U.S. patent application number 10/045674 was filed with the patent office on 2009-06-25 for novel methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries.
This patent application is currently assigned to Dyax Corp.. Invention is credited to Edward H. Cohen, Rene Hoet, Robert C. Ladner, Hendricus Renerus Jacobus Mattheus Hoogenboom, Horacio G. Nastri, Kristin L. Rookey.
Application Number | 20090162835 10/045674 |
Document ID | / |
Family ID | 32475265 |
Filed Date | 2009-06-25 |
United States Patent
Application |
20090162835 |
Kind Code |
A9 |
Ladner; Robert C. ; et
al. |
June 25, 2009 |
Novel methods of constructing libraries comprising displayed and/or
expressed members of a diverse family of peptides, polypeptides or
proteins and the novel libraries
Abstract
Methods useful in constructing libraries that collectively
display and/or express members of diverse families of peptides,
polypeptides or proteins and the libraries produced using those
methods. Methods of screening those libraries and the peptides,
polypeptides or proteins identified by such screens.
Inventors: |
Ladner; Robert C.;
(Ijamsville, MD) ; Cohen; Edward H.; (Belmont,
MA) ; Nastri; Horacio G.; (Newton, MA) ;
Rookey; Kristin L.; (Revere, MA) ; Hoet; Rene;
(Maastricht, NL) ; Mattheus Hoogenboom; Hendricus Renerus
Jacobus; (Maastricht, NL) |
Correspondence
Address: |
LOWRIE, LANDO & ANASTASI, LLP
ONE MAIN STREET, SUITE 1100
CAMBRIDGE
MA
02142
US
|
Assignee: |
Dyax Corp.
Cambridge
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20030232333 A1 |
December 18, 2003 |
|
|
Family ID: |
32475265 |
Appl. No.: |
10/045674 |
Filed: |
October 25, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10000516 |
Oct 24, 2001 |
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10045674 |
Oct 25, 2001 |
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09837306 |
Apr 17, 2001 |
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10000516 |
Oct 24, 2001 |
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60198069 |
Apr 17, 2000 |
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Current U.S.
Class: |
435/6.1 ;
435/6.18; 435/91.2 |
Current CPC
Class: |
C07K 2317/515 20130101;
C07K 2317/622 20130101; C12N 15/1093 20130101; C40B 50/06 20130101;
C07K 16/005 20130101; C07K 2317/55 20130101; C12N 15/10 20130101;
C12N 15/66 20130101; C12N 15/1037 20130101; C40B 40/02 20130101;
C07K 2317/51 20130101; C40B 40/08 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Claims
1. A method for cleaving single-stranded nucleic acid sequences at
a desired location, the method comprising the steps of: (i)
contacting the nucleic acid with a single-stranded oligonucleotide,
the oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired and including a
sequence that with its complement in the nucleic acid forms a
restriction endonuclease recognition site that on restriction
results in cleavage of the nucleic acid at the desired location;
and (ii) cleaving the nucleic acid solely at the recognition site
formed by the complementation of the nucleic acid and the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
2. A method for cleaving single-stranded nucleic acid sequences at
a desired location, the method comprising the steps of: (i)
contacting the nucleic acid with a partially double-stranded
oligonucleotide, the single-stranded region of the oligonucleotide
being functionally complementary to the nucleic acid in the region
in which cleavage is desired, and the double-stranded region of the
oligonucleotide having a restriction endonuclease recognition site;
and (ii) cleaving the nucleic acid solely at the restriction
endonuclease recognition site formed by the complementation of the
nucleic acid and the single-stranded region of the oligonucleotide;
the contacting and the cleaving steps being performed at a
temperature sufficient to maintain the nucleic acid in
substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
3. In a method for displaying a member of a diverse family of
peptides, polypeptides or proteins on the surface of a genetic
package and collectively displaying at least a part of the
diversity of the family, the improvement being characterized in
that the displayed peptide, polypeptide or protein is encoded at
least in part by a nucleic acid that has been cleaved at a desired
location by a method comprising the steps of: (i) contacting the
nucleic acid with a single-stranded oligonucleotide, the
oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired and including a
sequence that with its complement in the nucleic acid forms a
restriction endonuclease recognition site that on restriction
results in cleavage of the nucleic acid at the desired location;
and (ii) cleaving the nucleic acid solely at the recognition site
formed by the complementation of the nucleic acid and the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
4. In a method for displaying a member of a diverse family of
peptides, polypeptides or proteins on the surface of a genetic
package and collectively displaying at least a part of the
diversity of the family, the improvement being characterized in
that the displayed peptide, polypeptide or protein is encoded by a
DNA sequence comprising a nucleic acid that has been cleaved at a
desired location by (i) contacting the nucleic acid with a
partially double-stranded oligonucleotide, the single-stranded
region of the oligonucleotide being functionally complementary to
the nucleic acid in the region in which cleavage is desired, and
the double-stranded region of the oligonucleotide having a
restriction endonuclease recognition site; and (ii) cleaving the
nucleic acid solely at the restriction endonuclease recognition
cleavage site formed by the complementation of the nucleic acid and
the single-stranded region of the oligonucleotide; the contacting
and the cleaving steps being performed at a temperature sufficient
to maintain the nucleic acid in substantially single-stranded form,
the oligonucleotide being functionally complementary to the nucleic
acid over a large enough region to allow the two strands to
associate such that cleavage may occur at the chosen temperature
and at the desired location, and the cleavage being carried out
using a restriction endonuclease that is active at the chosen
temperature.
5. A method for displaying a member of a diverse family of
peptides, polypeptides or proteins on the surface of a genetic
package and collectively displaying at least a part of the
diversity of the family, the method comprising the steps of: (i)
preparing a collection of nucleic acids that code at least in part
for members of the diverse family; (ii) rendering the nucleic acids
single-stranded; (iii) cleaving the single-stranded nucleic acids
at a desired location by a method comprising the steps of: (a)
contacting the nucleic acid with a single-stranded oligonucleotide,
the oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired and including a
sequence that with its complement in the nucleic acid forms a
restriction endonuclease recognition site that on restriction
results in cleavage of the nucleic acid at the desired location;
and (b) cleaving the nucleic acid solely at the recognition site
formed by the complementation of the nucleic acid and the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature; and (iv) displaying a
member of the family of peptides, polypeptides or proteins coded,
at least in part, by the cleaved nucleic acids on the surface of
the genetic package and collectively displaying at least a portion
of the diversity of the family.
6. A method for displaying a member of a diverse family of
peptides, polypeptides or proteins on the surface of a genetic
package and collectively displaying at least a portion of the
diversity of the family, the method comprising the steps of: (i)
preparing a collection of nucleic acids that code, at least in
part, for members of the diverse family; (ii) rendering the nucleic
acids single-stranded; (iii) cleaving the single-stranded nucleic
acids at a desired location by a method comprising the steps of:
(a) contacting the nucleic acid with a partially double-stranded
oligonucleotide, the single-stranded region of the oligonucleotide
being functionally complementary to the nucleic acid in the region
in which cleavage is desired, and the double-stranded region of the
oligonucleotide having a restriction endonuclease recognition site;
and (b) cleaving the nucleic acid solely at the restriction
endonuclease recognition cleavage site formed by the
complementation of the nucleic acid and the single-stranded region
of the oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the restriction being carried out using a cleavage endonuclease
that is active at the chosen temperature; and (iv) displaying a
member of the family of peptides, polypeptides or proteins coded,
at least in part, by the cleaved nucleic acids on the surface of
the genetic package and collectively displaying at least a portion
of the diversity of the family.
7. In a method for expressing a member of a diverse family of
peptides, polypeptides or proteins and collectively expressing at
least a part of the diversity of the family, the improvement being
characterized in that the expressed peptide, polypeptide or protein
is encoded at least in part by a nucleic acid that has been cleaved
at a desired location by a method comprising the steps of: (i)
contacting the nucleic acid with a single-stranded oligonucleotide,
the oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired and including a
sequence that with its complement in the nucleic acid forms a
restriction endonuclease recognition site that on restriction
results in cleavage of the nucleic acid at the desired location;
and (ii) cleaving the nucleic acid solely at the recognition site
formed by the complementation of the nucleic acid and the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
8. In a method for expressing a member of a diverse family of
peptides, polypeptides or proteins and collectively expressing at
least a part of the diversity of the family, the improvement being
characterized in that the expressed peptide, polypeptide or protein
is encoded by a DNA sequence comprising a nucleic acid that has
been cleaved at a desired location by (i) contacting the nucleic
acid with a partially double-stranded oligonucleotide, the
single-stranded region of the oligonucleotide being functionally
complementary to the nucleic acid in the region in which cleavage
is desired, and the double-stranded region of the oligonucleotide
having a restriction endonuclease recognition site; and (ii)
cleaving the nucleic acid solely at the restriction endonuclease
recognition cleavage site formed by the complementation of the
nucleic acid and the single-stranded region of the oligonucleotide;
the contacting and the cleaving steps being performed at a
temperature sufficient to maintain the nucleic acid in
substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
9. A method for expressing a member of a diverse family of
peptides, polypeptides or proteins and collectively expressing at
least a part of the diversity of the family, the method comprising
the steps of: (i) preparing a collection of nucleic acids that code
at least in part for members of the diverse family; (ii) rendering
the nucleic acids single-stranded; (iii) cleaving the
single-stranded nucleic acids at a desired location by a method
comprising the steps of: (a) contacting the nucleic acid with a
single-stranded oligonucleotide, the oligonucleotide being
functionally complementary to the nucleic acid in the region in
which cleavage is desired and including a sequence that with its
complement in the nucleic acid forms a restriction endonuclease
recognition site that on restriction results in cleavage of the
nucleic acid at the desired location; and (b) cleaving the nucleic
acid solely at the recognition site formed by the complementation
of the nucleic acid and the oligonucleotide; the contacting and the
cleaving steps being performed at a temperature sufficient to
maintain the nucleic acid in substantially single-stranded form,
the oligonucleotide being functionally complementary to the nucleic
acid over a large enough region to allow the two strands to
associate such that cleavage may occur at the chosen temperature
and at the desired location, and the cleavage being carried out
using a restriction endonuclease that is active at the chosen
temperature; and (iv) expressing a member of the family of
peptides, polypeptides or proteins coded, at least in part, by the
cleaved nucleic acids and collectively expressing at least a
portion of the diversity of the family.
10. A method for expressing a member of a diverse family of
peptides, polypeptides or proteins and collectively expressing at
least a portion of the diversity of the family, the method
comprising the steps of: (i) preparing a collection of nucleic
acids that code, at least in part, for members of the diverse
family; (ii) rendering the nucleic acids single-stranded; (iii)
cleaving the single-stranded nucleic acids at a desired location by
a method comprising the steps of: (a) contacting the nucleic acid
with a partially double-stranded oligonucleotide, the
single-stranded region of the oligonucleotide being functionally
complementary to the nucleic acid in the region in which cleavage
is desired, and the double-stranded region of the oligonucleotide
having a restriction endonuclease recognition site; and (b)
cleaving the nucleic acid solely at the restriction endonuclease
recognition cleavage site formed by the complementation of the
nucleic acid and the single-stranded region of the oligonucleotide;
the contacting and the cleaving steps being performed at a
temperature sufficient to maintain the nucleic acid in
substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the restriction being carried out using a cleavage endonuclease
that is active at the chosen temperature; and (iv) expressing a
member of the family of peptides, polypeptides or proteins coded,
at least in part, by the cleaved nucleic acids and collectively
expressing at least a portion of the diversity of the family.
11. A library comprising a collection of genetic packages that
display a member of a diverse family of peptides, polypeptides or
proteins and collectively display at least a portion of the
diversity of the family, the library being produced using the
methods of claims 3, 4, 5 or 6.
12. A library comprising a collection of genetic packages that
display a member of a diverse family of peptides, polypeptides or
proteins and that collectively display at least a portion of the
family, the displayed peptides, polypeptides or proteins being
encoded by DNA sequences comprising at least in part sequences
produced by cleaving single-stranded nucleic acid sequences at a
desired location by a method comprising the steps of: (i)
contacting the nucleic acid with a single-stranded oligonucleotide,
the oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired and including a
sequence that with its complement in the nucleic acid forms a
restriction endonuclease recognition site that on restriction
results in cleavage of the nucleic acid at the desired location;
and (ii) cleaving the nucleic acid solely at the recognition site
formed by the complementation of the nucleic acid and the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
13. A library comprising a collection of genetic packages that
display a member of a diverse family of peptides, polypeptides or
proteins and that collectively display at least a portion of the
diversity of the family of the displayed peptides, polypeptides or
proteins being encoded by DNA sequences comprising at least in part
sequences produced by cleaving single-stranded nucleic acid
sequences at a desired location by a method comprising the steps
of: (i) contacting the nucleic acid with a partially
double-stranded oligonucleotide, the single-stranded region of the
oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired, and the
double-stranded region of the oligonucleotide having a restriction
endonuclease recognition site; and (ii) cleaving the nucleic acid
solely at the restriction endonuclease recognition cleavage site
formed by the complementation of the nucleic acid and the
single-stranded region of the oligonucleotide; the contacting and
the cleaving steps being performed at a temperature sufficient to
maintain the nucleic acid in substantially single-stranded form,
the oligonucleotide being functionally complementary to the nucleic
acid over a large enough region to allow the two strands to
associate such that cleavage may occur at the chosen temperature
and at the desired location, and the cleavage being carried out
using a restriction endonuclease that is active at the chosen
temperature.
14. A library comprising a collection of members of a diverse
family of peptides, polypeptides or proteins and collectively
comprising at least a portion of the diversity of the family, the
library being produced using the methods of claims 7, 8, 9 or
10.
15. A library comprising a collection of members of a diverse
family of peptides, polypeptides or proteins and collectively
comprising at least a portion of diversity of the family, the
peptides, polypeptides or proteins being encoded by DNA sequences
comprising at least in part sequences produced by cleaving
single-stranded nucleic acid sequences at a desired location by a
method comprising the steps of: (i) contacting the nucleic acid
with a single-stranded oligonucleotide, the oligonucleotide being
functionally complementary to the nucleic acid in the region in
which cleavage is desired and including a sequence that with its
complement in the nucleic acid forms a restriction endonuclease
recognition site that on restriction results in cleavage of the
nucleic acid at the desired location; and (ii) cleaving the nucleic
acid solely at the recognition site formed by the complementation
of the nucleic acid and the oligonucleotide; the contacting and the
cleaving steps being performed at a temperature sufficient to
maintain the nucleic acid in substantially single-stranded form,
the oligonucleotide being functionally complementary to the nucleic
acid over a large enough region to allow the two strands to
associate such that cleavage may occur at the chosen temperature
and at the desired location, and the cleavage being carried out
using a restriction endonuclease that is active at the chosen
temperature.
16. A library comprising a collection of members of a diverse
family of peptides, polypeptides or proteins and collectively
comprising at least a portion of the diversity of the family, the
peptides, polypeptides or proteins being encoded by DNA sequences
comprising at least in part sequences produced by cleaving
single-stranded nucleic acid sequences at a desired location by a
method comprising the steps of: (i) contacting the nucleic acid
with a partially double-stranded oligonucleotide, the
single-stranded region of the oligonucleotide being functionally
complementary to the nucleic acid in the region in which cleavage
is desired, and the double-stranded region of the oligonucleotide
having a restriction endonuclease recognition site; and (ii)
cleaving the nucleic acid solely at the restriction endonuclease
recognition cleavage site formed by the complementation of the
nucleic acid and the single-stranded region of the oligonucleotide;
the contacting and the cleaving steps being performed at a
temperature sufficient to maintain the nucleic acid in
substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
17. A library of claims 11, 12 or 13 wherein the genetic packages
are selected from the group of phage, phagemid or yeast.
18. A library of claims 17 wherein the genetic packages are
selected are phage or phagemid.
19. The methods or libraries according claims 2, 4, 6, 8, 10, 13 or
16 wherein in the restriction endonuclease recognition site is for
a Type II-S restriction endonuclease.
20. The methods or libraries according to claims 1 to 19, wherein
the nucleic acid is cDNA.
21. The methods or libraries according to any one of claims 1 to
20, wherein the nucleic acids encode at least a portion of an
immunoglobulin.
22. The methods or libraries according to claim 21, wherein the
immunoglobulin comprises a Fab or single chain Fv.
23. The methods or libraries according to claim 21 or 22, wherein
the immunoglobulin comprises at least portion of a heavy chain.
24. The method or libraries according to claim 23, wherein the
heavy chain is IgM, IgG, IgA, IgE or IgD.
25. The methods or libraries according to claim 23 or 24, wherein
at least a portion of the heavy chain is human.
26. The methods or libraries according to claim 21 or 22, wherein
the immunoglobulin comprises at least a portion of FR1.
27. The methods or libraries according to claim 26, wherein at
least a portion of the FR1 is human.
28. The methods or libraries according to claim 21 or 22, wherein
the immunoglobulin comprises at least a portion of a light
chain.
29. The methods or libraries according to claim 28, wherein at
least a portion of the light chain is human.
30. The methods or libraries according to any one of claims 1 to
16, wherein the nucleic acid sequences are at least in part derived
from patients suffering from at least one autoimmune disease and/or
cancer.
31. The methods or libraries according to claim 30, wherein the
autoimmune disease is selected from the group comprising lupus,
erythematosus, systemic sclerosis, rheumatoid arthritis,
antiphosolipid syndrome or vasculitis.
32. The methods or libraries according to claim 30, wherein the
nucleic acids are at least in part isolated from the group
comprising peripheral blood cells, bone marrow cells spleen cells
or lymph node cells.
33. The methods according to claim 5, 6, 9 or 10 further comprising
at least one nucleic acid amplification step between one or more of
steps (i) and (ii), steps (ii) and (iii) or between steps (iii) and
(iv).
34. The method according to claim 33, wherein amplification primers
for the amplification step are functionally complementary to a
constant region of the nucleic acids.
35. The method according to claim 34, wherein the constant region
is genetically constant in the nucleic acids.
36. The method according to claim 35, wherein the genetically
constant region is a part of the genome of immunoglobulin genes
selected from the group of IgM, IgG, IgA, IgE or IgD.
37. The method according to claim 34, wherein the constant region
is exogenous to the nucleic acids.
38. The methods according to claim 33, wherein the amplification
step uses geneRACE.TM..
39. The methods or libraries according to any one of claims 1 to
16, wherein the chosen temperature is between 37.degree. C. and
75.degree. C.
40. The methods or libraries according to claim 39, wherein the
chosen temperature is between 45.degree. C. and 75.degree. C.
41. The methods or libraries according to claim 40, wherein the
chosen temperature is between 50.degree. C. and 60.degree. C.
42. The methods or libraries according to claim 41, wherein the
chosen temperature is between 55.degree. C. and 60.degree. C.
43. The methods or libraries according to claim 1, 3, 5, 7, 9, 12
or 15, wherein the length of the single-stranded oligonucleotide is
between 17 and 30 bases.
44. The methods or libraries according to claim 43, wherein the
length of the single-stranded oligonucleotide is between 18 and 24
bases.
45. The methods or libraries according to claim 1, 3, 5, 7, 9, 12
or 15, wherein the restriction endonuclease is selected from the
group comprising MaeIII, Tsp45I, HphI, BsaJI, AluI, BlpI, DdeI,
BglII, MslI, BsiEI, EaeI, EagI, HaeIII, Bst4CI, HpyCH4III, HinfI,
MlyI, PleI, MnlI, HpyCH4V, BsmAI, BpmI, XmnI, or SacI.
46. The methods or libraries according to claim 45, wherein the
restriction endonuclease is selected from the group comprising
Bst4CI, TaaI, HpyCH4III, BlpI, HpyCH4V or MslI.
47. The methods or libraries according to claim 2, 4, 6, 8, 10, 13
or 16, wherein the length of the single-stranded region of the
partially double-stranded oligonucleotide is between 14 and 22
bases.
48. The methods or libraries according to claim 47, wherein the
length of the single-stranded region of the partially
double-stranded oligonucleotide is between 14 and 17 bases.
49. The methods or libraries according to claim 47, wherein the
length of the single-stranded region of the oligonucleotide is
between 18 and 20 bases.
50. The methods or libraries according to claim 2, 4, 6, 8, 10, 13
or 16, wherein the length of the double-stranded region of the
partially double-stranded oligonucleotide is between 10 and 14 base
pairs formed by a stem and its palindrome.
51. The methods or libraries according to claim 50 wherein, the
partially double-stranded oligonucleotide comprises a loop of 3 to
8 bases between the stem and the palindrome.
52. The methods or libraries according to claim 19 wherein the Type
II-S restriction endonuclease is selected from the group comprising
AarICAC, AceIII, Bbr7I, BbvI, BbvII, Bce83I, BceAI, BcefI, BciVI,
BfiI, BinI, BscAI, BseRI, BsmFI, BspMI, EciI, Eco57I, FauI, FokI,
GsuI, HgaI, HphI, MboII, MlyI, MmeI, MnlI, PleI, RleAI, SfaNI,
SspD5I, Sth132I, StsI, TaqII, Tth111II, or UbaPI.
53. The methods or libraries according to claim 52, wherein the
Type II-S restriction endonuclease is FokI.
54. A method for preparing single-stranded nucleic acids, the
method comprising the steps of: (i) contacting a single-stranded
nucleic acid sequence that has been cleaved with a restriction
endonuclease with a partially double-stranded oligonucleotide, the
single-stranded region of the oligonucleotide being functionally
complementary to the nucleic acids in the region that remains after
cleavage, the double-stranded region of the oligonucleotide
including any sequences necessary to return the sequences that
remain after cleavage into proper and original reading frame for
expression and containing a restriction endonuclease recognition
site 5' of those sequences; and (ii) cleaving the partially
double-stranded oligonucleotide sequence solely at the restriction
endonuclease recognition site contained within the double-stranded
region of the partially double-stranded oligonucleotide. the
contacting and the cleaving steps being performed at a temperature
sufficient to maintain the nucleic acid in substantially
single-stranded form, the oligonucleotide being functionally
complementary to the nucleic acid over a large enough region to
allow the two strands to associate such that cleavage may occur at
the chosen temperature and at the desired location, and the
cleavage being carried out using a restriction endonuclease that is
active at the chosen temperature.
55. The method according to claim 54, wherein the length of the
single-stranded portion of the partially double-stranded
oligonucleotide is between 2 and 15 bases.
56. The method according to claim 55, wherein the length of the
single-stranded portion of the partially double-stranded
oligonucleotide is between 7 and 10 bases.
57. The method according to claim 54, wherein the length of the
double-stranded portion of the partially double-stranded
oligonucleotide is between 12 and 100 base pairs.
58. The method according to claim 57, wherein the length of the
double-stranded portion of the partially double-stranded
oligonucleotide is between 20 and 100 base pairs.
59. A method for preparing a library comprising a collection of
genetic packages that display a member of a diverse family of
peptides, polypeptides or proteins and that collectively display at
least a portion of the family comprising the steps: (i) preparing a
collection of nucleic acids that code at least in part for members
of the diverse family; (ii) rendering the nucleic acids
single-stranded; (iii) cleaving the single-stranded nucleic acids
at a desired location by a method comprising the steps of: (a)
contacting the nucleic acid with a single-stranded oligonucleotide,
the oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired and including a
sequence that with its complement in the nucleic acid forms a
restriction endonuclease recognition site that on restriction
results in cleavage of the nucleic acid at the desired location;
and (b) cleaving the nucleic acid solely at the recognition site
formed by the complementation of the nucleic acid and the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature; (iv) contacting the
nucleic acid with a partially double-stranded oligonucleotide, the
single-stranded region of the oligonucleotide being functionally
complementary to the nucleic acids in the region that remains after
the cleavage in step (iii) has been effected, and the
double-stranded region of the oligonucleotide including any
sequences necessary to return the sequences that remain after
cleavage into proper and original reading frame for display and
containing a restriction endonuclease recognition site 5' of those
sequences that is different from the restriction site used in step
(iii); and (v) cleaving the nucleic acid solely at the restriction
endonuclease recognition cleavage site contained within the
double-stranded region of the partially double-stranded
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the restriction being carried out using a cleavage endonuclease
that is active at the chosen temperature; and (vi) displaying a
member of the family of peptides, polypeptides or proteins coded,
at least in part, by the cleaved nucleic acids on the surface of
the genetic package and collectively displaying at least a portion
of the diversity of the family.
60. A method for preparing a library comprising a collection of
members of a diverse family of peptides, polypeptides or proteins
and collectively comprising at least a portion of the family
comprising the steps: (i) preparing a collection of nucleic acids
that code at least in part for members of the diverse family; (ii)
rendering the nucleic acids single-stranded; (iii) cleaving the
single-stranded nucleic acids at a desired location by a method
comprising the steps of: (a) contacting the nucleic acid with a
single-stranded oligonucleotide, the oligonucleotide being
functionally complementary to the nucleic acid in the region in
which cleavage is desired and including a sequence that with its
complement in the nucleic acid forms a restriction endonuclease
recognition site that on restriction results in cleavage of the
nucleic acid at the desired location; and (b) cleaving the nucleic
acid solely at the recognition site formed by the complementation
of the nucleic acid and the oligonucleotide; the contacting and the
cleaving steps being performed at a temperature sufficient to
maintain the nucleic acid in substantially single-stranded form,
the oligonucleotide being functionally complementary to the nucleic
acid over a large enough region to allow the two strands to
associate such that cleavage may occur at the chosen temperature
and at the desired location, and the cleavage being carried out
using a restriction endonuclease that is active at the chosen
temperature; (iv) contacting the nucleic acid with a partially
double-stranded oligonucleotide, the single-stranded region of the
oligonucleotide being functionally complementary to the nucleic
acids in the region that remains after the cleavage in step (iii)
has been effected, and the double-stranded region of the
oligonucleotide including any sequence necessary to return the
sequences that remain after cleavage into proper and original
reading frame for expression and containing a restriction
endonuclease recognition site 5' of those sequences that is
different from the restriction site used in step (iii); and (v)
cleaving the nucleic acid solely at the restriction endonuclease
recognition cleavage site contained within the double-stranded
region of the partially double-stranded oligonucleotide; the
contacting and the cleaving steps being performed at a temperature
sufficient to maintain the nucleic acid in substantially
single-stranded form, the oligonucleotide being functionally
complementary to the nucleic acid over a large enough region to
allow the two strands to associate such that cleavage may occur at
the chosen temperature and at the desired location, and the
restriction being carried out using a cleavage endonuclease that is
active at the chosen temperature; and (vi) expressing a member of
the family of peptides, polypeptides or proteins coded, at least in
part, by the cleaved nucleic acids and collectively expressing at
least a portion of the diversity of the family.
61. The methods according to claim 59 or 60, further comprising at
least one nucleic acid amplification step between one or more of
steps (i) and (ii), steps (ii) and (iii), steps (iii) and (iv) and
steps (iv) and (v).
62. A library comprising a collection of genetic packages that
display a member of a diverse family of peptides, polypeptides or
proteins and collectively display at least a portion of the
diversity of the family, the library being produced using the
methods of claims 59 or 61.
63. A library comprising a collection of members of a diverse
family of peptides, polypeptides or proteins and collectively
comprise at least a portion of the diversity of the family, the
library being produced using the methods of claims 60 or 61.
64. The methods and libraries according to any one of claim 59 to
63, wherein the members of the library encode immunoglobulins.
65. The method and libraries according to claim 64, wherein the
double-stranded region of the oligonucleotide encodes at least a
part of a framework sequence of an immunoglobulin.
66. The method and libraries according to claim 65, wherein the
framework sequence comprises framework 1 of an antibody.
67. The method and libraries according to claim 66, wherein the
framework sequence comprises framework 1 of a variable domain of a
light chain.
68. The method and libraries according to claim 66, wherein the
framework sequence comprises framework 1 of a variable domain of a
heavy chain.
69. The method and libraries according to claim 65, wherein the
framework sequence comprises framework 3 of an antibody.
70. The method and libraries according to claim 69, wherein the
framework sequence comprises framework 3 of a variable domain of a
light chain.
71. The method and libraries according to claim 69, wherein the
framework sequence is framework 3 of a variable domain of a heavy
chain.
72. The method and libraries according to claim 66, wherein the 5'
primer is complementary to a region outside framework 1.
73. The method according to claim 61, wherein amplification primers
for the amplification step are functionally complementary to a
constant region of the nucleic acids.
74. The method according to claim 73, wherein the constant region
is genetically constant in the nucleic acids.
75. The method according to claim 74, wherein the genetically
constant region is part of the genome of immunoglobulin genes
selected from the group of IgM, IgG, IgA, IgE or IgD.
76. The method according to claim 73, wherein the constant region
is exogenous to the nucleic acids.
77. The methods according to claim 61, wherein the amplification
step uses geneRACE.TM..
78. A vector comprising: (i) a DNA sequence encoding an antibody
variable region linked to a version of PIII anchor which does not
mediate infection of phage particles; and (ii) wild-type gene
III.
79. The vector according to claim 78, wherein the DNA encodes a
Fab.
80. The vector according to claim 78, wherein the DNA encodes heavy
chain VHCH1.
81. The vector according to claim 80, wherein the heavy chain VHCH1
is linked to trpIII.
82. The vector according to claim 78, wherein the DNA encodes light
chain VLCL.
83. The vector according to claim 82, wherein the light chain VLCL
is linked to trpIII.
84. The vector according to claim 78, wherein the DNA encodes
scFv.
85. The vector according to claim 84, wherein the scFv is
VL-VH.
86. The vector according to claim 84, wherein the scFv is
VH-VL.
87. The vector according to claim 78, wherein the DNA sequence
encoding an antibody variable region linked to a version of PIII
anchor further comprises an inducible promoter.
88. The vector according to claim 87, wherein the inducible
promoter regulates expression of the DNA sequence encoding an
antibody variable region linked to a version of PIII anchor.
89. The vector according to claim 78, wherein the DNA sequence
encoding an antibody variable region linked to a version of PIII
anchor further comprises an amber stop codon.
90. The vector according to claim 89, wherein the DNA encoding the
amber stop codon is located between the antibody variable region
and the version of pIII.
91. The vector according to any one of claims 78 to 90 wherein the
vector is phage or phagemid.
92. A method for producing a population of immunoglobulin genes
that comprises steps of: (i) introducing synthetic diversity into
at least one of CDR1 or CDR2 of those genes; and (ii) combining the
diversity from step (i) with CDR3 diversity captured from B
cells.
93. The method according to claim 92, wherein synthetic diversity
is introduced into both CDR1 and CDR2.
94. A method for producing a library of immunoglobulin genes that
comprises (i) introducing synthetic diversity into at least one of
CDR1 or CDR2 of those genes; and (ii) combining the diversity from
step (i) with CDR3 diversity captured from B cells.
95. The method according to claim 94, wherein synthetic diversity
is introduced into both CDR1 and CDR2.
96. A library of immunoglobulins that comprise members with at
least one variable domain in which at least one of CDR1 and CDR2
contain synthetic diversity and CDR3 diversity is captured from B
cells.
97. A library according to claim 96, where both CDR1 and CDR2
contain synthetic diversity.
98. The vector according to claim 78, wherein the version of PIII
anchor is characterized by a wild type amino acid sequence and is
encoded by a non-wild type degenerate DNA sequence to a very high
extent.
99. In a method for displaying a member of a diverse family of
peptides, polypeptides or proteins on the surface of a genetic
package and collectively displaying at least a part of the
diversity of the family, the improvement being characterized in
that the displayed peptide, polypeptide or protein is encoded by a
DNA sequence comprising a nucleic acid that has been cleaved at a
desired location by (i) contacting the nucleic acid with a
partially double-stranded oligonucleotide, the single-stranded
region of the oligonucleotide being functionally complementary to
the nucleic acid at its 5' terminal and (ii) cleaving the nucleic
acid solely at a restriction endonuclease cleavage site located in
the double-stranded region of the oligonucleotide or amplifying the
nucleic acid using a primer at least in part functionally
complementary to at least a part of the double-stranded region of
the oligonucleotide, the primer also introducing on amplification
an endonuclease cleavage site and cleaving the amplified nucleic
acid sequence solely at that site; the contacting and the cleaving
steps being performed at a temperature sufficient to maintain the
nucleic acid in substantially single-stranded form, the
oligonucleotide being functionally complementary to the nucleic
acid over a large enough region to allow the two strands to
associate such that cleavage may occur at the chosen temperature
and at the desired location, and the cleavage being carried out
using a restriction endonuclease that is active at the chosen
temperature.
100. A method for displaying a member of a diverse family of
peptides, polypeptides or proteins on the surface of a genetic
package and collectively displaying at least a portion of the
diversity of the family, the method comprising the steps of: (i)
preparing a collection of nucleic acids that code, at least in
part, for members of the diverse family; (ii) rendering the nucleic
acids single-stranded; (iii) cleaving the single-stranded nucleic
acids at a desired location by a method comprising the steps of:
(a) contacting the nucleic acid with a partially double-stranded
oligonucleotide, the single-stranded region of the oligonucleotide
being functionally complementary to the nucleic acid at its 5'
terminal region; and (b) cleaving the nucleic acid solely at a
restriction endonuclease cleavage site located in the
double-stranded region of the oligonucleotide or amplifying the
nucleic acid using a primer at least in part functionally
complementary to at least a part of the double-stranded region of
the oligonucleotide, the primer also introducing on amplification
an endonuclease cleavage site and cleaving the amplified nucleic
acid sequence solely at that site; the contacting and the cleaving
steps being performed at a temperature sufficient to maintain the
nucleic acid in substantially single-stranded form, the
oligonucleotide being functionally complementary to the nucleic
acid over a large enough region to allow the two strands to
associate such that cleavage may occur at the chosen temperature
and at the desired location, and the restriction being carried out
using a cleavage endonuclease that is active at the chosen
temperature; and (iv) displaying a member of the family of
peptides, polypeptides or proteins coded, at least in part, by the
cleaved nucleic acids on the surface of the genetic package and
collectively displaying at least a portion of the diversity of the
family.
101. In a method for expressing a member of a diverse family of
peptides, polypeptides or proteins and collectively expressing at
least a part of the diversity of the family, the improvement being
characterized in that the expressed peptide, polypeptide or protein
is encoded by a DNA sequence comprising a nucleic acid that has
been cleaved at a desired location by (i) contacting the nucleic
acid with a partially double-stranded oligonucleotide, the
single-stranded region of the oligonucleotide being functionally
complementary to the nucleic acid at its 5' terminal region; and
(ii) cleaving the nucleic acid solely at the restriction
endonuclease cleavage site located in the double-stranded region of
the oligonucleotide or amplifying the nucleic acid using a primer
at least in part functionally complementary to at least a part of
the double-stranded region of the oligonucleotide, the primer also
introducing on amplification an endonuclease cleavage site and
cleaving the amplified nucleic acid sequence solely at that site;
the contacting and the cleaving steps being performed at a
temperature sufficient to maintain the nucleic acid in
substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
102. A method for expressing a member of a diverse family of
peptides, polypeptides or proteins and collectively expressing at
least a portion of the diversity of the family, the method
comprising the steps of: (i) preparing a collection of nucleic
acids that code, at least in part, for members of the diverse
family; (ii) rendering the nucleic acids single-stranded; (iii)
cleaving the single-stranded nucleic acids at a desired location by
a method comprising the steps of: (a) contacting the nucleic acid
with a partially double-stranded oligonucleotide, the
single-stranded region of the oligonucleotide being functionally
complementary to the nucleic acid at its 5' terminal region; and
(b) cleaving the nucleic acid solely at a restriction endonuclease
cleavage site located in the double-stranded region of the
nucleotide; or amplifying the nucleic acid using a primer at least
in part functionally complementary to at least a part of the
double-stranded region of the oligonucleotide, the primer also
introducing on amplification an endonuclease cleavage site and
cleaving the amplified nucleic acid sequence solely at that site;
the contacting and the cleaving steps being performed at a
temperature sufficient to maintain the nucleic acid in
substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the restriction being carried out using a cleavage endonuclease
that is active at the chosen temperature; and (iv) expressing a
member of the family of peptides, polypeptides or proteins coded,
at least in part, by the cleaved nucleic acids and collectively
expressing at least a portion of the diversity of the family.
103. A method for preparing a library comprising a collection of
genetic packages that display a member of a diverse family of
peptides, polypeptides or proteins and that collectively display at
least a portion of the family comprising the steps: (i) preparing a
collection of nucleic acids that code at least in part for members
of the diverse family; (ii) rendering the nucleic acids
single-stranded; (iii) cleaving the single-stranded nucleic acids
at a desired location by a method comprising the steps of: (a)
contacting the nucleic acid with a single-stranded oligonucleotide,
the oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired and including a
sequence that with its complement in the nucleic acid forms a
restriction endonuclease recognition site that on restriction
results in cleavage of the nucleic acid at the desired location;
and (b) cleaving the nucleic acid solely at the recognition site
formed by the complementation of the nucleic acid and the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature; (iv) contacting the
nucleic acid with a partially double-stranded oligonucleotide, the
single-stranded region of the oligonucleotide being functionally
complementary to the nucleic acids in the 5' terminal region that
remains after the cleavage in step (iii) has been effected, and the
double-stranded region of the oligonucleotide including any
sequences necessary to return the sequences that remain after
cleavage into proper and original reading frame for display; and
(v) cleaving the nucleic acid solely at a restriction endonuclease
cleavage site contained within the double-stranded region of the
partially double-stranded oligonucleotide, the site being different
from that used in step (iii) or amplifying the nucleic acid using a
primer at least in part functionally complementary to at least a
part of the double-stranded region of the oligonucleotide, the
primer also introducing on amplification an endonuclease cleavage
site and cleaving the amplified nucleic acid sequence solely at
that site; the contacting and the cleaving steps being performed at
a temperature sufficient to maintain the nucleic acid in
substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the restriction being carried out using a cleavage endonuclease
that is active at the chosen temperature; and (vi) displaying a
member of the family of peptides, polypeptides or proteins coded,
at least in part, by the cleaved nucleic acids on the surface of
the genetic package and collectively displaying at least a portion
of the diversity of the family.
104. A method for preparing a library comprising a collection of
members of a diverse family of peptides, polypeptides or proteins
and collectively comprising at least a portion of the family
comprising the steps: (i) preparing a collection of nucleic acids
that code at least in part for members of the diverse family; (ii)
rendering the nucleic acids single-stranded; (iii) cleaving the
single-stranded nucleic acids at a desired location by a method
comprising the steps of: (a) contacting the nucleic acid with a
single-stranded oligonucleotide, the oligonucleotide being
functionally complementary to the nucleic acid in the region in
which cleavage is desired and including a sequence that with its
complement in the nucleic acid forms a restriction endonuclease
recognition site that on restriction results in cleavage of the
nucleic acid at the desired location; and (b) cleaving the nucleic
acid solely at the recognition site formed by the complementation
of the nucleic acid and the oligonucleotide; the contacting and the
cleaving steps being performed at a temperature sufficient to
maintain the nucleic acid in substantially single-stranded form,
the oligonucleotide being functionally complementary to the nucleic
acid over a large enough region to allow the two strands to
associate such that cleavage may occur at the chosen temperature
and at the desired location, and the cleavage being carried out
using a restriction endonuclease that is active at the chosen
temperature; (iv) contacting the nucleic acid with a partially
double-stranded oligonucleotide, the single-stranded region of the
oligonucleotide being functionally complementary to the nucleic
acids in the 5' terminal region that remains after the cleavage in
step (iii) has been effected, and the double-stranded region of the
oligonucleotide including any sequence necessary to return the
sequences that remain after cleavage into proper and original
reading frame for expression; and (v) cleaving the nucleic acid
solely at a restriction endonuclease cleavage site contained within
the double-stranded region of the partially double-stranded
oligonucleotide, the site being different from that used in step
(iii) or amplifying the nucleic acid using a primer at least in
part functionally complementary to at least a part of the
double-stranded region of the oligonucleotide, the primer
introducing on amplification an endonuclease cleavage site and
cleaving the amplified nucleic acid sequence solely at that site;
the contacting and the cleaving steps being performed at a
temperature sufficient to maintain the nucleic acid in
substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the restriction being carried out using a cleavage endonuclease
that is active at the chosen temperature; and (vi) expressing a
member of the family of peptides, polypeptides or proteins coded,
at least in part, by the cleaved nucleic acids and collectively
expressing at least a portion of the diversity of the family.
105. A library of immunoglobins comprising members having at least
one variable domain in which one or both of the CDR 1 and CDR 2
have synthetic diversity and the CDR 3 has diversity captured from
B-Cells.
106. The library according to claim 104, wherein a first variable
domain has synthetic diversity in CDR 1 and CDR 2 and has diversity
in CDR 3 captured from B-cells and a second variable domain has
diversity captured from B-cells.
107. The library according to claim 104 or 105, wherein the
variable domain is selected from the group of VH or VL.
108. A method for cleaving a nucleic acid sequence at a desired
location, the method comprising the steps of: (i) contacting a
single-stranded nucleic acid sequence with a partially
double-stranded oligonucleotide, the single-stranded region of the
oligonucleotide being functionally complementary to the 5' terminal
region of the nucleic acid sequence, the double-stranded region of
the oligonucleotide including any sequences necessary to return the
sequence in the single-stranded nucleic acid sequence into proper
and original reading frame for expression; and (ii) cleaving the
partially double-stranded oligonucleotide-single-stranded nucleic
acid combination solely at a restriction endonuclease cleavage site
contained within the double-stranded oligonucleotide or amplifying
the combination using a primer at least in part functionally
complementary to at least part of the double-stranded region of the
oligonucleotide, the primer introducing during amplification an
endonuclease cleavage site and cleaving the amplified sequence
solely at the site.
109. The method according to claim 108, wherein the length of the
single-stranded portion of the partially double-stranded
oligonucleotide is between 2 and 15 bases.
110. The method according to claim 109, wherein the length of the
single-stranded portion of the partially double-stranded
oligonucleotide is between 7 and 10 bases.
111. The method according to claim 108, wherein the length of the
double-stranded portion of the partially double-stranded
oligonucleotide is between 12 and 100 base pairs.
112. The method according to claim 111, wherein the length of the
double-stranded portion of the partially double-stranded
oligonucleotide is between 20 and 100 base pairs.
113. The methods according to any one of claims 99 to 104 and 108,
further comprising at least one nucleic acid amplification step
between one or more of steps (i) and (ii), steps (ii) and (iii),
steps (iii) and (iv) and steps (iv) and (v).
114. A library comprising a collection of genetic packages that
display a member of a diverse family of peptides, polypeptides or
proteins and collectively display at least a portion of the
diversity of the family, the library being produced using the
methods of claims 99, 100, 103 or 113.
115. A library comprising a collection of members of a diverse
family of peptides, polypeptides or proteins and collectively
comprise at least a portion of the diversity of the family, the
library being produced using the methods of claims 101, 102, 104 or
113.
116. The methods and libraries according to any one of claims 99 to
104 or 113, wherein the members of the library encode
immunoglobulins.
Description
[0001] This application is a continuation-in-part of United States
provisional application No. 06/198,069, filed Apr. 17, 2000, a
continuation-in-part of U.S. patent application Ser. No.
09/837,306, filed on Apr. 17, 2001, and a continuation-in-part of
U.S. application Ser. No. XX/XXX,XXX, filed by Express
Mail(EI125454535US) on Oct. 25, 2001. All of the earlier
applications are specifically incorporated by reference herein.
[0002] The present invention relates to libraries of genetic
packages that display and/or express a member of a diverse family
of peptides, polypeptides or proteins and collectively display
and/or express at least a portion of the diversity of the family.
In an alternative embodiment, the invention relates to libraries
that include a member of a diverse family of peptides, polypeptides
or proteins and collectively comprise at least a portion of the
diversity of the family. In a preferred embodiment, the displayed
and/or expressed polypeptides are human Fabs.
[0003] More specifically, the invention is directed to the methods
of cleaving single-stranded nucleic acids at chosen locations, the
cleaved nucleic acids encoding, at least in part, the peptides,
polypeptides or proteins displayed on the genetic packages of,
and/or expressed in, the libraries of the invention. In a preferred
embodiment, the genetic packages are filamentous phage or phagemids
or yeast.
[0004] The present invention further relates to vectors for
displaying and/or expressing a diverse family of peptides,
polypeptides or proteins.
[0005] The present invention further relates to methods of
screening the libraries of the invention and to the peptides,
polypeptides and proteins identified by such screening.
BACKGROUND OF THE INVENTION
[0006] It is now common practice in the art to prepare libraries of
genetic packages that display, express or comprise a member of a
diverse family of peptides, polypeptides or proteins and
collectively display, express or comprise at least a portion of the
diversity of the family. In many common libraries, the peptides,
polypeptides or proteins are related to antibodies. Often, they are
Fabs or single chain antibodies.
[0007] In general, the DNAs that encode members of the families to
be displayed and/or expressed must be amplified before they are
cloned and used to display and/or express the desired member. Such
amplification typically makes use of forward and backward
primers.
[0008] Such primers can be complementary to sequences native to the
DNA to be amplified or complementary to oligonucleotides attached
at the 5' or 3' ends of that DNA. Primers that are complementary to
sequences native to the DNA to be amplified are disadvantaged in
that they bias the members of the families to be displayed. Only
those members that contain a sequence in the native DNA that is
substantially complementary to the primer will be amplified. Those
that do not will be absent from the family. For those members that
are amplified, any diversity within the primer region will be
suppressed.
[0009] For example, in European patent 368,684 B1, the primer that
is used is at the 5' end of the V.sub.H region of an antibody gene.
It anneals to a sequence region in the native DNA that is said to
be "sufficiently well conserved" within a single species. Such
primer will bias the members amplified to those having this
"conserved" region. Any diversity within this region is
extinguished.
[0010] It is generally accepted that human antibody genes arise
through a process that involves a combinatorial selection of V and
J or V, D, and J followed by somatic mutations. Although most
diversity occurs in the Complementary Determining Regions (CDRs),
diversity also occurs in the more conserved Framework Regions (FRs)
and at least some of this diversity confers or enhances specific
binding to antigens (Ag). As a consequence, libraries should
contain as much of the CDR and FR diversity as possible.
[0011] To clone the amplified DNAs of the peptides, polypeptides or
proteins that they encode for display on a genetic package and/or
for expression, the DNAs must be cleaved to produce appropriate
ends for ligation to a vector. Such cleavage is generally effected
using restriction endonuclease recognition sites carried on the
primers. When the primers are at the 5' end of DNA produced from
reverse transcription of RNA, such restriction leaves deleterious
5' untranslated regions in the amplified DNA. These regions
interfere with expression of the cloned genes and thus the display
of the peptides, polypeptides and proteins coded for by them.
SUMMARY OF THE INVENTION
[0012] It is an object of this invention to provide novel methods
for constructing libraries that display, express or comprise a
member of a diverse family of peptides, polypeptides or proteins
and collectively display, express or comprise at least a portion of
the diversity of the family. These methods are not biased toward
DNAs that contain native sequences that are complementary to the
primers used for amplification. They also enable any sequences that
may be deleterious to expression to be removed from the amplified
DNA before cloning and displaying and/or expressing.
[0013] It is another object of this invention to provide a method
for cleaving single-stranded nucleic acid sequences at a desired
location, the method comprising the steps of: [0014] (i) contacting
the nucleic acid with a single-stranded oligonucleotide, the
oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired and including a
sequence that with its complement in the nucleic acid forms a
restriction endonuclease recognition site that on restriction
results in cleavage of the nucleic acid at the desired location;
and [0015] (ii) cleaving the nucleic acid solely at the recognition
site formed by the complementation of the nucleic acid and the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
[0016] It is a further object of this invention to provide an
alternative method for cleaving single-stranded nucleic acid
sequences at a desired location, the method comprising the steps
of: [0017] (i) contacting the nucleic acid with a partially
double-stranded oligonucleotide, the single-stranded region of the
oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired, and the
double-stranded region of the oligonucleotide having a restriction
endonuclease recognition site; and [0018] (ii) cleaving the nucleic
acid solely at the cleavage site formed by the complementation of
the nucleic acid and the single-stranded region of the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
[0019] In an alternative embodiment of this object of the
invention, the restriction endonuclease recognition site is not
initially located in the double-stranded part of the
oligonucleotide. Instead, it is part of an amplification primer,
which primer is complementary to the double-stranded region of the
oligonucleotide. On amplification of the DNA-partially
double-stranded combination, the restriction endonuclease
recognition site carried on the primer becomes part of the DNA. It
can then be used to cleave the DNA.
[0020] Preferably, the restriction endonuclease recognition site is
that of a Type II-S restriction endonuclease whose cleavage site is
located at a known distance from its recognition site.
[0021] It is another object of the present invention to provide a
method of capturing DNA molecules that comprise a member of a
diverse family of DNAs and collectively comprise at least a portion
of the diversity of the family. These DNA molecules in
single-stranded form have been cleaved by one of the methods of
this invention. This method involves ligating the individual
single-stranded DNA members of the family to a partially duplex DNA
complex. The method comprises the steps of: [0022] (i) contacting a
single-stranded nucleic acid sequence that has been cleaved with a
restriction endonuclease with a partially double-stranded
oligonucleotide, the single-stranded region of the oligonucleotide
being functionally complementary to the nucleic acid in the region
that remains after cleavage, the double-stranded region of the
oligonucleotide including any sequences necessary to return the
sequences that remain after cleavage into proper reading frame for
expression and containing a restriction endonuclease recognition
site 5' of those sequences; and [0023] (ii) cleaving the partially
double-stranded oligonucleotide sequence solely at the restriction
endonuclease cleavage site contained within the double-stranded
region of the partially double-stranded oligonucleotide.
[0024] As before, in this object of the invention, the restriction
endonuclease recognition site need not be located in the
double-stranded portion of the oligonucleotide. Instead, it can be
introduced on amplification with an amplification primer that is
used to amplify the DNA-partially double-stranded oligonucleotide
combination.
[0025] It is another object of this invention to prepare libraries,
that display, express or comprise a diverse family of peptides,
polypeptides or proteins and collectively display, express or
comprise at least part of the diversity of the family, using the
methods and DNAs described above.
[0026] It is an object of this invention to screen those libraries
to identify useful peptides, polypeptides and proteins and to use
those substances in human therapy.
[0027] Additional objects of the invention are reflected in claims
1-116. Each of these claims is specifically incorporated by
reference in this specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a schematic of various methods that may be
employed to amplify VH genes without using primers specific for VH
sequences.
[0029] FIG. 2 is a schematic of various methods that may be
employed to amplify VL genes without using primers specific for VL
sequences.
[0030] FIG. 3 is a schematic of RACE amplification of antibody
heavy and light chains.
[0031] FIG. 4 depicts gel analysis of amplification products
obtained after the primary PCR reaction from 4 different patient
samples.
[0032] FIG. 5 depicts gel analysis of cleaved kappa DNA from
Example 2.
[0033] FIG. 6 depicts gel analysis of extender-cleaved kappa DNA
from Example 2.
[0034] FIG. 7 depicts gel analysis of the PCR product from the
extender-kappa amplification from Example 2.
[0035] FIG. 8 depicts gel analysis of purified PCR product from the
extender-kappa amplification from Example 2.
[0036] FIG. 9 depicts gel analysis of cleaved and ligated kappa
light chains from Example 2.
[0037] FIG. 10 is a schematic of the design for CDR1 and CDR2
synthetic diversity.
[0038] FIG. 11 is a schemaitc of the cloning schedule for
construction of the heavy chain repertoire.
[0039] FIG. 12 is a schematic of the cleavage and ligation of the
antibody light chain.
[0040] FIG. 13 depicts gel analysis of cleaved and ligated lambda
light chains from Example 4.
[0041] FIG. 14 is a schematic of the cleavage and ligation of the
antibody heavy chain.
[0042] FIG. 15 depicts gel analysis of cleaved and ligated lambda
light chains from Example 5.
[0043] FIG. 16 is a schematic of a phage display vector.
[0044] FIG. 17 is a schematic of a Fab cassette.
[0045] FIG. 18 is a schematic of a process for incorporating fixed
FR1 residues in an antibody lambda sequence.
[0046] FIG. 19 is a schematic of a process for incorporating fixed
FR1 residues in an antibody kappa sequence.
[0047] FIG. 20 is a schematic of a process for incorporating fixed
FR1 residues in an antibody heavy chain sequence.
TERMS
[0048] In this application, the following terms and abbreviations
are used: TABLE-US-00001 Sense strand The upper strand of ds DNA as
usually written. In the sense strand, 5'-ATG-3' codes for Met.
Antisense strand The lower strand of ds DNA as usually written. In
the antisense strand, 3'-TAC-5' would correspond to a Met codon in
the sense strand. Forward primer A "forward" primer is
complementary to a part of the sense strand and primes for
synthesis of a new antisense- strand molecule. "Forward primer" and
"lower-strand primer" are equivalent. Backward primer A "backward"
primer is complementary to a part of the antisense strand and
primes for synthesis of a new sense- strand molecule. "Backward
primer" and "top-strand primer" are equivalent. Bases Bases are
specified either by their position in a vector or gene as their
position within a gene by codon and base. For example, "89.1" is
the first base of codon 89, 89.2 is the second base of codon 89. Sv
Streptavidin Ap Ampicillin ap.sup.R A gene conferring ampicillin
resistance. RERS Restriction endonuclease recognition site RE
Restriction endonuclease - cleaves preferentially at RERS URE
Universal restriction endonuclease Functionally Two sequences are
sufficiently complementary complementary so as to anneal under the
chosen conditions. AA Amino acid PCR Polymerization chain reaction
GLGs Germline genes An Antibody: an immunoglobin. The term also
covers any protein having a binding domain which is homologous to
an immunoglobin binding domain. A few examples of antibodies within
this definition are, inter alia, immunoglobin isotypes and the Fab,
F(ab.sup.1).sub.2, scfv, Fv, dAb and Fd fragments. Fab Two chain
molecule comprising an An light chain and part of a heavy-chain.
scFv A single-chain Ab comprising either VH::linker::VL or
VL::linker::VH w.t. Wild type HC Heavy chain LC Light chain VK A
variable domain of a Kappa light chain. VH A variable domain of a
heavy chain. VL A variable domain of a lambda light chain.
[0049] In this application when it is said that nucleic acids are
cleaved solely at the cleavage site of a restriction endonuclease,
it should be understood that minor cleavage may occur at random,
e.g., at non-specific sites other than the specific cleavage site
that is characteristic of the restriction endonuclease. The skilled
worker will recognize that such non-specific, random cleavage is
the usual occurrence. Accordingly, "solely at the cleavage site" of
a restriction endonuclease means that cleavage occurs
preferentially at the site characteristic of that endonuclease.
[0050] As used in this application and claims, the term "cleavage
site formed by the complementation of the nucleic acid and the
single-stranded region of the oligonucleotide" includes cleavage
sites formed by the single-stranded portion of the partially
double-stranded ologonucleotide duplexing with the single-stranded
DNA, cleavage sites in the double-stranded portion of the partially
double-stranded oligonucleotide, and cleavage sites introduced by
the amplification primer used to amplify the single-stranded
DNA-partially double-stranded oligonucleotide combination.
[0051] In the two methods of this invention for preparing
single-stranded nucleic acid sequences, the first of those cleavage
sites is preferred. In the methods of this invention for capturing
diversity and cloning a family of diverse nucleic acid sequences,
the latter two cleavage sites are preferred.
[0052] In this application, all references referred to are
specifically incorporated by reference.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0053] The nucleic acid sequences that are useful in the methods of
this invention, i.e., those that encode at least in part the
individual peptides, polypeptides and proteins displayed, or
expressed in or comprising the libraries of this invention, may be
native, synthetic or a combination thereof. They may be mRNA, DNA
or cDNA. In the preferred embodiment, the nucleic acids encode
antibodies. Most preferably, they encode Fabs.
[0054] The nucleic acids useful in this invention may be naturally
diverse, synthetic diversity may be introduced into those naturally
diverse members, or the diversity may be entirely synthetic. For
example, synthetic diversity can be introduced into one or more
CDRs of antibody genes. Preferably, it is introduced into CDR1 and
CDR2 of immunoglobulins. Preferably, natural diversity is captured
in the CDR3 regions of the immunoglogin genes of this invention
from B cells. Most preferably, the nucleic acids of this invention
comprise a population of immunoglobin genes that comprise synthetic
diversity in at least one, and more preferably both of the CDR1 and
CDR2 and diversity in CDR3 captured from B cells.
[0055] Synthetic diversity may be created, for example, through the
use of TRIM technology (U.S. Pat. No. 5,869,644). TRIM technology
allows control over exactly which amino-acid types are allowed at
variegated positions and in what proportions. In TRIM technology,
codons to be diversified are synthesized using mixtures of
trinucleotides. This allows any set of amino acid types to be
included in any proportion.
[0056] Another alternative that may be used to generate diversified
DNA is mixed oligonucleotide synthesis. With TRIM technology, one
could allow Ala and Trp. With mixed oligonucleotide synthesis, a
mixture that included Ala and Trp would also necessarily include
Ser and Gly. The amino-acid types allowed at the variegated
positions are picked with reference to the structure of antibodies,
or other peptides, polypeptides or proteins of the family, the
observed diversity in germline genes, the observed somatic
mutations frequently observed, and the desired areas and types of
variegation.
[0057] In a preferred embodiment of this invention, the nucleic
acid sequences for at least one CDR or other region of the
peptides, polypeptides or proteins of the family are cDNAs produced
by reverse transcription from mRNA. More preferably, the mRNAs are
obtained from peripheral blood cells, bone marrow cells, spleen
cells or lymph node cells (such as B-lymphocytes or plasma cells)
that express members of naturally diverse sets of related genes.
More preferable, the mRNAs encode a diverse family of antibodies.
Most preferably, the mRNAs are obtained from patients suffering
from at least one autoimmune disorder or cancer. Preferably, mRNAs
containing a high diversity of autoimmune diseases, such as
systemic lupus erythematosus, systemic sclerosis, rheumatoid
arthritis, antiphospholipid syndrome and vasculitis are used.
[0058] In a preferred embodiment of this invention, the cDNAs are
produced from the mRNAs using reverse transcription. In this
preferred embodiment, the mRNAs are separated from the cell and
degraded using standard methods, such that only the full length
(i.e., capped) mRNAs remain. The cap is then removed and reverse
transcription used to produce the cDNAs.
[0059] The reverse transcription of the first (antisense) strand
can be done in any manner with any suitable primer. See, e.g., H J
de Haard et al., Journal of Biological Chemistry, 274(26):18218-30
(1999). In the preferred embodiment of this invention where the
mRNAs encode antibodies, primers that are complementary to the
constant regions of antibody genes may be used. Those primers are
useful because they do not generate bias toward subclasses of
antibodies. In another embodiment, poly-dT primers may be used (and
may be preferred for the heavy-chain genes). Alternatively,
sequences complementary to the primer may be attached to the
termini of the antisense strand.
[0060] In one preferred embodiment of this invention, the reverse
transcriptase primer may be biotinylated, thus allowing the cDNA
product to be immobilized on streptavidin (Sv) beads.
Immobilization can also be effected using a primer labeled at the
5' end with one of a) free amine group, b) thiol, c) carboxylic
acid, or d) another group not found in DNA that can react to form a
strong bond to a known partner on an insoluble medium. If, for
example, a free amine (preferably primary amine) is provided at the
5' end of a DNA primer, this amine can be reacted with carboxylic
acid groups on a polymer bead using standard amide-forming
chemistry. If such preferred immobilization is used during reverse
transcription, the top strand RNA is degraded using well-known
enzymes, such as a combination of RNAseH and RNAseA, either before
or after immobilization.
[0061] The nucleic acid sequences useful in the methods of this
invention are generally amplified before being used to display
and/or express the peptides, polypeptides or proteins that they
encode. Prior to amplification, the single-stranded DNAs may be
cleaved using either of the methods described before.
Alternatively, the single-stranded DNAs may be amplified and then
cleaved using one of those methods.
[0062] Any of the well known methods for amplifying nucleic acid
sequences may be used for such amplification. Methods that
maximize, and do not bias, diversity are preferred. In a preferred
embodiment of this invention where the nucleic acid sequences are
derived from antibody genes, the present invention preferably
utilizes primers in the constant regions of the heavy and light
chain genes and primers to a synthetic sequence that are attached
at the 5' end of the sense strand. Priming at such synthetic
sequence avoids the use of sequences within the variable regions of
the antibody genes. Those variable region priming sites generate
bias against V genes that are either of rare subclasses or that
have been mutated at the priming sites. This bias is partly due to
suppression of diversity within the primer region and partly due to
lack of priming when many mutations are present in the region
complementary to the primer. The methods disclosed in this
invention have the advantage of not biasing the population of
amplified antibody genes for particular V gene types.
[0063] The synthetic sequences may be attached to the 5' end of the
DNA strand by various methods well known for ligating DNA sequences
together. RT CapExtention is one preferred method.
[0064] In RT CapExtention (derived from Smart PCR(.TM.)), a short
overlap (5'- . . . GGG-3' in the upper-strand primer (USP-GGG)
complements 3'-CCC . . . . 5' in the lower strand) and reverse
transcriptases are used so that the reverse complement of the
upper-strand primer is attached to the lower strand.
[0065] FIGS. 1 and 2 show schematics to amplify VH and VL genes
using RT CapExtention. FIG. 1 shows a schematic of the
amplification of VH genes. FIG. 1, Panel A shows a primer specific
to the poly-dT region of the 3' UTR priming synthesis of the first,
lower strand. Primers that bind in the constant region are also
suitable. Panel B shows the lower strand extended at its 3' end by
three Cs that are not complementary to the mRNA. Panel C shows the
result of annealing a synthetic top-strand primer ending in three
GGGs that hybridize to the 3' terminal CCCs and extending the
reverse transcription extending the lower strand by the reverse
complement of the synthetic primer sequence. Panel D shows the
result of PCR amplification using a 5' biotinylated synthetic
top-strand primer that replicates the 5' end of the synthetic
primer of panel C and a bottom-strand primer complementary to part
of the constant domain. Panel E shows immobilized double-stranded
(ds) cDNA obtained by using a 5'-biotinylated top-strand
primer.
[0066] FIG. 2 shows a similar schematic for amplification of VL
genes. FIG. 2, Panel A shows a primer specific to the constant
region at or near the 3' end priming synthesis of the first, lower
strand. Primers that bind in the poly-dT region are also suitable.
Panel B shows the lower strand extended at its 3' end by three Cs
that are not complementary to the mRNA. Panel C shows the result of
annealing a synthetic top-strand primer ending in three GGGs that
hybridize to the 3' terminal CCCs and extending the reverse
transcription extending the lower strand by the reverse complement
of the synthetic primer sequence. Panel D shows the result of PCR
amplification using a 5' biotinylated synthetic top-strand primer
that replicates the 5' end of the synthetic primer of panel C and a
bottom-strand primer complementary to part of the constant domain.
The bottom-strand primer also contains a useful restriction
endonuclease site, such as AscI. Panel E shows immobilized ds cDNA
obtained by using a 5'-biotinylated top-strand primer.
[0067] In FIGS. 1 and 2, each V gene consists of a 5' untranslated
region (UTR) and a secretion signal, followed by the variable
region, followed by a constant region, followed by a 3'
untranslated region (which typically ends in poly-A). An initial
primer for reverse transcription may be complementary to the
constant region or to the poly A segment of the 3'-UTR. For human
heavy-chain genes, a primer of 15 T is preferred. Reverse
transcriptases attach several C residues to the 3' end of the newly
synthesized DNA. RT CapExtention exploits this feature. The reverse
transcription reaction is first run with only a lower-strand
primer. After about 1 hour, a primer ending in GGG (USP-GGG) and
more RTase are added. This causes the lower-strand cDNA to be
extended by the reverse complement of the USP-GGG up to the final
GGG. Using one primer identical to part of the attached synthetic
sequence and a second primer complementary to a region of known
sequence at the 3' end of the sense strand, all the V genes are
amplified irrespective of their V gene subclass.
[0068] In another preferred embodiment, synthetic sequences may be
added by Rapid Amplification of cDNA Ends (RACE) (see Frohman, M.
A., Dush, M. K., & Martin, G. R. (1988) Proc. Natl. Acad. Sci.
USA (85): 8998-9002).
[0069] FIG. 1 shows a schematic of RACE amplification of antibody
heavy and light chains. First, mRNA is selected by treating total
or poly(A+) RNA with calf intestinal phosphatase (CIP) to remove
the 5'-phosphate from all molecules that have them such as
ribosomal RNA, fragmented mRNA, tRNA and genomic DNA. Full length
mRNA (containing a protective 7-methyl cap structure) is
uneffected. The RNA is then treated with tobacco acid
pyrophosphatase (TAP) to remove the cap structure from full length
mRNAs leaving a 5'-monophosphate group. Next, a synthetic RNA
adaptor is ligated to the RNA population, only molecules which have
a 5-phosphate (uncapped, full length mRNAs) will accept the
adaptor. Reverse trascriptase reactions using an oligodT primer,
and nested PCR (using one adaptor primer (located in the 5'
synthetic adaptor) and one primer for the gene) are then used to
amplify the desired transcript.
[0070] In a preferred embodiment of this invention, the upper
strand or lower strand primer may be also biotinylated or labeled
at the 5' end with one of a) free amino group, b) thiol, c)
carboxylic acid and d) another group not found in DNA that can
react to form a strong bond to a known partner as an insoluble
medium. These can then be used to immobilize the labeled strand
after amplification. The immobilized DNA can be either single or
double-stranded.
[0071] After amplification (using e.g., RT CapExtension or RACE),
the DNAs of this invention are rendered single-stranded. For
example, the strands can be separated by using a biotinylated
primer, capturing the biotinylated product on streptavidin beads,
denaturing the DNA, and washing away the complementary strand.
Depending on which end of the captured DNA is wanted, one will
choose to immobilize either the upper (sense) strand or the lower
(antisense) strand.
[0072] To prepare the single-stranded amplified DNAs for cloning
into genetic packages so as to effect display of, or for expression
of, the peptides, polypeptides or proteins encoded, at least in
part, by those DNAs, they must be manipulated to provide ends
suitable for cloning and display and/or expression. In particular,
any 5' untranslated regions and mammalian signal sequences must be
removed and replaced, in frame, by a suitable signal sequence that
functions in the display or expression host. Additionally, parts of
the variable domains (in antibody genes) may be removed and
replaced by synthetic segments containing synthetic diversity. The
diversity of other gene families may likewise be expanded with
synthetic diversity.
[0073] According to the methods of this invention, there are two
ways to manipulate the single-stranded DNAs for display and/or
expression. The first method comprises the steps of: [0074] (i)
contacting the nucleic acid with a single-stranded oligonucleotide,
the oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired and including a
sequence that with its complement in the nucleic acid forms a
restriction endonuclease recognition site that on restriction
results in cleavage of the nucleic acid at the desired location;
and [0075] (ii) cleaving the nucleic acid solely at the recognition
site formed by the complementation of the nucleic acid and the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
[0076] In this first method, short oligonucleotides are annealed to
the single-stranded DNA so that restriction endonuclease
recognition sites formed within the now locally double-stranded
regions of the DNA can be cleaved. In particular, a recognition
site that occurs at the same position in a substantial fraction of
the single-stranded DNAs is identical.
[0077] For antibody genes, this can be done using a catalog of
germline sequences. See, e.g.,
"http://www.mrc-cpe.cam.ac.uk/imt-doc/restricted/ok.htm l." Updates
can be obtained from this site under the heading "Amino acid and
nucleotide sequence alignments." For other families, similar
comparisons exist and may be used to select appropriate regions for
cleavage and to maintain diversity.
[0078] For example, Table 1 depicts the DNA sequences of the FR3
regions of the 51 known human VH germline genes. In this region,
the genes contain restriction endonuclease recognition sites shown
in Table 2. Restriction endonucleases that cleave a large fraction
of germline genes at the same site are preferred over endonucleases
that cut at a variety of sites. Furthermore, it is preferred that
there be only one site for the restriction endonucleases within the
region to which the short oligonucleotide binds on the
single-stranded DNA, e.g., about 10 bases on either side of the
restriction endonuclease recognition site.
[0079] An enzyme that cleaves downstream in FR3 is also more
preferable because it captures fewer mutations in the framework.
This may be advantageous is some cases. However, it is well known
that framework mutations exist and confer and enhance antibody
binding. The present invention, by choice of appropriate
restriction site, allows all or part of FR3 diversity to be
captured. Hence, the method also allows extensive diversity to be
captured.
[0080] Finally, in the methods of this invention restriction
endonucleases that are active between about 37.degree. C. and about
75.degree. C. are used. Preferably, restriction endonucleases that
are active between about 45.degree. C. and about 75.degree. C. may
be used. More preferably, enzymes that are active above 50.degree.
C., and most preferably active about 55.degree. C., are used. Such
temperatures maintain the nucleic acid sequence to be cleaved in
substantially single-stranded form.
[0081] Enzymes shown in Table 2 that cut many of the heavy chain
FR3 germline genes at a single position include: MaeIII(24@4),
Tsp45I(21@4), HphI(44@5), BsaJI(23@65), AluI(23@47), BlpI(21@48),
DdeI(29@58), BglII(10@61), MslI(44@72), BsiEI(23@74), EaeI(23@74),
EagI(23@74), HaeIII(25@75), Bst4CI(51@86), HpyCH4III(51@86),
HinfI(38@2), MlyI(18@2), PleI(18@2), MnlI(31@67), HpyCH4V(21@44),
BsmAI(16@11), BpmI(19@12), XmnI(12@30), and SacI(11@51). (The
notation used means, for example, that BsmAI cuts 16 of the FR3
germline genes with a restriction endonuclease recognition site
beginning at base 11 of FR3.)
[0082] For cleavage of human heavy chains in FR3, the preferred
restriction endonucleases are: Bst4CI (or TaaI or HpyCH4III), BlpI,
HpyCH4V, and MslI. Because ACNGT (the restriction endonuclease
recognition site for Bst4CI, TaaI, and HpyCH4III) is found at a
consistent site in all the human FR3 germline genes, one of those
enzymes is the most preferred for capture of heavy chain CDR3
diversity. BlpI and HpyCH4V are complementary. BlpI cuts most
members of the VH1 and VH4 families while HpyCH4V cuts most members
of the VH3, VH5, VH6, and VH7 families. Neither enzyme cuts VH2s,
but this is a very small family, containing only three members.
Thus, these enzymes may also be used in preferred embodiments of
the methods of this invention.
[0083] The restriction endonucleases HpyCH4III, Bst4CI, and TaaI
all recognize 5'-ACnGT-3' and cut upper strand DNA after n and
lower strand DNA before the base complementary to n. This is the
most preferred restriction endonuclease recognition site for this
method on human heavy chains because it is found in all germline
genes. Furthermore, the restriction endonuclease recognition region
(ACnGT) matches the second and third bases of a tyrosine codon
(tay) and the following cysteine codon (tgy) as shown in Table 3.
These codons are highly conserved, especially the cysteine in
mature antibody genes.
[0084] Table 4 E shows the distinct oligonucleotides of length 22
(except the last one which is of length 20) bases. Table 5 C shows
the analysis of 1617 actual heavy chain antibody genes. Of these,
1511 have the site and match one of the candidate oligonucleotides
to within 4 mismatches. Eight oligonucleotides account for most of
the matches and are given in Table 4 F.1. The 8 oligonucleotides
are very similar so that it is likely that satisfactory cleavage
will be achieved with only one oligonucleotide (such as
H43.77.97.1-02#1) by adjusting temperature, pH, salinity, and the
like. One or two oligonucleotides may likewise suffice whenever the
germline gene sequences differ very little and especially if they
differ very little close to the restriction endonuclease
recognition region to be cleaved. Table 5 D shows a repeat analysis
of 1617 actual heavy chain antibody genes using only the 8 chosen
oligonucleotides. This shows that 1463 of the sequences match at
least one of the oligonucleotides to within 4 mismatches and have
the site as expected. Only 7 sequences have a second HpyCH4III
restriction endonuclease recognition region in this region.
[0085] Another illustration of choosing an appropriate restriction
endonuclease recognition site involves cleavage in FR1 of human
heavy chains. Cleavage in FR1 allows capture of the entire CDR
diversity of the heavy chain.
[0086] The germline genes for human heavy chain FR1 are shown in
Table 6. Table 7 shows the restriction endonuclease recognition
sites found in human germline genes FR1s. The preferred sites are
BsgI(GTGCAG;39@4), BsoFI(GCngc;43@6,11@9,2@3,1@12),
TseI(Gcwgc;43@6,11@9,2@3,1@12), MspA1I(CMGckg;46@7,2@1),
PvuII(CAGctg;46@7,2@1), AluI(AGct;48@82@2), DdeI(Ctnag;22@52,9@48),
HphI(tcacc;22@80), BssKI(Nccngg;35@39,2@40),
BsaJI(Ccnngg;32@40,2@41), BstNI(CCwgg;33@40),
ScrFI(CCngg;35@40,2@41), Eco0109I(RGgnccy;22@46, 11@43),
Sau96I(Ggncc;23@47,11@44), AvaII(Ggwcc;23@47,4@44),
PpuMI(RGgwccy;22@46,4@43), BsmFI(gtccc;20@48),
HinfI(Gantc;34@16,21@56,21@77), TfiI(21@77), MlyI(GAGTC;34@16),
MlyI(gactc;21@56), and AlwNI(CAGnnnctg;22@68). The more preferred
sites are MspAI and PvuII. MspAI and PvuII have 46 sites at 7-12
and 2 at 1-6. To avoid cleavage at both sites, oligonucleotides are
used that do not fully cover the site at 1-6. Thus, the DNA will
not be cleaved at that site. We have shown that DNA that extends 3,
4, or 5 bases beyond a PvuII-site can be cleaved efficiently.
[0087] Another illustration of choosing an appropriate restriction
endonuclease recognition site involves cleavage in FR1 of human
kappa light chains. Table 8 shows the human kappa FR1 germline
genes and Table 9 shows restriction endonuclease recognition sites
that are found in a substantial number of human kappa FR1 germline
genes at consistent locations. Of the restriction endonuclease
recognition sites listed, BsmAI and PflFI are the most preferred
enzymes. BsmAI sites are found at base 18 in 35 of 40 germline
genes. PflFI sites are found in 35 of 40 germline genes at base
12.
[0088] Another example of choosing an appropriate restriction
endonuclease recognition site involves cleavage in FR1 of the human
lambda light chain. Table 10 shows the 31 known human lambda FR1
germline gene sequences. Table 11 shows restriction endonuclease
recognition sites found in human lambda FR1 germline genes. HinfI
and DdeI are the most preferred restriction endonucleases for
cutting human lambda chains in FR1.
[0089] After the appropriate site or sites for cleavage are chosen,
one or more short oligonucleotides are prepared so as to
functionally complement, alone or in combination, the chosen
recognition site. The oligonucleotides also include sequences that
flank the recognition site in the majority of the amplified genes.
This flanking region allows the sequence to anneal to the
single-stranded DNA sufficiently to allow cleavage by the
restriction endonuclease specific for the site chosen.
[0090] The actual length and sequence of the oligonucleotide
depends on the recognition site and the conditions to be used for
contacting and cleavage. The length must be sufficient so that the
oligonucleotide is functionally complementary to the
single-stranded DNA over a large enough region to allow the two
strands to associate such that cleavage may occur at the chosen
temperature and at the desired location.
[0091] Typically, the oligonucleotides of this preferred method of
the invention are about 17 to about 30 nucleotides in length. Below
about 17 bases, annealing is too weak and above 30 bases there can
be a loss of specificity. A preferred length is 18 to 24 bases.
[0092] Oligonucleotides of this length need not be identical
complements of the germline genes. Rather, a few mismatches taken
may be tolerated. Preferably, however, no more than 1-3 mismatches
are allowed. Such mismatches do not adversely affect annealing of
the oligonucleotide to the single-stranded DNA. Hence, the two DNAs
are said to be functionally complementary.
[0093] The second method to manipulate the single-stranded DNAs of
this invention for display and/or expression comprises the steps
of: [0094] (i) contacting the nucleic acid with a partially
double-stranded oligonucleotide, the single-stranded region of the
oligonucleotide being functionally complementary to the nucleic
acid in the region in which cleavage is desired, and the
double-stranded region of the oligonucleotide having a restriction
endonuclease recognition site; and [0095] (ii) cleaving the nucleic
acid solely at the cleavage site formed by the complementation of
the nucleic acid and the single-stranded region of the
oligonucleotide; the contacting and the cleaving steps being
performed at a temperature sufficient to maintain the nucleic acid
in substantially single-stranded form, the oligonucleotide being
functionally complementary to the nucleic acid over a large enough
region to allow the two strands to associate such that cleavage may
occur at the chosen temperature and at the desired location, and
the cleavage being carried out using a restriction endonuclease
that is active at the chosen temperature.
[0096] As explained above, the cleavage site may be formed by the
single-stranded portion of the partially double-stranded
oligonucleotide duplexing with the single-stranded DNA, the
cleavage site may be carried in the double-stranded portion of the
partially double-stranded oligonucleotide, or the cleavage site may
be introduced by the amplification primer used to amplify the
single-stranded DNA-partially double-stranded oligonucleotide
combination. In this embodiment, the first is preferred. And, the
restriction endonuclease recognition site may be located in either
the double-stranded portion of the oligonucleotide or introduced by
the amplification primer, which is complementary to that
double-stranded region, as used to amplify the combination.
[0097] Preferably, the restriction endonuclease site is that of a
Type II-S restriction endonuclease, whose cleavage site is located
at a known distance from its recognition site.
[0098] This second method, preferably, employs Universal
Restriction Endonucleases ("URE"). UREs are partially
double-stranded oligonucleotides. The single-stranded portion or
overlap of the URE consists of a DNA adapter that is functionally
complementary to the sequence to be cleaved in the single-stranded
DNA. The double-stranded portion consists of a restriction
endonuclease recognition site, preferably type II-S.
[0099] The URE method of this invention is specific and precise and
can tolerate some (e.g., 1-3) mismatches in the complementary
regions, i.e., it is functionally complementary to that region.
Further, conditions under which the URE is used can be adjusted so
that most of the genes that are amplified can be cut, reducing bias
in the library produced from those genes.
[0100] The sequence of the single-stranded DNA adapter or overlap
portion of the URE typically consists of about 14-22 bases.
However, longer or shorter adapters may be used. The size depends
on the ability of the adapter to associate with its functional
complement in the single-stranded DNA and the temperature used for
contacting the URE and the single-stranded DNA at the temperature
used for cleaving the DNA with the restriction enzyme. The adapter
must be functionally complementary to the single-stranded DNA over
a large enough region to allow the two strands to associate such
that the cleavage may occur at the chosen temperature and at the
desired location. We prefer singe-stranded or overlap portions of
14-17 bases in length, and more preferably 18-20 bases in
length.
[0101] The site chosen for cleavage using the URE is preferably one
that is substantially conserved in the family of amplified DNAs. As
compared to the first cleavage method of this invention, these
sites do not need to be endonuclease recognition sites. However,
like the first method, the sites chosen can be synthetic rather
than existing in the native DNA. Such sites may be chosen by
references to the sequences of known antibodies or other families
of genes. For example, the sequences of many germline genes are
reported at
http://www.mrc-cpe.cam.ac.uk/imt-doc/restricted/ok.html. For
example, one preferred site occurs near the end of FR3--codon 89
through the second base of codon 93. CDR3 begins at codon 95.
[0102] The sequences of 79 human heavy-chain genes are also
available at http://www.ncbi.nlm.nih.gov/entre2/nucleotide.html.
This site can be used to identify appropriate sequences for URE
cleavage according to the methods of this invention. See, e.g.,
Table 12B.
[0103] Most preferably, one or more sequences are identified using
these sites or other available sequence information. These
sequences together are present in a substantial fraction of the
amplified DNAs. For example, multiple sequences could be used to
allow for known diversity in germline genes or for frequent somatic
mutations. Synthetic degenerate sequences could also be used.
Preferably, a sequence(s) that occurs in at least 65% of genes
examined with no more than 2-3 mismatches is chosen.
[0104] URE single-stranded adapters or overlaps are then made to be
complementary to the chosen regions. Conditions for using the UREs
are determined empirically. These conditions should allow cleavage
of DNA that contains the functionally complementary sequences with
no more than 2 or 3 mismatches but that do not allow cleavage of
DNA lacking such sequences.
[0105] As described above, the double-stranded portion of the URE
includes an endonuclease recognition site, preferably a Type II-S
recognition site. Any enzyme that is active at a temperature
necessary to maintain the single-stranded DNA substantially in that
form and to allow the single-stranded DNA adapter portion of the
URE to anneal long enough to the single-stranded DNA to permit
cleavage at the desired site may be used.
[0106] The preferred Type II-S enzymes for use in the URE methods
of this invention provide asymmetrical cleavage of the
single-stranded DNA. Among these are the enzymes listed in Table
13. The most preferred Type II-S enzyme is FokI.
[0107] When the preferred FokI containing URE is used, several
conditions are preferably used to effect cleavage: [0108] 1) Excess
of the URE over target DNA should be present to activate the
enzyme. URE present only in equimolar amounts to the target DNA
would yield poor cleavage of ssDNA because the amount of active
enzyme available would be limiting. [0109] 2) An activator may be
used to activate part of the FokI enzyme to dimerize without
causing cleavage. Examples of appropriate activators are shown in
Table 14. [0110] 3) The cleavage reaction is performed at a
temperature between 45.degree.-75.degree. C., preferably above
50.degree. C. and most preferably above 55.degree. C.
[0111] The UREs used in the prior art contained a 14-base
single-stranded segment, a 10-base stem (containing a FokI site),
followed by the palindrome of the 10-base stem. While such UREs may
be used in the methods of this invention, the preferred UREs of
this invention also include a segment of three to eight bases (a
loop) between the FokI restriction endonuclease recognition site
containing segments. In the preferred embodiment, the stem
(containing the FokI site) and its palindrome are also longer than
10 bases. Preferably, they are 10-14 bases in length. Examples of
these "lollipop" URE adapters are shown in Table 15.
[0112] One example of using a URE to cleave an single-stranded DNA
involves the FR3 region of human heavy chain. Table 16 shows an
analysis of 840 full-length mature human heavy chains with the URE
recognition sequences shown. The vast majority (718/840=0.85) will
be recognized with 2 or fewer mismatches using five UREs
(VHS881-1.1, VHS881-1.2, VHS881-2.1, VHS881-4.1, and VHS881-9.1).
Each has a 20-base adaptor sequence to complement the germline
gene, a ten-base stem segment containing a FokI site, a five base
loop, and the reverse complement of the first stem segment.
Annealing those adapters, alone or in combination, to
single-stranded antisense heavy chain DNA and treating with FokI in
the presence of, e.g., the activator FOKIact, will lead to cleavage
of the antisense strand at the position indicated.
[0113] Another example of using a URE(s) to cleave a
single-stranded DNA involves the FR1 region of the human Kappa
light chains. Table 17 shows an analysis of 182 full-length human
kappa chains for matching by the four 19-base probe sequences
shown. Ninety-six percent of the sequences match one of the probes
with 2 or fewer mismatches. The URE adapters shown in Table 17 are
for cleavage of the sense strand of kappa chains. Thus, the adaptor
sequences are the reverse complement of the germline gene
sequences. The URE consists of a ten-base stem, a five base loop,
the reverse complement of the stem and the complementation
sequence. The loop shown here is TTGTT, but other sequences could
be used. Its function is to interrupt the palindrome of the stems
so that formation of a lollypop monomer is favored over
dimerization. Table 17 also shows where the sense strand is
cleaved.
[0114] Another example of using a URE to cleave a single-stranded
DNA involves the human lambda light chain. Table 18 shows analysis
of 128 human lambda light chains for matching the four 19-base
probes shown. With three or fewer mismatches, 88 of 128 (69%) of
the chains match one of the probes. Table 18 also shows URE
adapters corresponding to these probes. Annealing these adapters to
upper-strand ssDNA of lambda chains and treatment with FokI in the
presence of FOKIact at a temperature at or above 45.degree. C. will
lead to specific and precise cleavage of the chains.
[0115] The conditions under which the short oligonucleotide
sequences of the first method and the UREs of the second method are
contacted with the single-stranded DNAs may be empirically
determined. The conditions must be such that the single-stranded
DNA remains in substantially single-stranded form. More
particularly, the conditions must be such that the single-stranded
DNA does not form loops that may interfere with its association
with the oligonucleotide sequence or the URE or that may themselves
provide sites for cleavage by the chosen restriction
endonuclease.
[0116] The effectiveness and specificity of short oligonucleotides
(first method) and UREs (second method) can be adjusted by
controlling the concentrations of the URE adapters/oligonucleotides
and substrate DNA, the temperature, the pH, the concentration of
metal ions, the ionic strength, the concentration of chaotropes
(such as urea and formamide), the concentration of the restriction
endonuclease(e.g., FokI), and the time of the digestion. These
conditions can be optimized with synthetic oligonucleotides having:
1) target germline gene sequences, 2) mutated target gene
sequences, or 3) somewhat related non-target sequences. The goal is
to cleave most of the target sequences and minimal amounts of
non-targets.
[0117] In accordance with this invention, the single-stranded DNA
is maintained in substantially that form using a temperature
between about 37.degree. C. and about 75.degree. C. Preferably, a
temperature between about 45.degree. C. and about 75.degree. C. is
used. More preferably, a temperature between 50.degree. C. and
60.degree. C., most preferably between 55.degree. C. and 60.degree.
C., is used. These temperatures are employed both when contacting
the DNA with the oligonucleotide or URE and when cleaving the DNA
using the methods of this invention.
[0118] The two cleavage methods of this invention have several
advantages. The first method allows the individual members of the
family of single-stranded DNAs to be cleaved preferentially at one
substantially conserved endonuclease recognition site. The method
also does not require an endonuclease recognition site to be built
into the reverse transcription or amplification primers. Any native
or synthetic site in the family can be used.
[0119] The second method has both of these advantages. In addition,
the preferred URE method allows the single-stranded DNAs to be
cleaved at positions where no endoniuclease recognition site
naturally occurs or has been synthetically constructed.
[0120] Most importantly, both cleavage methods permit the use of 5'
and 3' primers so as to maximize diversity and then cleavage to
remove unwanted or deleterious sequences before cloning, display
and/or expression.
[0121] After cleavage of the amplified DNAs using one of the
methods of this invention, the DNA is prepared for cloning, display
and/or expression. This is done by using a partially duplexed
synthetic DNA adapter, whose terminal sequence is based on the
specific cleavage site at which the amplified DNA has been
cleaved.
[0122] The synthetic DNA is designed such that when it is ligated
to the cleaved single-stranded DNA in proper reading frame so that
the desired peptide, polypeptide or protein can be displayed on the
surface of the genetic package and/or expressed. Preferably, the
double-stranded portion of the adapter comprises the sequence of
several codons that encode the amino acid sequence characteristic
of the family of peptides, polypeptides or proteins up to the
cleavage site. For human heavy chains, the amino acids of the 3-23
framework are preferably used to provide the sequences required for
expression of the cleaved DNA.
[0123] Preferably, the double-stranded portion of the adapter is
about 12 to 100 bases in length. More preferably, about 20 to 100
bases are used. The double-standard region of the adapter also
preferably contains at least one endonuclease recognition site
useful for cloning the DNA into a suitable display and/or
expression vector (or a recipient vector used to archive the
diversity). This endonuclease restriction site may be native to the
germline gene sequences used to extend the DNA sequence. It may be
also constructed using degenerate sequences to the native germline
gene sequences. Or, it may be wholly synthetic.
[0124] The single-stranded portion of the adapter is complementary
to the region of the cleavage in the single-stranded DNA. The
overlap can be from about 2 bases up to about 15 bases. The longer
the overlap, the more efficient the ligation is likely to be. A
preferred length for the overlap is 7 to 10. This allows some
mismatches in the region so that diversity in this region may be
captured.
[0125] The single-stranded region or overlap of the partially
duplexed adapter is advantageous because it allows DNA cleaved at
the chosen site, but not other fragments to be captured. Such
fragments would contaminate the library with genes encoding
sequences that will not fold into proper antibodies and are likely
to be non-specifically sticky.
[0126] One illustration of the use of a partially duplexed adaptor
in the methods of this invention involves ligating such adaptor to
a human FR3 region that has been cleaved, as described above, at
5'-ACnGT-3' using HpyCH4III, Bst4CI or TaaI.
[0127] Table 4 F.2 shows the bottom strand of the double-stranded
portion of the adaptor for ligation to the cleaved bottom-strand
DNA. Since the HpyCH4III-Site is so far to the right (as shown in
Table 3), a sequence that includes the AflII-site as well as the
XbaI site can be added. This bottom strand portion of the
partially-duplexed adaptor, H43.XAExt, incorporates both XbaI and
AflII-sites. The top strand of the double-stranded portion of the
adaptor has neither site (due to planned mismatches in the segments
opposite the XbaI and AflII-Sites of H43.XAExt), but will anneal
very tightly to H43.XAExt. H43AExt contains only the AflII-site and
is to be used with the top strands H43.ABr1 and H43.ABr2 (which
have intentional alterations to destroy the AflII-site).
[0128] After ligation, the desired, captured DNA can be PCR
amplified again, if desired, using in the preferred embodiment a
primer to the downstream constant region of the antibody gene and a
primer to part of the double-standard region of the adapter. The
primers may also carry restriction endonuclease sites for use in
cloning the amplified DNA.
[0129] After ligation, and perhaps amplification, of the partially
double-stranded adapter to the single-stranded amplified DNA, the
composite DNA is cleaved at chosen 5' and 3' endonuclease
recognition sites.
[0130] The cleavage sites useful for cloning depend on the phage or
phagemid or other vectors into which the cassette will be inserted
and the available sites in the antibody genes. Table 19 provides
restriction endonuclease data for 75 human light chains. Table 20
shows corresponding data for 79 human heavy chains. In each Table,
the endonucleases are ordered by increasing frequency of cutting.
In these Tables, Nch is the number of chains cut by the enzyme and
Ns is the number of sites (some chains have more than one
site).
[0131] From this analysis, SfiI, NotI, AflII, ApaLI, and AscI are
very suitable. SfiI and NotI are preferably used in pCES1 to insert
the heavy-chain display segment. ApaLI and AscI are preferably used
in pCES1 to insert the light-chain display segment.
[0132] BstEII-sites occur in 97% of germ-line JH genes. In
rearranged V genes, only 54/79 (68%) of heavy-chain genes contain a
BstEII-Site and 7/61 of these contain two sites. Thus, 47/79 (59%)
contain a single BstEII-Site. An alternative to using BstEII is to
cleave via UREs at the end of JH and ligate to a synthetic
oligonucleotide that encodes part of CH1.
[0133] One example of preparing a family of DNA sequences using the
methods of this invention involves capturing human CDR 3 diversity.
As described above, mRNAs from various autoimmune patients are
reverse transcribed into lower strand cDNA. After the top strand
RNA is degraded, the lower strand is immobilized and a short
oligonucleotide used to cleave the cDNA upstream of CDR3. A
partially duplexed synthetic DNA adapter is then annealed to the
DNA and the DNA is amplified using a primer to the adapter and a
primer to the constant region (after FR4). The DNA is then cleaved
using BstEII (in FR4) and a restriction endonuclease appropriate to
the partially double-stranded adapter (e.g., XbaI and AfilII (in
FR3)). The DNA is then ligated into a synthetic VH skeleton such as
3-23.
[0134] One example of preparing a single-stranded DNA that was
cleaved using the URE method involves the human Kappa chain. The
cleavage site in the sense strand of this chain is depicted in
Table 17. The oligonucleotide kapextURE is annealed to the
oligonucleotides (kaBR01UR, kaBR02UR, kaBR03UR, and kaBR04UR) to
form a partially duplex DNA. This DNA is then ligated to the
cleaved soluble kappa chains. The ligation product is then
amplified using primers kapextUREPCR and CKForeAsc (which inserts a
AscI site after the end of C kappa). This product is then cleaved
with ApaLI and AscI and ligated to similarly cut recipient
vector.
[0135] Another example involves the cleavage of lambda light
chains, illustrated in Table 18. After cleavage, an extender
(ON_LamEx133) and four bridge oligonucleotides (ON_LamB1-133,
ON_LamB2-133, ON_LamB3-133, and ON_LamB4-133) are annealed to form
a partially duplex DNA. That DNA is ligated to the cleaved
lambda-chain sense strands. After ligation, the DNA is amplified
with ON_Lam133PCR and a forward primer specific to the lambda
constant domain, such as CL2ForeAsc or CL7ForeAsc (Table 130).
[0136] In human heavy chains, one can cleave almost all genes in
FR4 (downstream, i.e., toward the 3' end of the sense strand, of
CDR3) at a BstEII-Site that occurs at a constant position in a very
large fraction of human heavy-chain V genes. One then needs a site
in FR3, if only CDR3 diversity is to be captured, in FR2, if CDR2
and CDR3 diversity is wanted, or in FR1, if all the CDR diversity
is wanted. These sites are preferably inserted as part of the
partially double-stranded adaptor.
[0137] The preferred process of this invention is to provide
recipient vectors (e.g., for display and/or expression) having
sites that allow cloning of either light or heavy chains. Such
vectors are well known and widely used in the art. A preferred
phage display vector in accordance with this invention is phage
MALIA3. This displays in gene III. The sequence of the phage MALIA3
is shown in Table 21A (annotated) and Table 21B (condensed).
[0138] The DNA encoding the selected regions of the light or heavy
chains can be transferred to the vectors using endonucleases that
cut either light or heavy chains only very rarely. For example,
light chains may be captured with ApaLI and AscI. Heavy-chain genes
are preferably cloned into a recipient vector having SfiI, NcoI,
XbaI, AflII, BstEII, ApaI, and NotI sites. The light chains are
preferably moved into the library as ApaLI-AscI fragments. The
heavy chains are preferably moved into the library as SfiI-NotI
fragments.
[0139] Most preferably, the display is had on the surface of a
derivative of M13 phage. The most preferred vector contains all the
genes of M13, an antibiotic resistance gene, and the display
cassette. The preferred vector is provided with restriction sites
that allow introduction and excision of members of the diverse
family of genes, as cassettes. The preferred vector is stable
against rearrangement under the growth conditions used to amplify
phage.
[0140] In another embodiment of this invention, the diversity
captured by the methods of the present invention may be displayed
and/or expressed in a phagemid vector (e.g., pCES1) that displays
and/or expresses the peptide, polypeptide or protein. Such vectors
may also be used to store the diversity for subsequent display
and/or expression using other vectors or phage.
[0141] In another embodiment of this invention, the diversity
captured by the methods of the present invention may be displayed
and/or expressed in a yeast vector.
[0142] In another embodiment, the mode of display may be through a
short linker to anchor domains--one possible anchor comprising the
final portion of M13 III ("IIIstump") and a second possible anchor
being the full length III mature protein.
[0143] The IIIstump fragment contains enough of M13 III to assemble
into phage but not the domains involved in mediating infectivity.
Because the w.t. III proteins are present the phage is unlikely to
delete the antibody genes and phage that do delete these segments
receive only a very small growth advantage. For each of the anchor
domains, the DNA encodes the w.t. AA sequence, but differs from the
w.t. DNA sequence to a very high extent. This will greatly reduce
the potential for homologous recombination between the anchor and
the w.t. gene that is also present (see Example 6).
[0144] Most preferably, the present invention uses a complete phage
carrying an antibiotic-resistance gene (such as an
ampicillin-resistance gene) and the display cassette. Because the
w.t. iii and possibly viii genes are present, the w.t. proteins are
also present. The display cassette is transcribed from a
regulatable promoter (e.g., P.sub.LacZ). Use of a regulatable
promoter allows control of the ratio of the fusion display gene to
the corresponding w.t. coat protein. This ratio determines the
average number of copies of the display fusion per phage (or
phagemid) particle.
[0145] Another aspect of the invention is a method of displaying
peptides, polypeptides or proteins (and particularly Fabs) on
filamentous phage. In the most preferred embodiment this method
displays FABs and comprises: [0146] a) obtaining a cassette
capturing a diversity of segments of DNA encoding the elements:
[0147] P.sub.reg::RBS1::SS1::VL::CL::stop::RBS2::SS2::VH::CH1::
linker::anchor::stop::, where P.sub.reg is a regulatable promoter,
RBS1 is a first ribosome binding site, SS1 is a signal sequence
operable in the host strain, VL is a member of a diverse set of
light-chain variable regions, CL is a light-chain constant region,
stop is one or more stop codons, RBS2 is a second ribosome binding
site, SS2 is a second signal sequence operable in the host strain,
VH is a member of a diverse set of heavy-chain variable regions,
CH1 is an antibody heavy-chain first constant domain, linker is a
sequence of amino acids of one to about 50 residues, anchor is a
protein that will assemble into the filamentous phage particle and
stop is a second example of one or more stop codons; and [0148] b)
positioning that cassette within the phage genome to maximize the
viability of the phage and to minimize the potential for deletion
of the cassette or parts thereof.
[0149] The DNA encoding the anchor protein in the above preferred
cassette should be designed to encode the same (or a closely
related) amino acid sequence as is found in one of the coat
proteins of the phage, but with a distinct DNA sequence. This is to
prevent unwanted homologous recombination with the w.t. gene. In
addition, the cassette should be placed in the intergenic region.
The positioning and orientation of the display cassette can
influence the behavior of the phage.
[0150] In one embodiment of the invention, a transcription
terminator may be placed after the second stop of the display
cassette above (e.g., Trp). This will reduce interaction between
the display cassette and other genes in the phage antibody display
vector.
[0151] In another embodiment of the methods of this invention, the
phage or phagemid can display and/or express proteins other than
Fab, by replacing the Fab portions indicated above, with other
protein genes.
[0152] Various hosts can be used the display and/or expression
aspect of this invention. Such hosts are well known in the art. In
the preferred embodiment, where Fabs are being displayed and/or
expressed, the preferred host should grow at 30.degree. C. and be
RecA.sup.- (to reduce unwanted genetic recombination) and
EndA.sup.- (to make recovery of RF DNA easier). It is also
preferred that the host strain be easily transformed by
electroporation.
[0153] XL1-Blue MRF' satisfies most of these preferences, but does
not grow well at 30.degree. C. XL1-Blue MRF' does grow slowly at
38.degree. C. and thus is an acceptable host. TG-1 is also an
acceptable host although it is RecA.sup.+ and EndA.sup.+. XL1-Blue
MRF' is more preferred for the intermediate host used to accumulate
diversity prior to final construction of the library.
[0154] After display and/or expression, the libraries of this
invention may be screened using well known and conventionally used
techniques. The selected peptides, polypeptides or proteins may
then be used to treat disease. Generally, the peptides,
polypeptides or proteins for use in therapy or in pharmaceutical
compositions are produced by isolating the DNA encoding the desired
peptide, polypeptide or protein from the member of the library
selected. That DNA is then used in conventional methods to produce
the peptide, polypeptides or protein it encodes in appropriate host
cells, preferably mammalian host cells, e.g., CHO cells. After
isolation, the peptide, polypeptide or protein is used alone or
with pharmaceutically acceptable compositions in therapy to treat
disease.
EXAMPLES
Example 1
RACE Amplification of Heavy and Light Chain Antibody Repertoires
from Autoimmune Patients.
[0155] Total RNA was isolated from individual blood samples (50 ml)
of 11 patients using a RNAzol.TM. kit (CINNA/Biotecx), as described
by the manufacturer. The patients were diagnosed as follows: [0156]
1. SLE and phospholipid syndrome [0157] 2. limited systemic
sclerosis [0158] 3. SLE and Sjogren syndrome [0159] 4. Limited
Systemic sclerosis [0160] 5. Reumatoid Arthritis with active
vasculitis [0161] 6. Limited systemic sclerosis and Sjogren
Syndrome [0162] 7. Reumatoid Artritis and (not active) vasculitis
[0163] 8. SLE and Sjogren syndrome [0164] 9. SLE [0165] 10. SLE and
(active) glomerulonephritis [0166] 11. Polyarthritis/Raynauds
Phenomen From these 11 samples of total RNA, Poly-A+ RNA was
isolated using Promega PolyATtract.RTM. mRNA Isolation kit
(Promega).
[0167] 250 ng of each poly-A+ RNA sample was used to amplify
antibody heavy and light chains with the GeneRAacer.TM. kit
(Invitrogen cat no. L1500-01). A schematic overview of the RACE
procedure is shown in FIG. 3.
[0168] Using the general protocol of the GeneRAacer.TM. kit, an RNA
adaptor was ligated to the 5' end of all mRNAs. Next, a reverse
transcriptase reaction was performed in the presence of oligo(dT15)
specific primer under conditions described by the manufacturer in
the GeneRAacer.TM. kit.
[0169] 1/5 of the cDNA from the reverse transcriptase reaction was
used in a 20 ul PCR reaction. For amplification of the heavy chain
IgM repertoire, a forward primer based on the CH1 chain of IgM
[HuCmFOR] and a backward primer based on the ligated synthetic
adaptor sequence [5'A] were used. (See Table 22).
[0170] For amplification of the kappa and lambda light chains, a
forward primer that contains the 3' coding-end of the cDNA [HuCkFor
and HuCLFor2+HuCLfor7] and a backward primer based on the ligated
synthetic adapter sequence [5'A] was used (See Table 22). Specific
amplification products after 30 cycles of primary PCR were
obtained.
[0171] FIG. 4 shows the amplification products obtained after the
primary PCR reaction from 4 different patient samples. 8 ul primary
PCR product from 4 different patients was analyzed on a agarose gel
[labeled 1, 2, 3 and 4]. For the heavy chain, a product of
approximately 950 nt is obtained while for the kappa and lambda
light chains the product is approximately 850 nt. M1-2 are
molecular weight markers.
[0172] PCR products were also analyzed by DNA sequencing [10 clones
from the lambda, kappa or heavy chain repertoires]. All sequenced
antibody genes recovered contained the full coding sequence as well
as the 5' leader sequence and the V gene diversity was the expected
diversity (compared to literature data).
[0173] 50 ng of all samples from all 11 individual amplified
samples were mixed for heavy, lambda light or kappa light chains
and used in secondary PCR reactions.
[0174] In all secondary PCRs approximately 1 ng template DNA from
the primary PCR mixture was used in multiple 50 ul PCR reactions
[25 cycles].
[0175] For the heavy chain, a nested biotinylated forward primer
[HuCm-Nested] was used, and a nested 5'end backward primer located
in the synthetic adapter-sequence [5'NA] was used. The 5'end
lower-strand of the heavy chain was biotinylated.
[0176] For the light chains, a 5'end biotinylated nested primer in
the synthetic adapter was used [5'NA] in combination with a 3'end
primer in the constant region of Ckappa and Clambda, extended with
a sequence coding for the AscI restriction site [kappa:
HuCkForAscI, Lambda: HuCL2-FOR-ASC+HuCL7-FOR-ASC]. [5'end Top
strand DNA was biotinylated]. After gel-analysis the secondary PCR
products were pooled and purified with Promega Wizzard PCR cleanup.
Approximately 25 ug biotinylated heavy chain, lambda and kappa
light chain DNA was isolated from the 11 patients.
Example 2
Capturing Kappa Chains with BsmAI.
[0177] A repertoire of human-kappa chain mRNAs was prepared using
the RACE method of Example 1 from a collection of patients having
various autoimmune diseases.
[0178] This Example followed the protocol of Example 1.
Approximately 2 micrograms (ug) of human kappa-chain (Igkappa) gene
PACE material with biotin attached to 5'-end of upper strand was
immobilized as in Example 1 on 200 microliters (.mu.L) of Seradyn
magnetic beads. The lower strand was removed by washing the DNA
with 2 aliquots 200 .mu.L of 0.1 M NaOH (pH 13) for 3 minutes for
the first aliquot followed by 30 seconds for the second aliquot.
The beads were neutralized with 200 .mu.L of 10 mM Tris (pH 7.5)
100 mM NaCl. The short oligonucleotides shown in Table 23 were
added in 40 fold molar excess in 100 .mu.L of NEB buffer 2 (50 mM
NaCl, 10 mM Tris-HCl, 10 mM MgCl.sub.2, 1 mM dithiothreitol pH 7.9)
to the dry beads. The mixture was incubated at 95.degree. C. for 5
minutes then cooled down to 55.degree. C. over 30 minutes. Excess
oligonucleotide was washed away with 2 washes of NEB buffer 3 (100
mM NaCl, 50 mM Tris-HCl, 10 MM MgCl.sub.2, 1 mM dithiothreitol pH
7.9). Ten units of BsmAI (NEB) were added in NEB buffer 3 and
incubated for 1 h at 55.degree. C. The cleaved downstream DNA was
collected and purified over a Qiagen PCR purification column (FIGS.
5 and 6).
[0179] FIG. 5 shows an analysis of digested kappa single-stranded
DNA. Approximately 151.5 pmol of adapter was annealed to 3.79 pmol
of immobilized kappa single-stranded DNA followed by digestion with
15 U of BsmAI. The supernatant containing the desired DNA was
removed and analyzed by 5% polyacrylamide gel along with the
remaining beads which contained uncleaved full length kappa DNA.
189 pmol of cleaved single-stranded DNA was purified for further
analysis. Five percent of the original full length ssDNA remained
on the beads.
[0180] FIG. 6 shows an analysis of the extender--cleaved kappa
ligation. 180 pmol of pre-annealed bridge/extender was ligated to
1.8 pmol of BsmAl digested single-stranded DNA. The ligated DNA was
purified by Qiagen PCR purification column and analyzed on a 5%
polyacrylamide gel. Results indicated that the ligation of extender
to single-stranded DNA was 95% efficient.
[0181] A partially double-stranded adaptor was prepared using the
oligonucleotide shown in Table 23. The adaptor was added to the
single-stranded DNA in 100 fold molar excess along with 1000 units
of T4 DNA ligase and incubated overnight at 16.degree. C. The
excess oligonucleotide was removed with a Qiagen PCR purification
column. The ligated material was amplified by PCR using the primers
kapPCRt1 and kapfor shown in Table 23 for 10 cycles with the
program shown in Table 24.
[0182] The soluble PCR product was run on a gel and showed a band
of approximately 700 n, as expected (FIGS. 7 and 8). The DNA was
cleaved with enzymes ApaLI and AscI, gel purified, and ligated to
similarly cleaved vector pCES1.
[0183] FIG. 7 shows an analysis of the PCR product from the
extender-kappa amplification. Ligated extender-kappa
single-stranded DNA was amplified with primers specific to the
extender and to the constant region of the light chain. Two
different template concentrations, 10 ng versus 50 ng, were used as
template and 13 cycles were used to generate approximately 1.5 ug
of dsDNA as shown by 0.8% agarose gel analysis.
[0184] FIG. 8 shows an analysis of the purified PCR product from
the extender-kappa amplification. Approximately 5 ug of PCR
amplified extender-kappa double-stranded DNA was run out on a 0.8%
agarose gel, cut out, and extracted with a GFX gel purification
column. By gel analysis, 3.5 ug of double-stranded DNA was
prepared.
[0185] The assay for capturing kappa chains with BsmA1 was repeated
and produced similar results. FIG. 9A shows the DNA after it was
cleaved and collected and purified over a Qiagen PCR purification
column. FIG. 9B shows the partially double-stranded adaptor ligated
to the single-stranded DNA. This ligated material was then
amplified (FIG. 9C). The gel showed a band of approximately 700
n.
[0186] Table 25 shows the DNA sequence of a kappa light chain
captured by this procedure. Table 26 shows a second sequence
captured by this procedure. The closest bridge sequence was
complementary to the sequence 5'-agccacc-3', but the sequence
captured reads 5'-Tgccacc-3', showing that some mismatch in the
overlapped region is tolerated.
Example 3
Construction of Synthetic CDR1 and CDR2 Diversity in V-3-23 VH
Framework.
[0187] Synthetic diversity in Complementary Determinant Region
(CDR) 1 and 2 was created in the 3-23 VH framework in a two step
process: first, a vector containing the 3-23 VH framework was
constructed; and then, a synthetic CDR 1 and 2 was assembled and
cloned into this vector.
[0188] For construction of the 3-23 VH framework, 8
oligonucleotides and two PCR primers (long
oligonucleotides--TOPFR1A, BOTFR1B, BOTFR2, BOTFR3, F06, BoTFR4,
ON-vgC1, and ON-vgC2 and primers--SFPRMET and BOTPcRPRIM, shown in
Table 27) that overlap were designed based on the Genebank sequence
of 3-23 VH framework region. The design incorporated at least one
useful restriction site in each framework region, as shown in Table
27. In Table 27, the segments that were synthesized are shown as
bold, the overlapping regions are underscored, and the PCR priming
regions at each end are underscored.
[0189] A mixture of these 8 oligos was combined at a final
concentration of 2.5 uM in a 20 ul PCR reaction. The PCR mixture
contained 200 uM dNTPs, 2.5 mM MgCl.sub.2, 0.02 U Pfu Turbo.TM. DNA
Polymerase, 1 U Qiagen HotStart Taq DNA Polymerase, and
1.times.Qiagen PCR buffer. The PCR program consisted of 10 cycles
of 94.degree. C. for 30s, 55.degree. C. for 30s, and 72.degree. C.
for 30s.
[0190] The assembled 3-23 VH DNA sequence was then amplified, using
2.5 ul of a 10-fold dilution from the initial PCR in 100 ul PCR
reaction. The PCR reaction contained 200 uM dNTPs, 2.5 mM
MgCl.sub.2, 0.02 U Pfu Turbo.TM. DNA Polymerase, 1 U Qiagen
HotStart Taq DNA Polymerase, 1.times.Qiagen PCR Buffer and 2
outside primers (SFPRMET and BOTPCRPRIM) at a concentration of 1
uM. The PCR program consisted of 23 cycles at 94.degree. C. for
30s, 55.degree. C. for 30s, and 72.degree. C. for 60s. The 3-23 VH
DNA sequence was digested and cloned into pCES1 (phagemid vector)
using the SfiI and BstEII restriction endonuclease sites. All
restriction enzymes mentioned herein were supplied by New England
BioLabs, Beverly, Mass. and used as per the manufacturer's
instructions.
[0191] Stuffer sequences (shown in Table 28 and Table 29) were
introduced into pCES1 to replace CDR1/CDR2 sequences (900 bases
between BspEI and XbaI RE sites) and CDR3 sequences (358 bases
between AflII and BstEII) prior to cloning the CDR1/CDR2 diversity.
This new vector was termed pCES5 and its sequence is given in Table
29.
[0192] Having stuffers in place of the CDRs avoids the risk that a
parental sequence would be over-represented in the library. The
stuffer sequences are fragments from the penicillase gene of E.
coli. The CDR1-2 stuffer contains restriction sites for BglII,
Bsu36I, BclI, XcmI, MluI, PvuII, HpaI, and HincII, the underscored
sites being unique within the vector pCES5. The stuffer that
replaces CDR3 contains the unique restriction endonuclease site
RsrII.
[0193] A schematic representation of the design for CDR1 and CDR2
synthetic diversity is shown FIG. 10. The design was based on the
presence of mutations in DP47/3-23 and related germline genes.
Diversity was designed to be introduced at the positions within
CDR1 and CDR2 indicated by the numbers in FIG. 10. The diversity at
each position was chosen to be one of the three following schemes:
1=ADEFGHIKLMNPQRSTVWY; 2=YRWVGS; 3=PS, in which letters encode
equimolar mixes of the indicated amino acids.
[0194] For the construction of the CDR1 and CDR2 diversity, 4
overlapping oligonucleotides (ON-vgC1, ON_Br12, ON_CD2Xba, and
ON-vgC2, shown in Table 27 and Table 30) encoding CDR1/2, plus
flanking regions, were designed. A mixture of these 4 oligos was
combined at a final concentration of 2.5 uM in a 40 ul PCR
reaction. Two of the 4 oligos contained variegated sequences
positioned at the CDR1 and the CDR2. The PCR mixture contained 200
uM dNTPs, 2.5 U Pwo DNA Polymerase (Roche), and 1.times.Pwo PCR
buffer with 2 mM MgSO.sub.4. The PCR program consisted of 10 cycles
at 94.degree. C. for 30s, 60.degree. C. for 30s, and 72.degree. C.
for 60s. This assembled CDR1/2 DNA sequence was amplified, using
2.5 ul of the mixture in 100 ul PCR reaction. The PCR reaction
contained 200 uM dNTPs, 2.5 U Pwo DNA Polymerase, 1.times.Pwo PCR
Buffer with 2 mM MgSO.sub.4 and 2 outside primers at a
concentration of 1 uM. The PCR program consisted of 10 cycles at
94.degree. C. for 30s, 60.degree. C. for 30s, and 72.degree. C. for
60s. These variegated sequences were digested and cloned into the
3-23 VH framework in place of the CDR1/2 stuffer.
[0195] We obtained approximately 7.times.10.sup.7 independent
transformants. CDR3 diversity either from donor populations or from
synthetic DNA can be cloned into the vector containing synthetic
CDR1 and CDR 2 diversity.
[0196] A schematic representation of this procedure is shown in
FIG. 11. A sequence encoding the FR-regions of the human V3-23 gene
segment and CDR regions with synthetic diversity was made by
oligonucleotide assembly and cloning via BspE1 and Xbal sites into
a vector that complements the FR1 and FR3 regions. Into this
library of synthetic VH segments, the complementary VH-CDR3
sequence (top right) was cloned via Xbal an BstEll sites. The
resulting cloned CH genes contain a combination of designed
synthetic diversity and natural diversity (see FIG. 11).
Example 4
Cleavage and Ligation of the Lambda Light Chains with HinfI.
[0197] A schematic of the cleavage and ligation of antibody light
chains is shown in FIGS. 12A and 12B. Approximately 2 ug of
biotinylated human Lambda DNA prepared as described in Example 1
was immobilized on 200 ul Seradyn magnetic beads. The lower strand
was removed by incubation of the DNA with 200 ul of 0.1 M NaOH
(pH=13) for 3 minutes, the supernatant was removed and an
additional washing of 30 seconds with 200 ul of 0.1 M NaOH was
performed. Supernatant was removed and the beads were neutralized
with 200 ul of 10 mM Tris (pH=7.5), 100 mM NaCl. 2 additional
washes with 200 ul NEB2 buffer 2, containing 10 mM Tris (pH=7.9),
50 mM NaCl, 10 mM MgCl2 and 1 mM dithiothreitol, were performed.
After immobilization, the amount of ssDNA was estimated on a 5%
PAGE-UREA gel.
[0198] About 0.8 ug ssDNA was recovered and incubated in 100 ul
NEB2 buffer 2 containing 80 molar fold excess of an equimolar mix
of ON_Lam1aB7, ON_Lam2aB7, ON_Lam31B7 and ON_Lam3rB7 [each oligo in
20 fold molar excess] (see Table 31).
[0199] The mixture was incubated at 95.degree. C. for 5 minutes and
then slowly cooled down to 50.degree. C. over a period of 30
minutes. Excess of oligonucleotide was washed away with 2 washes of
200 ul of NEB buffer 2. 4 U/ug of Hinf I was added and incubated
for 1 hour at 50.degree. C. Beads were mixed every 10 minutes.
[0200] After incubation the sample was purified over a Qiagen PCR
purification column and was subsequently analysed on a 5% PAGE-urea
gel (see FIG. 13A, cleavage was more than 70% efficient).
[0201] A schematic of the ligation of the cleaved light chains is
shown in FIG. 12B. A mix of bridge/extender pairs was prepared from
the Brg/Ext oligo's listed in Table 31 (total molar excess 100
fold) in 1000 U of T4 DNA Ligase (NEB) and incubated overnight at
16.degree. C. After ligation of the DNA, the excess oligonucleotide
was removed with a Qiagen PCR purification column and ligation was
checked on a Urea-PAGE gel (see FIG. 13B; ligation was more than
95% efficient).
[0202] Multiple PCRs were performed containing 10 ng of the ligated
material in an 50 ul PCR reaction using 25 pMol ON lamPlePCR and 25
pmol of an equimolar mix of Hu-CL2AscI/HuCL7AscI primer (see
Example 1).
[0203] PCR was performed at 60.degree. C. for 15 cycles using Pfu
polymerase. About 1 ug of dsDNA was recovered per PCR (see FIG.
13C) and cleaved with ApaL1 and AscI for cloning the lambda light
chains in pCES2.
Example 5
Capture of Human Heavy-chain CDR3 Population.
[0204] A schematic of the cleavage and ligation of antibody light
chains is shown in FIGS. 14A and 14B.
[0205] Approximately 3 ug of human heavy-chain (IgM) gene RACE
material with biotin attached to 5'-end of lower strand was
immobilized on 300 uL of Seradyn magnetic beads. The upper strand
was removed by washing the DNA with 2 aliquots 300 uL of 0.1 M NaOH
(pH 13) for 3 minutes for the first aliquot followed by 30 seconds
for the second aliquot. The beads were neutralized with 300 uL of
10 mM Tris (pH 7.5) 100 mM NaCl. The REdaptors (oligonucleotides
used to make single-stranded DNA locally double-stranded) shown in
Table 32 were added in 30 fold molar excess in 200 uL of NEB buffer
4 (50 mM Potasium Acetate, 20 mM Tris-Acetate, 10 mM Magnesuim
Acetate, 1 mM dithiothreitol pH 7.9) to the dry beads. The
REadaptors were incubated with the single-stranded DNA at 80
.degree. C. for 5 minutes then cooled down to 55 .degree. C. over
30 minutes. Excess REdaptors were washed away with 2 washes of NEB
buffer 4. Fifteen units of HpyCH4III (NEB) were added in NEB buffer
4 and incubated for 1 hour at 55.degree. C. The cleaved downstream
DNA remaining on the beads was removed from the beads using a
Qiagen Nucleotide removal column (see FIG. 15).
[0206] The Bridge/Extender pairs shown in Table 33 were added in 25
molar excess along with 1200 units of T4 DNA ligase and incubated
overnight at 16 .degree. C. Excess Bridge/Extender was removed with
a Qiagen PCR purification column. The ligated material was
amplified by PCR using primers H43.XAExtPCR2 and Hucumnest shown in
Table 34 for 10 cycles with the program shown in Table 35.
[0207] The soluble PCR product was run on a gel and showed a band
of approximately 500 n, as expected (see FIG. 15B). The DNA was
cleaved with enzymes SfiI and NotI, gel purified, and ligated to
similarly cleaved vector PCES1.
Example 6
Description of Phage Display Vector CJRA05, a Member of the Library
Built in Vector DY3F7.
[0208] Table 36 contains an annotated DNA sequence of a member of
the library, CJRA05, see FIG. 16. Table 36 is to be read as
follows: on each line everything that follows an exclamation mark
"!" is a comment. All occurrences of A, C, G, and T before "!" are
the DNA sequence. Case is used only to show that certain bases
constitute special features, such as restriction sites, ribosome
binding sites, and the like, which are labeled below the DNA.
CJRA05 is a derivative of phage DY3F7, obtained by cloning an ApaLI
to NotI fragment into these sites in DY3F31. DY3F31 is like DY3F7
except that the light chain and heavy chain genes have been
replaced by "stuffer" DNA that does not code for any antibody.
DY3F7 contains an antibody that binds streptavidin, but did not
come from the present library.
[0209] The phage genes start with gene ii and continue with genes
x, v, vii, ix, viii, iii, vi, i, and iv. Gene iii has been slightly
modified in that eight codons have been inserted between the signal
sequence and the mature protein and the final amino acids of the
signal sequence have been altered. This allows restriction enzyme
recognition sites EagI and XbaI to be present. Following gene iv is
the phage origin of replication (ori). After ori is bla which
confers resistance to ampicillin (ApR). The phage genes and bla are
transcribed in the same sense.
[0210] After bla, is the Fab cassette (illustrated in FIG. 17)
comprising: [0211] a) PlacZ promoter, [0212] b) A first Ribosome
Binding Site (RBS1), [0213] c) The signal sequence form M13 iii,
[0214] d) An ApaLI RERS, [0215] e) A light chain (a kappa L20::JK1
shortened by one codon at the V-J boundary in this case), [0216] f)
An AscI RERS, [0217] g) A second Ribosome Binding Site (RBS2),
[0218] h) A signal sequence, preferably PelB, which contains,
[0219] i) An SfiI RERS, [0220] j) A synthetic 3-23 V region with
diversity in CDR1 and CDR2, [0221] k) A captured CDR3, [0222] l) A
partially synthetic J region (FR4 after BstEII), [0223] m) CH1,
[0224] n) A NotI RERS, [0225] o) A His6 tag, [0226] p) A cMyc tag,
[0227] q) An amber codon, [0228] r) An anchor DNA that encodes the
same amino-acid sequence as codons 273 to 424 of M13 iii (as shown
in Table 37). [0229] s) Two stop codons, [0230] t) An AvrII RERS,
and [0231] u) A trp terminator.
[0232] The anchor (item r) encodes the same amino-acid sequence as
do codons 273 to 424 of M13 iii but the DNA is approximately as
different as possible from the wild-type DNA sequence. In Table 36,
the III' stump runs from base 8997 to base 9455. Below the DNA, as
comments, are the differences with wild-type iii for the comparable
codons with "!W.T" at the ends of these lines. Note that Met and
Trp have only a single codon and must be left as is. These AA types
are rare. Ser codons can be changed at all three base, while Leu
and Arg codons can be changed at two.
[0233] In most cases, one base change can be introduced per codon.
This has three advantages: 1) recombination with the wild-type gene
carried elsewhere on the phage is less likely, 2) new restriction
sites can be introduced, facilitating construction; and 3)
sequencing primers that bind in only one of the two regions can be
designed.
[0234] The fragment of M13 III shown in CJRA05 is the preferred
length for the anchor segment. Alternative longer or shorter anchor
segments defined by reference to whole mature III protein may also
be utilized.
[0235] The sequence of M13 III consists of the following elements:
Signal Sequence::Domain 1 (D1)::Linker 1 (L1)::Domain 2
(D2)::Linker 2 (L2)::Domain 3 (D3)::Transmembrane Segment
(TM)::Intracellular anchor (IC) (see Table 38).
[0236] The pIII anchor (also known as trpIII) preferably consists
of D2::L2::D3::TM::IC. Another embodiment for the pIII anchor
consists of D2'::L2::D3::TM::IC (where D2' comprises the last 21
residues of D2 with the first 109 residues deleted). A further
embodiment of the pIII anchor consists of D2'
(C>S)::L2::D3::TM::IC (where D2' (C>S) is D2' with the single
C converted to S), and d) D3::TM::IC.
[0237] Table 38 shows a gene fragment comprising the NotI site,
His6 tag, cMyc tag, an amber codon, a recombinant enterokinase
cleavage site, and the whole of mature M13 III protein. The DNA
used to encode this sequence is intentionally very different from
the DNA of wild-type gene iii as shown by the lines denoted "W.T."
containing the w.t. bases where these differ from this gene. III is
divided into domains denoted "domain 1", "linker 1", "domain 2",
"linker 2", "domain 3", "transmembrane segment", and "intracellular
anchor".
[0238] Alternative preferred anchor segments (defined by reference
to the sequence of Table 38) include: [0239] codons 1-29 joined to
codons 104-435, deleting domain 1 and retaining linker 1 to the
end; [0240] codons 1-38 joined to codons 104-435, deleting domain 1
and retaining the rEK cleavage site plus linker 1 to the end from
III; [0241] codons 1-29 joined to codons 236-435, deleting domain
1, linker 1, and most of domain 2 and retaining linker 2 to the
end; [0242] codons 1-38 joined to codons 236-435, deleting domain
1, linker 1, and most of domain 2 and retaining linker 2 to the end
and the rEK cleavage site; [0243] codons 1-29 joined to codons
236-435 and changing codon 240 to Ser(e.g., agc), deleting domain
1, linker 1, and most of domain 2 and retaining linker 2 to the
end; and [0244] codons 1-38 joined to codons 236-435 and changing
codon 240 to Ser(e.g., agc), deleting domain 1, linker 1, and most
of domain 2 and retaining linker 2 to the end and the rEK cleavage
site.
[0245] The constructs would most readily be made by methods similar
to those of Wang and Wilkinson (Biotechnigues 2001: 31(4)722-724)
in which PCR is used to copy the vector except the part to be
deleted and matching restriction sites are introduced or retained
at either end of the part to be kept. Table 39 shows the
oligonucleotides to be used in deleting parts of the III anchor
segment. The DNA shown in Table 38 has an NheI site before the
DINDDRMA recombinant enterokinase cleavage site (rEKCS). If NheI is
used in the deletion process with this DNA, the rEKCS site would be
lost. This site could be quite useful in cleaving Fabs from the
phage and might facilitate capture of very high-affinity
antibodies. One could mutagenize this sequence so that the NheI
site would follow the rEKCS site, an Ala Ser amino-acid sequence is
already present. Alternatively, one could use SphI for the
deletions. This would involve a slight change in amino acid
sequence but would be of no consequence.
Example 7
Selection of Antigen Binders from an Enriched Library of Human
Antibodies Using Phage Vector DY3F31.
[0246] In this example the human antibody library used is described
in de Haard et al., (Journal of Biological Chemistry, 274 (26):
18218-30 (1999). This library, consisting of a large non-immune
human Fab phagemid library, was first enriched on antigen, either
on streptavidin or on phenyl-oxazolone (phOx). The methods for this
are well known in the art. Two preselected Fab libraries, the first
one selected once on immobilized phOx-BSA (R1-ox) and the second
one selected twice on streptavidin (R2-strep), were chosen for
recloning.
[0247] These enriched repertoires of phage antibodies, in which
only a very low percentage have binding activity to the antigen
used in selection, were confirmed by screening clones in an ELISA
for antigen binding. The selected Fab genes were transferred from
the phagemid vector of this library to the DY3F31 vector via
ApaL1-Not1 restriction sites.
[0248] DNA from the DY3F31 phage vector was pretreated with ATP
dependent DNAse to remove chromosomal DNA and then digested with
ApaL1 and Not1. An extra digestion with AscI was performed in
between to prevent self-ligation of the vector. The ApaL1/NotI Fab
fragment from the preselected libraries was subsequently ligated to
the vector DNA and transformed into competent XL1-blue MRF'
cells.
[0249] Libraries were made using vector:insert ratios of 1:2 for
phOx-library and 1:3 for STREP library, and using 100 ng ligated
DNA per 50 .mu.l of electroporation-competent cells
(electroporation conditions : one shock of 1700 V, 1 hour recovery
of cells in rich SOC medium, plating on amplicillin-containing agar
plates).
[0250] This transformation resulted in a library size of
1.6.times.10.sup.6 for R1-ox in DY3F31 and 2.1.times.10.sup.6 for
R2-strep in DY3F31. Sixteen colonies from each library were
screened for insert, and all showed the correct size insert
(.+-.1400 bp) (for both libraries).
[0251] Phage was prepared from these Fab libraries as follows. A
representative sample of the library was inoculated in medium with
ampicillin and glucose, and at OD 0.5, the medium exchanged for
ampicillin and 1 mM IPTG. After overnight growth at 37 .degree. C.,
phage was harvested from the supernatant by PEG-NaCl precipitation.
Phage was used for selection on antigen. R1-ox was selected on
phOx-BSA coated by passive adsorption onto immunotubes and R2-strep
on streptavidin coated paramagnetic beads (Dynal, Norway), in
procedures described in de Haard et. al. and Marks et. al., Journal
of Molecular Biology, 222(3): 581-97 (1991). Phage titers and
enrichments are given in Table 40.
[0252] Clones from these selected libraries, dubbed R2-ox and
R3-strep respectively, were screened for binding to their antigens
in ELISA. 44 clones from each selection were picked randomly and
screened as phage or soluble Fab for binding in ELISA. For the
libraries in DY3F31, clones were first grown in 2TY-2% glucose-50
.mu.g/ml AMP to an OD600 of approximately 0.5, and then grown
overnight in 2TY-50 .mu.g/ml AMP +/-1 mM IPTG. Induction with IPTG
may result in the production of both phage-Fab and soluble Fab.
Therefore the (same) clones were also grown without IPTG. Table 41
shows the results of an ELISA screening of the resulting
supernatant, either for the detection of phage particles with
antigen binding (Anti-M13 HRP=anti-phage antibody), or for the
detection of human Fabs, be it on phage or as soluble fragments,
either with using the anti-myc antibody 9E10 which detects the
myc-tag that every Fab carries at the C-terminal end of the heavy
chain followed by a HRP-labeled rabbit-anti-Mouse serum (column
9E10/RAM-HRP), or with anti-light chain reagent followed by a
HRP-labeled goat-anti-rabbit antiserum(anti-CK/CL Gar-HRP).
[0253] The results shows that in both cases antigen-binders are
identified in the library, with as Fabs on phage or with the
anti-Fab reagents (Table 41). IPTG induction yields an increase in
the number of positives. Also it can be seen that for the
phOx-clones, the phage ELISA yields more positives than the soluble
Fab ELISA, most likely due to the avid binding of phage. Twenty
four of the ELISA-positive clones were screened using PCR of the
Fab-insert from the vector, followed by digestion with BstNI. This
yielded 17 different patterns for the phOx-binding Fab's in 23
samples that were correctly analyzed, and 6 out of 24 for the
streptavidin binding clones. Thus, the data from the selection and
screening from this pre-enriched non-immune Fab library show that
the DY3F31 vector is suitable for display and selection of Fab
fragments, and provides both soluble Fab and Fab on phage for
screening experiments after selection.
Example 8
Selection of Phage-antibody Libraries on Streptavidin Magnetic
Beads.
[0254] The following example describes a selection in which one
first depletes a sample of the library of binders to streptavidin
and optionally of binders to a non-target (i.e., a molecule other
than the target that one does not want the selected Fab to bind).
It is hypothesized that one has a molecule, termed a "competitive
ligand", which binds the target and that an antibody which binds at
the same site would be especially useful.
[0255] For this procedure Streptavidin Magnetic Beads (Dynal) were
blocked once with blocking solution (2% Marvel Milk, PBS (pH 7.4),
0.01% Tween-20 ("2% MPBST")) for 60 minutes at room temperature and
then washed five times with 2% MPBST. 450 .mu.L of beads were
blocked for each depletion and subsequent selection set.
[0256] Per selection, 6.25 .mu.L of biotinylated depletion target
(1 mg/mL stock in PBST) was added to 0.250 mL of washed, blocked
beads (from step 1). The target was allowed to bind overnight, with
tumbling, at 4.degree. C. The next day, the beads are washed 5
times with PBST.
[0257] Per selection, 0.010 mL of biotinylated target antigen (1
mg/mL stock in PBST) was added to 0.100 mL of blocked and washed
beads (from step 1). The antigen was allowed to bind overnight,
with tumbling, at 4.degree. C. The next day, the beads were washed
5 times with PBST.
[0258] In round 1, 2.times.10.sup.12 up to 10.sup.13 plaque forming
units (pfu) per selection were blocked against non-specific binding
by adding to 0.500 mL of 2% MPBS (=2% MPBST without Tween) for 1 hr
at RT (tumble). In later rounds, 1011 pfu per selection were
blocked as done in round 1.
[0259] Each phage pool was incubated with 50 .mu.L of depletion
target beads (final wash supernatant removed just before use) on a
Labquake rotator for 10 min at room temperature. After incubation,
the phage supernatant was removed and incubated with another 50
.mu.L of depletion target beads. This was repeated 3 more times
using depletion target beads and twice using blocked streptavidin
beads for a total of 7 rounds of depletion, so each phage pool
required 350 .mu.L of depletion beads.
[0260] A small sample of each depleted library pool was taken for
titering. Each library pool was added to 0.100 mL of target beads
(final wash supernatant was removed just before use) and allowed to
incubate for 2 hours at room temperature (tumble).
[0261] Beads were then washed as rapidly as possible (e.g.,3
minutes total) with 5.times.0.500 mL PBST and then 2.times. with
PBS. Phage still bound to beads after the washing were eluted once
with 0.250 mL of competitive ligand (.about.1 .mu..mu.M) in PBST
for 1 hour at room temperature on a Labquake rotator. The eluate
was removed, mixed with 0.500 mL Minimal A salts solution and
saved. For a second selection, 0.500 mL 100 mM TEA was used for
elution for 10 min at RT, then neutralized in a mix of 0.250 mL of
1 M Tris, pH 7.4+0.500 mL Min A salts.
[0262] After the first selection elution, the beads can be eluted
again with 0.300 mL of non-biotinylated target (1 mg/mL) for 1 hr
at RT on a Labquake rotator. Eluted phage are added to 0.450 mL
Minimal A salts.
[0263] Three eluates (competitor from 1st selection, target from
1st selection and neutralized TEA elution from 2nd selection) were
kept separate and a small aliquot taken from each for titering.
0.500 mL Minimal A salts were added to the remaining bead aliquots
after competitor and target elution and after TEA elution. Take a
small aliquot from each was taken for tittering.
[0264] Each elution and each set of eluted beads was mixed with
2.times.YT and an aliquot (e.g., 1 mL with 1. E 10/mL) of XL1-Blue
MRF' E. coli cells (or other F' cell line) which had been chilled
on ice after having been grown to mid-logarithmic phase, starved
and concentrated (see procedure below--"Mid-Log prep of XL-1 blue
MRF' cells for infection").
[0265] After approximately 30 minutes at room temperature, the
phage/cell mixtures were spread onto Bio-Assay Dishes
(243.times.243.times.18 mm, Nalge Nunc) containing 2.times.YT, 1 mM
IPTG agar. The plates were incubated overnight at 30.degree. C. The
next day, each amplified phage culture was harvested from its
respective plate. The plate was flooded with 35 mL TBS or LB, and
cells were scraped from the plate. The resuspended cells were
transferred to a centrifuge bottle. An additional 20 mL TBS or LB
was used to remove any cells from the plate and pooled with the
cells in the centrifuge bottle. The cells were centrifuged out, and
phage in the supernatant was recovered by PEG precipitation. Over
the next day, the amplified phage preps were titered.
[0266] In the first round, two selections yielded five amplified
eluates. These amplified eluates were panned for 2-3 more
additional rounds of selection using .about.1. E 12 input
phage/round. For each additional round, the depletion and target
beads were prepared the night before the round was initiated.
[0267] For the elution steps in subsequent rounds, all elutions up
to the elution step from which the amplified elution came from were
done, and the previous elutions were treated as washes. For the
bead infection amplified phage, for example, the competitive ligand
and target elutions were done and then tossed as washes (see
below). Then the beads were used to infect E. coli. Two pools,
therefore, yielded a total of 5 final elutions at the end of the
selection. [0268] 1st selection set [0269] A. Ligand amplified
elution: elute w/ligand for 1 hr, keep as elution [0270] B. Target
amplified elution: elute w/ligand for 1 hr, toss as wash elute
w/target for 1 hr, keep as elution [0271] C. Bead infect. amp.
elution: elute w/ligand for 1 hr, toss as wash elute w/target for 1
hr, toss as wash elute w/cell infection, keep as elution [0272] 2nd
selection set [0273] A. TEA amplified elution; elute w/TEA 10 min,
keep as elution [0274] B. Bead infect. amp. elution; elute w/TEA 10
min, toss as wash elute w/cell infection, keep as elution Mid-log
Prep of XL1 Blue MRF' Cells for Infection (Based on Barbas et al.
Phage Display Manual Procedure)
[0275] Culture XL1 blue MRF' in NZCYM (12.5 mg/mL tet) at
37.degree. C. and 250 rpm overnight. Started a 500 mL culture in 2
liter flask by diluting cells 1/50 in NZCYM/tet (10 mL overnight
culture added) and incubated at 37.degree. C. at 250 rpm until
OD600 of 0.45 (1.5-2 hrs) was reached. Shaking was reduced to 100
rpm for 10 min. When OD600 reached between 0.55-0.65, cells were
transferred to 2.times.250 mL centrifuge bottles, centrifuged at
600 g for 15 min at 4.degree. C. Supernatant was poured off.
Residual liquid was removed with a pipette.
[0276] The pellets were gently resuspended (not pipetting up and
down) in the original volume of 1.times.Minimal A salts at room
temp. The resuspended cells were transferred back into 2-liter
flask, shaken at 100 rpm for 45 min at 37.degree. C. This process
was performed in order to starve the cells and restore pili. The
cells were transferred to 2.times.250 mL centrifuge bottles, and
centrifuged as earlier.
[0277] The cells were gently resuspended in ice cold Minimal A
salts (5 mL per 500 mL original culture). The cells were put on ice
for use in infections as soon as possible.
[0278] The phage eluates were brought up to 7.5 mL with 2.times.YT
medium and 2.5 mL of cells were added. Beads were brought up to 3
mL with 2.times.YT and 1 mL of cells were added. Incubated at
37.degree. C. for 30 min. The cells were plated on 2.times.YT, 1 mM
IPTG agar large NUNC plates and incubated for 18 hr at 30.degree.
C.
Example 9
Incorporation of Synthetic Region in FR1/3 Region.
[0279] Described below are examples for incorporating of fixed
residues in antibody sequences for light chain kappa and lambda
genes, and for heavy chains. The experimental conditions and
oligonucleotides used for the examples below have been described in
previous examples (e.g., Examples 3 & 4).
[0280] The process for incorporating fixed FR1 residues in an
antibody lambda sequence consists of 3 steps (see FIG. 18): (1)
annealing of single-stranded DNA material encoding VL genes to a
partially complementary oligonucleotide mix (indicated with Ext and
Bridge), to anneal in this example to the region encoding residues
5-7 of the FR1 of the lambda genes (indicated with X . . . X;
within the lambda genes the overlap may sometimes not be perfect);
(2) ligation of this complex; (3) PCR of the ligated material with
the indicated primer (`PCRpr`) and for example one primer based
within the VL gene. In this process the first few residues of all
lambda genes will be encoded by the sequences present in the
oligonucleotides (Ext., Bridge or PCRpr). After the PCR, the lambda
genes can be cloned using the indicated restriction site for
ApaLI.
[0281] The process for incorporating fixed FR1 residues in an
antibody kappa sequence (FIG. 19) consists of 3 steps: (1)
annealing of single-stranded DNA material encoding VK genes to a
partially complementary oligonucleotide mix (indicated with Ext and
Bri), to anneal in this example to the region encoding residues
8-10 of the FR1 of the kappa genes (indicated with X . . . X;
within the kappa genes the overlap may sometimes not be perfect) ;
(2) ligation of this complex; (3) PCR of the ligated material with
the indicated primer (`PCRpr`) and for example one primer based
within the VK gene. In this process the first few (8) residues of
all kappa genes will be encode by the sequences present in the
oligonucleotides (Ext., Bridge or PCRpr.). After the PCR, the kappa
genes can be cloned using the indicated restriction site for
ApaLI.
[0282] The process of incorporating fixed FR3 residues in a
antibody heavy chain sequence (FIG. 20) consists of 3 steps: (1)
annealing of single-stranded DNA material encoding part of the VH
genes (for example encoding FR3, CDR3 and FR4 regions) to a
partially complementary oligonucleotide mix (indicated with Ext and
Bridge), to anneal in this example to the region encoding residues
92-94 (within the FR3 region) of VH genes (indicated with X . . .
X; within the VH genes the overlap may sometimes not be perfect);
(2) ligation of this complex; (3) PCR of the ligated material with
the indicated primer (`PCRpr`) and for example one primer based
within the VH gene (such as in the FR4 region). In this process
certain residues of all VH genes will be encoded by the sequences
present in the oligonucleotides used here, in particular from PCRpr
(for residues 70-73), or from Ext/Bridge oligonucleotides (residues
74-91). After the PCR, the partial VH genes can be cloned using the
indicated restriction site for XbaI.
[0283] It will be understood that the foregoing is only
illustrative of the principles of this invention and that various
modifications can be made by those skilled in the art without
departing from the scope of and sprit of the invention.
TABLE-US-00002 TABLE 1 Human GLG FR3 sequences ! VH1 ! 66 67 68 69
70 71 72 73 74 75 76 77 78 7980 agg gtc aco atg acc agg gac acg too
atc agc aca gcc tac atg ! 81 82 82a 82b 82c 83 84 85 86 87 88 89 90
91 92 gag ctg agc agg ctg aga tot gac gac acg gcc gtg tat tac tgt !
93 94 95 gcg aga ga ! 1-02# 1 aga gtc acc att acc agg gac aca too
gcg agc aca gcc tac atg gag ctg agc agc ctg aga tct gaa gac acg gct
gtg tat tac tgt gcg aga ga ! 1-03# 2 aga gto acc atg acc agg aac
acc too ata agc aca gcc tac atg gag ctg agc agc ctg aga tct gag gac
acg gcc gtg tat tac tgt gcg aga gg ! 1-08# 3 aga gtc acc atg acc
aca gac aca tcc acg agc aca gcc tac atg gag ctg agg agc ctg aga tct
gac gac acg gcc gtg tat tac tgt gcg aga ga ! 1-18# 4 aga gtc acc
atg acc gag gac aca tct aca gac aca gcc tac atg gag ctg agc agc ctg
aga tct gag gac acg gcc gtg tat tac tgt gca aca ga ! 1-24# 5 aga
gtc acc att acc agg gac agg tct atg agc aca gcc tac atg gag ctg agc
agc ctg aga tct gag gac aca gcc atg tat tac tgt gca aga ta ! 1-45#
6 aga gtc acc atg acc agg gac acg tcc acg agc aca gtc tac atg gag
ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt gcg aga ga
! 1-46# 7 aga gtc acc att acc agg gac atg tcc aca agc aca gcc tac
atg gag ctg agc agc ctg aga tcc gag gac acg gcc gtg tat tac tgt gcg
gca ga ! 1-58# 8 aga gtc acg att acc gcg gac gaa tcc acg agc aca
gcc tac atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac
tgt gcg aga ga ! 1-69# 9 aga gtc acg att acc gag gac aaa tcc acg
agc aca gcc tac atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg
tat tac tgt gcg aga ga ! 1-e# 10. aga gtc acc ata acc gcg gac acg
tct aca gac aca gcc tac atg gag ctg agc agc ctg aga tct gag gac acg
gcc gtg tat tac tgt gca aca ga ! 1-f# 11 ! VH2 agg ctc acc atc acc
aag gac acc tcc aaa aaccag gtg gtc ctt aca atg acc aac atg gac cct
gtg gac aca gcc aca tat tac tgt gca cac aga c ! 2-05# 12 agg ctc
acc atc tcc aag gac acc tcc aaa agc cag gtg gtc ctt acc atg acc aac
atg gac cct gtg gac aca gcc aca tat tac tgt gca cgg ata c ! 2-26#
13 agg ctc acc atc tcc aag gac acc tcc aaa aac cag gtg gtc ctt aca
atg acc aac atg gac cct gtg gac aca gcc acg tat tac tgt gca cgg ata
c ! 2-70# 14 ! VH3 cga ttc acc atc tcc aga gac aac gcc aag aac tca
ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac
tgt gcg aga ga ! 3-07# 15 cga ttc acc atc tcc aga gac aac gcc aag
aac tcc ctg tat ctg caa atg aac agt ctg aga gct gag gac acg gcc ttg
tat tac tgt gca aaa gat a ! 3-09# 16 cga ttc acc atc tcc aga gac
aac gcc aag aac tca ctg tat ctg caa atg aac agc ctg aga gcc gag gac
acg gcc gtg tat tac tgt gcg aga ga ! 3-11# 17 cga ttc acc atc tcc
aga gac aac gcc aag aac tca ctg tat ctg caa atg aac agc ctg aga gcc
gag gac acg gcc gtg tat tac tgt gcg aga ga ! 3-13# 18 aga ttc acc
atc tca aga gat gat tca aaa aac acg ctg tat ctg caa atg aac agc ctg
aaa acc gag gac aca gcc gtg tat tac tgt acc aca ga ! 3-15# 19 cga
ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg caa atg aac
agt ctg aga gcc gag gac acg gcc ttg tat cac tgt gcg aga ga ! 3-20#
20 cga ttc acc atc tcc aga gac aac gcc aag aac tca ctg tat ctg caa
atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt gcg aga ga
! 3-21# 21 cgg ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat
ctg caa atg aac agc ctg aga gcc gag gac acg gcc gta tat tac tgt gcg
aaa ga ! 3-23# 22 cga ttc acc atc tcc aga gac aat tcc aag aac acg
ctg tat ctg caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac
tgt gcg aaa ga ! 3-30# 23 cga ttc acc atc tcc aga gac aat tcc aag
aac acg ctg tat ctg caa atg aac agc ctg aga gct gag gac acg gct gtg
tat tac tgt gcg aga ga ! 3303# 24 cga tte acc atc tcc aga gac aat
tcc aag aac acg ctg tat ctg caa atg aac acc ctg aga gct gag gac acg
gct gtg tat tac tgt gcg aaa ga ! 3305# 25 cga ttc acc atc tcc aga
gac aat tcc aag aac acg ctg tat ctg caa atg aae agc ctg aga gcc gag
gac acg gct gtg tat tac tgt gcg aga ga ! 3-33# 26 cga ttc acc ate
tcc aga gac aae acc aaa aac tcc ctg tat ctg caa atg aac agt ctg aga
act gag gac aee gee ttg tat tac tgt gca aaa gat a! 3-43# 27 cga ttc
acc atc tcc aga gac aat gcc aag aac tca ctg tat ctg caa atg aac agc
ctg aga gac gag gac acg gct gtg tat tac tgt gcg aga ga ! 3-48# 28
aga ttc acc atc tca aga gat ggt tcc aaa agc atc gcc tat ctg caa atg
aae acc ctg aaa acc gag gac aca gcc gtg tat tac tgt act aga ga !
3-49# 29 cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat
ctt caa atg aac acc ctg aga gcc gag gac acg gcc gtg tat tac tgt gcg
aga ga ! 3-53# 30 aga tte acc atc tcc aga gac aat tcc aag aac acg
ctg tat ctt caa atg ggc acc ctg aga gct gag gac atg gct gtg tat tac
tgt gcg aga ga ! 3-64# 31 aga ttc acc atc tcc aga gac aat tcc aag
aac acg ctg tat ctt caa atg aac agc ctg aga gct gag gac acg gct gtg
tat tac tgt gcg aga ga ! 3-66# 32 aga ttc acc atc tca aga gat gat
tca aag aac tca ctg tat ctg caa atg aac agc ctg aaa acc gag gac acg
gcc gtg tat tac tgt gct aga ga ! 3-72# 33 agg ttc acc atc tcc aga
gat gat tca aag aac acg gcg tat ctg caa atg aac agc ctg aaa acc gag
gac acg gcc gtg tat tac tgt act aga ca ! 3-73# 34 cga ttc acc atc
tcc aga gac aac gcc aag aac acg ctg tat ctg caa atg aac agt ctg aga
gcc gag gac acg gct gtg tat tac tgt gea aga ga ! 3-74# 35 aga ttc
acc atc tcc aga gac aat tcc aag aac acg ctg eat ctt caa atg aac agc
ctg aga gct gag gac acg gct gtg tat tac tgt aag aaa ga ! 3-d# 36 !
VH4 cga gtc acc ata tca gta gac aag tcc aag aac cag ttc tcc ctg aag
ctg agc tct gtg acc gcc gcg gac acg gcc gtg tat tac tgt gcg aga ga
! 4-04# 37 cga gtc acc atg tca gta gac acg tcc aag aac cag ttc tcc
ctg aag ctg agc tct gtg acc gcc gtg gac acg gcc gtg tat tac tgt gcg
aga aa ! 4-28# 38 cga gtt acc ata tca gta gac acg tct aag aac cag
ttc tcc ctg aag ctg agc tct gtg act gcc gcg gac acg gce gtg tat tac
tgt gcg aga ga ! 4301# 39 cga gtc acc ata tca gta gac agg tcc aag
aac cag ttc tcc ctg aag ctg agc tct gtg acc gcc gcg gac acg gcc gtg
tat tac tgt gce aga ga ! 4302# 40 cga gtt acc ata tca gta gac acg
tcc aag aac cag ttc tcc ctg aag ctg agc tct gtg act gcc gca gac acg
gcc gtg tat tac tgt gcc aga ga ! 4304# 41 cga gtt acc ata tca gta
gac acg tct aag aac cag ttc tcc ctg aag ctg agc tct gtg act gcc gcg
gac acg gcc gtg tat tac tgt gcg aga ga ! 4-31# 42 cga gtc acc ata
tca gta gac acg tcc aag aac cag ttc tcc ctg aag ctg agc tct gtg acc
gcc gcg gac acg gct gtg tat tac tgt gcg aga ga ! 4-34# 43 cga gtc
acc ata tcc gta gac acg tcc aag aac cag ttc tcc ctg aag ctg agc tct
gtg acc gcc gca gac acg gct gtg tat tac tgt gcg aga ca ! 4-39# 44
cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg aag ctg
agc tct gtg acc gct gcg gac acg gcc gtg tat tac tgt gcg aga ga !
4-59# 45 cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc
ctg aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat tac tgt gcg
aga ga ! 4-61# 46 cga gtc acc ata tca gta gac acg tcc aag aac cag
ttc tcc ctg aag ctg agc tct gtg acc gcc gca gac acg gcc gtg tat tac
tgt gcg aga ga ! 4-b# 47 ! VH5 cag gtc acc atc tca gcc gac aag tcc
atc agc acc gcc tac ctg cag tgg agc agc ctg aag gcc teg gac acc gcc
atg tat tac tgt gcg aga ca ! 5-51# 48 cac gtc acc atc tca gct gac
aag tcc atc agc act gcc tac ctg cag tgg agc agc ctg aag gcc tcg gac
acc gcc atg tat tac tgt gcg aga ! 5-a# 49 ! VH6 cga ata acc atc aac
eca gac aca tcc aag aac cag ttc tcc ctg cag ctg aac tct gtg act ccc
gag gac acg gct gtg tat tac tgt gca aga ga ! 6-1# 50 ! VH7 cgg ttt
gtc ttc tcc ttg gac acc tct gtc agc acg gca tat ctg cag atc tgc agc
cta aag gct gag gac act gcc gtg tat tac tgt gcg aga ga ! 74.1#
51
[0284] TABLE-US-00003 TABLE 2 Enzymes that either cut 15 or more
human GLGs or have 5+-base recognition in FR3 Typical entry: REname
#sites Recognition GLGid#:base# GLGid#:base# GLGid#:base#. . .
BstEII Ggtnacc 2 1:3 48:3 There are 2 hits at base# 3 MaeIII gtnac
36 1:4 2:4 3:4 4:4 5:4 6:4 7:4 8:4 9:4 10:4 11:4 37:4 37:58 38:4
38:58 39:4 39:58 40:4 40:58 41:4 41:58 42:4 42:58 43:4 43:58 44:4
44:58 45:4 45:58 46:4 46:58 47:4 47:58 48:4 49:4 50:58 There are 24
hits at base# 4 Tsp45I gtsac 33 1:4 2:4 3:4 4:4 5:4 6:4 7:4 8:4 9:4
10:4 11:4 37:4 37:58 38:4 38:58 39:58 40:4 40:58 41:58 42:58 43:4
43:58 44:4 44:58 45:4 45:58 46:4 46:58 47:4 47:58 48:4 49:4 50:58
There are 21 hits at base# 4 HphI tcacc 45 1:5 2:5 3:5 4:5 5:5 6:5
7:5 8:5 11:5 12:5 12:11 13:5 14:5 15:5 16:5 17:5 18:5 19:5 20:5
21:5 22:5 23:5 24:5 25:5 26:5 27:5 28:5 29:5 30:5 31:5 32:5 33:5
34:5 35:5 36:5 37:5 38:5 40:5 43:5 44:5 45:5 46:5 47:5 48:5 49:5
There are 44 hits at base# 5 NlaIII CATG 26 1:9 1:42 2:42 3:9 3:42
4:9 4:42 5:9 5:42 6:42 6:78 7:9 7:42 8:21 8:42 9:42 10:42 11:42
12:57 13:48 13:57 14:57 31:72 38:9 48:78 49:78 There are 11 hits at
base# 42 There are 1 hits at base# 48 Could cause raggedness. BsaJI
Ccnngg 37 1:14 2:14 5:14 6:14 7:14 8:14 8:65 9:14 10:14 11:14 12:14
13:14 14:14 15:65 17:14 17:65 18:65 19:65 20:65 21:65 22:65 26:65
29:65 30:65 33:65 34:65 35:65 37:65 38:65 39:65 40:65 42:65 43:65
48:65 49:65 50:65 51:14 There are 23 hits at base# 65 There are 14
hits at base# 14 AluI AGct 42 1:47 2:47 3:47 4:47 5:47 6:47 7:47
8:47 9:47 10:47 11:47 16:63 23:63 24:63 25:63 31:63 32:63 36:63
37:47 37:52 38:47 38:52 39:47 39:52 40:47 40:52 41:47 41:52 42:47
42:52 43:47 43:52 44:47 44:52 45:47 45:52 46:47 46:52 47:47 47:52
49:15 50:47 There are 23 hits at base# 47 There are 11 hits at
base# 52 Only 5 bases from 47 BlpI GCtnagc 21 1:48 2:48 3:48 5:48
6:48 7:48 8:48 9:48 10:48 11:48 37:48 38:48 39:48 40:48 41:48 42:48
43:48 44:48 45:48 46:48 47:48 There are 21 hits at base# 48 MwoI
GCNNNNNnngc 19 1:48 2:28 19:36 22:36 23:36 24:36 25:36 26:36 35:36
37:67 39:67 40:67 41:67 42:67 43:67 44:67 45:67 46:67 47:67 There
are 10 hits at base# 67 There are 7 hits at base# 36 DdeI Ctnag 71
1:49 1:58 2:49 2:58 3:49 3:58 3:65 4:49 4:58 5:49 5:58 5:65 6:49
6:58 6:65 7:49 7:58 7:65 8:49 8:58 9:49 9:58 9:65 10:49 10:58 10:65
11:49 11:58 11:65 15:58 16:58 16:65 17:58 18:58 20:58 21:58 22:58
23:58 23:65 24:58 24:65 25:58 25:65 26:58 27:58 27:65 28:58 30:58
31:58 31:65 32:58 32:65 35:58 36:58 36:65 37:49 38:49 39:26 39:49
40:49 41:49 42:26 42:49 43:49 44:49 45:49 46:49 47:49 48:12 49:12
51:65 There are 29 hits at base# 58 There are 22 hits at base# 49
Only nine base from 58 There are 16 hits at base# 65 Only seven
bases from 58 BglII Agatct 11 1:61 2:61 3:61 4:61 5:61 6:61 7:61
9:61 10:61 11:61 51:47 There are 10 hits at base# 61 BstYI Rgatcy
12 1:61 2:61 3:61 4:61 5:61 6:61 7:61 8:61 9:61 10:61 11:61 51:47
There are 11 hits at base# 61 Hpy188I TCNga 17 1:64 2:64 3:64 4:64
5:64 6:64 7:64 8:64 9:64 10:64 11:64 16:57 20:57 27:57 35:57 48:67
49:67 There are 11 hits at base# 64 There are 4 hits at base# 57
There are 2 hits at base# 67 Could be ragged. MslI CAYNNnnRTG 44
1:72 2:72 3:72 4:72 5:72 6:72 7:72 8:72 9:72 10:72 11:72 15:72
17:72 18:72 19:72 21:72 23:72 24:72 25:72 26:72 28:72 29:72 30:72
31:72 32:72 33:72 34:72 35:72 36:72 37:72 38:72 39:72 40:72 41:72
42:72 43:72 44:72 45:72 46:72 47:72 48:72 49:72 50:72 51:72 There
are 44 hits at base# 72 BsiEI CGRYcg 23 1:74 3:74 4:74 5:74 7:74
8:74 9:74 10:74 11:74 17:74 22:74 30:74 33:74 34:74 37:74 38:74
39:74 40:74 41:74 42:74 45:74 46:74 47:74 There are 23 hits at
base# 74 EaeI Yggccr 23 1:74 3:74 4:74 5:74 7:74 8:74 9:74 10:74
11:74 17:74 22:74 30:74 33:74 34:74 37:74 38:74 39:74 40:74 41:74
42:74 45:74 46:74 47:74 There are 23 hits at base# 74 EagI Cggccg
23 1:74 3:74 4:74 5:74 7:74 8:74 9:74 10:74 11:74 17:74 22:74 30:74
33:74 34:74 37:74 38:74 39:74 40:74 41:74 42:74 45:74 46:74 47:74
There are 23 hits at base# 74 HaeIII GGcc 27 1:75 3:75 4:75 5:75
7:75 8:75 9:75 10:75 11:75 16:75 17:75 20:75 22:75 30:75 33:75
34:75 37:75 38:75 39:75 40:75 41:75 42:75 45:75 46:75 47:75 48:63
49:63 There are 25 hits at base # 75 Bst4CI ACNgt 65.degree. C. 63
Sites There is a third isoschismer 1:86 2:86 3:86 4:86 5:86 6:86
7:34 7:86 8:86 9:86 10:86 11:86 12:86 13:86 14:86 15:36 15:86 16:53
16:86 17:36 17:86 18:86 19:86 20:53 20:86 21:36 21:86 22:0 22:86
23:86 24:86 25:86 26:86 27:53 27:86 28:36 28:86 29:86 30:86 31:86
32:86 33:36 33:86 34:86 35:53 35:86 36:86 37:86 38:86 39:86 40:86
41:86 42:86 43:86 44:86 45:86 46:86 47:86 48:86 49:86 50:86 51:0
51:86 There are 51 hits at base# 86 All the other sites are well
away HpyCH4III ACNgt 63 1:86 2:86 3:86 4:86 5:86 6:86 7:34 7:86
8:86 9:86 10:86 11:86 12:86 13:86 14:86 15:36 15:86 16:53 16:86
17:36 17:86 18:86 19:86 20:53 20:86 21:36 21:86 22:0 22:86 23:86
24:86 25:86 26:86 27:53 27:86 28:36 28:86 29:86 30:86 31:86 32:86
33:36 33:86 34:86 35:53 35:86 36:86 37:86 38:86 39:86 40:86 41:86
42:86 43:86 44:86 45:86 46:86 47:86 48:86 49:86 50:86 51:0 51:86
There are 51 hits at base# 86 HinfI Gantc 43 2:2 3:2 4:2 5:2 6:2
7:2 8:2 9:2 9:22 10:2 11:2 15:2 16:2 17:2 18:2 19:2 19:22 20:2 21:2
23:2 24:2 25:2 26:2 27:2 28:2 29:2 30:2 31:2 32:2 33:2 33:22 34:22
35:2 36:2 37:2 38:2 40:2 43:2 44:2 45:2 46:2 47:2 50:60 There are
38 hits at base# 2 MlyI GAGTCNNNNNn 18 2:2 3:2 4:2 5:2 6:2 7:2 8:2
9:2 10:2 11:2 37:2 38:2 40:2 43:2 44:2 45:2 46:2 47:2 There are 18
hits at base# 2 PleI gagtc 18 2:2 3:2 4:2 5:2 6:2 7:2 8:2 9:2 10:2
11:2 37:2 38:2 40:2 43:2 44:2 45:2 46:2 47:2 There are 18 hits at
base# 2 AciI Ccgc 24 2:26 9:14 10:14 11:14 27:74 37:62 37:65 38:62
39:65 40:62 40:65 41:65 42:65 43:62 43:65 44:62 44:65 45:62 46:62
47:62 47:65 48:35 48:74 49:74 There are 8 hits at base# 62 There
are 8 hits at base# 65 There are 3 hits at base# 14 There are 3
hits at base# 74 There are 1 hits at base# 26 There are 1 hits at
base# 35 -''- Gcgg 11 8:91 9:16 10:16 11:16 37:67 39:67 40:67 42:67
43:67 45:67 46:67 There are 7 hits at base# 67 There are 3 hits at
base# 16 There are 1 hits at base# 91 BsiHKAI GWGCWc 20 2:30 4:30
6:30 7:30 9:30 10:30 12:89 13:89 14:89 37:51 38:51 39:51 40:51
41:51 42:51 43:51 44:51 45:51 46:51 47:51 There are 11 hits at
base# 51 Bsp1286I GDGCHc 20 2:30 4:30 6:30 7:30 9:30 10:30 12:89
13:89 14:89 37:51 38:51 39:51 40:51 41:51 42:51 43:51 44:51 45:51
46:51 47:51 There are 11 hits at base# 51 HgiAI GWGCWc 20 2:30 4:30
6:30 7:30 9:30 10:30 12:89 13:89 14:89 37:51 38:51 39:51 40:51
41:51 42:51 43:51 44:51 45:51 46:51 47:51 There are 11 hits at
base# 51 BsoFI GCngc 26 2:53 3:53 5:53 6:53 7:53 8:53 8:91 9:53
10:53 11:53 31:53 36:36 37:64 39:64 40:64 41:64 42:64 43:64 44:64
45:64 46:64 47:64 48:53 49:53 50:45 51:53 There are 13 hits at
base# 53 There are 10 hits at base# 64 TseI Gcwgc 17 2:53 3:53 5:53
6:53 7:53 8:53 9:53 10:53 11:53 31:53 36:36 45:64 46:64 48:53 49:53
50:45 51:53 There are 13 hits at base# 53 MnlI gagg 34 3:67 3:95
4:51 5:16 5:67 6:67 7:67 8:67 9:67 10:67 11:67 15:67 16:67 17:67
19:67 20:67 21:67 22:67 23:67 24:67 25:67 26:67 27:67 28:67 29:67
30:67 31:67 32:67 33:67 34:67 35:67 36:67 50:67 51:67 There are 31
hits at base# 67
HpyCH4V TGca 34 5:90 6:90 11:90 12:90 13:90 14:90 15:44 16:44 16:90
17:44 18:90 19:44 20:44 21:44 22:44 23:44 24:44 25:44 26:44 27:44
27:90 28:44 29:44 33:44 34:44 35:44 35:90 36:38 48:44 49:44 50:44
50:90 51:44 51:52 There are 21 hits at base# 44 There are 1 hits at
base# 52 AccI GTmkac 13 5-base recognition 7:37 11:24 37:16 38:16
39:16 40:16 41:16 42:16 43:16 44:16 45:16 46:16 47:16 There are 11
hits at base# 16 SacII CCGCgg 8 6-base recognition 9:14 10:14 11:14
37:65 39:65 40:65 42:65 43:65 There are 5 hits at base# 65 There
are 3 hits at base# 14 TfiI Gawtc 24 9:22 15:2 16:2 17:2 18:2 19:2
19:22 20:2 21:2 23:2 24:2 25:2 26:2 27:2 28:2 29:2 30:2 31:2 32:2
33:2 33:22 34:22 35:2 36:2 There are 20 hits at base# 2 BsmAI
Nnnnnngagac 19 15:11 16:11 20:11 21:11 22:11 23:11 24:11 25:11
26:11 27:11 28:11 28:56 30:11 31:11 32:11 35:11 36:11 44:87 48:87
There are 16 hits at base# 11 BpmI ctccag 19 15:12 16:12 17:12
18:12 20:12 21:12 22:12 23:12 24:12 25:12 26:12 27:12 28:12 30:12
31:12 32:12 34:12 35:12 36:12 There are 19 hits at base# 12 XmnI
GAANNnnttc 12 37:30 38:30 39:30 40:30 41:30 42:30 43:30 44:30 45:30
46:30 47:30 50:30 There are 12 hits at base# 30 BsrI NCcagt 12
37:32 38:32 39:32 40:32 41:32 42:32 43:32 44:32 45:32 46:32 47:32
50:32 There are 12 hits at base# 32 BanII GRGCYc 11 37:51 38:51
39:51 40:51 41:51 42:51 43:51 44:51 45:51 46:51 47:51 There are 11
hits at base# 51 Ecl136I GAGctc 11 37:51 38:51 39:51 40:51 41:51
42:51 43:51 44:51 45:51 46:51 47:51 There are 11 hits at base# 51
SacI GAGCTc 11 37:51 38:51 39:51 40:51 41:51 42:51 43:51 44:51
45:51 46:51 47:51 There are 11 hits at base# 51
[0285] TABLE-US-00004 TABLE 3 Synthetic 3-23 FR3 of human heavy
chains showning positions of possible cleavagc sites ! Sites
engineered into the synthetic gene are shown in upper case DNA !
with the RE name bctween vertical bars (as in | XbaI |). ! RERSa
frequently found in GLGs are shown below the synthetic sequence !
with the name to the right (as in gtn ac = MaeIII(24), indicating
that ! 24 of the 51 GLGs contain the site) ! ! |---FR3--- ! 89 90
(codon # in ! R F synthetic 3-23) |cgc|ttc| 6 ! Allowed DNA
|cgn|tty| ! |agr| ! ga ntc = HinfI(38) ! ga gtc = PleI(18) ! ga wtc
= TfiI(20) ! gtn ac = MaeIII(24) ! gts ac = Tsp45I(21) ! tc acc =
HphI(44) ! !
--------FR3-------------------------------------------------- ! 91
92 93 94 95 96 97 98 99 100 101 102 103 104 105 ! T I S R D N S K N
T L Y L Q M
|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| 51
!allowed|acn|ath|tcn|cgn|gay|aay|tcn|aar|aay|acn|ttr|tay|ttr|car|atg|
! |agy|agr| |agy| |ctn| |ctn| ! | ga|gac = BsmAI(16) ag ct =
AluI(23) ! c|tcc ag = BpmI(19) g ctn agc = BlpI(21) ! | | g aan nnn
ttt = XmnI(12) ! | XbaI | tg ca = HpyCH4V(21) ! !
---FR3----------------------------------------------------->| !
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 ! N S L
R A E D T A V Y Y C A K
|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa| 96
!allowed|aay|tcn|ttr|cgn|gcn|gar|gay|acn|gcn|gtn|tay|tay|tgy|gcn|aar|
! |agy|ctn|agr| | | ! | | cc nng g = BsaJI(23) ac ngt = Bst4CI(51)
! | aga tct = BglII(10) | ac ngt = HpyCH4III(51) ! | Rga tcY =
BstYI(11) | ac ngt = TaaI(51) ! | | c ayn nnn rtc = MslI(44) ! | |
cg ryc g = BsiEI(23) ! | | yg gcc r = EagI(23) ! | | cg gcc g =
EagI(23) ! | | |g gcc = HaeIII(25) ! | | gag g = MnlI(31)| ! |AflII
| | PstI |
[0286] TABLE-US-00005 TABLE 4 REdaptors, Extenders, and Bridges
used for Cleavacc and Capture of Human Heavy Chains in FR3. A:
HpyCH4V Probes of actual human HC genes !HpyCH4V in FR3 of human
HC, bases 35-56; only those with TGca site TGca; 10, RE
recognltion:tgca of length 4 is expected at 10 1 6-1
agttctccctgcagctgaactc 2 3-11,3-07, 3-21, 3-72,3-48
cactgtatctgcaaatgaacag 3 3-09, 3-43,3-20 ccctgtatctgcaaatgaacag 4
5-51 ccgcctacctgcagtggagcag 5
3-15,3-30,3-30.5,3-30.3,3-74,3-23,3-33 cgctgtatctgcaaatgaacag 6
7-4.1 cggcatatctgcagatctgcag 7 3-73 cggcgtatctgcaaatgaacag 8 5-a
actgcctacctgcagtggagcag 9 3-49 tcgcctatctgcaaatgaacag B: HpyCH4V
REdaptors, Extenders, and Bridges B.1 PEdaptors ! Cutting HC lower
strand: ! TmKeller for 100 mM NaCl, zero formamide ! Edapters for
cleavage T.sub.m.sup.W T.sub.m.sup.K (ON_HCFR36-1)
5'-agttctcccTGCAgctgaactc-3' 68.0 64.5 (ON_HCFR36-1A)
5'-ttctcccTGCAgctgaactc-3' 62.0 62.5 (ON_HCFR36-1B)
5'-ttctcccTGCAgctgaac-3' 56.0 59.9 (ON_HCFR33-15)
5'-cgctgtatcTGCAaatgaacag3' 64.0 60.8 (ON_HCFR33-15A)
5'-ctgtatcTGCAaatgaacag-3' 56.0 56.3 (ON_HCFR33-15B)
5'-tgtatcTGCAaatgaac-3' 50.0 53.1 (ON_HCFR33-11)
5'-cactgtatcTGCAaatgaacag-3' 62.0 58.9 (ON_HCFR35-51)
5'-ccgcctaccTGCAgtggagcag-3' 74.0 70.1 B.2 Segment of synthetic
3-23 gene into which captured CDR3 is to be cloned ! XbaI . . .
!D323* cgCttcacTaag tcT aga gac aaC tcT aag aaT acT ctC taC !
scab........designed gene 3-23 gene................ ! ! HpyCH4V !
.. .. AflII . . . ! Ttg caG atg aac agc TtA agG . . . !
.............................. . . . B.3 Extender and Bridges !
Extender (bottom strand): (ON_HCHpyEx01)
5'-cAAgTAgAgAgTATTcTTAgAgTTgTcTcTAgAcTTAgTgAAgcg-3' ! ON_HCHpyEx01
is the reverse complement of ! 5'-cgCttcacTaag tcT aga gac aaC tcT
aag aaT acT ctC taC Ttg -3' ! ! Bridges (top strand, 9-base
overlap) ! (ON_HCHpyBr016-1) 5'-cgCttcacTaag tcT aga gac aaC tcT
aag- aaT acT ctC taC Ttg CAgctgaac-3' {(3'-term C is blocked) ! !
3-15 et al. + 3-11 (ON_HCHpyBr023-15) 5'-cgCttcacTaag tcT aga gac
aaC tcT aag- aaT acT ctC taC Ttg CAaatgaac-3' {3'-term C is
blocked) ! ! 5-51 (ON_1-ICHpyBr045-51) 5'-cgCttcacTaag tcT aga gac
aaC tcT aag- aaT acT ctC taC Ttg CAgtggagc-3' (3'-term C is
blocked) ! ! PCR primer (top strand) ! (ON_HCHpyPCR)
5'-cgCttcacTaag tcT aca gac-3' ! C: BipI Probes from human HC GLGs
1 1-58, 1-03, 1-08, 1-69, 1-24, 1-45, 1-46, 1-f, 1-e
acatggaGCTGAGCagcctgag 2 1-02 acatggaGCTGAGCaggctqag 3 1-18
acatggagctgaggagcctgag 4 5-51, 5-a acctgcagtggagcagcctgaa 5 3-15,
3-73, 3-49, 3-72 atctgcaaatgaacagcctgaa 6
3303,3-33,3-07,3-11,3-30,3-21,3-23,3305,3-48 atctgcaaatgaacagcctgag
7 3-20,3-74,3-09,3-43 atctgcaaatgaacagtctgag 8 74.1
atctgcagatctgcagcctaaa 9 3-66, 3-13, 3-53, 3-d
atcttcaaatgaacagcctgag 10 3-64 atcttcaaatgggcagcctgag 11
4301,4-28,4302,4-04,4304,4-31,4-34,4-39,4-59,4-61,4-b
ccctgaaGCTGAGCtctgtgac 12 6-1 ccctgcagctgaactctgtgac 13 2-70,2-05
tccttacaatgaccaacatgga 14 2-26 tccttaccatgaccaacatgga D: BlpI
REdaptors, Extenders, and Bridges D.1 BEdaptors T.sub.m.sup.W
T.sub.m.sup.K (B1pF3HC1-58) 5'-ac atg gaG CTG AGC agcctg ag-3' 70
66. 4 (B1pF3HC6-1) 5'-cc ctg aag ctg agc tctgtg ac-3' 70 66. 4
B1pF3HC6-1 matches 4-30.1, not 6-1. D.2 Segment of synthetic 3-23
gene into which captured CDR3 is to be cloned ! Blp| ! ! XbaI . . .
... ... !D323* cgCttcacTaag TCT AGA gac aaC tcT aag aaT acT ctC taC
Ttg caG atg aac ! ! AtlII . . . ! agC TTA AGG D.3 Extender and
Bridges ! Bridges (BlpF3Br1) 5'-cgCttcacTcag tcT aga gaT aeC AGT
aaA aaT acT TtG- taC Ttg caG Ctg a|GC agc ctg-3' (BlpF3Br2)
5'-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG- taC Ttg caG
Ctg a|gc tct gtg-3' ! | lower strand is cut here ! Extender
(BlpF3Ext) 5'-
TcAgcTgcAAgTAcAAAgTATTTTTAcTgTTATcTcTAgAgAcTgAgTgAAgcg-3' !
BlpF3Ext is the reverse complement of: ! 5'-cgCttcacTcag tcT aga
gaT aaC AGT aaA aaT acT TtG taC Ttg caG Ctg a-3' ! (BlpF3PCR)
5'-cgCttcacTcag tcT aga gaT aaC-3' E: HpyCH4III Distinct GLG
sequences surrounding site, bases 77-98 1
102#118#4,146#7,169#9,1e#10,311#17,353#30,404#37,4301
ccgzqtattactgtgcgagaga 2
103#2,307#15,321#21,3303#24,333#26,348#28,364#31,366#32
ctgtgtattactgtgcgagaga 3 108#3 ccgtgtattactgtgcgagagg 4 124#5,
1f#11 ccgtgtattactgtgcaacaga 5 145#6 ccatgtattactgtgcaagata 158#8
ccgtgtattactgtgcggcaga 7 205#12 ccacatattactgtgcacacag 8 226#13
ccacatattactgtgcacggat 9 270#14 ccacgtattactgtgcacggat 10 309#16,
343#27 ccttgtattactgtgcaaaaga 11 313#18, 374#35, 61#50
ctgtgtattactgtgcaagaga 12 315#19 ccgtgtattactgtaccacaga 13 320#20
ccttgtatcactgtgcgagaga 14 323#22 ccgtatattactgtgcgaaaga 15 330#23,
3305#25 ctgtgtattactgtgcgaaaga 16 349#29 ccgtgtattactgtactagaga 17
372#33 ccgtgtattactgtgctagaga 18 373#34 ccgtgtattactgtactagaca 19
3d#36 ctgtgtattactgtaagaaaga 20 428#38 ccgtgtattactgtgcgagaaa 21
4302#40, 4304#41 ccgtgtattactgtgccagaga 22 439#44
ctgtgtattactgtgcgagaca 23 551#48 ccatgtattactgtgcgagaca 24 5a#49
ccatgtattactgtgcgaga F: HypCH4III PEdaptors, Extenders, and Bridges
F.1 PEdaptors ! ONs for cleavacc of HC(lower) in FR3(bases 77-97) !
For cleavacc with HpyCH4III, Bst4CI, or Taa| ! cleavage is in lower
chain before base 88. ! 77 788 888 888 889 999 999 9 ! 78 901 234
567 890 123 456 7 T.sub.m.sup.w T.sub.m.sup.k (H43.77.97.1-02#1)
5'-cc gtg tat tAC TGT gcg aga g-3' 6462.6 (H43.77.97.1-03#2) 5'-ct
gtg tat tAC TGT gcg aga g-3' 6260.6 (H43.77.97.108#3) 5'-cc gtg tat
tAC TGT gcg aga g-3' 6462.6 (H43.77.97.323#22) 5'-cc gta tat tac
tgt gcg aaa g-3' 6058.7 (H43.77.97.330#23) 5'-ct gtg tat tac tgt
gcg aaa g-3' 6058.7 (H43.77.97.439#44) 5'-ct gtg tat tac tgt gcg
aga c-3' 6260.6 (H43.77.97.551#48) 5'-cc atg tat tac tgt gcg aga
-3' 6260.6 (H43.77.97.5a#49) 5'-cc atg tat tAC TGT gcg aga -3'
5858.3 F.2 Extender and Bridges ! XbaI and Afill sites in bridges
are bunged (H43.XABr1) 5'-ggtgtagtga- |TCT|AGt|gac|aac|tct
|aag|aat|act|ctc|tac|ttg|cag|atg|-
|aac|agC|TTt|AGg|gct|gag|gac|aCT|GCA|Gtc|tac tat tgt gcg aga-3'
(H43.XABr2) 5'-ggtgtagtga- |TCT |AGt|gac|aac|tct
|aag|aat|act|ctc|tad ttg|cag|atg|-
|aac|agC|TTt|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat tgt gcg aaa-3'
(H43.XAExt) 5'-ATAgTAgAcT gcAgTgTccT cAgcccTTAA gcTgTTcATc
TgCAAgTAgA- gAgTATTcTT AgAgTTgTcT cTAgATcACT AcAcc-3' !H43.XAExt is
the reverse complement of ! 5'-ggtgtagtga- !
|TCT|AGA|gac|aac|tct|aag|aat|act|ctg|tac|ttg|cag|atg|- !
|aac|agC|TTt|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat-3' (H43.XAPCR)
5'-ggtgtagtga |TCT|AGA|gac|aac-3' ! XbaI and AflII sites in bridges
are bunged (H43.ABr1) 5'-ggtgtagtga-
|aac|agC|TTt|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat tgt gcg aga-3'
(H43.ABr2) 5'-ggtgtagtga-
|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat tgt gcg aaa-3'
(H43.AExt) 5'-ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTTcAcTAcAcc-3' !
(H43.AExt) is the reverse complement of 5'-ggtgtagtga- !
|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat-3' (H43.APCR)
5'-ggtgtagtga |aac|agC|TTA|AGg|gct|-3'
[0287] TABLE-US-00006 TABLE 5 Analysis of frequency of matching
REdaptors in actual V genes A: HpyCH4V in HC at bases 35-56 Number
of mismatches.....................Number Id Ntct 0 1 2 3 4 5 6 7 8
9 10 Cut Id Probe 1 510 5 11 274 92 61 25 22 11 1 3 5443 6-1
agttctcccTGCAgctgaactc 2 192 54 42 32 24 15 2 3 10 3 1 6 167 3-11
cactgtatcTGCAaatgaacag 3 58 19 7 17 6 5 1 0 1 0 2 0 543-09
ccctgtatcTGCAaatgaacag 4 267 42 33 9 8 8 82 43 22 8 11 1 100 5-51
ccgcctaccTGCAgtggagcag 5 250 111 59 41 24 7 5 1 0 0 2 0 242 3-15
cgctgtatcTGCAaatgaacag 6 7 0 2 0 1 0 0 0 0 0 4 0 3 74.1
cggcatatcTGCAgatctgcag 7 7 0 2 2 0 0 2 1 0 0 0 0 4 3-73
cggcgtatcTGCAaatgaacag 8 26 10 4 1 3 1 2 1 3 1 0 0 19 5-a
ctgcctaccTGCAgtggagcag 9 21 8 2 3 1 6 1 0 0 0 0 0 20 3-49
tcgcctatcTGCAaatgaacag 1338 249 162 379 149 103 120 71 47 13 23 12
1052 249 411 790 939 1162 1280 1316 1042 1233 1293 1338 Id Probe
dotted probe 6-1 agttctcccTGCAgctgaactc agttctcccTGCAgctgaactc 3-11
cactgtatcTGCAaatgaacag cac.g.at.....aa.....ag 3-09
ccctgtatcTGCAaatgaacag ccc.g.at.....aa.....ag 5-51
ccgcctaccTGCAgtggagcag ccgc..a.......tg..g.ag 3-15
cgctgtatcTGCAaatgaacag c.c.g.at.....aa.....ag 7-4.1
cggcatatcTGCAgatctgcag c.gca.at......a.ctg.ag 3-73
cggcgtatcTGCAaatgaacag c.gcg.at.....aa.....ag 5-a
ctgcctaccTGCAgtggaacag ctgc..a.......tg..g.ag 3-49
tcgcctatcTGCAaatgaacag tcgc..at.....aa.....ag Seqs with the
expected RE site only.......1004 (Counts only cases with 4 or fewer
mismatches) Seqs with only an unexpected site......... 0 Seqs with
both expected and unexpected.... 48 (Counts only cases with 4 or
fewer mismatches) Seqs with no sites........................ 0 B:
BlpI in HC Id Ntot 0 1 2 3 4 5 6 7 8 Ncut Name 1 133 73 16 11 13 6
9 1 4 0 119 1-58 acatggaGCTGAGCagcctgag 2 14 11 1 0 0 0 0 1 0 1 12
1-02 acatggagctgagcaggctgag 3 34 17 8 2 6 1 0 0 0 0 0 1-18
acatggagctgaggagcctgag 4 120 50 32 16 10 9 1 1 1 0 2 5-51
acctgcagtggagcagcctgaa 5 55 13 11 10 17 3 1 0 0 0 0 3-15
atctgcaaatgaacagcctgaa 6 340 186 88 41 15 6 3 0 1 0 0 3303
atctgcaaatgaacagcctgag 7 82 25 16 25 12 1 3 0 0 0 0 3-20
atctgcaaatgaacagtctgag 8 3 0 2 0 1 0 0 0 0 0 0 74.1
atctgcagatctgcagcctaaa 9 23 18 2 2 1 0 0 0 0 0 0 3-66
atcttcaaatgaacagcctgag 10 2 1 0 1 0 0 0 0 0 0 0 3-66
atcttcaaatgggcagcctgag 11 486 249 78 81 38 21 10 4 4 1 467 4301
ccctgaagctgagctctgtgac 12 16 6 3 1 0 1 1 3 1 0 1 6-1
ccctgcagctgaactctgtgac 13 28 15 8 2 2 1 0 0 0 0 0 2-70
tccttacaatgaccaacatgga 14 2 0 2 0 0 0 0 0 0 0 0 2-26
tccttaccatgaccaacatgga Name Full seguence Dot mode 1-58
acatggacCTGAGCagcctgag acatggaGCTGAGCagcctgag 1-02
acatggagctgagcaggctgag ...............g...... 1-18
acatggagctgaggagcctgag ............g......... 5-51
acctgcagtggagcagcctga& ..c..c..tg...........a 3-15
atctgcaaatgaacagcctgaa .tc..c.aa...a........a 3-30.3
atctgcaaatgaacagcctgag .tc..c.aa..a.......... 3-20
atctgcaaatgaacagtctgag .tc..c.aa...a...t..... 7-4.1
atctgcagatctgcagcctaaa .tc..c..a.ct.......a.a 3-66
atcttcaaatgaacagcctgag .tc.tc.aa...a......... 3-64
atcttcaaatgggcagcctgag .tc.tc.aa..g.......... 4-30.1
ccctgaagctgagctctgtgacc c.c..a........tctg...c 6-1
ccctgcagctgaactctgtgac c.c..c......a.tctg...c 2-70
tccttacaatgaccaacatggat t.c.tacaa...c..a.a..ga 2-26
tccttaccatgaccaacatggat t.c.tacca...c..a.a..ga Seqs with the
expected RE site only....... 597 (counting seguences with 4 or
fewer mismatches) Seqs with only an unexpected site......... 2 Seqs
with both expected and unexpected.... 2 Seqs with no
sites........................ 686 C. HpyCH4III, Bst4CI, or TaaI in
HC In scoring whether the RE site of interest is present, only ONs
that have 4 or fewer mismatches are counted Number of
sequences.......... 1617 Id Ntot 0 1 2 3 4 5 6 7 8 Ncut acngt acngt
1 244 78 92 43 18 10 1 2 0 0 241 102#1,1 ccgtgtattACTGTgcgagaga
ccgtgtattactgtgcgagaga 2 457 69 150 115 66 34 11 8 3 1 434 103#2,3
ctgtgtattactgtgcgagaga .t.................... 3 173 52 45 36 22 14
3 0 0 1 169 108#3 ccgtgtattactgtgcgagagg .....................g 4
16 0 3 2 2 1 6 0 1 1 8 124#5,1 ccgtgtattactgtgcaacaga
.................a.c.. 5 4 0 0 1 0 1 1 0 1 0 2 145#6
ccatgtattactgtgcaagata ..a..............a...t 6 15 1 0 1 0 6 4 1 1
1 8 158#8 ccgtgtattactgtgcggcaga ................gc.... 7 23 4 8 5
2 2 1 1 0 0 21 205#12 ccacatattactgtgcacacag ..aca...........acacag
8 9 1 1 1 0 3 2 1 0 0 6 226#13 ccacatattactgtgcacggat
..aca...........ac.gat 9 7 1 3 1 1 0 0 1 0 0 6 270#14
ccacgtattactgtgacggat ..ac............ac.gat 10 23 7 3 5 5 2 1 0 0
0 22 309#16, ccttgtattactgtgcaaaaga ..t.............a.a... 11 35 5
10 7 6 3 3 0 1 0 31 313#18, ctgtgtattactgtgcaagaga
.t..............a..... 12 18 2 3 2 2 6 1 0 2 0 15 315#19
ccgtgtattactgtaccacaga ..............a.c.c... 13 3 1 2 0 0 0 0 0 0
0 3 320#20 ccttgtatcactgtgcgagaga ..t......c............ 14 117 29
23 28 22 8 4 2 1 0 110 323#22 ccgtatattacttggcgaaaga
.....a................ 15 75 21 25 13 9 1 4 2 0 0 69 330#23,
ctgtgtattactgtgcgaaaga .t...............a.... 16 14 2 2 2 3 0 3 1 1
0 9 349#29 ccgtgtattactgtactagaga ..............a.t..... 17 2 0 0 1
0 0 1 0 0 0 1 372#33 ccgtgtattactgtgctagaga .................t....
18 1 0 0 1 0 0 0 0 0 0 1 373#34 ccgtgtattactgtaagaaaga
...............aa..a.. 19 2 0 0 0 0 0 0 0 0 2 0 3d#36
ctgtgtattactgtaagaaaga .t...........aa..a.... 20 34 4 9 9 4 5 3 0 0
0 31 428#38 ccgtgtattactgtgcgagaaa .....................a 21 17 5 4
2 2 3 1 0 0 0 16 4302#40 ccgtgtattactgtgcgagaca
................c..... 22 75 15 17 24 7 10 1 1 0 0 73 439#44
ctgtgtattactgtgcgagaca .t..................c. 23 40 14 15 4 5 1 0 1
0 0 39 551#48 ccatgtattactgtgagagaca ..a.................c. 24 213
26 56 60 42 20 7 2 0 0 204 5a#49 ccatgtattactgtgcgagaAA
..a.................AA Group 337 471 363 218 130 58 23 11 6
Cumulative 337 808 1171 1389 1519 1577 1600 1611 1617 Seqs with the
expected RE site only.......1511 Seqs with only en unexpected
site......... 0 Seqs with both expected and unexpected.... 8 Seqs
with no sites........................ 0
[0288] TABLE-US-00007 TABLE 5D Analysis repeated using only 8 best
REdaptors Id Ntot 0 1 2 3 4 5 6 7 8+ 1 301 78 101 54 32 16 9 10 1 0
281 102#1 ccgtgtattactgtgcgagaga 2 493 69 155 125 73 37 14 11 3 6
459 103#2 ctgtgtattactgtgcgagaga 3 189 52 45 38 23 18 5 4 1 3 176
108#3 ccgtgtattactgtgcgagagg 4 127 29 23 28 24 10 6 5 2 0 114
323#22 ccgtatattactgtgcgaaaga 5 78 21 25 14 11 1 4 2 0 0 72 330#23
ctgtgtattactgtgcgaaaga 6 79 15 17 25 8 11 1 2 0 0 76 439#44
ctgtgtattactgtgcgagaca 7 43 14 15 5 5 3 0 1 0 0 42 551#48
ccatgtattactgtgcgagaca 8 307 26 63 72 51 38 24 14 13 6 250 5a#49
ccatgtattactgtgcgaga 1 102#1 ccgtgtattactgtgcgagaga
ccgtgtattactgtgcgagaga 2 103#2 ctgtgtattactgtgcgagaga
.t.................... 3 108#3 ccgtgtattactgtgcgagagg
.....................g 4 323#22 ccgtatattactgtgcgaaaga
....a.............a... 5 330#23 ctgtgtattactgtgcgaaaga
.t.................a.. 6 439#44 ctgtgtattactgtgcgagaca
.t..................c. 7 551#48 ccatgtattactgtgcgagaca
..a.................c. 8 5a#49 ccatgtattactgtgcgagaAA
..a.................AA Seqs with the expected RE site
only.......1463/1617 Seqs with only an unexpected site......... 0
Seqs with both expected and unexpected.... 7 Seqs with no
sites........................ 0
[0289] TABLE-US-00008 TABLE 6 Human HC GLG FR1 Seguences VH
Exon--Nucleotide sequence alignment VH1 1-02 CAG GTG CAG CTG GTG
CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC TCA GTG AAG GTC TCC TGC
AAG GCT TCT GGA TAC ACC TTC ACC 1-03 cag gtC cag ctT gtg cag tct
ggg gct gag gtg aag aag cct ggg gcc toa gtg aag gtT tcc tgc aag gct
tct gga tac aoc ttc acT 1-08 cag gtg cag ctg gtg cag tct ggg gct
gag gtg aag aag cct ggg gcc tca gtg aag gtc tcc tgc aag gct tct gga
tac acc ttc acc 1-18 cag gtT cag ctg gtg cag tct ggA gct gag gtg
aag aag cct ggg gcc tca gtg aag gtc tcc tgc aag gct tct ggT tac acc
ttT acc 1-24 cag gtC cag ctg gtA cag tct ggg gct gag gtg aag aag
cct ggg gcc tca gtg aag gtc tcc tgc aag gTt tcC gga tac acc Ctc acT
1-45 cag Atg cag ctg gtg cag tct ggg gct gag gtg aag aag Act ggg
Tcc toa gtg aag gtT tcc tgc aag gct tcC gga tac acc ttc acc 1-46
cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg gcc tca
gtg aag gtT tcc tgc aag gcA tct gga tac acc ttc acc 1-58 caA Atg
cag ctg gtg cag tct ggg Cct gag gtg aag aag cct ggg Acc tca gtg aag
gtc tcc tgc aag gct tct gga tTc acc ttT acT 1-69 cag gtg cag ctg
gtg cag tct ggg gct gag gtg aag aag cct ggg Tcc tcG gtg aag gtc tcc
tgc aag gct tct gga GGo acc ttc aGc 1-e cag gtg cag ctg gtg cag tct
ggg gct gag gtg aag aag cct ggg Tcc tcG gtg aag gtc tcc tgc aag gct
tct gga GGc acc ttc aGc 1-f Gag gtC cag ctg gtA cag tct ggg gct gag
gtg aag aag cct ggg gcT Aca gtg aaA Atc tcc tgc aag gTt tct gga tac
acc ttc acc VH2 2-05 CAG ATC ACC TTG AAG GAG TCT GGT CCT ACG CTG
GTG AAA CCC ACA CAG ACC CTC ACG CTG ACC TGC ACC TTC TCT GGG TTC TCA
CTC AGC 2-26 cag Gtc acc ttg aag gag tct ggt cct GTg ctg gtg aaa
ccc aca Gag acc ctc acg ctg acc tgc acc Gtc tct ggg ttc tca ctc agc
2-70 cag Gtc acc ttg aag gag tct ggt cct Gcg ctg gtg aaa ccc aca
cag acc ctc acA ctg acc tgc acc ttc tct ggg ttc tca ctc agc VH3
3-07 GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC TTG GTC CAG CCT GGG
GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TTT AGT 3-09
gaA gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggC Agg tcc
ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt GAt 3-11 Cag gtg
cag ctg gtg gag tct ggg gga ggc ttg gtc Aag cct ggA ggg tcc ctg aga
ctc tcc tgt gca gcc tct gga ttc acc ttC agt 3-13 gag gtg cag ctg
gtg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tcc ctg aga ctc tcc
tgt gca gcc tct gga ttc acc ttC agt 3-15 gag gtg cag ctg gtg gag
tct ggg gga ggc ttg gtA Aag cct ggg ggg tcc otT aga ctc tcc tgt gca
gcc tct gga ttc acT ttC agt 3-20 gag gtg cag ctg gtg gag tct ggg
gga ggT Gtg gtA cGg cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct
gga ttc acc ttt GAt 3-21 gag gtg cag ctg gtg gag tct ggg gga ggc
Ctg gtc Aag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc
acc ttC agt 3-23 gag gtg cag ctg Ttg gag tct ggg gga ggc ttg gTA
cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt
agC 3-30 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct
ggg Agg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt
3-30.3 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg
Agg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 3-30.5
Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc
ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 3-33 Cag gtg
cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga
ctc tcc tgt gca gcG tct gga ttc acc ttC agt 3-43 gaA gtg cag ctg
gtg gag tct ggg gga gTc Gtg gtA cag cct ggg ggg tcc ctg aga ctc tcc
tgt gca gcc tct gga ttc acc ttt GAt 3-48 gag gtg cag ctg gtg gag
tct ggg gga ggc ttg gtA cag cct ggg ggg tcc ctg aga ctc tcc tgt gca
gcc tct gga ttc acc ttC agt 3-49 gag gtg cag ctg gtg gag tct ggg
gga ggc ttg gtA cag ccA ggg Cgg tcc ctg aga ctc tcc tgt Aca gcT tct
gga ttc acc ttt Ggt 3-53 gag gtg cag ctg gtg gag Act ggA gga ggc
ttg Atc cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct ggG ttc
acc GtC agt 3-64 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc
cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC
agt 3-66 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct
ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc Gtc agt
3-72 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggA
ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttC agt 3-73
gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg tcc
ctg aAa ctc tcc tgt gca gcc tct ggGttc acc ttC agt 3-74 gag gtg cag
ctg gtg gag tcC ggg gga ggc ttA gtT cag cct ggg ggg tcc ctg aga ctc
tcc tgt gca gcc tct gga ttc acc ttC agt 3-d gag gtg cag ctg gtg gag
tct Cgg gga gTc ttg gtA cag cct ggg ggg tcc ctg aga ctc tcc tgt gca
gcc tct gga ttc acc GtC agt VH4 4-04 CAG GTG CAG CTG CAG GAG TCG
GGC CCA GGA CTG GTG AAG CCT TCG GGG ACC CTG TCC CTC ACC TGC GCT GTC
TCT GGT GGC TCC ATC AGC 4-28 cag gtg cag ctg cag gag tcg ggc cca
gga ctg gtg aag cct tcg gAC acc ctg tcc ctc acc tgc gct gtc tct ggt
TAc tcc atc agc 4-30.1 cag gtg cag ctg cag gag tcg ggc cca gga ctg
gtg aag cct tcA CAg acc ctg tcc ctc acc tgc Act gtc tct ggt ggc tcc
atc agc 4-30.2 cag Ctg cag ctg cag gag tcC ggc Tca gga ctg gtg aag
cct tcA CAg acc ctg tcc ctc acc tgc gct gtc tct ggt ggc tcc atc agc
4-30.4 cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA
CAg acc ctg tcc ctc acc tgc Act gtc tct ggt ggc tcc atc agc 4-31
cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA CAg acc
ctg tcc ctc acc tgc Act gtc tct ggt ggc tcc atc agc 4-34 cag gtg
cag ctA cag Cag tGg ggc Gca gga ctg Ttg aag cct tcg gAg acc ctg tcc
ctc acc tgc gct gtc tAt ggt ggG tcc Ttc agT 4-39 cag Ctg cag ctg
cag gag tcg ggc cca gga ctg gtg aag cct tct gAg acc ctg tcc ctc acc
tgc Act gtc tct ggt ggc tcc atc agt 4-59 cag gtg cag ctg cag gag
tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc ctc acc tgc Act
gtc tct ggt ggc tcc atc agT 4-61 cag gtg cag ctg cag gag tcg ggc
cca gga ctg gtg aag cct tcg gAg acc ctg tcc ctc acc tgc Act gtc tct
ggt ggc tcc Gtc agc 4-b cag gtg cag ctg cag gag tcg ggc cca gga ctg
gtg aag cct tcg gAg acc ctg tcc ctc acc tgc gct gtc tct ggt TAc tcc
atc agc VH5 5-51 GAG GTG CAG CTG GTG CAG TCT GGA GCA GAG GTG AAA
AAG CCC GGG GAG TCT CTG AAG ATC TCC TGT AAG GGT TCT GGA TAC AGC TTT
ACC 5-a gaA gtg cag ctg gtg cag tct gga gca gag gtg aas aag ccc ggg
gag tct ctg aGg atc tcc tgt aag ggt tct gga tac agc ttt acc VH6 6-1
CAG GTA CAG CTG CAG CAG TCA GGT CCA GGA CTG GTG AAG CCC TCG CAG ACC
CTC TCA CTC ACC TGT GCC ATC TCC GGG GAC AGT GTC TCT VH7 7-4.1 CAG
GTG CAG CTG GTG CAA TCT GGG TCT GAG TTG AAG AAG CCT GGG GCC TCA GTG
AAG GTT TCC TGC AAG GCT TCT GGA TAC ACC TTC ACT
[0290] TABLE-US-00009 TABLE 7 RERS sites in Human HC GLG FR1s where
there are at least 20 GLGs cut BsgI GTGCAG 71 (cuts 16/14 bases to
right) 1:4 1:13 2:13 3:4 3:13 4:13 6:13 7:4 7:13 8:13 9:4 9:13 10:4
10:13 15:4 15:65 16:4 16:65 17:4 17:65 18:4 18:65 19:4 19:65 20:4
20:65 21:4 21:65 22:4 22:65 23:4 23:65 24:4 24:65 25:4 25:65 26:4
26:65 27:4 27:65 28:4 28:65 29:4 30:4 30:65 31:4 31:65 32:4 32:65
33:4 33:65 34:4 34:65 35:4 35:65 36:4 36:65 37:4 38:4 39:4 41:4
42:4 43:4 45:4 46:4 47:4 48:4 48:13 49:4 49:13 51:4 There are 39
hits at base# 4 There are 21 hits at base# 65 -''- ctgcac 9 12:63
13:63 14:63 39:63 41:63 42:63 44:63 45:63 46:63 BbvI GCAGC 65 1:6
3:6 6:6 7:6 8:6 9:6 10:6 15:6 15:67 16:6 16:67 17:6 17:67 18:6
18:67 19:6 19:67 20:6 20:67 21:6 21:67 22:6 22:67 23:6 23:67 24:6
24:67 25:6 25:67 26:6 26:67 27:6 27:67 28:6 28:67 29:6 30:6 30:67
31:6 31:67 32:6 32:67 33:6 33:67 34:6 34:67 35:6 35:67 36:6 36:67
37:6 38:6 39:6 40:6 41:6 42:6 43:6 44:6 45:6 46:6 47:6 48:6 49:6
50:12 51:6 There are 43 hits at base# 6 Bolded sites very near
sites listed below There are 21 hits at base# 67 -''- gctgc 13 37:9
38:9 39:9 40:3 40:9 41:9 42:9 44:3 44:9 45:9 46:9 47:9 50:9 There
are 11 hits at base# 9 BsoFI GCngc 78 1:6 3:6 6:6 7:6 8:6 9:6 10:6
15:6 15:67 16:6 16:67 17:6 17:67 18:6 18:67 19:6 19:67 20:6 20:67
21:6 21:67 22:6 22:67 23:6 23:67 24:6 24:67 25:6 25:67 26:6 26:67
27:6 27:67 28:6 28:67 29:6 30:6 30:67 31:6 31:67 32:6 32:67 33:6
33:67 34:6 34:67 35:6 35:67 36:6 36:67 37:6 37:9 38:6 38:9 39:6
39:9 40:3 40:6 40:9 41:6 41:9 42:6 42:9 43:6 44:3 44:6 44:9 45:6
45:9 46:6 46:9 47:6 47:9 48:6 49:6 50:9 50:12 51:6 There are 43
hits at base# 6 These often occur together. There are 11 hits at
base# 9 There are 2 hits at base# 3 There are 21 hits at base# 67
TseI Gcwgc 78 1:6 3:6 6:6 7:6 8:6 9:6 10:6 15:6 15:67 16:6 16:67
17:6 17:67 18:6 18:67 19:6 19:67 20:6 20:67 21:6 21:67 22:6 22:67
23:6 23:67 24:6 24:67 25:6 25:67 26:6 26:67 27:6 27:67 28:6 28:67
29:6 30:6 30:67 31:6 31:67 32:6 32:67 33:6 33:67 34:6 34:67 35:6
35:67 36:6 36:67 37:6 37:9 38:6 38:9 39:6 39:9 40:3 40:6 40:9 41:6
41:9 42:6 42:9 43:6 44:3 44:6 44:9 45:6 45:9 46:6 46:9 47:6 47:9
48:6 49:6 50:9 50:12 51:6 There are 43 hits at base# 6 Often
together. There are 11 hits at base# 9 There are 2 hits at base# 3
There are 1 hits at base# 12 There are 21 hits at base# 67 MspA1I
CMGckg 48 1:7 3:7 4:7 5:7 6:7 7:7 8:7 9:7 10:7 11:7 15:7 16:7 17:7
18:7 19:7 20:7 21:7 22:7 23:7 24:7 25:7 26:7 27:7 28:7 29:7 30:7
31:7 32:7 33:7 34:7 35:7 36:7 37:7 38:7 39:7 40:1 40:7 41:7 42:7
44:1 44:7 45:7 46:7 47:7 48:7 49:7 50:7 51:7 There are 46 hits at
base# 7 PvuII CAGctg 48 1:7 3:7 4:7 5:7 6:7 7:7 8:7 9:7 10:7 11:7
15:7 16:7 17:7 18:7 19:7 20:7 21:7 22:7 23:7 24:7 25:7 26:7 27:7
28:7 29:7 30:7 31:7 32:7 33:7 34:7 35:7 36:7 37:7 38:7 39:7 40:1
40:7 41:7 42:7 44:1 44:7 45:7 46:7 47:7 48:7 49:7 50:7 51:7 There
are 46 hits at base# 7 There are 2 hits at base# 1 AluI AGct 54 1:8
2:8 3:8 4:8 4:24 5:8 6:8 7:8 8:8 9:8 10:8 11:8 15:8 16:8 17:8 18:8
19:8 20:8 21:8 22:8 23:8 24:8 25:8 26:8 27:8 28:8 29:8 29:69 30:8
31:8 32:8 33:8 34:8 35:8 36:8 37:8 38:8 39:8 40:2 40:8 41:8 42:8
43:8 44:2 44:8 45:8 46:8 47:8 48:8 48:82 49:8 49:82 50:8 51:8
Threre are 48 hits at base# 8 There are 2 hits at base# 2 DdeI
Ctnag 48 1:26 1:48 2:26 2:48 3:26 3:48 4:26 4:48 5:26 5:48 6:26
6:48 7:26 7:48 8:26 8:48 9:26 10:26 11:26 12:85 13:85 14:85 15:52
16:52 17:52 18:52 19:52 20:52 21:52 22:52 23:52 24:52 25:52 26:52
27:52 28:52 29:52 30:52 31:52 32:52 33:52 35:30 35:52 36:52 40:24
49:52 51:26 51:48 There are 22 hits at base# 52 52 and 48 never
together. There are 9 hits at base# 48 There are 12 hits at base#
26 26 and 24 never together. HphI tcacc 42 1:86 3:86 6:86 7:86 8:80
11:86 12:5 13:5 14:5 15:80 16:80 17:80 18:80 20:80 21:80 22:80
23:80 24:80 25:80 26:80 27:80 28:80 29:80 30:80 31:80 32:80 33:80
34:80 35:80 36:80 37:59 38:59 39:59 40:59 41:59 42:59 43:59 44:59
45:59 46:59 47:59 50:59 There are 22 hits at base# 80 80 and 86
never together There are 5 hits at base# 86 There are 12 hits at
base# 59 BssKI Nccngg 50 1:39 2:39 3:39 4:39 5:39 7:39 8:39 9:39
10:39 11:39 15:39 16:39 17:39 18:39 19:39 20:39 21:29 21:39 22:39
23:39 24:39 25:39 26:39 27:39 28:39 29:39 30:39 31:39 32:39 33:39
34:39 35:19 35:39 36:39 37:24 38:24 39:24 41:24 42:24 44:24 45:24
46:24 47:24 48:39 48:40 49:39 49:40 50:24 50:73 51:39 There are 35
hits at base# 39 39 and 40 together twice. There are 2 hits at
base# 40 BsaJI Ccnngg 47 1:40 2:40 3:40 4:40 5:40 7:40 8:40 9:40
9:47 10:40 10:47 11:40 15:40 18:40 19:40 20:40 21:40 22:40 23:40
24:40 25:40 26:40 27:40 28:40 29:40 30:40 31:40 32:40 34:40 35:20
35:40 36:40 37:24 38:24 39:24 41:24 42:24 44:24 45:24 46:24 47:24
48:40 48:41 49:40 49:41 50:74 51:40 There are 32 hits at base# 40
40 and 41 together twice There are 2 hits at base# 41 There are 9
hits at base# 24 There are 2 hits at base# 47 BstNI CCwgg 44 PspGI
ccwgg ScrFI ($M.HpaII) CCwgg 1:40 2:40 3:40 4:40 5:40 7:40 8:40
9:40 10:40 11:40 15:40 16:40 17:40 18:40 19:40 20:40 21:30 21:40
22:40 23:40 24:40 25:40 26:40 27:40 28:40 29:40 30:40 31:40 32:40
33:40 34:40 35:40 36:40 37:25 38:25 39:25 41:25 42:25 44:25 45:25
46:25 47:25 50:25 51:40 There are 33 hits at base# 40 ScrFI CCngg
50 1:40 2:40 3:40 4:40 5:40 7:40 8:40 9:40 10:40 11:40 15:40 16:40
17:40 18:40 19:40 20:40 21:30 21:40 22:40 23:40 24:40 25:40 26:40
27:40 28:40 29:40 30:40 31:40 32:40 33:40 34:40 35:20 35:40 36:40
37:25 38:25 39:25 41:25 42:25 44:25 45:25 46:25 47:25 48:40 48:41
49:40 49:41 50:25 50:74 51:40 There are 35 hits at base# 40 There
are 2 hits at base# 41 EcoO109I RGgnccy 34 1:43 2:43 3:43 4:43 5:43
6:43 7:43 8:43 9:43 10:43 15:46 16:46 17:46 18:46 19:46 20:46 21:46
22:46 23:46 24:46 25:46 26:46 27:46 28:46 30:46 31:46 32:46 33:46
34:46 35:46 36:46 37:46 43:79 51:43 There are 22 hits at base# 46
46 and 43 never together There are 11 hits at base# 43 NlaIV GGNncc
71 1:43 2:43 3:43 4:43 5:43 6:43 7:43 8:43 9:43 9:79 10:43 10:79
15:46 15:47 16:47 17:46 17:47 18:46 18:47 19:46 19:47 20:46 20:47
21:46 21:47 22:46 22:47 23:47 24:47 25:47 26:47 27:46 27:47 28:46
28:47 29:47 30:46 30:47 31:46 31:47 32:46 32:47 33:46 33:47 34:46
34:47 35:46 35:47 36:46 36:47 37:21 37:46 37:47 37:79 38:21 39:21
39:79 40:79 41:21 41:79 42:21 42:79 43:79 44:21 44:79 45:21 45:79
46:21 46:79 47:21 51:43 There are 23 hits at base# 47 46 & 47
often together There are 17 hits at base# 46 There are 11 hits at
base# 43 Sau96I Ggncc 70 1:44 2:3 2:44 3:44 4:44 5:3 5:44 6:44 7:44
8:22 8:44 9:44 10:44 11:3 12:22 13:22 14:22 15:33 15:47 16:47 17:47
18:47 19:47 20:47 21:47 22:47 23:33 23:47 24:33 24:47 25:33 25:47
26:33 26:47 27:47 28:47 29:47 30:47 31:33 31:47 32:33 32:47 33:33
33:47 34:33 34:47 35:47 36:47 37:21 37:22 37:47 38:21 38:22 39:21
39:22 41:21 41:22 42:21 42:22 43:80 44:21 44:22 45:21 45:22 46:21
46:22 47:21 47:22 50:22 51:44 There are 23 hits at base# 47 These
do not occur together. There are 11 hits at base# 44 There are 14
hits at base# 22 These do occur together. There are 9 hits at base#
21 BsmAI GTCTCNnnnn 22 1:58 3:58 4:58 5:58 8:58 9:58 10:58 13:70
36:18 37:70 38:70 39:70 40:70 41:70 42:70 44:70 45:70 46:70 47:70
48:48 49:48 50:85 There are 11 hits at base# 70 -"- Nnnnnngagac 27
13:40 15:48 16:48 17:48 18:48 20:48 21:48 22:48 23:48 24:48 25:48
26:48 27:48 28:48 29:48 30:10 30:48 31:48 32:48 33:48 35:48 36:48
43:40 44:40 45:40 46:40 47:40 There are 20 hits at base# 48 AvaII
Ggwcc 44 Sau96I ($M.HaeIII) 44 Ggwcc 2:3 5:3 6:44 8:44 9:44 10:44
11:3 12:22 13:22 14:22 15:33 15:47 16:47 17:47 18:47 19:47 20:47
21:47 22:47 23:33 23:47 24:33 24:47 25:33 25:47 26:33 26:47 27:47
28:47 29:47 30:47 31:33 31:47 32:33 32:47 33:33 33:47 34:33 34:47
35:47 36:47 37:47 43:80 50:22 There are 23 hits at base# 47 44
& 47 never together There are 4 hits at base# 44
PpuMI RGgwccy 27 6:43 8:43 9:43 10:43 15:46 16:46 17:46 18:46 19:46
20:46 21:46 22:46 23:46 24:46 25:46 26:46 27:46 28:46 30:46 31:46
32:46 33:46 34:46 35:46 36:46 37:46 43:79 There are 22 hits at
base# 46 43 and 46 never occur together. There are 4 hits at base#
43 BsmFI GGGAC 3 8:43 37:46 50:77 -''- gtccc 33 15:48 16:48 17:48
1:0 1:0 20:48 21:48 22:48 23:48 24:48 25:48 26:48 27:48 28:48 29:48
30:48 31:48 32:48 33:48 34:48 35:48 36:48 37:54 38:54 39:54 40:54
41:54 42:54 43:54 44:54 45:54 46:54 47:54 There are 20 hits at
base# 48 There are 11 hits at base# 54 HinfI Gantc 80 8:77 12:16
13:16 14:16 15:16 15:56 15:77 16:16 16:56 16:77 17:16 17:56 17:77
18:16 18:56 18:77 19:16 19:56 19:77 20:16 20:56 20:77 21:16 21:56
21:77 22:16 22:56 22:77 23:16 23:56 23:77 24:16 24:56 24:77 25:16
25:56 25:77 26:16 26:56 26:77 27:16 27:26 27:56 27:77 28:16 28:56
28:77 29:16 29:56 29:77 30:56 31:16 31:56 31:77 32:16 32:56 32:77
33:16 33:56 33:77 34:16 35:16 35:56 35:77 36:16 36:26 36:56 36:77
37:16 38:16 39:16 40:16 41:16 42:16 44:16 45:16 46:16 47:16 48:46
49:46 There are 34 hits at base# 16 TfiI Gawtc 21 8:77 15:77 16:77
17:77 18:77 19:77 20:77 21:77 22:77 23:77 24:77 25:77 26:77 27:77
28:77 29:77 31:77 32:77 33:77 35:77 36:77 There are 21 hits at
base# 77 MlyI GAGTC 38 12:16 13:16 14:16 15:16 16:16 17:16 18:16
19:16 20:16 21:16 22:16 23:16 24:16 25:16 26:16 27:16 27:26 28:16
29:16 31:16 32:16 33:16 34:16 35:16 36:16 36:26 37:16 38:16 39:16
40:16 41:16 42:16 44:16 45:16 46:16 47:16 48:46 49:46 There are 34
hits at base# 16 -''- GACTC 21 15:56 16:56 17:56 18:56 19:56 20:56
21:56 22:56 23:56 24:56 25:56 26:56 27:56 28:56 29:56 30:56 31:56
32:56 33:56 35:56 36:56 There are 21 hits at base# 56 PleI gagtc 38
12:16 13:16 14:16 15:16 16:16 17:16 18:16 19:16 20:16 21:16 22:16
23:16 24:16 25:16 26:16 27:16 27:26 28:16 29:16 31:16 32:16 33:16
34:16 35:16 36:16 36:26 37:16 38:16 39:16 40:16 41:16 42:16 44:16
45:16 46:16 47:16 48:46 49:46 There are 34 hits at base# 16 -''
-gactc 21 15:56 16:56 17:56 18:56 19:56 20:56 21:56 22:56 23:56
24:56 25:56 26:56 27:56 28:56 29:56 30:56 31:56 32:56 33:56 35:56
36:56 There are 21 hits at base# 56 AlwNI CAGNNNctg 26 15:68 16:68
17:68 18:68 19:68 20:68 21:68 22:68 23:68 24:68 25:68 26:68 27:68
28:68 29:68 30:68 31:68 32:68 33:68 34:68 35:68 36:68 39:46 40:46
41:46 42:46 There are 22 hits at base# 68
[0291] TABLE-US-00010 TABLE 8 Kappa FR1 GLGs ! 1 2 3 4 5 6 7 8 9 11
12 GAC ATC CAG ATG ACC CAG TCT CCA TCC TGC CTG TCT ! 13 14 15 16 17
18 19 20 21 22 23 GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! O12
GAC ATC CAG ATG ACC CAG TCT CCA TGC TGC CTG TCT GCA TCT GTA GGA GAC
AGA GTC ACC ATC ACT TGC ! O2 GAC ATC CAG ATG ACC CAG TCT CCA TGC
TGC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ACC ACT TGC ! O18 GAC
ATC CAG ATG ACC CAG TCT CCA TGC TGC CTG TCT GCA TCT GTA GGA GAC AGA
GTC ACC ATC ACT TGC ! O8 GAC ATC CAG ATG ACC CAG TCT CCA TGC TGC
CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! A20 GAC ATC
CAG ATG ACC CAG TCT CCA TGC TGC CTG TCT GCA TCT GTA GGA GAC AGA GTC
ACC ATC ACT TGC ! A30 AAC ATC CAG ATG ACC CAG TCT CCA TCT GCC ATG
TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT ! L14 GAC ATC CAG
ATG ACC CAG TCT CCA TGC TCA CTG TCT CCA TCT GTA GGA GAC AGA GTC ACC
ATC ACT TGT ! L1 GAC ATC CAG ATC ACC CAG TCT CCA TGC TCA GTC TCT
CCA TCT CTA GGA GAC AGA GTC ACC ATC ACT TGT ! L15 GCC ATC CAG TTG
ACC CAG TCT CCA TGC TGC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC
ACT TGC ! L4 GCC ATC CAG TTG ACC CAG TCT CCA TGC TGC CTG TCT GCA
TCT CTA GGA GAC AGA GTC ACC ATC ACT TGC ! L18 GAC ATC CAG ATG ACC
CAG TCT CCA TCT TGC GTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT
TCT ! L5 GAC ATC CAG ATG ACC CAG TCT CCA TCT TCT GTG TCT CCA TCT
CTA CGA GAC ACA GTC ACC ATC ACT TGT ! L19 GAC ATC CAG TTG ACC CAG
TCT CCA TGC TTC GTC TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC
! L8 GAC ATC CCG ATC ACC CAG TCT CCA TTC TGC GTC TCT CCA TCT CTA
CGA GAC AGA GTC ACC ATC ACT TGC ! L23 GCC ATC CGG ATC ACC CAG TCT
CCA TCC TCA TTC TCT GCA TCT ACA GGA GAC AGA GTC ACC ATC ACT TCT !
L9 GTC ATC TGG ATG ACC CAG TCT CCA TGC TTA GTC TCT GCA TCT ACA GGA
GAC AGA GTC ACC ATC AGT TGT ! L24 GCC ATC CAG ATG ACC CAG TCT CCA
TGC TGC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! L11
GAC ATC CAG ATG ACC CAG TCT CCT TGC ACC CTG TCT GCA TCT GTA GGA GAC
AGA GTC ACC ATC ACT TGC ! L12 GAT ATT GTG ATG ACC CAG ACT CCA GTC
TGC CTG CCC GTC ACC CCT GGA GAG CCG GCC TGC ATC TGC TGC ! O11 GAT
ATT GTG ATG ACC CAG ACT CCA GTC TGC CTG CCC GTC ACC CCT GGA GAG CCG
GCC TGC ATC TGC TGC ! O1 GAT GTT GTG ATG ACT CAG TCT CCA GTC TGC
CTG CCC GTC ACC CTT GGA CAG CCG GCC TGC ATC TGC TGC ! A17 GAT GTT
GTG ATG ACT CAG TCT CCA GTC TGC CTG CCC GTC ACC CTT GGA CAG CCG GCC
TGC ATC TGC TGC ! A1 GAT ATT GTG ATG ACC CAG ACT CCA GTC TCT CTG
TGC GTC ACC CCT GGA CAG CCG GCC TGC ATC TGC TGC ! A18 GAT ATT GTG
ATG ACC CAG ACT CCA GTC TCT GTC TGC GTC ACC CCT GGA CAG CCG GCC TGC
ATC TGC TGC ! A2 GAT ATT GTG ATG ACT CAG TCT CCA GTC TGC CTG CCC
GTC ACC CCT GGA GAG CCG GCC TGC ATC TGC TGC ! A19 GAT ATT GTG ATG
ACT CAG TCT CCA GTC TGC CTG CCC GTC ACC CCT GGA GAG CCG GCC TGC ATC
TGC TGC ! A3 GAT ATT GTG ATG ACC CAG ACT CCA GTC TGC TCA CCT GTC
ACC CTT GGA CAG CCG GCC TGC ATC TGC TGC ! A23 GAA ATT GTG TTG ACG
CAG TCT CCA GGC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC GTC TGC
TGC ! A27 GAA ATT GTG TTG ACG CAG TCT CCA GCC ACC CTG TCT TTG TCT
CCA GGG GAA AGA GCC ACC GTC TGC TGC ! A11 GAA ATA GTG ATG ACG CAG
TCT CCA GCC ACC CTG TCT GTG TCT CCA GGG GAA AGA GCC ACC GTC TGC TGC
! L2 GAA ATA GTG ATG ACG CAG TCT CCA CCC ACC CTG TCT GTG TCT CCA
GGG GAA AGA CCC ACC GTC TGC TGC ! L16 GAA ATT GTG TTG ACA CAG TCT
CCA CCC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC GTC TGC TGC !
L6 GAA ATT GTG TTG ACA CAG TCT CCA CCC ACC GTC TCT TTG TCT CCA GGG
GAA AGA GCC ACC GTC TGC TGC ! L20 GAA ATT GTA ATG ACA CAG TCT CCA
GCC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC GTC TGC TGC ! L25
GAC ATC GTG ATG ACC CAG TCT CCA GAC TGC CTG GCT GTG TCT CTG GGC GAG
AGG GCC ACC ATC AAC TGC ! B3 GAA ACG ACA GTC ACG CAG TCT CCA GCA
TTC ATG TCA GCG ACT CCA GGA GAC AAA GTC AAC ATC TGC TGC ! B2 GAA
ATT GTG CTG ACT CAG TCT CCA GAC TTT CAG TCT GTG ACT CCA AAG GAG AAA
GTC ACC ATC ACC TGC ! A26 GAA ATT GTG CTG ACT CAG TCT CCA GAC TTT
CAG TCT GTG ACT CCA AAG GAG AAA GTC ACC ATC ACC TGC ! A10 GAT GTT
GTG ATG ACA CAG TCT CCA GCT TTC GTC TCT GTG ACT CCA GGG GAG AAA GTC
ACC ATC ACC TGC ! A14
[0292] TABLE-US-00011 TABLE 9 RERS sites found in Human Kappa FR1
GLGs FokI HpyCH MslI --> <-- --> PflFI BsrI BsmAI MnlI 4V
VKI O12 1-69 3 3 23 12 49 15 18 47 26 36 O2 101-169 103 103 123 112
149 115 118 147 126 136 O18 201--269 203 203 223 212 249 215 218
247 226 236 O8 301-369 303 303 323 312 349 315 318 347 326 336 A20
401-469 403 403 423 412 449 415 418 447 426 436 A30 501-569 503 503
523 512 549 515 518 547 526 536 L14 601-669 603 603 612 649 615 618
647 -- 636 L1 701-769 703 703 723 712 749 715 718 747 726 736 L15
801-869 803 803 823 812 849 815 818 847 826 836 L4 901-969 -- 903
923 912 949 906 915 918 947 926 936 L18 1001-1069 -- 1003 1012 1049
1006 1015 1018 1047 1026 1036 L5 1101-1169 1103 -- 1112 1149 1115
1118 1147 -- 1136 L19 1201-1269 1203 1203 1212 1249 1215 1218 1247
-- 1236 L8 1301-1369 -- 1303 1323 1312 1349 1306 1315 1318 1347 --
1336 L23 1401-1469 1403 1403 1408 1412 1449 1415 1418 1447 -- 1436
L9 1501-1569 1503 1503 1508 1523 1512 1549 1515 1518 1547 1526 1536
L24 1601-1669 1603 1608 1623 1612 1649 1615 1618 1647 -- 1636 L11
1701-1769 1703 1703 1723 1712 1749 1715 1718 1747 1726 1736 L12
1801-1869 1803 1803 1812 1849 1815 1818 1847 -- 1836 VKII O11
1901-1969 -- -- -- -- -- 1956 -- O1 2001-2069 -- -- -- -- 2056 --
A17 2101-2169 -- -- 2112 -- 2118 2156 -- A1 2201-2269 -- -- 2212 --
2218 2256 -- A18 2301-2369 -- -- -- -- -- 2356 -- A2 2401-2469 --
-- -- -- -- 2456 -- A19 2501-2569 -- -- 2512 -- 2518 2556 -- A3
2601-2669 -- -- 2612 -- 2618 2656 -- A23 2701-2769 -- -- -- -- --
2729 2756 -- VKIII A27 2801-2869 -- -- 2812 -- 2818 2839 2860 --
A11 2901-2969 -- -- 2912 -- 2918 2939 2960 -- L2 3001-3069 -- --
3012 -- 3018 3039 3060 -- L16 3101-3169 -- -- 3112 -- 3118 3139
3160 -- L6 3201-3269 -- -- 3212 -- 3218 3239 3260 -- L20 3301-3369
-- -- 3312 -- 3318 3339 3360 -- L25 3401-3469 -- -- 3412 -- 3418
3439 3460 -- VKIV B3 3501-3569 3503 -- 3512 3515 3518 3539 3551<
-- VKV B2 3601-3669 -- -- 3649 -- 3618 3647 -- VKVI A26 3701-3769
-- -- 3712 -- 3718 -- A10 3801-3869 -- -- 3812 -- 3818 -- A14
3901-3969 -- -- 3912 -- 3918 3930> -- MaeIII HpaII MlyI Tsp45I
same HphI MspI SfaNI SfcI HinfI --> --> <-- sites xx38
xx56 xx62 xx06 xx52 VKI O12 1-69 37 41 53 53 55 56 -- O2 101-169
137 141 153 153 155 156 -- O18 201-269 237 241 253 253 255 256 --
O8 301-369 337 341 353 353 355 356 -- A20 401-469 437 441 453 453
455 456 -- A30 501-569 537 541 553 553 555 556 -- L14 601-669 637
641 653 653 655 656 -- L1 701-769 737 741 753 753 755 756 -- L15
801-869 837 841 853 853 855 856 -- L4 901-969 937 941 953 953 955
956 -- L18 1001-1069 1037 1041 1053 1053 1055 1056 -- L5 1101-1169
1137 1141 1153 1153 1155 1156 -- L19 1201-1269 1237 1241 1253 1253
1255 1256 -- L8 1301-1369 1337 1341 1353 1353 1355 1356 -- L23
1401-1469 1437 1441 1453 1453 1455 1456 1406 L9 1501-1569 1537 1541
1553 1553 1555 1556 1506 L24 1601-1669 1637 1641 1653 1653 1655
1656 L11 1701-1769 1737 1741 1753 1753 1755 1756 L12 1801-1869 1837
1841 1853 1853 1855 1856 VKII O11 1901-1969 -- -- 1918 1918 1937
1938 1952 O1 2001-2069 -- -- 2018 2018 2037 2038 2052 A17 2101-2169
-- -- 2112 2112 2137 2138 2152 A1 2201-2269 -- -- 2212 2212 2237
2238 2252 A18 2301-2369 -- -- 2318 2318 2337 2338 2352 A2 2401-2469
-- -- 2418 2418 2437 2438 2452 A19 2501-2569 -- -- 2512 2512 2537
2538 2552 A3 2601-2669 -- -- 2612 2612 2637 2638 2652 A23 2701-2769
-- -- 2718 2718 2737 2731* 2738* -- VKIII A27 2801-2869 -- -- -- --
-- A11 2901-2969 -- -- -- -- -- L2 3001-3069 -- -- -- -- -- L16
3101-3169 -- -- -- -- -- L6 3201-3269 -- -- -- -- -- L20 3301-3369
-- -- -- -- -- L25 3401-3469 -- -- -- -- -- VKIV B3 3501-3569 -- --
3525 3525 -- VKV B2 3601-3669 -- -- 3639 3639 -- VKVI A26 3701-3769
-- -- 3712 3739 3712 3739 3737 3755 3756 3762 -- A10 3801-3869 --
-- 3812 3839 3812 3839 3837 3855 3856 3862 -- A14 3901-3969 -- --
3939 3939 3937 3955 3956 3962 -- BsrFI BpmI Cac8I BsaJI BssKI
(NstNI) xx20 xx41 xx44 NaeI xx29 xx42 xx43 xx22 xx30 xx43 -->
--> <-- NgoMIV HaeIII Tsp509I VKI O12 1-69 -- -- -- -- -- --
O2 101-169 -- -- -- -- -- -- O18 201-269 -- -- -- -- -- -- O8
301-369 -- -- -- -- -- -- A20 401-469 -- -- -- -- -- -- A30 501-569
-- -- -- -- -- -- L14 601-669 -- -- -- -- -- -- L1 701-769 -- -- --
-- -- -- L15 801-869 -- -- -- -- -- -- L4 901-969 -- -- -- -- -- --
L18 1001-1069 -- -- -- -- -- -- L5 1101-1169 -- -- -- -- -- -- L19
1201-1269 -- -- -- -- -- -- L8 1301-1369 -- -- -- -- -- -- L23
1401-1469 -- -- -- -- -- -- L9 1501-1569 -- -- -- -- -- -- L24
1601-1669 -- -- -- -- -- -- L11 1701-1769 -- -- -- -- -- -- L12
1801-1869 -- -- -- -- -- -- VKII O11 1901-1969 1942 1943 1944 1951
1954 -- O1 2001-2069 2042 2043 2044 2051 2054 -- A17 2101-2169 2142
-- -- 2151 2154 -- A1 2201-2269 2242 -- -- 2251 2254 -- A18
2301-2369 2342 2343 -- 2351 2354 -- A2 2401-2469 2442 2443 -- 2451
2454 -- A19 2501-2569 2542 2543 2544 2551 2554 -- A3 2601-2669 2642
2643 2644 2651 2654 -- A23 2701-2769 2742 -- -- 2751 2754 -- VKIII
A27 2801-2869 2843 2822 2843 2820 2841 -- -- 2803 A11 2901-2969
2943 2943 2920 2941 -- -- 2903 L2 3001-3069 3043 3043 3041 -- -- --
L16 3101-3169 3143 3143 3120 3141 -- -- -- L6 3201-3269 3243 3243
3220 3241 -- -- 3203 L20 3301-3369 3343 3343 3320 3341 -- -- 3303
L25 3401-3469 3443 3443 3420 3441 -- -- 3403 VKIV B3 3501-3569 3529
3530 3520 -- 3554 VKV B2 3601-3669 3643 3620 3641 -- -- VKVI A26
3701-3769 -- 3720 -- -- 3703 A10 3801-3869 -- 3820 -- -- 3803 A14
3901-3969 3943 3943 3920 3941 -- -- --
[0293] TABLE-US-00012 TABLE 10 Lambda FR1 GLG sequences ! VL1 CAG
TCT GTG CTG ACT CAG CCA CCC TCG GTG TCT GAA GCC CCC AGG CAG AGG GTC
ACC ATO TCC TGT ! 1a cag tct gtg ctg acG cag ccG ccc tcA gtg tct
gGG gcc ccA Ggg cag agg gtc acc atc tcc tgC ! 1e cag tct gtg ctg
act cag cca ccc tcA gcg tct gGG Acc ccc Ggg cag agg gtc acc atc tcT
tgt ! 1c cag tct gtg ctg act cag cca ccc tcA gCg tct gGG Acc ccc
Ggg cag agg gtc acc atc tcT tgt ! 1c cag tct gtg ctg acG cag ccG
ccc tcA gtg tot gGG gcc ccA GgA cag aAg gtc acc atc tcc tgC ! 1b !
VL2 CAG TCT GCC CTG ACT CAG CCT CCC TCC GCG TCC GGG TCT CCT GGA CAG
TCA GTC ACC ATC TCC TGC ! 2c cag tct gcc ctg act cag cct cGc tcA
gTg tcc ggg tct cct gga cag tca gtc acc atc tcc tgc! 2e cag tct gcc
ctg act cag cct Gcc tcc gTg tcT ggg tct cct gga cag tcG Atc acc atc
tcc tgc ! 2a2 cag tct gcc ctg act cag cct ccc tcc gTg tcc ggg tct
cct gga cag tca gtc acc atc tcc tgc ! 2d cag tct gcc ctg act cag
cct Gcc tcc gTg tcT ggg tct cct gga cag tcG Atc acc atc tcc tgc !
2b2 ! VL3 TCC TAT GAG CTG ACT CAG CCA CCC TCA GTG TCC GTG TCC CCA
GGA CAG ACA GCC AGC ATC ACC TGC! 3r tcc tat gag ctg act cag cca cTc
tca gtg tcA gtg Gcc cTG gga cag acG gcc agG atT acc tgT ! 3j tcc
tat gag ctg acA cag cca ccc tcG gtg toA gtg tcc cca gga caA acG gcc
agG atc acc tgc! 3p tcc tat gag ctg acA cag cca ccc tcG gtg tcA gtg
tcc cTa gga cag aTG gcc agG atc acc tgc ! 3a tcT tct gag ctg act
cag GAC ccT GcT gtg tcT gtg Gcc TTG gga cag aca gTc agG atc acA tgc
! 3l tcc tat gTg ctg act cag cca ccc tca gtg tcA gtg Gcc cca gga
Aag acG gcc agG atT acc tgT ! 3h tcc tat gag ctg acA cag cTa ccc
tcG gtg tcA gtg tcc cca gga cag aca gcc agG atc acc tgc ! 3e tcc
tat gag ctg aTG cag cca ccc tcG gtg tcA gtg tcc cca gga cag acG gcc
agG atc acc tgc ! 3m tcc tat gag ctg acA cag cca Tcc tca gtg tcA
gtg tcT ccG gga cag aca gcc agG atc acc tgc ! V2-19 ! VL4 CTG CCT
GTG CTG ACT CAG CCC CCG TCT GCA TCT GCC TTG CTG GGA GCC TCG ATC AAG
CTC ACC TGC ! 4c cAg cct gtg ctg act caA TcA TcC tct gcC tct gcT
tCC ctg gga Tcc tcg Gtc aag ctc acc tgc ! 4a cAg cTt gtg ctg act
caA TcG ccC tct gcC tct gcc tCC ctg gga gcc tcg Gtc aag ctc acc tgc
! 4b ! VL5 CAG CCT GTC CTG ACT CAG CCA CCT TCC TCC TCC GCA TCT CCT
GGA GAA TCC GCC AGA CTC ACC TGC ! 5e cag Gct gtg ctg act cag ccG
Gct tcc CTc tcT gca tct cct gga gCa tcA gcc agT ctc acc tgc ! 5c
cag cct gtg ctg act cag cca Tct tcc CAT tcT gca tct Tct gga gCa tcA
gTc aga ctc acc tgc ! 5b ! VL6 AAT TTT ATG CTG ACT CAG CCC CAC TCT
GTG TCG GAG TCT CCG GGG AAG ACG GTA ACC ATC TCC TGC ! 6a ! VL7 GAC
ACT GTG GTG ACT CAG GAG CCC TCA CTG ACT GTG TCC CCA GGA GGG ACA GTC
ACT CTC ACC TGT ! 7a cag Gct gtg gtg act cag gag ccc tca ctg act
gtg tcc cca gga ggg aca gtc act ctc acc tgt ! 7b ! VL8 CAG ACT GTG
GTG ACC CAG GAG CCA TCG TTC TCA GTG TCC CCT GGA GGG ACA GTC ACA CTC
ACT TGT ! 8a ! VL9 CAG CCT GTG CTG ACT CAG CCA CCT TCT GCA TCA GCC
TCC CTG GGA GCC TCG GTC ACA CTC ACC TGC ! 9a ! VL10 CAG GCA GGG CTG
ACT CAG CCA CCC TCG GTG TCC AAG GGC TTG AGA CAG ACC GCC ACA CTC ACC
TGC ! 10a
[0294] TABLE-US-00013 TABLE 11 RERSs found in human lambda FR1 GLGs
! There are 31 lambda GLGs MlyT NnnnnnGACTC 25 1: 6 3: 6 4: 6 6: 6
7: 6 8: 6 9: 6 10: 6 11: 6 12: 6 15: 6 16: 6 20: 6 21: 6 22: 6 23:
6 23: 50 24: 6 25: 6 25: 50 26: 6 27: 6 28: 6 30: 6 31: 6 There are
23 hits at base# 6 -''- GAGTCNNNNNn 1 26: 34 MwoI GCNNNNNnngc 20 1:
9 2: 9 3: 9 4: 9 11: 9 11: 56 12: 9 13: 9 14: 9 16: 9 17: 9 18: 9
19: 9 20: 9 23: 9 24: 9 25: 9 26: 9 30: 9 31: 9 There are 19 hits
at base# 9 HinfI Gantc 27 1: 12 3: 12 4: 12 6: 12 7: 12 8: 12 9: 12
10: 12 11: 12 12: 12 15: 12 16: 12 20: 12 21: 12 22: 12 23: 12 23:
46 23: 56 24: 12 25: 12 25: 56 26: 12 26: 34 27: 12 28: 12 30: 12
31: 12 There are 23 hits at base# 12 PleI gactc 25 1: 12 3: 12 4:
12 6: 12 7: 12 8: 12 9: 12 10: 12 11: 12 12: 12 15: 12 16: 12 20:
12 21: 12 22: 12 23: 12 23: 56 24: 12 25: 12 25: 56 26: 12 27: 12
28: 12 30: 12 31: 12 There are 23 hits at base# 12 -''- gagtc 1 26:
34 DdeI Ctnag 32 1: 14 2: 24 3: 14 3: 24 4: 14 4: 24 5: 24 6: 14 7:
14 7: 24 8: 14 9: 14 10: 14 11: 14 11: 24 12: 14 12: 24 15: 5 15:
14 16: 14 16: 24 19: 24 20: 14 23: 14 24: 14 25: 14 26: 14 27: 14
28: 14 29: 30 30: 14 31: 14 There are 21 hits at base# 14 BsaJI
Ccnngg 38 1: 23 1: 40 2: 39 2: 40 3: 39 3: 40 4: 39 4: 40 5: 39 11:
39 12: 38 12: 39 13: 23 13: 39 14: 23 14: 39 15: 38 16: 39 17: 23
17: 39 18: 23 18: 39 21: 38 21: 39 21: 47 22: 38 22: 39 22: 47 26:
40 27: 39 28: 39 29: 14 29: 39 30: 38 30: 39 30: 47 31: 23 31: 32
There are 17 hits at base# 39 There are 5 hits at base# 38 There
are 5 hits at base# 40 Makes cleavage ragged. MnlI cctc 35 1: 23 2:
23 3: 23 4: 23 5: 23 6: 19 6: 23 7: 19 8: 23 9: 19 9: 23 10: 23 11:
23 13: 23 14: 23 16: 23 17: 23 18: 23 19: 23 20: 47 21: 23 21: 29
21: 47 22: 23 22: 29 22: 35 22: 47 23: 26 23: 29 24: 27 27: 23 28:
23 30: 35 30: 47 31: 23 There are 21 hits at base# 23 There are 3
hits at base# 19 There are 3 hits at base# 29 There are 1 hits at
base# 26 There are 1 hits at base# 27 These could make cleavage
ragged. -''- gagg 7 1: 48 2: 48 3: 48 4: 48 27: 44 28: 44 29: 44
BssKI Nccnqg 39 1: 40 2: 39 3: 39 3: 40 4: 39 4: 40 5: 39 6: 31 6:
39 7: 31 7: 39 8: 39 9: 31 9: 39 10: 39 11: 39 12: 38 12: 52 13: 39
13: 52 14: 52 16: 39 16: 52 17: 39 17: 52 18: 39 18: 52 19: 39 19:
52 21: 38 22: 38 23: 39 24: 39 26: 39 27: 39 28: 39 29: 14 29: 39
30: 38 There are 21 hits at base# 39 There are 4 hits at base# 38
There are 3 hits at base# 31 There are 3 hits at base# 40 Ragged
BstNI CCwqg 30 1: 41 2:40 5: 40 6: 40 7: 40 8: 40 9: 40 10: 40 11:
40 12: 39 12: 53 13: 40 13: 53 14: 53 16: 40 16: 53 17: 40 17: 53
18: 40 18: 53 19: 53 21: 39 22: 39 23: 40 24: 40 27: 40 28: 40 29:
15 29: 40 30: 39 There are 17 hits at base# 40 There are 7 hits at
base# 53 There are 4 hits at base# 39 There are 1 hits at base# 41
Ragged PspGI ccwgg 30 1: 41 2: 40 5: 40 6: 40 7: 40 8: 40 9: 40 10:
40 11: 40 12: 39 12: 53 13: 40 13: 53 14: 53 16: 40 16: 53 17: 40
17: 53 18: 40 18: 53 19: 53 21: 39 22: 39 23: 40 24: 40 27: 40 28:
40 29: 15 29: 40 30: 39 There are 17 hits at base# 40 There are 7
hits at base# 53 There are 4 hits at base# 39 There are 1 hits at
base# 41 ScrFI CCngg 39 1: 41 2: 40 3: 40 3: 41 4: 40 4: 41 5: 40
6: 32 6: 40 7: 32 7: 40 8: 40 9: 32 9: 40 10: 40 11: 40 12: 39 12:
53 13: 40 13: 53 14: 53 16: 40 16: 53 17: 40 17: 53 18: 40 18: 53
19: 40 19: 53 21: 39 22: 39 23: 40 24: 40 26: 40 27: 40 28: 40 29:
15 29: 40 30: 39 There are 21 hits at base# 40 There are 4 hits at
base# 39 There are 3 hits at base# 41 MaeIII gtnac 16 1: 52 2: 52
3: 52 4: 52 5: 52 6: 52 7: 52 9: 52 26: 52 27: 10 27: 52 28: 10 28:
52 29: 10 29: 52 30: 52 There are 13 hits at base# 52 Tsp45I gtsac
15 1: 52 2: 52 3: 52 4: 52 5: 52 6: 52 7: 52 9: 52 27: 10 27: 52
28: 10 28: 52 29: 10 29: 52 30: 52 There are 12 hits at base# 52
HphI tcacc 26 1: 53 2: 53 3: 53 4: 53 5: 53 6: 53 7: 53 8: 53 9: 53
10: 53 11: 59 13: 59 14: 59 17: 59 18: 59 19: 59 20: 59 21: 59 22:
59 23: 59 24: 59 25: 59 27: 59 28: 59 30: 59 31: 59 There are 16
hits at base# 59 There are 10 hits at base# 53 BspMI ACCTGCNNNNn 14
11: 61 13:61 14: 61 17: 61 18: 61 19: 61 20: 61 21:61 22: 61 23: 61
24: 61 25: 61 30: 61 31:61 There are 14 hits at base# 61 Goes into
CDR1
[0295] TABLE-US-00014 TABLE 12 Matches to URE FR3 adapters in 79
human HC. A. List of Heavy-chains genes sampled AF008566 AF103367
HSA235674 HSU94417 S83240 AF035043 AF103368 HSA235673 HSU94418
SABVH369 AF103026 AF103369 HSA240559 H5U96389 SADEIGVH af103033
AF103370 HSCB201 HSU96391 SAH2IGVH AF103061 af103371 HSIGGVHC
HSU96392 SDA3IGVH Af103072 AF103372 HSU44791 HSU96395 SIGVHTTD
af103078 AF158381 HSU44793 HSZ93849 SUK4TGVH AF103099 E05213
HSU82771 HSZ93850 AF103102 E05886 HSU82949 HSZ93851 AF103103 E05887
HSU82950 HSZ93853 AF103174 HSA235661 HSU82952 HSZ93855 AF103186
HSA235664 HSU82961 HSZ93857 af103187 HSA235660 HSU86522 HSZ93860
AF103195 HSA235659 HSU86523 HSZ93863 af103277 HSA235678 HSU92452
MCOMFRAA af103286 HSA235677 HSU94412 MCOMFRVA AF103309 HSA235676
HSU94415 S82745 af103343 HSA235675 HSU94416 S82764
[0296] TABLE-US-00015 TABLE 12B Testing all distinct GLGs from
bases 89.1 to 93.2 of the heavy variable domain Id SEQ NO: Nb 0 1 2
3 4 ID 1 38 15 11 10 0 2 Seq1 gtgtattactgtgc 25 2 19 7 6 4 2 0 Seq2
gtAtattactgtgc 26 3 1 0 0 1 0 0 Seq3 gtgtattactgtAA 27 4 7 1 5 1 0
0 Seq4 gtgtattactgtAc 28 5 0 0 0 0 0 0 Seq5 Ttgtattactgtgc 29 6 0 0
0 0 0 0 Seq6 Ttgtatcactgtgc 30 7 3 1 0 1 1 0 Seq7 ACAtattactgtgc 31
8 2 0 2 0 0 0 Seq8 ACgtattactgtgc 32 9 9 2 2 4 1 0 Seq9
ATatattactatac 33 Group 26 26 21 4 2 Cumula- 26 52 73 77 79
tivet
[0297] TABLE-US-00016 TABLE 12C Most important URE recognition seqs
in FR3 Heavy 1 VRSzy1 GTGtattactgtgc (ON_SHC103) (SEQ ID NO:25) 2
VHSzy2 GTAtattactgtgc (ON_SHC323) (SEQ ID NO:26) 3 VHSzy4
GTGtattactgtac (ON_SHC349) (SEQ ID NO:28) 4 VHSzy9 ATGtattactgtgc
(ON_SHC5a) (SEQ ID NO:33)
[0298] TABLE-US-00017 TABLE 12D testing 79 human HC V genes with
four probes Number of sequences. . . 79 Number of bases. . . 29143
Number of mismatches Id Best 0 1 2 3 4 5 1 39 15 11 10 1 2 0 Seq1
gtgtattactgtgc (SEQ ID NO:25) 2 22 7 6 5 3 0 1 Seq2 gtAtattactgtgc
(SEQ ID NO:26) 3 7 1 5 1 0 0 0 Seq4 gtgtattactgtAc (SEQ ID NO:28) 4
11 2 4 4 1 0 0 Seq9 ATgtattactgtgc (SEQ ID NO:33) Group 25 26 20 5
2 Cumu- 25 51 71 76 78 lative One sequence has five mismatches with
sequences 2, 4, and 9; it is scored as best for 2. Id is the number
of the adapter. Best is the number of sequence for which the
identified adapter was the best available. The rest of the table
shows how well the sequences match the adapters. For example, there
are 10 sequences that match VHSzy1 (Id=1) with 2 mismatches and are
worse for all other adapters. In this sample, 90% come within 2
bases of one of the four adapters.
[0299] TABLE-US-00018 TABLE 13 The following list of enzymes was
taken from htttp//rebase.neb.com/cgi-bin/asymmlist. I have removed
the enzymes that a) cut within the recognition, b) cut on both
sides of the recognition, or c) have fewer than 2 bases between
recognition and closest cut site. REBASE Enzymes Apr. 13, 2001 Type
II restriction enzymes with asymmetric recognition sequences:
Enzymes Recognition Sequence Isoschizomers Suppliers AarI
CACCTGCNNNN{circumflex over ( )}NNNN.sub.-- -- y AceIII
CAGCTCNNNNNNN{circumflex over ( )}NNNN.sub.-- -- -- Bbr7I
GAAGACNNNNNNN{circumflex over ( )}NNNN.sub.-- -- -- BbvI
GCAGCNNNNNNNN{circumflex over ( )}NNNN.sub.-- -- y BbvII
GAAGACNN{circumflex over ( )}NNNN.sub.-- Bce83I
CTTGAGNNNNNNNNNNNNNN_NN{circumflex over ( )} -- -- BceAI
ACGGCNNNNNNNNNNNN{circumflex over ( )}NN.sub.-- -- y BcefI
ACGGCNNNNNNNNNNNN{circumflex over ( )}N.sub.-- -- -- BciVI
GTATCCNNNNN_N{circumflex over ( )} BfuI y BfiI
ACTGGGNNNN_N{circumflex over ( )} BmrI y BinI GGATCNNNN{circumflex
over ( )}N.sub.-- BscAI GCATCNNNN{circumflex over ( )}NN.sub.-- --
-- BseRI GAGGAGNNNNNNNN_NN{circumflex over ( )} -- y BsmFI
GGGACNNNNNNNNNN{circumflex over ( )}NNNN.sub.-- BspLU11III y BspMI
ACCTGCNNNN{circumflex over ( )}NNNN.sub.-- Acc36I y EciI
GGCGGANNNNNNNNN_NN{circumflex over ( )} -- y Eco57I
CTGAAGNNNNNNNNNNNNNN_NN{circumflex over ( )} BspKT5I y FauI
CCCGCNNNN{circumflex over ( )}NN.sub.-- BstFZ438I y FokI
GGATGNNNNNNNNN{circumflex over ( )}NNNN.sub.-- BstPZ418I y GsuI
CTGGAGNNNNNNNNNNNNNN_NN{circumflex over ( )} -- y HgaI
GACGCNNNNN{circumflex over ( )}NNNNN.sub.-- -- y HphI
GGTGANNNNNNN_N{circumflex over ( )} AsuHPI y MboII
GAAGANNNNNNN_N{circumflex over ( )} -- y MlyI GAGTCNNNNN{circumflex
over ( )} SchI y MmeI TCCRACNNNNNNNNNNNNNNNNNN.sub.--NN{circumflex
over ( )} -- -- MnlI CCTCNNNNNN_N{circumflex over ( )} -- y Plel
GAGTCNNNN{circumflex over ( )}N.sub.-- PpsI y RleAI
CCCACANNNNNNNNN_NNN{circumflex over ( )} -- SfaNI
GCATCNNNNN{circumflex over ( )}NNNN.sub.-- BspST5I y SspD5I
GGTGANNNNNNNN{circumflex over ( )} -- -- Sth132I
CCCGNNNN{circumflex over ( )}NNNN.sub.-- -- -- StsI
GGATGNNNNNNNNNN{circumflex over ( )}NNNN.sub.-- -- -- TaqII
GACCGANNNNNNNNN_NN{circumflex over ( )},
CACCCANNNNNNNNN_NN{circumflex over ( )} -- -- Tth111II
CAARCANNNNNNNNN_NN{circumflex over ( )} -- -- UbaPI CGAACG -- --
The notation is {circumflex over ( )} means cut the upper strand
and _ means cut the lower strand. If the upper and lower strand are
cut at the same place, then only {circumflex over ( )} appears.
[0300] TABLE-US-00019 TABLE 14 (FOKIact) 5'-cAcATccgTg TT
cAcggATgTg-3' (VHEx881) 5'-AATAgTAgAc TgcAgTgTcc TcAgcccTTA
AgcTgTTcAT cTgcAAgTAg- AgAgTATTcT TAgAgTTgTc TcTAgAcTTA gTgAAgcg-3'
! note that VHEx881 is the reverse complement of the ON below !
[RC] 5'-cgCttcacTaag- ! Scab........ ! Synthetic 3-23 as in Table
206 ! |TCT|AGA|gac|aac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|- !
XbaI. . . ! aac|agC|TTA|AGg|gct|gag|gac|aCT|GCC|Gtc|tac|tat|t-3' !
AflII . . . (VHBA881) 5'-cgCttcacTaag-
TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgt gcg ag-3'
(VHBB881) 5'-cgCttcacTaag-
TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgt Acg ag-3'
(VH881PCR) 5'-cgCttcacTaag|TCT|AGA|gac|aac -3'
[0301] TABLE-US-00020 TABLE 15 Use of FokI as "Universal
Restriction Enzyme" FokI -- for dsDNA, | represents sites of
cleavage sites of cleavage 5'-cacGGATGtg--nnnnnnn|nnnnnnn-3' (SEQ
ID NO:15) 3'-gtgCCTACac--nnnnnnnnnnn|nnn-5' (SEQ ID NO:16) RECOG
NITion of FokI Case I 5'-...gtg|tatt-actgtgc..Substrate....-3' (SEQ
ID NO:17) 3'-cac-ata|tgacacg--| gtGTAGGcac\ 5'-caCATCCgtg/ (SEQ ID
NO:18) Case II 5'-...gtgtatt|agac-tgc..Substrate....-3' (SEQ ID
NO:19) |--cacataa-tctg|acg-5' /gtgCCTACac \cacGGATGtg-3' (SEQ ID
NO:20) Case III (Case I rotated 180 degrees) /gtgCCTACac-5'
\cacGGATGtg--| gtgtctt|acag-tcc-3' Adapter (SEQ ID NO:21)
3'-...cacagaa-tgtc|agg..substrate....-5' (SEQ ID NO:22) Case IV
(Case II rotated 180 degrees) 3'- gtGTAGGcac\ (SEQ ID NO:23)
|--caCATCCgtg/ 5'-gag|tctc-actgagc Substrate
3'-...ctc-agag|tgactcg...-5' (SEQ ID NO:24) Improved FokI adapters
Fok-- for dsDNA, | represents sites of cleavage Case I Stem 11,
loop 5, stem 11, recognition 17
5'-...catgtg|tatt-actgtgc..Substrate....-3'
3'-gtacac-ataa|tgacacg--| |-T--| gtGTAGGcacG T 5'- caCATCCgtgc C
|-TT-| Case II Stem 10, loop 5, stem 10, recognition 18
5'-...gtgtatt|agac-tgctgcc..Substrate....-3' |-T-|
|--cacataa-tctg|acgacgg-5' T gtgCCTACac C cacGGATGtg-3' |-TT-| Case
III (Case I rotated 180 degrees) Stem 11, loop 5, stem 11,
recognition 20 |-T-| T TgtgCCTACac-5' G AcacGGATGtg--| |-TT-|
gtgtctt|acag-tccattctg-3' Adapter
3'-...cacagaa-tgtc|aggtaagac..substrate....-5' Case IV (Case II
rotated 180 degrees) Stem 11, loop 4, stem 11, recognition 17 |-T-|
3'- gtGTAGGcacc T |--caCATCCgtgg T 5'-atcgag|tctc-actgagc |-T-|
Substrate 3'-...tagctc-agag|tgactcg...-5' BseRI | sites of cleavage
5'-cacGAGGAGnnnnnnnnnn|nnnnn-3' 3'-gtgctcctcnnnnnnnn|nnnnnnn-5'
RECOG NITion of BseRI Stem 11, loop 5, stem 11, recognition 19
3'-.......gaacat|cg-ttaagccagta.....5' |-T-T-|
cttgta-gc|aattcggtcat-3' C GCTGAGGAGTC--| T cgactcctcag-5' An
adapter for BseRI to cleave the substrate above. |-T--|
[0302] TABLE-US-00021 TABLE 16 Human heavy chans bases 88.1 to 94.2
Number of sequences.......... 840 Number of Mismatchers.........
Probe Id Ntot 0 1 2 3 4 5 6 7 Name Sequence............ Dot
form............ 1 364 152 97 76 26 7 4 2 0 VHS881-1.1
gctgtgtattactgtgcgag gctgtgtattactgtgcgag 2 265 150 60 33 13 5 4 0
0 VHS881-1.2 gccgtgtattactgtgcgag ..c................. 3 96 14 34
16 10 5 7 9 1 VHS881-2.1 gccgtatattactgtgcgag ..c..a..............
4 20 0 3 4 9 2 2 0 0 VHS881-4.1 gccgtgtattactgtacgag
..c............a.... 5 95 25 36 18 11 2 2 0 1 VH5881-9.1
gccatgtattactgtgcgag ..ca................ 840 341 230 147 69 21 19
11 2 341 571 718 787 808 827 838 840 88 89 90 91 92 93 94 95 Codon
number as in Table 195 Recognition........... Stem...... Loop.
Stem...... (VHS881-1.1) 5'-gctgtgtat|tact-gtgcgag cAcATccgTg TTgTT
cAcggATgTg-3' (VHS881-1.2) 5'-gccgtgtat|tact-gtgcgag cAcATccgTg
TTgTT cAcggATgTg-3' (VHS881-2.1) 5'-gccgtatat|tact-gtgcgag
cAcATccgTg TTgTT cAcggATgTg-3' (VHS881-9.1)
5'-gccatgtat|tact-gtgcgag cAcATccgTg TTgTT cAcggATgTg-3' | site of
substrate cleavage (FOKIact) 5'cAcATccgTg TTgTT cAcggATgTg-3'
(VHEx881) 5'-AATAgTAgAc TgcAgTgTcc TcAgcccTTA AgcTgTTcAT
cTgcAAgTAg- AgAgTATTcT TAgAgTTgTc TcTAgAcTTA gTgAAgcg-3' ! note
that VHEx881 is the reverse complement of the ON below ! [RC]
5'cgCttcacTaag- ! Scab........ ! Synthetic 3-23 as in Table 206 !
|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|- ! XbaI... !
|aac|agC|TTA|AGg|gct|gag|aCT|GCA|Gtc|tac|tat|t-3' ! AflII...
(VHBA881) 5'-cgCttcacTaag-
|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgt gcg ag-3'
(VHBB881) 5'-cgCttcacTaag-
|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|-
|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgt Acg ag-3'
(VH881PCR) 5'-cgCttcacTaag|TCT|AGA|gac|aac-3'
[0303] TABLE-US-00022 TABLE 17 Kappa, bases 12-30 ! ! ID Ntot 0 1 2
3 4 5 6 Name Sequence........... Dot Form.......... ! 1 84 40 21 20
1 2 0 0 SK12O12 gaccagtctccatcctcc gacccagtctccatcctcc ! 2 32 19 3
6 2 1 0 1 SK12A17 gactcagtctccactctcc ...t.........ct.... ! 3 26 17
8 1 0 0 0 0 SK12A27 gacgcagtctccaggcacc ...g.........gg.aa.. ! 4 40
21 18 1 0 0 0 0 SK12A11 gacgcagtctccagccacc ...g.........g..a.. !
182 97 50 28 3 3 0 1 ! 97 147 175 178 181 181 182 ! URE adapters: !
Stem...... Loop. Stem...... Recognition........ (SzKB1230-O12)
5'-cAcATccgTg TTgTT cAcggATgTg ggAggATggAgAcTgggTc-3' ! [RC]
5'-gaccagtctccatcctcc cAcATccgTG AAcAA cAcggATgTg-3' !
Recognition........ Stem...... loop. Stem...... ! FokI. Foki. ! !
Stem...... Loop. Stem...... Recognition........ (SzKB1230-A17)
5'cAcATccgTg TTgTT cAcggATgTg ggAgAgTggAgAcTgAgTc-3' ! [RC]
5'-gactcagtctccactctcc cAcATccTg AAcAA cAcggATgTg-3' !
Recognition......... Stem...... loop. Stem...... ! FokI. FokI. ! !
Stem...... Loop. Stem...... Recognition........ (SzKB1230-A27)
5'-cAcATccgTg TTgTT cAcggATgTg ggTgccTggAgAcTgcgTc-3' ! [RC]
5'-gacgcagtctccaggcacc cAcATccgTg AAcAA cAcggATgTg-3' !
Recognition........ Stem...... loop. Stem...... ! FokI. FokI. ! !
Stem...... Loop. Stem...... Recognition........ (SzKB1230-A11)
5'-cAcAtccgTg TTgTT cAcggATgTg ggTggcTggAgAcTgcgTc-3' ! [RC]
5'-gacgcagtctccagccacc cAcATccgTg AAcAA cAcggATgTg-3' !
Recognition........ Stem...... loop. Stem...... ! FokI. FokI. What
happens in the upper strand: (SzKB1230-O12*) 5'-gac cca gtc|tcc
a-tc ctc c-3' ! | Site of cleavage in substrate ! (SzKB1230-A17*)
5'-gac tca gtc|tcc a-ct ctc c-3' ! (SzKB1230-A27*) 5'-gac gca
gtc|tcc a-gg cac c-3' ! (SzKB1230-A11*) 5'-gac gca gtc|tcc a-gc cac
c-3' (kapextURE) 5'-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg-3' !sense
strand Scab.............ApaLI. (kapextUREPCR)
5'-ccTctactctTgTcAcAgTg-3' Scab... ......... (kaBRO1UR)
5'-ggAggATggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3' ! [RC]
5'-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-tc ctc c-3' ON above
is R.C. of this one (kaBRO2UR) 5'-ggAgAgTggA cTggATgTcT TgTgcAcTgT
gAcAAgAgTA gAgg-3' ! [RC] 5'-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg
tcc a-ct ctc c-3' ON above is R.C. of this one (kaBRO3UR)
5'-ggTgccTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3' ! [RC]
5'-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-gg cac c-3' ON above
is R.C. of this one (kaBRO4UR) 5'-ggTggcTggA cTggATgTcT TgTgcAcTgT
gAcAAgAgTA gAgg-3' ! [RC] 5'-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg
tcc a-gc cac c-3' Scab.............ApaLI.
[0304] TABLE-US-00023 TABLE 18 Lambda URE adapters bases 13.3 to
19.3 ! ! Number of sequences.......... 128 ! ! Number of
mismatches.............. ! Id Ntot 0 1 2 3 4 5 6 7 8 Name
Sequence........... Dot form........... ! 1 58 45 7 1 0 0 0 2 2 1
VL133-2a2 gtctcctggacagtcgatc gtctcctggacagtcgatc ! 2 16 10 1 0 1 0
1 1 0 2 VL122-3I ggccttgggacagacagtc gcttg......a.ag.. ! 3 17 6 0 0
0 4 1 1 5 0 VL133-2c gtctcctggacagtcagtc ............ag.. ! 4 37 3
0 10 4 4 3 7 4 2 VL133-1c ggccccagggcagagggtc g.c..a..g...a..g.. !
128 64 8 11 5 8 5 11 11 5 ! 64 72 83 88 96 101 112 123 128 !
Stem...... loop. Stem...... Recognition........ VL133-2a2)
5'cAcATccgTg TTgTT cAcggATgTggATcgAcTgTccAggAgAc-3' ! [RC]
5'-gtctcctggacagtcgatc cAcATccTg AAcAA cAcggATgTg-3' !
Recognition........ Stem...... Loop. Stern...... ! Stern......
loop. Stem...... Recognition........ (VL133-31) 5'-cAcATccgTg TTgTT
cAcggATgTggAcTgTcTgTcccAAggcc-3' ! [RC] 5'-ggccttgggacagacagtc
cAcATccgTg AAcAA cAcggATgTg-3' ! Recognition........ Stern......
Loop. Stern...... ! Stern...... loop. Stern......
Recognition........ (VL133-2c) 5'cAcATccgTgTTgTT cAcggATgTg
gAcTgAcTgTccAggAgAc-3' ! [RC] 5'-gtctcctggacagtcagtc cAATccgTg
AAcAA cAcggATgTg-3' ! Recognition........ Stern...... Loop.
Stern...... ! Stern...... loop. Stern...... Recognition........
(VL133-1c) 5'-cAcATccgTg TTgTT cAcggATgTg gAcccTcTgcccTggggcc-3' !
[RC] 5'-ggccccagggcagagggtc cAcATccgTg AAcAA cacggATgTg-3' What
happens in the top strand: ! | site cleavage in the upper strand
(VL133-2a2*) 5'-g tct cct g|ga cag tcg atc ! (VL133-31*) 5'-g gcc
ttg g|ga cag aca gtc ! (VL133-2c*) 5'-g tct cct g|ga cag tca gtc !
(VL133-1c*) 5'-g gcc cca g|gg cag agg gtc ! ! The following
Extenders and Bridges all encode the AA sequence of 2a2 for codons
1-15 ! 1 (ON_LamEx133) 5'-ccTcTgAcTgAgT gcA cAg - ! 2 3 4 5 6 7 8 9
10 11 12 AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT- ! ! 13 14 15
tcC ccG g! 2a2 ! 1 (ON_LamB1-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg - !
! 2 3 4 5 6 7 8 9 10 11 12 AGt gcT TtA acC caA ccG gcT AGT gtT AGC
ggT- ! ! 13 14 15 tcC ccG g ga cag tcg at-3' !2a2 N.B. the sctuA
seq is the ! reverse complement of the ! one shown. !
(ON_LamB2-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg - ! ! 2 3 4 5 6 7 8 9
10 11 12 AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT- ! ! 13 14 15
tcC ccG g ga cag aca gt-3' |3l N.B. the actual seq is the ! reverse
complement of the ! one shown. ! ! (ON_LamB3-133) [RC]
5'-ccTcTgAcTgAgT gcA cAg - ! ! 2 3 4 5 6 7 8 9 10 11 12 AGt gcT TtA
acC caA ccG gcT AGT gtT AGC ggT- ! 13 14 15 tcC ccG g ga cag tcagt
-3'! 2c N.B. the actual seq is the ! reverse complement of the !
one shown. !(ON_LamE4-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg - ! ! 2 3
4 5 6 7 8 9 10 11 12 AGt gcT TtA acC caA ccG gcT AGT gtT AGC gqT-s
! ! 13 14 15 tcC ccG g gg cag agg gt-3' ! 1c N.B. the actual seq is
the ! reverse complement off the ! one shown. (ON_Lam133PCR)
5'-ccTcTgAcTgAgT gcA cAg AGt gc-3'
[0305] TABLE-US-00024 TABLE 19 Cleavage of 75 human light chains.
Planned location of Enzyme Recognition Nch Ns site AfeI AGCgct 0 0
AflII Cttaag 0 0 HC FR3 Agel Accggt 0 0 AscI GGcgcgcc 0 0 After LC
BglII Agatct 0 0 BsiWI Cgtacg 0 0 BspDI ATcgat 0 0 BssHII Gcgcgc 0
0 BstBI TTcgaa 0 0 DraIII CACNNNgtg 0 0 EagI Cggccg 0 0 FseI
GGCCGGcc 0 0 FspI TGCgca 0 0 HpaI GTTaac 0 0 HfeI Caattg 0 0 MC FRi
MluI Acgcgt 0 0 NcoI Ccatgg 0 0 Heavy chain signal NheI Gctagc 0 0
HC/anchor linker NotI GCggccgc 0 0 In linker after MC NruI TCGcga 0
0 Pad TTAATtaa 0 0 PmeI GTTTaaac 0 0 PmlI CACgtg 0 0 PvuI CGATcg 0
0 SacII CCGCgg 0 0 SaiI Gtcgac 0 0 SfiI GGCC1-NNNnggcc 0 0 Heavy
Chain signal SgfI GCGATcgc 0 0 SnaBI TACgta 0 0 StuI AGGcct 0 0
XbaI Tctaga 0 0 MC FR3 AatII GACGTc 1 1 AciI AAcgtt 1 1 AseI ATtaat
1 1 BsmI GAATGCN 1 1 EspEI Tccgga 1 1 MC FR1 BstXI CCANNNNNntgg 1 1
MC FR2 DrdI GACNNNNnngtc 1 1 HindIII Aagctt 1 1 PciI Acatgt 1 1
SapI gaagagc 1 1 Scal AGTact 1 1 SexAl Accwggt 1 1 Spel Actagt 1 1
TiiI Ctcgag 1 1 XhoI Ctcgag 1 1 BcgI cgannnnnntgc 2 2 BlpI GCtnagc
2 2 BssSI Ctcgtg 2 2 EstAPI GCANNNNntgc 2 2 EspI GCtnagc 2 2 KasI
Ggcgcc 2 2 PflMI CCANNNNntgg 2 2 XrnnI GAANNnnttc 2 2 ApaLI Gtgcac
3 3 LC signal seq NaeI GCCggc 3 3 NgoMI Gccggc 3 3 PvuII CAGctg 3 3
RsrII CGgwccg 3 3 BsrBI GAGcgg 4 4 BsrDI GCAPLTGNNn 4 4 BstZl7I
GTAtac 4 4 EcoRI Gaattc 4 4 SphI GCATGc 4 4 SspI AATatt 4 4 AccI
GTmkac 5 5 BclI Tgatca 5 5 BsmBI Nnnnnngagacg 5 5 BsrGI Tgtaca 5 5
Dral TTTaaa 6 6 NdeI CAtatg 6 6 HC FR4 Swal ATTTaaat 6 6 BamHI
Ggatcc 7 7 Sad GAGCTc 7 7 BciVI GTATCCNNNNNN 8 8 BsaBI GATNNnnatc 8
8 NsiI ATGCAt 8 8 Espl20I Gggccc 9 9 CH1 Apal GGGCCc 9 9 CH3.
PspOOMI Gggccc 9 9 BspHI Tcatga 9 11 EcoRV GATatc 9 9 AhdI
GACNNNnngtC 11 11 BbsI GAAGAC 11 14 PsiI TTAtaa 12 12 BsaI
GGTCTCNnnnn 13 15 XmaI Cccggg 13 14 Aval Cycgrg 14 16 BglI
GCCNNNNnggc 14 17 AiwNI CAGNNNctg 16 16 BspMI ACCTGC 17 19 XcmI
CCANNNNNnnnntgg 17 26 EstEZI Ggtnacc 19 22 HC FR4 Sse8387I CCTGCAgg
20 20 AvrII Cctagg 22 22 HincII GTYraC 22 22 EsgI GTGCAG 27 29 MscI
TGGcca 30 34 BseRI NNnnnnnnnnctcctc 32 35 Bsu36I CCtnagg 35 37 PstI
CTGCAg 35 40 EciI nnnnnnnnntccgcc 38 40 PpuMI RGgwccy 41 50 StyI
Ccwwgg 44 73 EcoO109I RGgnccy 46 70 Acc65I Ggtacc 50 51 KpnI GGTACc
50 51 BpmI ctccag 53 82 AvaII Ggwcc 71 124 *cleavage occurs in the
top strand after the last upper-case base. For REs that cut
palindromic sequences, the lower strand is cut at the symmetrical
site.
[0306] TABLE-US-00025 TABLE 20 Cleavage of 79 human heavy chains
Planned location of Enzyme Recognition Nch Ns site AfeI AGCgct 0 0
AflII Cttaag 0 0 NC FR3 AscI GGcgcgcc 0 0 After LC BsiWI Cgtacg 0 0
BspDI ATcgat 0 0 BssHII Gcgcgc 0 0 FseI GGCCGGcc 0 0 HpaI GTTaac 0
0 NheI Gctagc 0 0 NC Linker NotI GCggccgc 0 0 In linker, NC/anchor
NruI TCGcga 0 0 NsiI ATGCAt 0 0 PacT TTAATtaa 0 0 PciI Acatgt 0 0
PmeI GTTTaaac 0 0 PvuI CGATcg 0 0 RsrII CGgwccg 0 0 SapI gaagagc 0
0 SfiI GGCCNNNNnggcc 0 0 NC signal seq SgfI GCGATcgc 0 0 SwaT
ATTTaaat 0 0 AcII AAcgtt 1 1 AgeT Accggt 1 1 AseI ATtaat 1 1 AvrII
Cctagg 1 1 EsmI GAATGCN 1 1 BsrBI GAGcgg 1 1 BsrDI GCAATGNNn 1 1
DraI TTTaaa 1 1 FspI TGCgca 1 1 HindIII Aagctt 1 1 MfeI Caattg 1 1
NC FR1 NaeI GCCggc 1 1 NgoMI Gccggc 1 1 SpeI Actagt 1 1 Acc65I
Ggtacc 2 2 BstEI TTcgaa 2 2 KpnI GGTACc 2 2 MIuI Acgcgt 2 2 NcoI
Ccatgg 2 2 In NC signal seq NdeI CAtatg 2 2 NC FR4 PmlT CACgtg 2 2
XcmI CCANNNNNnnnntgg 2 2 BcgI cgannnnnntgc 3 3 EdI Tgatca 3 3 EglI
GCCNNNNnggc 3 3 BsaBI GATNNnnatc 3 3 EsrGI Tgtaca 3 3 SnaBI TACgta
3 3 SseE387I CCTGCAgg 3 3 ApaLI Gtgcac 4 4 LC Signal/FRi EspHI
Tcatga 4 4 BssSI Ctcgtg 4 4 PsiI TTAtaa 4 5 SphI GCATGc 4 4 AhciI
GACNNNnngtc 5 5 EspHI Tccgga 5 5 HC FR1 MscI TGGcca 5 5 Sad GAGCTc
5 5 Scal AGTact 5 5 SexAl Accwggt 5 6 SspI AATatt 5 5 TliI Ctcgag 5
5 XhoI Ctcgag 5 5 BbsI GAAGAC 7 8 EstAPI GCANNNNntgc 7 8 BstZl7I
GTAtac 7 7 EcoRV GATatc 7 7 EcoRI Gaattc 8 8 SipI GCtnagc 9 9
Bsu36I CCtnagg 9 9 DraIII CACNNNgtg 9 9 EspI GCtnagc 9 9 StuI
AGGcct 9 13 XbaI Totaga 9 9 HC FR3 Bsp120I Gggccc 10 11 CH1 ApaI
GGGCCC10 11 CH1 PspOOMI Gggccc 10 11 BciVI GTATCCNNNNNN 11 11 Sail
Gtcgac 11 12 DrdI GACNNNNnngtc 12 12 KasI Ggcgcc 12 12 XmaI Cccggg
12 14 BglII Agatot 14 14 HincII GTYrac 16 18 BainHI Ggatcc 17 17
Pf1MI CCANNNNntgg 17 18 BsmBI Nnnnnngagacg 18 21 BstXI CCANNNNNntgg
18 19 HC FR2 XmnI GAANNnnttc 18 18 SaclI CCGCgg 19 19 PstI CTGCAg
20 24 PvuII CAGctg 20 22 AvaI Cycgrg 21 24 EagI Cggccg 21 22 AatII
GACGTc 22 22 BspMI ACCTGC 27 33 AccI GTmkac 30 43 StyI Ccwwgg 36 49
AlWNI CAGNNNctg 38 44 BsaI GGTCTCNnnnn 38 44 PpuMI RGgwccy 43 46
BsgI GTGCAG 44 54 BseRI NNnnnnnnnctcctc 48 60 EciI nnnnnnnntccgcc
52 57 EstEII Ggtnacc 54 61 HC Fr4, 47/79 have one EcoO109I RGgnccy
54 86 BpmI ctccag 60 121 AvaII Ggwcc 71 140
[0307] TABLE-US-00026 TABLE 21 MALIA3, annotated ! MALIA3 9532
bases
!----------------------------------------------------------------------
1 aat gct act act att agt aga att gat gcc acc ttt tca gct cgc gcc !
gene ii continued 49 cca aat gaa aat ata gct aaa cag gtt att gac
cat ttg cga aat gta 97 tct aat ggt caa act aaa tct act cgt tcg cag
aat tgg gaa tca act 145 gtt aca tgg aat gaa act ttc aga cac cgt act
tta gtt gca tat tta 193 aaa cat gtt gag cta cag cac cag att cag caa
tta agc tct aag cca 241 ttc gca aaa atg acc tct tat caa aag gag caa
tta aag gta ctc tct 289 aat cct gac ctg ttg gag ttt gct ttc ggtctg
gtt cgc ttt gaa gct 337 cga att aaa acg cga tat ttg aag tct ttc ggg
ctt cct ctt aat ctt 385 ttt gat gca atc cgc ttt gct tct gac tat aat
agt cag ggtaaa gac 433 ctg att ttt gat tta tgg tca ttc tcg ttt tct
gaa ctg ttt aaa gca 481 ttt gag ggg gat tca ATG aat att tat gac gat
ttc gca gta ttg gac ! RES?...... Start gene x, ii continues 529 gct
atc cag tct aaa cat ttt act att acc ccc tct ggc aaa act tct 577 ttt
gca aaa gcc tct cgc tat ttt ggtttt tat cgt cgt ctg gta aac 625 gag
ggttat gat agt gtt gct ctt act atg cct cgt aat ttc ttt tgg 673 cgt
tat gta tct gca tta gtt gaa tgt ggtatt cct aaa tct caa ctg 721 atg
aat ctt tct acc tgt aat aat gtt ggt ccg tta gtt cgt ttt att 769 aac
gta gat ttt tct ttc caa cgt cct gac tgg tat aat gag cca gtt 817 ctt
aaa atc gca TAA ! End X & II 832 ggtaattca ca ! ! M1 ES Q10 T15
843 ATG att aaa gtt gaa att aaa cca tct caa gcc caa ttt act act cgt
! Start gene V ! ! S17 S20 P25 E30 891 tct ggt gtt tct cgt cag ggc
aag cct tat tca ctg aat gag cag ctt ! ! V35 E40 V45 939 tgt tac gtt
gat ttg ggtaat gaa tat ccg gtt ctt gtc aag att act ! ! D50 A55 L60
987 ctt gat gaa ggtcag cca gcc tat gcg cct ggtcTG TAC Acc gtt cat !
BsrGI... ! L65 V70 S75 R80 1035 ctg ttc tct ttc aaa gtt ggtcag ttc
ggtttc ctt atg att gac cgt ! ! P85 K87 end of V 1083 ctg cgc ctc
gtt ccg gct aag TAA C ! 1108 ATG gag cag gtc gcg gat ttc gac aca
att tat cag gcg atg ! Start gene VII ! 1150 ata caa atc ttc gtt gta
ctt tgt ttc gcg ctt ggt ata atc ! ! VII and IX overlap. ! S2 V3 L4
V5 S10 1192 gct ggg ggt caa agA TGA gt gtt tta gtg tat tct ttc gcc
tct ttc gtt ! End VII ! |start IX ! L13 W15 G20 T25 E29 1242 tta
ggt tgg tgc ctt cgt agt ggc att acg tat ttt acc cgt tta atg gaa !
1293 act ttc tc ! ! .... stop of IX, IX and VIII overlap by four
bases 1301 ATG aaa aag tct tta gtc ctc aaa gcc tct gta gcc gtt gct
acc ctc ! Start signal sequence of viii. ! 1349 gtt cog atg ctg tct
ttc gct gct gag ggt gac gat ccc gca aaa gcg ! mature VIII --->
1397 gcc ttt aac tcc ctg caa gcc tca gcg acc gaa tat atc ggt tat
gcg 1445 tgg gcg atg gtt ggt gtc att 1466 gtc ggc gca act atc ggt
atc aag ctg ttt aag 1499 aaa ttc acc tcg aaa gca ! 1515 !
|........... -35 .. ! 1517 agc tga taaaccgat acaattaaag gctccttttg
! ..... -10 ... ! 1552 gagccttttt ttttGGAGAt ttt ! S.D. underlined
! ! >----------- III signal sequence
-----------------------------> ! M K K L L F A I P L V 1575 caac
GTG aaa aaa tta tta ttc gca att cct tta gtt ! 1611 ! ! V P F Y S H
S A Q 1612 gtt cct ttc tat tct cac aGT gcA Cag tCT ! ApaLI... !
1642 GTC GTG ACG CAG CCG CCC TCA GTG TCT GGG GCC CCA GGG CAG AGG
GTC ACC ATC TCC TGC ACT GGG AGC AGC TCC AAC ATC GGG GCA ! BstEII...
1729 GGT TAT GAT GTA CAC TGG TAC CAG CAG CTT CCA GGA ACA GCC CCC
AAA 1777 CTC CTC ATC TAT GGT AAC AGC AAT CGG CCC TCA GGG GTC CCT
GAC CGA 1825 TTC TCT GGC TCC AAG TCT GGC ACC TCA GCC TCC CTG GCC
ATC ACT 1870 GGG CTC CAG GCT GAG GAT GAG GCT GAT TAT 1900 TAC TGC
CAG TCC TAT GAC AGC AGC CTG AGI 1930 GGC CTT TAT GTC TTC GGA ACT
GGG ACC AAG GTC ACC GTC ! BstEII... 1969 CTA GGT CAG CCC AAG GCC
AAC CCC ACT GTC ACT 2002 CTG TTC CCG CCC TCC TCT GAG GAG CTC CAA
GCC AAC AAG GCC ACA CTA 2050 GTG TGT CTG ATC AGT GAC TTC TAC CCG
GGA GCT GTG ACA GTG GCC TGG 2098 AAG GCA GAT AGC AGC CCC GTC AAG
GCG GGA GTG GAG ACC ACC ACA CCC 2146 TCC AAA CAA AGC AAC AAC AAG
TAC GCG GCC AGC AGC TAT CTG AGC CTG 2194 ACG CCT GAG CAG TGG AAG
TCC CAC AGA AGC TAC AGC TGC CAG GTC ACG 2242 CAT GAA GGG AGC ACC
GTG GAG AAG ACA GTG GCC CCT ACA GAA TGT 2290 TAA TAA ACCG
CCTCCACCGG GCGCGCCAAT TCTATTTCAA GGAGACAGTC ATA ! AscI..... ! !
PelB signal----------------------------------------> ! M K Y L L
P T A A A G L L L L 2343 ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT
GGA TTG TTA TTA CTC ! ! 16 17 18 19 20 21 22 ! A A Q P A M A 2388
gcG GCC cag ccG GCC atg gcc ! SfiI............. ! NgoMI...(1/2) !
NcoI......... ! ! FR1 (DP47/V3-23)--------------- ! 23 24 25 26 27
28 29 30 ! E V Q L L E S G 2409 gaa|gtt|CAA|TTG|tta|gag tct|ggt| !
|MfeI | ! !
--------------FR1-------------------------------------------- ! 31
32 33 34 35 36 37 38 39 40 41 42 43 44 45 ! G G L V Q P G G S L R L
S C A 2433
|ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct| ! !
----FR1---------------->|...CDR1................|---FR2------ !
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ! A S G F I F S S Y A
M S W V R 2478
|gct|TCC|GGA|ttc|act|ttc|tct|tCG|TAC|Gct|atg|tct|tgg|gtt|cgc| ! |
BspEI | | BsiwW| |BstXI. ! !
-------FR2-------------------------------->|...CDR2........ ! 61
62 63 64 65 66 67 68 69 70 71 72 73 74 75 ! Q A P G K G L K W V S A
I S G 2523
|CAa|gct|ccT|GGt|aaa|ggt|ttg|gag|tgg|gtt|tct|gct|atc|tct|ggt| !
...BstXI | ! !
.....CDR2............................................|---FR3--- !
76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 ! S G G S T Y Y A D S
K G R F 2568
|tct|ggt|ggc|agt|act|tac|tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc| ! ! !
--------FR3-------------------------------------------------- ! 91
92 93 94 95 96 97 98 99 100 101 102 103 104 105 ! T I S R D N S K N
I L Y L Q M 2613
|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| ! |
XbaI | ! !
---FR3----------------------------------------------------->| !
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 ! N S L
R A E D T A V Y Y C A K 2658
|aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa| !
|AflII | | PstI | ! !
.......CDR3.................|----FR4------------------------- ! 121
122 123 124 125 126 127 128 129 130 131 132 133 134 135 ! D Y E G T
G Y A F D I W G Q G 2703
|gac|tat|gaa|ggt|act|ggt|tat|gct|ttc|gaC|ATA|TGg|ggt|caa|ggt| ! |
NdeI |(1/4) ! ! --------------FR4---------->| ! 136 137 138 139
140 141 142 ! T M V I V S S 2748 |act|atG|GTC|ACC|gtc|tct|agt ! |
BstEII | ! From BstEII onwards, pV323 is same as pCES1, except as
noted. ! EstEII sites may occur in light chains; not likely to be
unique in final ! vector. ! ! 143 144 145 146 147 148 149 150 151
152 ! A S I K G P S V F P 2769 gcc ttc acc aaG GGC CCa tcg GTC TTC
ccc ! Bsp120I. BbsI...(2/2) ! ApaI.... ! ! 153 154 155 156 157 158
159 160 161 162 163 164 165 166 167 ! L A P S S K S T S G G T A A L
2799 ctg gca ccc TCC TCc aag agc acc tct ggg ggc aca gcg gcc ctg !
BseRI...(2/2) ! ! 168 169 170 171 172 173 174 175 176 177 178 179
180 181 182 ! G C L V K D Y F P E P 2844 ggc tgc ctg GTC AAG GAC
TAC TTC CCC gaA CCG GTg acg gtg tcg ! AgeI.... ! ! 183 184 185 186
187 188 189 190 191 192 193 194 195 196 197 ! W N S G A L I S G V H
T F P A 2889 tgg aac tca GGC GCC ctg acc agc ggc gtc cac acc ttc
ccg gct ! KasI...(1/4) ! ! 198 199 200 201 202 203 204 205 206 207
208 209 210 211 212 ! V L Q S S G L Y S L S S V V T 2934 gtc cta
cag tCt agc GGa ctc tac ttc ctc agc agc gta gtg acc !
(Bsu36I...)(knocked out) ! ! 213 214 215 216 217 218 219 220 221
222 223 224 225 226 227 ! V P S S S L G T Q T Y I C N V 2979 gtg
CCC tCt tct agc tTG Ggc acc cag acc tac ctc tgc cac gtg !
(BstXI...........)N.B. destruction of BstXI & BpmI sites. ! !
228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 ! N H K
P S N T K V D K 3024 aat cac aag ccc agc aac acc acg gtg gac aag
aaa gtt gag ccc ! ! 243 244 245 ! K S C A A A H H H H H H S A 3069
aaa tct tgt GCG GCC GCt cat cac cac cat cat cac tct gct !
NotI......
! ! E Q K L I S E E D L N G A A 3111 gaa caa aaa ctc ctc tca gaa
gag gct ctg act ggt gcc gcc ! ! ! D I N D D R M A S G A 3153 GAT
ATC acc gat gat cgt atg gct AGC ggc gcc ! rEK cleavage
site.......... NheI... KasI... ! EcoRV.. ! ! Domain 1
------------------------------------------------------------ ! A E
I V E S C L A 3183 gct gaa act gtt gac cgt tgt tta gca ! ! ! K P H
T E I S F 3210 caa ccc cat aca gcc aat tca ttt ! ! T N V W K D D K
T 3234 aCT AAC GTC TGG AAA GAC GAC AAA ACt ! ! L D R Y A N Y E G C
L W N A T G V 3261 tta gat cgt tac gct aac tat gag ggttgt ctg tgG
AAT GCt aca ggc gtt ! BsmI____ ! ! V V C T G D E T Q C Y G T W V P
I 3312 gta gtt tgt act ggt GAC GAA ACT CAG TGT TAC GGT ACA TGG GTT
cct att ! ! G L A I P E N 3363 ggg ctt gct atc cct gaa aat ! ! L1
linker ------------------------------------ ! E G G G S E G G G S
3384 gag ggt ggt ggc tct gag ggt ggc ggt tct ! ! E C G G S E G G G
S 3414 gag ggt ggc ggt tct gag ggt ggc ggt act ! ! Domain 2
------------------------------------ 3444 aaa cct cct gag tac
ggtgat aca cct att ccg ggc tat act tat atc aac 3495 cct ctc gac ggc
act tat ccg cct ggtact gag caa aac ccc gct aat cct 3546 aat cct tct
ctt GAG GAG tct cag cct ctt aat act ttc atg ttt cag aat ! EseRI
3597 aat agg ttc cga aat agg cag ggg gca tta act gtt tat acg ggc
act 3645 gtt act caa ggc act gac ccc gtt aaa act tat tac cag tac
act cct 3693 gta tca tca aaa gcc atg tat gac gct tac tgg aac ggt
aaa ttC AGA ! AlwNI 3741 GAC TGc gct ttc cat tct ggc ttt aat gaa
gat cca ttc gtt tgt gaa ! AlwNI 3789 tat caa ggc caa tcg tct gac
ctg cct caa cct cct gto aat gct ! 3834 ggc ggc ggc tct ! start L2
------------------------------------------------------------ 3846
ggt ggt ggt tct 3858 ggt ggc ggc tct 3870 gag ggt ggt ggc tct gag
ggt ggc ggt tct 3900 gag ggt ggc ggc tct gag gga ggc ggt tcc 3930
ggt ggt ggc tct ggt ! end L2 ! ! Domain 3
-------------------------------------------------------------- ! S
G D F D Y E K M A N A N K G A 3945 tcc ggt gat ttt gat tat gaa aag
atg gca aac gct aat aag ggg gct ! ! M T E N A D E N A L Q S D A K G
3993 atg acc gaa aat gcc gat gaa aac gcg cta cag tct gac gct aaa
ggc ! ! K L D S V A I D Y G A A I D G F 4041 aaa ctt gat tct gtc
gct act gat tac ggt gct gct atc gat ggt ttc ! ! I C D V S G L A N G
N G A T G D 4089 att ggt gac gtt tcc ggc ctt gct aat ggt aat ggt
gct act ggt gat ! ! F A C S N S Q M A Q V G D G D N 4137 ttt gct
ggc tct aat tcc caa atg gct caa gtc ggt gac ggt gat aat ! ! S P L M
N N F R Q Y L P S L P Q 4185 tca cct tta atg aat aat ttc cgt caa
tat tta cct tcc ctc cct caa ! ! S V K C R P F V F S A G K P Y E
4233 tcg gtt gaa tgt cgc cct ttt gtc ttt agc gct ggt aaa cca tat
gaa ! ! F S I U C U K I N L F R 4281 ttt tct att gat tgt gac aaa
ata aac tta ttc cgt ! End Domain 3 ! ! G V F A F L L Y V A T F M Y
V F140 4317 ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat
gta ttt ! start transmembrane segment ! ! S T F A N I L 4365 tct
acg ttt gct aac ata ctg ! ! R N K E S 4386 cgt aat aag gag tct TAA
! stop of iii ! Intracellular anchor. ! ! M1 P2 V L L5 G I P L L10
L R F L G15 4404 tc ATG cca gtt ctt ttg ggt att ccg tta tta ttg cgt
ttc ctc ggt ! Start VI ! 4451 ttc ctt ctg gta act ttg ttc ggc tat
ctg ctt act ttt ctt aaa aag 4499 ggc ttc ggt aag ata gct att gct
att tca ttg ttt ctt gct ctt att 4547 att ggg ctt aac tca att ctt
gtg ggt tat ctc tct gat att agc gct 4595 caa tta ccc tct gac ttt
gtt cag ggt gtt cag tta att ctc ccg tct 4643 aat gcg ctt ccc tgt
ttt tat gtt att ctc tct gta aag gct gct att 4691 ttc att ttt gac
gtt aaa caa aaa atc gtt tct tat ttg gat tgg gat ! ! M1 A2 V3 F5 L10
G13 4739 aaa TAA t ATG gct gtt tat ttt gta act ggc aaa tta ggc tct
gga ! end VI Start gene I ! ! 14 15 16 17 18 19 20 21 22 23 24 25
26 27 28 ! K T L V S V G K I Q D K I V A 4785 aag acg ctc gtt agc
gtt ggt acg att cag gat aaa att gta gct ! ! 29 30 31 32 33 34 35 36
37 38 39 40 41 42 43 ! G C K I A I N L D L P L Q N L 4830 ggg tgc
aaa ata gca act aat ctt gat tta agg ctt caa aac ctc ! ! 44 45 46 47
48 49 50 51 52 53 54 55 56 57 58 ! P Q V G R F A K T P R V L R I
4875 ccg caa gtc ggg agg ttc gct aaa acg cct cgc gtt ctt aga ata !
! 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 ! P D K P S I S D L
L A I G R G 4920 ccg gat aag cct tct ata tct gat ttg ctt gct att
ggg cgc ggt ! ! 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 ! N D
S Y D E N K N G L L V L V D 4965 aat gat tcc tac gat gaa aat aaa
aac ggc ttg ctt gtt ctc gat ! ! 89 90 91 92 93 94 95 96 97 98 99
100 101 102 103 ! E C G T W F N T R S W N D K E 5010 gag tgc ggt
act tgg ttt aat acc cgt tct tgg aat gat aag gaa ! ! 104 105 106 107
108 109 110 111 112 113 114 115 116 117 118 ! R Q P I I D W F L H A
R K L G 5055 aga cag ccg att att gat tgg ttt cta cat gct cgt aaa
tta gga ! ! 119 120 121 122 123 124 125 126 127 128 129 130 131 132
133 ! W D I I F L V Q D L S I V D K 5100 tgg gat att att ttt ctt
gtt cag gac tta tct att gtt gat aaa ! ! 134 135 136 137 138 139 140
141 142 143 144 145 146 147 148 ! Q A R S A L A E H V V Y C R R
5145 cag gcg cgt tct gca tta gct gaa cat gtt ggt tat tgt cgt cgt !
! 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 ! L D
R I T L P F V G I L Y S L 5190 ctg gac aga att act tta cct ttt gtc
ggt act tta tat tct ctt ! ! 164 165 166 167 168 169 170 171 172 173
174 175 176 177 178 ! I I G S K M P L P K L H V G V 5235 att act
ggc tcg aaa atg cct ctg cct aaa tta cat gtt ggc gtt ! ! 179 180 181
182 183 184 185 186 187 188 189 190 191 192 193 ! V K Y G D S Q L S
P I V E R W 5280 gtt aaa tat ggc gat tct caa tta agc cct act gtt
gag cgt tgg ! ! 194 195 196 197 198 199 200 201 202 203 204 205 206
207 208 ! L Y T G K N L Y N A Y D T K Q 5325 ctt tat act ggt aag
aat ttg tat aac gca tat gat act aaa cag ! ! 209 210 211 212 213 214
215 216 217 218 219 220 221 222 223 ! A F S S N Y D S G V Y S Y L I
5370 gct ttt tct agt aat tat gat ttc ggt gtt tat tct tat tta acg !
! 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 ! P Y
L S H G R Y F K P L N L G 5415 cct tat tta tca cac ggt cgg tat ttc
aaa cca tta aat tta ggt ! ! 239 240 241 242 243 244 245 246 247 248
249 250 251 252 253 ! Q K M K L I K I Y L K K F S R 5460 cag aag
atg aaa tta act aaa ata tat ttg aaa aag ttt tct cgc ! ! 254 255 256
257 258 259 260 261 262 263 264 265 266 267 268 ! V L C L A I G F A
S A F I Y S 5505 gtt ctt tgt ctt gcg att gga ttt gca tca gca ttt
aca tat agt ! ! 269 270 271 272 273 274 275 276 277 278 279 280 281
282 283 ! Y I T Q P K P E V K K V V S Q 5550 tat ata acc caa cct
aag ccg gag gtt aaa aag gta gtc tct cag ! ! 284 285 286 287 288 289
290 291 292 293 294 295 296 297 298 ! T Y D F D K F T I D S S Q R L
5595 acc tat gat ttt gat aaa ttc act att gac tct tct cag cgt ctt !
! 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 ! N L
S Y P Y V F K D S K G K L 5640 aat cta agc tat cgc tat gtt ttc aag
gat tct aag gga aaa TTA ! PacI ! ! 314 315 316 317 318 319 320 321
322 323 324 325 326 327 328 ! I N S D D L Q K Q G Y S L I Y 5685
ATT AAt agc gac gat tta cag aag caa ggt tat tca ctc aca tat ! PacI
49 ! 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 !
i I D L C I V S I K K G N S N E ! iv M1 K 5730 att gat tta tgt act
gtt ttc att aaa aaa ggt aat tca aAT Gaa ! Start IV ! ! 344 345 346
347 348 349 ! i I V K C N .End of I ! iv L3 L N5 V 17 N F V10 5775
att gtt aaa tgt aat TAA T TTT GTT ! IV continued..... 5800 ttc ttg
atg ttt ggt tca tca tct tct ttt gct cag gta att gaa atg 5848 aat
aat tcg cct ctg cgc gat ttt gta act tgg tat tca aag caa tca 5896
ggc gaa ttc ggt att ggt tct ccc gat gta aaa ggt act gtt act gta
5944 tat tca tct gac ggt aaa cct gaa aat cta cgc aat ttc ttt att
tct 5992 ggt tta cgt gct aat aat ttt gat atg ggt ggt tca att cct
tcc ata 6040 att cag aag tat aat cca aac aat cag gat tat att gat
gaa ttg cca 6088 tca tct gat aat cag gaa tat gat gat aat ttc gct
cct tct ggt ggt 6136 ttc ttt ggt ccg caa aat gat aat ggt act caa
act ttt aaa att att 6184 aac ggt cgg gca aag gat tta ata cga gtt
gtc gaa ttg ttt gta aag 6280 cta tta gtt ggt TCT gca cct aaa gat
att tta gat aac ctt cct caa ! ApaLI removed 6328 ttc ctt tct act
gtt gat ttg cca act gac cag ata ttg att gag ggt 6376 ttg ata ttt
gag gtt cag caa ggt gat gct tta gat ttt tca ttt gct 6424 gct ggc
tct cag cgt ggc act gtt gca ggc ggt gtt aat act gac cgc 6472 ctc
acc tct gtt tta tct tct gct ggt ggt tcg ttc ggt att ttt aat 6520
ggc gat gtt tta ggg cta tca gtt cgc gca tta aag act aat agc cat
6568 tca aaa ata ttg tct gtg cca cgt att ctt acg ctt tca ggt cag
aag 6616 ggt tct atc tct gtT GGC CAg aat gtc cct ttt att act ggt
cgt gtg ! MscI 6664 act ggt gaa tct gcc aat gta aat aat cca ttt cag
acg att gag cgt 6712 caa aat gta ggt att ttc atg agc gtt ttt cct
gtt gca atg gct ggc 6760 ggt aat att gtt ctg gat att acc agc aag
gcc gat agt ttg agt tct 6808 tct act cag gca agt gat gtt att act
aat caa aga agt att gct aca 6856 acg gtt aat ttg cgt gat gga cag
act ctt tta ctc ggt ggc ctc act 6904 gat tat aaa aac act tct caa
gat tct ggc gta ccg ttc ctg tct aaa 6952 atc oct tta atc ggc ctc
ctg ttt agc ttc cgc tct gat ttc aac gag 7000 gaa agc acg tta tac
gtg ctc gtc aaa gca acc ata gta cgc gcc ctg 7048 TAG cggcgcatt !
End IV 7060 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
gcgccctagc 7120 gcccgctcct ttcgctttct tcccttcctt tctcgccacg
ttcGCCGGCt ttccccgtca ! NgoMI_ 7180 agctctaaat cgggggctcc
ctttagggtt ccgatttagt gctttacggc acctcgaccc 7240 caaaaaactt
gatttgggtg atggttCACG TAGTGggcca tcgccctgat agacggtttt ! DraIII
7300 tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga ctcttgttcc
aaactggaac ! DrdI 7360 aacactcaac cctatctcgg gctattcttt tgatttataa
gggattttgc cgatttcgga 7420 accaccatca aacaggattt tcgcctgctg
gggcaaacca gcgtggaccg cttgctgcaa 7480 ctctctcagg gccaggcggt
gaagggcaat CAGCTGttgc cCGTCTCact ggtgaaaaga ! PvuII. BsmBI. 7540
aaaaccaccc tGGATCC AAGCTI ! BamHI HindIII (1/2) ! Insert carrying
bla gene 7563 gcaggtg gcacttttcg gggaaatgtg cgcggaaccc 7600
ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga caataaccct !
BciVI 7660 gataaatgct tcaataatat tgaaaaAGGA AGAgt ! RBSsI.?... !
Start bla gene 7695 ATG agt att caa cat ttc cgt gtc gcc ctt att ccc
ttt ttt gcg gca ttt 7746 tgc ctt cct gtt ttt gct cac cca gaa acg
ctg gtg aaa gta aaa gat gct 7797 gaa gat cag ttg ggc gCA CGA Gtg
ggt tac atc gaa ctg gat ctc aac agc ! BssSI... ! ApaLI removed 7848
ggt aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc
7899 act ttt aaa gtt ctg cta tgt cat aca cta tta ttc cgt att gac
gcc ggg 7950 caa gaG CAA CTC GGT CGc cgg gcg cgg tat tct cag aat
gac ttg gtt gAG ! BcgI ScaI 8001 TAC Tca cca gtc aca gaa acg cat
ctt acg gat ggc atg aca gta aga gaa ! ScaI 8052 tta tgc agt gct gcc
ata acc atg agt gat aac act gcg gcc aac tta ctt 8103 ctg aca aCG
ATC Gga gga ccg aag gag cta acc gct ttt ttg cac aac atg ! PvuI 8154
ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc
8205 ata cca aac gac gag cgt gac acc acg atg cct gta gca atg cca
aca acg 8256 tTG CGC Aaa cta tta act ggc gaa cta ctt act cta gct
ttc cgg caa caa ! FspI.... ! 8307 tta ata gac tgg atg gag gcg gat
aaa gtt gca gga cca ctt ctg cgc tcg 8358 GCC ctt ccG GOt ggc tgg
ttt att gct gat aaa tct gga gcc ggt gag cgt ! BglI 8409 gGG TCT Cgc
ggt atc att gca gca ctg ggg cca gat ggt aag ccc ttc cgt ! BsaI 8460
atc gta gtt atc tac acG ACg ggg aGT Cag gca act atg gat gaa cga aat
! AhdI 8511 aga cag atc gct gag ata ggt gcc tca ctg att aag cat tgg
TAA ctgt ! stop 8560 cagaccaagt ttactcatat atactttaga ttgatttaaa
acttcatttt taatttaaaa 8620 ggatctaggt gaagatcctt tttgataatc
tcatgaccaa aatcccttaa cgtgagtttt 8680 cgttccactg tacgtaagac cccc
8704 AAGCTT GTCGAC tgaa tggcgaatgg cgctttgcct ! HindIII SalI.. !
(2/2) HincII 8740 ggtttccggc accagaagcg gtgccggaaa gctggctgga
gtgcgatctt ! 8790 CCTGAGG ! Bsu36I_ 8797 ccgat actgtcgtcg
tcccctcaaa ctggcagatg 8832 cacggt tacg atgcgcccat ctacaccaac
gtaacctatc ccattacggt caatccgccg 8892 tttgttccca cggagaatcc
gacgggttgt tactcgctca catttaatgt tgatgaaagc 8952 tggctacagg
aaggccagac gcgaattatt tttgatggcg ttcctattgg ttaaaaaatg 9012
agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaATTTAAA !
SwaI... 9072 Tatttgctta tacaatcttc ctgtttttgg ggcttttctg attatcaacc
GGGGTAcat ! RBS 9131 ATG att gac atg cta gtt tta cga tta ccg ttc
atc gat tct ctt gtt tgc ! Start gene II 9182 ttc aga ctc tca ggc
aat gac ctg ata gcc ttt gtA GAT CTc tca aaa ata ! BglII... 9233 gct
acc ctc ttc ggc atg aat tta tca gct aga acg gtt gaa tat cat att
9284 gat ggt gat ttg act gtc ttc ggc ctt tct cac cct ttt gaa tct
tta cct 9335 aca cat tac tca ggc att gca ttt aaa ata tat gag ggt
tct aaa aat ttt 9386 tat cct tgc gtt gaa ata aag gct tct ccc gca
aaa gta tta cag ggt cat 9437 aat gtt ttt ggt aca acc gat tta gct
tta tgc tct gag gct tta ttg ctt 9488 aat ttt gct aat tct ttg cct
tgc ctg tat gat tta ttg gat gtt ! 9532 ! gene II continues
[0308] TABLE-US-00027 TABLE 212 Sequence of MALIA3, condensed LOCUS
ORIGIN MALIA3 9532 CIRCULAR 1 AATGCTACTA CTATTAGTAG AATTGATGCC
ACCTTTTCAG CTCGCGCCCC AAATGAAAAI 61 ATAGCTAAAC AGGTTATTGA
CCATTTGCGA AATGTATCTA ATGGTCAAAC TAAATCTACT 121 CGTTCGCAGA
ATTGGGAATC AACTGTTACA TGGAATGAAA CTTCCAGACA CCGTACTTTA 181
GTTGCATATT TAAAACATGT TGAGCTACAG CACCAGATTC AGCAATTAAG CTCTAAGCCA
241 TCCGCAAAAA TGACCTCTTA TCAAAAGGAG CAATTAAAGG TACTCTCTAA
TCCTGACCTG 361 TCTTTCGGGC TTCCTCTTAA TCTTTTTGAT GCAATCCGCT
TTGCTTCTGA CTATAATAGT 421 CAGGGTAAAG ACCTGATTTT TGATTTATGG
TCATTCTCGT TTTCTGAACT GTTTAAAGCA 481 TTTGAGGGGG ATTCAATGAA
TATTTATGAC GATTCCGCAG TATTGGACGC TATCCAGTCT 541 AAACATTTTA
CTATTACCCC CTCTGGCAAA ACTTCTTTTG CAAAAGCCTC TCGCTATTTT 601
GGTTTTTATC GTCGTCTGGT AAACGAGGGT TATGATAGTG TTGCTCTTAC TATGCCTCGT
661 AATTCCTTTT GGCGTTATGT ATCTGCATTA GTTGAATGTG GTATTCCTAA
ATCTCAACTG 721 ATGAATCTTT CTACCTGTAA TAATGTTGTT CCGTTAGTTC
&TTTTATTAA CGTAGATTTT 781 TCTTCCCAAC GTCCTGACTG GTATAATGAG
CCAGTTCTTA AAATCGCATA AGGTAATTCA 841 CAATGATTAA AGTTGAAATT
AAACCATCTC AAGCCCAATT TACTACTCGT TCTGGTGTTT 901 CTCGTCAGGG
CAAGCCTTAT TCACTGAATG AGCAGCTTTG TTACGTTGAT TTGGGTAATG 961
AATATCCGGT TCTTGTCAAG ATTACTCTTG ATGAAGGTCA GCCAGCCTAT GCGCCTGGTC
1021 TGTACACCGT TCATCTGTCC TCTTTCAAAG TTGGTCAGTT CGGTTCCCTT
ATGATTGACC 1081 GTCTGCGCCT CGTTCCGGCT AAGTAACATG GAGCAGGTCG
CGGATTTCGA CACAATTTAT 1141 CAGGCGATGA TACAAATCTC CGTTGTACTT
TGTTTCGCGC TTGGTATAAT CGCTGGGGGT 1201 CAAAGATGAG TGTTTTAGTG
TATTCTTTCG CCTCTTTCGT TTTAGGTTGG TGCCTTCGTA 1261 GTGGCATTAC
GTATTTTACC CGTTTAATGG AAACTTCCTC ATGAAAAAGT CTTTAGTCCT 1321
CAAAGCCTCT GTAGCCGTTG CTACCCTCGT TCCGATGCTG TCTTTCGCTG CTGAGGGTGA
1381 CGATCCCGCA AAAGCGGCCT TTAACTCCCT GCAAGCCTCA GCGACCGAAT
ATATCGGTTA 1441 TGCGTGGGCG ATGGTTGTTG TCATTGTCGG CGCAACTATC
GGTATCAAGC TGTTTAAGAA 1501 ATTCACCTCG AAAGCAAGCT GATAAACCGA
TACAATTAAA GGCTCCTTTT GGAGCCTTTT 1561 TTTTTGGAGA TTTTCAACGT
GAAAAAATTA TTATTCGCAA TTCCTTTAGT TGTTCCTTTC 1621 TATTCTCACA
GTGCACAGTC TGTCGTGACG CAGCCGCCCT CAGTGTCTGG GGCCCCAGGG 1681
CAGAGGGTCA CCATCTCCTG CACTGGGAGC AGCTCCAACA TCGGGGCAGG TTATGATGTA
1741 CACTGGTACC AGCAGCTTCC AGGAACAGCC CCCAAACTCC TCATCTATGG
TAACAGCAAT 1801 CGGCCCTCAG GGGTCCCTGA CCGATTCTCT GGCTCCAAGT
CTGGCACCTC AGCCTCCCTG 1861 GCCATCACTG GGCTCCAGGC TGAGGATGAG
GCTGATTATT ACTGCCAGTC CTATGACAGC 1921 AGCCTGAGTG GCCTTTATGT
CTTCGGAACT GGGACCAAGG TCACCGTCCT AGGTCAGCCC 1981 AAGGCCAACC
CCACTGTCAC TCTGTTCCCG CCCTCCTCTG AGGAGCTCCA AGCCAACAAG 2041
GCCACACTAG TGTGTCTGAT CAGTGACTTC TACCCGGGAG CTGTGACAGT GGCCTGGAAG
2101 GCAGATAGCA GCCCCGTCAA GGCGGGAGTG GAGACCACCA CACCCTCCAA
ACAAAGCAAC 2161 AACAAGTACG CGGCCAGCAG CTATCTGAGC CTGACGCCTG
AGCAGTGGAA GTCCCACAGA 2221 AGCTACAGCT GCCAGGTCAC GCATGAAGGG
AGCACCGTGG AGAAGACAGT GGCCCCTACA 2281 GAATGTTCAT AATAAACCGC
CTCCACCGGG CGCGCCAATT CTATTTCAAG GAGACAGTCA 2341 TAATGAAATA
CCTATTGCCT ACGGCAGCCG CTGGATTGTT ATTACTCGCG GCCCAGCCGG 2401
CCATGGCCGA AGTTCAATTG TTAGAGTCTG GTGGCGGTCT TGTTCAGCCT GGTGGTTCTT
2461 TACGTCTTTC TTGCGCTGCT TCCGGATTCA CTTTCTCTTC GTACGCTATG
TCTTGGGTTC 2521 GCCAAGCTCC TGGTAAAGGT TTGGAGTGGG TTTCTGCTAT
CTCTGGTTCT GGTGGCAGTA 2581 CTTACTATGC TGACTCCGTT AAAGGTCGCT
TCACTATCTC TAGAGACAAC TCTAAGAATA 2641 CTCTCTACTT GCAGATGAAC
AGCTTAAGGG CTGAGGACAC TGCAGTCTAC TATTGCGCTA 2701 AAGACTATGA
AGGTACTGGT TATGCTTTCG ACATATGGGG TCAAGGTACT ATGGTCACCG 2761
TCTCTAGTGC CTCCACCAAG GGCCCATCGG TCTTCCCCCT GGCACCCTCC TCCAAGAGCA
2821 CCTCTGGGGG CACAGCGGCC CTGGGCTGCC TGGTCAAGGA CTACTTCCCC
GAACCGGTGA 2881 CGGTGTCGTG GAACTCAGGC GCCCTGACCA GCGGCGTCCA
CACCTTCCCG GCTGTCCTAC 2941 AGTCTAGCGG ACTCTACTCC CTCAGCAGCG
TAGTGACCGT GCCCTCTTCT AGCTTGGGCA 3001 CCCAGACCTA CATCTGCAAC
GTGAATCACA AGCCCAGCAA CACCAAGGTG GACAAGAAAG 3061 TTGAGCCCAA
ATCTTGTGCG GCCGCTCATC ACCACCATCA TCACTCTGCT GAACAAAAAC 3121
TCATCTCAGA AGAGGATCTG AATGGTGCCG CAGATATCAA CGATGATCGT ATGGCTGGCG
3181 CCGCTGAAAC TGTTGAAAGT TGTTTAGCAA AACCCCATAC AGAAAATTCA
TTTACTAACG 3241 TCTGGAAAGA CGACAAAACT TTAGATCGTT ACGCTAACTA
TGAGGGTTGT CTGTGGAATG 3301 CTACAGGCGT TGTAGTTTGT ACTGGTGACG
AAACTCAGTG TTACGGTACA TGGGTTCCTA 3361 TTGGGCTTGC TATCCCTGAA
AATGAGGGTG GTGGCTCTGA GGGTGGCGGT TCTGAGGGTG 3421 GCGGTTCTGA
GGGTGGCGGT ACTAAACCTC CTGAGTACGG TGATACACCT ATTCCGGGCT 3481
ATACTTATAT CAACCCTCTC GACGGCACTT ATCCGCCTGG TACTGAGCAA AACCCCGCTA
3541 ATCCTAATCC TTCTCTTGAG GAGTCTCAGC CTCTTAATAC TTTCATGTTT
CAGAATAATA 3601 GGTTCCGAAA TAGGCAAGGG GCATTAACTG TTTATACGGG
CACTGTTACT CAAGGCACTG 3661 ACCCCGTTAA AACTTATTAC CAGTACACTC
CTGTATCATC AAAAGCCATG TATGACGCTT 3721 ACTGGAACGG TAAATTCAGA
GACTGCGCTT TCCATTCTGG CTTTAATGAA GATCCATTCG 3781 TTTGTGAATA
TCAAGGCCAA TCGTCTGACC TGCCTCAACC TCCTGTCAAT GCTGGCGGCG 3841
GCTCTGGTCG TGGTTCTGGT GGCGGCTCTG AGGGTGGTGG CTCTGAGGGT GGCGGTTCTG
3901 AGGGTGGCGG CTCTGAGGGA GGCGGTTCCG GTGGTGGCTC TGGTTCCGGT
GATTTTGATT 3961 ATGAAAAGAT GGCAAACGCT AATAAGGGGG CTATGACCGA
AAATGCCGAT GAAAACGCGC 4021 TACAGTCTGA CGCTAAAGGC AAACTTGATT
CTGTCGCTAC TGATTACGGT GCTGCTATCG 4081 ATGGTTTCAT TGGTGACGTT
TCCGGCCTTG CTAATGGTAA TGGTGCTACT GGTGATTTTG 4141 CTGGCTCTAA
TTCCCAAATG GCTCAAGTCG GTGACGGTGA TAATTCACCT TTAATGAATA 4201
ATTTCCGTCA ATATTTACCT TCCCTCCCTC AATCGGTTGA ATGTCGCCCT TTTGTCTTTA
4261 GCGCTGGTAA ACCATATGAA TTTTCTATTG ATTGTGACAA AATAAACTTA
TTCCGTGGTG 4321 TCTTTGCGTT TCTTTTATAT GTTGCCACCT TTATGTATGT
ATTTTCTACG TTTGCTAACA 4381 TACTGCGTAA TAAGGAGTCT TAATCATGCC
AGTTCTTTTG GGTATTCCGT TATTATTGCG 4441 TTTCCTCGGT TTCCTTCTGG
TAACTTTGTT CGGCTATCTG CTTACTTTTC TTAAAAAGGG 4501 CTTCGGTAAG
ATAGCTATTG CTATTTCATT GTTTCTTGCT CTTATTATTG GGCTTAACTC 4561
AATTCTTGTG GGTTATCTCT CTGATATTAG CGCTCAATTA CCCTCTGACT TTGTTCAGGG
4621 TGTTCAGTTA ATTCTCCCGT CTAATGCGCT TCCCTGTTTT TATGTTATTC
TCTCTGTAAA 4681 GGCTGCTATT TTCATTTTTG ACGTTAAACA AAAAATCGTT
TCTTATTTGG ATTGGGATAA 4741 ATAATATGGC TGTTTATTTT GTAACTGGCA
AATTAGGCTC TGGAAAGACG CTCGTTAGCG 4801 TTGGTAAGAT TCAGGATAAA
ATTGTAGCTG GGTGCAAAAT AGCAACTAAT CTTGATTTAA 4861 GGCTTCAAAA
CCTCCCGCAA GTCGGGAGGT TCGCTAAAAC GCCTCGCGTT CTTAGAATAC 4921
CGGATAAGCC TTCTATATCT GATTTGCTTG CTATTGGGCG CGGTAATGAT TCCTACGATG
4981 AAAATAAAAA CGGCTTGCTT GTTCTCGATG AGTGCGGTAC TTGGTTTAAT
ACCCGTTCTT 5041 GGAATGATAA GGAAAGACAG CCGATTATTG ATTGGTTTCT
ACATGCTCGT AAATTAGGAT 5101 GGGATATTAT TTTTCTTGTT CAGGACTTAT
CTATTGTTGA TAAACAGGCG CGTTCTGCAT 5161 TAGCTGAACA TGTTGTTTAT
TGTCGTCGTC TGGACAGAAT TACTTTACCT TTTGTCGGTA 5221 CTTTATATTC
TCTTATTACT GGCTCGAAAA TGCCTCTGCC TAAATTACAT GTTGGCGTTG 5281
TTAAATATGG CGATTCTCAA TTAAGCCCTA CTGTTGAGCG TTGGCTTTAT ACTGGTAAGA
5341 ATTTGTATAA CGCATATGAT ACTAAACAGG CTTTTTCTAG TAATTATGAT
TCCGGTGTTT 5401 ATTCTTATTT AACGCCTTAT TTATCACACG GTCGGTATTT
CAAACCATTA AATTTAGGTC 5461 AGAAGATGAA ATTAACTAAA ATATATTTGA
AAAAGTTTTC TCGCGTTCTT TGTCTTGCGA 5521 TTGGATTTGC ATCAGCATTT
ACATATAGTT ATATAACCCA ACCTAAGCCG GAGGTTAAAA 5581 AGGTAGTCTC
TCAGACCTAT GATTTTGATA AATTCACTAT TGACTCTTCT CAGCGTCTTA 5641
ATCTAAGCTA TCGCTATGTT TTCAAGGATT CTAAGGGAAA ATTAATTAAT AGCGACGATT
5701 TACAGAAGCA AGGTTATTCA CTCACATATA TTGATTTATG TACTGTTTCC
ATTAAAAAAG 5761 GTAATTCAAA TGAAATTGTT AAATGTAATT AATTTTGTTT
TCTTGATGTT TGTTTCATCA 5821 TCTTCTTTTG CTCAGGT0AT TGAAATGAAT
AATTCGCCTC TGCGCGATTT TGTAACTTGG 5881 TATTCAAAGC AATCAGGCGA
ATCCGTTATT GTTTCTCCCG ATGTAAAAGG TACTGTTACT 5941 GTATATTCAT
CTGACGTTAA ACCTGAAAAT CTACGCAATT TCTTTATTTC TGTTTTACGT 6001
GCTAATAATT TTGATATGGT TGGTTCAATT CCTTCCATAA TTCAGAAGTA TAATCCAAAC
6061 AATCAGGATT ATATTGATGA ATTGCCATCA TCTGATAATC AGGAATATGA
TGATAATTCC 6121 GCTCCTTCTG GTGGTTTCTT TGTTCCGCAA AATGATAATG
TTACTCAAAC TTTTAAAATT 6181 AATAACGTTC GGGCAAAGGA TTTAATACGA
GTTGTCGAAT TGTTTGTAAA GTCTAATACT 6241 TCTAAkTCCT CAAATGTATT
ATCTATTGAC GGCTCTAATC TATTAGTTGT TTCTGCACCT 6301 AAAGATATTT
TAGATAACCT TCCTCAATTC CTTTCTACTG TTGATTTGCC AACTGACCAG 6361
ATATTGATTG AGGGTTTGAT ATTTGAGGTT CAGCAAGGTG ATGCTTTAGA TTTTTCATTT
6421 GCTGCTGGCT CTCAGCGTGG CACTGTTGCA GGCGGTGTTA ATACTGACCG
CCTCACCTCT 6481 GTTTTATCTT CTGCTGGTGG TTCGTTCGGT ATTTTTAATG
GCGATGTTTT AGGGCTATCA 6541 GTTCGCGCAT TAAAGACTAA TAGCCATTCA
AAAATATTGT CTGTGCCACG TATTCTTACG 6601 CTTTCAGGTC AGAAGGGTTC
TATCTCTGTT GGCCAGAATG TCCCTTTTAT TACTGGTCGT 6661 GTGACTGGTG
AATCTGCCAA TGTAAATAAT CCATTTCAGA CGATTGAGCG TCAAAATGTA 6721
GGTATTTCCA TGAGCGTTTT TCCTGTTGCA ATGGCTGGCG GTAATATTGT TCTGGATATT
6781 ACCAGCAAGG CCGATAGTTT GAGTTCTTCT ACTCAGGCAA GTGATGTTAT
TACTAATCAA 6841 AGAAGTATTG CTACAACGGT TAATTTGCGT GATGGACAGA
CTCTTTTACT CGGTGGCCTC 6901 ACTGATTATA AAAACACTTC TCAAGATTCT
GGCGTACCGT TCCTGTCTAA AATCCCTTTA 6961 ATCGGCCTCC TGTTTAGCTC
CCGCTCTGAT TCCAACGAGG AAAGCACGTT ATACGTGCTC 7021 GTCAAAGCAA
CCATAGTACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGG GTGTGGTGGT 7081
TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT TCGCTTTCTT
7141 CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA GCTCTAAATC
GGGGGCTCCC 7201 TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC
AAAAAACTTG ATTTGGGTGA 7261 TGGTTCACGT AGTGGGCCAT CGCCCTGATA
GACGGTTTTT CGCCCTTTGA CGTTGGAGTC
7321 CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA ACACTCAACC
CTATCTCGGG 7381 CTATTCTTTT GATTTATAAG GGATTTTGCC GATTTCGGAA
CCACCATCAA ACAGGATTTT 7441 CGCCTGCTGG GGCAAACCAG CGTGGACCGC
TTGCTGCAAC TCTCTCAGGG CCAGGCGGTG 7501 AAGGGCAATC AGCTGTTGCC
CGTCTCACTG GTGAAAAGAA AAACCACCCT GGATCCAAGC 7561 TTGCAGGTGG
CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA 7621
TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT
7681 GAAAAAGGAA GAGTATGAGT ATTCAACATT TCCGTGTCGC CCTTATTCCC
TTTTTTGCGG 7741 CATTTTGCCT TCCTGTTTTT GCTCACCCAG AAACGCTGGT
GAAAGTAAAA GATGCTGAAG 7801 ATCAGTTGGG CGCACGAGTG GGTTACATCG
AACTGGATCT CAACAGCGGT AAGATCCTTG 7861 AGAGTTTTCG CCCCGAAGAA
CGTTTTCCAA TGATGAGCAC TTTTAAAGTT CTGCTATGTC 7921 ATACACTATT
ATCCCGTATT GACGCCGGGC AAGAGCAACT CGGTCGCCGG GCGCGGTATT 7981
CTCAGAATGA CTTGGTTGAG TACTCACCAG TCACAGAAAA GCATCTTACG GATGGCATGA
8041 CAGTAAGAGA ATTATGCAGT GCTGCCATAA CCATGAGTGA TAACACTGCG
GCCAACTTAC 8101 TTCTGACAAC GATCGGAGGA CCGAAGGAGC TAACCGCTTT
TTTGCACAAC ATGGGGGATC 8161 ATGTAACTCG CCTTGATCGT TGGGAACCGG
AGCTGAATGA AGCCATACCA AACGACGAGC 8221 GTGACACCAC GATGCCTGTA
GCAATGCCAA CAACGTTGCG CAAACTATTA ACTGGCGAAC 8281 TACTTACTCT
AGCTTCCCGG CAACAATTAA TAGACTGGAT GGAGGCGGAT AAAGTTGCAG 8341
GACCACTTCT GCGCTCGGCC CTTCCGGCTG GCTGGTTTAT TGCTGATAAA TCTGGAGCCG
8401 GTGAGCGTGG GTCTCGCGGT ATCATTGCAG CACTGGGGCC AGATGGTAAG
CCCTCCCGTA 8461 TCGTAGTTAT CTACACGACG GGGAGTCAGG CAACTATGGA
TGAACGAAAT AGACAGATCG 8521 CTGAGATAGG TGCCTCACTG ATTAAGCATT
GGTAACTGTC AGACCAAGTT TACTCATATA 8581 TACTTTAGAT TGATTTAAAA
CTTCATTTTT AAATTTAAAG GATCTAGGTG AAGATCCTTT 8641 TTGATAATCT
CATGACCAAA ATCCCTTAAC GTGAGTTTTC GTTCCACTGT ACGTAAGACC 8701
CCCAAGCTTG TCGACTGAAT GGCGAATGGC GCTTTGCCTG GTTTCCGGCA CCAGAAGCGG
8761 TGCCGGAAAG CTGGCTGGAG TGCGATCTTC CTGAGGCCGA TACTGTCGTC
GTCCCCTCAA 8821 ACTGGCAGAT GCACGGTTAC GATGCGCCCA TCTACACCAA
CGTAACCTAT CCCATTACGG 8881 TCAATCCGCC GTTTGTTCCC ACGGAGAATC
CGACGGGTTG TTACTCGCTC ACATTTAATG 8941 TTGATGAAAG CTGGCTACAG
GAAGGCCAGA CGCGAATTAT TTTTGATGGC GTTCCTATTG 9001 GTTAAAAAAT
GAGCTGATTT AACAAAAATT TAACGCGAAT TTTAACAAAA TATTAACGTT 9061
TACAATTTAA ATATTTGCTT ATACAATCTT CCTGTTTTTG GGGCTTTTCT GATTATCAAC
9121 CGGGGTACAT ATGATTGACA TGCTAGTTTT ACGATTACCG TTCATCGATT
CTCTTGTTTG 9181 CTCCAGACTC TCAGGCAATG ACCTGATAGC CTTTGTAGAT
CTCTCAAAAA TAGCTACCCT 9241 CTCCGGCATG AATTTATCAG CTAGAACGGT
TGAATATCAT ATTGATGGTG ATTTGACTGT 9301 CTCCGGCCTT TCTCACCCTT
TTGAATCTTT ACCTACACAT TACTCAGGCA TTGCATTTAA 9361 AATATATGAG
GGTTCTAAAA ATTTTTATCC TTGCGTTGAA ATAAAGGCTT CTCCCGCAAA 9421
AGTATTACAG GGTCATAATG TTTTTGGTAC AACCGATTTA GCTTTATGCT CTGAGGCTTT
9481 ATTGCTTAAT TTTGCTAATT CTTTGCCTTG CCTGTATGAT TTATTGGATG TT
[0309] TABLE-US-00028 TABLE 22 Primers used in RACE amplification:
Heavy chain Huc.mu.-FOR (1st PCR) 5'-TGG AAG AGG CAC GTT CTT TTC
TTT-3' HuC.mu.-Nested (2nd PCR) 5'-CTT TTC TTT GTT GCC GTT GGG
GTG-3' Kappa light chain HuCkFor (1st PCR) 5'-ACA CTC TCC CCT GTT
GAA GCT CTT-3' HuCkForAscI (2nd PCR) 5'-ACC GCC TCC ACC GGG CGC GCC
TTA TTA ACA CTC TCC CCT GTT GAA GCT CTT-3' Lamba light chain
HuClambdaFor (1st PCR) HuCL2-FOR 5'-TGA ACA TTC TGT AGG GGC CAC
TG-3' HuCL7-FOR 5'-AGA GCA TTC TGC AGG GGC CAC TG-3'
HuClambdaForAscI (2nd PCR) HuCL2-FOR-ASC 5'-ACC GCC TCC ACC GGG CGC
GCC TTA TTA TGA ACA TTC TGT AGG GGC CAC TG-3' HuCL7-FOR-ASC 5'-ACC
GCC TCC ACC GGG CGC GCC TTA TTA AGA GCA TTC TGC AGG GGC CAC TG-3'
GeneRAcer 5' Primers provided with the kit (Invitrogen) 5'A 1st PCR
5'CGACTGGAGCACGAGGACACTGA 3' 5'NA 2nd pCR
5'GGACACTGACATGGACTGAAGGAGTA-3'
[0310] TABLE-US-00029 TABLE 23 ONs used in Capture of kappa light
chains using CJ method and BsmAI REdapters (6) ON_20SK15012
gggAggATggAgAcTgggTc ON_20SK15L12 gggAAgATggAgAcTgggTc ON_20SK15A17
gggAgAgTggAgAcTgAgTc ON_20SK15A27 gggTgccTggAgAcTgcgTc ON_20SK15A11
gggTggcTggAgAcTgcgTc ON_20SK15B gggAgTcTggAgAcTgggTc Bridges (6)
kapbri1012 gggAggATggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
kapbrilL12 gggAAgATggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
kapbrilA17 gggTgccTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
kapbrilA27 gggTgccTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
kapbrilA11 gggTggcTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
kapbri1B3 gggAgTcTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg
Extender (5' biotinylated) kapextlbio
ccTcTgTcAcAgTgcAcAAgAcATccAgATgAcccAgTcTcc Primers kaPCRt1
ccTcTgTcAcAgTgcAcAAgAc kapfor 5'-aca ctc tcc cct gtt gaa gct ctt-3'
All ONs are written 5' to 3'.
[0311] TABLE-US-00030 TABLE 24 PCR program for amplification of
kappa DNA 95.degree. C. 5 minutes 95.degree. C. 15 seconds
65.degree. C. 30 seconds 72.degree. C. 1 minute 72.degree. C. 7
minutes 4.degree. C. hold Reagents (100 ul reaction): Template 50
ng 10x turbo PCR buffer 1x turbo Pfu 4U dNTPs 200 .mu.M each
kaPCRt1 300 nM kapfor 300 nM
[0312] TABLE-US-00031 TABLE 25 h3401-h2 captured Via Cl with BsmAI
! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ! S A Q D I Q M T Q S P A T L
S aGT GCA Caa gac atc cag atg ace cag tct cca gcc acc ctg tct !
ApaLI... a gcc acc ! L25,L6,L20,L2,L16,A11 !
Extender.................................Bridge... ! 16 17 18 19 20
21 22 23 24 25 26 27 28 29 30 ! I V S P G E R A T L S C R A S Q gtg
tct cca ggg gaa agg gcc acc ctc ttc tgc agg gcc agt cag ! 31 32 33
34 35 36 37 38 39 40 41 42 43 44 45 ! S V S N N L A W Y Q Q K P G Q
agt gtt agt aac aac tta gcc tgg tac cag cag aaa cct ggc cag ! 46 47
48 49 50 51 52 53 54 55 56 57 58 59 60 ! V P R L L I Y G A S T R A
T D gtt ccc agg ctc ctc atc tat ggt gca ttc acc agg gcc act gat !
61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 ! I P A R F S G S G S
G T D F T atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc
act ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 ! L T I S R L E
P E D F A V Y Y ctc acc atc agc aga ctg gag cct gaa gat ttt gca gtg
tat tac ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 ! C Q
R Y G S S P G W T F G Q G tgt cag cgg tat ggt agc tca ccg ggg tgg
acg ttc ggc caa ggg ! 106 107 108 109 110 111 112 113 114 115 116
117 118 119 120 ! T K V E I K R T V A A P S V F acc aag gtg gaa atc
aaa cga act gtg gct gca cca tct gtc ttc ! 121 122 123 124 125 126
127 128 129 130 131 132 133 134 135 ! I F P P S D E Q L K S G T A S
atc ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct ! 136
137 138 139 140 141 142 143 144 145 146 147 148 149 150 ! V V C L L
N N F Y P R E A K V gtt gtg tgc ctg clg aat aac ttc tat ccc aga gag
gcc aaa gta ! 151 152 153 154 155 156 157 158 159 160 161 162 163
164 165 ! Q W K V D N A L Q S G N S Q E cag tgg aag gtg gat aac gcc
ctc caa tcg ggt aac ttc cag gag ! 166 167 168 169 170 171 172 173
174 175 176 177 178 179 180 ! S V T E Q D S K D S T Y S L S agt gtc
aca gag cag gac agc aag gac agc acc tac agc ctc agc ! 181 182 183
184 185 186 187 188 189 190 191 192 193 194 195 ! S T L T L S K A D
Y E K H K V agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa
gtc ! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 !
Y A C E V T H Q G L S S P V I tac gcc tgc gaa gtc acc cat cag ggc
ctg agc tcg cct gtc aca ! 211 212 213 214 215 216 217 218 219 220
221 222 223 ! K S F N K G E C K G E F A aag agc ttc aac aaa gga gag
tgt aag ggc gaa ttc gc.....
[0313] TABLE-US-00032 TABLE 26 h3401-d8 KAPPA captured with CJ and
BsmAI ! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ! S A Q D I Q M T Q S P
A T L S aGT GCA Caa gac atc cag atg acc cag tct cct gcc acc ctg tct
! ApaLI...Extender...........................a gcc acc !
L25,L6,L20,L2,L16,A11 ! A GCC ACC CTG TCT ! L2 ! 16 17 18 19 20 21
22 23 24 25 26 27 28 29 30 ! V S P G E R A T L S C R A S Q gtg tct
cca ggt gaa aga gcc acc dc ttc tgc agg gcc agt cag ! GTG TCT CCA
GGG GAA AGA GCC ACC CTC TCC TGC ! L2 ! 31 32 33 34 35 36 37 38 39
40 41 42 43 44 45 ! N L L S N L A W Y Q Q K P G Q aat cct ctc agc
aac tta gcc tgg tac cag cag aaa cct ggc cag ! 46 47 48 49 50 51 52
53 54 55 56 57 58 59 60 ! A P R L L I Y G A S T G A I G gct ccc agg
ctc ctc atc tat ggt gct ttc acc ggg gcc att ggt ! 61 62 63 64 65 66
67 68 69 70 71 72 73 74 75 ! I P A R F S G S G S G T E F I atc cca
gcc agg ttc agt ggc agt ggg tct ggg aca gag ttc act ! 76 77 78 79
80 81 82 83 84 85 86 87 88 89 90 ! L T I S S L Q S E D F A V Y F
ctc acc atc agc agc ctg cag tct gaa gat ttt gca gtg tat ttc ! 91 92
93 94 95 96 97 98 99 100 101 102 103 104 105 ! C Q Q Y G T S P P T
F G G G I tgt cag cag tat ggt acc tca ccg ccc act ttc ggc gga ggg
acc ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 !
K V E I K R T V A A P S V F I aag gtg gag atc aaa cga act gtg gct
gca cca tct gtc ttc atc ! 121 122 123 124 125 126 127 128 129 130
131 132 133 134 135 ! F P P S D E Q L K S G T A S V ttc ccg cca tct
gat gag cag ttg aaa tct gga act gcc tct gtt ! 136 137 138 139 140
141 142 143 144 145 146 147 148 149 150 ! V C P L N N F Y P R E A K
V Q gtg tgc ccg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag !
151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 ! W K V
D N A L Q S G N S Q E S tgg aag gtg gat aac gcc ctc caa tcg ggt aac
ttc cag gag agt ! 166 167 168 169 170 171 172 173 174 175 176 177
178 179 180 ! V T E Q D N K D S T Y S L S S gtc aca gag cag gac aac
aag gac agc acc tac agc ctc agc agc ! 181 182 183 184 185 186 187
188 189 190 191 192 193 194 195 ! T L T L S K V D Y E K H E Y acc
ctg acg ctg agc aaa gta gac tac gag aaa cac gaa gtc tac ! 196 197
198 199 200 201 202 203 204 205 206 207 208 209 210 ! A C E V T H Q
G L S S P V T K gcc tgc gaa gtc acc cat cag ggc ctt agc tcg ccc gtc
acg aag ! 211 212 213 214 215 216 217 218 219 220 221 222 223 ! S F
N R G E C K K E F V agc ttc aac agg gga gag tgt aag aaa gaa ttc gtt
t
[0314] TABLE-US-00033 TABLE 27 V3-23 VH framework with variegated
codons shown ! ! 17 18 19 20 21 22 ! A Q P A M A 5'-ctg tct gaa cG
GCC cag ccG GCC atg gcc 29 3'-gac aga ctt gc cgg gtc ggc cgg tac
cgg ! Scab.........SfiI............. ! NgoMI... ! NcoI... ! !
FR1(DP47/V3-23)--------------- ! 23 24 25 26 27 28 29 30 ! E V Q L
L E S G gaa|gtt|CAA|TTG|tta|gag|tct|ggt| 53 !
ctt|caa|gtt|aac|aat|ctc|aga|cca| ! |MfeI | ! !
--------------FR1-------------------------------------------- ! 31
32 33 34 35 36 37 38 39 40 41 42 43 44 45 ! G G L V Q P G G S L R L
S C A |ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct|
98 ! |ccg|cca|gaa|caa|gtc|gga|cca|cca|aga|aat|gca|gaa|aga|acg|cga|
! ! Sites to be varied---> *** *** *** !
----FR1---------------->|...CDR1................|---FR2------ !
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ! A S G F T F S S Y A
M S W V R
|gct|TCC|GGA|ttc|act|ttc|tct|tCG|TAC|Gct|atg|tct|tgg|gtt|cgC| 143 !
|cga|agg|cct|aag|tga|aag|aga|agc|atg|cga|tac|aga|acc|caa|gcg| ! |
BspEI | |BsiWI| |BstXI. ! ! Sites to be varies-->*** *** *** !
61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 ! Q A P G K G L E W V
S A I S G
|CAa|gct|ccT|GGt|aaa|ggt|ttg|gag|tgg|gtt|tct|gct|atc|tct|ggt| 188 !
|gtt|cga|gga|cca|ttt|cca|aac|ctc|acc|caa|aga|cga|tag|aga|cca| !
...BstXI | ! ! *** *** !
.....CDR2............................................|---FR3--- !
76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 ! S G G S T Y Y A D S
V K G R F
|tct|ggt|ggc|agt|act|tac|tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc| 233 !
|aga|cca|ccg|tca|tga|atg|ata|cga|ctg|agg|caa|ttt|cca|gcg|aag| ! !
--------FR3-------------------------------------------------- ! 91
92 93 94 95 96 97 98 99 100 101 102 103 104 105 ! T I S R D N S K N
T L Y L Q M
|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| 278 !
|tga|tag|aga|tct|ctg|ttg|aga|ttc|tta|tga|gag|atg|aac|gtc|tac| ! |
XbaI | ! !
---FR3----------------------------------------------------->| !
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 ! N S L
R A E D T A V Y Y C A K !
|acc|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa| 323 !
|ttg|tcg|aat|tcc|cga|ctc|ctg|tga|cgt|cag|atg|ata|acg|cga|ttt| !
|AflII | | PstI | ! ! .....
CDR3.................|----FR4------------------------- ! 121 122
123 124 125 126 127 128 129 130 131 132 133 134 135 ! D Y E G T G Y
A F D I W G Q G
|gac|tat|gaa|ggt|act|ggt|tat|gct|ttc|gaC|ATA|TGg|ggt|caa|ggt| 368 !
|ctg|ata|ctt|cca|tga|cca|ata|cga|aag|ctg|tat|acc|cca|gtt|cca| ! |
NdeI | ! ! --------------FR4---------->| ! 136 137 138 139 140
141 142 ! T M V T V S S |act|atG|GTC|ACC|gtc|tct|agt- 389 !
tga|tac|cag|tgg|cag|aga|tca- ! | BstEII | ! ! 143 144 145 146 147
148 149 150 151 152 ! A S T K G P S V F P gcc tcc ace aaG GGC CCa
tcg GTC TTC ccc-3' 419 ! cgg agg tgg ttc ccg ggt agc cag aag ggg-5'
! Bsp120I. BbsI...(2/2) ! ApaI.... (SFPRMET) 5'-ctg tct gaa cG GCC
cag ccG-3' (TOPFRIA) 5'-ctg tct gaa cG GCC cag ccG GCC atg gcc-
|gaa|gtt|CAA|TTG|tta|gag|tct|ggt|-
|ggc|ggt|ctt|gtt|cag|cct|ggt|tct|tta-3' (BOTFR1B)
3'-caa|gtc|gga|cca|cca|aga|aat|gca|gaa|aga|acg|cga|-
|cga|agg|tga|aag|tga|aag-5' ! bottom strand (BOTFR2)
3'-acc|caa|gcg|- |gtt|cga|gga|cca|ttt|cca|aac|ctc|acc|caa|aga|-5' !
bottom strand (BOTFR3) 3'-a|cga|ctg|agg|caa|ttt|cca|gcg|aag|-
|tga|tag|aga|tct|ctg|ttg|aga|ttc|tta|tga|gag|atg|aac|gtc|tac|
|ttg|tcg|aat|tgc|cga|ctc|ctg|tga-5' (F06)
5'-gC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa|-
|gac|tat|gaa|ggt|act|ggt|tat|gct|ttc|gaC|ATA|TGg|ggt|c-3' (BOTFR4)
3'-cga|aag|ctg|tat|acc|cca|gtt|cca- |tga|tac|cag|tgg|cag|aga|tca-
cgg agg tgg ttc ccg ggt agc cag aag ggg-5' ! bottom strand
(BOTPRCPRIM) 3'-gg ttc ccg ggt agc cag aag ggg-5' ! ! CDR1
diversity ! (ON-vgC1)
5'-|gct|TCC|GGA|ttc|act|ttc|tct|<|>|TAC|<|>|atg<-
|>|- ! CDR1...................6859
|tgg|gtt|cuG|CAa|gct|ccT|GG-3' ! !<|> stands for an equimolar
mix of {ADEFGHIKLMNPQRSTVWY}; no C ! (this is not a sequence) ! !
CDR2 diversity ! (ON-vgc2)
5'-ggt|ttg|gag|tgg|gtt|tct|<2>|atc|>2>|<3>|- !
CDR2............
tct|ggt|ggc|<1>|act|<|>|tat|gct|gac|tcc|gtt|aaa|ggg-3-
' ! CDR2................................................ !
<1> is an equimolar mixture of {ADEFGHIIKLMNPQRSTVWY}; no C !
<2> is an equimolar mixture of {YRWVGS}; no ACDEFHIKLMNPQT !
<3> is an equimolar mixture of {PS}; no
ACDEFGHIKLMNQRTVWY
[0315] TABLE-US-00034 TABLE 28 Stuffer used in VH 1 TCCGGAGCTT
CAGATCTGTT TGCCTTTTTG TGGGGTGGTG CAGATCGCGT TACGGAGATC 61
GACCGACTGC TTGAGCAAAA GCCACGCTTA ACTGCTGATC AGGCATGGGA TGTTATTCGC
121 CAAACCAGTC GTCAGGATCT TAACCTGAGG CTTTTTTTAC CTACTCTGCA
AGCAGCGACA 181 TCTGGTTTGA CACAGAGCGA TCCGCGTCGT CAGTTGGTAG
AAACATTAAC ACGTTGGGAT 241 GGCATCAATT TGCTTAATGA TGATGGTAAA
ACCTGGCAGC AGCCAGGCTC TGCCATCCTG 301 AACGTTTGGC TGACCAGTAT
GTTGAAGCGT ACCGTAGTGG CTGCCGTACC TATGCCATTT 361 GATAAGTGGT
ACAGCGCCAG TGGCTACGAA ACAACCCAGG ACGGCCCAAC TGGTTCGCTG 421
AATATAAGTG TTGGAGCAAA AATTTTGTAT GAGGCGGTGC AGGGAGACAA ATCACCAATC
481 CCACAGGCGG TTGATCTGTT TGCTGGGAAA CCACAGCAGG AGGTTGTGTT
GGCTGCGCTG 541 GAAGATACCT GGGAGACTCT TTCCAAACGC TATGGCAATA
ATGTGAGTAA CTGGAAAACA 601 CCTGCAATGG CCTTAACGTT CCGGGCAAAT
AATTTCTTTG GTGTACCGCA GGCCGCAGCG 661 GAAGAAACGC GTCATCAGGC
GGAGTATCAA AACCGTGGAA CAGAAAACGA TATGATTGTT 721 TTCTCACCAA
CGACAAGCGA TCGTCCTGTG CTTGCCTGGG ATGTGGTCGC ACCCGGTCAG 781
AGTGGGTTTA TTGCTCCCGA TGGAACAGTT GATAAGCACT ATGAAGATCA GCTGAAAATG
841 TACGAAAATT TTGGCCGTAA GTCGCTCTGG TTAACGAAGC AGGATGTGGA
GGCGCATAAG 901 GAGTCGTCTA GA
[0316] TABLE-US-00035 TABLE 29 DNA sequence of pCES5 ! pCES5 6680
bases = pCes4 with stuffers in CDR1-2 and CDR3 2000.12.13 ! ! Ngene
= 6680 !.phi.Useful REs (cut MAnoLI fewer than 3 times) 2000.06.05
! ! Non-cutters !Acc65I Ggtacc AfeI AGCgct AvrII Cctagg !BsaBI
GATNNnnatc BsiWI Cgtacg BsmFI Nnnnnnnnnnnnnnngtccc !BsrGI Tgtaca
BstAPI GCANNNNntgc BstBI TTcgaa !BstZI7I GTAtac BtrI CACgtg Ec1136I
GAGctc !EcoRV GATatc FseI GGCCGGcc KpnI GGTACc !MscI TGGcca NruI
TCGcga NsiI ATGCAt !PacI TTAATtaa PmeI GTTTaaac PmlI CACgtg PpuMI
RGgwccy PshAI GACNNnngtc SacI GAGCTc !SacIl CCGCgg SbfI CCTGCAgg
SexAI Accwggt !SgfI GCGATcgc SnaBI TACgta SpeI Actagt !SphI GCATGc
SseS387I CCTGCAgg StuI AGGect !SwaI ATTTanat XmaI Cccggg ! !
cutters ! Enzymes that cut more than 3 times !ALwNI CAGNNNctg 5
!BsgI ctgcac 4 !BsrFI Rccggy 5 !EarI CTCTTCNnnn 4 !FauI
nNNNNNNGCGGG 10 ! ! Enzymes that cut from 1 to 3 times. ! !EcoO109I
RGgnccy 3 7 2636 4208 !BssSI Ctcgtg 1 12 !-''- Cacgag 1 1703 !BspHI
Tcatga 3 43 148 1156 !AatII GACGTc 1 65 !BciVI GTATCCNNNNNN 2 140
1607 !Eco57I CTGAAG 1 301 !-''- cttcag 2 1349 !AvaI Cycgrg 3 319
2347 6137 !BsiHKAI GWGCWc 3 401 2321 4245 !HgiAI GWGCWc 3 401 2321
4245 !BcgI gcannnnnntcg 1 461 !ScaI AGTact 1 505 !PvuI CGATcg 3 616
3598 5926 !FspI TGCgca 2 763 5946 !BglI GCCNNNNnggc 3 864 2771 5952
!BpmI CTGGAG 1 898 !-''- ctccag 1 4413 !BsaI GGTCTCNnnnn 1 916
!AhdI GACNNNnngtc 1 983 !Eaml 1051 GACNNNnngtc 1 983 !DrdI
GACNNNNnngtc 3 1768 6197 6579 !SapI gaagagc 1 1998 !PvuII CACctg 3
2054 3689 5896 !PfIMI CCANNNNntgg 3 2233 3943 3991 !HindIII Aagctt
1 2235 !ApaLI Gtgcac 1 2321 !BspMI Nnnnnnnnnngcaggt 1 2328 !-''-
ACCTGCNNNNn 2 3460 !PstI CTGCAg 1 2335 !AccI GTmkac 2 2341 2611
!HincII GTYrac 2 2341 3730 !SalI Gtcgac 1 2341 !ThiI Ctcgag 1 2347
!XhoI Ctcgag 1 2347 !BbsI gtcttc 2 2383 4219 !BlpI GCtnagc 1 2580
!ElpI GCtnagc 1 2580 !SgrAI CRccggyg 1 2648 !AgeI Acggt 2 2649 4302
!AscI GCcgcgcc 1 2689 !BssHII Gcgcgc 1 2690 !SfiI GGCCNNNNnggcc 1
2770 !NaeI GCGggc 2 2776 6349 !NgoMIV Gccggc 2 2776 6349 !BtgI
Ccrygg 3 2781 3553 5712 !DsaI Ccrygg 3 2781 3553 5712 !NcoI Ccatgg
1 2781 !StyI Ccwwagg 3 2781 4205 4472 !MfeI Caattg 2795 !BspEI
Tccgga 1 2861 !BglII Agatct 1 2872 !BclI Tgatca 1 2956 !Bsu36I
CCtnagg 3 3003 4143 4373 !XcmI CCANNNNNnnnntgg 1 3215 !MluI Acgcgt
1 3527 !HpaI GTTaac 1 3730 !XbaI Tctaga 1 3767 ! !AflII Cttaag 1
3811 !BsmI NGcattc 1 3821 !-''- GAATGCN 1 4695 !RsrII CGgwccg 1
3827 !NheI Gctagc 1 4166 !BstEII Ggtnacc 1 4182 !BsmBI CGTCTCNnnnn
2 4188 6625 !-''- Nnnnnngagacg 1 6673 !ApaI GGGCCc 1 4209 !BanII
GRCCYc 3 4209 4492 6319 Bsp120I Gggccc 1 4209 !PspOMI Gggccc 1 4209
!BseRI NNnnnnnnnnctcctc 1 4226 !-''- GAGGAGNNNNNNNNN 1 4957 !EcoNI
CCTNNnnnagg 1 4278 !PflFI GACNnngtc 1 4308 !Tth111I GACNnngtc 1
4308 !KasI Ggcgcc 2 4327 5967 !BstXI CCANNNNNNntgg 1 4415 !NotI
GCggccgc 1 4507 !EagI Cggccg 1 4508 !BamHI Ggatcc 1 5169 !BspDI
ATcgat 1 5476 !NdeI CAtatg 1 5672 !EcoRI Gaattc 1 5806 !PsiI TTAtaa
1 6118 !DraIII CACNNNNgtg 1 6243 !BsaAI YACgtr 1 6246
!--------------------------------------------------------------------------
--- 1 gacgaaaggg cCTCGTGata cgcctatttt tataggttaa tgtcatgata
ataatggttt BssSL(1/2) 61 cttaGACGTC aggtggcact tttcggggaa
atgtgcgcgg aacccctatt tgtttatttt ! AatII. 121 tctaaataca ttcaaatatG
TATCCgctca tgagacaata accctgataa atgcttcaat ! BeiVI..(1 of 2) 181
aatattgaaa aaggaagagt ! Base # 201 to 1061 = ApR gene from pUC119
with some RE sites removed ! ! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
! fM S T Q H G R V A L A L I P F F A 201 atg agt att caa cat ttc
cgt gtc gcc ctt att ccc ttt ttt gcg ! ! 16 17 18 19 20 21 22 23 24
25 26 27 28 29 30 ! A F C L P V F A H P E T L V K 246 gca ttt tgc
ctt ctt gtt ttt gct cac cca gaa acg ctg gtg aaa ! ! 31 32 33 34 35
36 37 38 39 40 41 42 43 44 45 ! V K D A E D Q L G A R V G Y I 291
gta aaa gat gct gaa gat cag ttg ggt gcc cga gtg ggt tac atc ! 46 47
48 49 50 51 52 53 54 55 56 57 58 59 60 ! E L D L N S G K I L E S F
R P 336 gaa ctg gat ctc aac agc ggt aag atc ctt gag agt ttt cgc ccc
! ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 ! E E R F P M M S
T F K V L L C 381 gaa gaa cgt ttt cca atg atg agc act ttt aaa gtt
ctg cta tgt
! ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 ! G A V L S R I D
A G Q E Q L G 426 ggc gcg gta tta tcc cgt att gac gcc ggg caa gaG
CAa ctc ggT ! BcgI............ ! ! 91 92 93 94 95 96 97 98 99 100
101 102 103 104 105 ! R R I H Y S Q N D L V E Y S P 471 CGc cgc ata
cac tat tct cag aat gac ttg gtt gAG TAC Tca cca !..BcgI......
ScaI.... ! ! 106 107 108 109 110 112 113 114 115 116 117 118 119
120 ! V T E K H L T D G M T V R E L 516 gtc aca gaa aag cat ctt acg
gat ggc atg aca gta aga gaa tta ! ! 121 122 123 124 125 126 127 128
129 130 131 132 133 134 135 ! C S A A A I T M S D N T A A N L 561
tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac tta ! ! 136
137 138 139 140 141 142 143 144 145 146 147 148 149 150 ! L L T T I
G G P K E L T A F L 606 ctt ctg aca aCG ATC Gga gga ccg aag gag cta
acc gct ttt ttg ! PvuI....(1/2) ! ! 151 152 153 154 155 156 157 158
159 160 161 162 163 164 165 ! H N M G D H V T R L D R W E P 651 cac
aac atg ggg gat cat gta act gcg ctt gat cgt tgg gaa ccg ! ! 166 167
168 169 170 171 172 173 174 175 176 177 178 179 180 ! E L N E A I P
N D E R D T T M 696 gag ctg aat gaa gcc ata cca aac gac gag cgt gac
acc acg atg ! ! 181 182 183 184 185 186 187 188 189 190 191 192 193
194 195 ! P V A M A T T L R K L L T G E 741 cct gta GCA ATG gca aca
acg tTG CGC Aaa cta tta act ggc gaa ! BsrDI..(1/2) FspI....(1/2) !
! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 ! L L
T L A S R Q Q L I D W M E 786 cta ctt act cta gct tcc cgg caa caa
tta ata gac tgg atg gag ! ! 211 212 213 214 215 216 217 218 219 220
221 222 223 224 225 ! A D K V A G A P L L R S A L P A 831 gcg gat
aaa gtt gca gga cca ctt ctg cgc tcg gcc ctt ccg gct ! ! 226 227 228
229 230 231 232 233 234 235 236 240 ! G W F I A D K S G A G E R G S
876 ggc tgg ttt att gct gat aaa tCT GGA Gcc ggt gag cgt gGG TCT !
BpmI....(1/2) BsaI.... ! ! 241 242 243 244 245 246 247 248 249 250
251 252 253 254 255 ! R G I I A A L G P D G K P S R 921 Cgc ggt atC
ATT GCa gca ctg ggg cca gat ggt aag ccc tcc cgt ! BsaI......
BsrDI...(2/2) ! ! 256 257 258 259 260 261 262 263 264 265 266 267
268 269 270 ! I V V I Y T T G S Q A T M D E 966 atc gta gtt atc tac
acG ACg ggg aGT Cag gca act atg gat gaa ! AdhI........... ! ! 271
272 273 274 275 276 277 278 279 280 281 282 283 284 285 ! R N R Q I
A E I G A S L I K H 1011 cga aat aga cag atc gct gag ata ggt gcc
tca ctg att aag cat ! ! 286 287 ! W 1056 tgg taa 1062 ctgtcagac
caagtttact 1081 catatact ttagattgattaaaacttc atttttaatt taaaaggatc
taggtgaaga 1141 tccttttttga tatctcatg accaaaatcc cttaacgtga
gttttcgttc cactgagct 1201 cagacccccgt agaaaagatc aaaggatctt
cttgagatcc tttttttctg cgcgtaatct 1261 gctgcttgca aacaaaaaaa
ccaccgctac cagcggtggt ttgtttgccg gatcaaagagc 1321 taccaactct
ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 1381
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
tgtcttaccg 1501 ggttggactc aagacgatag ttaccggata aggcgcagcg
gtcggggctga acggggggtt 1561 cgtgcataca gcccagcttg gagcgaagga
cctacaccga actgagatac ctacagcgtg 1621 agcattgaga aagcgccacg
cttcccgaag ggagaaaggc ggacagGTAT CCggtaagcg ! BciVI..(2 of 2) 1681
gcagggtcgg aacaggagag cgCACGAGgg agcttccagg gggaaacgcc tggtatctt !
BssSI.(2/2) 1741 atagtcctgt cgggtttcgc cacctcgac ttgagcgtcg
atttttgtga tgctcgtcag 1801 ggggcggag cctatggaaa aacgccagca
acgcggcctt tttacggttc ctggcctttt 1861 gctggccttt tgctcACATG
Ttctttcctg cgttatcccc tgattctgtg gataaccgta ! PciI... 1921
ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgagcgagt
1981 cagtgagcga ggaagcgGAA GAGCgcccaa tacgcaaacc gcctctccccc
gcgcgttggc ! SapI... 2041 cgattcatta atgCAGCTGg cacgacaggt
ttcccgactg gaaagcgggc agtgagcgca ! PvuIL.(1/3) 2101 acgcaatTAA
TGTgagttag ctcactcatt aggacccca ggcTTACAc tttatgcttc ! ..-35.. Plac
..-10. 2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacaCAGGA
AACAGCTATG ! M13Rev_seq_primer 2221 ACcatgatta cgCCAAGCTT TGG
agcctttt tttttggaga ttttcaac ! PflMI..... ! Hind3. !
signal:linker::CLight ! ! 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 2269 gtg aaa aaa tta tta ttc gca att
cct tta gtt gtt cct ttc tat ! ! Linker...........
................End of FR4 ! 16 17 18 19 20 21 22 23 24 25 26 27 28
29 30 ! S H S A Q V Q L Q V D L E I K 2314 tct cac aGT GCA Cag gtc
caa CTG CAG GTC GAC CTC GAG atc aaa ! ApaLI...... PstI... BspMI...
! SalI... ! AccI...(1/2) ! HincIII.(1/2) ! ! Vlight domains could
be cloned in as ApaLI-XhoI fragments. ! VL-CL (kappa) segments can
be cloned in as ApaLI-AscI fragments <-------- ! !
Ckappa---------------------------------------------------- ! 31 32
33 34 35 36 37 38 39 40 41 42 43 44 45 ! R G T V A A P S V F I F P
P S 2359 cgt gga act gtg gct gca cca tct GTC TTC atc ttc ccg cca
tct ! BbsI...(1/2) ! ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
! D E Q L K S G T A S V V C L L 2404 gat gag cag ttg aaa tct gga
act gcc tct gtt gtg tgc ctg ctg ! ! 61 62 63 64 65 66 67 68 69 70
71 72 73 74 75 ! N N F Y P R E A K V Q W K V D 2449 aat aac ttc tat
ccc aga gag gcc aaa gta cag tgg aag gtg gat ! ! 76 77 78 79 80 81
82 83 84 85 86 87 88 89 90 ! N A L Q S G N S Q E S V T E Q 2494 aac
gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag ! ! 91 92
93 94 95 96 97 98 99 100 101 102 103 104 105 ! D S K D S T Y S L S
S T L T L 2539 gac agc aag gac agc acc tac agc ctc agc acc ctg acG
CTG ! EspI... ! ! 106 107 108 109 110 111 112 113 114 115 116 117
118 119 120 ! S K A D Y E K H K V Y A C E V 2584 AGC aaa gca gac
tac gag aaa cac aaa GTC TAC gcc tgc gaa gtc ! ...ESpI....
AccI...(2/2) ! ! 121 122 123 124 125 126 127 128 130 131 132 133
134 135 ! T H Q G L S S P V T K S F N R 2629 acc cat cag ggc ctg
agt tCA CCG GTg aca aag agc ttc aac agg ! AgeI....(1/2) ! ! 136 137
138 139 140 ! G E C 2674 gga gag tgt taa taa GG CGCGCCaatt !
AscI..... ! BssHII. ! 2701 ctatttcaag gagacagtca ta ! !
PelB::3-23(stuffed)::CH1::III fusion gene ! ! 1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 ! M K Y L L P T A A A G L L L L 2723 atg aaa tac cta
ttg cct acg gca gcc gct gga ttg tta tta ctc !
!--------------------------- ! ! 16 17 18 19 20 21 22 ! A A Q P A M
A M A 2768 gcG GCC cag ccG GCC atg gcc ! SfiI............. !
NgoMIV..(1/2) ! NcoI.... ! ! Fr1(dp47/V3-23)--------------- ! 23 24
25 26 27 28 29 30 ! E V Q L L E S G 2789
gaa|gtt|CAA|TTG|tta|gag|tct|ggt| ! | MfeI | ! !
--------------FR1-------------------------------------------- ! 31
32 33 34 35 36 37 38 39 40 41 42 43 44 45 ! G G L V Q P G G S L R L
S C A 2813
|ggc|ggt|ctt|gtt|cag|cct|ggt|tct|tta|cgt|ctt|tct|tgc|gct| ! !
----FR1----- ! 46 47 48 ! A S G 2858 |gct|TCC|GGA| ! | BspEI | ! !
Stuffer for CDR1, FR2, and
CDR2---------------------------------> ! There are no stop
codons in this stuffer. 2867 gcttcAGATC Tgtttgcctt ! BglII.. 2887
tttgtggggt ggtgcagatc gcgttacgga gatcgaccga ctgcttgagc aaaagccacg
2947 cttaactgcT GATCAggcat gggatgttat tcgccaaaacc agtcgtcagg
atcttaacct ! BclI... 3007 gaggcttttt ttacctactc tgcaagcagc
gacatctggt ttgacacaga gcgatccgcg 3067 tcgtcagttg gtagaaacat
taacagttg ggatggcatc aatttgctta atgatgatgg 3127 taaaacctgg
cagcagccag gctctgccat cctgaacgtt tggctgacca gtatgttgaa 3187
gcgtaccgta gtggctgccg tacctatgCC Atttgataag TGGtacagcg ccagtggcta !
XcmI............. 3247 cgaaacaacc caggacggcc caactggttc gctgaatata
agtgttggag caaaaaattt 3307 gtatgaggcg gtgcagggag acaaatcacc
aatcccacag gcggttgatc tgtttgctgg 3367 gaaaccacag caggaggttg
tgttggctgc gctggaagat acctgggaga ctctttccaa 3427 acgctatggc
aataatgtga gtaactggaa aacacctgca atggccttaa cgttccgggc 3487
aaataatttc tttggtgtac cgcaggccgc agcggaagaa ACGCGTcatc aggcggagta !
MluI.. 3547 tcaaaaaccgt ggacagaaa acgatatgat tgttttctca ccaacgacaa
gcgatcgtcc 3607 tgtgcttgcc tgggatgtgg tcgcacccgg tcagagtggg
tttattgctc ccgatggaac 3667 agttgataag cactatgaag atcagctgaa
aatgtacgaa aattttggcc gtaagtcgct ! PvuII. 3727 ctgGTTAACg
aagcaggatg tggagggca taaggagtcg ! HpaI ! HindII(2/2) ! !
--------FR3-------------------------------------------------- ! 4 5
6 7 8 9 10 11 12 13 14 15 16 ! 93 94 95 96 97 98 99 100 101 102 103
104 105 ! S R D N S K N T L Y L Q M 3767
|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| ! | XbaI |
!
! ---FR3----------------------------------------------------->|
! 17 18 19 20 ! 106 107 108 109 ! N S L s l s i r s g 3806
|aac|agC|TTA|AG t ctg agc att CGG TCC G ! |AfIII | RsrII.. ! ! q h
s p t 3834 gg caa cat tct cca aac tga ccagacga cacaaacggc 3872
ttacgctaaa tcccgcgcat gggatggtaa agaggtggcg tctttgctgg cctggactca
3932 tcagatgaag gccaaaaatt ggcaggagtg gacacagcag gcagcgaaac
aagcactgac 3992 catcaactgg tactatgctg atgtaaacgg caatattggt
tatgttcata ctggtgctta 4052 tccagatcgt caatcaggcc atgatccgcg
attacccgtt cctggtacgg gaaaatggga 4112 ctggaaaggg ctattgcctt
ttgaaatgaa ccctaaggtg tataaccccc ag 4164 aa GCTAGC ctgcggcttc !
NHeI.. ! 4182 G|GTC|AAC| gtc tca agc ! | BstEII | ! ! 136 137 138
139 140 141 142 143 144 145 146 147 148 149 150 ! A S T K G P S V F
P L A P S S 4198 gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca
ccc tcc tcc ! ! 151 152 153 154 155 156 157 158 159 160 161 162 163
164 165 ! K S T S G G T A A L G C L V K 4243 aag agc acc tct ggg
ggc aca gcg gcc ctg ggc tgc ctg gtc aag ! ! 166 167 168 169 170 171
172 173 174 175 176 177 178 179 180 ! D Y G P E P V T V S W N S G A
4288 gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc !
! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 ! L T
S G V H T G P A V L Q S S 4333 ctg acc agc ggc gtc cac acc ttc ccg
gct gtc cta cag tcc tca ! ! 196 197 198 199 190 200 201 202 203 204
205 206 207 208 209 210 ! G L Y S L S S V V T V P S S S 4378 gga
ctc tac tcc ctc agc agc gta gtg acc gtg ccc tcc agc agc ! ! 211 212
213 214 215 216 217 218 219 220 221 222 223 224 225 ! L G T Q T Y I
C N V N H K P S 4423 ttg ggc acc cag acc tac atc tgc aac gtg aat
cac aag ccc agc ! ! 226 227 228 229 230 231 232 233 234 235 236 237
238 ! N T K V D K K V E P K S C 4468 aac acc aag gtg gac aaG AAA
GTT GAG CCC AAA TCT TGT ! ON-TQHCforw...................... ! !
Poly His linker ! 139 140 141 142 143 144 145 146 147 148 149 150 !
A A A H H H H H H G A A 4507 GCG GCC GCa cat cat cat cac cat cac
ggg gcc gca ! NotI...... ! EagI.... ! ! 151 152 153 154 155 156 157
158 159 160 161 162 163 164 165 ! E Q K L I S E E D L N G A A 4543
gaa caa aaa ctc atc tca gaa gag gat ctg aat ggg gcc gca tag ! !
Mature III------------------------------------------------>... !
166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 ! T V E
S C L A K P H T E N S F 4588 act gtt gaa agt tgt tta gca aaa cct
cat aca gaa aat tca ttt ! ! 181 182 183 184 185 186 187 188 189 190
191 192 193 194 195 ! T N V W K D D K T L D R Y A N 4633 act aac
gtc tgg aaa gac gac aaa act ta gat cgt tac gct aac ! ! 196 197 198
199 200 201 202 203 204 205 206 207 208 209 210 ! Y E G C L W N A T
G V V V C T 4678 tat gag ggc tgt ctg tgG AAT GCt aca ggc gtt gtg
gtt tgt act ! BsmI.... ! ! 211 212 213 214 215 216 217 218 219 220
221 222 223 224 225 ! G D E T Q C Y G T W V P I G L 4723 ggt gac
gaa act cag tgt tac ggt aca tgg gtt cct att ggg ctt ! ! 226 227 228
229 230 231 232 233 234 235 236 237 238 239 240 ! A I P E N E G G G
S E G G G S 4768 gct atc cct gaa aat gag ggt ggt ggc tct gag ggt
ggc ggt tct ! ! 241 242 243 244 245 246 247 248 249 250 251 252 253
254 255 ! E G G G S E G G G T K P P E Y 4813 gag ggt ggc ggt tct
gag ggt ggc ggt act aaa cct cct gag tac ! ! 256 257 258 259 260 261
262 263 264 265 266 267 268 269 270 ! G D T P I P G T Y I N P L D
4858 ggt gat aca cct att ccg ggc tat act tat atc aac cct ctg gac !
! 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 ! G T
Y P P G T E Q N P A N P N 4903 ggc act tat ccg cct ggt act gag caa
aac ccc gct aat cct aat ! ! 286 287 288 289 290 291 292 293 294 295
296 297 298 299 300 ! P S L E E S Q P L N T F M F Q 4948 cct tct
ctt GAG GAG tct cag cct ctt aat act ttc atg ttt cag ! BseRI..(2/2)
! ! 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 ! N
N R F R N R Q G A L T V Y T 4993 aat aat agg ttc cga aat agg cag
ggt gca tta act gtt tat acg ! ! 316 317 318 319 320 321 322 323 324
325 326 327 328 329 330 ! G T V T Q G T D P V K T Y Y Q 5038 ggc
act gtt act caa ggc act gac ccc gtt aaa act tat tac cag ! ! 331 332
333 334 335 336 337 338 339 340 341 342 343 344 345 ! Y T P V S S K
A M Y D A Y W N 5083 tac act cct gta tca tca aaa gcc atg tat gac
gct tac tgg aac ! ! 346 347 348 349 350 351 352 353 354 355 356 357
358 359 360 ! G K F R D C A F H S G F N E D 5128 ggt aaa ttc aga
gac tgc gct ttc cat tct ggc ttt aat gaG GAT ! BamHI.. ! ! 361 362
363 364 365 366 367 368 369 370 371 372 373 374 375 ! P F V C E Y Q
G Q S S D L P Q 5173 CCa ttc gtt tgt gaa tat caa ggc caa tcg tct
gAC CTG Cct caa ! BamHI... BspMI...(2/2) ! ! 376 377 378 379 380
381 382 383 384 385 386 387 388 389 390 ! P P V N A G G G S G G G S
G G 5218 cct cct gtc aat gct ggc ggc ggc tct ggt ggt ggt tct ggt
ggc ! ! 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405
! G S E G G G S E G G G S E G G 5263 ggc tct gag ggt ggc ggc tct
gag ggt ggc ggt tct gag ggt ggc ! ! 406 407 408 409 410 411 412 413
414 415 416 417 418 419 420 ! G S E G G G S G G G S G S G D 5308
ggc tct gag ggt ggc ggt tcc ggt ggc ggc tcc ggt tcc ggt gat ! ! 421
422 423 424 425 426 427 428 429 430 431 432 433 434 435 ! F D Y E K
M A N A N K G A M T 5353 ttt gat tat gaa aaa atg gca aac gct aat
aag ggg gct atg acc ! ! 436 437 438 439 440 441 442 443 444 445 446
447 448 449 450 ! E N A D E N A L Q S D A K G K 5398 gaa aat gcc
gat gaa aac gcg cta cag tct gac gct aaa ggc aaa ! ! 451 452 453 454
455 456 457 458 459 460 461 462 463 464 465 ! L D S V A T D Y G A A
I D G F 5443 ctt gat tct gtc gct act gat tac ggt gct gct ATC GAT
ggt ttc ! BspDI.. ! ! 466 467 468 469 470 471 472 473 474 475 476
477 478 479 480 ! I G D V S G L A N G N G A T G 5488 att ggt gac
gtt tcc ggc ctt gct aat ggt aat ggt gct act ggt ! ! 481 482 483 484
485 486 487 488 489 490 491 492 493 494 495 ! D F A G S N S Q M A Q
V G D G 5533 gat ttt gct ggc tct aat tcc caa atg gct caa gtc ggt
gac ggt ! ! 496 497 498 500 501 502 503 504 505 506 507 508 509 510
! D N S P L M N N F R Q Y L P S 5578 gat aat tca cct tta atg aat
aat ttc cgt caa tat tta cct tct ! ! 511 512 513 514 515 516 517 518
519 520 521 522 523 524 525 ! L P Q S V E C R P Y V F G A G 5623
ttg cct cag tcg gtt gaa tgt cgc cct tat gtc ttt ggc gct ggt ! ! 526
527 528 529 530 531 532 533 534 535 536 537 538 539 540 ! K P Y E F
S I D C D K I N L F 5668 aaa cCA TAT Gaa ttt tct att gat tgt gac
aaa ata aac tta ttc ! NdeI.. . ! ! 541 542 543 544 545 546 547 548
549 550 551 552 553 554 555 ! R G V F A F L L Y V A T F M Y 5713
cgt ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat ! ! 556
557 558 559 560 561 562 563 563 564 565 566 567 568 569 570 ! V F S
T F A N I L R N K E S 5758 gta ttt tcg acg ttt gct aac ata ctg cgt
aat aag gag tct taa ! ! 571 ! ! 5803 taa GAATTC ! EcoRI 5812
actggccgt cgttttacaa cgtcgtgact gggaaaaacc tggcgttacc caattaatc
5871 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
cgcacCGATC ! PvuI.. 5931 Gcccttccca acagtTGCGC Agcctgaatg
gcgaatGGCG CCgatgcgg tattttctcc ! ...PvuI...(3/3) FspI..(2/2)
KasI...(2/2) 5991 ttacgcatct gtgcggtatt tcacaccgca tataaattgt
aaacgttaat attttgttaa 6051 aattcgcgtt aaatttttgt taaatcagct
cattttttaa ccaataggcc gaaatcggca 6111 aaatcccTTA TAAatcaaaa
gaatagcccg agatagggtt gagtgggtt ccagtttgga ! PsiI... 6171
acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
6231 agggcgatgg ccCACtacGT Gaaccatcac ccaaatcaag tttttggggg
tcgaggtgcc ! DraIII.... 6291 gtaaagcact aaatcggaac cctaaaggga
gcccccgatt tagagcttga cgggaaaGC ! NgoMIV.. 6351 GCCGgaacgt
ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg !
..NgoMIV(2/2) 6411 caagtgtagc ggtcacgctg cgcgtaacca ccacacccaga
cgcgcttaat gcgccgctac 6471 agggcgcgta ctatggttgc tttgacgggt
gcagtctcag tacaatctgc tctgatgccg 6531 catagttaag ccagccccga
cacccgccaa cacccgctga cgcgccctga cgggcttgtc 6591 tgctcccggc
atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 6651
ggttttcacc gtcatcaccg aaacgcgcga
[0317] TABLE-US-00036 TABLE 30 Oligonucleotides used to clone
CDR1/2 diversity All sequences are 5' to 3'. 1) ON_CDLBsp, 30 bases
A c c T c A c T g g c T T c c g g A 1 2 3 4 5 6 7 8 9 10 11 12 13
14 15 16 17 18 T T c A c T T T c T c T 19 20 21 22 23 24 25 26 27
28 29 30 2) ON_Br12, 42 bases A g A A A c c c A c T c c A A A C c 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 T T T A c c A g g A g c
T T g g c g 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 A
A c c c A 37 38 39 40 41 42 3) ON.sub.13 CD2Xba, 51 bases g g A A g
g c A g T g A T C T A g A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
18 g A T A g T g A A g c g A c c T T T 19 20 21 22 23 24 25 26 27
28 29 30 31 32 33 34 35 36 A A c g g A g T c A g c A T A 37 38 39
40 41 42 43 44 45 46 47 48 49 50 51 4) ON_BotXba, 23 bases g g A A
g g c A g T g A T c T A g A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
17 18 g A T A g 19 20 21 22 23
[0318] TABLE-US-00037 TABLE 31 Bridge/Extender Oligonucleotides
ON_Lam1aB7(rc) .........................GTGCTGACTCAGCCACCCTC. 20
ON_Lam2aB7(rc) ........................GCCCTGACTCAGCCTGCCTC. 20
ON_Lam31B7(rc) .......................GAGCTGACTCAGGACCCTGC 20
ON_Lam3rB7(cc) ........................GAGCTGACTGAGCCACGCTC. 20
ON_LamHf1cBrg(rc) CCTCGACAGCGAAGTGCACAGAGCGTCTTGACTGAGCC....... 38
ON_LamHf1cExc CCTCGACAGCGAAGTGCACAGAGCGTCTTG............... 30
ON_LamNf2b2Brg(rc) CCTCGACAGCGAAGTGCACAGAGCGCTTTGACTCAGCC....... 38
ON_LamNf2b2Ext CCTCGACAGCGAGTGCACAGAGCGCTTTG............... 30
ON_LarnHf2dBrg(rc) CCTCGACAGCTAAGTGCACAGAGCGCTTTGACTCAGCC....... 38
ON_LamNf2dExt CCTCGACAGCGAAGTGCACAGAGCGCTTTG............... 30
ON_LarnH3rBrg(rc) CCTCGACAGCGAAGTGCACAGAGCGAATTGACTCAGCC....... 38
ON_LamHf3rExt CCTCCACAGCGAAGTGCACAGAGCCAATTG............... 30
ON_LamNf3rBrg(rc) CCTCGACAGCGAAGTGCACAGTACGAATTGACTCAGCC....... 38
ON_LemHf3rExt CCTCGACAGCGAAGTGCACAGTACGAATTG............... 30
ON_lamPlePCR CCTCGACAGCGAACTGCACAG........................ 21
Consensus
[0319] TABLE-US-00038 TABLE 32 Oligonucleotides used to make SSDNA
locally double-stranded Adapters (8) H43 HF3.1-02#1 5'-cc gtg tat
tac tgt gcg aga g-3' H43.77.97.1-03#2 5'-ct gtg tat tac tgt gcg aga
g-3' H43.77.97.323 #22 5'-cc gta tat tac tgt gcg aaa g-3'
H43.77.97.330 #23 5'-ct gtg tat tac tgt gcg aaa c-3' H43.77.97.439
#44 5'-ct gtg tat tac tgt gcg aga c-3' H43.77.97.551 #48 5'-cc atg
tat tac tgt gcg aga c-3'
[0320] TABLE-US-00039 TABLE 33 Bridge/extender pairs Bridges (2)
H43.XABr1 5'ggtgtagtgaTCTAGtgacaactctaagaatactctctacttgcagat
gaacagCTTtAGggctgaggacaCTGCAGtctactattgtgcgaga-3' H43.XABr2
5'ggtgtagtgaTCTAGtgacaactctaagaatactctctacttgcagat
gaacagCTTtAGggctgaggacaCTGCACtctactattgtgcgaaa-3' Extender
H-43.XAExt 5'ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTcATcTgcAAgTA
gAgAgTATTcTTAgAgTTgTcTcTAgATcAcTAcAcc-3'
[0321] TABLE-US-00040 TABLE 34 PCR primers Primers H43.XAPCR2
gactgggTgTAgTgATCTAg Hucmnest cttttctttgtgccgttggggtg
[0322] TABLE-US-00041 TABLE 35 PCR program for amplification of
heavy chain CDR3 DNA 95 degrees C. 5 minutes 95 degrees C. 20
seconds 60 degrees C. 30 seconds repeat 20x 72 degrees C. 1 minute
72 degrees C. 7 minutes 4 degrees C. hold Reagents (100 ul
reaction) Template 5 ul ligation mix 10x PCR buffer 1x Taq 5U dNTPs
200 uM each MgCl.sub.2 2 mM H43.XAPCR2-biotin 400 nM Hucmnest 200
nM
[0323] TABLE-US-00042 !TABLE 36 Annotated sequence of CJR
DY3F7(CJR-A05) 10251 bases ! ! Non-cutters ! !BclI Tgatca BsiWI
Cgtacg BssSI Cacgag !BstZ17I GTAtac BtrI CACgrg EcoRV GATatc !FseI
GGCCGGcc HpaI GTTaac MluT Acgcgt !PmeI GTTTaaac PmlI CACgtg PpuMI
RGgwccy !RsrII CGgwccg SapI GCTCTTC SexAI Accwggt !SgfI GCGATcgc
SgrAI CRccgggg SphI GCATGc !StuI AGGcct XmaI Cccggg ! ! cutters ! !
Enzymes that cut from 1 to 4 times and other features !End of genes
II and X 829 !Start gene V 843 !BsrGI Tgtaca 1 1021 !BspMI
Nnnnnnnnngcaggt 3 1104 5997 9183 !-''- ACCTGCNNNNn 1 2281 !End of
gene V 1106 !Start gene VII 1108 !BsaBI GATNNnnatc 2 1149 3967
!Start gene IX 1208 !End gene VII 1211 !SnaBI TACgta 2 1268 7133
!BspHI Tcatga 3 1299 6085 7093 !Start gene VIII 1301 !End gene IX
1304 !End gene VIII 1522 !Start gene III 1578 !EagI Cggccg 2 1630
8905 !XbaI Tctaga 2 1643 8436 !KasI Ggcgcc 4 1650 8724 9039 9120
!BsmI GAATGCN 2 1769 9065 !BseRI GAGGAGNNNNNNNNNN 2 2031 8516 !-''-
NNnnnnnnnnctcctc 2 7603 8623 !AlwNI CAGNNNctg 3 2210 8072 8182
!BspDI ATcgat 2 2520 9883 !NdeI CAtatg 3 2716 3796 9847 !End gene
III 2846 !Start gene VI 2848 !AfeI AGCgct 1 3032 !End gene VI 3187
!Start gene I 3189 !EarI CTCTTCNnnn 2 4067 9274 !-''- Nnnnngaagag 2
6126 8953 !PacI TTAATtaa 1 4125 !Start gene IV 4213 !End gene I
4235 !BsmFI Nnnnnnnnnnnnnnngtccc 2 5068 9515 !MscI TGGcca 3 5073
7597 9160 !PsiI TTAtaa 2 5349 5837 !End gene IV 5493 !Start on 5494
!NgoMIV Gccggc 3 5606 8213 9315 !BanII GRGCYc 4 5636 8080 8606 8889
!DraIII CACNNNgtg 1 5709 !DrdI GACNNNNnngtc 1 5752 !AvaI Cycgrg 2
5818 7240 !PvuII CAGctg 1 5953 !BsmBI CGTCTCNnnnn 3 5964 8585 9271
!End ori region 5993 !BamHI Ggatcc 1 5994 !HindIII Aagctt 3 6000
7147 7384 !BciVI GTATCCNNNNNN 1 6077 !Start bla 6138 !Eco57I CTGAAG
2 6238 7716 !SpeI Actagt 1 6257 !BcgI gcannnnnntcg 1 6398 !ScaI
AGTact 1 6442 !PvuI CGATcg 1 6553 !FspI TGCgca 1 6700 !BglI
GCCNNNNnggc 3 6801 8208 8976 !BsaI GGTCTCNnnnn 1 6853 !AhdI
GACNNNnngtc 1 6920 !Eam1105I GACNNNnngtc 1 6920 !End bla 6998 !AccI
GTmkac 2 7153 8048 !HincII GTYrac 1 7153 !SalI Gtcgac 1 7153 !XhoI
Ctcgag 1 7240 !Start PlacZ region 7246 !End PlacZ region 7381
!PflMI CCANNNNntgg 1 7382 !RBS1 7405 !start M13-iii signal seq for
LC 7418 !ApaLI Gtgcac 1 7470 !end M13-iii signal seq 7471 !Start
light chain kappa L20:JK 1 7472 !PflFI GACNnngtc 3 7489 8705 9099
!SbfI CCTGCAgg 1 7542 !PstI CTGCAg 1 7543 !KpnI GGTACc 1 7581 !XcmI
CCANNNNNnnnntgg 2 7585 9215 !NsiI ATGCAt 2 7626 9503 !BsgI ctgcac 1
7809 !BbsI gtcttc 2 7820 8616 !BlpI GCtnagc 1 8017 !EspI GCtnagc 1
8017 !EcoO109I RGgnccy 2 8073 8605 !Ecl136I GACctc 1 8080 !SacT
GASCTc 1 8080 !End light chain 8122 !AscI GGcgcgcc 1 8126 !BssHII
Gcgcgc 1 8127 !RBS2 8147 !SfiI GGCCNNNNnggcc 1 8207 !NcoI Ccatgg 1
8218 !Start 3-23, FR1 8226 !MfeI Caattg 1 6232 !BspEI Tccgga 1 8298
!Start CDR1 8316 !Start FR2 8331 !BstXI CCANNNNNntgg 2 8339 8812
!EcoNI CCTNNnnnagg 2 8346 8675 !Start FR3 8373 !XhaI Tctaga 2 8436
1643 !AflII Cttaag 1 8480 !Start CDR3 8520 !AatII GACGTc 1 8556
!Start FR4 8562 !PshAI GACNNnngcc 2 8573 9231 !BstEII Ggtnacc 1
8579 !Start CH1 8595 !ApaI GGGCCc 1 8606 !Bsp120I Gggccc 1 8606
!PspOMI Gggccc 1 8606 !AgeI Accggt 1 8699 !Bsu36I CCtnagg 2 8770
9509 !End of CH1 8903 !NotI GCggccgc 1 8904 !Start His6 tag 8913
!Start cMyc tag 8931 !Amber codon 8982 !NheI Gctagc 1 8985 !Start
M13 III Domain 3 8997 !Nrul TCGcga 1 9106 !BstEI TTcgaa 1 9197
!EcoRI Gaattc 1 9200 !XcmI CCANNNNNnnnntgg 1 9215 !BstAPI
GCANNNNntgc 1 9337 !SacII CCGCgg 1 9365 !End IIIstump anchor 9455
!AvrII Cctagg 1 9462 !trp terminator 9470 !SwaI ATTTaaat 1 9784
!Start gene II 9850 !BglII Agatct 1 9936
!----------------------------------------------------------------------
1 aat gct act act att agt aga att gat gcc acc ttt tca gct cgc gcc !
gene ii continued 49 cca aat gaa aat ata gct aaa cag gtt att gac
cat ttg cga aat gta 97 tct aat ggt caa act aaa tct act cgt tcg cag
aat tgg gaa tca act 145 gtt aTa tgg aat gaa act tcc aga cac cgt act
tta gtt gca tat tta 193 aaa cat gtt gag cta cag caT TaT att cag caa
tta agc tct aag cca 241 tcc gca aaa atg acc tct tat caa aag gag caa
tta aag gta ctc tct 289 aat cct gac ctg ttg gag ttt gct tcc ggt ctg
gtt cgc ttt gaa gct 337 cga att aaa acg cga tat ttg aag tct ttc ggg
ctt cct ctt aat ctt 385 ttt gat gca atc cgc ttt gct tct gac tat aat
agt cag ggt aaa gac 433 ctg att ttt gat tta tgg tca ttc tcg ctt tct
gaa ctg ttt aaa gca 481 ttt gag ggg gat tca ATG aat att tat gac gat
tcc gca gta ttg gac ! Start gene x, ii continues 529 gct atc cag
tct aaa cat ttt act att acc ccc tct ggc aaa act tct 577 ttt gca aaa
gcc tct cgc tat ttt ggt ttc tat cgt cgt ctg gta aac 625 gag ggt tat
gac agt gtt gct ctt act acg cct cgt aat tcc ttt tgg 673 cgt tat gta
tct gca tta gtt gaa tgt ggt att cct aaa tct caa ctg 721 atg aat cct
tct acc tgt aat aat gtt gtt ccg tta gtt cgt ttt att 789 aac gta gat
ttt tct tcc caa cgt cct gac tgg tat aat gag cca gtt 817 ctt aaa atc
gca TAA ! End X & II 832 ggtaattca ca ! ! M1 ES Q10 T15 843 ATG
att aaa gtc gaa act aaa oca tct caa gcc caa ttt act act cgt ! Start
gene V ! ! S17 S20 P25 E30 891 tct ggt gtt tct cgt cag ggc aag cct
tat tca ctg aac gag cag ctt ! ! V35 E40 V45 939 cgt tac ggt gat tcg
ggt aac gaa tat ccg gtt gtt gtc aag att act ! ! D50 A55 L60 987 ctt
gac gaa ggc ccg cca gcc tat gcg cct ggt cTG TAC Acc gtt cat !
BsrGI... ! L85 V70 S75 R80 1035 ctg tcc tct ttc aaa gtt ggt cag ttc
ggt tcc ctt atg att gac cgt ! ! P85 K87 end of V 1083 ctg cgc ctc
gtt ccg gct aag TAA C ! 1108 ATG gag cag gtc gcg gat ttc gac aca
att tat cag gcg atg ! Start gene VII ! 1150 ata caa atc tcc gtt gta
ctt tgt ttc gcg ctt ggt ata atc ! ! VII end IX overlap. ! ..... S2
V3 L4 V5 S10 1192 gct ggg ggt caa agA TGA gt gtt tta gtg tat cct
ttT gcc tct ttc gtt ! End VII ! |start IX ! L13 W15 G20 T25 E29
1242 tta ggt tgg tgc ctt cgt agt ggc att acg tat ttt acc cgt tta
atg gaa ! 1293 act tcc tc ! ! .... stop of IX, IX end VIII overlap
by four bases 1301 ATG aaa aag tct tta gtc ctc aaa gcc tcc gta gcc
gtt gct acc ctc ! Start signal sequence of viii. ! 1349 gtt ccg atg
ctg tct ttc gct gct gag ggt gac get ccc gca aaa gcg ! mature VIII
---> 1397 gcc ttt aac tcc ctg caa gcc tca gcg acc gaa tat atc
ggt tat gcg 1445 tgg gcg atg gtt gtt gtc att 1468 gtc ggc gca act
atc ggt atc aag ctg ttt aag ! ! bases 1499-1539 are probable
promoter for iii 1499 aaa ttc acc tcg aaa gca ! 1515 ! ...........
-35 .. ! 1517 agc tga oaaaccgat acaattaaag gctcctttcg
! ..... -10 ... ! 1552 gagccttttt ttt SCAGAt ttt ! S.D. uppercase,
there may be 9 Ts ! ! <------ III signal sequence
-----------------------------> ! M K K L L F A I P L V V P F
1574 caac GTG aaa aaa tta tta ttc gca att ccc tta gct gct cct ttc !
1620 ! ! Y S G A A S S H L D S A 1620 tat tct ggc gCG GCC Gaa tca
caT CTA SAc ggc gcc ! EagI.... XbaI.... ! ! Domain 1
------------------------------------------------------------ ! A S
T V K S C L A 1656 gct gaa act gct gaa agt tgt tta gca ! ! K S H T
K I S F T N V W K D D K T 1693 aaA Tcc cat aca gaa aat tca tta aCT
AAC GTC TGG AAA GAC SAC AAA ACt ! ! L D R Y A N Y E G S L W N A T G
V 1734 tta gat cgt tac gct aac tat gag ggC tgt ctg tgG AAT SCt aca
ggc gtt ! BsmI.... ! ! V V C T S D K T Q C Y S T W V P I 1785 gta
gtt tgt act ggt SAC GAA ACT CAG TGT TAC GGT ACA TSG STT ccc att ! !
G L A I P K N 1836 ggg ctt gct atc cct gaa aat ! L1 linker
------------------------------------ ! E S S G S K G G S S 1857 gag
ggt ggt ggc tct gag ggt ggc ggt tct ! ! E G G G S K G S G T 1887
gag ggt ggc ggt tct gag ggt ggc ggt act ! Domain 2
------------------------------------ 1917 aaa cct cct gag tac ggt
gat aca cct att ccg ggc tat act tat atc aac 1968 cct ctc gac ggc
act tat ccg cct ggt act gag caa aac ccc gct aat cct 2019 aat cct
tct cct GAG GAG tcc cag cct ctt aat act ttc atg ttt cag aac !
BseRI.. 2070 aat agg tcc cga aat agg cag ggg gca tta act gtt cat
acg ggc acc 2118 gct act caa ggc act gac ccc gtt aaa act tac tac
cag tac act cct 2166 gta tca tca aaa gcc atg tat gac gct tac tgg
tac ggt aaa ttC AGA ! AlwNI 2214 GAG TGc gct ttc cat tct ggc tct
aac gaG gat TTa ttT gtt cgc gaa ! AlwNI 2262 tat caa ggc caa tcg
tct gac ctg cct caa cct cct gtc aat gct ! 2307 ggc ggc ggc tct !
start L2
------------------------------------------------------------- 2319
ggt ggt ggt tct 2331 ggt ggc ggc tct 2343 gag ggc ggt ggc tct gag
gga ggc ggt 2373 ggt ggt ggc tct ggt ! end L2 ! ! Many published
sequences of M13-derived phage have a longer linker ! than shown
here by repeats of the EGGGS motif two more times. ! ! Domain
3-------------------------------------------------------------- ! S
G D B D Y E K M A N A N K G A 2388 tcc ggt gat tct gat tat gaa aag
atg gca aac gct aat aag ggg gct ! ! M T K N A D K N A I Q S D A K G
2436 atg acc gaa aat gcc gat gaa aac gcg cta cag tct gac gct aaa
ggc ! ! K L D S V A T D Y C A A M D G F 2484 aaa ctt gat tct gtc
gct act gat tac ggt gct gct atc gat ggt ttc ! ! I C D V S G L A N G
N C A T C D 2532 att ggt gac gtt tcc ggc otc gct aat ggt ast ggt
gct gct ggt gat ! ! F A G S N S Q I A Q V G D G D N 2580 ttt gct
ggc tct aat tcc cac atg gct caa gtc ggt gac ggt gat aat ! ! S P I N
N B R Q V L P S I P Q 2628 tca cct tta atg aat aat ttc cgt caa tat
tta cct tcc ctc cct cac ! ! S V K C P P B V B C A C K P Y E 2676
tcg gtt gaa tgt cgc cct ttt gcc ctt ggc gct ggc asa cca tat gaa ! !
I B S I D C D K I N L B R 2724 ttt tct att gat tgt gac aaa ata aac
tta ttc cgt ! End Domain 3 ! ! G V B A B I I V V A T B M V V F140
2760 ggc gtc ttt gcg ttt ctt tta tat gtc gcc acc ttt atg tat gta
ctt ! start transmembrane segment ! ! S T B A N I L 2808 tct acg
ttt gct aac ata ctg ! ! R N K F S 2829 cgt aat aag gag tct TAA !
step of iii Intracellular anchor. ! M1 P2 V L L5 G I P L L10 L R F
L G15 2847 tc ATG cca gtt ctt ttg ggt att ccg tca tta ttg cgt ttc
ctc ggt ! Start VI ! 2894 ttc ctt ctg gta act ttg ttc ggc tat ctg
ctt act ttt ctt aaa aag 2942 ggc ttc ggt aag ata gct att gct att
tca ttg ttt ctt gct ctt atc 2990 atc ggg ctc aac cca att ctt gtg
ggc tac ctc tct gat att agc gct 3038 caa tta ccc tct gac ttt gct
cag ggc gct cag tta att ctc ccg tct 3086 aat gcg ctt ccc tgt ttt
tac gtc att ccc cct gta aag gcc gct act 3134 tcc att tct gac gtt
aaa caa aaa atc gtt tct tat ttg gat tgg gac ! ! M1 A2 V3 F5 L10 G13
3182 aaa TAA t ATG gct gtt tat ttt gta act ggc aaa tta ggc tct gga
! end VI Start gene I ! ! K T L V S V G K I Q D K I V A 3228 aag
acg ctc gct agc gtt ggc aag att cag gac aaa att gta gct ! ! G C K I
A T N L D L K L Q N L 3273 ggg tgc aca cta gca act aat ctt gct tta
cgg ctt ccc ccc ctc ! P Q V C R F A K T P K V L K I 3318 ccg ccc
gtc ggg cgg ttc gct caa ccg cct cgc gtt cct aga ata ! ! P D K P S I
S D L L A I G K C 3363 ccg gct aag cct tct cta tct gct ttg ctc gct
act ggg cgc ggt ! ! N D S V D E N K N C L L V L D 3408 aat gct tcc
tac gct gcc aat cca ccc ggc ctg ctt gtt ctc gct ! ! E C G T W F N T
R S W N D K E 3453 gag tgc ggt act tgg ttt aat acc cgt tct tgg aat
gct aag gcc ! ! R Q P I I D W F L H A K K L G 3498 agc cag ccg att
gct tgg ttt cta aat gct cgt caa cta gga ! ! W D I I F L V Q D L S I
V D K 3543 tgg gct ctt cct ttt ctc gtt ccg gac tcc ccc cct gtt gac
cac ! ! Q A R S A L A E H V V Y C R R 3588 ccg gcg cgc tct gca ttc
gct gcc ccc gtt gtt aat tgt cgt cgt ! ! L D K I T L P F V G T L Y S
L 3633 ctg gac agc ctc act tta cct ttt gtc ggt acc tta tat tct cct
! ! I T G S K N P L P K L H V C V 3678 att act ggc tcg cca atg cct
ctg ccc cca tta aat gtt ggc gct ! ! V K V G D S Q L S P T V K K N
3723 gtt aaa tat ggc gat tct caa tta agc cct act gtt gag cgt tgg !
! L Y T G K N L V N A V D T K Q 3768 ctt tat act ggt aag aat ttg
tat aac gca tac gat acc aaa cag ! ! A F S S N V D S G V Y S Y L T
3813 gct ttt tct agt aat tat gat tcc ggc gtt tat tct tat tta acg !
! P V L S H G R V F K P L N L G 3858 cct tat tta tca cac ggt cgg
tat ttc aaa cca tta aat tta ggt ! ! Q K M K L T K I V L K K F S R
3903 cag aag atg aaa tta act aaa ata tat ctg aaa aag ttt tct cgc !
! V L C L A I C F A S A F T Y S 3948 gtt ctt tgc ctt gcg att gga
ttt gca tca gca ttt aca tat agt ! ! V I T Q P K F F V K K V V S Q
3993 tat aca acc caa cct aag ccg gag gct aaa aag gta gtc tct cag !
! T Y D F D K F T I B S S Q K L 4038 acc tat gat ttt gat aaa ttc
act att gac tct tct cag cgt ctt ! ! N L S V K V V F K B S K G K L
4083 aat cta agc tat cgc tat gtt ttc aag gat tct aag gga aaa TTA !
PacI ! ! I N S D D L Q K Q G V S L T Y 4128 ATT AAt agc gac gat cta
cag aag caa ggt tat tca ctc aca tat ! PacI ! ! i I D L C T V S I K
K G N S N E ! iv M1 K 4173 att gat tta tgt act gtt tcc att aaa aaa
ggt aat tca aAT Gaa ! Start IV ! 49 i I V K C N End of I ! iv L3 L
N5 V I7 N F V10 4218 att gtt aaa tgt aat TAA T TTT GTT ! IV
continued..... 4243 ttc ttg acg ttt gtt tca tca tct tct ttc gct cag
gta att gaa atg 4291 aat aat tcg cct ctg cgc gat ttt gta act tgg
tac tca aag caa tca 4339 ggc gaa tcc gtt att gtt tct ccc gat gta
aaa ggt act gtt act gta 4387 aat cca tct gac gtt aaa cct gaa aat
cta cgc aat tcc ctt att tct 4435 gct tta cgc gcA aac aat ctc gat
atg gtA ggt tcT aAC cct tcc act 4483 att cag aag tat aat cca aac
aat cag gat tat atc gat gaa ttg cca 4531 tca tct gat aat cag gaa
tat gat gat aac ccc gct ccc cct ggc ggt 4579 ttc ttt gtt ccg caa
aat gat aat gtt act caa act tct aaa act aat 4627 aac gct cgg gca
aag gat cta ata cga gtt gtc gaa ttg ctc gta aag 4675 tct aat acc
tct aaa tcc tca aat gta tta tct att gac ggc tct aat 4723 cta cta
gtt gtt agt gcT cct aaa gat atc tta gat aac ctc ccc caa 4771 ttc
ctt tcA act gtt gat ttg cca act gac cag ata ttg att gag ggt 4819
ttg ata ttt gag gtt cag caa ggt gat gct tta gac ttt tca ttt gct
4867 gct ggc tct cag cgt ggc act gtt gca ggc ggt gtt aat act gac
cgc 4915 ctc acc tct gtt tta tct tct gct ggt ggt tcg ttc ggt att
ttt aat 4963 ggc gat gtt tta ggg cta tca gtt cgc gcc tta aag act
aat agc aat 5011 tca aaa ata ttg tct gtg cca cgt att cct aag ctt
tca ggt cag aag 5059 ggt tcc agc tct gtT GGC CAg aat gtc cct ctt
att act ggt ctg gcg ! MscI.... 5107 act ggt gaa tcc gcc aat gta aat
aat cca ttc cag aag att gag ctg 5155 caa aat gta ggt att tcc atg
agc gct ctt cct gct gca atg gct ggc 5203 ggt aat att gtt ctg gat
att acc agc aag gcc gat agt ttg agt tct 5251 cct act cag gca agt
gat gtt att act aat caa aga agt att gct acc 5299 aag gtt aat ttg
cgc gat gga cag act ctt cta ctc ggt ggc ctc act 5347 gat tat aaa
aac act tct caG gat tct ggc gta ccg ttc ctg tct aaa 5395 atc cct
tta agc ggc ctc ctg ttt agc tcc cgc tct gat teT aac gag
5443 gaa agc aag tta tac gtg ctc gtc aaa gca acc ata gta cgc gcc
ctg 5491 TAG cggcgcatt ! End IV 5503 aagcgcggeg ggtgtggtgg
ttccgcgcag cgtgaccgct acacttgcca gcgccctagc 5563 gcccgctcct
ttcgctttct tcccttcctt tctcgccaag ttcGCCGGCt ttccccgcca ! NgoMI.
5623 agctctaaat cgggggcccc ccttagggtt ccgatttagt gctttacggc
acctcgaccc 5683 caaaaaactt gattcgggtg atggttCACG TAGTGggcca
tcgccctgat agacggtttt ! DraIII.... 5743 tcgccctttG ACGTTGGAGT
Ccaagttctt taatagtgga ctcttgttcc aaactggaac ! DrdI.......... 5903
aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga
5863 accacaatca aacaggattt tcgcctgctg gggcaaacca gctgggaccg
cttgccgcaa 5923 ctctctcagg gccaggcggt gaagggcaat CAGCTGctgc
cCGTCTCact ggtgaaaaga ! PvuII. BsmBI. 5983 aaaaccaccc tGGATCC
AAGCTT ! BamHI HindIII (1/2) ! Insert carrying bla gene 6006
gcaggtg gcacttctcg gggaaatgtg cgcggcaccc 6043 ctatttgctt atttctctaa
atacatcccc atatGTATCC gctcatgaga caataacct ! BciVI 6103 gacaaacgct
ccaataatat tgaaaaAGGA AGAgt ! RBS.?... ! Start bla gene 6138 ATG
agt att caa aat ttc cgt gtc gcc ctt att ccc ttt ttt gag gca ttt
6189 tgc ctt cct gtt ttt gct ccc cca gaa acg ctg gtg aaa gta aaa
gat gct 6240 gaa gat cag ttg ggC gcA CTA GTg ggt tac atc gaa ctg
gat atc aac agc ! SpeI.... ! ApaLI & BssSI Removed 6291 ggt aag
atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc 6342
act ttt aaa gtc ctg cta tgt GGC GcG Gta tta tcc cgt att gac gcc ggg
6393 caa gaG CAA CTC GGT CGc agc ATA cAC tat tct cag aat gac ttg
gtt gAG ! BcgI............ ScaI 6444 TAC Tca cca gtc aca gaa aag
aat ctt acg gat ggc atg aca gta aga gaa ! Scal. 6495 tta cgc agt
gct gcc ata acc atg agc gat aac act gcg gcc aac tta ctt 6546 ctg
aca aCG ATC Gga gga ccg aag gag cta acc gct ttt ttg cac aac atg !
PvuI.... 6597 ggg gat aat gta act agc ctt gat cgt tgg gaa ccg gag
ctg aat gaa gac 6648 ata cca aac gac gag cgt gac acc aag atg aat
gta gca atg Gca aca aag 6699 tTG CGC Aaa cta tta act ggc gaa cta
ctt act ata gct tcc cgg aaa caa ! FspI.... ! 6750 tta ata gac tgg
atg gag gcg gat aaa gtt gca gga cca ctt ctg agc tcg 6801 GCC ctt
ccG GCt ggc tgg ttt att gct gat aaa tat gga gac ggt gag agt !
BglI.......... 6852 gGG TCT Cgc ggt atc att gca gca ctg ggg cca gat
ggt aag ccc tcc agt ! BsaI.... 6903 atc gta gtt atc tac acG ACg ggg
aGT Cag gca act atg gat gaa cga aat ! AhdI........... 6954 aga cag
atc gct gag ata ggt gcc tca ccg att aag aat tgg TAA ctgt ! stop
7003 cagaccaagc ttactaatat atactttaga ttgatttaaa acttaatttt
taatttaaaa 7063 ggatataggt gaagatcctc tttgataatc tcatgaccaa
aatccattaa cgtgagtttt 7123 cgttccactg tacgtaagac cccc 7147 AAGCTT
GTCGAG tgaa tggcgaatgg cgctttgcct ! HindIII SalI.. ! (2/2) HincII
7183 ggtttccggc accagaagcg gcgccggaaa gctggctgga gcgcgatctt ! !
Start of Fab-display cacsettc, the Fab DSR-A05, selected for !
binding to a protein antigen. ! 7233 CCTGAcG CTCGAG ! xBsu36I
XhoI.. ! ! PlacZ promoter is in the following block 7246 cgcaacgc
aactaatgtg agttagctca 7274 ctcattaggc accccaggct ttacacttta
tgcttccggc tcgtatgttg 7324 tgtggaattg tgagcggata acaatttccc
acaggaaaca gctatgacca 7374 tgattacgCC AagcttTGGa gccttttttt
tggagatttt caac ! PflMI ! Hind3. (there are 3) ! Gene iii signal
sequence: ! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ! M K K L L F A I P
L V Y 7418 gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc
tat ! ! 16 17 18 Start light chain (L20:JK1) ! S H S A Q D I Q M T
Q S P A 7463 tct cac aGT GCA Caa aat atc tac acc act tac cct cca
gct ! ApaLI... ! Sequence supplied by extender............ ! ! T L
S L 7505 acc ctg tct ttg ! ! S P G E R A T L S C R A A S Q G 7517
tcc cca ggg gaa agc gct act ctt tcc tgc agg gct ctg tag Ggt ! V S S
Y L A N Y Q Q K P G Q A 7562 gtt agc agc tat tta gct tgg tac cag
cag aaa cct ggc tag gct ! ! P R L L I V D A S S R A T G I 7607 ccc
agg ctt ctt atc tat gAt gtc tcc aAc agg gct act ggc atc ! ! P A R F
S G S G F G T D F T L 7652 tcc gct cgg ttc ctg ggc ctg ggg Cct ggg
acc gac ttc act ctt ! ! T I S S L E P E D F A V Y Y C 7697 ctc ctc
cgc agC ctA gag tct gcc gct ttt gtc gtT tat tac tgt ! ! Q Q R S N H
P W I F G Q G T R 7742 cag cag CGt aAc tgg aat ccg tgg ACG TTC GGC
CAA GGG ACC AAG ! ! V E I K K T V A A P S V F I F 7787 gtg gcc ctc
aaa cga act gtg gCT GCA Cca tct gtc ttc ctc ttc ! BsgI.... ! ! P P
S D E Q L K S G T A S V V 7832 ccg tcc tct gct gag cag ttg ccc tct
gga act gct tct gtt gtg ! ! C L L N N F Y P K E A K V Q W 7877 tgc
ctg ctg aat ccc ttc tat ccc agc gag gcc aaa gta cag tgg ! ! K V D N
A L Q S G N S Q E S V 7922 aag gtg gct aat gct ctt tcc tcg ggt ccc
tcc cag gag agt gct ! ! T E R D D S K D S T Y S L S S T 7967 aca
gag cgg gct agc aag gac agc acc tac agc ctt agc agc ctc ! ! L T L S
K A D Y F K H K V Y A 8012 ctg acG CTG AGc aaa gca gac tac gag caa
ccc ccc gtc tac gct ! EspI..... ! ! C E V T H Q G L S S P V T K S
8057 tgc gaa gtc acc aat cag ggc ctG AGC Tcg ccc gtc aca aag agc !
SacI.... ! ! F N R G E C . . 8102 ttc aac agg gga gag tgt caa taa
8128 GGCGCG CCaattctat ttcaaGGAGA cagtaata ! AscI..... RBS2. ! !
PelB signal sequence------(22 codons)-----> ! 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 ! M K Y L L P T A A A A L L L L 8160 aag aaa tac
cta ttg cct aag gca gcc gct gga ttg tta tta ctc ! ! ...PelB
signal------------> Scare VH, FR1-----------------> ! 16 17
18 19 20 21 22 23 24 25 26 27 28 29 30 ! A A Q P A M A K V Q L L E
S G 8205 gcG GCC cag ccG GCC atg gcc gaa gtt CAA TTG tta gag tct
ggt ! SfiI............. MfeI... ! NcoI.... ! ! 31 32 33 34 35 36 37
38 39 40 41 42 43 44 45 ! G G L V Q P G G S L R L S C A 8250 ggc
ggt ctt gtt cag cct ggt ggt tct tta cgt ctt tct tgc gct ! !
...FR1--------------------> CDR1-------------->
FR2--------> ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ! A
S G F I F S T V K N R W V R 8295 gct TCC GGA ttc act ttc tct act
tac gag atg ctg tgg gtt cgC ! BspE71.. BstXI... ! !
FR2--------------------------------------> CDR2 ----------> !
61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 ! Q A P G K G L K W V
S Y I A P 8340 CAa gct ccT GGt aaa ggt ttg gag tgg gtc tct tat agc
gct cct ! BstXI................ ! !
...CDR2--------------------------------------------->
FR3----> ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 ! S G G
D T A Y A D S V K G R F 8385 tct ggt ggc gat aet gct tat gct gae
ccc gtt aaa ggt cgc ttc ! ! 91 92 93 94 95 96 97 98 99 100 101 102
103 104 105 ! T I S R D N S K N T L V L Q N 8430 act atc TGT AGA
gac aac tct aag aat act ctc tac ttg cag atg ! XbaI... ! Supplied by
extender------------------------------- ! !
-----------------------------------------FR3--------------> !
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 ! N S L
R A K D T A V Y Y C A R 8475 aac agC TTA AGg gct gag gac act gca
gtc tac tat tgt gcg agg ! AflII... ! from
extender---------------------------------> ! !
CDR3-------------------------------------------------->
FR4--> ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134
135 ! R L D G Y I S Y Y V G M D V W 8520 agg ctc gat ggc tat att
tcc tac tac tac ggt atg GAC GTC tgg ! AatII.. ! ! 136 137 138 139
140 141 142 143 144 145 ! G Q C T T V T V S S 8565 ggc caa ggg acc
acG GTC ACC gtc tca agc ! BstEII... ! ! CH1 of IgG1----------> !
A S T K G P S V F P L A P S S 8595 gcc tcc acc aag ggc cca tcg gcc
ttc ccc ctg gca ccc tcc ccc ! ! K S T S G G T A A L G C L V K 8640
aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag ! ! D V
F P E P V I V S W N S G A 8685 gac tac ttc ccc gaa ccg gtg acg gtg
tcg tgg aac tca ggc gcc ! ! L T S G V H T F P A V L Q S S 8730 ctg
acc agc ggc gtc cac acc ctc ccg gct gtc cta cag tcc TCA Bsu36I....
! ! G L Y S L S S V V I V P S S S 8775 GGa ctc tac tcc ctc agc agc
gta gtg acc gtg ccc tcc agc agc ! Bsu36I.... ! ! L G T Q T Y I C N
V N H K P S 8820 ttg ggc acc cag acc tac agc tgc aac gtg aat cac
aag ccc agc ! ! N T K V D K K V F P K S C A A 8865 aac acc aag gtg
gac aag aaa gtt gag ccc aaa tcc tgt GCG GCC NotI...... ! ! A H H H
H H H G A A F Q K L 8910 GCa aat aat aat cac aat cac ggg gcc gca
gaa caa aaa ctc agc ! ..NoeI.... H6 tag.................Myc-
Tag........................ ! ! S E E D L N G A A q A S S A 8955
tca gaa gag gat ctg aat ggg gcc gca cag GCT AGC tct gct !
Myc-Taq.................... ... NheI... ! Amber
! ! III'stump ! ! Domain 3 of III
------------------------------------------------------- ! ! S G D F
D Y E K N A N A N K G A 8997 agc ggc gac ttc gac tac gag aaa atg
gct aat gcc aac aaa GGC GCC ! tcc t t t t t a g a c t t g g t !W.T.
! KasI...(2/4) ! ! M T K N A D E N A L Q S D A K G 9045 atG ACT GAG
AAC GCT GAC GAG aat gct ttg caa agc gat gcc aag ggt ! c a t c t a c
g c a g t c t c t a c !W.T. ! ! K L D S V A T D Y G A A I D G F
9093 aag tta gac agc gTC GCG Aoo gac tat GGC GOD gcc ATO GAc ggc
ttt ! a c t t tct t t t c t t t t t c !W.T. ! NruI.... KasI...(3/4)
! ! I G I D V S G L A N G N G A T G D 9141 atc ggc gat gcc agt ggt
tTG GCC Aac ggc aac gga gcc acc gga gac ! t t c t tcc c c t t t t t
t t t t t !W.T. ! MscI....(3/3) ! ! F A G S N S Q M A Q V G D G D N
9189 ttc GCA GGT tcG AAT TGt cag atg gcC CAG GTT GGA GAT GGg gac
aac ! t t c t c a t a c t c t t t !W.T. ! BspMI.. (2/2)
XcmI................ ! EcoRI... ! ! S P L N N N F K Q Y L P S L P Q
9237 agt ccg ctt atg aac aac ttt aga cag tac ctt ccg tct ctt ccg
cag ! tca t t a t t c c t a t t a t c c t a !W.T. ! ! S V E C R P F
V F S A G K P Y E 9285 agt gtc gag tgc cgt cca ttc gtt ttc tcc gcc
ggc aag cct tac gag ! tcg t a t c t t c t agc t t a a t a !W.T. ! !
F S I D C I D K I N L F R 9333 ttc aGC Atc gac TGC gat aag atc aat
ctt ttC CGC ! t tct t t t c a a c t a c t !W.T. ! BatAPI........
SacII... ! End Domain 3 ! ! G V F A F L L V V A T F N V V F 9369
GGc gtt ttc gct tcc ttg cta tac gtc gct act ttc atg tac gtt ttc ! t
c t g t c t t a t t c c t t a t !W.T. ! start transmembrane segment
! ! S T F A N I L R N K E S 9417 aCC ACT TTC GCC AAT ATT TTA Cgc
aac aaa gaa agc ! tct g t t c a c g t t g g tct !W.T. !
Intraccellular anchor. ! ! . . 9453 tag tga tct CCT AGG ! AvrII.. !
9468 aag ccc gcc taa tga gcg ggc ctt tct ttt ct ggt ! | Trp
terminator | ! ! End Fab cassette ! 9503 ATGCAT CCTGAGC ccgat
actgtcgtcg tcccctcaaa ccggcagatg ! NsiI.. Bsu36I.(3/3) 9551
cacggtcacg atgcgcccat ctacaccaac gcgacctatc ccactacggt caacccgccg
9611 tttgttccca cggagaatcc gacgggttgt tactcgctca aatttaatgt
tgatgaaagc 9671 tggctacagg aaggccagac gcgaattatt tttgatggcg
ttcctattgg tcagaaaatg 9731 agctgactta accaaaattt aaTgcgaatt
ttaacaaaat attaacgttt acaATTTAAA ! SwaI... 9791 Tatttgctta
tacaatcttc ctgtctctgg ggcttttctg attatcaacc GGGGTAaat 9850 ATG att
gac atg cta gtt tta cga tta ccg ttc atc gat tct ctt gtt tgc ! Start
gene II 9901 tcc aga ctc tca ggc aat gac ctg ata gcc ttt gtA GAT
CTc tca aaa ata ! BglII... 9952 gct acc ctc tcc ggc atT aat tta tca
gct aga acg gct gaa tat aat att 10003 gat ggt gat ttg act gtc tcc
ggc ctc cct cac ccc ctt gaa tct tta cct 10054 aca aat tac tca ggc
att gca ctt aaa ata tat gag ggt tct aaa aat ttt 10105 tat cct tgc
gtt gaa ata aag gct tct ccc gca aaa gta tta cag ggt aat 10156 aat
gtt ttt ggt aca acc gat tta gct cta tgc tct gag gct tta ttg ctt
10207 aat ttt gct aat tct ttg cct tgc ctg tat gat tta ttg gat gtt !
! gene II continues !------------------------ End of Table
-------------------------------
[0324] TABLE-US-00043 !TABLE 37 DNA seq of w.t. M13 gene 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
1579 gtg aaa aaa tta tta ctc gca att act tta gtt gtt cct ttc tat !
Signal sequence............................................ ! ! 16
17 18 19 20 21 22 23 24 25 26 27 28 29 30 ! S H S A E T V E S C L A
K P H 1624 tct cac tcc gct gaa act gtt gaa agt tgt tta gca aaa ccc
cat ! Signal sequence> Domain
1--------------------------------------- ! ! 31 32 33 34 35 36 37
38 39 40 41 42 43 44 45 ! T E N S F T N V N K D D K T L 1669 aca
gaa aat tca ctt act aac gtc tgg aaa gac gac aaa act tta ! Domain
1--------------------------------------------------- ! ! 46 47 48
49 50 51 52 53 54 55 56 57 58 59 60 ! D R Y A N Y S G C L N N A T G
1714 gat cgt tac gct aac tat gag ggc tgt ctg tgG AAT GCt aca ggc !
BsmI.... ! Domain
1--------------------------------------------------- ! ! 61 62 63
64 65 66 67 68 69 70 71 72 73 74 75 ! V V V C T G D E T Q C Y C T N
1759 gtt gta gtt tgt act ggt gac gaa act cag tgt tac ggt aca tgg !
Domain 1--------------------------------------------------- ! ! 76
77 78 79 80 81 82 83 84 85 86 87 88 89 90 ! V P I G L A I P E N S G
G G S 1804 gtt cct att ggg ctt gct agc cct gaa aat gag ggt ggt ggc
tct ! Domain 1------------------------------> Linker
1----------- ! ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105
! S G C C S E G G G S S G G G T 1849 gag ggt ggc ggt tct gag ggc
ggc ggt tct gag ggt ggc ggt act ! Linker
1--------------------------------------------------> ! ! 106 107
108 109 110 111 112 113 114 115 116 117 118 119 120 ! K P P E Y G D
T P I P G Y T Y 1894 aaa cct ccc gag tac ggt gat acc cct att ccg
ggc tat act aat ! Domain
2--------------------------------------------------- ! ! 121 122
123 124 125 126 127 128 129 130 131 132 133 134 135 ! I N P L D G T
Y P P G T S Q N 1939 agc aac ccc ctc gac ggc act taT CCG CGt ggt
act gag caa aac ! EciI.... ! Domain
2--------------------------------------------------- ! ! 136 137
138 139 140 141 142 143 144 145 146 147 148 149 150 ! P A N P N P E
L F S S Q P L N 1984 ccc gct aat cct aat cct tct ctt GAG GAG tct
cag cct ctt aac ! BseRI.. ! Domain
2--------------------------------------------------- ! ! 151 152
153 154 155 156 157 158 159 160 161 162 163 164 165 ! T F N F Q N N
R F R N R Q S A 2029 act ttc atg ctt cag aat aat agg ttc ega aat
agg cag ggg gca ! Domain
2--------------------------------------------------- ! ! 166 167
168 169 170 171 172 173 174 175 176 177 178 179 180 ! L T V Y I G T
V T Q G T D P V 2074 tta act gtt tat aag ggc act gtt act caa ggc
act gac ccc gtt ! Domain
2--------------------------------------------------- ! ! 181 182
183 184 185 188 187 188 189 190 191 192 193 194 195 ! K T V Y Q V T
P V S S K A N Y 2119 aaa act aat tac cag tec act ccc gta tca tca
aaa gcc atg tat ! Domain
2--------------------------------------------------- ! ! 196 197
198 199 200 201 202 203 204 205 206 207 208 209 210 ! D A Y W N G K
F R V C A F H S 2164 gac gct tac tgg aac ggt aaa ttC AGa gaC TGc
gct ttc aat tct ! AlwNI....... ! Domain
2--------------------------------------------------- ! ! 211 212
213 214 215 216 217 218 219 220 221 222 223 224 225 ! G F N E D P F
V C E Y Q G Q S 2209 ggc ttt aat gaG GAT CCa ctc gtt tgt caa ggc
caa ggc caa tcg ! BamHI... ! Domain
2--------------------------------------------------- ! ! 226 227
228 229 230 231 232 233 234 235 236 237 238 239 240 ! S D L P Q P P
V N A G G C S C 2254 tct gac ctg cct caa cct cct gcc aat gct ggc
ggc ggc tct ggt ! Domain 2------------------------------> Linker
2----------- ! ! 241 242 243 244 245 246 247 248 249 250 251 252
253 254 255 ! G G S C G G S K C C G S K G C 2299 ggt ggt Oct ggt
ggc ggc tct gag ggt ggt ggc tct gag ggt ggc ! Linker
2--------------------------------------------------- ! ! 256 257
258 259 260 261 262 263 264 265 266 267 268 269 270 ! G S K G G G S
E G C G S G G G 2344 ggt tct gag ggt ggc ggc tct gag gga ggc ggt
tcc ggt ggt ggc ! Linker
2--------------------------------------------------- ! ! 271 272
273 274 275 276 277 278 279 280 281 282 283 284 285 ! S G S C G F D
Y K K N A N A N 2389 tct ggt tcc ggt gct ctt gat tat gaa aag atg
gca aac gct aat !Linker 2> Domain
3------------------------------------------- ! ! 286 287 288 289
290 291 292 293 294 295 296 297 298 299 300 ! K G A M T K N A D E N
A L Q S 2434 aag ggg gct atg acc gaa aat gcc gct gaa aac gcg cta
cag tct ! Domain
3--------------------------------------------------- ! ! 301 302
303 304 305 306 307 308 309 310 311 312 313 314 315 ! D A K C K L D
S V A T D Y G A 2479 gac gct aea ggc caa ctt gct tct gtc gct act
gct tac ggt gct ! Domain
3--------------------------------------------------- ! ! 316 317
318 319 320 321 322 323 324 325 326 327 328 329 330 ! A I D G F I G
D V S G L A N G 2524 gct ctc gct ggt tcc att ggt gcc gtt tcc ggc
cct gct aat ggt ! Domain
3--------------------------------------------------- ! ! 331 332
333 334 335 336 337 338 339 340 341 342 343 344 345 ! N C A T G D F
A G S N S Q M A 2569 aat ggt gct act ggt gct ttt gct ggc tct aat
tcc ccc atg gcc ! Domain
3--------------------------------------------------- ! ! 346 347
348 349 350 351 352 353 354 355 356 357 358 359 360 ! Q V G D G D N
S P L M N N F R 2614 caa gtc ggt gac ggt gat aat cca cct tta atg
aat aat ttc cgt ! Domain
3--------------------------------------------------- ! ! 361 362
363 364 365 366 367 368 369 370 371 372 373 374 375 ! Q Y L P S L P
Q S V K C R P F 2659 caa tat tta cct tcc ctc cct caa rcg gct gaa
tgt cgc cct ttt ! Domain
3--------------------------------------------------- ! ! 376 377
378 379 380 381 382 383 384 385 386 387 388 389 390 ! V F S A G K P
Y K F S I D C D 2704 gtc ttc agc gct ggt aaa cca tat gaa ttc tct
att gat tgt gac ! Domain
3--------------------------------------------------- ! 391 392 393
394 395 396 397 398 399 400 401 402 403 404 405 ! K I N L F R G V F
A F L L Y V 2749 aaa ata aac tca tcc cgt ggt gcc ttt gcg ttt ctt
tta tac gct ! Domain 3--------------> Transmembrane
segment-------------- ! ! 406 407 408 409 410 411 412 413 414 415
416 417 418 419 420 ! A T F M Y V F S T F A N I L R 2794 gcc acc
ttt atg tat gta ttt tct acg ttt gct aac ata ctg cgt ! Transmembrane
segment---------------------------------> ICA-- ! ! 421 422 423
424 425 ! N K K S . 2839 aat aag gag cct taa ! 2853 !
ICA-----------> ICA = intracellular anchor ! !------------------
End of Table -----------------------------------------
[0325] TABLE-US-00044 TABLE 38 Whole mature III anchor M13-III
derived anchor with racorded DNA ! ! 1 2 3 ! A A A 1 GCG gcc gca !
NotI...... ! ! 4 5 6 7 8 9 10 11 12 13 14 15 16 17 ! H H H H H H C
A A E Q K L I 10 cat cat cat cac aat cac ggg gcc gca gaa caa aaa
ctc atc ! ! 18 19 20 21 22 23 24 25 26 27 28 29 ! S E S D L N G A A
. A S 52 tca gaa gag gct ctg aat ggg gcc gca Tag GCT AGC ! NheI...
! ! 30 31 32 33 34 35 36 37 38 39 ! D I N D D R M A S T 88 GAT ATC
cac gat gat cgt atg gct tct act ! (ON_C37hor) [RC] 5-c aac gat gat
cgt atg gcG CAt Gct gcc gag aca g-3' ! EcoRV.. ! Enterokinase
cleavage site. ! ! Start mature III (recorded) Domain 1 ----> !
40 41 42 43 ! A S T V 118 |gcC|gaG|acA|gtC| ! t a t t ! W.T. ! ! 44
45 48 47 48 49 50 51 52 53 54 55 58 57 58 ! E S C L A K P H I S N S
F I N 130
|gaa|TCC|tgC|CTG|GCC|AaG|ccT|caC|acT|gaG|aat|AGT|ttC|aCA|Aat| ! agt
t t a a a c t a a tca t t c ! W.T. ! MscI.... ! ! 59 60 61 62 63 64
65 66 67 68 69 70 71 72 73 ! V W K D D K T L D R V A N V S 175
|gtg|TGG|aaG|gaT|gaT|aaG|acC|CtT|gAT|CGA|TaT|gcC|aaT|taC|gcA| ! c a
c c a t t a t c t c t g ! W.T. ! BspDI... ! ! 74 75 76 77 78 79 80
81 82 83 84 85 86 87 88 ! G C L N N A I G V V V C I C D 220
|ggC|tgC|Tta|tgg|aat|gcC|ACC|GGC|GtC|gtT|gtC|TGC|ACG|ggC|gaT| ! t t
c g t a t a t t t t c ! W.T. ! SgrAI...... BsgI.... ! ! 89 90 91 92
93 94 95 96 97 98 99 100 101 102 103 ! E T Q C V G I N V P I G L A
I 265 |gaG|acA|caA|tgC|taT|ggC|ACG|TCg|gtC|ccG|atA|gGC|TTA|GCC|atA|
! a t g t c t a t t t g c t t c ! W.T. ! PmlI.... BlpI..... ! !
Domain 1-----> Linker 1----------------> ! 104 105 106 107
108 109 110 111 112 113 114 115 116 117 118 ! P E N E G G C S F G G
G S K G 310
|ccG|gaG|aaC|gaA|ggC|ggC|ggT|AGC|gaA|ggC|ggT|ggC|AGC|gaA|ggC| ! t a
t g t t c tct g t c t tct g t ! W.T. ! ! Linker
1----------------------> Domain 2---------------> ! 119 120
121 122 123 124 125 126 127 128 129 130 131 132 133 ! G C S F C G G
T K P P F Y G D 355
|ggT|GGA|TGC|gaA|ggA|ggT|ggA|acC|aaG|ccG|ccG|gaA|taT|ggC|gaC| ! c t
t g t c t t a t t g c t t ! W.T. ! BamHI..(2/2) ! ! 134 135 136 137
138 139 140 141 142 143 144 145 146 147 148 ! T P I P G Y T Y I N P
L D G T 400
|acT|ccC|atA|CCT|GCT|taC|acC|taC|atT|aaT|ccG|TtA|gaT|ggA|acC| ! a t
t g c c t t c a t c c a a t ! W.T. ! SexAI.... ! ! 149 150 151 152
153 154 155 156 157 158 159 160 161 162 163 ! Y P P G T F Q N P A N
P N P S 445
|taC|caT|ccG|ggC|acC|gaA|caC|aaT|ccT|gcC|aaC|caG|caC|acA|AGC| ! T G
t t t g a c a t t t t ttc ! W.T. ! HindIII... ! ! 164 165 166 167
168 169 170 171 172 173 174 175 176 177 178 ! L F E S Q P L N T F M
F Q N N 490
|TTA|gaA|gaA|AGC|caA|aaG|TaA|aaC|acC|ttT|atg|ttC|caA|aaC|aaC| ! c t
G G tct g t c t t t a t g t t ! W.T. ! HindIII. ! ! 179 180 181 182
183 184 185 186 187 188 189 190 191 192 193 ! Y F P N P Q G A L T V
Y T G T 535
|CgT|ttT|AgG|aaC|CgT|CaA|gCT|GCT|CtT|acC|gTG|TAC|AcT|ggA|acC| ! a g
c c a t a g g g a t a t t t g c t ! W.T. ! HgiAI... BsrGI... ! !
194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 ! V T Q
G T D P V K T Y Y Q Y T 580
|gtC|aaC|aaG|GGT|ACC|gaT|ccT|gtC|aaC|taC|taT|caA|taT|aaC| ! t t a c
t a a t a t t a g a t ! W.T. ! KpnI... ! ! 209 210 211 212 213 214
215 216 217 218 219 220 221 222 223 ! P V S S K A N Y D A Y N N G K
625 |caG|gtC|TCG|AGt|aaG|gcT|atg|taC|gaT|gcC|taT|tgg|aaT|ggC|aaC| !
t a a tca a c t a t a a t a ! W.T. ! BsaI.... ! XhoI.... ! ! 224
225 226 227 228 229 230 231 232 233 234 235 236 237 238 ! F R D C A
F H S G F N F D P F 670
|ttT|CgT|gaT|tgT|gcC|taT|caC|AGC|ggT|ttC|aaC|gaa|gac|CCt|ttT| ! C A
a C a t c t tct c t t G T a c ! W.T. ! ! 239 240 241 242 243 244
245 246 247 248 249 250 251 252 253 ! V C E Y Q G Q S S D L P Q P P
715 |gtC|tgC|gaG|taC|caG|ggT|caG|AGT|AGC|gaT|TcA|ccG|caG|ccA|CCG| !
t t a t a c a tcg tct c c g t a t t ! W.T. ! DrdI..... AgeI..... !
Domain 2--------> Linker 2---------------------> ! 254 255
256 257 258 259 260 261 262 263 264 265 266 267 268 ! V N A G G G S
G G G S G G G S 760
|GTT|AAC|gcG|ggT|ggT|ggT|AGC|ggC|ggA|ggC|AGC|ggC|ggT|ggT|AGC| ! c t
t c c c tct t t t t tct t c c tct ! W.T. ! AgeI..... ! Hpa... !
HincII. ! ! Linker
2----------------------------------------------> Domain 3-->
! 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 ! E G
G G S K G G G S G G G S C 805
|gaA|ggC|ggA|ggT|AGC|gaA|ggA|ggT|ggC|AGC|ggA|ggC|ggT|AGC|ggC| ! g t
t c tct g t c t tct g t c tct t ! W.T. ! ! ------------Domain
3-------------------> ! 284 285 286 287 288 289 290 291 292 293
294 295 296 297 298 ! S G D F D Y K K N A N A N K G 850
|AGT|ggC|gac|ttc|gac|tac|gag|aaa|atg|gct|aat|gcc|aac|aaa|GGC| ! tcc
t t t t t a g a c t t g g ! W.T. ! Kas.... ! ! 299 300 301 302 303
304 305 306 307 308 309 310 311 312 313 ! A M K N A D K N A L Q S D
A 895 |GCC|atg|act|gag|aac|gcc|gac|gaG|AAT|GCA|ctg|caa|agt|gat|gCC|
! t c a t c t a c g a g tct c t ! W.T. ! Kas.... BsmI.... Sty... !
! 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 ! K G
K L D S V A T D Y C A A I 940
|AAG|GGt|aag|tta|gac|agc|gTC|GCc|Aca|gac|tat|ggT|GCt|gac|atc| ! a c
a c t tct t t t c t ! W.T. ! Sty...... PflFI...... ! ! 329 330 331
332 333 334 335 336 337 338 339 340 341 342 343 ! D C F G D V S C L
A N C N G 985
|gac|ggc|ttt|atc|ggc|gat|gtc|agt|ggt|ctg|gct|aac|ggc|aac|gga| ! t t
c t t c t tcc c c t t t t t t ! W.T. ! ! 344 345 346 347 348 349
350 351 352 353 ! A T G D F A G S N S 1030
|gcc|acc|gga|gac|ctc|GCA|GGT|tcG|AAT|TCt| ! t t t t t t c t c !
W.T. ! BstBI... ! EcoRI... ! BspMI.. ! ! 354 355 356 357 358 359
360 361 362 363 ! Q M A Q V G D G D N 1060 cag atg gcC CAG GTT GGA
GAT GGg gac aac ! a t a c t c t t t ! W.T. ! XcmI................ !
! 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 !
S P L K N N F R Q Y L P S L P Q 1090 agt ccg ctt atg cac aac tct
aga cag cac ctt ccg cct ctt ccg cag ! tca t t a t t c c t a t t a t
c c t a ! W.T. ! ! 380 381 382 383 384 385 386 387 388 389 390 391
392 393 394 395 ! S V K C R P F V F S A G K P Y E 1138 agt gtc gag
tgc cgt cca ttc gtt ttc tct gcc ggc aag cct tac gag ! tcg t a t c t
t c t agc t t a a t a ! W.T. ! ! Domain
3-------------------------------> ! 396 397 398 399 400 401 402
403 404 405 406 407 ! F S I D C D K N L F K 1186 ttc aGC Atc gac
TGC gct cag atc aat ctt ttC CGC ! t tct t t t c a a c t a t !
BstAPI........ SacII... ! ! transmembrane segment------------->
! 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 !
G V F A F L L Y V A T F K V V F 1222 GGc gtt ctc gct ttc ttg cta
tac gcc gct act ttc atg tac gtt ttc ! t c t g t c t t a t t c c t t
a t ! W.T. ! ! 424 425 426 427 428 429 430 431 432 433 434 435 ! S
T F A N L R N K E S 1270 aGC ACT TTC GCC AAT ATT TTA Cgc aac aaa
gaa agc ! tct g t t c a c g t t g g tct ! W.T. ! Intracellular
anchor. ! ! . . 1306 tag tga tct CCT AGG ! AvrII.. ! 1321 aag ccc
gcc taa tga gcg ggc ttt ttt ttt ct ggt ! | Trp terminator | ! ! End
Feb cassette !---------------------------- End of Table
-------------------------
[0326] TABLE-US-00045 TABLE 39 ONs to make deletions in III ! ONs
for use with NheI ! (ON_G29bot) 5'-c gTT gAT ATc gcT Agc cTA Tgc-3'
! 22 ! this is the reverse complement of 5'-gca cag gct agc gat atc
aac g-3' ! NheI... scab......... (ON_G104top)
5'-g|ata|ggc|tta|gcT|aGC|ccg|gag|aac|gaa|gg-3' ! 30 !
Scab..........NheI... 104 105 106 107 108 (ON_G236top)
5'-c|ttt|cac|agc|ggt|ttc|GCT|AGC|gac|ccc|ttt|gtc|tgc-3' ! 37 !
NheI... 236 237 238 239 240 (ON_G236tCS)
5'-c|ttt|cac|agc|ggt|tcc|GCT|AGC|gac|cct|ctc|gcc|Agc- ! NheI... 236
237 238 239 240 gag|tac|cag|ggt|c-3' ! 50 ! ONs for use with SphI G
CAT Gc (ON_X37bot) 5'-gAc TgT cTc ggc Agc ATg cgc cAT Acg ATc ATc
gTT g 3' ! 37 ! N D D R M A H A !(ON_X37bot)=[RC] 5'-c cac gat gat
cgt atg gcG CAt Gct gcc gag aca gtc -3' ! SphI....Scab...........
(ON_X104top) 5'-g|gtG|ccg|agc|ggc|ttG|CAT|GCa|ccg|gag|ccc|gaa|gg-3'
! 36 ! Scab................SphI.... 104 105 106 107 108
(ON_X236top)
5'-c|ttt|ccc|cgc|ggt|ttG|CaT|gCa|gac|cct|ttt|gtc|tgc-3' ! 37 !
Sph.... 236 237 238 239 240 (ON_X236tCS)
5'-c|ttt|ccc|agc|ggt|ttG|CaT|gCa|gac|ccc|ttt|gtc|Agc- NheI... 236
237 238 239 240 gag|tac|cag|ggt|c-3' ! 50
[0327] TABLE-US-00046 TABLE 40 Phage titers and enrichments of
selections with a DY3F31-based human Fab library Output/input Input
(total cfu) Output (total cfu) ratio R1-ox selected on 4.5 .times.
10.sup.12 3.4 .times. 10.sup.5 7.5 .times. 10.sup.-8 phOx-BSA
R2-Strep selected 9.2 .times. 10.sup.12 3 .times. 10.sup.8 3.3
.times. 10.sup.-5 on Strep-beads
[0328] TABLE-US-00047 TABLE 41 Frequency of ELISA positives in
DY3F31-based Fab libraries Anti-M13 9E10/RAM- Anti-CK/CL HRP HRP
Gar-HRP P2-ox (with IPTG induction) 18/44 10/44 10/44 P2-ox
(without IPTG) 13/44 ND ND R3-strep (with IPTG) 39/44 38/44 36/44
R3-strep (without IPTG) 33/44 ND ND
* * * * *
References