertline.- .vertline. G .vertline. L .vertline. C .vertline. Q
.vertline. T .vertline. F .vertline. V .vertline. Y .vertline. G
.vertline. G .vertline. .vertline. 51.vertline. 52.vertline.
53.vertline. 54.vertline. 55.vertline. 56.vertline. 57.vertline.
58.vertline. 59.vertline. 60.vertline. A.vertline.GGC.vertline.C-
TG.vertline.TGC.vertline.CAG.vertline.ACC.vertline.TTT.vertline.GTA.vertli-
ne.TAC.vertline.GGT.vertline.GGT.vertline.- .vertline. Stu
I.vertline. .vertline. Acc I .vertline. .vertline. Xca I .vertline.
.vertline. C .vertline. R .vertline. A .vertline. K .vertline. R
.vertline. N .vertline. N .vertline. F .vertline. K .vertline.
.vertline. 61.vertline. 62.vertline. 63.vertline. 64.vertline.
65.vertline. 66.vertline. 67.vertline. 68.vertline. 69.vertline.
.vertline.TGC.vertline.CGT.vertline.GCT.vertline.AAG.vertline.CGT.vertli-
ne.AAC.vertline.AAC.vertline.TTT.vertline.AAA.vertline.- .vertline.
Esp I .vertline. .vertline. S .vertline. A .vertline. E .vertline.
D .vertline. C .vertline. M .vertline. R .vertline. T .vertline. C
.vertline. G .vertline. .vertline. 70.vertline. 71.vertline.
72.vertline. 73.vertline. 74.vertline. 75.vertline. 76.vertline.
77.vertline. 78.vertline. 79.vertline.
.vertline.TCG.vertline.GCC.vertline.GAA.vertline.GAT.vertline.TGC.vertli-
ne.ATG.vertline.CGT.vertline.ACC.vertline.TGC.vertline.GGT.vertline.-
.vertline.XmaIII.vertline. .vertline.Sph I .vertline. -------------
BPTI insert------------------ BPTI/M13 boundary .dwnarw. .vertline.
G .vertline. A .vertline. A .vertline. E .vertline. G .vertline. D
.vertline. D .vertline. P .vertline. A .vertline. K .vertline. A
.vertline. A .vertline. .vertline. 80.vertline. 81.vertline.
82.vertline. 83.vertline. 84.vertline. 85.vertline. 86.vertline.
87.vertline. 88.vertline. 89.vertline. 90.vertline. 91.vertline.
.vertline.GGC.vertline.GCC.vertline.GCT.vertline.GAA.vertline.GGT.vertli-
ne.GAT.vertline.GAT.vertline.CCG.vertline.GCC.vertline.AAG.vertline.GCG.ve-
rtline.GCC.vertline.- .vertline. Bbe I .vertline. .vertline. Sfi I
.vertline. .vertline. Nar I .vertline. --
BPTI-->.vertline.<----- mature gene VIII coat protein ----
.vertline. F .vertline. N .vertline. S .vertline. L .vertline. Q
.vertline. A .vertline. S .vertline. A .vertline. T .vertline.
.vertline. 92.vertline. 93.vertline. 94.vertline. 95.vertline.
96.vertline. 97.vertline. 98.vertline. 99.vertline.100.vertline.
.vertline.TTC.vertline.AAT.vertline.T-
CT.vertline.CTG.vertline.CAA.vertline.GCT.vertline.TCT.vertline.GCT.vertli-
ne.ACC.vertline.- .vertline.Hind 3.vertline. .vertline. E
.vertline. Y .vertline. I .vertline. G .vertline. Y .vertline. A
.vertline. W .vertline.
.vertline.101.vertline.102.vertline.103.vertline.104.vertline.105.vertlin-
e.106.vertline.107.vertline. .vertline.GAG.vertline.TAT.vertline-
.ATT.vertline.GGT.vertline.TAC.vertline.GCG.vertline.TGG.vertline.-
.vertline. A .vertline. M .vertline. V .vertline. V .vertline. V
.vertline. I .vertline. V .vertline. G .vertline. A .vertline.
.vertline.108.vertline.109.vertline.110.vertline.-
111.vertline.112.vertline.113.vertline.114.vertline.115.vertline.116.vertl-
ine. .vertline.GCC.vertline.ATG.vertline.GTG.vertline.GTG.ver-
tline.GTT.vertline.ATC.vertline.GTT.vertline.GGT.vertline.GCT.vertline.-
.vertline. BstX I .vertline. .vertline. Nco I.vertline. .vertline.
T .vertline. I .vertline. G .vertline. I .vertline.
.vertline.117.vertline.118.vertline.119.vertline.120.vertline.
.vertline.ACC.vertline.ATC.vertline.GGG.vertline.ATC.vertline.-
.vertline. K .vertline. L .vertline. F .vertline. K .vertline. K
.vertline. F .vertline. T .vertline. S .vertline. K .vertline. A
.vertline. .vertline.121.vertline.122.vertline.123-
.vertline.124.vertline.125.vertline.126.vertline.127.vertline.128.vertline-
.129.vertline.130.vertline. .vertline.AAA.vertline.CTG.vertline.-
TTC.vertline.AAG.vertline.AAG.vertline.TTT.vertline.ACT.vertline.TCG.vertl-
ine.AAG.vertline.GCG.vertline.- .vertline.Asu II.vertline.
.vertline. S .vertline. . .vertline. . .vertline. . .vertline.
.vertline.131.vertline.132- .vertline.133.vertline.134.vertline.
.vertline.TCT.vertline.TAA.- vertline.TGA.vertline.TAG.vertline.
GGTTACC- BstE II AGTCTA AGCCCGC CTAATGA GCGGGCT TTTTTTTT-
terminator aTCGA GACctgca GGTCGAC-3' .vertline.SalI.vertline.
[1475]
94TABLE 114 Neutralization of Phage Titer Using Agarose-immobilized
Anhydro-Trypsin Percent Residual Titer As a Function of Time
(hours) Phase Type Addition 1 2 4 MK-BPTI 5 .mu.l IS 99 104 105 2
.mu.l IAT 82 71 51 5 .mu.l IAT 57 40 27 10 .mu.l IAT 40 30 24 MK 5
.mu.l IS 106 96 98 2 .mu.l IAT 97 103 95 5 .mu.l IAT 110 111 96 10
.mu.l IAT 99 93 106 Legend: IS = Immobilized streptavidin IAT =
Immobilized anhydro-trypsin
[1476]
95TABLE 115 Affinity Selection of MK-BPTI Phage on Immobilized
Anhydro-Trypsin Percent of Total Phage Phage Type Addition
Recovered in Elution Buffer MK-BPTI 5 .mu.l IS <<1.sup.a 2
.mu.l IAT 5 5 .mu.l IAT 20 10 .mu.l IAT 50 MK 5 .mu.l IS
<<1.sup.a 2 .mu.l IAT <<1 5 .mu.l IAT <<1 10
.mu.l IAT <<1 Legend: IS = Immobilized streptavidin IAT =
Immobilized anhydro-trypsin .sup.anot detectable.
[1477]
96TABLE 116 translation of Signal-III::bpti::mature- -III 1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 fM K K L L F A I P L V V P F Y GTG AAA
AAA TTA TTA TTC GCA ATT CCT TTA GTT GTT CCT TTC TAT
.vertline.<------- gene III signal peptide
------------------------- 16 17 18 19 20 21 22 23 24 25 26 27 28 29
30 S G A R P D F C L E P P Y T G TCT GCC GCC cgt ccg gat ttc tgt
ctc gag cca cca tac act ggg ----------->.vertline.<----- BPTI
insertion ------------------------ 31 32 33 34 35 36 37 38 39 40 41
42 43 44 45 P C K A R I I R Y F Y N A K A ccc tgc aaa gcg cgc atc
atc cgc tat ttc tac aat gct aaa gca 46 47 48 49 50 51 52 53 54 55
56 57 58 59 60 G L C Q T F V Y G G C R A K R ggc ctg tgc cag acc
ttt gta tac ggt ggt tgc cgt gct aag cgt 61 62 63 64 65 66 67 68 69
70 71 72 73 74 75 N N F K S A E D C M R T C G G aac aac ttt aaa tcg
gcc gaa gat tgc atg cgt acc tgc ggt ggc 76 77 78 79 80 81 82 83 84
85 86 87 88 89 90 A G A A E T V E S C L A K P H gcc GGC GCC GCT GAA
ACT GTT GAA AGT TGT TTA GCA AAA CCC CAT .vertline.<-------
mature gene III protein --------------------- 91 92 93 94 95 96 97
98 99 100 101 102 103 104 105 T E N S F T N V W K D D K T L ACA GAA
AAT TCA TTT ACT AAC GTC TGG AAA GAC GAC AAA ACT TTA 106 107 108 109
110 111 112 113 114 115 116 117 118 119 120 D R Y A N Y E G C L W N
A T G GAT CGT TAC GCT AAC TAT GAG GGT TGT CTG TGG AAT GCT ACA GGC
121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 V V V C
T G D E T Q C Y G T W GTT GTA GTT TGT ACT GGT GAC GAA ACT CAG TGT
TAC GGT ACA TGG 136 137 138 139 140 141 142 143 144 145 146 147 148
149 150 V P I G L A I P E N E G G G S GTT CCT ATT GGG CTT GCT ATC
CCT GAA AAT GAG GGT GGT GGC TCT 151 152 153 154 155 156 157 158 159
160 161 162 163 164 165 E G G G S E G G G S E G G G T GAG GGT GGC
GGT TCT GAG GGT GGC GGT TCT GAG GGT GGC GGT ACT 166 167 168 169 170
171 172 173 174 175 176 177 178 179 180 K P P E Y G D T P I P G Y T
Y AAA CCT CCT GAG TAC GGT GAT ACA CCT ATT CCG GGC TAT ACT TAT 181
182 183 184 185 186 187 188 189 190 191 192 193 194 195 I N P L D G
T Y P P G T E Q N ATC AAC CCT CTC GAC GGC ACT TAT CCG CCT GGT ACT
GAG CAA AAC 196 197 198 199 200 201 202 203 204 205 206 207 208 209
210 P A N P N P S L E E S Q P L N CCC GCT AAT CCT AAT CCT TCT CTT
GAG GAG TCT CAG CCT CTT AAT 211 212 213 214 215 216 217 218 219 220
221 222 223 224 225 T F M F Q N N R F R N R Q G A ACT TTC ATG TTT
CAG AAT AAT AGG TTC CGA AAT AGG CAG GGG GCA 226 227 228 229 230 231
232 233 234 235 236 237 238 239 240 L T V Y T G T V T Q G T D P V
TTA ACT GTT TAT ACG GGC ACT GTT ACT CAA GGC ACT GAC CCC GTT 241 242
243 244 245 246 247 248 249 250 251 252 253 254 255 K T Y Y Q Y T P
V S S K A H Y AAA ACT TAT TAC CAG TAC ACT CCT GTA TCA TCA AAA GCC
ATG TAT 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270
D A Y W N G K F R D C A F H S GAC GCT TAC TGG AAC GGT AAA TTC AGA
GAC TGC GCT TTC CAT TCT 271 272 273 274 275 276 277 278 279 280 281
282 283 284 285 G F N E D P F V C E Y Q G Q S GGC TTT AAT GAG GAT
CCA TTC GTT TGT GAA TAT CAA GGC CAA TCG 286 287 288 289 290 291 292
293 294 295 296 297 298 299 300 S D L P Q P P V N A G G G S G TCT
GAC CTG CCT CAA CCT CCT GTC AAT GCT GGC GGC GGC TCT GGT 301 302 303
304 305 306 307 308 309 310 311 312 313 314 315 G G S G G G S E G G
G S E G G GGT GGT TCT GGT GGC GGC TCT GAG GGT GGT GGC TCT GAG GGT
GGC 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 G S
E G G G S E G G G S G G G GGT TCT GAG GGT GGC GGC TCT GAG GGA GGC
GGT TCC GGT GGT GGC 331 332 333 334 335 336 337 338 339 340 341 342
343 344 345 S G S G D F D Y E K M A N A N TCT GGT TCC GGT GAT TTT
GAT TAT GAA AAG ATG GCA AAC GCT AAT 346 347 348 349 350 351 352 353
354 355 356 357 358 359 360 K G A M T E N A D E N A L Q S AAG GGG
GCT ATG ACC GAA AAT GCC GAT GAA AAC GCG CTA CAG TCT 361 362 363 364
365 366 367 368 369 370 373 372 373 374 375 D A K G K L D S V A T D
Y G A GAC GCT AAA GGC AAA CTT GAT TCT GTC GCT ACT GAT TAC GGT GCT
376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 A I D G
F I G D V S G L A N G GCT ATC GAT GGT TTC ATT GGT GAC GTT TCC GGC
CTT GCT AAT GGT 391 392 393 394 395 396 397 398 399 400 401 402 403
404 405 N G A T G D F A G S N S Q M A AAT GGT GCT ACT GGT GAT TTT
GCT GGC TCT AAT TCC CAA ATG GCT 406 407 408 409 410 411 412 413 414
415 416 417 418 419 420 Q V G D G D N S P L M N N F R CAA GTC GGT
GAC CGT GAT AAT TCA CCT TTA ATG AAT AAT TTC CGT 421 422 423 424 425
426 427 428 429 430 431 432 433 434 435 Q Y L P S L P Q S V E C R P
F CAA TAT TTA CCT TCC CTC CCT CAA TCG GTT GAA TGT CGC CCT TTT 436
437 438 439 440 441 442 443 444 445 446 447 448 449 450 V F S A C K
P Y E F S I D C D GTC TTT AGC GCT GGT AAA CCA TAT GAA TTT TCT ATT
GAT TGT GAC 451 452 453 454 455 456 457 458 459 460 461 462 463 464
465 K I N L F R C V F A F L L Y V AAA ATA AAC TTA TTC CGT GGT GTC
TTT CCC TTT CTT TTA TAT GTT .vertline.<----- uncharged anchor
region ----- 466 467 468 469 470 471 472 473 474 475 476 477 478
479 480 A T F M Y V F S T F A N I L R GCC ACC TTT ATG TAT GTA TTT
TCT ACG TTT GCT AAC ATA CTG CCT --------- uncharged anchor region
continues --------->.vertline. 481 482 483 484 485 N K E S . AAT
AAG GAG TCT TAA Molecular weight of peptide = 58884 Charge on
peptide = -20 [A + G + P] = 143 [C + F + H + I + L + M + V + W + Y]
= 140 [D + E + K + R + N + Q + S + T + .] = 202 Second Base t c a g
t 15 21 15 8 t 12 5 10 6 c 10 4 0 0 a 0 3 0 4 g c 6 20 2 8 t 3 4 0
3 c 1 4 9 1 a 4 3 7 0 g a 5 19 21 1 t 5 4 11 1 c 2 4 16 1 a 8 2 4 2
g g 13 22 14 41 t 6 7 12 29 c 4 5 12 1 a 1 3 16 4 g AA # AA # AA #
AA # A 37 C 14 D 26 E 28 F 27 G 75 H 2 I 12 K 20 L 24 M 9 N 32 P 31
Q 16 R 15 S 35 T 29 V 23 W 4 Y 25 . 1
[1478]
97TABLE 130 Sampling of a Library encoded by (NNK).sup.6 A. Numbers
of hexapeptides in each class total = 64,000,000 stop-free
sequences. .alpha. can be one of [WMFYCIKDENHQ] .PHI. can be one of
[PTAVG] .OMEGA. can be one of [SLR]
.alpha..alpha..alpha..alpha..alpha..al- pha. = 2985984.
.PHI..alpha..alpha..alpha..alpha..alpha. = 7464960.
.OMEGA..alpha..alpha..alpha..alpha..alpha. = 4478976.
.PHI..PHI..alpha..alpha..alpha..alpha. = 7776000.
.PHI..OMEGA..alpha..alpha..alpha..alpha. = 9331200.
.OMEGA..OMEGA..alpha..alpha..alpha..alpha. = 2799360.
.PHI..PHI..PHI..alpha..alpha..alpha. = 4320000.
.PHI..PHI..OMEGA..alpha..- alpha..alpha. = 7776000.
.PHI..OMEGA..OMEGA..alpha..alpha..alpha. = 4665600.
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha. = 933120.
.PHI..PHI..PHI..PHI..alpha..alpha. = 1350000.
.PHI..PHI..PHI..OMEGA..alph- a..alpha. = 3240000.
.PHI..PHI..OMEGA..OMEGA..alpha..alpha. = 2916000.
.PHI..OMEGA..OMEGA..OMEGA..alpha..alpha. = 1166400.
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha. = 174960.
.PHI..PHI..PHI..PHI..PHI..alpha. = 225000. .PHI..PHI..PHI..PHI..OM-
EGA..alpha. = 675000. .PHI..PHI..PHI..OMEGA..OMEGA..alpha. =
810000. .PHI..PHI..OMEGA..OMEGA..OMEGA..alpha. = 486000.
.PHI..OMEGA..OMEGA..OME- GA..OMEGA..alpha. = 145800.
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alp- ha. = 17496.
.PHI..PHI..PHI..PHI..PHI..PHI. = 15625.
.PHI..PHI..PHI..PHI..PHI..OMEGA. = 56250.
.PHI..PHI..PHI..PHI..OMEGA..OME- GA. = 84375.
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA. = 67500.
.PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA. = 30375.
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA. = 7290.
.OMEGA..OMEGA..OMEGA..OM- EGA..OMEGA..OMEGA. = 729.
.PHI..PHI..OMEGA..OMEGA..alpha..alpha., for example, stands for the
set of peptides having two amino acids from the .alpha. class, two
from .PHI., and two from .OMEGA. arranged in any order. There are,
for example, 729 = 3.sup.6 sequences composed entirely of S, L, and
R. B. Probability that any given stop-free DNA sequence will encode
a hexapeptide from a stated class. P % of class
.alpha..alpha..alpha..alpha..alpha..alpha.. . . 3.364E-03
(1.13E-07) .PHI..alpha..alpha..alpha..alpha..alpha.. . . 1.682E-02
(2.25E-07) .OMEGA..alpha..alpha..alpha..alpha..alpha.. . .
1.514E-02 (3.38E-07) .PHI..PHI..alpha..alpha..alpha..alpha.. . .
3.505E-02 (4.51E-07) .PHI..OMEGA..alpha..alpha..alpha..alpha.. . .
6.308E-02 (6.76E-07) .OMEGA..OMEGA..alpha..alpha..alpha..alpha.. .
. 2.839E-02 (1.01E-06) .PHI..PHI..PHI..alpha..alpha..alpha.. . .
3.894E-02 (9.01E-07) .PHI..PHI..OMEGA..alpha..alpha..alpha.. . .
1.051E-01 (1.35E-06) .PHI..OMEGA..OMEGA..alpha..alpha..alpha.. . .
9.463E-02 (2.03E-06) .OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.. .
. 2.839E-02 (3.04E-06) .PHI..PHI..PHI..PHI..alpha..alpha.. . .
2.434E-02 (1.80E-06) .PHI..PHI..PHI..OMEGA..alpha..alpha.. . .
8.762E-02 (2.70E-06) .PHI..PHI..OMEGA..OMEGA..alpha..alpha.. . .
1.183E-01 (4.06E-06) .PHI..OMEGA..OMEGA..OMEGA..alpha..alpha.. . .
7.097E-02 (6.08E-06) .OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.. .
. 1.597E-02 (9.13E-06) .PHI..PHI..PHI..PHI..PHI..alpha.. . .
8.113E-02 (3.61E-06) .PHI..PHI..PHI..PHI..OMEGA..alpha.. . .
3.651E-02 (5.41E-06) .PHI..PHI..PHI..OMEGA..OMEGA..alpha.. . .
6.571E-02 (8.11E-06) .PHI..PHI..OMEGA..OMEGA..OMEGA..alpha.. . .
5.914E-02 (1.22E-05) .PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . .
2.661E-02 (1.83E-05) .OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. .
. 4.790E-03 (2.74E-05) .PHI..PHI..PHI..PHI..PHI..PHI.. . .
1.127E-03 (7.21E-06) .PHI..PHI..PHI..PHI..PHI..OMEGA.. . .
6.083E-03 (1.08E-05) .PHI..PHI..PHI..PHI..OMEGA..OMEGA.. . .
1.369E-02 (1.62E-05) .PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.. . .
1.643E-02 (2.43E-05) .PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.. . .
1.109E-02 (3.65E-05) .PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . .
3.992E-03 (5.48E-05) .OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. .
. 5.988E-04 (8.21E-05) C. Number of different stop-free amino-acid
sequences in each class expected for various library sizes Library
size = 1.0000E+06 total = 9.7446E+05 % sampled = 1.52 Class Number
% Class Number % .alpha..alpha..alpha..alpha..alpha..alpha- .. . .
3362.6 (.1) .PHI..alpha..alpha..alpha..alpha.. . . 16803.4 (.2)
.OMEGA..alpha..alpha..alpha..alpha..alpha.. . . 15114.6 (.3)
.PHI..alpha..alpha..alpha..alpha.. . . 34967.8 (.4)
.PHI..OMEGA..alpha..alpha..alpha..alpha.. . . 62871.1 (.7)
.OMEGA..OMEGA..alpha..alpha..alpha.. . . 28244.3 (1.0)
.PHI..PHI..PHI..alpha..alpha..alpha.. . . 38765.7 (.9)
.PHI..PHI..OMEGA..alpha..alpha..alpha.. . . 104432.2 (1.3)
.PHI..OMEGA..OMEGA..alpha..alpha..alpha.. . . 93672.7 (2.0)
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.. . . 27960.3 (3.0)
.PHI..PHI..PHI..PHI..alpha..alpha.. . . 24119.9 (1.8)
.PHI..PHI..PHI..OMEGA..alpha..alpha.. . . 86442.5 (2.7)
.PHI..PHI..OMEGA..OMEGA..alpha..alpha.. . . 115915.5 (4.0)
.PHI..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 68853.5 (5.9)
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 15261.1 (8.7)
.PHI..PHI..PHI..PHI..OMEGA..alpha.. . . 7968.1 (3.5)
.PHI..PHI..PHI..PHI..OMEGA..alpha.. . . 35537.2 (5.3)
.PHI..PHI..PHI..OMEGA..OMEGA..alpha.. . . 63117.5 (7.8)
.PHI..PHI..OMEGA..OMEGA..OMEGA..alpha.. . . 55684.4 (11.5)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 24325.9 (16.7)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 4190.6 (24.0)
.PHI..PHI..PHI..PHI..PHI..PHI.. . . 1087.1 (7.0)
.PHI..PHI..PHI..PHI..PHI..OMEGA.. . . 5767.0 (10.3)
.PHI..PHI..PHI..PHI..OMEGA..OMEGA.. . . 12637.2 (15.0)
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.. . . 14581.7 (21.6)
.PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.. . . 9290.2 (30.6)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 3073.9 (42.2)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 408.4 (56.0)
Library size = 3.000E+06 total = 2.7885E+06 % sampled = 4.36
.alpha..alpha..alpha..alpha..alpha..alpha.. . . 10076.4 (.3)
.PHI..alpha..alpha..alpha..alpha..alpha.. . . 50296.9 (.7)
.OMEGA..alpha..alpha..alpha..alpha..alpha.. . . 45190.9 (1.0)
.PHI..PHI..alpha..alpha..alpha..alpha.. . . 104432.2 (1.3)
.PHI..OMEGA..alpha..alpha..alpha..alpha.. . . 187345.5 (2.0)
.OMEGA..OMEGA..alpha..alpha..alpha..alpha.. . . 83880.9 (3.0)
.PHI..PHI..PHI..alpha..alpha..alpha.. . . 115256.6 (2.7)
.PHI..PHI..OMEGA..alpha..alpha..alpha.. . . 309107.9 (4.0)
.PHI..OMEGA..OMEGA..alpha..alpha..alpha.. . . 275413.9 (5.9)
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.. . . 81392.5 (8.7)
.PHI..PHI..PHI..PHI..alpha..alpha.. . . 71074.5 (5.3)
.PHI..PHI..PHI..OMEGA..alpha..alpha.. . . 252470.2 (7.8)
.PHI..PHI..OMEGA..OMEGA..alpha..alpha.. . . 334106.2 (11.5)
.PHI..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 194606.9 (16.7)
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 41905.9 (24.0)
.PHI..PHI..PHI..PHI..PHI..alpha.. . . 23067.8 (10.3)
.PHI..PHI..PHI..PHI..OMEGA..alpha.. . . 101097.3 (15.0)
.PHI..PHI..PHI..OMEGA..OMEGA..alpha.. . . 174981.0 (21.6)
.PHI..PHI..OMEGA..OMEGA..OMEGA..alpha.. . . 148643.7 (30.6)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 61478.9 (42.2)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 9801.0 (56.0)
.PHI..PHI..PHI..PHI..PHI..PHI.. . . 3039.6 (19.5)
.PHI..PHI..PHI..PHI..PHI..OMEGA.. . . 15587.7 (27.7)
.PHI..PHI..PHI..PHI..OMEGA..OMEGA.. . . 32516.8 (38.5)
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.. . . 34975.6 (51.8)
.PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.. . . 20215.5 (66.6)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 5879.9 (80.7)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 667.0 (91.5)
Library size = 1.000E+07 total = 8.1204E+06 % sampled = 12.69
.alpha..alpha..alpha..alpha..alpha..alpha.. . . 33455.9 (1.1)
.PHI..alpha..alpha..alpha..alpha..alpha.. . . 166342.4 (2.2)
.OMEGA..alpha..alpha..alpha..alpha..alpha.. . . 148871.1 (3.3)
.PHI..PHI..alpha..alpha..alpha..alpha.. . . 342685.7 (4.4)
.PHI..OMEGA..alpha..alpha..alpha..alpha.. . . 609987.6 (6.5)
.OMEGA..OMEGA..alpha..alpha..alpha..alpha.. . . 269958.3 (9.6)
.PHI..PHI..PHI..alpha..alpha..alpha.. . . 372371.8 (8.6)
.PHI..PHI..OMEGA..alpha..alpha..alpha.. . . 983416.4 (12.6)
.PHI..OMEGA..OMEGA..alpha..alpha..alpha.. . . 856471.6 (18.4)
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.. . . 244761.5 (26.2)
.PHI..PHI..PHI..PHI..alpha..alpha.. . . 222702.0 (16.5)
.PHI..PHI..PHI..OMEGA..alpha..alpha.. . . 767692.5 (23.7)
.PHI..PHI..OMEGA..OMEGA..alpha..alpha.. . . 972324.6 (33.3)
.PHI..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 531651.3 (45.6)
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 104722.3 (59.9)
.PHI..PHI..PHI..PHI..PHI..alpha.. . . 68111.0 (30.3)
.PHI..PHI..PHI..PHI..OMEGA..alpha.. . . 281976.3 (41.8)
.PHI..PHI..PHI..OMEGA..OMEGA..alpha.. . . 450120.2 (55.6)
.PHI..PHI..OMEGA..OMEGA..OMEGA..alpha.. . . 342072.1 (70.4)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 122302.6 (83.9)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 16364.0 (93.5)
.PHI..PHI..PHI..PHI..PHI..PHI.. . . 8028.0 (51.4)
.PHI..PHI..PHI..PHI..PHI..OMEGA.. . . 37179.9 (66.1)
.PHI..PHI..PHI..PHI..OMEGA..OMEGA.. . . 67719.5 (80.3)
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.. . . 61580.0 (91.2)
.PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.. . . 29586.1 (97.4)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 7259.5 (99.6)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 728.8 (100.0)
Library size = 3.0000E+07 total 1.8633E+07 % sampled = 29.11
.alpha..alpha..alpha..alpha..alpha..alpha.. . . 99247.4 (3.3)
.PHI..alpha..alpha..alpha..alpha..alpha.. . . 487990.0 (6.5)
.OMEGA..alpha..alpha..alpha..alpha..alpha.. . . 431933.3 (9.6)
.PHI..PHI..alpha..alpha..alpha..alpha.. . . 983416.5 (12.6)
.PHI..OMEGA..alpha..alpha..alpha..alpha.. . . 1712943.0 (18.4)
.OMEGA..OMEGA..alpha..alpha..alpha..alpha.. . . 734284.6 (26.2)
.PHI..PHI..PHI..alpha..alpha..alpha.. . . 1023590.0 (23.7)
.PHI..PHI..OMEGA..alpha..alpha..alpha.. . . 2592866.0 (33.3)
.PHI..OMEGA..OMEGA..alpha..alpha..alpha.. . . 2126605.0 (45.6)
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.. . . 558519.0 (59.9)
.PHI..PHI..PHI..PHI..alpha..alpha.. . . 563952.6 (41.8)
.PHI..PHI..PHI..OMEGA..alpha..alpha.. . . 1800481.0 (55.6)
.PHI..PHI..OMEGA..OMEGA..alpha..alpha.. . . 2052433.0 (70.4)
.PHI..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 978420.5 (83.9)
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 163640.3 (93.5)
.PHI..PHI..PHI..PHI..PHI..alpha.. . . 148719.7 (66.1)
.PHI..PHI..PHI..PHI..OMEGA..alpha.. . . 541755.7 (80.3)
.PHI..PHI..PHI..OMEGA..OMEGA..alpha.. . . 738960.1 (91.2)
.PHI..PHI..OMEGA..OMEGA..OMEGA..alpha.. . . 473377.0 (97.4)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 145189.7 (99.6)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 17491.3 (100.0)
.PHI..PHI..PHI..PHI..PHI..PHI.. . . 13829.1 (88.5)
.PHI..PHI..PHI..PHI..PHI..OMEGA.. . . 54058.1 (96.1)
.PHI..PHI..PHI..PHI..OMEGA..OMEGA.. . . 83726.0 (99.2)
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.. . . 67454.5 (99.9)
.PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.. . . 30374.5 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 7290.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 729.0 (100.0)
Library size = 7.6000E+07 total = 3.2125E+07 % sampled = 50.19
.alpha..alpha..alpha..alpha..alpha..alpha.. . . 245057.8 (8.2)
.PHI..alpha..alpha..alpha..alpha..alpha.. . . 1175010.0 (15.7)
.OMEGA..alpha..alpha..alpha..alpha..alpha.. . . 1014733.0 (22.7)
.PHI..PHI..alpha..alpha..alpha..alpha.. . . 2255280.0 (29.0)
.PHI..OMEGA..alpha..alpha..alpha..alpha.. . . 3749112.0 (40.2)
.OMEGA..OMEGA..alpha..alpha..alpha..alpha.. . . 1504128.0 (53.7)
.PHI..PHI..PHI..alpha..alpha..alpha.. . . 2142478.0 (49.6)
.PHI..PHI..OMEGA..alpha..alpha..alpha.. . . 4993247.0 (64.2)
.PHI..OMEGA..OMEGA..alpha..alpha..alpha.. . . 3666785.0 (78.6)
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.. . . 840691.9 (90.1)
.PHI..PHI..PHI..PHI..alpha..alpha.. . . 1007002.0 (74.6)
.PHI..PHI..PHI..OMEGA..alpha..alpha.. . . 2825063.0 (87.2)
.PHI..PHI..OMEGA..OMEGA..alpha..alpha.. . . 2782358.0 (95.4)
.PHI..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 1154956.0 (99.0)
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 174790.0 (99.9)
.PHI..PHI..PHI..PHI..PHI..alpha.. . . 210475.6 (93.5)
.PHI..PHI..PHI..PHI..OMEGA..alpha.. . . 663929.3 (98.4)
.PHI..PHI..PHI..OMEGA..OMEGA..alpha.. . . 808298.6 (99.8)
.PHI..PHI..OMEGA..OMEGA..OMEGA..alpha.. . . 485953.2 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 145799.9 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 17496.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..PHI.. . . 15559.9 (99.6)
.PHI..PHI..PHI..PHI..PHI..OMEGA.. . . 56234.9 (100.0)
.PHI..PHI..PHI..PHI..OMEGA..OMEGA.. . . 84374.6 (100.0)
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.. . . 67500.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.. . . 30375.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 7290.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 729.0 (100.0)
Library size = 1.0000E+08 total = 3.6537E+07 % sampled = 57.09
.alpha..alpha..alpha..alpha..alpha..alpha.. . . 318185.1 (10.7)
.PHI..alpha..alpha..alpha..alpha..alpha.. . . 1506161.0 (20.2)
.OMEGA..alpha..alpha..alpha..alpha..alpha.. . . 1284677.0 (28.7)
.PHI..PHI..alpha..alpha..alpha..alpha.. . . 2821285.0 (36.3)
.PHI..OMEGA..alpha..alpha..alpha..alpha.. . . 4585163.0 (49.1)
.OMEGA..OMEGA..alpha..alpha..alpha..alpha.. . . 1783932.0 (63.7)
.PHI..PHI..PHI..alpha..alpha..alpha.. . . 2566085.0 (59.4)
.PHI..PHI..OMEGA..alpha..alpha..alpha.. . . 5764391.0 (74.1)
.PHI..OMEGA..OMEGA..alpha..alpha..alpha.. . . 4051713.0 (86.8)
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.. . . 888584.3 (95.2)
.PHI..PHI..PHI..PHI..alpha..alpha.. . . 1127473.0 (83.5)
.PHI..PHI..PHI..OMEGA..alpha..alpha.. . . 3023170.0 (93.3)
.PHI..PHI..OMEGA..OMEGA..alpha..alpha.. . . 2865517.0 (98.3)
.PHI..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 1163743.0 (99.8)
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 174941.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..alpha.. . . 2188866.6 (97.3)
.PHI..PHI..PHI..PHI..OMEGA..alpha.. . . 671976.9 (99.6)
.PHI..PHI..PHI..OMEGA..OMEGA..alpha.. . . 809757.3 (100.0)
.PHI..PHI..OMEGA..OMEGA..OMEGA..alpha.. . . 485997.5 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 145800.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 17496.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..PHI.. . . 15613.5 (99.9)
.PHI..PHI..PHI..PHI..PHI..OMEGA.. . . 56248.9 (100.0)
.PHI..PHI..PHI..PHI..OMEGA..OMEGA.. . . 84375.0 (100.0)
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.. . . 67500.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.. . . 30375.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 7290.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 729.0 (100.0)
Library size = 3.0000E+08 total = 5.2634E+07 % sampled = 82.24
.alpha..alpha..alpha..alpha..alpha..alpha.. . . 856451.3 (28.7)
.PHI..alpha..alpha..alpha..alpha..alpha.. . . 3668130.0 (49.1)
.OMEGA..alpha..alpha..alpha..alpha..alpha.. . . 2854291.0 (63.7)
.PHI..PHI..alpha..alpha..alpha..alpha.. . . 5764391.0 (74.1)
.PHI..OMEGA..alpha..alpha..alpha..alpha.. . . 8103426.0 (86.8)
.OMEGA..OMEGA..alpha..alpha..alpha..alpha.. . . 2665753.0 (95.2)
.PHI..PHI..PHI..alpha..alpha..alpha.. . . 4030893.0 (93.3)
.PHI..PHI..OMEGA..alpha..alpha..alpha.. . . 7641378.0 (98.3)
.PHI..OMEGA..OMEGA..alpha..alpha..alpha.. . . 4654972.0 (99.8)
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.. . . 933018.6 (100.0)
.PHI..PHI..PHI..PHI..alpha..alpha.. . . 1343954.0 (99.6)
.PHI..PHI..PHI..OMEGA..alpha..alpha.. . . 3239029.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..alpha..alpha.. . . 2915985.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 1166400.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 174960.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..alpha.. . . 224995.5 (100.0)
.PHI..PHI..PHI..PHI..OMEGA..alpha.. . . 674999.9 (100.0)
.PHI..PHI..PHI..OMEGA..OMEGA..alpha.. . . 810000.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..OMEGA..alpha.. . . 486000.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 145800.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 17496.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..PHI.. . . 15625.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..OMEGA.. . . 56250.0 (100.0)
.PHI..PHI..PHI..PHI..OMEGA..OMEGA.. . . 84375.0 (100.0)
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.. . . 67500.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.. . . 30375.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 7290.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 729.0 (100.0)
Library size = 1.0000E+09 total = 6.1999E+07 % sampled = 96.87
.alpha..alpha..alpha..alpha..alpha..alpha.. . . 2018278.0 (67.6)
.PHI..alpha..alpha..alpha..alpha..alpha.. . . 6680917.0 (89.5)
.OMEGA..alpha..alpha..alpha..alpha..alpha.. . . 4326519.0 (96.6)
.PHI..PHI..alpha..alpha..alpha..alpha.. . . 7690221.0 (98.9)
.PHI..OMEGA..alpha..alpha..alpha..alpha.. . . 9320389.0 (99.9)
.OMEGA..OMEGA..alpha..alpha..alpha..alpha.. . . 2799250.0 (100.0)
.PHI..PHI..PHI..alpha..alpha..alpha.. . . 4319475.0 (100.0)
.PHI..PHI..OMEGA..alpha..alpha..alpha.. . . 7775990.0 (100.0)
.PHI..OMEGA..OMEGA..alpha..alpha..alpha.. . . 4665600.0 (100.0)
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.. . . 933120.0 (100.0)
.PHI..PHI..PHI..PHI..alpha..alpha.. . . 1350000.0 (100.0)
.PHI..PHI..PHI..OMEGA..alpha..alpha.. . . 3240000.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..alpha..alpha.. . . 2916000.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 1166400.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 174960.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..alpha.. . . 225000.0 (100.0)
.PHI..PHI..PHI..PHI..OMEGA..alpha.. . . 675000.0 (100.0)
.PHI..PHI..PHI..OMEGA..OMEGA..alpha.. . . 810000.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..OMEGA..alpha.. . . 486000.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 145800.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 17496.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..PHI.. . . 15625.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..OMEGA.. . . 56250.0 (100.0)
.PHI..PHI..PHI..PHI..OMEGA..OMEGA.. . . 84375.0 (100.0)
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.. . . 67500.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.. . . 30375.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 7290.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 729.0 (100.0)
Library size = 3.0000E+09 total 6.3890E+07 % sampled = 99.83
.alpha..alpha..alpha..alpha..alpha..alpha.. . . 2884346.0 (96.6)
.PHI..alpha..alpha..alpha..alpha..alpha.. . . 7456311.0 (99.9)
.OMEGA..alpha..alpha..alpha..alpha..alpha.. . . 4478800.0 (100.0)
.PHI..PHI..alpha..alpha..alpha..alpha.. . . 7775990.0 (100.0)
.PHI..OMEGA..alpha..alpha..alpha..alpha.. . . 9331200.0 (100.0)
.OMEGA..OMEGA..alpha..alpha..alpha..alpha.. . . 2799360.0 (100.0)
.PHI..PHI..PHI..alpha..alpha..alpha.. . . 4320000.0 (100.0)
.PHI..PHI..OMEGA..alpha..alpha..alpha.. . . 7776000.0 (100.0)
.PHI..OMEGA..OMEGA..alpha..alpha..alpha.. . . 4665600.0 (100.0)
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.. . . 933120.0 (100.0)
.PHI..PHI..PHI..PHI..alpha..alpha.. . . 1350000.0 (100.0)
.PHI..PHI..PHI..OMEGA..alpha..alpha.. . . 3240000.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..alpha..alpha.. . . 2916000.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 1166400.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.. . . 174960.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..alpha.. . . 225000.0 (100.0)
.PHI..PHI..PHI..PHI..OMEGA..alpha.. . . 675000.0 (100.0)
.PHI..PHI..PHI..OMEGA..OMEGA..alpha.. . . 810000.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..OMEGA..alpha.. . . 486000.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 145800.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.. . . 17496.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..PHI.. . . 15625.0 (100.0)
.PHI..PHI..PHI..PHI..PHI..OMEGA.. . . 56250.0 (100.0)
.PHI..PHI..PHI..PHI..OMEGA..OMEGA.. . . 84375.0 (100.0)
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.. . . 67500.0 (100.0)
.PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.. . . 30375.0 (100.0)
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 7290.0 (100.0)
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.. . . 729.0 (100.0) D.
Formulae for tabulated quantities. Lsize is the number of
independent transformants. 31**6 is 31 to sixth power; 6*3 means 6
times 3. A = Lsize/(31**6) .alpha. can be one of [WMFYCIKDENHQ.]
.PHI. can be one of [PTAVG] .OMEGA. can be one of [SLR] F0 = (12)
**6 F1 = (12) **5 F2 = (12) **4 F3 = (12) **3 F4 = (12) **2 F5 =
(12) F6 = 1 .alpha..alpha..alpha..alpha..alpha..alpha. = F0 *
(1-exp(-A)) .PHI..alpha..alpha..alpha..alpha..alpha. = 6 * 5 * F1 *
(1-exp(-2*A)) .OMEGA..alpha..alpha..alpha..alpha..alpha. = 6 * 3 *
F1 * (1-exp(-3*A)) .PHI..PHI..alpha..alpha..alpha..alpha. = (15) *
5**2 * F2 * (1-exp(-4*A)) .PHI..OMEGA..alpha..alpha..alpha..alpha.
= (6*5)*5*3 *F2 * (1-exp(-6*A))
.OMEGA..OMEGA..alpha..alpha..alpha..- alpha. = (15) * 3**2 * F2 *
(1-exp(-9*A)) .PHI..PHI..PHI..alpha..al- pha..alpha. = (20)*(5**3)
* F3 * (1-exp(-8*A)) .PHI..PHI..OMEGA..alpha..alpha..alpha. =
(60)*(5*5*3)*F3* (1-exp(-12*A))
.PHI..OMEGA..OMEGA..alpha..alpha..alpha. = (60)*(5*3*3)*F3*(1-exp(-
-18*A)) .OMEGA..OMEGA..OMEGA..alpha..alpha..alpha. =
(20)*(3)**3*F3*(1-exp(-27*A)) .PHI..PHI..PHI..PHI..alpha..alpha. =
(15)*(5)**4*F4*(1-exp(-16*A)) .PHI..PHI..PHI..OMEGA..alpha..alpha.
= (60)*(5)**3*3*F4*(1-exp(-24*A)) .PHI..PHI..OMEGA..OMEGA..alpha..-
alpha. = (90)*(5*5*3*3)*F4*(1-exp(-36*A))
.PHI..OMEGA..OMEGA..OMEGA- ..alpha..alpha. =
(60)*(5*3*3*3)*F4*(1-exp(-54*A))
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha. = (15)*(3)**4 * F4
*(1-exp(-81*A)) .PHI..PHI..PHI..PHI..PHI..alpha. = (6)*(5)**5 * F5
* (1-exp(-32*A)) .PHI..PHI..PHI..PHI..OMEGA..alpha. =
30*5*5*5*5*3*F5*(1-exp(-48*A)) .PHI..PHI..PHI..OMEGA..OMEGA..alpha-
. = 60*5*5*5*3*3*F5*(1-exp(-72*A))
.PHI..PHI..OMEGA..OMEGA..OMEGA..- alpha. =
60*5*5*3*3*3*F5*(1-exp(-108*A)) .PHI..OMEGA..OMEGA..OMEGA.-
.OMEGA..alpha. = 30*5*3*3*3*3*F5*(1-exp(-162*A))
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha. =
6*3*3*3*3*3*F5*(1-exp(-243*A- )) .PHI..PHI..PHI..PHI..PHI..PHI. =
5**6 * (1-exp(-64*A)) .PHI..PHI..PHI..PHI..PHI..OMEGA. =
6*3*5**5*(1-exp(-96*A)) .PHI..PHI..PHI..PHI..OMEGA..OMEGA. =
15*3*3*5**4*(1-exp(-144*A)) .PHI..PHI..PHI..OMEGA..OMEGA..OMEGA. =
20*3**3*5**3*(1-exp(-216*A)) .PHI..PHI..OMEGA..OMEGA..OMEGA..OMEGA.
= 15*3**4*5**2*(1-exp(-324*A))
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA. = 6*3**5*5*(1-exp(-486*A))
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA. = 3**6*(1-exp(-729*A))
total = .alpha..alpha..alpha..alpha..alpha..alpha.+
.PHI..alpha..alpha..alpha..alpha..alpha.+
.OMEGA..alpha..alpha..alpha..al- pha..alpha.+
.PHI..PHI..alpha..alpha..alpha..alpha.+
.PHI..OMEGA..alpha..alpha..alpha..alpha.+
.OMEGA..OMEGA..alpha..alpha..al- pha..alpha.+
.PHI..PHI..PHI..alpha..alpha..alpha.+ .PHI..PHI..OMEGA..alpha-
..alpha..alpha.+ .PHI..OMEGA..OMEGA..alpha..alpha..alpha.+
.OMEGA..OMEGA..OMEGA..alpha..alpha..alpha.+
.PHI..PHI..PHI..PHI..alpha..a- lpha.+
.PHI..PHI..PHI..OMEGA..alpha..alpha.+
.PHI..PHI..OMEGA..OMEGA..alpha..alpha.+
.PHI..OMEGA..OMEGA..OMEGA..alpha.- .alpha.+
.OMEGA..OMEGA..OMEGA..OMEGA..alpha..alpha.+
.PHI..PHI..PHI..PHI..PHI..alpha.+ .PHI..PHI..PHI..PHI..OMEGA..alp-
ha.+ .PHI..PHI..PHI..OMEGA..OMEGA..alpha.+
.PHI..PHI..OMEGA..OMEGA..OMEGA.- .alpha.+
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..alpha.+
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..alpha.+
.PHI..PHI..PHI..PHI..PHI..PHI- .+ .PHI..PHI..PHI..PHI..PHI..OMEGA.+
.PHI..PHI..PHI..PHI..OMEGA..OMEGA.+
.PHI..PHI..PHI..OMEGA..OMEGA..OMEGA.+
.PHI..PHI..OMEGA..OMEGA..OMEG- A..OMEGA.+
.PHI..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.+
.OMEGA..OMEGA..OMEGA..OMEGA..OMEGA..OMEGA.
[1479]
98TABLE 131 Sampling of a Library Encoded by (NNT).sup.4(NNG).sup.2
X can be F, S, Y, C, L, P, H, R, I, T, N, V, A, D, G .GAMMA.can be
L.sup.2, R.sup.2, S, W, P, Q, M, T, K, V, A, E, G Library comprises
8.55 .multidot. 10.sup.6 amino-acid sequences; 1.47 .multidot.
10.sup.7 DNA sequences. Total number of possible aa sequences =
8,555,625 x LVPTARGFYCHIND S S .THETA. VPTAGWQMKES .OMEGA. LR The
first, second, fifth, and sixth positions can hold x or S; the
third and fourth position can hold .THETA. or .OMEGA.. I have
lumped sequences by the number of xs, Ss, .THETA.s, and .OMEGA.s.
For example xx.THETA..OMEGA.SS stands for: [xx.THETA..OMEGA.SS,
xS.THETA..OMEGA.xS, xS.THETA..OMEGA.Sx, SS.THETA..OMEGA.xx,
Sx.THETA..OMEGA.xS, Sx.THETA..OMEGA.Sx, xx.OMEGA..THETA.SS,
xS.OMEGA..THETA.xS, xS.OMEGA..THETA.Sx, SS.OMEGA..THETA.xx,
Sx.OMEGA..THETA.xS, Sx.OMEGA..THETA.Sx] The following table shows
the likelihood that any particular DNA sequence will fall into one
of the defined classes. Library size = 1.0 Sampling = .00001% total
1.0000E+00 % sampled 1.1688E-07 xx.THETA..THETA.xx 3.1524E-01
xx.THETA..OMEGA.xx 2.2926E-01 xx.OMEGA..OMEGA.xx 4.1684E-02
xx.THETA..THETA.xS 1.8013E-01 xx.THETA..OMEGA.xS 1.3101E-01
xx.OMEGA..OMEGA.xS 2.3819E-02 xx.THETA..OMEGA.SS 3.8600E-02
xx.THETA..OMEGA.SS 2.8073E-02 xx.OMEGA..OMEGA.SS 5.1042E-03
xS.THETA..THETA.SS 3.6762E-03 xS.THETA..OMEGA.SS 2.6736E-03
xS.OMEGA..OMEGA.SS 4.8611E-04 SS.THETA..THETA.SS 1.3129E-04
SS.THETA..OMEGA.SS 9.5486E-05 SS.OMEGA..OMEGA.SS 1.7361E-05 The
following sections show how many sequences of each class are
expected for libraries of different sizes. Type Number % Type
Number % Library size = 1.0000E+05 total 9.9137E+04 fraction
sampled = 1.1587E-02 xx.THETA..THETA.xx 31416.9 (.7)
xx.THETA..OMEGA.xx 22771.4 (1.3) xx.OMEGA..OMEGA.xx 4112.4 (2.7)
xx.THETA..THETA.xS 17891.8 (1.3) xx.THETA..OMEGA.xS 12924.6 (2.7)
xx.OMEGA..OMEGA.xS 2318.5 (5.3) xx.THETA..THETA.SS 3808.1 (2.7)
xx.THETA..OMEGA.SS 2732.5 (5.3) xx.OMEGA..OMEGA.SS 483.7 (10.3)
xS.THETA..THETA.SS 357.8 (5.3) xS.THETA..OMEGA.SS 253.4 (10.3)
xS.OMEGA..OMEGA.SS 43.7 (19.5) SS.THETA..THETA.SS 12.4 (10.3)
SS.THETA..OMEGA.SS 8.6 (19.5) SS.OMEGA..OMEGA.SS 1.4 (35.2) Library
size = 1.0000E+06 total 9.2064E+05 fraction sampled = 1.0761E-01
xx.THETA..THETA.xx 304783.9 (6.6) xx.THETA..OMEGA.xx 214394.0
(12.7) xx.OMEGA..OMEGA.xx 36508.6 (23.8) xx.THETA..THETA.xS
168452.5 (12.7) xx.THETA..OMEGA.xS 114741.4 (23.8)
xx.OMEGA..OMEGA.xS 18383.8 (41.9) xx.THETA..THETA.SS 33807.7 (23.8)
xx.THETA..OMEGA.SS 21666.6 (41.9) xx.OMEGA..OMEGA.SS 3114.6 (66.2)
xS.THETA..THETA.SS 2837.3 (41.9) xS.THETA..OMEGA.SS 1631.5 (66.2)
xS.OMEGA..OMEGA.SS 198.4 (88.6) SS.THETA..THETA.SS 80.1 (66.2)
SS.THETA..OMEGA.SS 39.0 (88.6) SS.OMEGA..OMEGA.SS 3.9 (98.7)
Library size = 3.0000E+06 total 2.3880E+06 fraction sampled =
2.7912E-01 xx.THETA..THETA.xx 855709.5 (18.4) xx.THETA..OMEGA.xx
565051.6 (33.4) xx.OMEGA..OMEGA.xx 85564.7 (55.7)
xx.THETA..THETA.xS 443969.1 (33.4) xx.THETA..OMEGA.xS 268917.8
(55.7) xx.OMEGA..OMEGA.xS 35281.3 (80.4) xx.THETA..THETA.SS 79234.7
(55.7) xx.THETA..OMEGA.SS 41581.5 (80.4) xx.OMEGA..OMEGA.SS 4522.6
(96.1) xS.THETA..THETA.SS 5445.2 (80.4) xS.THETA..OMEGA.SS 2369.0
(96.1) xS.OMEGA..OMEGA.SS 223.7 (99.9) SS.THETA..THETA.SS 116.3
(96.1) SS.THETA..OMEGA.SS 43.9 (99.9) SS.OMEGA..OMEGA.SS 4.0
(100.0) Library size = 8.5556E+06 total 4.9303E+06 fraction sampled
= 5.7626E-01 xx.THETA..THETA.xx 2046301.0 (44.0) xx.THETA..OMEGA.xx
1160645.0 (68.7) xx.OMEGA..OMEGA.xx 138575.9 (90.2)
xx.THETA..THETA.xS 911935.6 (68.7) xx.THETA..OMEGA.xS 435524.3
(90.2) xx.OMEGA..OMEGA.xS 43480.7 (99.0) xx.THETA..THETA.SS
128324.1 (90.2) xx.THETA..OMEGA.SS 51245.1 (99.0)
xx.OMEGA..OMEGA.SS 4703.6 (100.0) xS.THETA..THETA.SS 6710.7 (99.0)
xS.THETA..OMEGA.SS 2463.8 (100.0) xS.OMEGA..OMEGA.SS 224.0 (100.0)
SS.THETA..THETA.SS 121.0 (100.0) SS.THETA..OMEGA.SS 44.0 (100.0)
SS.OMEGA..OMEGA.SS 4.0 (100.0) Library size = 1.0000E+07 total
5.3667E+06 fraction sampled = 6.2727E-01 xx.THETA..THETA.xx
2289093.0 (49.2) xx.THETA..OMEGA.xx 1254877.0 (74.2)
xx.OMEGA..OMEGA.xx 143467.0 (93.4) xx.THETA..THETA.xS 985974.9
(74.2) xx.THETA..OMEGA.xS 450896.3 (93.4) xx.OMEGA..OMEGA.xS
43710.7 (99.6) xx.THETA..THETA.SS 132853.4 (93.4)
xx.THETA..OMEGA.SS 51516.1 (99.6) xx.OMEGA..OMEGA.SS 4703.9 (100.0)
xS.THETA..THETA.SS 6746.2 (99.6) xS.THETA..OMEGA.SS 2464.0 (100.0)
xS.OMEGA..OMEGA.SS 224.0 (100.0) SS.THETA..THETA.SS 121.0 (100.0)
SS.THETA..OMEGA.SS 44.0 (100.0) SS.OMEGA..OMEGA.SS 4.0 (100.0)
Library size = 3.0000E+07 total 7.8961E+06 fraction sampled =
9.2291E-01 xx.THETA..THETA.xx 4040589.0 (86.9) xx.THETA..OMEGA.xx
1661409.0 (98.3) xx.OMEGA..OMEGA.xx 153619.1 (100.0)
xx.THETA..THETA.xS 1305393.0 (98.3) xx.THETA..OMEGA.xS 482802.9
(100.0) xx.OMEGA..OMEGA.xS 43904.0 (100.0) xx.THETA..THETA.SS
142254.4 (100.0) xx.THETA..OMEGA.SS 51744.0 (100.0)
xx.OMEGA..OMEGA.SS 4704.0 (100.0) xS.THETA..THETA.SS 6776.0 (100.0)
xS.THETA..OMEGA.SS 2464.0 (100.0) xS.OMEGA..OMEGA.SS 224.0 (100.0)
SS.THETA..THETA.SS 121.0 (100.0) SS.THETA..OMEGA.SS 44.0 (100.0)
SS.OMEGA..OMEGA.SS 4.0 (100.0) Library size = 5.0000E+07 total
8.3956E+06 fraction = 9.8130E-01 xx.THETA..THETA.xx 4491779.0
(96.6) xx.THETA..OMEGA.xx 1688387.0 (99.9) xx.OMEGA..OMEGA.xx
153663.8 (100.0) xx.THETA..THETA.xS 1326590.0 (99.9)
xx.THETA..OMEGA.xS 482943.4 (100.0) xx.OMEGA..OMEGA.xS 43904.0
(100.0) xx.THETA..OMEGA.SS 142295.8 (100.0) xx.THETA..OMEGA.SS
51744.0 (100.0) xx.OMEGA..OMEGA.SS 4704.0 (100.0)
xS.THETA..THETA.SS 6776.0 (100.0) xS.THETA..OMEGA.SS 2464.0 (100.0)
xS.OMEGA..OMEGA.SS 224.0 (100.0) SS.THETA..THETA.SS 121.0 (100.0)
SS.THETA..OMEGA.SS 44.0 (100.0) SS.OMEGA..OMEGA.SS 4.0 (100.0)
Library size = 1.0000E+08 total 8.5503E+06 fraction = 9.9938E-01
xx.THETA..THETA.xx 4643063.0 (99.9) xx.THETA..OMEGA.xx 1690302.0
(100.0) xx.OMEGA..OMEGA.xx 153664.0 (100.0) xx.THETA..THETA.xS
1328094.0 (100.0) xx.THETA..OMEGA.xS 482944.0 (100.0)
xx.OMEGA..OMEGA.xS 43904.0 (100.0) xx.THETA..THETA.SS 142296.0
(100.0) xx.THETA..OMEGA.SS 51744.0 (100.0) xx.OMEGA..OMEGA.SS
4704.0 (100.0) xS.THETA..THETA.SS 6776.0 (100.0) xS.THETA..OMEGA.SS
2464.0 (100.0) xS.OMEGA..OMEGA.SS 224.0 (100.0) SS.THETA..THETA.SS
121.0 (100.0) SS.THETA..OMEGA.SS 44.0 (100.0) SS.OMEGA..OMEGA.SS
4.0 (100.0)
[1480]
99TABLE 132 Relative efficiencies of various simple variegation
codons Number of codons 5 6 7 #DNA/#AA #DNA/#AA #DNA/#AA [#DNA]
[#DNA] [#DNA] vgCodon (#AA) (#AA) (#AA) NNK 8.95 13.86 21.49
assuming [2.86 .multidot. 10.sup.7] [8.87 .multidot. 10.sup.8]
.sup. [2.75 .multidot. 10.sup.10] stops vanish (3.2 .multidot.
10.sup.6) (6.4 .multidot. 10.sup.7) (1.28 .multidot. 10.sup.9) NNT
1.38 1.47 1.57 [1.05 .multidot. 106].sup. [1.68 .multidot.
10.sup.7] [2.68 .multidot. 10.sup.8] (7.59 .multidot. 10.sup.5)
(1.14 .multidot. 10.sup.7) (1.71 .multidot. 10.sup.8) NNG 2.04 2.36
2.72 assuming [7.59 .multidot. 10.sup.5] [1.14 .multidot. 10.sup.6]
[1.71 .multidot. 10.sup.8] stops vanish (3.7 .multidot. 10.sup.5)
(4.83 .multidot. 10.sup.6) (6.27 .multidot. 10.sup.7)
[1481]
100TABLE 140 Affect of anti BPTI IgG on phase titer. Phage
+Anti-BPTI Strain Input +Anti-BPTI +Protein A(a) Eluted Phage
M13MP18 100 (b) 98 92 7 .multidot. 10.sup.-4 BPTI.3 100 26 21 6
M13MB48 (c) 100 90 36 0.8 M13MB48 (d) 100 60 40 2.6 (a)Protein
A-agarose beads. (b) Percentage of input phage measured as plaque
forming units (c) Batch number 3 (d) Batch number 4
[1482]
101TABLE 141 Affect of anti-BPTI or protein A on phage titer.
+Anti- No +Anti- +Protein A BPTI Strain Input Addition BPTI (a)
+Protein A M13MP18 100 (b) 107 105 72 65 M13MB48 100 92 7.10.sup.-3
58 <10.sup.-4 (b) (a) Protein A-agarose beads (b) Percentage of
input phage measured as plaque forming units (c) Batch number 5
[1483]
102TABLE 142 Affect of anti-BPTI and non-immnune serum on phage
titer +Anti- BPTI +NRS +Anti- +NRS +Protein A +Protein Strain Input
BPTI (a) (b) A M13MP18 100 (c) 65 104 71 88 M13MB48 (d) 100 30 125
13 121 M13MB48 (e) 100 2 105 0.7 110 (a) Purified IgG from normal
rabbit serum. (b) Protein A-agarose beads. (c) Percentage of input
phage measured as plaque forming units (d) Batch number 4 (e) Batch
number 5
[1484]
103TABLE 143 Loss in titer of display phage with anhydrotrypsin.
Anhydrotrypsin Streptavidin Beads Beads Post Post Strain Start
Incubation Start Incubation M13MP18 100 (a) 121 ND ND M13MB48 100
58 100 98 5AA Pool 100 44 100 93 (a) Plaque forming units expressed
as a percentage of input.
[1485]
104TABLE 144 Binding of Display Phage to Anhydrotrypsin. Relative
to Strain Eluted Phage (a) M13MP18 Experiment 1. M13MP18 .sup. 0.2
(a) 1.0 BPTI-IIIMK 7.9 39.5 M13MB48 11.2 56.0 Experiment 2. M13mp18
0.3 1.0 BPTI-IIIMK 12.0 40.0 M13MB56 17.0 56.7 (a) Plaque forming
units acid eluted from beads, expressed as a percentage of the
input.
[1486]
105TABLE 145 Binding of Display Phage to Anhydrotrypsin or Trypsin.
Anhydrotrypsin Beads Eluted Trypsin Beads Phage Relative Eluted
Relative Strain (a) Binding (b) Phage Binding M13MP18 0.1 1 2.3
.times. 10.sup.-4 1.0 BPTI-IIIMK 9.1 91 1.17 5 .times. 10.sup.3
M13.3X7 25.0 250 1.4 6 .times. 10.sup.3 M13.3X11 9.2 92 0.27 1.2
.times. 10.sup.3 (a) Plaque forming units eluted from beads,
expressed as a percentage of the input. (b) Relative to the
non-display phage, M13MP18.
[1487]
106TABLE 146 Binding of Display Phage to Trypsin or Human
Neutrophil Elastase. Trypsin Beads HNE Beads Eluted Phage Relative
Eluted Relative Strain (a) Binding(b) Phage Binding M13MP18 5
.times. 10.sup.-4 1 3 .times. 10.sup.-4 1.0 BPTI-IIIMK 1.0 2000 5
.times. 10.sup.-3 16.7 M13MB48 0.13 260 9 .times. 10.sup.-3 30.0
M13.3X7 1.15 2300 1 .times. 10.sup.-3 3.3 M13.3X11 0.8 1600 2
.times. 10.sup.-3 6.7 BPTI3.CL 1 .times. 10.sup.-3 2 4.1 1.4
.times. 10.sup.4 (c) (a) Plague forming units acid eluted from the
beads, expressed as a percentage of input. (b)Relative to the
non-display phage, M13MP18. (c) BPTI-IIIMK (K15L MGNG)
[1488]
107TABLE 155 Distance in .ANG. between alpha carbons in
octapeptides: 1 2 3 4 5 6 7 8 Extended Strand: angle of
C.sub..alpha.1-C.sub..alpha.2-C.sub..alpha.3 = 138.degree. 1 -- 2
3.8 -- 3 7.1 3.8 -- 4 10.7 7.1 3.8 -- 5 14.2 10.7 7.1 3.8 -- 6 17.7
14.1 10.7 7.1 3.8 -- 7 21.2 17.7 14.1 10.6 7.0 3.8 -- 8 24.6 20.9
17.5 13.9 10.6 7.0 3.8 -- Reverse turn between residues 4 and 5. 1
-- 2 3.8 -- 3 7.1 3.8 -- 4 10.6 7.0 3.8 -- 5 11.6 8.0 6.1 3.8 -- 6
9.0 5.8 5.5 5.6 3.8 -- 7 6.2 4.1 6.3 8.0 7.0 3.8 -- 8 5.8 6.0 9.1
11.6 10.7 7.2 3.8 -- Alpha helix: angle of
C.sub..alpha.1-C.sub..alpha.2-C.sub..alpha.3 = 93.degree. 1 -- 2
3.8 -- 3 5.5 3.8 -- 4 5.1 5.4 3.8 -- 5 6.6 5.3 5.5 3.8 -- 6 9.3 7.0
5.6 5.5 3.8 -- 7 10.4 9.3 6.9 5.4 5.5 3.8 -- 8 11.3 10.7 9.5 6.8
5.6 5.6 3.8 --
[1489]
108TABLE 156 Distances between alpha carbons in closed
mini-proteins of the form disulfide cyclo(CXXXXC) 1 2 3 4 5 6
Minimum distance 1 -- 2 3.8 -- 3 5.9 3.8 -- 4 5.6 6.0 3.8 -- 5 4.7
5.9 6.0 3.8 -- 6 4.8 5.3 5.1 5.2 3.8 -- Average distance 1 -- 2 3.8
-- 3 6.3 3.8 -- 4 7.5 6.4 3.8 -- 5 7.1 7.5 6.3 3.8 -- 6 5.6 7.5 7.7
6.4 3.8 -- Maximum distance 1 -- 2 3.8 -- 3 6.7 3.8 -- 4 9.0 6.9
3.8 -- 5 8.7 8.8 6.8 3.8 -- 6 6.6 9.2 9.1 6.8 3.8 --
[1490]
109TABLE 160 pH Profile of BPTI-III MK phage and Shad 1 phage
binding to Cat G beads. pH Total pfu in Fraction Percentage of
Input BPTI-IIIMK 7 3.7 .times. 10.sup.5 3.7 .times. 10.sup.-2 6 3.1
.times. 10.sup.5 3.1 .times. 10.sup.-2 5 1.4 .times. 10.sup.5 1.4
.times. 10.sup.-2 4.5 3.1 .times. 10.sup.4 3.1 .times. 10.sup.-3 4
7.1 .times. 10.sup.3 7.1 .times. 10.sup.-4 3.5 2.6 .times. 10.sup.3
2.6 .times. 10.sup.-4 3 2.5 .times. 10.sup.3 2.5 .times. 10.sup.-4
2.5 8.8 .times. 10.sup.2 8.8 .times. 10.sup.-5 2 7.6 .times.
10.sup.2 7.6 .times. 10.sup.-5 (total input = 1 .times. 10.sup.9
phage) Shad 1 7 2.5 .times. 10.sup.5 1.1 .times. 10.sup.-2 6 6.3
.times. 10.sup.4 2.7 .times. 10.sup.-3 5 7.4 .times. 10.sup.4 3.1
.times. 10.sup.-3 4.5 7.1 .times. 10.sup.4 3.0 .times. 10.sup.-3 4
4.1 .times. 10.sup.4 1.7 .times. 10.sup.-3 3.5 3.3 .times. 10.sup.4
1.4 .times. 10.sup.-3 3 2.5 .times. 10.sup.3 1.1 .times. 10.sup.-4
2.5 1.4 .times. 10.sup.4 5.7 .times. 10.sup.-4 2 5.2 .times.
10.sup.3 2.2 .times. 10.sup.-4 (total input = 2.35 .times. 10.sup.8
phage)
[1491]
110TABLE 201 Elution of Bound Fusion Phage from Immobilized Active
Trypsin Total Plaque- Forming Units Percent of Type of Recovered in
Input Phage Phage Buffer Elution Buffer Recovered Ratio BPTI-III MK
CBS 8.80 .multidot. 10.sup.7 4.7 .multidot. 10.sup.-1 1675 MK CBS
1.35 .multidot. 10.sup.6 2.8 .multidot. 10.sup.-4 BPTI-III MK TBS
1.32 .multidot. 10.sup.8 7.2 .multidot. 10.sup.-1 2103 MK TBS 1.48
.multidot. 10.sup.6 -3.4 .multidot. 10.sup.-4 The total input for
BPTI-III MK phage was 1.85 .multidot. 10.sup.10 plaque-forming
units while the input for MK phage was 4.65 .multidot. 10.sup.11
plaque-forming units.
[1492]
111TABLE 202 Elution of BPTI-III MK and BPTI(K15L)-III MA Phage
from Immobilized Trypsin and HNE Total Plaque- Forming Units
Percentage of Type of Immobilized in Elution Input Phage Phage
Protease Fraction Recovered BPTI-III Trypsin 2.1 .multidot.
10.sup.7 4.1 .multidot. 10.sup.-1 MK BPTI-III HNE 2.6 .multidot.
10.sup.5 5 .multidot. 10.sup.-3 MK BPTI (K15L)- Trypsin 5.2
.multidot. 10.sup.4 5 .multidot. 10.sup.-3 III MA BPTI (K15L)- HNE
1.0 .multidot. 10.sup.6 1.0 .multidot. 10.sup.-1 III MA The total
input of BPTI-III MK phage was 5.1 .multidot. 10.sup.9 pfu and the
input of BPTI (K15L)-III MA phage was 9.6 .multidot. 10.sup.8
pfu.
[1493]
112TABLE 203 Effect of pH on the Disociation of Bound BPTI-III MK
and BPTI (K15L)-III MA Phage from Immobilized HNE BPTI-III MK BPTI
(K15L)-III MA Total Plaque- % Total Plaque- % Forming Units of
Input Forming Units of Input pH in Fraction Phage in Fraction Phage
7.0 5.0 .multidot. 10.sup.4 2 .multidot. 10.sup.-3 1.7 .multidot.
10.sup.5 3.2 .multidot. 10.sup.-2 6.0 3.8 .multidot. 10.sup.4 2
.multidot. 10.sup.-3 4.5 .multidot. 10.sup.5 8.6 .multidot.
10.sup.-2 5.0 3.5 .multidot. 10.sup.4 1 .multidot. 10.sup.-3 2.1
.multidot. 10.sup.6 4.0 .multidot. 10.sup.-1 4.0 3.0 .multidot.
10.sup.4 1 .multidot. 10.sup.-3 4.3 .multidot. 10.sup.6 8.2
.multidot. 10.sup.-1 3.0 1.4 .multidot. 10.sup.4 1 .multidot.
10.sup.-3 1.1 .multidot. 10.sup.6 2.1 .multidot. 10.sup.-1 2.2 2.9
.multidot. 10.sup.4 1 .multidot. 10.sup.-3 5.9 .multidot. 10.sup.4
1.1 .multidot. 10.sup.-2 Percentage of Percentage of Input Phage =
8.0 .multidot. 10.sup.-3 Input Phage = 1.56 Recovered Recovered The
total input of BPTI-III MK phage was 0.030 ml .times. (8.6
.multidot. 10.sup.10 pfu/ml) = 2.6 .multidot. 10.sup.9. The total
input of BPTI (K15L)-III MA phage was 0.030 ml .times. (1.7
.multidot. 10.sup.10 pfu/ml) = 5.2 .multidot. 10.sup.8. Given that
the infectivity of BPTI (K15L)-III MA phage is 5 fold lower than
that of BPTI-III MK phage, the phage inputs utilized above ensure
that an equivalent number of phage particles are added to the
immobilized HNE.
[1494]
113TABLE 204 Effect of Mutation of Residues 39 to 42 of BPTI on the
ability of BPTI (K15L)-III MA to Bind to Immobilized HNE BPTI
(K15L)-III MA BPTI (K15L, MGNG)-III MA Total Plaque- % Total
Plaque- % pH Forming Units Input Forming Units Input 7.0 3.0
.multidot. 10.sup.5 8.2 .multidot. 10.sup.-2 4.5 .multidot.
10.sup.5 1.63 .multidot. 10.sup.-1 6.0 3.6 .multidot. 10.sup.5 1.00
.multidot. 10.sup.-1 6.3 .multidot. 10.sup.5 2.27 .multidot.
10.sup.-1 5.5 5.3 .multidot. 10.sup.5 1.46 .multidot. 10.sup.-1 7.3
.multidot. 10.sup.5 2.64 .multidot. 10.sup.-1 5.0 5.6 .multidot.
10.sup.5 1.52 .multidot. 10.sup.-1 8.7 .multidot. 10.sup.5 3.16
.multidot. 10.sup.-1 4.75 9.9 .multidot. 10.sup.5 2.76 .multidot.
10.sup.-1 1.3 .multidot. 10.sup.6 4.60 .multidot. 10.sup.-1 4.5 3.1
.multidot. 10.sup.5 8.5 .multidot. 10.sup.-2 3.6 .multidot.
10.sup.5 1.30 .multidot. 10.sup.-1 4.25 5.2 .multidot. 10.sup.5
1.42 .multidot. 10.sup.-1 5.0 .multidot. 10.sup.5 1.80 .multidot.
10.sup.-1 4.0 5.1 .multidot. 10.sup.4 1.4 .multidot. 10.sup.-2 1.3
.multidot. 10.sup.5 4.8 .multidot. 10.sup.-2 3.5 1.3 .multidot.
10.sup.4 4 .multidot. 10.sup.-3 3.8 .multidot. 10.sup.4 1.4
.multidot. 10.sup.-2 Total Total Percentage = 1.00 Percentage =
1.80 Recovered Recovered The total input of BPTI (K15L)-III MA
phage was 0.030 ml .times. (1.2 .multidot. 10.sup.10 pfu/ml) = 3.6
.multidot. 10.sup.8 pfu. The total input of BPTI (K15L, MGNG)-III
MA phage was 0.030 ml .times. (9.2 .multidot. 10.sup.9 pfu/ml) =
2.8 .multidot. 10.sup.8 pfu.
[1495]
114TABLE 205 Fractionation of a Mixture of BPTI-III MK and BPTI
(K15L, MGNG)-III MA Phage on Immobilized HNE BPTI-III MK BPTI
(K15L, MGNG)-III MA Total Total Kanamycin Ampicillin Transducing %
Transducing % pH Units of Input Units of Input 7.0 4.01 .multidot.
10.sup.3 4.5 .multidot. 10.sup.-3 1.39 .multidot. 10.sup.5 3.13
.multidot. 10.sup.-1 6.0 7.06 .multidot. 10.sup.2 8 .multidot.
10.sup.-4 7.18 .multidot. 10.sup.4 1.62 .multidot. 10.sup.-1 5.0
1.81 .multidot. 10.sup.3 2.0 .multidot. 10.sup.-3 1.35 .multidot.
10.sup.5 3.04 .multidot. 10.sup.-1 4.0 1.49 .multidot. 10.sup.3 1.7
.multidot. 10.sup.-3 7.43 .multidot. 10.sup.5 1.673 The total input
of BPTI-III MK phage was 0.015 ml .times. (5.94 .multidot. 10.sup.9
kanamycin transducing units/ml) = 8.91 .multidot. 10.sup.7
kanamycin transducing units. The total input of BPTI (K15L,
MGNG)-III MA phage was 0.015 ml .times. (2.96 .multidot. 10.sup.9
ampiciliin transducing units/ml) = 4.44 .multidot. 10.sup.7
ampicillin transducing units.
[1496]
115TABLE 206 Characterization of the Affinity of BPTI (K15V,
R17L)-III MA Phage for Immobilized HNE BPTI (K15V, R17L)-III MA
BPTI (K15L, MGNG)-III MA Total Plaque- Percentage Total Plaque-
Percentage Forming Units of Input Forming Units of Input pH
Recovered Phage Recovered Phage 7.0 3.19 .multidot. 10.sup.6 8.1
.multidot. 10.sup.-2 9.42 .multidot. 10.sup.4 4.6 .multidot.
10.sup.-2 6.0 5.42 .multidot. 10.sup.6 1.38 .multidot. 10.sup.-1
1.61 .multidot. 10.sup.5 7.9 .multidot. 10.sup.-2 5.0 9.45
.multidot. 10.sup.6 2.41 .multidot. 10.sup.-1 2.85 .multidot.
10.sup.5 1.39 .multidot. 10.sup.-1 4.5 1.39 .multidot. 10.sup.7
3.55 .multidot. 10.sup.-1 4.32 .multidot. 10.sup.5 2.11 .multidot.
10.sup.-1 4.0 2.02 .multidot. 10.sup.7 5.15 .multidot. 10.sup.-1
1.42 .multidot. 10.sup.5 6.9 .multidot. 10.sup.-2 3.75 9.20
.multidot. 10.sup.6 2.35 .multidot. 10.sup.-1 -- -- 3.5 4.16
.multidot. 10.sup.6 1.06 .multidot. 10.sup.-1 5.29 .multidot.
10.sup.4 2.6 .multidot. 10.sup.-2 3.0 2.65 .multidot. 10.sup.6 6.8
.multidot. 10.sup.-2 -- -- Total Input = 1.73 Total Input = 0.57
Recovered Recovered Total input of BPTI (K15L, R17L)-III MA phage
was 0.040 ml .times. (9.80 .multidot. 10.sup.10 pfu/ml) = 3.92
.multidot. 10.sup.9 pfu. Total input of BPTI (K15L, MGNG)-III MA
phage was 0.040 ml .times. (5.13 .multidot. 10.sup.9 pfu/ml) = 2.05
.multidot. 10.sup.8 pfu.
[1497]
116TABLE 207 Sequence of the EpiNE.alpha. Clone Selected From the
Mini-Library 1 1 1 1 1 1 1 2 2 3 4 5 6 7 8 9 0 1 P C V A M F Q R Y
CCT.TGC.GTG.GCT.ATG.TTC.CAA.CGC- .TAT
[1498]
117TABLE 208 SEQUENCES OF THE EpiNE CLONES IN THE P1 REGION CLONE
IDENTI- FIERS SEQUENCE 3, 9 1 1 1 1 1 1 1 2 2 EpiNE3 16, 17 3 4 5 6
7 8 9 0 1 18 ,19 P C V G F F S R Y
CCT.TGC.GTC.GGT.TTC.TTC.TCA.CGC.TAT 6 1 1 1 1 1 1 1 2 2 EpiNE6 3 4
5 6 7 8 9 0 1 P C V G F F Q R Y CCT.TGC.GTC.GGT.TTC.TTC.CAA.CGC.TAT
7, 13 1 1 1 1 1 1 2 2 2 EpiNE7 14, 15 3 4 5 6 7 8 9 0 1 20 P C V A
N F P R Y CCT.TGC.GTC.GCT.ATG.TTC.CCA.CGC.TAT 4 1 1 1 1 1 1 1 2 2
EpiNE4 3 4 5 6 7 8 9 0 1 P C V A I F P R Y
CCT.TGC.GTC.GCT.ATC.TTC.CCA.CG- C.TAT 8 1 1 1 1 1 1 1 2 2 EpiNE8 3
4 5 6 7 8 9 0 1 P C V A I F K R S
CCT.TGC.GTC.GCT.ATC.TTC.AAA.CGC.TCT 1, 10 1 1 1 1 1 1 1 2 2 EpiNE1
11, 12 3 4 5 6 7 8 9 0 1 P C I A F F P R Y
CCT.TCC.ATC.GCT.TTC.TTC.CCA.CGC.TAT 5 1 1 1 1 1 1 1 2 2 EpiNE5 3 4
5 6 7 8 9 0 1 P C I A F F Q R Y CCT.TGC.ATC.GCT.TTC.TTC.CAA.C-
GC.TAT 2 1 1 1 1 1 1 1 2 2 EpiNE2 3 4 5 6 7 8 9 0 1 P C I A L F K R
Y CCT.TGC.ATC.GCT.TTG.TTC.AAA.CGC.TAT
[1499]
118TABLE 209 DNA sequences and predicted amino acid sequences
around the P1 region of BPTI analogues selected for binding to
Cathepsin G. P1 Clone 15 16 17 18 19 BPTI AAA . GCG . CGC . ATC .
ATC LYS ALA ARG ILE ILE EpiC 1 ATG . GGT . TTC . TCC . AAA (a) MET
GLY PHE SER LYS EpiC 7 ATG . GCT . TTG . TTC . AAA MET ALA LEU PHE
LYS EpiC 8 TTC . GCT . ATC . ACC . CCA (b) PHE ALA ILE THR PRO EpiC
10 ATG . GCT . TTG . TTC . CAA MET ALA LEU PHE GLN EpiC 20 ATG .
GCT . ATC . TCC . CCA MET ALA ILE SER PRO (a) Clones 11 and 31 also
had the identical sequence. (b) Clone 8 also contained the mutation
Tyr 10 to ASN.
[1500]
119TABLE 210 Derivatives of EpiNE7 Obtained by Variegation at
positions 34, 36, 39, 40 and 41 EpiNE7
.diamond-solid..diamond-solid..diamond-solid..diamond-solid-
..diamond-solid. **** RPDFCLEPPYTGPCvAmfpRYFYNAK-
AGLCQTFVYGGCmgngNNFKSAEDCMRTCGGA 1 2 3 4 5
12345678901234567890123456789012345678901234567890- 12345678
.diamond-solid.0 EpiNE7.6 .dwnarw..dwnarw..dwnarw..dwnarw..dwnarw.
.diamond-solid. .diamond-solid.
.diamond-solid..diamond-solid..diamond-solid..dwnarw.
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFlYgGCkgkGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.8, EpiNE7.9, and EpiNE7.31
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFeYgGCwakGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.11 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQ-
TFgYaGCrakGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.7
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFeYgGChaeGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.4 and EpiNE7.14
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFlYgGCwaqGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.5 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQT-
FrYgGClaeGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.10 and EpiNE7.20
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFdYgGChadGNNFKSAEDCMRTCG- GA
.diamond-solid.0 EpiNE7.1 RPDFCLJPPYTGPCvAmfpRYFY-
NAKAGLCQTFkYgGClahGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.16
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFtYgGCwanGNNFKSAEDCMRTCG- GA
.diamond-solid.0 EpiNE7.19
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFnYgGCegkGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.12 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQ-
TFqYgGCegyGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.17
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFqYgGClgeGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.21 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQ-
TFhYgGCwgqGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7
.diamond-solid..diamond-solid..diamond-solid..diamond-solid..diamond-soli-
d. **** RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFVYGGCmgn-
gNNFKSAEDCMRTCGGA 1 2 3 4 5
1234567890123456789012345678901234567890123456789012345678
.diamond-solid.0 EpiNE7.22 .dwnarw..dwnarw..dwnarw..dwnarw..-
dwnarw. .diamond-solid. .diamond-solid.
.diamond-solid..diamond-solid..diamond-solid..dwnarw.
RPDFcLEPPYTGPCvAmfpRYFYNAKAGLCQTFhYgGCwgeGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.23 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQ-
TFkYgGCwgkGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.24
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFkYgGChgnGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.25 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQ-
TFpYgGCwakGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.26
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFkYgGCwghGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.27 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQ-
TFnYgGCwgkGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.28
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFtYgGClghGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.29 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQ-
TFtYgGClgyGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.30, EpiNE7.34,
and EpiNE7.35 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFkYgGCwae-
GNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.32
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFgYgGCwgeGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.33 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQ-
TFeYgGCwanGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.36
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFvYgGChgdGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.37 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQ-
TFmYgGCqgkGNNFKSAEDCMRTCGGA .diamond-solid.0 EpiNE7.38
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFyYgGCwakGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7
.diamond-solid..diamond-solid..diamond-solid..diamond-solid..diamond-soli-
d. **** RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFVYGGCmgn-
gNNFKSAEDCMRTCGGA 1 2 3 4 5
1234567890123456789012345678901234567890123456789012345678
.diamond-solid.0 EpiNE7.39 .dwnarw..dwnarw..dwnarw..dwnarw..-
dwnarw. .diamond-solid. .diamond-solid.
.diamond-solid..diamond-solid..diamond-solid..dwnarw.
RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQTFmYgGCwgdGNNFKSAEDCMRTCGGA
.diamond-solid.0 EpiNE7.40 RPDFCLEPPYTGPCvAmfpRYFYNAKAGLCQ-
TFtYgGChgnGNNFKSAEDCMRTCGGA Notes: a) .diamond-solid. indicates
variegated residue. * indicates imposed change. .dwnarw. indicates
carry over from EpiNE7. b) The sequence M.sub.39-GNG in EpiNE7
(indicated by *) was imposed to increase similarity to ITI-D1. b)
Lower case letters in EpiNE7.6 to 7.38 indicate changes from BPTI
that were selected in the first round (residues 15-19) or positions
where the PBD was variegated in the second round (residues 34, 36,
39, 40, and 41). c) All EpiNE7 derivatives have G.sub.42.
.diamond-solid.ps TABLE 211 Effects of antisera on phage
infectifity Phage (dilution Incubation Relative of stock)
Conditions pfu/ml Titer MA-ITI PBS 1.2 .multidot. 10.sup.11 1.00
(10.sup.-1) NRS 6.8 .multidot. 10.sup.10 0.57 anti-ITI 1.1
.multidot. 10.sup.10 0.09 MA-ITI PBS 7.7 .multidot. 10.sup.8 1.00
(10.sup.-3) NRS 6.7 .multidot. 10.sup.8 0.87 anti-ITI 8.0
.multidot. 10.sup.6 0.01 MA PBS 1.3 .multidot. 10.sup.12 1.00
(10.sup.-1) NRS 1.4 .multidot. 10.sup.12 1.10 anti-ITI 1.6
.multidot. 10.sup.12 1.20 MA PBS 1.3 .multidot. 10.sup.10 1.00
(10.sup.-3) NRS 1.2 .multidot. 10.sup.10 0.92 anti-ITI 1.5
.multidot. 10.sup.10 1.20
[1501]
120TABLE 212 Fractionation of EpiNE-7 and MA-ITI phage on HNE beads
EpiNE-7 MA-ITI Total pfu Fraction Total pfu Fraction Sample in
sample of input in sample of input INPUT 3.3 .multidot. 10.sup.9
1.00 3.4 .multidot. 10.sup.11 1.00 Final 3.8 .multidot. 10.sup.5
1.2 .multidot. 10.sup.-4 1.8 .multidot. 10.sup.6 5.3 .multidot.
10.sup.-6 TBS-TWEEN Wash pH 7.0 6.2 .multidot. 10.sup.5 1.8
.multidot. 10.sup.-4 1.6 .multidot. 10.sup.6 4.7 .multidot.
10.sup.-6 pH 6.0 1.4 .multidot. 10.sup.6 4.1 .multidot. 10.sup.-4
1.0 .multidot. 10.sup.6 2.9 .multidot. 10.sup.-6 pH 5.5 9.4
.multidot. 10.sup.5 2.8 .multidot. 10.sup.-4 1.6 .multidot.
10.sup.6 4.7 .multidot. 10.sup.-6 pH 5.0 9.5 .multidot. 10.sup.5
2.9 .multidot. 10.sup.-4 3.1 .multidot. 10.sup.5 9.1 .multidot.
10.sup.-7 pH 4.5 1.2 .multidot. 10.sup.6 3.5 .multidot. 10.sup.-4
1.2 .multidot. 10.sup.5 3.5 .multidot. 10.sup.-7 pH 4.0 1.6
.multidot. 10.sup.6 4.8 .multidot. 10.sup.-4 7.2 .multidot.
10.sup.4 2.1 .multidot. 10.sup.-7 pH 3.5 9.5 .multidot. 10.sup.5
2.9 .multidot. 10.sup.-4 4.9 .multidot. 10.sup.4 1.4 .multidot.
10.sup.-7 pH 3.0 6.6 .multidot. 10.sup.5 2.0 .multidot. 10.sup.-4
2.9 .multidot. 10.sup.4 8.5 .multidot. 10.sup.-8 pH 2.5 1.6
.multidot. 10.sup.5 4.8 .multidot. 10.sup.-5 1.4 .multidot.
10.sup.4 4.1 .multidot. 10.sup.-8 pH 2.0 3.0 .multidot. 10.sup.5
9.1 .multidot. 10.sup.-5 1.7 .multidot. 10.sup.4 5.0 .multidot.
10.sup.-8 SUM* 6.4 .multidot. 10.sup.6 3 .multidot. 10.sup.-3 5.7
.multidot. 10.sup.6 2 .multidot. 10.sup.-5 *SUM is the total pfu
(or fraction of input) obtained from all pH elution fractions
[1502]
121TABLE 213 Fractionation of EpiC-10 and MA-ITI phage on Cat-G
beads EpiC-10 MA-ITI Total pfu Fraction Total pfu Fraction Sample
in sample of input in sample of input INPUT 5.0 .multidot.
10.sup.11 1.00 4.6 .multidot. 10.sup.11 1.00 Final 1.8 .multidot.
10.sup.7 3.6 .multidot. 10.sup.-5 7.1 .multidot. 10.sup.6 1.5
.multidot. 10.sup.-5 TBS-TWEEN Wash pH 7.0 1.5 .multidot. 10.sup.7
3.0 .multidot. 10.sup.-5 6.1 .multidot. 10.sup.6 1.3 .multidot.
10.sup.-5 pH 6.0 2.3 .multidot. 10.sup.7 4.6 .multidot. 10.sup.-5
2.3 .multidot. 10.sup.6 5.0 .multidot. 10.sup.-6 pH 5.5 2.5
.multidot. 10.sup.7 5.0 .multidot. 10.sup.-5 1.2 .multidot.
10.sup.6 2.6 .multidot. 10.sup.-6 pH 5.0 2.1 .multidot. 10.sup.7
4.2 .multidot. 10.sup.-5 1.1 .multidot. 10.sup.6 2.4 .multidot.
10.sup.-6 pH 4.5 1.1 .multidot. 10.sup.7 2.2 .multidot. 10.sup.-5
6.7 .multidot. 10.sup.5 1.5 .multidot. 10.sup.-6 pH 4.0 1.9
.multidot. 10.sup.6 3.8 .multidot. 10.sup.-6 4.4 .multidot.
10.sup.5 9.6 .multidot. 10.sup.-7 pH 3.5 1.1 .multidot. 10.sup.6
2.2 .multidot. 10.sup.-6 4.4 .multidot. 10.sup.5 9.6 .multidot.
10.sup.-7 pH 3.0 4.8 .multidot. 10.sup.5 9.6 .multidot. 10.sup.-7
3.6 .multidot. 10.sup.5 7.8 .multidot. 10.sup.-7 pH 2.5 2.0
.multidot. 10.sup.5 4.0 .multidot. 10.sup.-7 2.7 .multidot.
10.sup.5 5.9 .multidot. 10.sup.-7 pH 2.0 2.4 .multidot. 10.sup.5
4.8 .multidot. 10.sup.-7 3.2 .multidot. 10.sup.5 7.0 .multidot.
10.sup.-7 SUM* 9.9 .multidot. 10.sup.7 2 .multidot. 10.sup.-4 1.4
.multidot. 10.sup.7 3 .multidot. 10.sup.-5 *SUM is the total pfu
(or fraction of input) obtained from all pH elution fractions
[1503]
122TABLE 214 Abbreviated fractionation of display phage on HNE
beads DISPLAY PHAGE EpiNE-7 MA-ITI 2 MA-ITI-E7 1 MA-ITI-E7 2 INPUT
1.00 1.00 1.00 1.00 (pfu) (1.8 .multidot. 10.sup.9) (1.2 .multidot.
10.sup.10) (3.3 .multidot. 10.sup.9) (1.1 .multidot. 10.sup.9) WASH
6 .multidot. 10.sup.-5 1 .multidot. 10.sup.-5 2 .multidot.
10.sup.-5 2 .multidot. 10.sup.-5 pH 7.0 3 .multidot. 10.sup.-4 1
.multidot. 10.sup.-5 2 .multidot. 10.sup.-5 4 .multidot. 10.sup.-5
pH 3.5 3 .multidot. 10.sup.-3 3 .multidot. 10.sup.-6 8 .multidot.
10.sup.-5 8 .multidot. 10.sup.-5 pH 2.0 1 .multidot. 10.sup.-3 1
.multidot. 10.sup.-6 6 .multidot. 10.sup.-6 2 .multidot. 10.sup.-5
SUM* 4.3 .multidot. 10.sup.-3 1.4 .multidot. 10.sup.-5 1.1
.multidot. 10.sup.-4 1.4 .multidot. 10.sup.-4 *SUM is the total
fraction of input pfu obtained from all pH elution fractions
[1504]
123TABLE 215 Fractionation of EpiNE-7 and MA-ITI-E7 phage on HNE
beads EpiNE-7 MA-ITI-E7 Total pfu Fraction Total pfu Fraction
Sample in sample of input in sample of input INPUT 1.8 .multidot.
10.sup.9 1.00 3.0 .multidot. 10.sup.9 1.00 pH 7.0 5.2 .multidot.
10.sup.5 2.9 .multidot. 10.sup.-4 6.4 .multidot. 10.sup.4 2.1
.multidot. 10.sup.-5 pH 6.0 6.4 .multidot. 10.sup.5 3.6 .multidot.
10.sup.-4 4.5 .multidot. 10.sup.4 l.5 .multidot. 10.sup.-5 pH 5.5
7.8 .multidot. 10.sup.5 4.3 .multidot. 10.sup.-4 5.0 .multidot.
10.sup.4 1.7 .multidot. 10.sup.-5 pH 5.0 8.4 .multidot. 10.sup.5
4.7 .multidot. 10.sup.-4 5.2 .multidot. 10.sup.4 1.7 .multidot.
10.sup.-5 pH 4.5 1.1 .multidot. 10.sup.6 6.1 .multidot. 10.sup.-4
4.4 .multidot. 10.sup.4 1.5 .multidot. 10.sup.-5 pH 4.0 1.7
.multidot. 10.sup.6 9.4 .multidot. 10.sup.-4 2.6 .multidot.
10.sup.4 8.7 .multidot. 10.sup.-6 pH 3.5 1.1 .multidot. 10.sup.6
6.1 .multidot. 10.sup.-4 1.3 .multidot. 10.sup.4 4.3 .multidot.
10.sup.-6 pH 3.0 3.8 .multidot. 10.sup.5 2.1 .multidot. 10.sup.-4
5.6 .multidot. 10.sup.3 1.9 .multidot. 10.sup.-6 pH 2.5 2.8
.multidot. 10.sup.5 1.6 .multidot. 10.sup.-4 4.9 .multidot.
10.sup.3 1.6 .multidot. 10.sup.-6 pH 2.0 2.9 .multidot. 10.sup.5
1.6 .multidot. 10.sup.-4 2.2 .multidot. 10.sup.3 7.3 .multidot.
10.sup.-7 SUM* 7.6 .multidot. 10.sup.6 4.1 .multidot. 10.sup.-4 3.1
.multidot. 10.sup.5 1.1 .multidot. 10.sup.-4 *SUM is the total pfu
(or fraction of input) obtained from all pH elution fractions
CITATIONS
[1505] AKOH72:
[1506] Ako, H, R J Foster, and C A Ryan, "The preparation of
anhydro-trypsin and its reactivity with naturally occurring
proteinase inhibitors", Biochem Biophys Res Commun (USA)(1972),
47(6)1402-7.
[1507] ALBR83a:
[1508] Albrecht, G, K Hochstrasser, and O L Schonberger,
"Kunitz-type proteinase inhibitors derived by limited proteolysis
of the inter-.alpha.-trypsin inhibitor, IX: isolation and
characterization of the inhibitory parts of inter-.alpha.-trypsin
inhibitors from several mammalian sera", Hoppe-Seyler's Z Physiol
Chem (1983), 364:1697-1702.
[1509] ALBR83b:
[1510] Albrecht, G J, K Hochstrasser, and J-P Salier, "Elastase
inhibition by the inter-.alpha.-trypsin inhibitor and derived
inhibitors of man and cattle", Hoppe-Seyler's Z Physiol chem
(1983), 364:1703-1708.
[1511] ALMA83a:
[1512] Almassy, R C, J C Fontecilla-Camps, F L Suddath, and C E
Bugg, "Structure of scorpion neurotoxin at 1.8 .ANG. resolution",
Entry 1SN3 in Brookhaven Protein Data Bank, (1983).
[1513] ALMA83b:
[1514] Almassy, R C, J C Fontecilla-Camps, F L Suddath, and C E
Bugg, "Structure of variant-3 scorpion neurotoxin from Centruroides
Sculpturatus ewing refined at 1.8 .ANG. resolution", J Mol Biol
(1983), 170:497ff.
[1515] ALMQ89:
[1516] Almquist, R G, S R Kadambi, D M Yasuda, F L Weitl, W E
Polgar, and L R Toll, "Paralytic activity of (des-Glul)conotoxin GI
analogs in the mouse diaphragm", Int J Pept Protein Res, (Dec
1989), 34(6)455-62.
[1517] ANFI73:
[1518] Anfinsen, C B, "Principles that govern the folding of
protein chains", Science (1973), 181(96)223-30.
[1519] ARG087:
[1520] Argos, P, "Analysis of Sequence-similar Pentapeptides in
Unrelated Protein Tertiary Structures", J Mol Biol (1987),
197:331-348.
[1521] ARAK90:
[1522] Araki, K, M Kuwada, O Ito, J Kuroki, and S Tachibana, "Four
disulfide bonds allocation of Na.sup.+, K.sup.+-ATPase inhibitor
(SPAI)", Biochem Biophys Res Comm (1990), 172(1)42-46.
[1523] ARMS81:
[1524] Armstrong, J, R N Perham, and J E Walker, "Domain structure
of Bacteriophage fd Adsorption Protein", FEBS Lett (1981),
135(1)167-172.
[1525] ARMS83:
[1526] Armstrong, J, J A Hewitt, and R N Perham, "Chemical
modification of the coat protein in bacterio-phage fd and
orientation of the virion during assembly and disassembly", EMBO J
(1983), 2(10)1641-6.
[1527] ARNA90:
[1528] Arnaout, M A, "Leukocyte Adhesion Molecules Deficiency: Its
STructural Basis, Pathophysiology and Implications for Modulating
the Inflammatory Response", Immunological Reviews (1990),
114:______
[1529] AUER87:
[1530] Auerswald, E-A, W Schroeder, and M Kotick, "Synthesis,
Cloning and Expression of Recombinant Aprotin-in", Biol Chem
Hoppe-Seyler (1987), 368:1413-1425.
[1531] AUER88:
[1532] Auerswald, E-A, D Hoerlein, G Reinhardt, W Schroder, and E
Schnabel, "Expression Isolation, and Characterization of
Recombinant [Arg.sup.15, Glu.sup.52]Aprotinin", Bio Chem
Hoppe-Seyler (1988), 369(Supplement):27-35.
[1533] AUER89:
[1534] Auerswald, E-A, W Bruns, D Hoerlein, G Reinhardt, E
Schnabel, and W Schroder, "Variants of bovine pancreatic trypsin
inhibitor produced by recombinant DNA technology", UK Patent
Application GB 2,208,511 A.
[1535] AUER90:
[1536] Auerswald, E-A, W Schroeder, E Schnabel, W Bruns, G
Reinhard, and M Kotick, "Homologs of Aprotinin produced from a
recombinant host, process ecpression vector and recombinant host
therefor and pharmaceutical use thereof", U.S. Pat. No. 4,894,436
(Jan. 16, 1990).
[1537] AUSU87:
[1538] Ausubel, F M, R Brent, R E Kingston, D D Moore, J G Seidman,
J A Smith, and K Struhl, Editors Current Protocols in Molecular
Biology, Greene Publishing Associates and Wiley-Interscience,
Publishers: John Wiley & Sons, New York, 1987.
[1539] BAKE87:
[1540] Baker, K, N Mackman, and I B Holland, "Genetics and
Biochemistry of the Assembly of Proteins into the Outer Membrane of
E. coli", Prog Biophys molec Biol (1987), 49:89-115.
[1541] BALD85:
[1542] Balduyck, M, M Davril, C Mizon, M Smyrlaki, A Hayem, and J
Mizon, "Human urinary proteinase inhibitor: inhibitory properties
and interaction with bovine trypsin", Biol Chem Hoppe-Seyler
(1985), 366:9-14.
[1543] BANN81:
[1544] Banner, D W, C Nave, and D A Marvin, "Structure of the
protein and DNA in fd filamentous bacterial virus", Nature (1981),
289:814-816.
[1545] BARB85:
[1546] Barbe, J, J A Vericat, M Llagostera, and R Guerrero,
"Expression of the SOS genes of Escherichia coli in Salmonella
tyPhimurium", Microbiologia (1985), 1(1-2)77-87.
[1547] BECK80:
[1548] Beck, E, "Nucleotide sequence of the gene ompA coding the
outer membrane protein II* of Escherichia coli K-12", Nucl Acid Res
(1980), 8(13)3011-3024.
[1549] BECK83:
[1550] Beckwith, J, and T J Silhavy, "Genetic Analysis of Protein
Export in Escherichia coli", Methods in Enzymology (1983),
97:3-11.
[1551] BECK88b:
[1552] Beckmann, J, A Mehlich, W Schroeder, H R Wenzel, and H
Tschesche, Preparation of chemically `mutated` aprotinin homologues
by semisynthesis: P1 substitutions change inhibitory specificity",
Eur J Biochem (1988), 176:675-82.
[1553] BECK89a:
[1554] Beckmann, J, A Mehlich, W Schroeder, H R Wenzel, and H
Tschesche, "Semisynthesis of Arg.sup.15, Glu.sup.15, Met.sup.15,
and Nle.sup.15-Aprotinin Involving Enzymatic Peptide Bond
Resynthesis", J Protein Chem (1989), 8(1)101-113.
[1555] BECK89b:
[1556] Becker, S, E Atherton, H Michel, and R D Gordon, "Synthesis
and characterization of conotoxin IIIa", J Protein Chem, (Jun
1989), 8(3)393-4.
[1557] BECK89c:
[1558] Becker, S, E Atherton, and R D Gordon, "Synthesis and
characterization of mu-conotoxin IIIa", Eur J Biochem, (Oct. 20
1989), 185(1)79-84.
[1559] BENS84:
[1560] Benson, S A, E Bremer, and T J Silhavy, "Intragenic regions
required for LamB export", Proc Natl Acad Sci USA (1984),
81:3830-34.
[1561] BENS87b:
[1562] Benson, S A, and E Bremer, "In vivo selection and
characterization of internal deletions in the lamB::lacZ gene
fusion", Gene (1987), 52(2-3)165-73.
[1563] BENS87c:
[1564] Benson, S A, M N Hall, and B A Rasmussen, "Signal Sequence
Mutations That Alter coupling of Secretion and Translation of an
Escherichia coli Outer Membrane Protein", J Bacteriol (1987),
169(10)4686-91.
[1565] BENS88:
[1566] Benson, S A, J L Occi, B A Sampson, "Mutations that alter
the pore function of the OmpF porin of Escherichia coli K12", J Mol
Biol (1988) 203(4)961-70.
[1567] BENZ88a:
[1568] Benz, R, and K Bauer, "Permeation of hydrophilic molceules
through the outer membrane of gram-negative bacteria", Eur J
Biochem (1988), 176:1-19.
[1569] BENZ88b:
[1570] Benz, R, "Structure and Fucntion of Porins from
Gram-Negative Bacteria", Ann Rev Microbiol (1988), 42:359-93.
[1571] BERG88:
[1572] Berg, J M, "Proposed structure for the zinc-binding domains
from transcription factor IIIA and related proteins", Proc Natl
Acad Sci USA (1988), 85:99-102.
[1573] BETT88:
[1574] Better, M, C P Chang, R R Robinson, and A H Horwitz,
"Escherichai coli Secretion of an Active Chimeric Antibody
Fragment", Science (1988), 240:1041-1043.
[1575] BHAT86:
[1576] Bhatnagar, P K, and J C Frantz, "Synthesis and Antigenic
activity of E. coli ST and its analogues", Develop biol Standard
(1986), 63:79-87.
[1577] BIRD67:
[1578] Birdsell, D C, and E H Cota-Robles, "Production and
Ultrastructure of lysozyme and ethylenediaminetetraacetate-lysozyme
spheroplasts of E. coli", J Bacteriol (1967), 93:427-437.
[1579] BIET86:
[1580] Bieth, J G, "Elastase: Catalytic and Biological Properties",
pp. 217-320 in Regulation of Matrix Accumulation, Editor: RP
Mecham, Academic Press, Orlando, 1986.
[1581] BLOW72:
[1582] Blow &al., J Mol Biol (1972), 69:137ff.
[1583] BODE89:
[1584] Bode, W, H J Greyling, R Huber, J Otlewski, and T Wilusz,
"The refined 2.0 A X-ray crystal structure of the complex formed
between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from
squash seeds (Cucurbita maxima). Topological similarity of the
squash seed inhibitors with the carboxypeptidase A inhibitor from
potatoes", FEBS Lett (Jan. 2 1989), 242(2)285-92.
[1585] BOEK80:
[1586] Boeke, J D, M Russel, and P Model, "Processing of
Filamentous Phage Pre-coat Protein: Effect of Sequence Variations
near the Signal Peptidase Cleavage Site", J Mol Biol (1980),
144:103-116.
[1587] BOEK82:
[1588] Boeke, J D, P Model, and N D Zinder, "Effects fo
Bacteriophage f1 Gene III Protein on the Host Cell Membrane", Molec
and Gen Genet, (1982), 186:185-192.
[1589] BOQU87:
[1590] Boquet, P L, C Manoil, and J Beckwith, "Use of TnphoA to
Detect Genes for Exported Proteins in Escherichia coli:
Identification of the Plasmid-Encoded Gene for a Periplasmic Acid
Phosphatase", J Bacteriol (1987), 169:1663-1669.
[1591] BOTS85:
[1592] Botstein, D, and D Shortle, "Strategies and applications of
in vitro mutagenesis", Science, (1985), 229(4719)1193-201.
[1593] BOUG84:
[1594] Bouges-Bocquet, B, H Villarroya, and M Hofnung, "Linker
Mutagenesis in the Gene of an Outer Membrane Protein of Escherichia
coli, LamB", J Cellular Biochem (1984), 24:217-28.
[1595] BOUL86a:
[1596] Boulain, J C, A Charbita and M Hofnung, "Mutagenesis by
random linker insertion into the lamB gene of Escherichia coli
K12", Mol Gen Genet, (1986), 205(2)339-48.
[1597] BRAW87:
[1598] Brawerman, G, "Determinants of messenger RNA stability",
Cell (1987), 48(1)5-6.
[1599] CALA90:
[1600] Calamia, J, and C Manoil, "lac permease of Escherichia coli:
topology and sequence elements promoting membrane insertion", Proc
Natl Acad Sci USA, (Jul 1990), 87(13)4937-41.
[1601] CAMP90:
[1602] Campanelli, D, M Melchior, Yiping Fu, M Nakata, H Shuman, C
Nathan, and J E Gabay, "Cloning of cDNA for Proteinase 3: A Serine
Protease, Antibiotic, and Autoantigen from Human Neutrophils", J
Exp Med (Dec 1990), 172:1709-15.
[1603] CARM90:
[1604] Carmel, G, D Hellstern, D Henning, and J W Coulton,
"Insertion mutagenesis of the gene encoding the ferrichrome-iron
receptor of Escherichia coli K-12", J Bacteriol, (April 1990),
172(4)1861-9.
[1605] CARU85:
[1606] Caruthers, M H, "Gene Synthesis Machines: DNA Chemistry and
Its Uses", Science (1985), 230:281-285.
[1607] CARU87:
[1608] Caruthers, M H, P Gottlieb, L P Bracco, and L Cummings, "The
Thymine 5-Methyl Group: A Protein-DNA Contact Site Useful for
Redesigning Cro Repressor to Recognize a New Operator", in Protein
Structure, Folding, and Design 2, 1987, Ed. D Oxender (New York, AR
Liss Inc) p.9ff.
[1609] CAST79:
[1610] Castillo, M J, K Nakajima, M Zimmerman, and J C Powers,
"Sensitive substrates for human leukocyte and porcine pancreatic
elastase: a study of the merits of various chromophoric and
fluorogenic leaving groups in assays for serine proteases", Anal
Biochem (1979), 99(1)53-64.
[1611] CATR87:
[1612] Catron, K M, and C A Schnaitman, "Export of Protein in
Escherichia coli: a Novel Mutation in ompC Affects Expression of
Other Major Outer Membrane Proteins", J Bacteriol (1987),
169:4327-34.
[1613] CHAM82:
[1614] Chambers, R W, I Kucan, and Z Kucan, "Isolation and
characterization of phi-X174 mutants carrying lethal missense
mutations in gene G", Nucleic Acids Res (1982), 10(20)6465-73.
[1615] CHAN79:
[1616] Chang, C N, P Model, and G Blobel, "Membrane biogenesis:
Cotranslational integration of the bacteriophage f1 coat protein
into an Escherichia coli membrane fraction", Proc Natl Acad Sci USA
(1979), 76:1251-1255.
[1617] CHAP90:
[1618] Chapot, M P, Y Eshdat, S Marullo, J G Guillet, A Charbit, A
D Strosberg, and C Delavier-Klutchko, "Localization and
characterization of three different beta-adrenergic receptors
expressed in Escherichia coli", Eur J Biochem (1990),
187(1)137-44.
[1619] CHAR84:
[1620] Charbit, A, J-M Element, and M Hofnung, "Further Sequence
Analysis of the Phage Lambda Receptor Site", J Mol Biol (1984),
175:395-401.
[1621] CHAR86a:
[1622] Charbit, A, J C Boulain, A Ryter, and M Hofnung, "Probing
the topology of a bacterial membrane protein by genetic insertion
of a foreign epitope; expression at the cell surface", EMBO J,
(1986), 5(11)3029-37.
[1623] CHAR86b:
[1624] Charbit, A, J-C Boulain, and M Hofnung, "Une methode
genetique pur exposer un epitope choisi a la surface de la bacteria
Escherichia coli. Perspectives [A genetic method to expose a chosen
epitope on the surface of the bacteria E. coli]", Comptes Rendu
Acad Sci, Paris, (1986), 302:617-24.
[1625] CHAR87:
[1626] Charbit, A, E Sobczak, M L Michel, A Molla, P Tiollais, and
M Hofnung, "Presentation of two epitopes of the preS2 region of
hepatitis B virus on live recombinant bacteria", J Immunol (1987),
139:1658-64.
[1627] CHAR88a:
[1628] Charbit, A, K Gehring, H Nikaido, T Ferenci, and M Hofnung,
"Maltose transport and starch binding in phage-resistant point
mutants of maltoporin. Functional and topological implications", J
Mol Biol (1988), 201(3)487-96.
[1629] CHAR88b:
[1630] Charbit, A, A Molla, W Saurin, and M Hofnung, "Versatility
of a vector for expressing foreign polypeptides at the surface of
gram-negative bacteria", Gene (1988), 70(1)181-9.
[1631] CHAR88c:
[1632] Charbit, A, S Van der Werf, V Mimic, J C Boulain, M Girard,
and M Hofnung, "Expression of a poliovirus neutralization epitope
at the surface of recombinant bacteria: first immunization
results", Ann Inst Pasteur Microbiol (1988), 139(1)45-58.
[1633] CHAR90:
[1634] Charbit, A, A Molla, J Ronco, J M Element, V Favier, EM
Bahraoui, L Montagnier, A Leguern, and M Hofnung, "Immunogenicity
and antigenicity of conserved peptides from the envelope of HIV-1
expressed at the surface of recombinant bacteria", AIDS (1990),
4(6)545-51.
[1635] CHAV88:
[1636] Chavrier, P, P Lemaire, O Revelant, R Bravo, and P Charnay,
"Characterization of a Mouse Multigene Family That Encodes Zinc
Finger Structures", Molec Cell Biol (1988), 8(3)1319-26.
[1637] CHAZ85:
[1638] Chazin, W J, DP Goldenberg, T E Creighton, and K Wuthrich,
"Comparative studies of conformation and internal mobility in
native and circular basic pancreatic trypsin inhibitor by .sup.1H
nuclear magnetic resonance in solution", Eur J Biochem (1985),
152:(2)429-37.
[1639] CHOT75:
[1640] Chothia, C, and J Janin, "Principles of protein-protein
recognition", Nature (1975), 256:705-708.
[1641] CHOT76:
[1642] Chothia, C, S Wodak, and J Janin, "Role of subunit
interfaces in the allosteric mechanism of hemoglobin", Proc Natl
Acad Sci USA (1976), 73:3793-7.
[1643] CHOU74:
[1644] Chou, P Y, and G D Fasman, "Prediction of protein
conformation" Biochemistry (1974), 13:(2)222-45.
[1645] CHOU78a:
[1646] Chou, P Y, and G D Fasman, "Prediction of the secondary
structure of proteins from their amino acid sequence", Adv Enzymol
(1978), 47:45-148.
[1647] CHOU78b:
[1648] Chou, P Y, and G D Fasman, "Empirical predictions of protein
conformation" Annu Rev Biochem (1978), 47:251-76.
[1649] CHOW87:
[1650] Chowdhuury, K, U Deutsch, and P Gruss, "A Multigene Family
Encoding Several `Finger` Structures Is Present and Differentially
Active in Mammalian Genomes", Cell (1987), 48:771-778.
[1651] CLEM81:
[1652] Element, J M, and M Hofnung, "The sequence of the lambda
receptor, an outer membrane protein of E. coli K12", Cell (1981),
27:507-514.
[1653] CLEM83:
[1654] Element J M, E Lepouce, C Marchal, and M Hofnung, "Genetic
Study of a membrane protein: DNA sequence alterations due to 17
LamB point mutations affecting adsorption of phage lambda", EMBO J
(1983), 2:77-80.
[1655] CLIC88:
[1656] Click, E M, G A McDonald, and C A Schnaitman, "Translational
Control of Exported Proteins That Results from OmpC Porin
Overexpression", J Bacteriol (1988), 170:2005-2011.
[1657] CLOR86:
[1658] Clore, G M, A T Brunger, M Karplus, A M Gronenborn,
"Application of Molecular Dynamics with Interproton Distance
Restraints to Three-dimensional Protein Structure Determination: A
model study of Crambin", J Mol Biol (1986), 191:523-551.
[1659] CLOR87a:
[1660] Clore, G M, A M Gronenborn, M Kjaer, and F M Poulsen, "The
determination of the three-dimensional structure of barley serine
proteinase inhibitor 2 by nuclear magnetic resonance distance
geometry and restrained molecular dynamics", Protein Engineering
(1987), 1(4)305-311.
[1661] CLOR87b:
[1662] Clore, G M, A M Gronenborn, M N G James, M Kjaer, C A
McPhalen, and F M Poulsen, "Comparison of the solution and X-ray
structures of barley serine proteinase inhibitor 2", Protein
Engineering (1987), 1(4)313-318.
[1663] CLUN84:
[1664] Clune, A, K-S Lee, and T Ferenci, "Affinity Engineering of
Maltoporin: Variants with Enhanced Affinity for Particular
Ligands", Biochem and Biophys Res Comm (1984), 121:34-40.
[1665] CREI74:
[1666] Creighton, T E, "Intermediates in the Refolding of Reduced
Pancreatic Trypsin Inhibitor", J Mol Biol (1974), 87:579-602.
[1667] CREI77a:
[1668] Creighton, T E, "Conformational Restrictions on the Pathway
of Folding and Unfolding of the Pancreatic Trypsin Inhibitor", J
Mol Biol (1977), 113:275-293.
[1669] CREI77b:
[1670] Creighton, T E, "Energetics of Folding and Unfolding of
Pancreatic Trypsin Inhibitor", J Mol Biol (1977), 113:295-312.
[1671] CREI80:
[1672] Creighton, T E, "Role of the Environment in the Refolding of
Reduced Pancreatic Trypsin Inhibitor", J Mol Biol (1980),
144:521-550.
[1673] CREI84:
[1674] Creighton, T E, Proteins: Structures and Molecular
Principles, W H Freeman & Co, New York, 1984.
[1675] CREI87:
[1676] Creighton, T E, and I G Charles, "Biosynthesis, Processing,
and Evolution of Bovine Pancreatic Trypsin Inhibitor", Cold Spring
Harb Symp Quant Biol (1987), 52:511-519.
[1677] CREI88:
[1678] Creighton, T E, "Disulphide Bonds and Protein Stability",
BioEssays (1988), 8(2)57-63.
[1679] CRIS84:
[1680] Crissman, J W, and GP Smith, "Gene-III Protein of
Filamentous Phages: Evidence for a Carboxyl-Terminal Domain with a
Role in Morphogenesis", Virology (1984), 132:445-55.
[1681] CRUZ85:
[1682] Cruz, I J, W R Gray, B M Olivera, R D Zeikus, L Kerr, D
Yoshikami, and E Moczydlowski, "Conus geographus toxins that
discriminate between neuronal and muscle sodium channels", J Biol
Chem, (1985), 260(16)9280-8.
[1683] CRUZ89:
[1684] Cruz, L J, G Kupryszewski, G W LeCheminant, W R Grey, B M
Oliveria, and J Rivier, "mu-Conotoxin GIIIA, a Peptide Ligand for
Muscle Scodium Channels: Chemical Synthesis, Radiolabeling, and
Receptor Characterization", Biochem (1989), 28:3437-3442.
[1685] CWIR90:
[1686] Cwirla, S E, E A Peters, R W Barrett, and W J Dower,
"Peptides on Phage: A vast library of peptides for identifying
ligands", Proc Natl Acad Sci USA, (August 1990), 87:6378-6382.
[1687] DAIL90:
[1688] Dailey, D, G L Schieven, M Y Lim, H Marquardt, T Gilmore, J
Thorner, and G S Martin, "Novel yeast protein kinase (YPK1 gene
product) is a 40-kilodalton phosphotyrosyl protein associated with
protein-tyrosine kinase activity", Mol Cell Biol (December 1990),
10(12)6244-56.
[1689] DALL90:
[1690] Dallas, W S, "The Heat-Stable Toxin I Gene from Escherichia
coli 18D", J Bacteriol (1990), 172(9)5490-93.
[1691] DARG88:
[1692] Dargent, B, A Charbit, M Hofnung, and F Pattus, "Effect of
point mutations on the in-vitro pore properties of maltoporin, a
protein of Escherichia coli outer membrane", J Mol Biol (1988),
201(3)497-506.
[1693] DAWK86:
[1694] Dawkins, R, The Blind Watchmaker, W W Norton & Co, New
York, 1986.
[1695] DAYL88:
[1696] Day, L A, C J Marzec, S A Reisberg, and A Casadevall, "DNA
Packing in Filamentous Bacteriophage", Ann Rev Biophys Biophys Chem
(1988), 17:509-39.
[1697] DAYR86:
[1698] Dayringer, H, A Tramantano, and R Fletterick, "Proteus
Software for Molecular Modeling" p.5-8 in Computer Graphics and
Molecular Modeling, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1986.
[1699] DEBR86:
[1700] Debro, L, P C Fitz-James, and A Aronson, "Two different
parasporal inclusions are produced by Bacillus thuringiensis subsp.
finitimus.", J Bacteriol (1986), 165:258-68.
[1701] DEGE84:
[1702] de Geus, P, H M Verheij, N H Reigman, W P M Hoekstra, and G
H de Haas, "The pro- and mature forms of the E. coli K-12 outer
memberane phospholipase A are identical", EMBO J (1984),
3(8)1799-1802.
[1703] DEGR87:
[1704] DeGrado, W F, L Regan, and S P Ho, "The Design of a
Four-helix Bundle Protein", Cold Spring Harbor Symp Quant Biol,
(1987), 52:521-6.
[1705] DELA88:
[1706] de la Cruz, V F, A A Lal and T F McCutchan, "Immunogenicity
and epitope mapping of foreign sequences via genetically engineered
filamentous phage", J Biol Chem, (1988), 263(9)4318-22.
[1707] DENH78:
[1708] Denhardt, D T, D Dressler, and D S Ray editors, The
Single-Stranded DNA Phages, Cold Spring Harbor Laboratory,
1978.
[1709] DEVL90:
[1710] Devlin, J J, L C Panganiban, and P E Devlin, "Random Peptide
Libraries: A Source of Specific Protein Binding Molecules",
Science, (Jul. 27, 1990), 249:404-406.
[1711] DEVO78:
[1712] DeVore, D P, and R J Gruebel, "Dityrosine in adhesive formed
by the sea mussel, Mytilus edulis", Biochem Biophys Res Commun
(1978), 80(4)993-9.
[1713] DEVR84:
[1714] de Vries, G, C K raymond, and R A Ludwig, "Extension of
bacteriophage .lambda. host range: Selection, cloning, and
characterization of a constitutive .lambda. receptor gene", Proc
Natl Acad Sci USA (1984), 81:6080-4.
[1715] DIAR90:
[1716] Diarra-Mehrpour, M, J Bourguignon, R Sesboue, J-P Salier, T
Leveillard and J-P Martin, "Structural analysis of the human
inter-.alpha.-trypsin inhibitor light-chain gene", Eur J Biochem
(1990), 191:131-139.
[1717] DICK83:
[1718] Dickerson, R E, and I Geis, Hemoglobin: Structure, Function,
Evolution, and Pathology, The Bejamin/Cummings Publishing Co, Menlo
Park, Calif., 1983.
[1719] DILL87:
[1720] Dill, K A, "Protein Surgery", Protein Engineering (1987),
1:369-371.
[1721] DOUG84:
[1722] Dougan, G, and P Morrissey, "Molecular analysis of the
virulence determinants of enterotoxigenic Escherichia coli isolated
from domestic animals: applications for vaccine development", Vet
Microbiol (1984/5), 10:241-57.
[1723] DON087
[1724] Donovan, W, Z Liangbiao, K Sandman, and R Losick, "Genes
Encoding Spore Coat Polypeptides from Bacillus subtilis", J Mol
Biol (1987), 196:1-10.
[1725] DUCH88:
[1726] Duchene, M, A Schweized, F Lottspeich, G Krauss, M Marget, K
Vogel, B-U von Specht, and H Domdey, "Sequence and Transcriptional
Start Site of the Pseudomonas aeruginosa Outer Membrane Porin
Protein F Gene", J Bacteriol (1987), 170:155-162.
[1727] DUFT85:
[1728] Dufton, M J, "Proteinase inhibitors and dendrotoxins", Eur J
Biochem (1985), 153:647-654.
[1729] DULB86:
[1730] Dulbecco, R, "Viruses with Recombinant Surface Proteins",
U.S. Pat. No. 4,593,002, Jun, 3, 1986.
[1731] DUPL88:
[1732] Duplay, P, and M Hofnung, "Two Regions of Mature Periplasmic
Maltose-Binding Protein of Escherichia coli Involved in Secretion",
J Bacteriol (1988), 170(10)4445-50.
[1733] DWAR89:
[1734] Dwarakanath, P, S S Viswiswariah, Y V B K Subrahmanyam, G
Shanthi, H M Jagannatha, and T S Balganesh, "Cloning and
hyperexpression of a gene encoding the heat-stable toxin of
Escherichia coli", Gene (1989), 81:219-226.
[1735] EHRM90:
[1736] Ehrmann, M, D Boyd, and J Beckwith, "Genetic analysis of
membrane protein topology by a sandwich gene fusion approach", Proc
Natl Acad Sci USA, (October 1990), 87(19)7574-8.
[1737] EIGE90:
[1738] Eigenbrot, C, M Randal, and A A Kossiakoff, "Structural
effects induced by removal of a disulfide-bridge: the X-ray
structure of the C30A/C51A mutant of basic pancreatic trypsin
inhibitor at 1.6 .ANG.", Protein Engineering (1990),
3(7)591-598.
[1739] EISE85:
[1740] Eisenbeis, S J, M S Nasoff, S A Noble, L P Bracco, D R
Dodds, M H Caruthers, "Altered Cro Repressors from engineered
mutagenesis of a synthetic cro gene", Proc Natl Acad Sci USA
(1985), 82:1084-1088.
[1741] ELLE88:
[1742] Elleman, T C, "Pilins of Bacteroides nodosus: molecular
basis of serotypic variation and relationships to other bacterial
pilins", Microbiol Rev (1988), 52(2)233-47.
[1743] EMPI82:
[1744] Empie, M W, and M Laskowski, Jr, "Thermodynamics and
Kinetics fo Single Residue Replacements in Avian Ovomucoid Third
Domains: Effect on Inhibitor Interactions with Serine Proteinases",
Biochemistry (1982), 21:2274-84.
[1745] ENGH89:
[1746] Enghild, J J, I B Thogersen, S V Pizzo, and G Salvesen,
"Anallysis of inter-.alpha.-trypsin inhibitor and a novel
inhibitor, pre-.alpha.-trypsin inhibitor, from human plasma:
polypeptide chain stoichiometry and assembly by glycan", J Biol
Biochem (1989), 264:15975-15981.
[1747] EPST63:
[1748] Epstein , C J, R F Goldberger, and C B Anfinsen, Cold Spr
Harb Symp Quant Biol (1963), 28:439ff.
[1749] ERIC86:
[1750] Erickson, B W, S B Daniels, P A Reddy, C G Unson, J S
Richardson, and D C Richardson, "Betabellin: An Engineered
Protein", Current Communications in Molecular Biology: Computer
Graphics and Molecular Modeling, Cold Spring Harbor Laboratoary,
Cold Spring Harbor, N.Y., 1986, Fletterick, R and M Zoller,
Editors.
[1751] EVAN88:
[1752] Evans, R M, and S M Hollenberg, "Zinc Fingers: Gilt by
Association", Cell (1988), 52:1-3.
[1753] FAVE89:
[1754] Favel, A, D Le-Nguyen, M A Coletti-Previero, and C Castro,
"Active site chemical mutagenesis of Ecbalium elaterium Trypsin
Inhibitor II: New microproteins inhibiting elastase and
chymotrypsin", Biochem Biophys Res Comm (1989), 162:79-82.
[1755] FERE80c:
[1756] Ferenci, T, "The recognition of maltodextrins by Escherichia
coli", Eur J Biochem (1980), 108:631-6.
[1757] FERE82a:
[1758] Ferenci, T, "Affinity-chromatographic Studies based on the
Binding-specificity of the Lambda Receptor of Escherichia coli",
Ann Microbiol (Inst Pasteur) (1982), 133A:167-169.
[1759] FERE82b:
[1760] Ferenci, T, and K-S Lee, "Directed Evolution of the Lambda
Receptor of Escherichia coli through Affinity Chromatographic
Selection", J Mol Biol (1982), 160:431-444.
[1761] FERE83:
[1762] Ferenci, T, and K S Lee, "Isolation by affinity
chromatography, of mutant Escherichia coli cells with novel
regulation of lamB expression", J Bacteriol (1983),
154:984-987.
[1763] FERE84:
[1764] Ferenci, T, "Genetic manipulation of bacterial surfaces
through affinity-chromatographic selection", Trends in Biological
Science (1984) Vol. ?:44-48.
[1765] FERE86a:
[1766] Ferenci, T, and K-S Lee, "Temperature-Sensitive Binding of
a-Glucans by Bacillus stearothermophilus", J Bacteriol (1986),
166:95-99.
[1767] FERE86b:
[1768] Ferenci, T, M Muir, K-S Lee, and D Maris, "Substrate
specificity of the Escherichia coli maltodextrin transport system
and its component proteins.", Biochimica et Biophysica Acta (1986),
860:44-50.
[1769] FERE89a:
[1770] Ferenci, T, and K S Lee, "Channel architecture in
maltoporin: dominance studies with lamB mutations influencing
maltodextrin binding provide evidence for independent selectivity
filters in each subunit", J Bacteriol (1989) 171(2)855-61.
[1771] FERE89b:
[1772] Ferenci, T, and S Stretton, "Cysteine-22 and cysteine-38 are
not essential for the function of maltoporin (LamB protein)", FEMS
Microbiol Lett (1989), 52(3)335-9.
[1773] FERR90:
[1774] Ferrer-Lopez, P, P Renesto, M Schattner, S Bassot, P
Laurent, and M Chignard, "Activation of human platelets by
C5a-stimulated neutrophils: a role for cathepsin G", American J
Physiology (1990) 258:C1100-C1107.
[1775] FIOR85:
[1776] Fioretti, E, G Iacopino, M Angeletti, D Barra, F Bossa, and
F Ascoli, "Primary Structure and Antiproteolytic Activity of a
Kunitz-type Inhibitor from Bovine Spleen", J Biol Chem (1985),
260:11451-11455.
[1777] FIOR88:
[1778] Fioretti, E, M Angeletti, L Fiorucci, D Barra, F Bossa, and
F Ascoli, "Aprotinin-Like Isoinhibitors in Bovine Organs", Biol
Chem Hoppe-Seyler (1988), 369(Suppl)37-42.
[1779] FRAN87:
[1780] Frankel, A D, J M Berg, and C O Pabo, "Metal-dependent
folding of a single zinc finger from transcription factor IIIA",
Proc Natl Acad Sci USA (1987), 84:4841-45.
[1781] FRAN88:
[1782] Frankel, A, and C O Pabo, "Fingering Too Many Proteins",
Cell (1988), 53:675.
[1783] FRAN89:
[1784] Franconi, G M, P D Graf, S C Lazarus, J A Nadel, G H
Caughey, "Mast Cell Tryptase and Chymase Reverse Airway Smooth
Muscle Relaxation Induced by Vasoactive Intestinal Peptide in the
Ferret", J Pharmacol and Exp Therap (1989), 248(3)947-51.
[1785] FREI90:
[1786] Freimuth, P I, J W Taylor, and E T Kaiser, "Introduction of
Guest Peptides into Escherichia coli Alkaline Phosphatase", J Biol
Chemistry, (Jan. 15, 1990), 265(2)896-901.
[1787] FREU89:
[1788] Freudl, R, H Schwarz, M Degen, and U Henning, "A lower size
limit exists for export of fragments of an outer membrane protein
(OmpA) of Escherichia coli K-121", J Mol Biol (1989),
205(4)771-5.
[1789] FRIT85:
[1790] Fritz, H-J, "The Oligonucleotide-directed Construction of
Mutations in Recombinant Filamentous Phage", DNA Cloning, Editor:
DM Glover, IRL Press, Oxford, UK, 1985.
[1791] GARI84:
[1792] Gariepy, J, P O'Hanley, S A Waldman, F Murad, and G K
Schoolnik, "A common antigenic determinant found in two
functionally unrelated toxins", J Exp Med, (1984),
160(4)1253-8.
[1793] GARI86:
[1794] Gariepy, J, A Lane, F Frayman, D Wilbur, W Robien, G
Schoolnik, and O Jardetzky, "Structure of the Toxic Domain of the
Eshcerichia coli Heat-Stable Enterotoxin ST I", Biochem (1986),
25:7854-7866.
[1795] GARI87:
[1796] Gariepy, J, A K Judd, and G K Schoolnik, "Importance of
disulfide bridges in the structure and activity of Escherichia coli
enterotoxin ST1b", Proc Natl Acad Sci USA (1987), 84:8907-11.
[1797] GAUS87:
[1798] Gauss, P, K B Krassa, D S McPheeters, M A Nelson, and L
Gold, "Zinc(II) and the single-strnaded DNA binding protein of
bacteriophage T4", Proc Natl Acad Sci USA (1987), 84:8515-19.
[1799] GEBH86:
[1800] Gebhard, W, and K Hochstrasser, "Inter-.alpha.-trypsin
inhibitor and its close relatives", in Barret and Salvesen (eds.)
Protease Inhibitors (1986) Elsevier Science Publishers BV
(Biomedical Division) pp.389-401.
[1801] GEBH90:
[1802] Gebhard, W, K Hochstrasser, H Fritz, J J Enghild, S V Pizzo,
and G Salvesen, "Structure of the inter-.alpha.-inhibitor
(inter-.alpha.-trypsin inhibitor) and pre-.alpha.-inhibitor:
current state and proposition of a new terminology", Biol Chem
Hoppe-Seyler (1990), 371,suppl 13-22.
[1803] GEHR87:
[1804] Gehring, K, A Charbit, E Brissaud, and M Hofnung,
"Bacteriophage lambda receptor site on the Escherichia coli K-12
LamB protein", J Bacteriol (1987), 169(5)2103-6.
[1805] GERD84:
[1806] Gerday, C, M Herman, J Olivy, N Gerardin-Otthiers, D Art, E
Jacquemin, A Kaeckenbeeck, and J van Beeumen, "Isolation and
characterization of the Heat Stable enterotoxin for a pathogenic
bovine strain of Escherichia coli", Vet Microbiol (1984),
9:399-414.
[1807] GETZ88:
[1808] Getzoff, E D, H E Parge, D E McRee, and J A Tainer,
"Understanding the Structure and Antigenicity of Gonococcal Pilil",
Rev Infect Dis (1988), l0(Suppl 2)S296-299.
[1809] GIBS88:
[1810] Gibson, T J, J P M Postma, R S Brown, and P Argos, "A model
for the tertiary structure of the 28 residue DNA-binding motif
(`Zinc finger`) common to many eukaryotic transcriptional
regulatory proteins", Protein Engineering (1988), 2(3)209-218.
[1811] GIRA89:
[1812] Girard, T J, L A Warren, W F Novotny, K M Likert, S G Brown,
J P Miletich, and G J Broze Jr, "Functional significance of the
Kunitz-type inhibitory domains of lipoprotein-associated
coagulation inhibitor", Nature (1989), 338:518-20.
[1813] GOLD83:
[1814] Goldenberg, D P, and T E Creighton, "Circular and circularly
permuted forms of bovine pancreatic trypsin inhibitor.", J Mol Biol
(1983), 165(2)407-13.
[1815] GOLD84:
[1816] Goldenberg, D P, and T E Creighton, "Folding Pathway of a
circular Form of Bovine Pancreatic Trypsin Inhibitor", J Mol Biol
(1984), 179:527-45.
[1817] GOLD85:
[1818] Goldenberg, D P, "Dissecting the Roles of Individual
Interactions in Protein Stability: Lessons From a Circularized
Protein", J Cellular Biochem (1985), 29:321-335.
[1819] GOLD87:
[1820] Gold, L, and G Stormo, "Translation Initiation", Volume 2,
Chapter 78, p 1302-1307, Escherichia coli and Salmonella
typhimurium: Cellular and Molecular Biology, Neidhardt, F C,
Editor-in-Chief, Amer Soc for Microbiology, Washington, D.C.,
1987.
[1821] GOLD88:
[1822] Goldenberg, D P, "Kinetic Analysis of the Folding and
Unfolding of a Mutant Form of Bovine Pancreatic Trypsin Inhibitor
Lacking the Cysteine-14 and -38 Thiols", Biochem (1988),
27:2481-89.
[1823] GOTT87:
[1824] Gottesman, S, "Regulation by Proteolysis", Volume 2, chapter
79, p 1308-1312. Escherichia coli and Salmonella typhimurium:
Cellular and Molecular Biology, Neidhardt, F C, Editor-in-Chief,
Amer Soc for Microbiology, Washington, D.C., 1987.
[1825] GRAY81a:
[1826] Gray, W R, A Luque, B M Olivera, J Barrett, and L J Cruz,
"Peptide Toxins from Conus geographicus Venom", J Biol Chem (1981),
256:4734-40.
[1827] GRAY81b:
[1828] Gray, C W, R S Brown, and D A Marvin, "Adsorption Complex of
Filamentous Virus", J Mol Biol (1981), 146:621-627.
[1829] GRAY83:
[1830] Gray, W R, J E Rivier, R Galyean, L J Cruz, and B M Olivera,
"Conotoxin MI. Disulfide bonding and conformational states", J Biol
Chem, (1983), 258(20)12247-51.
[1831] GRAY84:
[1832] Gray, W R, F A Luque, R Galyean, E Atherton, and R C
Sheppard, B L Stone, A Reyes, J Alford, M McIntosh, B M Olivera et
al. "Conotoxin GI: disulfide bridges, synthesis, and preparation of
iodinated derivatives", Biochemistry, (1984), 23(12)2796-802.
[1833] GRAY88:
[1834] Gray, W R, and B M Olivera, "Peptide Toxins from Venomous
Conus Snails", Ann Rev Biochem (1988), 57:665-700.
[1835] GREC79:
[1836] Greco, W R, and M T Hakala, "Evaluation of Methods for
Estimating the Dissociation Constant of Tight Binding Enzyme
Inhibitors", J Biol Chem (1979), 254:12104-109.
[1837] GREE53:
[1838] Green, N M, and E Work, "Pancreatic Trypsin Inhibitor: 2.
Reactions with Trypsin", Biochem J (1953), 54:347-52.
[1839] GUAR89:
[1840] Cuarino, A, R Giannella, and M R Thompson, "Citrobacter
freundii Produces an 18-Amino-Acid Heat-Stable Enterotoxin
Identical to the 18-amino-acid Escherichia coli Heat-Stable
Enterotoxin (ST Ia)", Infection And Immunity (1989),
57(2)649-52.
[1841] GUDM89:
[1842] Gudmundsdottir, A, P E Bell, M D Lundrigan, and C Bradbeer,
and R J Kadner, "Point mutations in a conserved region (TonB box)
of Escherichia coli outer membrane protein BtuB affect vitamin B12
transport", J Bacteriol, (December 1989), 171(12)6526-33.
[1843] GUPT90:
[1844] Gupta, S K, J L Niles, R T McCluskey, M A Arnaout, "Identity
of Wegener's autoantigen (p29) with proteinase 3 and myeloblastin",
Blood (Nov. 15 1990), 76(10)2162.
[1845] GUSS88:
[1846] Guss, J M, E A Merritt, R P Phizackerley, R Hedman, M
Murata, K O Hodgson, H C Freeman, "Phase Determination by
Multiple-Wavelength X-ray Diffraction: Crystal Structure of a Basic
"Blue" Copper Protein from Cucumbers", Science (1988),
241:806-11.
[1847] GUZM87:
[1848] Guzman-Verduzco, L-M, and Y M Kupersztoch, "Fusion of
Escherichia coli Heat-Stable Enterotoxin and Heat-Labile
Enterotoxin B Subunit", J Bacteriol (1987), 169:5201-8.
[1849] GUZM89:
[1850] Guzman-Verduzco, L-M, and Y M Kupersztoch, "Rectification of
Two Escherichia coli Heat-Stable Enterotoxin Allel Sequences and
Lack of Biological Effect of Changing the Carboxy-Terminal Tyrosine
to Histidine", Infection and Immunity (1989), 57(2)645-48.
[1851] GUZM90:
[1852] Guzman-Verduzco, L-M, and Y M Kupersztoch, "Export and
processing analysis of a fusion between the extracellular
heat-stable enterotoxin and the periplasmic B subunti of the
heat-labile enterotoxin in Escherichia coli", Molec Microbiol
(1990), 4:253-64.
[1853] HALL82:
[1854] Hall, M N, M Schwartz, and T J Silhavy, "Sequence
Information within the lamB Gene is Required for Proper Routing of
the Bacteriophage .lambda. Receptor Protein to the Outer Membrane
of Escherichia coli K-12", J Mol Biol (1982), 156:93-112.
[1855] HANC87:
[1856] Hancock, R E W, "Role of Porins in Outer Membrane
Permeability", J Bacteriol (1987), 169:929-33.
[1857] HARD90:
[1858] Hard, T, E Kellenbach, R Boelens, B A Maler, K Dahlman, LP
Freedman, J Carlstedt-Duke, K R Yamamoto, J-A Gustafsson, and R
Kaptein, "Solution Sturcture of the Glucocorticoid Receptor
DNA-Binding Domain", Science (Jul. 13, 1990), 249:157-60.
[1859] HARK86:
[1860] Harkki, A, T R Hirst, J Holmgren, and E T Palva, "Expression
of the Escherichia coli lamB gene in Vibrio cholerae", Microb
Pathog (1986), 1(3)283-8.
[1861] HARK87:
[1862] Harkki, A, H Karkku, and E T Palva, "Use of lambda vehicles
to isolate ompC-lacZ gene fusions in Salmonella typhimurium LT2",
Mol Gen Genet (1987), 209(3)607-11.
[1863] HASH85:
[1864] Hashimoto, K, S Uchida, H Yoshida, Y Nishiuchi, S
Sakakibara, and K Yukari, "Structure-activity relations of
conotoxins at the neuromuscular junction", Eur J Pharmacol (1985),
118(3)351-4.
[1865] HATA90:
[1866] Hatanaka, Y, E Yoshida, H Nakayama, and Y Kanaoka,
"Synthesis of mu-conotoxin GIIIA: a chemical probe for sodium
channels", Chem Pharm Bull (Tokyo), (Jan 1990), 38:236-8.
[1867] HECH90:
[1868] Hecht, M H, J S Richardson, D C Richardson, and R C Ogden,
"De Novo Design, Expression, and Characterization of Felix: A
Four-Helix Bundle Protein of Native-Like Sequence", Science, (Aug.
24, 1990), 249:884-91.
[1869] HEDE89:
[1870] Hedegaard, L, and P Klemm, "Type 1 fimbriae of Escherichia
coli as carriers of heterologous antigenic sequences", Gene, (Dec.
21, 1989), 85(1)115-24.
[1871] HEIJ90:
[1872] Heijne, G von, and C Manoil, "Review: Membrane proteins:
from sequence to structure", Protein Engineering (1990),
4(2)109-112.
[1873] HEIN87:
[1874] Heine, H G, J Kyngdon, and T Ferenci, "Sequence determinants
in the lamB gene of Escherichia coli influencing the binding and
pore selectivity of maltoporin.", Gene (1987), 53:287-92.
[1875] HEIN88:
[1876] Heine, H G, G Francis, K S Lee, and T Ferenci, "Genetic
analysis of sequences in maltoporin that contribute to binding
domains and pore structure.", J Bacteriol (April 1988),
170:1730-8.
[1877] HEIT89:
[1878] Heitz, A, L Chiche, D Le-Nguyen, and B Castro, ".sup.1H 2D
NMR and Distance Geometry Study of the Folding of Ecballium
elaterium Trypsin Inhibitor, a Member of the Squash Inhibitor
Family", Biochem (1989), 28:2392-98.
[1879] HENR87:
[1880] Henriksen, A Z, and J A Maeland, "The Porin Protein of the
Outer Membrane of Escherichia coli: Reactivity in Immunoblotting,
Antibody-binding by the Native Protein, and Cross-Reactivity with
other Enteric Bacteria", Acta path microbiol immunol scand, Sect B
(1987), 95:315-321.
[1881] HIDA90:
[1882] Hidaka, Y, K Sato, H Nakamura, J Kobayashi, Y Ohizumi, and Y
SHimonishi, "Disulfide Pairings in geographutoxin I, a peptide
neurotoxin from Conus geographus", FEBS Lett (1990),
264(1)29-32.
[1883] HILL89:
[1884] Hillyard, D R, B M Olivera, S Woodward, G P Corpuz, W R
Gray, C A Ramilo, L J Cruz, "A Molluscivorus Conus Toxin: Conserved
Framework in Conotoxins", Biochem (1989), 28:358-61.
[1885] HINE80:
[1886] Hines, J C, and D S Ray, "Construction and characterization
of new coliphage M13 cloning vectors.", Gene (1980),
11:(3-4)207-18.
[1887] HOCH84:
[1888] Hoschstrasser, K, and E Wachter, "Elastase inhibitors, a
process for their preparation and medicaments containing these
inhibitors", US Patent 4,485,100 (Nov. 27, 1984).
[1889] HOCJ85:
[1890] Ho, C, M Jasin, and P Schimmel, "Amino acid replacements
that compensate for a large polypeptide deletion in an enzyme",
Science (1985), 229:389-93.
[1891] HOJI82:
[1892] Hojima, Y, J V Pierce, and J J Pisano, "Pumpkin Seed
Inhibitor of Human Factor XIIa (activated Hageman Factor) and
Bovine Trypsin", Biochem (1982), 21:3741-46.
[1893] HOLA89a:
[1894] Holak, T A, D Gondol, J Otlewski, and T Wilusz,
"Determination of the Complete Three-Dimensional Structure of the
Trypsin Inhibitor from Squash Seeds in Aqueous Solution by Nuclear
Magnetic Resonance and a Combination of Distance Geometry and
Dynamic Simulated Annealing", J Mol Biol (1989), 210:635-648.
[1895] HOLA89b:
[1896] Holak, T A, W Bode, R Huber, J Otlewski, and T Wilusz,
"Nuclear magnetic resonance solution and X-ray structures of squash
trypsin inhibitor exhibit the same conformation of the proteinase
binding loop", J Mol Biol (Dec. 5, 1989), 210(3)649-54.
[1897] HORV89:
[1898] Horvat, S, B Grgas, N Raos, and V I Simeon, "Synthesis and
acid ionization constants of cyclic cystine peptides 62
[1899] (n=0-4)", Int J Peptide Protein Res (1989), 34:346-51.
[1900] HOOP87:
[1901] Hoopes, B C, and W R McClure, "Strategies in Regulation of
Transcription Initiation", Volume 2, Chapter 75, p 1231-1240,
Escherichia coli and Salmonella typhimurium: Cellular and Molecular
Biology, Neidhardt, F C, Editor-in-Chief, Amer Soc for
Microbiology, Washington, D.C., 1987.
[1902] HOUG84:
[1903] Houghten, R A, J M Ostresh, and F A Klipstein, "Chemical
synthesis of an octadecapeptide with the biological and
immunological properties of human heat-stable Escherichia coli
enterotoxin", Eur J Biochem (1984), 145:157-162.
[1904] HUBB86:
[1905] Hubbard, R C, and R G Crystal, "Antiproteases and
Antioxidants: Strategies for the Pharmacologic Prevention of Lung
Destruction", Respiration (1986), 50(Suppl 1)56-73.
[1906] HUBB89:
[1907] Hubbard, R C, M A Casolaro, M Mitchell, S E Sellers, F
Arabia, M A Matthay, and R G Crystal, "Fate of aerosolized
recombinant DNA-produced .alpha.-1-antitrypsin: Use of the
epithelial surface of the lower respiratory tract to administer
proteins of therapeutic importance", Proc Natl Acad Sci USA (1989),
86:680-4.
[1908] HUBE74:
[1909] Huber, R, D Kukla, W Bode, P Schwager, K Bartels, J
Deisenhofer, and W Steigemann, "Structure of the Complex formed by
Bovine Trypsin and Bovine Pancreatic Tryspin Inhibitor", J Mol Biol
(1974), 89:73-101.
[1910] HUBE75:
[1911] Huber, R, W Bode, D Kukla, and U Kohl, "The Structure of the
Complex Formed by Bovine Trypsin and Bovine Pancreatic Trypsin
Inhibitor: III. Structure of the Anhydrotrypsin-Inhibitor Complex",
Biophys Struct Mechan (1975), 1:189-201.
[1912] HUBE77:
[1913] Huber, R, W Bode, D Kukla, U Kohl, C A Ryan, "The structure
of the complex formed by bovine trypsin and bovine pancreatic
trypsin inhibitor III. Structure of the anhydro-trypsin-inhibitor
complex.", Biophys Struct Mech (1975), 1(3)189-201.
[1914] HUTC87:
[1915] Hutchinson, D C S, "The role of proteases and antiproteases
in bronchial secretions", Eur J Respir Dis (1987),
71(Suppl.153)78-85.
[1916] HYNE90:
[1917] Hynes, T R, M Randal, L A Kenedy, C Eigenbrot, and A A
Kossiakoff, "X-ray crystal structure of the protease inhibitor
domain of Alzheimer's amyloid beta-protein precursor", Biochemistry
(1990), 29:10018-10022.
[1918] ILIC89:
[1919] Il'ichev, AA, O O Minenkova, S I Tat'kov, N N Karpyshev, A M
Eroshkin, V A Petrenko, and L S Sandakhchiev, "[Production of a
viable variant of the M13 phage with a foreign peptide inserted
into the basic coat protein]<Original>Poluchenie
zhiznesposobnogo varianta faga M13 so vstroennym chuzherodnym
peptidom v osnovnoi belok obolochki", Dokl Akad Nauk SSSR, (1989),
307(2)481-3.
[1920] INOU82:
[1921] Inouye, H, W Barnes, and J Beckwith, "Signal Sequence of
Alkaline Phosphatase of Escherichia coli", J Bacteriol (1982),
149(2)434-439.
[1922] INOU86:
[1923] Inouye, M, and R Sarma, Editors, Protein Engineering:
Applications in Science, Medicine, and Industry., Academic Press,
New York, 1986.
[1924] ITOK79:
[1925] Ito, K, G Mandel, and W Wickner, "Soluble precursor of an
integral membrane protein: Synthesis of procoat protein in
Escherichia coli infected with bacteriophage M13.", Proc Natl Acad
Sci USA (1979), 76:1199-1203.
[1926] JANA89:
[1927] Janatova, J, K B M Reid, and A C Willis, "Disulfide Bonds
Are Localized within the Short Consensus Repeat Units of Complement
Regulatory Proteins: C4b-Binding Protein", Biochem (1989),
28:4754-61.
[1928] JANI85:
[1929] Janin, J, and C Chothia, "Domains in Proteins: Definitions,
Location, and Structural Principles", Methods in Enzymology (1985),
115(28)420-430.
[1930] JENN89:
[1931] Jennings, P A, M M Bills, D O Irving, and J S Mattick,
"Fimbriae of Bacteroides nodosus: protein engineering of the
structural subunit for the production of an exogenous peptide",
Protein Eng, (January 1989), 2(5)365-9.
[1932] JERI74a,
[1933] Jering, H, and H Tschesche, "Replacement of Lysine by
Arginine, Phenylalanine, and Tryptophan in the Reactive Site of the
Trypsin-Kallikrein Inhibitor (Kunitz)", Angew Chem internat Edit
(1974), 13:662-3.
[1934] JERI76b:
[1935] Jering, H, and H Tschesche, "Replacement of Lysine by
Arginine, Phenylalanine, and Tryptophan in the Reactive Site of the
Bovine Trypsin-Kallekrein Inhibitor (Kunitz) and Change of the
Inhibitory Properties", Eur J Biochem (1976), 61:453-63.
[1936] JOUB84:
[1937] Joubert, F J, "Trypsin Isoinhibitors from Momordica Repens
Seeds", Phytochemistry (1984), 23:1401-6.
[1938] JUDD85:
[1939] Judd, R C, "Structure and surface exposure of protein IIs of
Neisseria gonorrhoeae JS3", Infect Immun (1985), 48(2)452-7.
[1940] JUDD86:
[1941] Judd, R C, "Evidence for N-terminal exposure of the protein
IA subclass of Neisseria gonorrhoeae protein I", Infect Immun
(1986), 54(2)408-14.
[1942] KABS84:
[1943] Kabsch, W, and C Sander, "On the use of sequence homologies
to predict protein structure: identical pentapeptides can have
completely different conformations", Proc Natl Acad Sci USA (1984),
81(4)1075-8.
[1944] KAIS87a:
[1945] Kaiser, C A, D Preuss, P Grisafi, and D Botstein, "Many
Random Sequences Functionally Replace the Secretion Signal Sequence
of Yeast Invertase", Science (1987), 235:312-7.
[1946] KAOR88:
[1947] Kao, R C, N G Wehner, K M Skubitz, B H Gray, and J R Hoidal,
"Proteinase 3, A Distinct Human Polymorphonuclear Leukocyte
Proteinase that Produces Emphysema in Hamsters", J Clin Invest
(1988), 82:1963-73.
[1948] KAPL78:
[1949] Kaplan, D A, L Greenfield, and G Wilcox, "Molecular Cloning
of Segments of the M13 Genome.", in The Single-Stranded DNA Phages,
Denhardt, D T, D Dressler, and D S Ray editors, Cold Spring Harbor
Laboratory, 1978., p461-467.
[1950] KATZ86:
[1951] Katz, B A, and A Kossiakoff, "The Crystallographically
Determined Structures of Atypical Stained Disulfides Engineered
into Subtilisin", J Biol Chem (1986), 261(33)15480-85.
[1952] KATZ90:
[1953] Katz, B, and A A Kossiakoff, "Crystal Structures of
Subtilisin BPN' Variants Containing Disulfide Bonds and Cavities:
Concerted Structural Rearrangements Induced by Mutagenesis",
Proteins, Struct, Funct, and Genet (1990), 7:343-57.
[1954] KAUM86:
[1955] Kaumerer, J F, J O Polazzi, and M P Kotick, "The mRNA for a
proteinase inhibitor related to the HI-30 domain of
inter-.alpha.-trypsin inhibitor also encodes
.alpha..sub.1-microglobulin (protein HC)", Nucleic Acids Res
(1986), 14:7839-7850.
[1956] KIDO88:
[1957] Kido, H, Y Yokogoshi, and N Katunuma, "Kunitz-type Protease
Inhibitor Found in Rat Mast Cells", J Biol Chem (1988),
263:18104-7.
[1958] KIDO90:
[1959] Kido, H, A Fukutomi, J Schelling, Y Wang, B Cordell, and N
Katunuma, "Protease-Specificity of Kunitz Inhibitor Domain of
Alzheimer's Disease Amyloid Protein Precursor", Biochem &
Biophys Res Comm (Mar. 16, 1990), 167(2)716-21.
[1960] KING86:
[1961] King, T C, R Sirdeskmukh, and D Schlessinger, "Nucleolytic
processing of ribonucleic acid transcripts in procaryotes",
Microbiol Rev (1986), 50(4)428-51.
[1962] KISH85:
[1963] Kishore, R, and P Balaram, "Stablization of gamma-Turn
Conformations in Peptides by Disulfide Bridges", Biopolymers
(1985), 24:2041-43.
[1964] KOBA89:
[1965] Kobayashi, Y, T Ohkubo, Y Kyogoku, Y Nishiuchi, S
Sakakibara,--W Braun, nad N Go, "Solution Conformation of Conotoxin
GI Determined by .sup.1H Nuclear Magnetic Resonance Spectroscopy
and Distance Geometry Calculations", Biochemistry (1989),
28:4853-60.
[1966] KUBO89:
[1967] Kubota, H, Y Hidaka, H Ozaki, H Ito, T Hirayama, Y Takeda,
and Y Shimonishi, "A Long-acting Heat-Stable Enterotoxin Analog of
Enterotoxigenic Esherichia coli with a Single D-Amino Acid.",
Biochem Biophys Res Comm (1989), 161:229-235.
[1968] KUHN85a:
[1969] Kuhn, A, and W Wickner, "Conserved Residues of the Leader
Peptide Are Essential for Cleavage by Leader Peptidase.", J Biol
Chem (1985), 260:15914-15918.
[1970] KUHN85b:
[1971] Kuhn, A, and W Wickner, "Isolation of Mutants in M13 Coat
Protein That Affect Its Synthesis, Processing, and Assembly into
Phage.", J Biol Chem (1985), 260:15907-15913.
[1972] KUHN87:
[1973] Kuhn, A, "Bacteriophage M13 Procoat Protein Inserts into the
Plasma Membrane as a Loop Structure.", Science (1987),
238:1413-1415.
[1974] KUHN88:
[1975] Kuhn, A, "Alterations in the extracellular domain of M13
procoat protein make its membrane insertion dependent on secA and
secy", Eur J Biochem (1988), 177(2)267-71.
[1976] KUKS89:
[1977] Kuks, P F M, C Creminon, A-M Leseney, J Bourdais, A Morel,
and P Cohen, "Xenopus laevis Skin
Arg-Xaa-Val-Arg-Gly-endoprotease", J Biol Chem (1989),
264(25)14609-12.
[1978] KUOM90:
[1979] Kuo, M D, S S Huang, and J S Huang, "Acidic fibroblast
growth factor receptor purified from bovine liver is a novel
protein tyrosine kinase." J Biol Chem (1990), 265(27)16455-63.
[1980] KUPE90:
[1981] Kupersztoch, Y M, K Tachias, C R Moomaw, L A Dreyfus, R
Urban, C Slaughter, and S Whipp, "Secretion of Methanol-Insoluble
Heat-Stable Enterotoxin (STB): Energy- and secA-Dependent
Conversion of Pre-STB to an Intermediate Indistingurisable from the
Extracellular Toxin", J Bacteriol (1990), 172(5)2427-32.
[1982] LAMB90:
[1983] Lambert, P, H Kuroda, N Chino, TX Watanabe, T Kimura, and S
Sakakibara, "Solution Synthesis of Charybdotoxin (ChTX), A K.sup.+
Channel Blocker", Biochem Biophys Res Comm (1990),
170(2)684-690.
[1984] LAND87:
[1985] Landick, R, and C Yanofsky, "Transcription Attenuation",
Volume 2, Chapter 77, p 1276-1301, Escherichia coli and Salmonella
typhimurium: Cellular and Molecular Biology, Neidhardt, F C,
Editor-in-Chief, Amer Soc for Microbiology, Washington, D.C.,
1987.
[1986] LASK80:
[1987] Laskowski, M, Jr, and I Kato, "Protein Inhibitors of
Proteases", Ann Rev Biochem (1980), 49:593-626.
[1988] LAZU83:
[1989] Lazure, C, NG Seidah, M Chretien, R Lallier, and S
St-Pierre, "Primary structure determination of Escherichia coli
heat-stable enterotoxin of porcine origin", Canadian J Biochem Cell
Biol (1983), 61:287-92.
[1990] LECO87:
[1991] Lecomte, J T J, D Kaplan, M Llinas, E Thunberg, and G
Samuelsson, "Proton Magnetic Resonance Characterization of
Phoratoxins and Homologous Proteins Related to Crambin",
Biochemistry (1987), 26:1187-94.
[1992] LEEB71:
[1993] Lee, B, and FM Richards, "The interpretation of protein
structures: estimation of static accessibility.", J Mol Biol
(1971), 55:(3)379-400,
[1994] LEEC83:
[1995] Lee, C H, S L Moseley, H W Moon, S C Whipp, C L Gyles, and M
So, "Characterization of the Gene Encoding Heat-Stable Toxin II and
Preliminary Molecular Epidemiological Studies of Enterotoxigenic
Escherichia coli Heat-Stable Toxin II Producers", Infection and
Immunity (1983), 42:264-268.
[1996] LEEC86:
[1997] Lee, C, and J Beckwith, "Cotranssational and
Posttranslational Protein Translocation in Prokaryotic Systems.",
Ann Rev Cell Biol (1986), 2:315-336.
[1998] LENG89b:
[1999] Le-Nguyen, D, D Nalis, and B Castro, "Solid phase synthesis
of a trypsin inhibitor isolated from the Cucurbitaceae Ecballium
elaterium", Int J Peptide Protein Res (1989), 34:492-97.
[2000] LISS85:
[2001] Liss, L R, B L Johnson, and D B Oliver, "Export defect
adjacent to the processing site of staphylococcal nuclease is
suppressed by a prlA mutation", J Bacteriol (1985),
164(2)925-8.
[2002] LOPE85a:
[2003] Lopez, J, and R E Webster, "Assembly site of bacteriophage
f1 corresponds to adhesion zones between the inner and outer
membranes of the host cell", J Bacteriol (1985), 163(3)1270-4.
[2004] LOPE85b:
[2005] Lopez, J, and R E Webster, "fipB and fipC: two bacterial
loci required for morphogenesis of the filamentous bacteriophage
f1", J Bacteriol (1985), 163(3)900-5.
[2006] LOSI86:
[2007] Losick, R, P Youngman, and P J Piggot, "Genetics of
Endospore formation in Bacillus subtilis", Ann Rev Genet (1986),
20:625-669.
[2008] LUGT83:
[2009] Lugtenberg, B, and L van Alphen, "Molecular Architecture and
Function-of the-Outer Membrane of Escherichia coli and other
Gram-Negative Bacteria", Biochim Biophys Acta (1983),
737:51-115.
[2010] LUIT83:
[2011] Luiten, R G M, J G G Schoenmakers, and R N H Konings, "The
major coat protein gene of the filamentous Pseudomonas aeruginosa
phage Pf3: absence of an N-terminal leader signal sequence",
Nucleic Acids Research (1983), 11(22)8073-85.
[2012] LUIT85:
[2013] Luiten, R G M, D G Putterman, J G G Schoenmakers, R N H
Konings, and L A Day, "Nucleotide Sequence of the Genome of Pf3, an
IncP-1 Plasmid-Specific Filamentous Bacteriophage of Pseudomonas
aeruginosa", J Virology, (1985), 56(1)268-276.
[2014] LUIT87:
[2015] Luiten, R G M, R I L Eggen, J G G Schoenmakers, and R N H
Konings, "Spontaneous Deletion Mutants of Bacteriophage Pf3:
Mapping of Signals Involved in Replication and Assembly", DNA
(1987), 6(2)129-37.
[2016] LUND86:
[2017] Lundeen, M, "Preferences of the Side Chains in Proteins for
Helix, Beta Strand, Turn, and Other Conformations. Secondary
Structures of Copper Proteins", J inorgan Biochem (1986),
27:151-62.
[2018] MACH89:
[2019] Machleidt, W, U Thiele, B Laber, I Assfalg-Machleidt, A
Esterl, G Wiegand, J Kos, V Turk, and W Bode, "Mechanism of
inhibition of papain by chicken egg white cystatin", FEBS Lett
(1989), 243(2)234-8.
[2020] MACI88:
[2021] MacIntyre, S, R Freudl, ML Eschbach, and U Henning, "An
artificial hydrophobic sequence functions as either an anchor or a
signal sequence at only one of two positions within the Escherichia
coli outer membrane protein OmpA", J Biol Chem (1988),
263(35)19053-9.
[2022] MAKO80:
[2023] Makowski, L, D L D Caspar, and DA Marvin, "Filamentous
Bacteriophage Pf1 Structure Determined at 7 A Resolution by
Refinement of Models for the alpha-Helical Subunit.", J Mol Biol
(1980), 140:149-181.
[2024] MALA64:
[2025] Malamay, M H, and B L Horecker, "Release of alkaline
phosphotase from cells of E. coli upon lysozyme spheroplast
formation", Biochem (1964), 3:1889-1893.
[2026] MANI82:
[2027] Maniatis, T, E F Fritsch, and J Sambrook, Molecular Cloning,
Cold Spring Harbor Laboratory, 1982.
[2028] MANO86:
[2029] Manoil, C, and J Beckwith, "A Genetic Approach to Analyzing
Membrane Protein Topology", Science (1986), 233:1403-1408.
[2030] MANO88:
[2031] Manoil, C, D Boyd, and J Beckwith, "Molecular genetic
analysis of membrane protein topology", Topics in Genetics (1988),
4(8)223-6.
[2032] MARK86:
[2033] Marks, CB, M Vasser, P Ng, W Henzel, and S Anderson,
"Production of native, correctly folded bovine pancreatic trypsin
inhibitor in Escherichia coli", J Biol Chem (1986),
261:7115-7118.
[2034] MARK87:
[2035] Marks, C B, H Naderi, P A Kosen, I D Kuntz, and S Anderson,
"Mutants of Bovine Pancreatic Trypsin Inhibitor Lacking Cysteines
14 and 38 Can Fold Properly", Science (1987), 235:1370-1373.
[2036] MARQ83:
[2037] Marquart, M, J Walter, J Deisinhoffer, W Bode, and R Huber,
"The geometry of the reactive site and of the peptide groups in
trypsin, trypsinogen, and its complexes with inhibitors", Acta
Cryst, B (1983), 39:480ff.
[2038] MARV75:
[2039] Marvin, D A and E J Wachtel, "Structure and assembly of
filamentous bacterial viruses", Nature (1975), 253:19-23.
[2040] MARV78:
[2041] Marvin, D A, "Structure of the Filamentous Phage Virion.",
in The Single-Stranded DNA Phages, Denhardt, D T, D Dressler, and D
S Ray editors, Cold Spring Harbor Laboratory, 1978., p583-603.
[2042] MARV80:
[2043] Marvin, D, and L Makowski, "Helical Viruses", Progr Clin
Biol Res (1980), 40:347-48.
[2044] MASS90:
[2045] Massefski, W, Jr, A G Redfield, D R Hare, and C Miller,
"Molecular Structure of Charybdotoxin, a Pore-Directed Inhibitor of
Potassium Ion Channels", Science (Aug 3, 1990), 249:521-524.
[2046] MATS89:
[2047] Matsumura, M, W J Becktel, M Levitt, and B W Matthews,
"Stabilization of phage T4 lysozyme by engineered disulfide bonds",
Proc Natl Acad Sci USA (1989), 86:6562-6.
[2048] MCCA90:
[2049] McCafferty, J, A D Griffiths, G Winter, and D J Chiswell,
"Phage antibodies: filamintous phage displaying antibody variable
domains", Nature, (Dec. 6, 1990), 348:552-4.
[2050] MCKE85:
[2051] McKern, N M, I J O'Donnell, D J Stewart, and B L Clark,
"Primary structure of pilin protein from Bacteroides nodosus strain
216: comparison with the corresponding protein from strain 198", J
Gen Microbiol (1985), 131(Pt 1)1-6.
[2052] MCPH85:
[2053] McPhalen, C A, H P Schnebli, and M N G James, "Crystal and
molecular structure of the inhibitor eglin from leeches in complex
with subtilisin Carlsberg", FEBS Lett (1985), 188(1)55-8.
[2054] MCWH89:
[2055] McWherter, C A, W F Walkenhorst, E J Campbell, and G I
Glover, "Novel Inhibitors of Human Leukocyte Elastase and Cathepsin
G. Sequence Variants of Squash Seed Protease Inhibitor with Altered
Protease Selectivity", Biochemistry (1989), 28:5708-14.
[2056] MEDV89:
[2057] Medved, L V, T F Busby, and K C Ingham, "Calorimetric
Investigation of the Domain Structure of Human Complement
C1s{overscore (:)} Reversible Unfolding of the Short Consensus
Repeat Units", Biochem (1989), 28:5408-14.
[2058] MESS77:
[2059] Messing, J, B Gronenborn, B Muller-Hill, and P H
Hofschneider, "Filamentous coliphage M13 as a cloning vehicle:
insertion of a HindII fragment of the lac regulatory region in M13
replicative form in vitro.", Proc Natl Acad Sci USA (1977),
74:3642-6.
[2060] MESS78:
[2061] Messing, J, and B Gronenborn, "The Filamentous Phage M13 as
a Carrier DNA for Operon Fusions In Vitro.", in The Single-Stranded
DNA Phages, Denhardt, D T, D Dressler, and D S Ray editors, Cold
Spring Harbor Laboratory, 1978.,p449-453.
[2062] MILL87a:
[2063] Miller, S, J Janin, A M Lesk, and C Chothia, "Interior and
Surface Monomeric Proteins", J Mol Biol (1987), 196:641-656.
[2064] MILL87b:
[2065] Miller, E S, J Karam, M Dawson, M Trojanowska, P Gauss, and
L Gold, "Translational repression: biological activity of
plasmid-encoded bacteriophage T4 RegA protein.", J Mol Biol (1987),
194:397-410.
[2066] MISR88a:
[2067] Misra, R, and SA Benson, "Genetic identification of the pore
domain of the OmpC porin of Escherichia coli K-12", J Bacteriol
(1988), 170(8)3611-7.
[2068] MISR88b:
[2069] Misra, R, and S A Benson, "Isolation and Characterization of
OmpC Porin Mutants with Altered Pore Properties", J Bacteriol
(1988), 170:528-33.
[2070] MOLL89:
[2071] Molla, A, A Charbit, A Le Guern, A Ryter, and M Hofnung,
"Antibodies against synthetic peptides and the-topology of LamB, an
outer membrane protein from Escherichia coli K12", Biochem (1989),
28(20)8234-41.
[2072] MORS87:
[2073] Morse, S A, T A Mietzner, G Bolen, A Le Faou, and G
Schoolnik, "Characterization of the major iron-regulated protein of
Neisseria gonorrhoeae and Neisseria meningitidis", Antonie Van
Leeuwenhoek (1987), 53(6)465-9.
[2074] MORS88:
[2075] Morse, S A, C-Y Chen, A LeFaou, and T A Meitzner, "A
Potential Role for the Major Iron-Regulated Protein Expressed by
Pathogenic Neisseria Species", Rev Infect Dis (1988), 10(Suppl
2)S306-10.
[2076] MOSE82:
[2077] Moses, P B, and K Horiuchi, "Effects of Transposition and
Delection upon Coat Protein Gene Expression in Bacteriophage f1",
Virology (1982), 119:231-244.
[2078] MOSE83:
[2079] Moser, R, R M Thomas, and B Gutte, "An Artificial
Crystalline DDT-binding polypeptide", FEBS Letters (1983),
157:247-251.
[2080] MOSE85:
[2081] Moser, R, S Klauser, T Leist, H Langen, T Epprecht, and B
Gutte, "Applications of Synthetic Peptides", Angew Chemie, Int
Edition English (1985), 24(9)719-27.
[2082] MOSE87:
[2083] Moser, R, S Frey, K Muenger, T Hehlgans, S Klauser, H
Langen, E-L Winnacker, R Mertz, and B Gutte, "Expression of the
synthetic gene of an artificial DDT-binding polypeptide in
Escherichia coli", Protein Engineering (1987), 1:339-343.
[2084] NADE87:
[2085] Nadel, J A, and B Borson, "Secretion and ion transport in
airways during inflammation", Biorheology (1987), 24:541-549.
[2086] NADE90:
[2087] Nadel, J A, "Neutrophil Proteases and Mucus Secretion", 1990
Cystic Fibrosis Meeting, Arlington, Va., p156.
[2088] NAKA81:
[2089] Nakashima, Y, B Frangione, R L Wiseman, W H Konigsberg,
"Primary Structure of the Major Coat Protein of the Filamentous
Bacterial Viruses, If1 and Ike", J Biol Chem (1981),
256(11)5792-7.
[2090] NAKA86a:
[2091] Nakae, T, J Ishii, and T Ferenci, "The Role of the
Maltodextrin-binding Site in Determining the Transport Properties
of the LamB Protein", J Biol Chem (1986), 261:622-26.
[2092] NAKA86b:
[2093] Nakae, T, "Outer-Membrane Permeability of Bacteria", CRC
Crit Rev Microbiol (1986), 13:1-62.
[2094] NAKA87:
[2095] Nakamura, T, T Hirai, F Tokunaga, S Kawabata, and S Iwanaga,
"Purification and Amino Acid Sequence of Kunitz-type Protease
Inhibitor Found in the Hemocytes of Horseshoe Crab (Tachypleus
tridentatus)", J Biochem (1987), 101:1297-1306.
[2096] NICH88:
[2097] Nicholson, H, W J Becktel, and B W MAtthews, "Enhanced
protein thermostability from desgined mutations that interact with
a-helix dipoles", Nature (1988), 336:651-56.
[2098] NIKA84:
[2099] Nikaido, H, and H C P Wu, "Amino acid sequence homology
among the major outer membrane proteins of Escherichia coli", Proc
Natl Acad Sci USA (1984), 81:1048-52.
[2100] NILE89:
[2101] Niles, J L, R T McCluskey, M F Ahmad, and M A Arnaout,
"Wgener's Granulomatosis Autoantigen Is a Novel Neutrophil Serine
Proteinase", Blood (1989), 74(6)1888-93.
[2102] NISH82:
[2103] Nishiuchi, Y, and S Sakakibara, "Primary and secondary
structure of conotoxin GI, a neurotoxic tridecapeptide from a
marine snail", FEBS Lett (1982), 148:260-2.
[2104] NISH86:
[2105] Nishiuchi, Y, K Kumagaye, Y Noda, T X Watanabe, and S
Sakakibara, "Synthesis and secondary-structure determination of
omega-conotoxin GVIA: a 27-peptide with three intramolecular
disulfide bonds", Biopolymers, (1986), 25:S61-8.
[2106] NORR89a:
[2107] Norris, K, and L C Petersen, "Aprotinin analogues and
process for the production thereof", European Patent Application 0
339 942 A2.
[2108] NORR89b:
[2109] Norris, K, F Norris, S BJorn, "Aprotinin Homologues and
Process for the Production of Aprotinin and aprotinin homologues in
Yeast", PCT patent application W089/01968.
[2110] OAST88:
[2111] Oas, T G, and P S Kim, "A peptide model of a protein folding
intermediate", Nature (1988), 336:42-48.
[2112] ODOM90:
[2113] Odom, L, "Inter-.alpha.-trypsin inhibitor: a plasma
proteinase inhibitor with a unique chemical structure", Int J
Biochem (1990), 22:925-930.
[2114] OHKA81:
[2115] Ohkawa, I, and R E Webster, "The Orientation of the Major
Coat Protein of Bacteriophage f1 in the Cytoplasmic Membrane of
Esherichia coli.", J Biol Chem (1981), 256:9951-9958.
[2116] OKAM87:
[2117] Okamoto, K, K Okamoto, J Yukitake, Y Kawamoto, and A Miyama,
"Substitutions of Cysteine Residues of Escherichia coli Heat-Stable
Enterotoxin by Oligonucleotide-Directed Mutagenesis", Infection and
Immunity (1987), 55:2121-2125.
[2118] OKAM88:
[2119] Okamoto, K, K Okamoto, J Yukitake, and A Miyama, "Reduction
of Enterotoxic Activity of Escherichia coli Heat-Stable Enterotoxin
by Substitution for an Aspartate Residue", Infection and Immunity
(1988), 56:2144-8.
[2120] OKAM90:
[2121] Okamoto, K, and M Takahara, "Synthesis of Escherichia coli
Heat-Stable Enterotoxin STp as a Pre-Pro Form and Role of the Pro
Sequence in Secretion", J Bacteriol (1990), 172(9)5260-65.
[2122] OLIP86:
[2123] Oliphant, AR, AL Nussbaum, and K Struhl, "Cloning of
random-sequence oligodeoxynucleotides", Gene (1986),
44:177-183.
[2124] OLIP87:
[2125] Oliphant, AR, and K Struhl "The Use of Random-Sequence
Oligonucleotides for Determining Consensus Sequences", in Methods
in Enzymolocy 155 (1987)568-582. Editor Wu, R; Academic Press, New
York.
[2126] OLIV85a:
[2127] Oliver, D, "Protein Secretion in Escherichia coli.", Ann Rev
Microbiol (1985), 39:615-648.
[2128] OLIV85b:
[2129] Olivera, B M, W R Gray, R Zeikus, J M McIntosh, J Varga, J
Rivier, V de Santos, and L J Cruz, "Peptide Neurotoxins from Fish
Hunting Cone Snails", Science (1985), 230:1338-43.
[2130] OLIV87b:
[2131] Olivera, B M, L J Cruz, V de Santos, G W LeCheminant, D
Griffin, R Zeikus, J M McIntosh, R Galyean, J Varga, W R Gray, et
al. "Neuronal calcium channel antagonists. Discrimination between
calcium channel subtypes using omega-conotoxin from Conus magus
venom", Biochemistry, (1987), 26(8)2086-90.
[2132] OLIV90a:
[2133] Olivera, B M, J Rivier, C Clark, C A Ramilo, G P Corpuz, F C
Abogadie, E E Mena, S R Woodward, D R Hillyard, L J Cruz,
"Diversity of Conus Neuropeptides", Science, (Jul. 20, 1990),
249:257-263.
[2134] OLIV90b:
[2135] Olivera, B M, D R Hillyard, J Rivier, S Woodward, W R Gray,
G Corpuz, L J Cruz, "Conotoxins: Targeted Peptide Ligands from
Snail Venoms", Chapter 20 in Marine Topxins, American Chemical
Society, 1990.
[2136] OLTE89:
[2137] Oltersdorf, T, L C Fritz, D B Schenk, I Lieberburg, K L
Johnson-Wood, E C Beattie, P J Ward, R W Blacher, H F Dovey, and S
Sinha, "The Secreted form of the Alzheimer's amyloid precursor
protein with the Kunitz domain is protease nexin-II", Nature
(1989), 341:144-7.
[2138] ORND85:
[2139] Orndorff, P E, and S Falkow, "Nucleotide Sequence of pilA,
the Gene Encoding the Structural Component of Type 1 Pili in
Escherichia coli", J Bacteriol (1985), 162:454-7.
[2140] OTLE85:
[2141] Otlewski, J, and T Wilusz, "The Serine Proteinase Inhibitor
from Summer Squash (Cucurbita pepo): Some Structural Features,
Stability and Proteolytic Degradation", Acta Biochim Polonica
(1985), 32(4)285-93.
[2142] OTLE87:
[2143] Otlewski, J, H Whatley, A Polanowski, and T Wilusz,
"Amino-Acid Sequences of Trypsin Inhibitors from Watermelon
(Citrullus vulgaris) and Red Bryony (Bryonia dioica) Seeds", Biol
Chem Hoppe-Seyler (1987), 368:1505-7.
[2144] PABO79:
[2145] Pabo, C O, R T Sauer, J M Sturtevant, and M Ptashne, "The
Lambda Repressor Contains Two Domains.", Proc Natl Acad Sci USA
(1979), 76:1608-1612.
[2146] PABO86:
[2147] Pabo, C O, and E G Suchanek, "Computer-Aided Model Building
Strategies for Protein Design", Biochem (1986), 25:5987-91.
[2148] PAGE88:
[2149] Pages, J M, and J M Bolla, "Assembly of the OmpF porin of
Escherichia coli B. Immunological and kinetic studies of the
integration pathway", Eur J Biochem (1988), 176(3)655-60.
[2150] PAGE90:
[2151] Pages, J M, J M Bolla, A Bernadac, and D Fourel,
"Immunological approach of assembly and topology of OmpF, an outer
membrane protein of Escherichia coli", Biochimie (1990),
72:169-76.
[2152] PAKU86:
[2153] Pakula, A A, V B Young, and R T Sauer, "Bacteriophage
.lambda. cro mutations: Effects on activity and intracellular
degradation.", Proc Natl Acad Sci USA (1986), 83:8829-8833.
[2154] PANT87:
[2155] Pantoliano, M W, R C Ladner, P N Bryan, M L Rollence, J F
Wood, and T L Poulos, "Protein Engineering of Subtilisin BPN':
Enhanced Stabilization through the Introduction of Two Cysteines To
Form a Disulfide Bond", Biochem (1987), 26:2077-82.
[2156] PANT90:
[2157] Pantoliano, M W, and R C Ladner, "Computer Designed
Stabilized Proteins and Method for Producing Same", U.S. Pat. No.
4,908,773, Mar. 13, 1990.
[2158] PAOL86:
[2159] Paoletti, E, and D Panicali, "Modified Vaccinia Virus", U.S.
Pat. No. 4,603,112, Jul. 29, 1986.
[2160] PAPA82:
[2161] Papamokos, E, E Weber, W Bode, R Huber, M Empie, I Kato, and
M Laskowski Jr, "Crystallographic Refinement of Japanese Quail
Ovomucoid, a Kazal-type Inhibitor, and Model Building Studies of
Complexes with Serine Proteases", J Mol Biol (1982),
158:515-537.
[2162] PARD89:
[2163] Pardi, A, A Galdes, J Florance, and D Maniconte, "Solution
Structres of .alpha.-Conotoxin G1 Determined by Two-Dimensional NMR
Spectroscopy", Biochemistry (1989), 28:5494-5501.
[2164] PARG87:
[2165] Parge, H E, D E McRee, M A Capozza, S L Bernstein, E D
Getzoff, and J A Tainer, "Three dimensional structure of bacterial
pili", Antonie Van Leeuwenhoek (1987), 53(6)447-53.
[2166] PARM88:
[2167] Parmley, S F, and G P Smith, "Antibody-selectable
filamentous fd phage vectors: affinity purification of target
genes", Gene (1988), 73:305-318.
[2168] PARR88:
[2169] Parraga, G, S J Horvath, A Eisen, W E Taylor, L Hood, E T
Young, R E Klevit, "Zinc-Dependent Structures of a Single-Finger
Domain of Yeast ADR1", Science (1988), 241:1489-92.
[2170] PEAS88:
[2171] Pease, J H B, and D E Wemmer, Biochem (1988),
27:8491-99.
[2172] PEAS90:
[2173] Pease, J H B, R W Storrs, and D E Wemmer, "Folding and
activity of hybrid sequence, disuylfide-stabilized peptides", Proc
Natl Acad Sci USA (1990), 87:5643-47.
[2174] PEET85:
[2175] Peeters, B P H, R M Peters, J G G Schoenmakers, and R N H
Konings, "Nucleotide Sequence and Genetic Organization of the
Genome of the N-Specific Filamentous Bacteriophage Ike: Comparison
with the Genome of the F-Specific Filamentous Phages M13, fd, and
f1", J Mol Biol (1985), 181:27-39.
[2176] PEET87:
[2177] Peeters, B P H, J G G Schoenmakers, and R N H Konings,
"Comparison of the DNA Sequences Involved in Replication and
Packaging of the Filamentous Phages IKe, and Ff (M13, fd, and f1)",
DNA (1987), 6(2)139-147.
[2178] PERR84:
[2179] Perry, L J, and R Wetzel, "Disulfide Bond Engineered into T4
Lysozyme: Stablilation of the Protein Toward Thermal Inactivation",
Science (1984), 226:555-7.
[2180] PERR86:
[2181] Perry, L J, and R Wetzel, "Unpaired Cysteine-54 Interferes
with the Ability of an Engineered Disulfide To Stabilize T4
Lysozyme", Biochem (1986), 25:733-39.
[2182] PETE89:
[2183] Peterson, M W, "Neutrophil cathepsin G increases
transendothelial albumin flux", J Lab Clin Med (1989),
113(3)297-308.
[2184] PONT88:
[2185] Ponte, P, P Gonzalez-DeWhitt, J Schilling, J Miller, D Hsu,
B Greenberg, K Davis, W Wallace, I Liederburg, F Fuller, and B
Cordell, "A new A4 amyloid mRNA contains a domain homologous to
serine proteinase inhibitors", Nature (1988), 331:525-7.
[2186] POTE83:
[2187] Poteete, A R, "Domain Structure and Quaternary Organization
of the Bacteriophage P22 Erf Protein.", J Mol Biol (1983),
171:401-418.
[2188] QUIO87:
[2189] Quiocho, F A, N K Vyas, JS Sack and M A Storey, "Periplasmic
Binding Proteins: Structure and New Understanding of Protein-Ligand
Interactions.", in Crystallography in Molecular Biology, Moras, D.
et al., editors, Plenum Press, 1987.
[2190] RAND87:
[2191] Randall, L L, S J S Hardy, and J R Thom, "Export of Protein:
A Biochemical View", Ann Rev Microbiol (1987), 41:507-41.
[2192] RASC86:
[2193] Rasched, I, and E Oberer, "Ff Coliphages: Structural and
Functional Relationships", Microbiol Rev (1986) 50:401-427.
[2194] RASH84:
[2195] Rashin, A, "Prediction of Stabilities of Thermolysin
Fragments", Biochemistry (1984), 23:5518.
[2196] RAYC87:
[2197] Ray, C, K M Tatti, C H Jones, and C P Moran Jr, "Genetic
Analysis of RNA Polymerase-Promoter Interaction during Sporulation
in Bacillus subtilis", J Baceriol (1987), 169(5)1807-1811.
[2198] REID88a:
[2199] Reidhaar-Olson, J F, and R T Sauer, "Combinatorial Cassette
Mutagenesis as a Probe of the Information Content of Protein
Sequences", Science (1988), 241:53-57.
[2200] REID88b:
[2201] Reid, J, H Fung, K Gehring, P E Klebba, and H Nikaido,
"Targeting of porin to the outer membrane of Escherichia coli. Rate
of trimer assembly and identification of a dimer intermediate", J
Biol Chem (1988), 263(16)7753-9.
[2202] REST88:
[2203] Rest, R F, "Human Neutrophil and Mast Cell Proteases
Implicated in Inflammation", Meth Enzymol (1988), 163:309-27.
[2204] RICH81:
[2205] Richardson, J S, "The Anatomy and Taxonomy of Protein
Structure", Adv Protein Chemistry (1981), 34:167-339.
[2206] RICH86:
[2207] Richards, J H, "Cassette mutagenesis shows its strength.",
Nature (1986), 323:187.
[2208] RITO83:
[2209] Ritonja, A, B Meloun, and F Gubensek, "The Primary Structure
of Vipera ammodytes venom chymotrypsin inhibitor", Biochim Biophys
Acta (1983), 746:138-145.
[2210] RIVI87b:
[2211] Rivier, J, R Galyean, W R Gray, A Azimi-Zonooz, J M
McIntosh, I J Cruz, and B M Olivera, "Neuronal calcium channel
inhibitors. Synthesis of omega-conotoxin GVIA and effects on 45Ca
uptake by synaptosomes", J Biol Chem, (1987), 262(3)1194-8.
[2212] ROBE86:
[2213] Roberts, S, and A R Rees "The cloning and expression of an
anti-peptide antibody: a system for rapid analysis of the binding
properties of engineered antibodies.", Protein Engineering (1986),
1:59-65.
[2214] RONC90:
[2215] Ronco, J, A Charbit, and M Hofnung, "Creation of targets for
proteolytic cleavage in the LamB protein of E coli K12 by genetic
insertion of foreign sequences: implications for topological
studies", Biochimie (1990), 72(2-3)183-9.
[2216] ROSE85:
[2217] Rose, G D, "Automatic Recognition of Domains in Globular
Proteins", Methods in Enzymololgy (1985), 115(29)430-440.
[2218] ROSS81:
[2219] Rossman, M, and P Argos, "Protein Folding.", Ann Rev Biochem
(1981), 50:497-532.
[2220] RUEH73:
[2221] Ruehlmann, A, D Kukla, P Schwager, K Bartels, and R Huber,
"Structure of the Complex formed by Bovine Trypsin and Bovine
Pancreatic Trypsin Inhibitor: Crystal Structure Determination and
Stereochemistry of the Contact Region", J Mol Biol (1973),
77:417-436.
[2222] RUSS81:
[2223] Russel, M, and P Model, "A mutation dowanstream from the
signal peptidase cleavage site affects cleavage but not membrane
insertion of phage coat protein.", Proc Natl Acad Sci USA (1981),
78:1717-1721.
[2224] SALI64:
[2225] Salivar, W O, H Tzagoloff, and D Pratt, "Some physical,
chemical, and biological properties of the rod-shaped-coliphage
M13", Virology (1964), 24:359-71.
[2226] SALI87:
[2227] Salier, J P, M Diarra-Mehrpour, R Sesboue, J Bourguignon, R
Benarous, I Ohkubo, S Kurachi, K Kurachi, and J P Martin,
"Isolation and characterization of cDNAs encoding the heavy chain
of human inter-alphy-trypsin inhibitor (IaTI): Unambiguous evidence
for multipolypeptide chain sturcture of IaTI", Proc Nat Acad Sci
USA (1987), 84:8271-8276.
[2228] SALI88:
[2229] Sali, D, M Bycroft, and A R Fersht, "Stabilization of
protein structure by interaction of .alpha.-helix dipole with a
charged side chain", Nature (1988), 335:740-3.
[2230] SALI90:
[2231] Salier, J-P, "Inter-.alpha.-trypsin inhibitor: emergence of
a family within the Kunitz-type protease inhibitor superfamily",
TIBS (1990), 15:435-439.
[2232] SALV87:
[2233] Salvesen, G, D Farley, J Shuman, A Przybyla, C Reilly, and J
Travis, "Molecular Cloning of Human Cathepsin G: Structural
Similarity to Mast Cell and Cytotoxic T Lymphocyte Proteinases",
Biochem (1987), 26:2289-93.
[2234] SAMB89:
[2235] Sambrook, J, E F Fritsch, and T Maniatis, Molecular Cloning,
A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory,
1989.
[2236] SASA84:
[2237] Sasaki, T, "Amino Acid Sequence of a Novel Kunitz-type
chymotrypsin inhibitor from hemolymph of silkworm larvae, Bombyx
mori", FEBS Lett (1984), 168:227-230.
[2238] SAUE86:
[2239] Sauer, R T, K Hehir, R S Stearman, M A Weiss, A
Jeitler-Nilsson, E G Suchanek, and C O Pabo, "An Engineered
Intersubunit Disulfide Enhances the Stability and DNA Binding of
the N-Terminal Domain of .lambda. Repressor", Biochem (1986),
25:5992-98.
[2240] SCHA78:
[2241] Schaller, H, E Beck, and M Takanami, "Sequence and
Regulatory Signals of the Filamentous Phage Genome.", in The
Single-Stranded DNA Phages, Denhardt, D. T., D. Dressler, and D.S.
Ray editors, Cold Spring Harbor Laboratory, 1978., p139-163.
[2242] SCHN86:
[2243] Schnabel, E, W Schroeder, and G Reinhardt,
"(Ala.sub.2.sup.14,38]Ap- rotinin: Preparation by Partial
Desulphurization of Aprotinin by Means of Raney Nickel and
Comparison with other Aprotinin Derivatives", Biol Chem
Hoppe-Seyler (1986), 367:1167-76.
[2244] SCHN88a:
[2245] Schnabel, E, G Reinhardt, W Schroeder, H Tschesche, HR
Wenzel, and A Mehlich, "Enzymatic Resynthesis of the `Reactive
Site` Bond in the Modified Aprotinin Derivatives
[Seco-15/16]Aprotinin and [Di-seco-15/16,39/40]Aprotinin", Biol
Chem Hoppe-Seyler (1988), 369:461-8.
[2246] SCHU79:
[2247] Schulz, G E, and R H Schirmer, Principles of Protein
Structure, Springer-Verlag, New York, 1979.
[2248] SCHW87:
[2249] Schwarz, H, H J Hinz, A Mehlich, H Tschesche, and H R
Wenzel, "Stability studies on derivatives of the bovine pancreatic
trypsin inhibitor.", Biochemistry (1987), 26:(12)p3544-51.
[2250] SCOT87a:
[2251] Scott, M J, C S Huckaby, I Kato, W J Kohr, M Laskowski Jr.,
M-J Tsai and B W O'Malley, "Ovoinhibitor Introns Specify Functional
Domains as in the Related and Linked Ovomucoid Gene", J Biol Chem
(1987), 262(12)5899-5907.
[2252] SCOT87b:
[2253] Scott, C F, H R Wenzel, H R Tschesche, and R W Colman,
"Kinetics of Inhibition of Human Plasma Kallikrein by a
Site-Specific Modified Inhibitor Arg.sup.15-Aprotinin: Evaluation
Using a Microplate System and Comparison With Other Proteases",
Blood (1987), 69:1431-6.
[2254] SCOT90:
[2255] Scott, J K, and G P Smith, "Searching for Peptide Ligands
with an Epitope Library", Science, (27 July 1990), 249:386-390.
[2256] SEKI85:
[2257] Sekizaki, T, H Akaski, and N Terakado, "Nucleotide sequences
of the genes for Escherichia coli heat-stable enterotoxin I of
bovine, avian, and porcine origins", Am J Vet Res (1985),
46:909-12.
[2258] SELL87:
[2259] Selloum, L, M Davril, C Mizon, M Balduyck, and J Mizon, "The
effect of the glycosaminoglycan chain removal on some properties of
the human urinary trypsin inhibitor", Biol Chem Hoppe-Seyler
(1987), 368:47-55.
[2260] SERW87:
[2261] Serwer, P, "Review: Agarose Gel Electrophoresis of
Bacteriophages and Related Particles", J Chromatography (1987),
418:345-357.
[2262] SHIM87:
[2263] Shimonishi, Y, Y Hidaka, M Koizumi, M Hane, S Aimoto, T
Takeda, T Miwatani, and Y Takeda, "Mode of disulfide bond formation
of a heat-stable enterotoxin (STh) produced by a human strain of
enterotoxigenic Escherichia coli", FEBS Lett (1987),
215:165-170.
[2264] SHOR81:
[2265] Shortle, D, D Koshland, G M Weinstock, and D Botstein,
"Segment-directed mutagenesis: Construction in vitro of point
mutations limited to a small predetermined region of a circular DNA
molecule", Proc Natl Acad Sci USA (1980), 77:5375-79.
[2266] SHOR85:
[2267] Shortle, D, and B Lin, "Genetic Analysis of Staphylococcal
Nuclease: Identification of Three Intragenic `Global` Suppressors
of Nuclease-Minus Mutations.", Genetics (1985), 110:539-555.
[2268] SIEK87:
[2269] Siekmann, J, HR Wenzel, W Schroeder, H Schutt, E Truscheit,
A Arens, E Rauenbusch, WH CHazin, K Wutrich, and H Tschesche,
"Pyroglutamul-aprotinin, a new aprotinin homologue from bovine
lungs-isolation, properties, sequence analysis nad characterization
using .sup.1H nuclear magnetic resonance in solution", Biol Chem
Hoppe-Seyler (1987), 368:1589-96.
[2270] SIEK88:
[2271] Siekmann, J, H R Wenzel, W Schroeder, and H Tschesche,
"Characterization and Sequence Determination of Six-Aprotinin
homologues from bovine lungs", Biol Chem Hoppe-Seyler (1988),
369:157-163.
[2272] SIEK89:
[2273] Siekmann, J, J Beckmann, A Mehlich, H R Wenzel, H Tschesche,
E Schnabel, W Mueller-Esterl, "Immunological Characterization of
Natural and Semisynthetic Aprotinin Variants", Biol Chem
Hoppe-Seyler (1989), 370:677-81.
[2274] SILH77:
[2275] Silhavy, T J, H A Shuman, J Beckwith, and M Schwartz, "Use
of gene fusions to study outer membrane protein localization in
Escherichia coli", Proc Natl Acad Sci USA (1977),
74(12)5411-5415.
[2276] SILH85:
[2277] Silhavy, T J, and J R Beckwith, "Uses of lac Fusions for the
Study of Biological Problems", Microbiol Rev (1985),
49(4)398-418.
[2278] SINH90:
[2279] Sinha, S, H F Dovey, P Seubert, P J Ward, R W Blacher, M
Blaber, R A Bradshaw, M Arici, W C Mobley, and I Lieberburg, "The
Protease Inhibitory Properties of the Alzheimer's beta-amyloid
Precursor Protein", J Biol Chem (1990), 265(16)8983-5.
[2280] SMIT85:
[2281] Smith G P, "Filamentous Fusion Phage: Novel Expression
Vectors That Display Cloned Antigens on the Virion Surface",
Science (1985), 228:1315-1317.
[2282] SMIT88a:
[2283] Smith, G P, "Filamentous Phage Assembly: Morphogenetically
Defective Mutants That Do Not Kill the Host", Virology (1988),
167:156-165.
[2284] SMIT88b:
[2285] Smith, G P, "Filamentous Phages as Cloning Vectors", Chapter
3 in Vectors: A Survey of Molecular Cloning Vectors and Their Uses,
Editors: R L Rodriguez and D T Denhardt, Butterworth, Boston,
1988.
[2286] SODE85:
[2287] Sodergren, E J, J Davidson, R K Taylor, and T J Silhavy,
"Selection for Mutants Altered in the Expression or Export of Outer
Membrane Porin OmpF", J Bacteriol (1985), 162(3)1047-1053.
[2288] SOME85:
[2289] So, M, E Billyard, C Deal, E Getzoff, P Hagblom, T F Meyer,
E Segal, and J Tainer, "Gonococcal Pilus: Genetics and Structure",
Curr Top in Microbiol & Immunol (1985), 118:13-28.
[2290] SOMM89:
[2291] Sommerhoff, C P, G H Caughey, W E Finkbeiner, S C Lazarus, C
B Basbaum, and J A Nadel, "A Potent Secretagogue for Airway Gland
Serous Cells", J Immunol (1989), 142:2450-56.
[2292] SOMM90:
[2293] Sommerhoff, C P, J A Nadel, C B Basbaum, and G H Caughey,
"Neutrophil Elastase and Cathepsin G Stimulate Secretion from
Cultured Bovine Airway Gland Serous Cells", J Clin Invest (March
1990), 85:682-689.
[2294] STAD86:
[2295] Stader, J, S A Benson, and T J Silhavy "Kinetic analysis of
lamB mutants suggests the signal sequence plays multiple roles in
protein export", J Biol Chem (1986), 261(32)15075-80.
[2296] STAD89:
[2297] Stader, J, L J Gansheroff, and T J Silhavy, "New suppressors
of signal-sequence mutations, pr1G, are linked tightly to the secE
gene of Escherichia coli", Genes & Develop (1989),
3:1045-1052.
[2298] STAT87:
[2299] States, D J, T E Creighton, C M Dobson, and M Karplus,
"Conformations of intermediates in the folding of the pancreatic
trypsin inhibitor.", J Mol Biol (1987), 195(3)731-9.
[2300] STEI85:
[2301] Steiner, BioScience Repts. (1985), 5:973ff.
[2302] STUB90:
[2303] Stubbs, M T, B Laber, W Bode, R Huber, R Jerala, B Lenarcic,
and V Turk, "The refined 2.4 .ANG. X-ray crystal structure of
recombinant human stefin B in complex with the cysteine proteinase
papain: a novel type of proteinase inhibitor interaction", EMBO J
(1990), 9(6)1939-47.
[2304] SUNX87:
[2305] Sun, X P, H Takeuchi, Y Okano, and Y Nozawa, "Effects of
synthetic omega-conotoxin GVIA (omega-CgTX GVIA) on the membrane
calcium current of an identifiable giant neurone, d-RPLN, of an
African giant snail (Achatina fulica Ferussac), measured under the
voltage clamp condition", Comp Biochem Physiol [C], (1987),
87(2)363-6.
[2306] SUTC87a:
[2307] Sutcliffe, M J, I Haneef, D Carney, and T L Blundell,
"Knowledge based modelling of homologous proteins, part I:
three-dimensional frameworks derived from the simultaneous
superposition of multiple structures", Protein Engineering (1987),
1:377-384.
[2308] SUTC87b:
[2309] Sutcliffe, M J, F R F Hayes, and T L Blundell, "Knowledge
based modelling of homologous proteins, part II: rules for the
conformations of substituted sidechains", Protein Engineering
(1987), 1:385-392.
[2310] SVEN82:
[2311] Svendsen, I B, "Amino Acid Sequence of Serine Protease
Inhibitor CI-1 from Barley. Homology with Barley Inhibitor CI-2,
Potato Inhibitor I, and Leech Elgin", Carlsberg Res Comm (1982),
47:45-53.
[2312] SWAI88:
[2313] Swaim, M W, and S V Pizzo, "Modification of the tandem
reactive centres of human inter-.alpha.-trypsin inhibitor with
butanedione and cis-dichlorodiammineplatinum(II)", Biochem J
(1988), 254:171-178.
[2314] TAKA74:
[2315] Takahashi, H, S Iwanage, T Kitagawa, Y Hokama, and T Suzuki,
"Snake venom proteinase inhibitors. II. Chemical structure of
inhibitor II isolated from the venom of Russell's viper (Vipera
russelli).", J Biochem (1974), 76:721-733.
[2316] TAKA85:
[2317] Takao, T, N Tominaga, S Yoshimura, Y Shimonishi, S Hara, T
Inoue, and A Miyama, "Isolation, primary structure and synthesis of
heat-stable enterotoxin produced by Yersinia enterocolitica", Eur J
Biochem (1985), 152:199-206.
[2318] TAKE90:
[2319] Takeda, T, G B Nair, K Suzuki, and Y Shimonishi, "Production
of a Monoclonal Antibody to Vibrio cholerae Non-O1 Heat-Stable
Enterotoxin (ST) Which is Cross-Reactive with Yersinia
enterocolitica ST", Infection and Immunity (1990), 58(9)2755-9.
[2320] TANK77:
[2321] Tan, N H, and E T Kaiser, "Synthesis and Characterization of
a Pancreatic Trypsin Inhibitor Homologue and a Model Inhibitor",
Biochemistry, (1977), 16:1531-41.
[2322] THER88:
[2323] Theriault, N Y, J B Carter, and S P Pulaski, "Optimization
of Ligation Reaction Conditions in Gene Synthesis", BioTechniques
(1988), 6(5)470-473.
[2324] THOM83:
[2325] Thomas, G J, B Prescott, and L A Day, "Structure Similarity,
Difference and Variability in the Filamentous Viruses fd, If1, Ike,
Pf1, and Xf", J Mol Biol (1983), 165:321-56.
[2326] THOM85a:
[2327] Thompson, M R, M Luttrell, G Overmann, R A Giannella,
"Biological and Immunological Characteristics of .sup.125I-4Tyr and
-18Tyr Escherichia coli Heat-Stable Enterotoxin Species Purified by
High-Performance Liquid Chromatography", Analytical Biochem (1985),
148:26-36.
[2328] THOM85b:
[2329] Thompson, M R, and R A Giannella, "Revised Amino Acid
Sequence for a Heat-Stable Enterotoxin Produced by an Escherichia
coli Strain (18D) that is Pathogenic for Humans", Infection &
Immunity (1985), 47:834-36.
[2330] THOM86:
[2331] Thompson, R C, and K Ohlsson, "Isolation, properties, and
complete amino acid sequence of human secretory leukocyte protease
inhibitor, a potent inhibitor or leukocyte elastase", Proc Natl
Acad Sci USA (1986), 83:6692-96.
[2332] THOM88a:
[2333] Thomas, G J, Jr, B Prescott, S J Opella, and L A Day, "Sugar
Pucker and Phosphodiester Conformations in Viral Genomes of
Filamentous Bacteriophages: fd, If1, IKe, Pf1, Xf, and Pf3",
Biochem (1988), 27:4350-57.
[2334] THOR88:
[2335] Thornton, J M, B L Sibinda, M S Edwards, and D J Barlow,
"Analysis, Design, and Modification of Loop Regions in Proteins.",
BioEssays (?) SKG 3039 ??????
[2336] TOMM82:
[2337] Tommassen, J, P van der Ley, A van der Ende, H Bergmans, and
B Lugtenberg, "Cloning of ompF, the Structural Gene for an Outer
Membrane Pore Protein of E. coli K12: Physical Localization and
Homology with the phoE Gene", Mol gen Genet (1982),
185:105-110.
[2338] TOMM85:
[2339] Tommassen, J, P van der Ley, M van Zeijl, and M Agterberg,
"Localization of functional domains in E. coli K-12 outer membrane
porins", EMBO J (1985), 4(6)1583-7.
[2340] TRAB86:
[2341] Traboni, C, R Cortese, "Sequence of a full length cDNA
coding for human protein HC (.alpha..sub.1 microglobulin)", Nucleic
Acids Res (1986), 14(15)6340.
[2342] TRIA88:
[2343] Trias, J, E Y Rosenberg, and H Nikaido, "Specificity of the
glucose channel formed by protein D1 of Pseudomonas aeruginosa",
Biochim Biophys Acta (1988), 938:493-496.
[2344] TSCH86:
[2345] Tschesche, H, H Wenzel, R Schmuck, and E Schnabel,
"Homologues of Aprotinin with, in place of lysine, other amino
acids in position 15, process for their preparation and their use
as medicaments", U.S. Pat. No. 4,595,674 (Jun. 17, 1986).
[2346] TSCH87:
[2347] Tschesch, H, J Beckmann, A Mehlich, E Schnabel, E Truscheit,
and H R Wenzel, "Semisynthetic engineering of proteinase inhibitor
homologues", Biochimica et Biophysica Acta (1987), 913:97-101.
[2348] VAND86:
[2349] van der Ley, P, M Struyve, and J Tommassen, "Topology of
outer membrane pore protein PhoE of Escherichia coli.
Identification of cell surface-exposed amino acids with the aid of
monoclonal antibodies", J Biol Chem (1986), 261(26)12222-5.
[2350] VAND89:
[2351] Vanderslcie, P, C S Craik, J A Nadel, G H Caughey,
"Molecular Cloning of Dog Mast Cell Tryptase and a Related
Protease: Structural Evidence of a Unique Mode of Serine Protease
Activation", Biochem (1989), 28:4148-55.
[2352] VAND90:
[2353] van der Werf, S, A Charbit, C Leclerc, V Mimic, J Ronco, M
Girard, and M Hofnung, "Critical role of neighbouring sequences on
the immunogenicity of the C3 poliovirus neutralization epitope
expressed at the surface of recombinant bacteria", Vaccine (1990),
8(3)269-77.
[2354] VERS86a:
[2355] Vershon, A K, K Blacker, and R T Sauer, "Mutagenesis of the
Arc Repressor Using Synthetic Primers with Random Nucleotide
Substitutions", pp243-256 in Protein Engineering, Applications in
Science, Medicine, and Industry, Academic Press, 1986.
[2356] VERS86b:
[2357] Vershon, A K, J U Bowie, T M Karplus, and R T Sauer,
"Isolation and Analysis of Arc Repressor Mutants: Evidence for an
Unusual Mechanism of DNA Binding", pp302-311 in Proteins:
Structure, Function, and Genetics, Alan R. Liss, Inc., 1986.
[2358] VINC72:
[2359] Vincent &al, Biochem (1972), 11:2967ff.
[2360] VINC74:
[2361] Vincent &al., Biochem (1974), 13:4205.
[2362] VITA84:
[2363] Vita, C, D Dalzoppo, and A Fontana, "Independent Folding of
the Carboxyl-Terminal Fragment 228-316 of Thermolysin",
Biochemistry (1984), 23:5512-5519.
[2364] VOGE86:
[2365] Vogel, H, and F Jahnig, "Models for the structure of outer
membrane proteins of E. coli derived from Raman spectroscopy and
prediction methods", J Mol Biol (1986), 190:191-99.
[2366] VOND86:
[2367] Vonderviszt, F, G Y Matrai, and I Simon, "Characteristic
sequential residue environment of amino acids in proteins", Int J
Peptide Protein Res (1986), 27:483-92.
[2368] WACH79:
[2369] Wachter, E, K Hochstrasser, G Bretzel, and S Heindl,
"Kunitz-Type Proteinase Inhibitors Derived by Limited Proteolysis
of the Inter-.alpha.-trypsin Inhibitor, II. Characterization of a
Second Inhibitory Inactive Domain by Amino Acid Sequence
Determination", Hoppe-Seyler Z Physiol Chem (1979),
360:1297-1303.
[2370] WACH80:
[2371] Wachter, E, K Deppner, and K Hochstrasser, "A New
Kunitz-type Inhibitor from Bovine Serum, Amino Acid Sequence
Determination.", FEBS Letters (1980), 119:58-62.
[2372] WAGN78:
[2373] Wagner, G, K Wuthrich, and H Tschesche, "A H
Nuclear-Magnetic-Resonance Study of the Solution Conformation of
the Isoinhibitor K from Helix pomatia.", Eur J Biochem (1978),
89:367-377.
[2374] WAGN79:
[2375] Wanger, G, H Tschesche, and K Wuthrich, "The Influence of
Localized Chemical Modifications of the Basic Pancreatic Trypsin
Inhibitor on Static and Dynamic Aspects of the Molecular
Conformation in Solution", Eur J Biochem (1979), 95:239-248.
[2376] WANG87:
[2377] Wagner, G, D Bruhwiler, and K Wuthrich, "Reinvestigation of
the aromatic side-chains in the basic pancreatic trypsin inhibitor
by heteronuclear two-dimensional nuclear magnetic resonance.", J
Mol Biol (1987), 196(1)227-31.
[2378] WAIT83:
[2379] Waite, J H, "Evidence for a repeating
3,4-dihydroxyphenylalanine- and hydroxyproline-containing
decapeptide in the adhesive protein of the mussel, Mytilus edulis
L.", J Biol Chem (1983), 258(5)2911-5.
[2380] WAIT85:
[2381] Waite, J H, T J Housley, and M L Tanzer, "Peptide repeats in
a mussel glue protein: theme and variations.", Biochemistry (1985),
24(19)5010-4.
[2382] WAIT86:
[2383] Waite, J H, "Mussel glue from Mytilus californianus Conrad:
a comparative study.", J Comp Physiol [B] (1986), 156(4)491-6.
[2384] WATS87:
[2385] Molecular Biology of the Gene, Fourth Edition, Watson, J D,
N H Hopkins, J W Roberts, J A Steitz, and A M Weiner,
Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif.,
1987.
[2386] WEBS78:
[2387] Webster, R E, and J S Cashman, "Morphogenesis of the
Filamentous Single-stranded DNA Phages.", in The Single-Stranded
DNA Phages, Denhardt, D T, D Dressler, and D S Ray editors, Cold
Spring Harbor Laboratory, 1978., p557-569.
[2388] WEHM89:
[2389] Wehmeier, U, G A Sprenger, and J W Lengeler, "The use of
lambda plac-Mu hybrid phages in Klebsiella pneumoniae and the
isolation of stable Hfr strains", Mol Gen Genet (1989),
215(3)529-36.
[2390] WEIN83:
[2391] Weinstock, G M, C ap Rhys, M L Berman, B Hampar, D Jackson,
T J Silhavy, J Weisemann, and M Zweig, "Open reading frame
expression vectors: A general method for antigen production in
Escherichia coli using protein fusions to beta-galactosidase", Proc
Natl Acad Sci USA (1983), 80:4432-4436.
[2392] WELL86:
[2393] Wells, J A, and D B Powers, "In vivo Formation and Stability
of Engineered Disulfide Bonds in Subtilisin", J Biol Chem (1986),
261:6564-70.
[2394] WELL87a:
[2395] Wells, J A, B C Cunningham, T P Graycar, and D A Estell,
"Recruitment of substrate-specificity properties from one enzyme
into a related one by protein engineering", Proc Natl Acad Sci USA
(1987), 84:5167-71.
[2396] WELL87b:
[2397] Wells, J A, D B Powers, R R Bott, T P Graycar, and D A
Estell, "Designing substrate specificity by protein engineering of
electrostatic interactions", Proc Natl Acad Sci USA (1987),
84:1219-23.
[2398] WEMM83:
[2399] Wemmer, D, and N R Kallenbach, Biochem (1983),
22:1901-6.
[2400] WENZ80:
[2401] Wenzel, H R, and H Tschesche, Hoppe-Seyler Z Physiol Chem
(1980), 361:345.
[2402] WENZ81:
[2403] Wenzel, H R, and H Tschesche, "`Chemical Mutation` by Amino
Acid Exchange in the Reactive Site of a Proteinase Inhibitor and
Alteration of Its Inhibitor Specificity", Angew Chem Int Ed Engl
(1981), 20(3)295-6.
[2404] WETZ88:
[2405] Wetzel, R, et al., Proc Natl Acad Sci USA (1988),
85:401-5.
[2406] WEWE87:
[2407] Wewers, M D, M A Casolaro, S E Sellers, S C Swayze, K M
McPhaul, J T Wittes, and R G Crystal, "Replacement therapy for
.alpha.-1-antitrypsin deficiency associated with emphysema", New
Engl J Med (1987), 316(17)1055-62.
[2408] WHAR86:
[2409] Wharton, R P, The Binding Specificity Determinants of 434
Repressor., Harvard U. PhD Thesis, 1986, University Microfilms, Ann
Arbor, Mich.
[2410] WIEC85:
[2411] Wieczorek, M, J Otlewski, J Cook, K Parks, J Leluk, A
Wilimowska-Pelc, A Polanowski, T Wilusz, and L Laskowski, Jr, "The
Squash Family of Serine Protease Inhibitors. Amino Acid Sequences
and association equilibrium constants of inhibitors from squash,
summer squash, zucchini, and cucumber seeds", Biochem Biophys Res
Comm (1985), 126(2)646-652.
[2412] WILK84:
[2413] Wilkinson, A J, A R Fersht, D M Blow, P Carter, and G
Winter, "A large increase in enzyme-substrate affinity by protein
engineering.", Nature (1984), 307:187-188.
[2414] WINT87b:
[2415] Winter, A J, "Outer membrane proteins of Brucella", Ann Inst
Pasteur Microbiol (1987), 138(1)87-9.
[2416] WLOD84:
[2417] Wlodawer, A, J Walter, R Huber, and L Sjolin, "Structure of
bovine pancreatic trypsin inhibitor. Results of joint neutron and
X-ray refinement of crystal form II.", J Mol Biol (1984),
180(2)301-29.
[2418] WLOD87a:
[2419] Wlodawer, A, J Nachman, G L Gilliland, W Gallagher, and C
Woodward, "Structure of form III crystals of bovine pancreatic
trypsin inhibitor.", J Mol Biol (1987), 198(3)469-80.
[2420] WLOD87b:
[2421] Wlodawer, A, J Deisenhofer, and R Huber, "Comparison of two
highly refined structures of bovine pancreatic trypsin inhibitor.",
J Mol Biol (1987), 193(1)145-56.
[2422] WOOD90:
[2423] Woodward, S R, L J Cruz, B M Olivera, and D R Hillyard,
"Constant and hypervariable regions in conotoxin propeptides", EMBO
J (1990), 9:1015-1020.
[2424] WUNT88:
[2425] Wun, T-C, K K Kretzmer, T J Girard, J P Miletich, and G J
Broze, Jr, "Cloning and Characterization of a cDNA Coding for the
Lipoprotein-associated Coagulation Inhibitor Shows That It Consists
of Three Tandem Kunitz-type Inhibitory Domains", J Biol Chem
(1988), 263:6001-4.
[2426] YAGE87:
[2427] Yager, T D, and P H von Hippel, "Transcription Elongation
and Termination in E. coli", Volume 2, Chapter 76, p 1241-1275,
Escherichia coli and Salmonella typhimurium: Cellular and Molecular
Biology, Neidhardt, F C, Editor-in-Chief, Amer Soc for
Microbiology, Washington, D.C., 1987.
[2428] YANI85:
[2429] Yanisch-Perron, C, J Vieira, and J Messing, "Improved M13
phage cloning vectors and host strains: nucleotide sequeices of the
M13mp18 and pUC19 vectors", Gene, (1985), 33:103-119.
[2430] YOKO77:
[2431] Yokosawa, H, and S-I Ishii, "Anhydrotrypsin: New Features in
Ligand Interactions Revealed by Affininty Chromatography and
Thionine Replacement", J Biochem (1977), 81:647-56.
[2432] YOSH85:
[2433] Yoshimura, S, H Ikemura, H Watanabe, S Aimoto, Y Shimonishi,
S Hara, T Takeda, T Miwatani, and Y Takeda, "Essential structure
for full enterotoxigenic activity of heat-stable enterotoxin
produced by enterotoxigenic Escherichia coli", FEBS Lett (1985),
181:138-42.
[2434] ZAFA88:
[2435] Zafaralla, G C, C Ramilo, W R Gray, R Karlstrom, B M
Olivera, and L J Cruz, "Phylogenetic specificity of cholinergic
ligands: .alpha.-conotoxin SI", Biochemistry, (1988),
27(18)7102-5.
[2436] ZIMM82:
[2437] Zimmermann, R, C Watts, and W Wickner, "The Biosynthesis of
Membrane-bound M13 Coat Protein: Energetics and Assembly
Intermediates.", J Biol Chem (1982), 257:6529-6536.
[2438] ZOLL84:
[2439] Zoller, M J, and M Smith, "Oligonucleotide-Directed
Mutagenesis: A Simple Method Using Two oligonucleotide Primers and
a Single-Stranded DNA Template.", DNA (1984), 3(6)479-488.
* * * * *