U.S. patent application number 13/725184 was filed with the patent office on 2013-06-27 for processes using vlps with capsids resistant to hydrolases.
This patent application is currently assigned to APSE, LLC. The applicant listed for this patent is Apse, LLC. Invention is credited to Juan P. Arhancet, Juan Pedro Humberto Arhancet, Kimberly Delaney, Kathleen B. Hall, Neena Summers.
Application Number | 20130167267 13/725184 |
Document ID | / |
Family ID | 48655925 |
Filed Date | 2013-06-27 |
United States Patent
Application |
20130167267 |
Kind Code |
A1 |
Arhancet; Juan Pedro Humberto ;
et al. |
June 27, 2013 |
PROCESSES USING VLPS WITH CAPSIDS RESISTANT TO HYDROLASES
Abstract
Novel processes and compositions are described which use viral
capsid proteins resistant to hydrolases to prepare virus-like
particles to enclose and subsequently isolate and purify target
cargo molecules of interest including nucleic acids such as siRNA's
and shRNA's, and small peptides.
Inventors: |
Arhancet; Juan Pedro Humberto;
(Creve Coeur, MO) ; Arhancet; Juan P.; (Creve
Coeur, MO) ; Delaney; Kimberly; (St. Louis, MO)
; Hall; Kathleen B.; (St. Louis, MO) ; Summers;
Neena; (St. Charles, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Apse, LLC; |
Creve Coeur |
MO |
US |
|
|
Assignee: |
APSE, LLC
Creve Coeur
MO
|
Family ID: |
48655925 |
Appl. No.: |
13/725184 |
Filed: |
December 21, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61607900 |
Mar 7, 2012 |
|
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61578706 |
Dec 21, 2011 |
|
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61661688 |
Jun 19, 2012 |
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Current U.S.
Class: |
800/298 ;
424/418; 424/491; 435/252.3; 435/254.11; 435/254.2; 435/262;
435/320.1; 435/325; 435/419; 435/69.1; 504/234; 514/1.1; 514/44A;
536/23.1; 536/24.1; 536/24.5 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2795/18122 20130101; C07K 14/01 20130101; C12N 2310/121
20130101; C12N 2330/51 20130101; A01N 25/28 20130101; C12N 2310/14
20130101; C12N 2795/18123 20130101; C12N 7/04 20130101; C12N
2310/123 20130101; C12N 15/111 20130101; C12N 2310/16 20130101;
A01N 25/26 20130101; C12N 2310/128 20130101; C12N 15/113 20130101;
A01N 25/28 20130101; C12N 2310/14 20130101; C12N 7/00 20130101;
C12N 2795/18142 20130101; A01N 47/40 20130101; A01N 57/12 20130101;
A01N 57/14 20130101; A01N 43/70 20130101; A01N 57/30 20130101; C12N
2310/531 20130101 |
Class at
Publication: |
800/298 ;
424/418; 536/24.1; 536/23.1; 536/24.5; 504/234; 435/419; 435/320.1;
435/252.3; 435/325; 435/254.11; 435/254.2; 435/262; 435/69.1;
424/491; 514/44.A; 514/1.1 |
International
Class: |
C07K 14/01 20060101
C07K014/01; C12N 15/113 20060101 C12N015/113; A01N 25/26 20060101
A01N025/26 |
Claims
1. A virus-like particle (VLP) comprising a capsid enclosing at
least one heterologous cargo molecule and a packing sequence.
2. A VLP according to claim 1, further comprising at least one
ribozyme enclosed by the capsid.
3. A VLP according to claim 2, wherein the heterologous cargo
molecule comprises an oligonucleotide.
4. A VLP according to claim 3, wherein the heterologous cargo
molecule comprises an oligoribonucleotide is a short RNA selected
from siRNA, shRNA, sshRNA, lshRNA and miRNA.
5. A VLP according to claim 4, wherein the ribozyme is flanked by
the packing sequence and the oligoribonucleotide to form a nucleic
acid construct.
6. A VLP according to claim 5, comprising at least two ribozymes,
wherein each ribozyme is selected to cut one end of the short
RNA.
7. A VLP according to claim 4, further comprising a linker
consisting of at least 1 to 100 nucleotides, the linker comprising
at least 40% A's or at least 40% U's, wherein the linker links the
oligoribonucleotide and the packing sequence, or the
oligoribonucleotide and the ribozyme,
8. A VLP according to claim 2, wherein the ribozyme is selected
from a Hammerhead ribozyme and a Hepatitis Delta V ribozyme.
9. A VLP according to claim 2, wherein the ribozyme is a Hammerhead
ribozyme variant having a contiguous set of nucleotides
complementary to at least 6 contiguous nucleotides of the
oligoribonucleotide.
10. A VLP according to claim 2, wherein the ribozyme is a mutant
Hepatitis Delta V ribozyme capable of cleaving its connection with
the oligoribonucleotide at a rate at most about 50% the rate of a
wildtype Hepatitis Delta V ribozyme, or a mutant HDV ribozyme
having a nucleic acid sequence selected from SEQ ID Nos: 10-18.
11. A VLP according to claim 1, wherein the capsid comprises a wild
type viral capsid which is resistant to hydrolysis catalyzed by a
peptide bond hydrolase category EC 3.4.
12. A VLP according to claim 1, wherein the capsid comprises a
capsid protein having at least 40% sequence identity with the amino
acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID
NO: 3) and is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4.
13. A VLP according to claim 1, wherein the capsid comprises a
capsid protein having at least 86% sequence identity with the amino
acid sequence of wild type Enterobacteria phage MS2 capsid protein
(SEQ ID NO: 3) and is resistant to hydrolysis catalyzed by a
peptide bond hydrolase category EC 3.4.
14. A VLP according to claim 1, wherein the capsid comprises a wild
type Enterobacteria phage MS2 capsid protein having the amino acid
sequence of SEQ ID NO: 3.
15. A VLP according to claim 1, wherein the heterologous cargo
molecule comprises a peptide or a polypeptide.
16. A VLP according to claim 15, further comprising an
oligonucleotide linker coupling the heterologous cargo molecule and
the viral capsid.
17. A VLP according to claim 16, wherein the oligonucleotide linker
is an oligoribonucleotide comprising a ribozyme sequence.
18. A VLP according to claim 1, wherein the heterologous cargo
molecule comprises a bi-molecular cargo molecule comprising a
bifunctional polynucleotide comprising a first apatamer sequence
which specifically binds a bioactive small molecule having a
molecular weight of about 1,500 Da or less and a second aptamer
sequence for binding a packing sequence of the capsid.
19. A VLP according to claim 18, further comprising the bioactive
small molecule bound to the first aptameric sequence, wherein the
bioactive small molecule comprises an herbicide or a pesticide.
20. A VLP according to claim 19, wherein the bioactive small
molecule is selected from the group consisting of: atrazine,
acetamipridphorate, profenofos, isocarbophos and omethoateas.
21. A nucleic acid construct comprising a nucleotide sequence that
encodes a short RNA, a ribozyme and a packing sequence.
22. A nucleic acid construct according to claim 21, wherein the
short RNA is an siRNA or an shRNA.
23. A nucleic acid construct according to claim 21, further
comprising a linking nucleotide sequence of 4 to 100 nucleotides of
which at least 40% are A's, at least 40% are T's, or at least 40%
are U's, wherein the linking nucleotide sequence is flanked by the
ribozyme and the short RNA-encoding sequence
24. A nucleic acid construct according to claim 21, wherein the
ribozyme is flanked by the short RNA and the packing sequence.
25. A vector comprising a nucleic acid construct according to claim
21.
26. A host cell comprising the vector according to claim 25.
27. A host cell according to claim 37, wherein the host cell is
stably transfected with the vector.
28. A host cell according to claim 37, which is selected from a
bacterial cell, a plant cell, a mammalian cell, a fungal cell, and
a yeast cell.
29. A host cell according to claim 27, wherein the host cell is
further stably transfected with a second vector comprising a second
nucleic acid sequence encoding a viral capsid which is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC
3.4.
30. A host cell according to claim 29, wherein the second nucleic
acid sequence encodes a viral protein encoding a viral capsid
having at least 40% sequence identity with the amino acid sequence
of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3)
and is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4.
31. A host cell according to claim 29, wherein the second nucleic
acid sequence encodes a wild type Enterobacteria phage MS2 capsid
protein (SEQ ID NO: 3).
32. A nucleic acid construct according to claim 21, wherein the
ribozyme is a Hammerhead ribozyme or a Hepatitis Delta V
ribozyme.
33. A nucleic acid construct according to claim 32, wherein the
ribozyme is a Hammerhead ribozyme variant having a contiguous set
of nucleotides complementary to at least 6 contiguous nucleotides
of the short RNA.
34. A nucleic acid construct according to claim 32, wherein the
ribozyme is a mutant Hepatitis Delta V ribozyme capable of cleaving
its connection with the short RNA at a rate at most 50% the rate of
a wildtype Hepatitis Delta V ribozyme, or a mutant HDV ribozyme
having a nucleic acid sequence selected from SEQ ID NOs: 10-18.
35. A plant or plant tissue transformed to contain the nucleic acid
construct according to claim 21.
36. A seed or progeny of the plant or plant tissue of claim 35,
wherein the seed or progeny comprises the nucleic acid
construct.
37. A composition comprising: a) a plurality of virus-like
particles each comprising a viral capsid enclosing at least one
heterologous cargo molecule; and b) one or more cell lysis products
present in an amount of less than 4 grams for every 100 grams of
capsid present in the composition, wherein the cell lysis products
are selected from proteins, polypeptides, peptides and any
combination thereof.
38. A composition according to claim 37, wherein the capsid is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4.
39. A composition according to claim 37, wherein the capsid
comprises a capsid protein having at least 40% sequence identity
with the amino acid sequence of wild type Enterobacteria phage MS2
capsid (SEQ ID NO: 3) and is resistant to hydrolysis catalyzed by a
peptide bond hydrolase category EC 3.4.
40. A composition according to claim 37, wherein the capsid
comprises a wild type Enterobacteria phage MS2 capsid protein (SEQ
ID NO: 3).
41. A composition according to claim 37, wherein the heterologous
cargo molecule comprises an oligonucleotide.
42. A composition according to claim 41, wherein the heterologous
cargo molecule comprises an oligoribonucleotide selected from
siRNA, shRNA, sshRNA, lshRNA and miRNA.
43. A composition according to claim 42, wherein each virus-like
particle further comprises at least one ribozyme, wherein the
ribozyme is flanked by the packing sequence and the
oligoribonucleotide to form a nucleic acid construct.
44. A composition according to claim 42, wherein the ribozyme is
selected from a Hammerhead ribozyme and a Hepatitis Delta V
ribozyme.
45. A composition according to claim 42, wherein the ribozyme is a
Hammerhead ribozyme variant having a contiguous set of nucleotides
complementary to at least 6 contiguous nucleotides of the
oligoribonucleotide.
46. A composition according to claim 42, wherein the ribozyme is a
mutant Hepatitis Delta V ribozyme capable of cleaving its
connection with the oligoribonucleotide at a rate at most about 50%
the rate of a wildtype Hepatitis Delta V ribozyme, or a mutant HDV
ribozyme having a nucleic acid sequence selected from SEQ ID NOS:
10-18.
47. A composition according to claim 42, wherein the
oligoribonucleotide and the packing sequence are linked by a
nucleotide sequence of 1 to 100 nucleotides in length, such linking
sequence comprising more than 40% A's or more than 40% of U's.
48. A composition according to claim 41, wherein the heterologous
cargo molecule comprises a peptide or a polypeptide.
49. A composition according to claim 48, further comprising an
oligonucleotide linker coupling the heterologous cargo molecule and
the viral capsid.
50. A composition according to claim 49, wherein the
oligonucleotide linker is an oligoribonucleotide comprising a
ribozyme sequence.
51. A method for isolating and purifying a target cargo molecule,
the method comprising: (a) obtaining a whole cell lysate comprising
a plurality of virus-like particles (VLPs) each comprising a capsid
enclosing at least one target cargo molecule, wherein the capsids
are resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4; (b) subjecting the VLP's to hydrolysis using a
peptide bond hydrolase category EC 3.4, for a time and under
conditions sufficient for at least 60, at least 70, at least 80, or
at least 90 of every 100 individual polypeptides present in the
whole cell lysate but not enclosed by the capsids to be cleaved,
while at least 60, at least 70, at least 80, or at least 90 of
every 100 capsids present in the whole cell lysate before such
hydrolysis remain intact following the hydrolysis.
52. A method according to claim 51, wherein the capsids each
comprises a viral capsid protein having at least 40% sequence
identity with the amino acid sequence of wild type Enterobacteria
phage MS2 capsid protein (SEQ ID NO: 3) and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC
3.4.
53. A method according to claim 51, wherein the capsids each
comprise a wild type Enterobacteria phage MS2 capsid protein (SEQ
ID NO: 3).
54. A method according to claim 51, wherein the target cargo
molecule comprises an oligonucleotide.
55. A method according to claim 51, wherein the target cargo
molecule comprises an oligoribonucleotide selected from siRNA,
shRNA, sshRNA, lshRNA and miRNA.
56. A method according to claim 51, wherein each virus-like
particle further comprises a ribozyme, wherein the ribozyme is
flanked by the packing sequence and the oligoribonucleotide to form
a nucleic acid construct.
57. A method according to claim 51, further comprising purification
of the capsids following hydrolysis.
58. A method according to claim 51, wherein the target cargo
molecule comprises a peptide or a polypeptide.
59. A composition produced by the method according to claim 51.
60. A method for protecting a target molecule from hydrolysis in a
whole cell lyste following intracellular production of the target
molecule in a host cell, the method comprising: (a) selecting a
viral capsid which is resistant to hydrolysis catalyzed by a
peptide bond hydrolase category EC 3.4; (b) stably transfecting the
host cell with a first vector comprising a nucleic acid sequence
encoding a viral protein forming the viral capsid, and a second
vector comprising a nucleic acid sequence comprising a ribozyme
flanked by a packing sequence and an siRNA sequence; and (c)
maintaining the cells for a time and under conditions sufficient
for the transformed cells to express and assemble capsids
encapsidating the ribozyme flanked by the packing sequence and the
siRNA sequence.
61. A process for purifying VLP's enclosing at least one
heterologous cargo molecule, the process comprising: (a) obtaining
a cell lysate comprising a plurality of the VLP's; (b) contacting
the cell lysate with a protease for a time and under conditions
sufficient to hydrolyze cell lysis products other than the VLP's to
form a hydrolysate; and (c) isolating the VLP's from the
hydrolsyate.
62. The process according to claim 61, wherein step (c) comprises
(i) performing a first precipitation with ammonium sulfate followed
by a first centrifugation to obtain a first precipitate and a first
supernatant; and (ii) performing a second precipitation on the
first supernatant with ammonium sulfate followed by a second
centrifugation to obtain a second precipitate, wherein the second
precipitate comprises at least about 90% by weight of the
VLP's.
63. The process according to claim 61, wherein step (c) comprises
(i) performing a first precipitation with ethanol followed by a
first centrifugation to obtain a first precipitate and a first
supernatant; and (ii) performing a second precipitation on the
first supernatant with ammonium sulfate followed by a second
centrifugation to obtain a second precipitate, wherein the second
precipitate comprises at least about 90% by weight of the
VLP's.
64. The process according to claim 61, wherein step (c) comprises
ultracentrifuging the hydrolysate to obtain a precipitate
comprising at least about 90% by weight of the VLP's.
65. The process according to claim 61, wherein the VLP's each
comprise a capsid which is resistant to hydrolysis catalyzed by a
peptide bond hydrolase category EC 3.4.
66. The process according to claim 61, wherein the VLP's each
comprise a capsid which comprises a capsid protein having at least
40% sequence identity with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO: 3) and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC
3.4.
67. The process according to claim 61, wherein the VLP's each
comprise a wild type Enterobacteria phage MS2 capsid protein (SEQ
ID NO: 3).
68. The process according to claim 61, wherein step (b) is
performed for at least about 30 minutes at about 37.degree. C.
69. The process according to claim 61, further comprising before
step (b), contacting the cell lysate with at least one of a
nuclease, an amylase and a lypase for at least about 30 minutes at
about 37.degree. C.
70. The process according to claim 61, wherein the protease is a
peptide bond hydrolase category EC 3.4.
71. The process according to claim 61, wherein the protease is
selected from Proteinase K, Protease from Streptomyces griseus, and
Protease from Bacillus lichenformis, pepsin and papain.
72. The process according to claim 61, wherein the heterologous
cargo molecule comprises an oligonucleotide.
73. The process according to claim 61, wherein the heterologous
cargo molecule comprises an oligoribonucleotide selected from
siRNA, shRNA, sshRNA, lshRNA and miRNA.
74. The process according to claim 61, wherein the VLP's each
further comprise a ribozyme flanked by a packing sequence and the
oligoribonucleotide to form a nucleic acid construct.
75. The process according to claim 74, wherein the ribozyme is
selected from a Hammerhead ribozyme and a Hepatitis Delta V
ribozyme.
76. The process according to claim 74, wherein the ribozyme is a
Hammerhead ribozyme variant having a contiguous set of nucleotides
complementary to at least 6 contiguous nucleotides of the
oligoribonucleotide.
77. The process according to claim 74, wherein the ribozyme is a
mutant Hepatitis Delta V ribozyme capable of cleaving its
connection with the oligoribonucleotide at a rate at most about 50%
the rate of a wildtype Hepatitis Delta V ribozyme, or a mutant HDV
ribozyme having a nucleic acid sequence selected from SEQ ID Nos:
10-18.
78. The process according to claim 74, wherein the
oligoribonucleotide and the packing sequence are linked by a linker
sequence of at least 1 to 100 nucleotides, and comprising more than
40% A's or more than 40% of U's.
79. The process according to claim 61, wherein the heterologous
cargo molecule comprises a peptide or a polypeptide.
80. The process according to claim 79, wherein the VLP's each
further comprise an oligonucleotide linker coupling the
heterologous cargo molecule and the viral capsid.
81. The process according to claim 80, wherein the oligonucleotide
linker is an oligoribonucleotide comprising a ribozyme
sequence.
82. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) except that the A
residue at position 1 is deleted, and is resistant to hydrolysis
catalyzed by a peptide bond hydrolase category EC 3.4.
83. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) except that the A
residue at position 1 is deleted and the S residue at position 2 is
deleted, and is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4.
84. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) except that the A
residue at position 1 is deleted, the S residue at position 2 is
deleted and the N residue at position 3 is deleted, and is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4.
85. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) except that the Y
reside at position 129 is deleted, and is resistant to hydrolysis
catalyzed by a peptide bond hydrolase category EC 3.4.
86. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) having a single (1)
amino acid deletion in the 112-117 segment and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC
3.4.
87. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) having a single (1)
amino acid deletion in the 112-117 segment and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC
3.4.
88. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) having a 1-2 residue
insertion in the 65-83 segment and is resistant to hydrolysis
catalyzed by a peptide bond hydrolase category EC 3.4.
89. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) having a 1-2 residue
insertion in the 44-55 segment and is resistant to hydrolysis
catalyzed by a peptide bond hydrolase category EC 3.4.
90. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) having a single (1)
residue insertion in the 33-43 segment and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC
3.4.
91. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) having a 1-2 residue
insertion in the 24-30 segment and is resistant to hydrolysis
catalyzed by a peptide bond hydrolase category EC 3.4.
92. A VLP according to claim 1, wherein the capsid comprises a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) having a single (1)
residue insertion in the 10-18 segment and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC
3.4.
93. A VLP according to claim 1, wherein the capsid comprises a
capsid protein monomer sequence concatenated with a second capsid
monomer sequence which assembles into a capsid which resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC
3.4.
94. A VLP according to claim 1, wherein the capsid comprises a
capsid protein monomer sequence whose C-terminus is extended with a
0-6 residue linker segment whose C-terminus is concatenated with a
second capsid monomer sequence, all of which assembles into a
capsid which resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4.
95. A VLP according to claim 109, wherein the linker sequence is
-(Gly).sub.x-, wherein x=0-6, or a Gly-Ser linker selected from
-Gly-Gly-Ser-Gly-Gly-, -Gly-Gly-Ser and -Gly-Ser-Gly-.
96. A VLP according to claim 109, wherein the capsid comprises a
capsid protein concatenated with a third capsid monomer sequence
which assembles into a capsid which resistant to hydrolysis
catalyzed by a peptide bond hydrolase category EC 3.4.
97. A VLP according to claim 109, wherein the capsid comprises a
capsid protein wherein the C-terminus is extended with a 0-6
residue linker segment whose C-terminus s concatenated with a third
capsid monomer sequence, all of which assembles into a capsid which
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4.
98. A VLP according to claim 109, wherein the capsid comprises a
capsid protein in which one or both linker sequences is
-(Gly).sub.x-, wherein x=0-6, or a Gly-Ser linker selected from
-Gly-Gly-Ser-Gly-Gly-, -Gly-Gly-Ser and -Gly-Ser-Gly- which
assembles into a capsid which is resistant to hydrolysis catalyzed
by a peptide bond hydrolase category EC 3.4.
99. A VLP according to claim 109, wherein the capsid comprises a
capsid protein of in which one or both linker sequences is
-(Gly).sub.x-, x=1. which assembles into a capsid which is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4.
100. A VLP according to claim 136, wherein the capsid comprises a
capsid protein of in which one or both linker sequences is
-(Gly).sub.x-, x=2. which assembles into a capsid which is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4.
101. A VLP according to claim 109, wherein the capsid comprises a
capsid protein of in which one or both linker sequences is
-(Gly).sub.x-, x=3. which assembles into a capsid which is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4.
102. A VLP according claim 1, wherein one or more coat protein
sequences is N-terminally truncated by 1-3 residues and a linker
sequence is lengthened by the number of residues deleted, and which
is resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4, wherein the linker sequence is -(Gly).sub.x-,
wherein x=0-6.
103. A VLP according claim 1, wherein one or more coat protein
sequences is C-terminally truncated by 1 residue and a linker
sequence is lengthened by the one residue and which is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4,
wherein the linker sequence is -(Gly).sub.x-, wherein x=0-6.
104. A VLP according claim 1, wherein the first coat protein
sequence in a concatenated dimer is C-terminally truncated by 1
residue and a linker sequence is lengthened by the one residue or
wherein the first and/or second coat protein sequence in a
concatenated trimer is C-terminally truncated by 1 residues and
which is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4, wherein the linker sequence is
-(Gly).sub.x-, wherein x=0-6.
105. A VLP according claim 1, containing N- and C-terminal
truncations and which is resistant to hydrolysis catalyzed by
peptide bond hydrolase category EC 3.4.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of U.S. provisional
application No. 61/578,706, filed Dec. 21, 2011, U.S. provisional
application No. 61/607,900, filed Mar. 7, 2012, and U.S.
provisional application No. 61/661,688, filed Jun. 19, 2012, the
entire disclosures of which are hereby incorporated by
reference.
INCORPORATION OF SEQUENCE LISTING
[0002] The entire contents of a paper copy of the "Sequence
Listing" and a computer readable form of the sequence listing on
diskette, containing the file named
450061_SequenceListing_ST25.txt, which is 77 kilobytes in size and
was created on Dec. 20, 2012, are herein incorporated by
reference.
TECHNICAL FIELD
[0003] The invention relates to virus-like particles, and in
particular to methods and compositions using viral capsids as
nanocontainers for producing, isolating and purifying heterologous
nucleic acids and proteins.
BACKGROUND OF THE INVENTION
[0004] Virus-like particles (VLPs) are particles derived in part
from viruses through the expression of certain viral structural
proteins which make up the viral envelope and/or capsid, but VLPs
do not contain the viral genome and are non-infectious. VLPs have
been derived for example from the Hepatitis B virus and certain
other viruses, and have been used to study viral assembly and in
vaccine development.
[0005] Viral capsids are composed of at least one protein, several
copies of which assemble to form the capsid. In some viruses, the
viral capsid is covered by the viral envelope. Such viral envelopes
are comprised of viral glycoproteins and portions of the infected
host's cell membranes, and shield the viral capsids from large
molecules that would otherwise interact with them. The capsid is
typically said to encapsidate the nucleic acids which encode the
viral genome and sometimes also proteins necessary for the virus'
persistence in the natural environment. For the viral genome of a
virus to enter a new host, the capsid must be disassembled. Such
disassembly happens under conditions normally used by the host to
degrade its own as well as foreign components, and most often
involves proteolysis. Viruses take advantage of normal host
processes such as proteolytic degradation to enable that critical
part of their cycle, i.e. capsid disassembly and genome
release.
[0006] It is therefore unsurprising that the literature has not
previously described capsids resistant to hydrolases that act on
peptide bonds. A very limited number of certain specific peptide
sequences which are part of larger proteins are known to be
somewhat resistant to certain proteases, but the vast majority of
peptide sequences are not. Viruses that resist proteolysis have
been reported, but these are all enveloped viruses, in which the
capsid is shielded by the viral envelope. In such viruses the
capsids are not in contact with, i.e. they are shielded from, the
proteases described. Thus the role, if any, of the proteolytical
stability of the virus capsid in such cases is unknown.
[0007] In large-scale manufacturing of recombinant molecules such
as proteins, ultrafiltration is often used to remove molecules
smaller than the target protein in the purification steps leading
to its isolation. Purification methods also often involve
precipitation, solvent extraction, and crystallization techniques.
These separation techniques are inherently simple and low cost
because, in contrast to chromatography, they are not based on
surface but on bulk interactions. However, these techniques are
typically limited to applications to simple systems, and by the
need to specify a different set of conditions for each protein and
expression system. Yet each target recombinant protein presents a
unique set of binding interactions, thereby making its isolation
process unique and complex. The separation efficiency for
recombinant proteins using these simple isolation processes is
therefore low.
[0008] Nucleic acids, including siRNA and miRNA, have for the most
part been manufactured using chemical synthesis methods. These
methods are generally complex and high cost because of the large
number of steps needed and the complexity of the reactions which
predispose to technical difficulties, and the cost of the
manufacturing systems. In addition, the synthetic reagents involved
are costly and so economy of scale is not easily obtained by simply
increasing batch size.
BRIEF SUMMARY OF THE INVENTION
[0009] In one aspect the present disclosure provides a virus-like
particle (VLP) comprising a capsid enclosing at least one
heterologous cargo molecule and a packing sequence. A VLP may
further comprise at least one ribozyme enclosed by the capsid. The
heterologous cargo molecule may comprise an oligonucleotide, or an
oligoribonucleotide. A VLP may comprise a one or more ribozymes,
and a ribozyme may be flanked by the packing sequence and the
oligoribonucleotide to form a nucleic acid construct. A VLP may
comprise a plurality of the nucleic acid constructs. In a VLP
comprising an oligoribonucleotide, oligoribonucleotide may be a
short RNA selected from siRNA, shRNA, sshRNA, lshRNA and miRNA. A
VLP may comprise at least two ribozymes, wherein each ribozyme is
selected to cut one end of the short RNA. A VLP may further
comprise a linker consisting of at least 1 to 100 nucleotides, the
linker comprising at least 40% A's or at least 40% U's, wherein the
linker links the oligoribonucleotide and the packing sequence, or
the oligoribonucleotide and the ribozyme. Ribozymes may be selected
for example from a Hammerhead ribozyme and a Hepatitis Delta V
ribozyme. A Hammerhead ribozyme may be a Hammerhead ribozyme
variant having a contiguous set of nucleotides complementary to at
least 6 contiguous nucleotides of the oligoribonucleotide.
Alternatively, the ribozyme may be a mutant Hepatitis Delta V
ribozyme capable of cleaving its connection with the
oligoribonucleotide at a rate at most about 50% the rate of a wild
type Hepatitis Delta V ribozyme. Such a mutant HDV ribozyme may
have for example a nucleic acid sequence selected from SEQ ID Nos:
10-18.
[0010] VLP's according to the present disclosure may comprise a
capsid which comprises a wild type viral capsid which is resistant
to hydrolysis catalyzed by a peptide bond hydrolase category EC
3.4, or a capsid protein having at least 15%, at least 16%, at
least 21%, at least 40%, at least 41%, at least 45%, at least 52%,
at least 53%, at least 56%, at least 59% or at least 86% sequence
identity with the amino acid sequence of wild type Enterobacteria
phage MS2 capsid (SEQ ID NO: 3) and is resistant to hydrolysis
catalyzed by a peptide bond hydrolase category EC 3.4. The capsid
may comprise a wild type Enterobacteria phage MS2 capsid protein
having the amino acid sequence of SEQ ID NO: 3.
[0011] VLP's according to the present disclosure may comprise a
heterologous cargo molecule comprising a peptide or polypeptide. A
VLP may further comprise an oligonucleotide linker coupling the
heterologous cargo peptide or polypeptide molecule and the viral
capsid. The oligonucleotide linker may be an oligoribonucleotide
comprising a ribozyme sequence. Alternatively, the heterologous
cargo molecule may comprise a bi-molecular cargo molecule
comprising a bifunctional polynucleotide comprising a first aptamer
sequence which specifically binds a bioactive small molecule having
a molecular weight of about 1,500 Da or less and a second aptamer
sequence for binding a packing sequence of the capsid. The VLP may
further comprise the bioactive small molecule bound to the first
aptameric sequence. The bioactive small molecule may comprise and
herbicide or a pesticide, which may selected for example from
atrazine, acetamipridphorate, profenofos, isocarbophos and
omethoateas.
[0012] In another aspect, the present disclosure provides a nucleic
acid construct comprising a nucleotide sequence that encodes a
short RNA, a ribozyme and a packing sequence. The short RNA may be
for example an siRNA or an shRNA. The nucleic acid construct may
further comprise a linking nucleotide sequence of 4 to 100
nucleotides of which at least 40% are A's or at least 40% are T's,
wherein the linking nucleotide sequence is flanked by the
packing-coding sequence and by the short RNA-coding sequence. The
nucleic acid construct may further comprise a linking nucleotide
sequence of 4 to 100 nucleotides of which at least 40% are A's or
at least 40% are U's, wherein the linking nucleotide sequence is
flanked by the ribozyme and the short RNA-encoding sequence. The
ribozyme sequence may be flanked by the short RNA and the packing
sequence. The present disclosure also encompasses a vector
comprising any such nucleic acid constructs, and host cells
comprising such a vector, as well as host cell stably transformed
with such a vector. Host cells may be a bacterial cell, such as but
not limited to an Escherichia coli cell, a plant cell, a mammalian
cell, an insect cell, a fungal cell or a yeast cell. A host cell
may further be stably transfected with a second vector comprising a
second nucleic acid sequence encoding a viral capsid which is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. The second nucleic acid sequence may encode for
example a viral protein encoding a viral capsid having at least 40%
sequence identity with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3) and is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. A nucleic acid construct as described herein may
also encode a wild type Enterobacteria phage MS2 capsid protein
(SEQ ID NO: 3). The ribozyme in such a nucleic acid construct may
be for example a Hammerhead ribozyme, .a Hammerhead ribozyme
variant having a contiguous set of nucleotides complementary to at
least 6 contiguous nucleotides of the short RNA, a Hepatitis Delta
V ribozyme, or a mutant Hepatitis Delta V ribozyme capable of
cleaving its connection with the short RNA at a rate at most 50%
the rate of a wildtype Hepatitis Delta V ribozyme. An non-limiting
but exemplary mutant HDV ribozyme has a nucleic acid sequence
selected from SEQ ID NOs: 10-18. The present disclosure also
encompasses a plant or plant tissue transformed to contain a
nucleic acid construct described herein, and seed or progeny of
such a plant or plant tissue, wherein the seed or progeny comprises
the nucleic acid construct.
[0013] In another aspect, the present disclosure provides a
composition comprising: a) a plurality of virus-like particles each
comprising a viral capsid enclosing at least one heterologous cargo
molecule; and b) one or more cell lysis products present in an
amount of less than 4 grams for every 100 grams of capsid present
in the composition, wherein the cell lysis products are selected
from proteins, polypeptides, peptides and any combination thereof.
In the composition, the capsid is for example resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
The capsid may comprise a capsid protein having at least 15%, at
least 16%, at least 21%, at least 40%, at least 41%, at least 45%,
at least 52%, at least 53%, at least 56%, at least 59% or at least
86% sequence identity with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO: 3) and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
The capsid may comprises a wild type Enterobacteria phage MS2
capsid protein (SEQ ID NO: 3). In the composition, the heterologous
cargo molecule may comprise an oligonucleotide which may be an
oligoribonucleotide. An oligoribonucleotide may be selected for
example from siRNA, shRNA, sshRNA, lshRNA and miRNA. In the
composition, each virus-like particle may further comprise at least
one ribozyme, wherein the ribozyme is flanked by the packing
sequence and the oligoribonucleotide to form a nucleic acid
construct, and each virus-like particle may comprise a plurality of
the nucleic acid constructs. In the VLP's of such a composition,
the ribozyme may be for example a Hammerhead ribozyme, .a
Hammerhead ribozyme variant having a contiguous set of nucleotides
complementary to at least 6 contiguous nucleotides of the short
RNA, a Hepatitis Delta V ribozyme, or a mutant Hepatitis Delta V
ribozyme capable of cleaving its connection with the short RNA at a
rate at most 50% the rate of a wildtype Hepatitis Delta V ribozyme.
An non-limiting but exemplary mutant HDV ribozyme has a nucleic
acid sequence selected from SEQ ID NOs: 10-18. The VLP's in such a
composition may further comprise a linking nucleotide sequence of 4
to 100 nucleotides of which at least 40% are A's or at least 40%
are T's, wherein the linking nucleotide sequence is flanked by the
packing-coding sequence and by the short RNA-coding sequence, or a
linking nucleotide sequence of 4 to 100 nucleotides of which at
least 40% are A's or at least 40% are U's, wherein the linking
nucleotide sequence is flanked by the ribozyme and the short
RNA-encoding sequence. The ribozyme sequence may be flanked by the
short RNA and the packing sequence. The VLP's in such a composition
may f comprise a heterologous cargo molecule comprising a peptide
or polypeptide. Such VLP's in a composition may further comprise an
oligonucleotide linker coupling the heterologous cargo molecule and
the viral capsid. The oligonucleotide linker may be an
oligoribonucleotide comprising a ribozyme sequence. In such a
composition, the cell lysis products may be present in an amount of
less than 0.5 grams, less than 0.2 grams or less than 0.1
grams.
[0014] In another aspect, the present disclosure provides method
for isolating and purifying a target cargo molecule, the method
comprising: (a) obtaining a whole cell lysate comprising a
plurality of virus-like particles (VLPs) each comprising a capsid
enclosing at least one target cargo molecule, wherein the capsids
are resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4; (b) subjecting the VLP's to hydrolysis using a
peptide bond hydrolase category EC 3.4, for a time and under
conditions sufficient for at least 60, at least 70, at least 80, or
at least 90 of every 100 individual polypeptides present in the
whole cell lysate but not enclosed by the capsids to be cleaved,
while at least 60, at least 70, at least 80, or at least 90 of
every 100 capsids present in the whole cell lysate before such
hydrolysis remain intact following the hydrolysis. In the method,
the capsids may each comprises a viral capsid protein having at
least 15%, at least 16%, at least 21%, at least 40%, at least 41%,
at least 45%, at least 52%, at least 53%, at least 56%, at least
59% or at least 86% sequence identity with the amino acid sequence
of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3)
and is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4. The capsids may each comprise a wild
type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). In the
method, the heterologous cargo molecule may comprise an
oligonucleotide which may be an oligoribonucleotide, or a peptide
or a polypeptide. An oligoribonucleotide may be selected for
example from siRNA, shRNA, sshRNA, lshRNA and miRNA. In the method,
each virus-like particle may further comprise a ribozyme, wherein
the ribozyme is flanked by the packing sequence and the
oligoribonucleotide to form a nucleic acid construct. The method
may further comprise purification of the capsids following
hydrolysis. Purification may include at least one of a
liquid-liquid extraction step, a crystallization step, a fractional
precipitation step, and an ultra filtration step. The present
disclosure also encompasses a composition produced by such a
method.
[0015] In another aspect, the present disclosure provides a method
for protecting a target molecule from hydrolysis in a whole cell
lyste following intracellular production of the target molecule in
a host cell, the method comprising: (a) selecting a viral capsid
which is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4; (b) stably transfecting the host cell
with a first vector comprising a nucleic acid sequence encoding a
viral protein forming the viral capsid, and a second vector
comprising a nucleic acid sequence comprising a ribozyme flanked by
a packing sequence and an siRNA sequence; and (c) maintaining the
cells for a time and under conditions sufficient for the
transformed cells to express and assemble capsids encapsidating the
ribozyme flanked by the packing sequence and the siRNA sequence. In
the process, the capsids may each comprises a viral capsid protein
having at least 15%, at least 16%, at least 21%, at least 40%, at
least 41%, at least 45%, at least 52%, at least 53%, at least 56%,
at least 59% or at least 86% sequence identity with the amino acid
sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ
ID NO: 3) and is resistant to hydrolysis catalyzed by a peptide
bond hydrolase category EC 3.4.
[0016] In another aspect, the present disclosure provides a process
for purifying VLP's enclosing at least one heterologous cargo
molecule, the process comprising: (a) obtaining a cell lysate
comprising a plurality of the VLP's; (b) contacting the cell lysate
with a protease for a time and under conditions sufficient to
hydrolyze cell lysis products other than the VLP's to form a
hydrolysate; and (c) isolating the VLP's from the hydrolsyate. Step
(c) may comprise (i) performing a first precipitation with ammonium
sulfate followed by a first centrifugation to obtain a first
precipitate and a first supernatant; and (ii) performing a second
precipitation on the first supernatant with ammonium sulfate
followed by a second centrifugation to obtain a second precipitate,
wherein the second precipitate comprises at least about 70%, 80% or
90% by weight of the VLP's. Step (c) may comprise (i) performing a
first precipitation with ethanol followed by a first centrifugation
to obtain a first precipitate and a first supernatant; and (ii)
performing a second precipitation on the first supernatant with
ammonium sulfate followed by a second centrifugation to obtain a
second precipitate, wherein the second precipitate comprises at
least about 70%, 80% or 90% by weight of the VLP's. Step (c) may
comprise ultracentrifuging the hydrolysate to obtain a precipitate
comprising at least about 70%, 80% or 90% by weight of the VLP's.
In the process, the VLP's may each comprise a capsid which is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4., which can comprise a capsid protein having at
least 15%, at least 16%, at least 21%, at least 40%, at least 41%,
at least 45%, at least 52%, at least 53%, at least 56%, at least
59% or at least 86% sequence identity with the amino acid sequence
of wild type Enterobacteria phage MS2 capsid (SEQ ID NO: 3) and is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. Te VLP's may each comprise a wild type
Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). In the
process, step (b) can be performed for at least about 30 minutes at
about 37.degree. C. The process may further comprise, before step
(b), contacting the cell lysate with at least one of a nuclease, an
amylase and a lypase for at least about 30 minutes at about
37.degree. C. In the process, the protease can be fore example a
peptide bond hydrolase category EC 3.4, which can be selected for
example from Proteinase K, Protease from Streptomyces griseus,
Protease from Bacillus lichenformis, pepsin and papain. In the
process, the heterologous cargo molecule enclosed by the VLP's may
comprise an oligonucleotide which may be an oligoribonucleotide, or
a peptide or a polypeptide. An oligoribonucleotide may be selected
for example from siRNA, shRNA, sshRNA, lshRNA and miRNA. In the
process, the VLP's may each further comprise a ribozyme as
described herein, flanked by a packing sequence and the
oligoribonucleotide to form a nucleic acid construct. The
oligoribonucleotide and the packing sequence may be linked by a
linker sequence of at least 1 to 100 nucleotides, and comprising
more than 40% A's, more than 40% of U's, or more than 40% T's. The
process may further comprise preparing the cell lyaste before step
(a) by centrifuging cells following expression of the VLPs in the
cells; resuspending the cells; lysing the cells and centrifuging
the cell lysate to obtain a supernatant, wherein the supernatant is
used as the cell lysate for step (a).
[0017] In another aspect, the present disclosure provides VLP's
comprising a capsid enclosing at least one heterologous cargo
molecule and a packing sequence wherein the capsid comprises a
capsid protein which is a variant of wild type Enterobacteria phage
MS2 capsid (SEQ ID NO: 3). The capsid protein may be one which has
the amino acid sequence of wild type Enterobacteria phage MS2
capsid (SEQ ID NO: 3) except that the A residue at position 1 is
deleted, and is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4. The capsid protein may be one which has
the amino acid sequence of wild type Enterobacteria phage MS2
capsid (SEQ ID NO:3) except that the A residue at position 1 is
deleted and the S residue at position 2 is deleted, and is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. The capsid protein may be one which has the amino
acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID
NO: 3) except that the A residue at position 1 is deleted, the S
residue at position 2 is deleted and the N residue at position 3 is
deleted, and is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4. The capsid protein may be one which has
the amino acid sequence of wild type Enterobacteria phage MS2
capsid (SEQ ID NO: 3) except that the Y reside at position 129 is
deleted, and is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4. The capsid protein may be one which has
the amino acid sequence of wild type Enterobacteria phage MS2
capsid (SEQ ID NO:3) but having a single (1) amino acid deletion in
the 112-117 segment and is resistant to hydrolysis catalyzed by a
peptide bond hydrolase category EC 3.4. The capsid protein may be
one which has the amino acid sequence of wild type Enterobacteria
phage MS2 capsid (SEQ ID NO:3) but having a single (1) amino acid
deletion in the 112-117 segment and is resistant to hydrolysis
catalyzed by a peptide bond hydrolase category EC 3.4. The capsid
protein may be one which has the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a 1-2
residue insertion in the 65-83 segment and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
The capsid protein may be one which has the amino acid sequence of
wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having
a 1-2 residue insertion in the 44-55 segment and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
The capsid protein may be one which has the amino acid sequence of
wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having
a single (1) residue insertion in the 33-43 segment and is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. The capsid protein may be one which has the amino
acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID
NO:3) but having a 1-2 residue insertion in the 24-30 segment and
is resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. The capsid protein may be one which has the amino
acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID
NO:3) but having a single (1) residue insertion in the 10-18
segment and is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4. The capsid may comprise a capsid protein
monomer sequence concatenated with a second capsid monomer sequence
which assembles into a capsid which resistant to hydrolysis
catalyzed by a peptide bond hydrolase category EC 3.4. The capsid
may comprise a capsid protein monomer sequence whose C-terminus is
extended with a 0-6 residue linker segment whose C-terminus is
concatenated with a second capsid monomer sequence, all of which
assembles into a capsid which resistant to hydrolysis catalyzed by
a peptide bond hydrolase category EC 3.4. A linker segment may have
a sequence such as, for example, -(Gly).sub.x, wherein x=0-6.
including -Gly-; -Gly-Gly-; and -Gly-Gly-Gly-. A linker segment may
be a Gly-Ser linker selected from -Gly-Gly-Ser-Gly-Gly-,
-Gly-Gly-Ser and -Gly-Ser-Gly- The capsid may comprise the capsid
protein concatenated with a third capsid monomer sequence which
assembles into a capsid which resistant to hydrolysis catalyzed by
a peptide bond hydrolase category EC 3.4. The capsid may comprise a
capsid protein wherein the C-terminus is extended with a 0-6
residue linker segment whose C-terminus s concatenated with a third
capsid monomer sequence, all of which assembles into a capsid which
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. The capsid may comprise a capsid protein wherein
the capsid comprises a capsid protein in which one or both linker
sequences is -(Gly).sub.x, wherein x=0-6, including -Gly-;
-Gly-Gly-; and -Gly-Gly-Gly-. A linker segment may be a Gly-Ser
linker selected from -Gly-Gly-Ser-Gly-Gly-, -Gly-Gly-Ser and
-Gly-Ser-Gly-. Such a capsid protein assembles for example into a
capsid which is resistant to hydrolysis catalyzed by a peptide bond
hydrolase category EC 3.4. For example, the capsid may comprise a
capsid protein in which one or both linker sequences is
-(Gly).sub.x-, x=1, which assembles into a capsid which is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. The capsid may comprise a capsid protein in which
one or both linker sequences is -(Gly).sub.x-, x=2, which assembles
into a capsid which is resistant to hydrolysis catalyzed by a
peptide bond hydrolase category EC 3.4. The capsid may comprise a
capsid protein in which one or both linker sequences is
-(Gly).sub.x-, x=3, which assembles into a capsid which is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. The capsid may comprise one or more coat protein
sequences which is N-terminally truncated by 1-3 residues and a
linker segment as described herein is lengthened by the number of
residues deleted, and which is resistant to hydrolysis catalyzed by
a peptide bond hydrolase category EC 3.4. The capsid may comprise
one or more coat protein sequences which is C-terminally truncated
by 1 residue, and linker segments as described herein are
lengthened by the one residue, wherein the capsid is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
The capsid may comprise a first coat protein sequence in a
concatenated dimer which is C-terminally truncated by 1 residue and
the linker segments lengthened by the one residue or wherein the
first and/or second coat protein sequence in a concatenated trimer
is C-terminally truncated by 1 residues and which is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
The capsid may comprise a capsid protein having N- and C-terminal
truncations and which is resistant to hydrolysis catalyzed by
peptide bond hydrolase category EC 3.4.
REFERENCE TO COLOR FIGURES
[0018] The application file contains at least one photograph
executed in color. Copies of this patent application publication
with color photographs will be provided by the Office upon request
and payment of the necessary fee.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a plot of Optical Density (OD; filled diamonds)
and pH (open squares) over time, showing propagation of wild type
MS2 bacteriophage (ATCC No. 15597-B1, from American Type Culture
Collection, Rockville, Md.) in its E. Coli host (ATCC No.
15669).
[0020] FIG. 2 is a gel showing results of SDS-PAGE analysis of MS2
bacteriophage samples obtained following propagation in E. Coli and
purified using Proteinase K and ultrafiltration, showing that
Proteinase K purification yields phage purified to higher than 99%
(band at 14 kDa corresponds to MS2 bacteriophage coat protein).
[0021] FIG. 3 is a gel showing results of SDS-PAGE analysis of
partially purified MS2, showing complete degradation of the phage
and results obtained after 1.times. or 2.times. ultrafiltration of
the lysate (Lanes 4 and 6).
[0022] FIG. 4 is a gel showing results of SDS-PAGE analysis of MS2
samples purified using ultrafiltration and Proteinase K
treatment.
[0023] FIG. 5 is a gel showing results of SDS-PAGE analysis of MS2
samples purified using Proteinase K treatment, precipitation at
acidic conditions, precipitation using ethanol at basic and acidic
conditions, and ultrafiltration.
[0024] FIG. 6 is a graph showing the UV spectrum of MS2 samples
purified using Proteinase K treatment, precipitation at acidic
conditions, precipitation using ethanol at basic and acidic
conditions, and ultrafiltration.
[0025] FIG. 7 is a chromatograph of PCR products obtained from an
MS2 sample following purification described for FIGS. 5 and 6,
chromatographed in 1.5% agarose gel stained with Ethidium Bromide
(1.2 kbp for primers F1201.sub.--1223-R1979.sub.--2001 in Lane
1,800 bp for primers F1201.sub.--1223-R1979.sub.--2001 in Lane 2,
and 304 bp for primers F1401.sub.--1426-R1680.sub.--1705 in Lane
3), showing consistency with an intact MS2 bacteriophage
genome.
[0026] FIG. 8 is a plot of Optical Density (OD; filled diamonds)
over time, obtained with a control sample (open diamonds) and an
MS2 sample following purification described for FIGS. 5 and 6
(filled squares), showing that the purified sample contained phage
that retained high infectivity.
[0027] FIG. 9 is a gel showing results of SDS-PAGE analysis of MS2
samples following expression of MS2 capsids encapsidating RNA
coding for the coat protein attached to a coat-specific 19-mer RNA
hairpin.
[0028] FIG. 10 is a chromatograph of PCR products from PCR
interrogation of an MS2 sample for presence or absence of a section
of the MS2 capsid following purification, chromatographed in 2%
agarose gel stained with Ethidium Bromide (304 bp in Lane 1; the
leftmost Lane corresponds to 1 kb plus ladder from Life
Technologies), showing consistency with an intact MS2 coat
gene.
[0029] FIG. 11 is a gel showing results of SDS-PAGE analysis of MS2
samples following simple precipitation with ethanol for
purification of MS2 Virus-Like Particles (VLPs).
[0030] FIG. 12 is a gel showing results of SDS-PAGE analysis of MS2
samples following use of Proteinase K (PK) and simple precipitation
with ethanol for purification of MS2 VLPs.
[0031] FIG. 13 is a gel showing results of SDS-PAGE analysis of MS2
samples following use of constitutive hydrolases (CH), fractional
precipitation with ethanol, and ultrafiltration for purification of
MS2 VLPs.
[0032] FIG. 14 is a gel showing results of SDS-PAGE analysis of MS2
samples following use of various hydrolases, and factional
precipitation with ammonium sulfate for purification of MS2
VLPs.
[0033] FIG. 15 is a gel showing results of PAGE analysis of RNA
obtained from RNA encapsidated in MS2 capsids.
[0034] FIG. 16 is a gel showing results of PAGE analysis of RNA
products produced following in vitro transcriptions using Hepatitis
Delta Virus (HDV) ribozyme.
[0035] FIG. 17 is a gel showing results of PAGE analysis of siRNA
products obtained during in vitro transcriptions using long
flanking Hammerhead ribozymes.
[0036] FIG. 18 is a gel showing results of PAGE analysis of RNA
products obtained from RNA encapsidated in VLPs, following
purification of the VLP's and isolation of the RNA from the
VLPs.
[0037] FIG. 19 is a series of gels showing results of SDS-PAGE
analyses of VLP's comprising MS2 capsids, following purification
and suspension of the VLPs, and exposure to various proteases for 1
hour and 4 hours of incubation.
[0038] FIG. 20 is an alignment of selected enterobacteria phage MS2
capsid proteins.
[0039] FIG. 21 is an alignment of complete leviviridae viral coat
protein sequences retrieved from the UniProt database and aligned
using their BLAST multiple alignment with default values for
weighting array choice, gap penalties, etc.
[0040] FIG. 22 is a graphic illustration of backbone superposition
of 1AQ3 chain B (leviviridae coat protein monomer) with 1 QBE chain
C (alloleviridae coat protein monomer).
[0041] FIG. 23 is a graphic illustration of an alternative view of
the backbone superposition of 1AQ3 chain B (leviviridae coat
protein monomer) with 1 QBE chain C (alloleviridae coat protein
monomer) shown in FIG. 22.
[0042] FIG. 24 is a graphic illustration of another alternative
view of the backbone superposition of 1AQ3 chain B (leviviridae
coat protein monomer) with 1QBE chain C (alloleviridae coat protein
monomer) shown in FIG. 22.
[0043] FIG. 25 is a graphic illustration of another alternative
view of the backbone superposition of 1AQ3 chain B (leviviridae
coat protein monomer) with 1QBE chain C (alloleviridae coat protein
monomer) shown in FIG. 22.
[0044] FIG. 26 is a structural sequence alignment of 1AQ3, 2VTU and
1QBE.using jFATCAT rigid.
[0045] FIG. 27 is an alignment of complete alloleviviridae viral
coat protein sequences retrieved from the UniProt database and
aligned using their BLAST multiple alignment with default values
for weighting array choice, gap penalties, etc.
[0046] FIG. 28 is a graphic illustration showing another 60 of the
180 monomers forming the isosahedral levi- and alloleviviridae
capsid. The backbone of each monomer of represented by a ribbon of
a different color. Backbone hydrogen bonds are represented by cyan
lines. The isosahedral three-fold axis is in the center of the
figure. Monomer-monomer contacts do not fill the central circle
outlined by hydrogen bonds connecting the tips of flexible loops
67-81.
[0047] FIG. 29 is a graphic illustration showing 2 MS2 monomers
(ribbons representing backbone colored dark and pale blue)
surrounded by monomers in contact in the isosahedral capsid
(ribbons representing monomer backbones in brown and navy). The
alloleviviridae Qbeta has a two residue deletion with respect to
leviviridae between residues 72 & 73 (red, bottom center). The
central void is immediately below this deletion site (FIG. 5). The
deletion causes it to slightly expand. The Qbeta deletion at 126
(red central left) removes the excursion from the segment but
extensive contacts between the sheets of neighboring monomers
essentially holds the monomers in place. MS2 sequence numbering is
used.
[0048] FIG. 30 is a graphic illustration of 2 MS2 monomers (ribbons
representing backbone colored dark and pale blue) surrounded by
monomers in contact in the isosahedral capsid (ribbons representing
monomer backbones in brown and navy). The alloleviviridae Qbeta has
a one residue insertion with respect to leviviridae between
residues 12 & 13 (yellow, top left center), a flexible loop
that extends from the outer capsid surface into solvent; a two
residue insertion between residues 53 & 54 (yellow, lower left
central) at the end a stand connection extending into the interior
cargo; a one-residue insertion between residues 27 & 28 is also
at the end of a beta-strand connector extending into the capsid
cargo space. None of these insertions require movement in the
monomer fold or between neighbors
[0049] FIG. 31 is a graphic illustration of 2 MS2 monomers (ribbons
representing backbone colored dark and pale blue) surrounded by
monomers in contact in the isosahedral capsid (ribbons representing
monomer backbones in brown and navy). The alloleviviridae Qbeta has
a one residue insertion with respect to leviviridae between
residues 36 & 37 (yellow, center right). The loop packs against
the end of the adjacent helix but inserted residues can extend into
the central space above the flexible loop immediately below.
[0050] FIG. 32 is a graphic illustration of backbone ribbons of 3
noncovalent Enterobacteria phage MS2 noncovalent dimers packed
around a symmetry point in the assembled capsid, with all of the
N-termini colored green, the C-termini red.
DETAILED DESCRIPTION OF THE INVENTION
[0051] Section headings as used in this section and the entire
disclosure herein are not intended to be limiting.
A. DEFINITIONS
[0052] As used herein, the singular forms "a," "an" and "the"
include plural referents unless the context clearly dictates
otherwise. For the recitation of numeric ranges herein, each
intervening number there between with the same degree of precision
is explicitly contemplated. For example, for the range 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
[0053] The use of "or" means "and/or" unless stated otherwise.
Furthermore, the use of the term "including", as well as other
forms, such as "includes" and "included", is not limiting.
[0054] Unless otherwise defined herein, scientific and technical
terms used in connection with the present disclosure shall have the
meanings that are commonly understood by those of ordinary skill in
the art. For example, any nomenclatures used in connection with,
and techniques of, animal and cellular anatomy, cell and tissue
culture, biochemistry, molecular biology, immunology, and
microbiology described herein are those that are well known and
commonly used in the art. The meaning and scope of the terms should
be clear; in the event however of any latent ambiguity, definitions
provided herein take precedent over any dictionary or extrinsic
definition. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular.
[0055] A wide variety of conventional techniques and tools in
chemistry, biochemistry, molecular biology, and immunology are
employed and available for practicing the methods and compositions
described herein, are within the capabilities of a person of
ordinary skill in the art and well described in the literature.
Such techniques and tools include those for generating and
purifying VLP's including those with a wild type or a recombinant
capsid together with the cargo molecule(s), and for transforming
host organisms and expressing recombinant proteins and nucleic
acids as described herein. See, e.g., MOLECULAR CLONING, A
LABORATORY MANUAL 2.sup.nd ed. 1989 (Sambrook et al., Cold Spring
Harbor Laboratory Press); and CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY (Eds. Ausubel et al., Greene Publ. Assoc.,
Wiley-Interscience, NY) 1995. The disclosures in each of these are
herein incorporated by reference.
[0056] As used herein, the term "cargo molecule" refers to an
oligonucleotide, polypeptide or peptide molecule, which is or may
be enclosed by a capsid.
[0057] As used herein, the term "oligonucleotide" refers to a short
polymer of at least two, and no more than about 70 nucleotides,
preferably no more than about 55 nucleotides linked by
phosphodiester bonds. An oligonucleotide may be an
oligodeoxyribonucleotide (DNA) or a oligoribonucleotide (RNA), and
encompasses short RNA molecules such as but not limited to siRNA,
shRNA, sshRNA, and miRNA.
[0058] As used herein, the term "peptide" refers to a polymeric
molecule which minimally includes at least two amino acid monomers
linked by peptide bond, and preferably has at least about 10, and
more preferably at least about 20 amino acid monomers, and no more
than about 60 amino acid monomers, preferably no more than about 50
amino acid monomers linked by peptide bonds. For example, the term
encompasses polymers having about 10, about 20, about 30, about 40,
about 50, or about 60 amino acid residues.
[0059] As used herein, the term "polypeptide" refers to a polymeric
molecule including at least one chain of amino acid monomers linked
by peptide bonds, wherein the chain includes at least about 70
amino acid residues, preferably at least about 80, more preferably
at least about 90, and still more preferably at least about 100
amino acid residues. As used herein the term encompasses proteins,
which may include one or more linked polypeptide chains, which may
or may not be further bound to cofactors or other proteins. The
term "protein" as used herein is used interchangeably with the term
"polypeptide."
[0060] As used herein, the term "variant" with reference to a
molecule is a sequence that is substantially similar to the
sequence of a native or wild type molecule. With respect to
nucleotide sequences, variants include those sequences that may
vary as to one or more bases, but because of the degeneracy of the
genetic code, still encode the identical amino acid sequence of the
native protein. Variants include naturally occurring alleles, and
nucleotide sequences which are engineered using well-known
techniques in molecular biology, such as for example site-directed
mutagenesis, and which encode the native protein, as well as those
that encode a polypeptide having amino acid substitutions.
Generally, nucleotide sequence variants of the invention have at
least 40%, at least 50%, at least 60%, at least 70% or at least 80%
sequence identity to the native (endogenous) nucleotide sequence.
The present disclosure also encompasses nucleotide sequence
variants having at least about 85% sequence identity, at least
about 90% sequence identity, at least about 85%, 86%, 87%, 88%,
89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
[0061] Sequence identity of amino acid sequences or nucleotide
sequences, within defined regions of the molecule or across the
full-length sequence, can be readily determined using conventional
tools and methods known in the art and as described herein. For
example, the degree of sequence identity of two amino acid
sequences, or two nucleotide sequences, is readily determined using
alignment tools such as the NCBI Basic Local Alignment Search Tool
(BLAST) (Altschul et al., 1990), which are readily available from
multiple online sources. Algorithms for optimal sequence alignment
are well known and described in the art, including for example in
Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Pearson and
Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988). Algorithms
for sequence analysis are also readily available in programs such
as blastp, blastn, blastx, tblastn and tblastx. For the purposes of
the present disclosure, two nucleotide sequences may be also
considered "substantially identical" when they hybridize to each
other under stringent conditions. Stringent conditions including a
high hybridization temperature and low salt in hybridization
buffers which permit hybridization only between nucleic acid
sequences that are highly similar. Stringent conditions are
sequence-dependent and will be different in different circumstance,
but typically include a temperature at least about 60.degree.,
which is about 10.degree. C. to about 15.degree. C. lower than the
thermal melting point (Tm) for the specific sequence at a defined
ionic strength and pH. Salt concentration is typically about 0.02
molar at pH 7.
[0062] As used herein with respect to a given nucleotide sequence,
the term "conservative variant" refers to a nucleotide sequence
that encodes an identical or essentially identical amino acid
sequence as that of a reference sequence. Due to the degeneracy of
the genetic code, whereby almost always more than one codon may
code for each amino acid, nucleotide sequences encoding very
closely related proteins may not share a high level of sequence
identity. Moreover, different organisms have preferred codons for
many amino acids, and different organisms or even different strains
of the same organism, e.g., E. coli strains, can have different
preferred codons for the same amino acid. Thus, a first nucleotide
acid sequence which encodes essentially the same polypeptide as a
second nucleotide acid sequence is considered substantially
identical to the second nucleotide sequence, even if they do not
share a minimum percentage sequence identity, or would not
hybridize to one another under stringent conditions. Additionally,
it should be understood that with the limited exception of ATG,
which is usually the sole codon for methionine, any sequence can be
modified to yield a functionally identical molecule by standard
techniques, and such modifications are encompassed by the present
disclosure. As described herein below, the present disclosure
specifically contemplates protein variants of a native protein,
which have amino acid sequences having at least 15%, at least 16%,
at least 21%, at least 40%, at least 41%, at least 52%, at least
53%, at least 56%, at least 59% or at least 86% sequence identity
to a native nucleotide sequence.
[0063] The degree of sequence identity between two amino acid
sequences may be determined using the BLASTp algorithm of Karlin
and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1993). The
percentage of sequence identity is determined by comparing two
optimally aligned sequences over a comparison window, wherein the
portion of the amino acid sequence in the comparison window may
comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which an
identical amino acid occurs in both sequences to yield the number
of matched positions, dividing the number of matched positions by
the total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0064] One of skill will recognize that polypeptides may be
"substantially similar" in that an amino acid may be substituted
with a similar amino acid residue without affecting the function of
the mature protein. Polypeptide sequences which are "substantially
similar" share sequences as noted above except that residue
positions, which are not identical, may have conservative amino
acid changes. Conservative amino acid substitutions refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Preferred conservative
amino acid substitution groups include: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine.
[0065] A nucleic acid encoding a peptide, polypeptide or protein
may be obtained by screening selected cDNA or genomic libraries
using a deduced amino acid sequence for a given protein.
Conventional procedures using primer extension procedures, as
described for example in Sambrook et al., can be used to detect
precursors and processing intermediates.
B. VIRUS-LIKE PARTICLES (VLP'S) COMPOSED OF A CAPSID ENCLOSING A
CARGO MOLECULE
[0066] The methods and compositions described herein are the result
in part of the appreciation that certain viral capsids can be
prepared and/or used in novel manufacturing and purification
methods to improve commercialization procedures for nucleic acids.
The methods described herein use recombinant viral capsids which
are resistant to readily available hydrolases, to enclose
heterologous cargo molecules such as nucleic acids, peptides, or
polypeptides including proteins.
[0067] The capsid may be a wild type capsid or a mutant capsid
derived from a wild type capsid, provided that the capsid exhibits
resistance to hydrolysis catalyzed by at least one hydrolase acting
on peptide bonds when the capsids are contacted with the hydrolase.
As used interchangeably herein, the phrases "resistance to
hydrolysis" and "hydrolase resistant" refer to any capsid which,
when present in a whole cell lysate also containing polypeptides
which are cell lysis products and not enclosed in the capsids, and
subjected to hydrolysis using a peptide bond hydrolase category EC
3.4 for a time and under conditions sufficient for at least 60, at
least 70, at least 80, or at least 90 of every 100 individual
polypeptides present in the lysate (which are cell lysis products
and not enclosed in the capsids) to be cleaved (i.e. at least 60%,
at least 70%, at least 80%, or at least 90% of all individual
unenclosed polypeptides are cleaved), yet at least 60, at least 70,
at least 80, or at least 90 of every 100 capsids present before
such hydrolysis remain intact following the hydrolysis. Hydrolysis
may be conducted for a period of time and under conditions
sufficient for the average molecular weight of cell proteins
remaining from the cell line following hydrolysis is less than
about two thirds, less than about one half, less than about one
third, less than about one fourth, or less than about one fifth, of
the average molecular weight of the cell proteins before the
hydrolysis is conducted. Methods may further comprise purifying the
intact capsid remaining after hydrolysis, and measuring the weight
of capsids and the weight of total dry cell matter before and after
hydrolysis and purification, wherein the weight of capsids divided
by the weight of total dry cell matter after hydrolysis and
purification is at least twice the weight of capsids divided by the
weight of total dry cell matter measured before the hydrolysis and
purification. The weight of capsids divided by the weight of total
dry cell matter after hydrolysis and purification may be at least
10 times more than, preferably 100 times more than, more preferably
1,000 times more than, and most preferably 10,000 times more than
the weight of capsids divided by the weight of total dry cell
matter measured before such hydrolysis and purification.
[0068] Hydrolases are enzymes that catalyze hydrolysis reactions
classified under the identity number EC 3 by the European
Commission. For example, enzymes that catalyze hydrolysis of ester
bonds have identity numbers starting with EC 3.1. Enzymes that
catalyze hydrolysis of glycosidic bonds have identity numbers
starting with EC 3.2. Enzymes that catalyze hydrolysis of peptide
bonds have identity numbers starting with EC 3.4. Proteases, which
are enzymes that catalyze hydrolysis of proteins, are classified
using identity numbers starting with EC 3.4, including but not
limited to Proteinase K and subtilisin. For example, Proteinase K
has identity number EC 3.4.21.64. The present disclosure
encompasses VLP's which are resistant, in non-limiting example,
Proteinase K, Protease from Streptomyces griseus, Protease from
Bacillus lichenformis, pepsin and papain, and methods and processes
of using such VLP's.
[0069] The Nomenclature Committee of the International Union of
biochemistry and Molecular Biology also recommends naming and
classification of enzymes by the reactions they catalyze. Their
complete recommendations are freely and widely available, and for
example can be accessed online at http://enzyme.expasy.org and,
www.chem.qmul.ac.uk/iubmb/enzyme/, among others. The IUBMB
developed a shorthand for describing what sites each enzyme is
active against. Enzymes that indescriminately cut are referred to
as broadly specific. Cleavage patterns for the other enzymes are
described as Xaa|Yaa, where | represents the cleavage site,
Xaa={set of residues preferred by the enzyme on the N-terminal side
of the cleavage}, and Yaa={set of residues preferred by the enzyme
on the C-terminal side of the cleavage}. Some enzymes have more
binding requirements than this so the description can become more
complicated. For an enzyme that catalyzes a very specific reaction,
for example an enzyme that processes prothrombin to active
thrombin, then that activity is the basis of the cleavage
description. In certain instances the precise activity of an enzyme
may not be clear, and in such cases, cleavage results against
standard test proteins like B-chain insulin are reported. As an
alternative to using enzymes that catalyze hydrolysis of peptide
bonds which have identity numbers starting with EC 3.4, broadly
specific enzymes can be used which have Xaa|Yaa preferences where
the enzyme has reported P1 pocket binding preferences Xaa but no
preference for binding the P1' pocket Yaa and conversely, where the
enzyme has reported P1' pocket binding preferences Yaa but no
preference for binding the P1 pocket Xaa.
[0070] The capsids can be further selected and/or prepared such
that they can be isolated and purified using simple isolation and
purification procedures, as described in further detail herein. For
example, the capsids can be selected or genetically modified to
have significantly higher hydrophobicity than a surrounding matrix
as described herein, so as to selectively partition into a
non-polar water-immiscible phase into which they are simply
extracted. Alternatively, a capsid may be selected or genetically
modified for improved ability to selectively crystallize from
solution.
[0071] Use of simple and effective purification processes using the
capsids is enabled by the choice of certain wild type capsids, or
modifications to the amino acid sequence of proteins comprising the
wild type capsids, such that the capsid exhibits resistance to
hydrolysis catalyzed by at least one hydrolase acting on peptide
bonds as described herein above. Such wild type capsids, such as
the wild type MS2 capsid, can be used in a purification process in
which certain inexpensive enzymes such as Proteinase K or
subtilisin are used for proteolysis. A non-limiting example is the
Enterobacteria phage MS2 (SEQ ID NO: 1, whole MS2 wild type genome;
SEQ ID NO: 2, MS2 wild type coat protein, DNA sequence; and SEQ ID
NO: 3, MS2 wild type coat protein, amino acid sequence.
TABLE-US-00001 (SEQ. ID NO: 1) Whole MS2 Genome, wild type, coat
protein sequence in bold:
GGGTGGGACCCCTTTCGGGGTCCTGCTCAACTTCCTGTCGAGCTAATGCC
ATTTTTAATGTCTTTAGCGAGACGCTACCATGGCTATCGCTGTAGGTAGC
CGGAATTCCATTCCTAGGAGGTTTGACCTGTGCGAGCTTTTAGTACCCTT
GATAGGGAGAACGAGACCTTCGTCCCCTCCGTTCGCGTTTACGCGGACGG
TGAGACTGAAGATAACTCATTCTCTTTAAAATATCGTTCGAACTGGACTC
CCGGTCGTTTTAACTCGACTGGGGCCAAAACGAAACAGTGGCACTACCCC
TCTCCGTATTCACGGGGGGCGTTAAGTGTCACATCGATAGATCAAGGTGC
CTACAAGCGAAGTGGGTCATCGTGGGGTCGCCCGTACGAGGAGAAAGCCG
GTTTCGGCTTCTCCCTCGACGCACGCTCCTGCTACAGCCTCTTCCCTGTA
AGCCAAAACTTGACTTACATCGAAGTGCCGCAGAACGTTGCGAACCGGGC
GTCGACCGAAGTCCTGCAAAAGGTCACCCAGGGTAATTTTAACCTTGGTG
TTGCTTTAGCAGAGGCCAGGTCGACAGCCTCACAACTCGCGACGCAAACC
ATTGCGCTCGTGAAGGCGTACACTGCCGCTCGTCGCGGTAATTGGCGCCA
GGCGCTCCGCTACCTTGCCCTAAACGAAGATCGAAAGTTTCGATCAAAAC
ACGTGGCCGGCAGGTGGTTGGAGTTGCAGTTCGGTTGGTTACCACTAATG
AGTGATATCCAGGGTGCATATGAGATGCTTACGAAGGTTCACCTTCAAGA
GTTTCTTCCTATGAGAGCCGTACGTCAGGTCGGTACTAACATCAAGTTAG
ATGGCCGTCTGTCGTATCCAGCTGCAAACTTCCAGACAACGTGCAACATA
TCGCGACGTATCGTGATATGGTTTTACATAAACGATGCACGTTTGGCATG
GTTGTCGTCTCTAGGTATCTTGAACCCACTAGGTATAGTGTGGGAAAAGG
TGCCTTTCTCATTCGTTGTCGACTGGCTCCTACCTGTAGGTAACATGCTC
GAGGGCCTTACGGCCCCCGTGGGATGCTCCTACATGTCAGGAACAGTTAC
TGACGTAATAACGGGTGAGTCCATCATAAGCGTTGACGCTCCCTACGGGT
GGACTGTGGAGAGACAGGGCACTGCTAAGGCCCAAATCTCAGCCATGCAT
CGAGGGGTACAATCCGTATGGCCAACAACTGGCGCGTACGTAAAGTCTCC
TTTCTCGATGGTCCATACCTTAGATGCGTTAGCATTAATCAGGCAACGGC
TCTCTAGATAGAGCCCTCAACCGGAGTTTGAAGCATGGCTTCTAACTTTA
CTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCC
CCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCTAACTCGCG
TTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGA
ATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAACCCAGACT
GTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTACTTAAATAT
GGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTG
TTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCA
ATCGCAGCAAACTCCGGCATCTACTAATAGACGCCGGCCATTCAAACATG
AGGATTACCCATGTCGAAGACAACAAAGAAGTTCAACTCTTTATGTATTG
ATCTTCCTCGCGATCTTTCTCTCGAAATTTACCAATCAATTGCTTCTGTC
GCTACTGGAAGCGGTGATCCGCACAGTGACGACTTTACAGCAATTGCTTA
CTTAAGGGACGAATTGCTCACAAAGCATCCGACCTTAGGTTCTGGTAATG
ACGAGGCGACCCGTCGTACCTTAGCTATCGCTAAGCTACGGGAGGCGAAT
GGTGATCGCGGTCAGATAAATAGAGAAGGTTTCTTACATGACAAATCCTT
GTCATGGGATCCGGATGTTTTACAAACCAGCATCCGTAGCCTTATTGGCA
ACCTCCTCTCTGGCTACCGATCGTCGTTGTTTGGGCAATGCACGTTCTCC
AACGGTGCTCCTATGGGGCACAAGTTGCAGGATGCAGCGCCTTACAAGAA
GTTCGCTGAACAAGCAACCGTTACCCCCCGCGCTCTGAGAGCGGCTCTAT
TGGTCCGAGACCAATGTGCGCCGTGGATCAGACACGCGGTCCGCTATAAC
GAGTCATATGAATTTAGGCTCGTTGTAGGGAACGGAGTGTTTACAGTTCC
GAAGAATAATAAAATAGATCGGGCTGCCTGTAAGGAGCCTGATATGAATA
TGTACCTCCAGAAAGGGGTCGGTGCTTTCATCAGACGCCGGCTCAAATCC
GTTGGTATAGACCTGAATGATCAATCGATCAACCAGCGTCTGGCTCAGCA
GGGCAGCGTAGATGGTTCGCTTGCGACGATAGACTTATCGTCTGCATCCG
ATTCCATCTCCGATCGCCTGGTGTGGAGTTTTCTCCCACCAGAGCTATAT
TCATATCTCGATCGTATCCGCTCACACTACGGAATCGTAGATGGCGAGAC
GATACGATGGGAACTATTTTCCACAATGGGAAATGGGTTCACATTTGAGC
TAGAGTCCATGATATTCTGGGCAATAGTCAAAGCGACCCAAATCCATTTT
GGTAACGCCGGAACCATAGGCATCTACGGGGACGATATTATATGTCCCAG
TGAGATTGCACCCCGTGTGCTAGAGGCACTTGCCTACTACGGTTTTAAAC
CGAATCTTCGTAAAACGTTCGTGTCCGGGCTCTTTCGCGAGAGCTGCGGC
GCGCACTTTTACCGTGGTGTCGATGTCAAACCGTTTTACATCAAGAAACC
TGTTGACAATCTCTTCGCCCTGATGCTGATATTAAATCGGCTACGGGGTT
GGGGAGTTGTCGGAGGTATGTCAGATCCACGCCTCTATAAGGTGTGGGTA
CGGCTCTCCTCCCAGGTGCCTTCGATGTTCTTCGGTGGGACGGACCTCGC
TGCCGACTACTACGTAGTCAGCCCGCCTACGGCAGTCTCGGTATACACCA
AGACTCCGTACGGGCGGCTGCTCGCGGATACCCGTACCTCGGGTTTCCGT
CTTGCTCGTATCGCTCGAGAACGCAAGTTCTTCAGCGAAAAGCACGACAG
TGGTCGCTACATAGCGTGGTTCCATACTGGAGGTGAAATCACCGACAGCA
TGAAGTCCGCCGGCGTGCGCGTTATACGCACTTCGGAGTGGCTAACGCCG
GTTCCCACATTCCCTCAGGAGTGTGGGCCAGCGAGCTCTCCTCGGTAGCT
GACCGAGGGACCCCCGTAAACGGGGTGGGTGTGCTCGAAAGAGCACGGGT
GCGAAAGCGGTCCGGCTCCACCGAAAGGTGGGCGGGCTTCGGCCCAGGGA
CCTCCCCCTAAAGAGAGGACCCGGGATTCTCCCGATTTGGTAACTAGCTG
CTTGGCTAGTTACCACCCA (SEQ. ID NO: 2) MS2 coat protein DNA sequence,
wild type: ATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGG
CGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGA
TCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGT
CAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAA
AGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGC
GTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCC
GACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAA
CCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG (SEQ. ID NO: 3) MS2
wild type coat protein, amino acid sequence:
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVR
QSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNS
DCELIVKAMQGLLKDGNPIPSAIAANSGIY
[0072] Surprisingly, the unmodified, wild type MS2 capsid though
lacking an envelope is resistant to a variety of category EC 3.4
hydrolases, including but not limited to Proteinase K and
subtilisin, such that a highly purified composition of the capsid,
which may contain a cargo molecule, can be prepared from a whole
cell lysate. Accordingly, the present disclosure provides VLPs
comprising viral capsids comprising the wild type MS2 capsid
protein, and/or capsid proteins sharing homology with wild type MS2
capsid proteins, which viral capsids encapsidate the cargo
molecule. The cargo molecule may comprise one or more heterologous
nucleic acids, peptides, polypeptides or proteins. These VLP's can
then be isolated and purified from a whole cell lysate after a
hydrolysis step using a category EC 3.4 hydrolase, to produce a
composition of VLP's of high purity, for example at least 60%, at
least 70%, a least 80%, or at least 85% by weight VLP's.
Compositions having a purity of at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, and 98% by weight of VLP's are expressly
contemplated.
[0073] The present disclosure encompasses a composition comprising:
a) a plurality of virus-like particles each comprising a wild type
viral capsid and at least one target heterologous cargo molecule
enclosed in the wild type viral capsid; and b) one or more cell
lysis products present in an amount of less than 40 grams, less
than 30 grams, less than 20 grams, less than 15 grams, less than 10
grams, and preferably less than 9, 8, 7, 6, 5, 4, 3, more
preferably less than 2 grams, and still more preferably less than 1
gram, for every 100 grams of capsid present in the composition,
wherein the cell lysis products are selected from proteins,
polypeptides, peptides and any combination thereof. Subsequently
the cargo molecules can be readily harvested from the capsids.
Accordingly, such compositions are highly desirable for all
applications where high purity and/or high production efficiency is
required.
[0074] Hydrolase resistant capsids as described herein may be used
to enclose different types of cargo molecules to form a virus-like
particle. The cargo molecule can be but is not limited to any one
or more oligonucleotide or oligoribonucleotide (DNA, RNA, LNA, PNA,
siRNA, shRNA, sshRNA, lshRNA or miRNA, or any oligonucleotide
comprising any type of non-naturally occurring nucleic acid), any
peptide, polypeptide or protein. A cargo molecule which is an
oligonucleotide or oligoribonucleotide may be enclosed in a capsid
with or without the use of a linker. A capsid can be triggered for
example to self-assemble from capsid protein in the presence of
nucleotide cargo, such as an oligoribonucleotide. In non-limiting
example, a capsid as described herein may enclose a target
heterologous RNA strand, such as for example a target heterologous
RNA strand containing a total of between 1,800 and 2,248
ribonucleotides, including the 19-mer pack site from Enterobacteria
phage MS2, such RNA strand transcribed from a plasmid separate from
a plasmid coding for the capsid proteins, as described by Wei, Y.
et al. (2008) J. Clin. Microbiol. 46:1734-1740.
[0075] RNA interference (RNAi) is a phenomenon mediated by short
RNA molecules such as siRNA molecules, which can be used for
selective suppression of a target gene of interest, and has
multiple applications in biotechnology and medicine. For example,
short RNA molecules can be employed to target a specific gene of
interest in an organism to obtain a desirable phenotype. Short RNA
molecules, including siRNA, are however easily degraded by
ubiquitous enzymes called RNAses. Capsids, such as those described
herein, protect encapsidated RNA from enzymatic degradation. A
capsid as described herein may however enclose an RNA strand
containing one or more ribozymes, either self-cleaving ribozymes
(cis-acting), or in certain cases capable of cleaving bonds in
other RNA (trans-acting). One or more ribozymes may be included for
example to specifically cut RNA sequence(s) to produce a
specifically tailored RNA molecule, such as for example but not
limited to an siRNA molecule. For example, variants of Hammerhead
and Hepatitis Delta Virus ribozymes are known and can be used to
cut long RNA sequences. The present disclosure describes novel VLPs
comprising a capsid encapsidating one or more ribozymes attached to
pack sequences as described above (i.e., RNA sequences with strong
affinity to the interior wall of a capsid), and the ribozymes used
to cut short RNA sequences from packing sequences attached to the
ribozymes.
[0076] The present disclosure thus also encompasses the novel use
of ribozymes to isolate short or small" RNA sequences such as
siRNA, shRNA, sshRNA, and miRNA sequences from the packing
sequence(s) used to encapsidate them. It should be understood that,
unless expressly indicated otherwise, the term short RNA
encompasses short single stranded and short hairpin (stem loop) RNA
sequences having a double stranded stem and a single-stranded loop
or hairpin. A short RNA is any RNA single strand having no more
than 30 nucleotides, preferably no more than 25 nucleotides, and
more preferably no more than 22 nucleotides; or a hairpin RNA
having a stem of no more than 30 nucleotides base pairs, preferably
no more than 25 nucleotide base pairs, and more preferably no more
than 22 nucleotide base pairs in the stem.
[0077] A challenge in using a ribozyme which is highly active to
isolate such short RNA sequences from packing sequences, is that
the ribozyme may works so fast as to liberate the short RNA from
the packing sequence before encapsidation of the RNA is achieved.
Additionally, it has been discovered that the three dimensional
structures of short RNA such as an siRNA, or the hairpin packing
sequences, can interfere with the proper functioning of the
ribozyme. These problems can be overcome by 1) using ribozyme
mutants which demonstrate a slower rate of activity, to avoid
liberation of the short RNA from the packing sequence before
encapsidation of the short RNA is achieved, and/or 2) increasing
the number of nucleotides in the ribozyme that form Watson-Crick
pairs with the short RNA. Additionally, trans-acting ribozymes can
be used advantageously to increase the percentage of RNA
encapsidated into VLPs as short RNA, if the short RNA sequence(s)
are flanked not by complete ribozymes but rather shorter sequences
that are targets of trans-acting ribozymes, also encapsidated into
the same VLP.
[0078] One or more short RNA sequences can also be encapsidated
into a viral capsid, either wild type or genetically modified,
which has been modified to insert an external peptide tag, to
deliver a protein or drug molecule to a specific class of cell.
Wild type capsids may also be genetically modified to insert
external peptide sequences acting as ligands for certain surface
protein cell receptors can be advantageously used to encapsidate
short RNA sequences aimed at inducing RNAi in specific target
cells. Such compositions are much simpler, less expensive and more
reliably manufactured than current alternatives for short RNA
delivery.
[0079] Non-limiting examples of useful VLP's which can be prepared
include a capsid enclosing an RNA strand comprising:
[0080] (i) at least one packing sequence and from 1 to 100
identical or different siRNAs flanked by one single stranded
(non-hybridyzing) RNA spacer, where every single stranded RNA
spacer has between 4 and 40 nucleotides (SEQ ID NO: 30);
[0081] (ii) one (1) ribozyme and one single stranded
(non-hybridizing) RNA sequence per siRNA, where every single
stranded RNA sequence has between 4 and 40 nucleotides (SEQ ID NO:
28; HDV ribozymes);
[0082] (iii) two (2) ribozymes per siRNA;
[0083] (iv) one (1) T7 start site, one (1) ribozyme, one (1)
packing site and one (1) transcription termination site; or
[0084] (v) one (1) T7 start site, one (1) packing site, and one (1)
transcription termination site.
[0085] (vi) four (4) ribozymes per siRNA (SEQ ID NO: 29)
[0086] In VLP's which include one or more ribozymes, the disclosure
further contemplates VLPs containing the resulting products after
the ribozymes have cut the RNA. VLP's which include a transcription
terminator can use for example the T7 transcription terminator as
described in Studier F W, Moffatt B A. (1986) J. Mol. Biol. May 5;
189(1):113-30; and R Sousa, D Patra, E M Lafer (1992) J. Mol. Biol.
224:319-334. For example, the following two sequences are suitable
transcription terminators for T7 RNA polymerase:
TABLE-US-00002 (SEQ ID NO: 4) CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTG
or (SEQ ID NO: 5)
TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG.
[0087] VLPs as described herein may alternatively enclose at least
one target peptide, polypeptide or protein. When the target
heterologous cargo molecule is a peptide, polypeptide or protein,
an oligonucleotide linker can be used to couple the target
heterologous cargo molecule and the viral capsid. A cargo molecule
which is a peptide, polypeptide or protein, preferably is packaged
in a capsid using a linker. The packaging process is promoted by
the linker, consisting of a short RNA aptamer sequence, which forms
a link between the coat protein and a peptide tag fused to the
target cargo molecule. (See Fiedler, J. et al., RNA-Directed
Packaging of Enzymes within Virus-like Particles, Angew. Chem. Int.
Ed. 49: 9648-9651 (2010)). The oligonucleotide linker may consist
of DNA, RNA, LNA, PNA, and the like. The linker is for example a
50- to 100-mer having a short sequence, for example about 20 nt
long, at a first end with binding specificity for the inside of the
capsid coat, and another sequence, for example about 70 nt long, at
the second, opposite end which has a binding specificity for the
cargo peptide, polypeptide or protein. Additionally, a slow
ribozyme may be incorporated into a linker consisting of RNA. For
example, a slow ribozyme can be incorporated between the packing
sequence (binding to the coat protein) and the aptamer (binding to
the tag of target protein). Upon activation, the ribozyme will
separate the coat protein from the target protein. Alternatively, a
capsid as described herein may enclose at least one target protein
N-terminally tagged with a peptide able to non-covalently bind to
an aptamer- and capsid pack sequence-containing RNA strand, for
example an N-terminal tag and aptamer- and pack sequence-containing
RNA strand as described by Fiedler, J. et al. (2010).
[0088] A cargo molecule can be a bi-molecular cargo molecule, and
capsids described herein may also encapsidate a bi-molecular cargo
molecule, which may or may not include one or more ribozymes. A
bi-molecular cargo molecule may comprise an aptamer linked to a
bifunctional polynucleotide. The aptamer may have a sequence
specifically selected using SELEX to exhibit specific binding to a
bioactive small molecule, i.e., a molecule having a low molecular
weight, preferably lower than 1,500 Da. The bifunctional
polynucleotide has both a first aptameric activity for binding the
low-molecular weight bioactive cargo molecule, and a second
aptameric activity for binding a packing sequence of a capsid. The
bifunctional polynucleotide linked to the bioactive cargo molecule
forms the bi-molecular cargo molecule which can then be linked to
the capsid. Such a cargo molecule can be used to bind the bioactive
small molecule, and thus load the VLP with the small molecule. The
present disclosure thus also encompasses a VLP comprising a capsid
linked to such a synthetic bi-molecular cargo molecule. Examples of
low molecular weight bioactives which can be loaded into a VLP by
binding to an RNA aptamer include: atrazine (herbicide),
acetamipridphorate, profenofos, isocarbophos and omethoateas
(insecticides), as described by Sett et al. (2012) Open Journal of
Applied Biosensor, 1:p. 9-19.
[0089] Examples of low molecular weight bioactives which can be
loaded into a VLP by binding to an RNA aptamer include herbicides
such as 2,4-D (2,4-Dichlorophenoxyacetic acid), December
((3,6-dichloro-2-methoxybenzoic acid), Paraquat
(N,N'-dimethyl-4,4'-bipyridinium dichloride), Oryzalin
(4-(dipropylamino)-3,5-dinitrobenzenesulfonamide), DCMU
(3,4-dichlorophenyl)-1,1-dimethylurea), Trifluralin
(2,6-Dinitro-N,N-dipropyl-4-(trifluoromethyl)aniline), Imazapic
(-methyl-2-[4-methyl-5-oxo-4-(propan-2-yl)-4,5-dihydro-1H-imidazol-2-yl]p-
yridine-3-carboxylic acid), Aminopyralid
(4-amino-3,6-dichloropyridine-2-carboxylic acid), Clopyralid
(3,6-dichloro-2-pyridinecarboxylic acid), Metolachlor
((RS)-2-Chloro-N-(2-ethyl-6-methyl-phenyl)-N-(1-methoxypropan-2-yl)acetam-
ide), Pendimethalin
(3,4-Dimethyl-2,6-dinitro-N-pentan-3-yl-aniline), Picloram
(4-Amino-3,5,6-trichloro-2-pyridinecarboxylic acid), Propanil
(N-(3,4-Dichlorophenyl)propanamide), Triclopyr
([(3,5,6-Trichloro-2-pyridinyl)oxy]acetic acid), and Atrazine
(2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine), among
other listed for example by Roberts et al. (1998) Metabolic
Pathways of Agrochemicals: Part 1: Herbicides and Plant Growth
Regulators. Published by Royal Society of Chemistry (Great Britain)
ISBN 978-1-84755-138-2. For example, an RNA aptamer binding
Atrazine was described by Sinha et al. (2010) Nature Chemical
Biology, 6:p. 464-470.
[0090] RNA aptamers can also be used to bind insecticides such as,
Propargite (2-(4-tert-butylphenoxy)cyclohexyl
prop-2-yne-1-sulfonate), Chlorpyrifos (O,O-diethyl
O-3,5,6-trichloropyridin-2-yl phosphorothioate), Cypermethrin,
Phosmet
(2-Dimethoxyphosphinothioylthiomethyl)isoindoline-1,3-dione),
Permethrin (3-Phenoxybenzyl
(1RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate-
), Diazinon (O,O-Diethyl
O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl]phosphorothioate),
Methylparathion (O,O-Dimethyl O-(4-nitrophenyl)phosphorothioate),
and Acetamiprid
(N-[(6-chloro-3-pyridyl)methyl]-N'-cyano-N-methyl-acetamidine), and
fungicides such as Chlorothalonil
(2,4,5,6-tetrachloroisophthalonitrile), Captan
((3aR,7aS)-2-[(trichloromethyl)sulfanyl]-3a,4,7,7a-tetrahydro-1H-i-
soindole-1,3(2H)-dione), Boscalid
(2-chloro-N-(4'-chlorobiphenyl-2-yl)nicotinamide), Iprodione
(3-(3,5-dichlorophenyl)-N-isopropyl-2,4-dioxoimidazolidine-1-carboxamide)-
, Azoxystrobin (Methyl
(2E)-2-(2-{[6-(2-cyanophenoxy)pyrimidin-4-yl]oxy}phenyl)-3-methoxyacrylat-
e), Pyraclostrobin, (methyl
2-[1-(4-chlorophenyl)pyrazol-3-yloxymethyl]-N-methoxycarbanilate),
Cyprodinil (4-cyclopropyl-6-methyl-N-phenylpyrimidin-2-amine),
among other listed for example by Roberts et al. (1999) Metabolic
Pathways of Agrochemicals: Part 2: Insecticides and Fungicides.
Published by Royal Society of Chemistry (Great Britain) ISBN
978-1-84755-137-5. For example, aptamers have been described to
bind acetamiprid, phorate, profenofos, isocarbophos and omethoate,
as exemplified by Sett et al. (2012) Open Journal of Applied
Biosensor, 1:p. 9-19 using DNA aptamers built in a similar manner
as RNA aptamers are built using SELEX.
[0091] These herbicides, insecticides or fungicides are bioactive
small molecules, i.e., molecules having a low molecular weight,
preferably lower than 1,500 Da. Due to their small size they can
permeate capsids forming VLPs of the current disclosure, as
exemplified by Wu et al. (2005) Delivery of antisense
oligonucleotides to leukemia cells by RNA bacteriophage capsids,
Nanomedicine: Nanotechnology, Biology and Medicine, 1:p. 67-76.
These small bioactive molecules are added to VLPs of the current
disclosure which encapsidate aptamers designed using SELEX to bind
the small bioactive molecules, after such VLPs have been formed,
either before or after purification. The addition of these small
bioactive molecules is done, for example by adding them to a
solution of the VLPs and incubation, for example at room
temperature for a time between 30 minutes and 10 hours. These small
bioactive molecules enter the VLPs by diffusion through the pores
at the particle symmetry axes and are retained inside due to their
binding to the enclosed aptamers. Suitable solvents used for
loading the small bioactive molecules into the VLPs range from
polar such as water and water-ethanol blends to non-polar such as,
for example, isooctane, toluene, dichloromethane, or chloroform.
Using non-polar solvents for the dissolution of VLPs is done, for
example, as described by Johnson et al. (2006), Solubilization and
stabilization of bacteriophage MS2 in organic solvents,
Biotechnology and bioengineering, 2007. 97(2): p. 224-34, with the
help of surfactants like Aerosol OT. Use of non-polar solvents for
loading small bioactive molecules is preferred since their
solubility in polar solvents is, in most cases, poor.
[0092] VLPs encapsidating both siRNA and small bioactive molecules
are preferred in applications where a synergistic effect is
achieved between the two bioactive ingredients, for example in
those cases where the targeted plant, insect or fungus is resistant
to the small bioactive molecule. In such cases, the siRNA is
designed to target the biologic pathway that confers the plant,
insect or fungus resistance to the small bioactive molecule, as
exemplified by Sammons et al., Polynucleotide molecules for gene
regulation in plants, US 2011/0296556.
[0093] Alternatively, the bi-functional polynucleotide may encode
at least one siRNA, shRNA, sshRNA, lshRNA or miRNA, and the cargo
molecule can be a small (low molecular weight) protein or peptide.
Accordingly, a bi-molecular cargo molecule can be capable of
binding a low molecular bioactive protein or peptide. Such a
bi-molecular cargo molecule may comprise a biologically active
protein or peptide, coupled to a polynucleotide encoding at least
one siRNA or shRNA or sshRNA or lshRNA of miRNA, and having a first
aptameric activity for binding the bioactive protein or peptide
cargo molecule and a second aptameric activity for binding a
packing sequence of a capsid. The polynucleotide is linked to the
protein or peptide cargo molecule and is capable of linking to
packing sequence of a capsid.
[0094] A bifunctional polynucleotide as described above may
optionally include one or more ribozyme sequences. A VLP including
a bi-molecular cargo molecule including a bifunctional
polynucleotide as described above may optionally include one or
more ribozymes. The present disclosure also encompasses a VLP
comprising a capsid and reaction products of the bi-molecular cargo
molecule after at least one ribozyme has reacted with bimolecular
cargo molecule to cut the cargo molecule into constituent parts
including the aptamer.
[0095] VLPs as described herein may be assembled by any available
method(s) which produces a VLP with an assembled, hydrolase
resistant capsid encapsidating one or more cargo molecule(s), and
optionally any linker, packing sequence, one or more ribozymes, or
tags. For example, capsids and cargo molecules may be co-expressed
in any expression system. Recombinant DNA encoding one or more
capsid proteins, one or more cargo molecule(s), and optionally any
linker, packing sequence, ribozyme(s) or tags can be readily
introduced into the host cells, e.g., bacterial cells, plant cells,
yeast cells, fungal cells, and animal cells (including insect and
mammalian) by transfection with one or more expression vectors by
any procedure useful for introducing such a vector into a
particular cell, and stably transfecting the cell to yield a cell
which expresses the recombinant sequence(s).
[0096] The host cell is preferably of eukaryotic origin, e.g.,
plant, mammalian, insect, yeast or fungal sources, but
non-eukaryotic host cells may also be used. Suitable expression
systems include but are not limited to microorganisms such as
bacteria (e.g., E. coli) transformed with recombinant bacteriophage
DNA, plasmid DNA or cosmid DNA expression vectors containing the
coding sequences for the VLP elements. In non-limiting example, for
VLPs using the MS2 capsid protein, expression in E. coli is a
suitable expression system.
[0097] The present disclosure expressly contemplates plant cells
which have been transformed using a nucleic acid construct as
described herein, and which expresses a capsid coat protein, cargo
molecule and a and optionally any linker, packing sequence, one or
more ribozymes, or tags. Means for transforming cells including
plant cells and preparing transgenic cells are well known in the
art. Vectors, plasmids, cosmids, YACs (yeast artificial
chromosomes) and DNA segments can be used to transform cells and
will as generally recognized include promoters, enhancers, and/or
polylinkers. Transgenic cells specifically contemplated include
transgenic plant cells including but not limited to cells obtained
from corn, soybean, wheat, vegetables, grains, legumes, fruit
trees, and so on, or any plant which would benefit from
introduction of a VLP as described herein. Also contemplated are
plants, plant tissue obtained from cells transformed as described
herein, and the seed or progeny of the plant or plant tissue.
[0098] Expression of assembled VLPs can be obtained for example by
constructing at least one expression vector including sequences
encoding all elements of the VLP. Sometimes two vectors are used, a
first which includes a sequence encoding the cargo molecule(s) and
optionally any linker, packing sequence, one or more ribozymes, or
tags; and a second vector which includes a sequence encoding the
capsid protein. In an exemplary process for generating exemplary
VLPs including siRNA, two vectors may be co-expressed in the host
cell for generation of the VLP, as further detailed in the
Examples. Methods and tools for constructing such expression
vectors containing the coding sequences and transcriptional and
translational control sequences are well known in the art.
Vector(s) once constructed are transferred to the host cells also
using techniques well known in the art, and the cells then
maintained under culture conditions for a time sufficient for
expression and assembling of the VLP's to occur, all using
conventional techniques. The present disclosure thus encompasses
host cells containing any such vectors, and cells which have been
transformed by such vectors, as well as cells containing the
VLP's.
[0099] When the VLP's have been expressed and assembled in the host
cells, they may be isolated and purified using any method known in
the art for virus purification. For example, the cells can be lysed
using conventional cell lysis techniques and agents, and the cell
lysate subjected to hydrolysis using at least one peptide bond
hydrolase category EC 3.4 such as but not limited to Proteinase K
or subtilisin. Intact capsids remaining in the cell lysate
following hydrolysis can be removed and purified using conventional
protein isolation techniques.
[0100] Purification of capsids, VLPs or proteins may also include
methods generally known in the art. For example, following capsid
expression and cell lysis, the resulting lysate can be subjected to
one or more isolation or purification steps. Such steps may include
for example enzymatic lipolysis, DNA hydrolysis, and proteolysis
steps. A proteolysis step may be performed for example using a
blend of endo- and exo-proteases. For example, after cell lysis and
hydrolytic disassembly of most cell components, such capsids with
their cargo molecules can be separated from surrounding matrix by
extraction, for example into a suitable non-polar water-immiscible
solvent, or by crystallization from a suitable solvent. For
example, hydrolysis and/or proteolysis steps transform contaminants
from the capsid that are contained in the lysate matrix into small,
water soluble molecules. Hydrophobic capsids may then be extracted
into an organic phase such as 1,3-bis(trifluoromethyl)benzene.
Purification of capsids, VLPs or proteins may include for example
at least one liquid-liquid extraction step, at least one fractional
precipitation step, at least one ultrafiltration step, or at least
one crystallization step. A liquid-liquid extraction may comprise
for example use of an immiscible non-aqueous non-polar solvent,
such as but not limited to benzene, toluene, hexane, heptane,
octane, chloroform, dichloromethane, or carbon tetrachloride.
Purifying may include at least one crystallization step. Use of one
or more hydrolytic steps, and especially of one or more proteolytic
steps, eliminates certain problems observed with current separation
processes used for cargo molecules, which are mainly result from
the large number and varying degree of binding interactions which
take place between cargo molecules and components derived from the
cell culture in which they are produced. The capsids described
herein resist hydrolytic steps such that the matrix which results
after hydrolysis includes intact capsids which safely partition any
cargo molecules from the surrounding matrix, thereby interrupting
the troublesome binding interactions which interfere with current
purification processes.
[0101] Following purification, the capsid can be opened to obtain
the cargo molecule, which maybe a protein or polypeptide, a
peptide, or a nucleic acid molecule as described herein. Capsids
can be opened using any one of several possible procedures known in
the art, including for example heating in an aqueous solution above
50.degree. C.; repeated freeze-thawing; incubating with denaturing
agents such as formamide; by incubating with one or more proteases;
or by a combination of any of these procedures.
[0102] Capsid proteins which are resistant to hydrolases and useful
in the VLPs and methods according to the present disclosure can
also be variants of, or derived from the wild type MS2 capsid
protein. Capsid proteins may comprise, for example, at least one
substitution, deletion or insertion of an amino acid residue
relative to the wild type MS2 capsid amino acid sequence. Such
capsid proteins may be naturally occurring variants or can be
obtained by genetically modifying the MS2 capsid protein using
conventional techniques, provided that the variant or modified
capsid protein forms a non-enveloped capsid which is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4 as
described herein.
[0103] Genetically modified MS2 capsid proteins which can assemble
into capsids which are resistant to hydrolysis as described herein
can be engineered by making select modifications in the amino acid
sequence according to conventional and well-known principles in
physical chemistry and biochemistry to produce a protein which
retains resistance to hydrolysis as described herein and in the
Examples herein below.
[0104] It is common knowledge for example that the shape or global
fold of a functional protein is determined by the amino acid
sequence of the protein, and that the fold defines the protein's
function. The global fold is comprised of one or more folding
domains. When more than one folding domain exists in the global
fold, the domains generally bind together, loosely or tightly along
a domain interface. The domain fold can be broken down into a
folding core of tightly packed, well-defined secondary structure
elements which is primarily responsible for the domain's shape and
a more mobile outer layer typically comprised of turns and loops
whose conformations are influenced by interactions with the folding
core as well as interactions with nearby domains and other
molecules, including solvent and other proteins. An extensive
public domain database of protein folds, the Structural
Classification of Proteins (SCOP) database (Alexey G Murzin, Curr
Opin Struct Biol (1996) 6, 386-394) of solved protein structures in
the public domain is maintained online at http://scop.berkeley.edu
and regularly expanded as new solved structures enter the public
domain (Protein Data Bank (F. C. Bernstein, T. F. Koetzle, G. J.
Williams, E. E. Meyer Jr., M. D. Brice, J. R. Rodgers, O. Kennard,
T. Shimanouchi, M. Tasumi, "The Protein Data Bank: A Computer-based
Archival File For Macromolecular Structures," J. of. Mol. Biol.,
112 (1977): 535), http://www.rcsb.org) database. Members of a
family which are evolutionarily distant, yet have the same shape
and very similar function, commonly retain as few as 30% identical
residues at topologically and/or functionally equivalent positions.
In some families, sequences of distant members have as few as 20%
of their residues unchanged with respect to each other, e.g. levi-
and alloleviviridae capsid proteins. Further, the fold and function
of a protein is remarkably tolerant to change via directed or
random mutation, even of core residues (Peter O. Olins, S.
Christopher Bauer, Sarah Braford-Goldberg, Kris Sterbenz, Joseph O.
Polazzi, Maire H. Caparon, Barbara K. Klein, Alan M. Easton, Kumnan
Paik, Jon A. Klover, Barrett R. Thiele, and John P. McKearn (1995)
J Biol Chem 270, 23754-23760; Yiqing Feng, Barbara K. Klein and
Charles A. McWherter (1996), J Mol Biol 259, 524-541; Dale Rennell,
Suzanne E. Bouvier, Larry W. Hardy and Anthony R. Poteetel (1991) J
Mol Biol 222, 67-87), insertion/deletion of one or more residues
(Yiqing Feng, Barbara K. Klein and Charles A. McWherter (1996), J
Mol Biol 259, 524-541), permutation of the sequence
(Multi-functional chimeric hematopoietic fusion proteins between
sequence rearranged c-mpl receptor agonists and other hematopoietic
factors, U.S. Pat. No. 6,066,318), concatenation via the N- or
C-terminus or both (to copies of itself or other peptides or
proteins) (Multi-functional chimeric hematopoietic fusion proteins
between sequence rearranged g-csf receptor agonists and other
hematopoietic factors, US20040171115; Plevka, P., Tars, K., Liljas,
L. (2008) Protein Sci. 17: 173) or covalent modification, e.g.,
glycosylation, pegylation, SUMOylation or the addition of peptidyl
or nonpeptidyl affinity tags as long as the residues critical to
maintaining the fold and/or function are spared.
[0105] VLPs according to the present disclosure and as used in any
of the methods and processes, thus encompass those comprising a
capsid protein having at least 15%, 16%, 21%, 40%, 41%, 52%, 53%,
56%, 59% or at least 86% sequence identity with the amino acid
sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ
ID NO: 3) and is resistant to hydrolysis catalyzed by a peptide
bond hydrolase category EC 3.4. Such VLPs include for example a VLP
comprising a capsid protein having at least 52% sequence identity
with SEQ ID NO: 3) as described above. Also included is a VLP
comprising a capsid protein having at least 53% sequence identity
to SEQ ID NO:3, which can be obtained substantially as described
above but not disregarding the FR capsid sequence, representing 53%
sequence identity to wild-type enterobacteria phage MS2 capsid
protein (SEQ ID NO:3). Also included is a VLP comprising a capsid
protein having at least 56% sequence identity to SEQ ID NO:3, when
it is considered that when the structures identified as 1AQ3 (van
den Worm, S. H., Stonehouse, N. J., Valegard, K., Murray, J. B.,
Walton, C., Fridborg, K., Stockley, P. G., Liljas, L. (1998)
Nucleic Acids Res. 26: 1345-1351), 1GAV (Tars, K., Bundule, M.,
Fridborg, K., Liljas, L. (1997) J. Mol. Biol. 271: 759-773), 1FRS
(Liljas, L., Fridborg, K., Valegard, K., Bundule, M., Pumpens, P.
(1994) J. Mol. Biol. 244: 279-290) and 2VTU (Plevka, P., Tars, K.,
Liljas, L. (2008) Protein Sci. 17: 1731) (Protein Data Bank
identifiers described above), only 56% of the sequence positions
have identical sequence and topologically equivalent positions with
respect to the backbone overlays when all three sequences are
considered together. Also included is a VLP comprising a capsid
protein having at least 59% sequence identity to SEQ ID NO:3, when
it is considered that the sequence of the MS2 viral capsid protein
compared to that of the GA viral capsid protein is 59%. Also
included is a VLP comprising a capsid protein having at least 86%
sequence identity to SEQ ID NO:3, when it is considered that the
sequence of the MS2 viral capsid protein compared to that of the FR
capsid protein is 86%. VLPs according to the present disclosure
thus encompass those comprising a capsid protein having at least
15%, 16%, or 21% sequence identity with the amino acid sequence of
wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) based on a
valid structure anchored alignment and is resistant to hydrolysis
catalyzed by a peptide bond hydrolase category EC 3.4.
[0106] A VLP may thus comprise any of the MS2 capsid protein
variants as described herein. Genetically modified capsid proteins
consistent with those described herein can be produced for example
by constructing at least one DNA plasmid encoding at least one
capsid protein having at least one amino acid substitution,
deletion or insertion relative to the amino acid sequence of the
wild type MS2 capsid protein, making multiple copies of each
plasmid, transforming a cell line with the plasmids; maintaining
the cells for a time and under conditions sufficient for the
transformed cells to express and assemble capsids encapsidating
nucleic acids; lysing the cells to form a cell lysate; subjecting
the cell lysate to hydrolysis using at least one peptide bond
hydrolase, category EC 3.4; and removing intact capsids remaining
in the cell lysate following hydrolysis to obtain capsids having
increased resistance to at least one hydrolase relative to the wild
type capsid protein. Following purification of the resulting,
intact capsids, an amino acid sequence for each capsid protein may
be determined according to methods known in the art.
[0107] The specialized capsids described herein can be used in
research and development and in industrial manufacturing facilities
to provide improved yields, since the purification processes used
in both settings have the same matrix composition. Having such same
composition mainly depends on using the same cell line in both
research and development and manufacturing processes. However,
differences in matrix composition due to using different cell lines
are greatly reduced after proteolytic steps used in both research
and development and manufacturing stages. This feature enables use
of different cell lines in both stages with a minimal manufacturing
yield penalty.
EXAMPLES
[0108] The following non-limiting examples are included to
illustrate various aspects of the present disclosure. It will be
appreciated by those of skill in the art that the techniques
disclosed in the following examples represent techniques discovered
by the Applicants to function well in the practice of the
invention, and thus can be considered to constitute preferred modes
for its practice. However, those of skill in the art should, in
light of the instant disclosure, appreciate that many changes can
be made in the specific examples described, while still obtaining
like or similar results, without departing from the scope of the
invention. Thus, the examples are exemplary only and should not be
construed to limit the invention in any way. To the extent
necessary to enable and describe the instant invention, all
references cited are herein incorporated by reference.
Example A
Propagation of MS2 Bacteriophage
[0109] MS2 bacteriophage (ATCC No. 15597-B1, from American Type
Culture Collection, Rockville, Md.) and its E. Coli host (ATCC No.
15669) were obtained from ATCC and propagated using the procedure
described by Strauss and Sinsheimer (1963) J. Mol. Biol. 7:43-54 J.
Mol. Biol. 7:43-54. Results are plotted in FIG. 1. Optical Density
(OD) at 600 nm and pH were followed during the reaction. ODi
represents OD immediately after inoculation with host. Infection
was done at 2.3 hours. Ln(OD/ODi) was plotted on the left axis
(full diamonds) and pH was plotted on the right axis (open
squares). This experiment was ended 5.3 hours after inoculation
with host. Lysate obtained was centrifuged at 2,000 g and filtered
through a 0.2 .mu.m membrane to eliminate remaining bacteria and
bacterial debris.
Example B
Purification of MS2 Bacteriophage Using Proteinase K and
Ultrafiltration
[0110] Purification of MS2 bacteriophage was conducted as follows.
Samples were taken during purification and SDS PAGE analysis was
run on the samples. Results obtained are shown in FIG. 2.
8 mL lysate obtained at end of Example A (sample in Lane 1, FIG. 2)
was filtered through a 300 kDa membrane (Vivaspin 2, from Sartorius
Stedim, Bohemia, N.Y.) and the filtrate was filtered through a 100
kDa membrane, from which 1 mL of retentate was obtained (sample in
Lane 2, FIG. 2). This retentate was divided in two equal parts. To
one half (control) 2064, mM CaCl.sub.2 aqueous solution at pH=7.5
was added. To the second half (Proteinase) 0.15 mg Proteinase K
(Sigma Aldrich, St. Louis, Mo.) dissolved in 2064, 20 mM CaCl.sub.2
aqueous solution at pH=7.5 and was added. Both tubes were incubated
at 37.degree. C. and after 1 hour they were placed in an ice-water
bath. Samples were then taken and analyzed: control sample in Lane
3, FIG. 2, and Proteinase sample in Lane 5, FIG. 2. Each product
was then diluted to 2 mL with deionized (DI) water and filtered
through a 100 kDa membrane. Each retentate (150 .mu.L) was diluted
to 2 mL with DI water and filtered again through the same membrane.
Dilution and ultrafiltration was repeated one more time for each
product. Samples of each retentate were then taken and analyzed:
control sample in Lane 4, FIG. 2, and Proteinase sample in Lane 6,
FIG. 2. Band at 14 kDa corresponds to MS2 bacteriophage's coat
protein. Band at 30 kDa corresponds to Proteinase K. Product from
control experiment yields a highly impure phage. Product from
Proteinase experiment yields a product containing phage with purity
higher than 99%.
Example C
Degradation of MS2 Bacteriophage
[0111] Treatment of MS2 bacteriophage was conducted as follows.
Samples were taken during treatment and SDS PAGE analysis was run
on the samples. Results obtained are shown in FIG. 3. 4 mL lysate
obtained at end of Example A was partially purified by
precipitation using ammonium sulfate and extraction using
trichlorofluoromethane (Freon 11) as described by Strauss &
Sinsheimer (1963) J. Mol. Biol. 7:43-54. A sample of the aqueous
solution after extraction with Freon 11 was taken and analyzed
(sample in Lane 1, FIG. 3). To the partially purified phage
solution (130 .mu.L) 370 .mu.L of 20 mMCaCl.sub.2 aqueous solution
was added. The mixture was incubated at 37.degree. C. and after 1
hour it was placed in an ice-water bath. A sample was then taken
and analyzed: sample in Lane 2, FIG. 3. The incubation product was
diluted to 2 mL with deionized (DI) water and filtered through 100
kDa membrane. The retentate (150 .mu.L) was diluted to 2 mL with DI
water and filtered again through the same membrane. Dilution and
ultrafiltration of the retentate was repeated one more time. A
sample of the retentate was then taken and analyzed: sample in Lane
3, FIG. 3. Only weak bands at lower than 10 kDa were observed,
indicating complete degradation of phage.
Example D
Purification of MS2 Bacteriophage Using Ultrafiltration
[0112] Purification of MS2 bacteriophage was conducted as follows.
Samples were taken during purification and SDS PAGE analysis was
run on the samples. Results obtained are shown in FIG. 3. 4 mL
lysate obtained at end of Example A was partially purified by
precipitation using ammonium sulfate and extraction using
trichlorofluoromethane (Freon 11) as described by Strauss &
Sinsheimer (1963) J. Mol. Biol. 7:43-54. The aqueous solution
containing partially purified phage was diluted to 2 mL with
deionized water, filtered through a 300 kDa membrane and the
filtrate was filtered through a 100 kDa membrane, from which 1504,
of retentate was obtained. The retentate was then diluted to 2 mL
with deionized (DI) water and filtered through the same 100 kDa
membrane. Dilution and ultrafiltration of the retentate (150 .mu.L)
was repeated one more time. A sample of the retentate was then
taken and analyzed: sample in Lane 4, FIG. 3.370 .mu.L of 20
mMCaCl.sub.2 aqueous solution was added to the retentate (130
.mu.L). The mixture was incubated at 37.degree. C. and after 1 hour
it was placed in an ice-water bath. A sample was then taken and
analyzed: sample in Lane 5, FIG. 3. The product was then diluted to
2 mL with deionized (DI) water and filtered through a 100 kDa
membrane. The retentate (150 .mu.L) was diluted to 2 mL with DI
water and filtered again through the same membrane. Dilution and
ultrafiltration of the retentate was repeated one more time. A
sample of the retentate was then taken and analyzed: sample in Lane
6, FIG. 3. MS2's coat protein, of 14 kDa, retained by a membrane
through which permeate proteins with less than 100 kDa molecular
weight, is clearly visible, indicating the presence of intact MS2
capsids. The product obtained contained phage with purity higher
than 99%.
Example E
Purification of MS2 Bacteriophage Using Proteinase K, and
Ultrafiltration
[0113] Purification of MS2 bacteriophage was conducted as follows.
Samples were taken during purification and SDS PAGE analysis was
run on the samples. Results obtained are shown in FIG. 4.
4 mL lysate obtained at end of Example A was partially purified by
precipitation using ammonium sulfate and extraction using
trichlorofluoromethane (Freon 11) as described by Strauss &
Sinsheimer (1963) J. Mol. Biol. 7:43-54. The aqueous solution
containing partially purified phage was diluted to 2 mL with
deionized water, filtered through a 100 kDa membrane, from which
150 .mu.L of retentate was obtained. The retentate was then diluted
to 2 mL with deionized (DI) water and filtered through the same 100
kDa membrane. Dilution and ultrafiltration of the retentate (150
.mu.L) was repeated one more time. A sample of the retentate was
then taken and analyzed: sample in Lane 1, FIG. 4. 0.15 mg of
Proteinase K dissolved in 370 .mu.L of 20 mMCaCl.sub.2 aqueous
solution was added to the retentate (130 .mu.L). The mixture was
incubated at 37.degree. C. and after 1 hour it was placed in an
ice-water bath. A sample was then taken and analyzed: sample in
Lane 2, FIG. 4. The product was then diluted to 2 mL with deionized
(DI) water and filtered through a 100 kDa membrane. The retentate
(150 .mu.L) was diluted to 2 mL with DI water and filtered again
through the same membrane. Dilution and ultrafiltration of the
retentate was repeated one more time. A sample of the retentate was
then taken and analyzed: sample in Lane 3, FIG. 4. The product
obtained contained phage with purity higher than 99%.
Example F
Purification of MS2 Bacteriophage Using Proteinase K, Precipitation
at Acidic Conditions, Precipitation Using Ethanol at Basic and
Acidic Conditions, and Ultrafiltration
[0114] Purification of MS2 bacteriophage was conducted as follows.
Samples were taken during purification and SDS PAGE analysis was
run on the samples. Results obtained are shown in FIG. 5. 50 mL
lysate obtained at end of Example A was partially purified by
precipitation using ammonium sulfate and extraction using
trichlorofluoromethane (Freon 11) as described by Strauss &
Sinsheimer (1963) J. Mol. Biol. 7:43-54. A sample of the aqueous
solution after extraction with Freon 11 was taken and analyzed
(sample in Lane 1, FIG. 5). To the partially purified phage
solution (1.2 mL) 0.9 mg of Proteinase K dissolved in 1.24 mL of 20
mMCaCl.sub.2 aqueous solution was added. The mixture was incubated
at 37.degree. C. and after 1 hour 60 .mu.L of 0.2M
Phenylmethanesulfonyl fluoride (PMSF) solution in ethanol was added
to inactivate Proteinase K. The mixture was then placed in an
ice-water bath. A sample was taken and analyzed: sample in Lane 2,
FIG. 5.0.68 mL of 0.1% phosphoric acid aqueous solution was slowly
added with vigorous agitation in an ice/water bath to bring the pH
of the liquid to 4. The liquid was kept at 0.degree. C. for 30
minutes and centrifuged at 16,000 g at 4.degree. C. for 30 min. The
supernatant was allowed to reach room temperature and 130 .mu.L of
1% NaOH was added to bring the pH of the liquid to 8. 0.81 mL of
ethanol at room temperature was slowly added with vigorous
agitation to bring the ethanol concentration in the liquid to 20%.
The liquid was kept at room temperature for 30 min and centrifuged
at 16,000 g at room temperature for 30 min. The supernatant was
placed in an ice/water bath for 15 min and 1.3 mL of 1% acetic acid
was slowly added at 0.degree. C. with vigorous agitation to bring
the pH of the liquid to 4. 1.5 mL of ethanol at 0.degree. C. was
slowly added with vigorous agitation to bring the ethanol
concentration in the liquid to 34%. The liquid was kept at
0.degree. C. for 30 minutes and centrifuged at 16,000 g at
4.degree. C. for 30 min. The pellet was resuspended in 200 .mu.L of
DI water and a 20 .mu.L sample was taken and analyzed: Lane 3, FIG.
5. The rest (180 .mu.L) was diluted with DI water to 2 mL and
filtered through 100 kDa membrane. The retentate (150 .mu.L) was
diluted to 2 mL with DI water and filtered again through the same
membrane. Dilution and ultrafiltration of the retentate was
repeated one more time. A sample of the retentate was then taken
and analyzed by SDS PAGE: sample in Lane 4, FIG. 5. MS2's coat
protein, of 14 kDa, retained by a membrane through which proteins
with less than 100 kDa molecular weight are able to permeate, is
clearly visible, consistent with the presence of intact MS2
capsids. A UV spectrum on the same retentate is shown in FIG. 6,
which is consistent with results published by G. F. Rohrmann and R.
G. Krueger, (1970) J. Virol., 6(3):26 for pure MS2 phage. A
Superdex 200 (GE Healthcare, Piscataway, N.J.) size exclusion
chromatography was run on the same retentate using Tris-buffered
saline at pH 7.4 and 150 mM NaCl. It showed 280 nm absorbance only
at the void volume of the column. There was no absorbance in the
elution volume for proteins of 600 kD to 2 kD. This test is
consistent with intact phage particles. RNA was isolated from
another sample of the same retentate using a QIAamp Viral RNA Mini
Kit (Qiagen, Valencia, Calif.) and a DNA-free kit (Life
Technologies, Grand Island, N.Y.), and reverse transcribed using a
High Capacity cDNA Reverse Transcription Kit (Life Technologies).
The presence or absence of three different sections of the MS2
genome was then interrogated in PCR experiments. The following
pairs of primers were used, each primer named for the position of
its first and last base in the MS2 genome, forward (F) and reverse
(R) respectively: F1001.sub.--1021-R2180.sub.--2201,
F1201.sub.--1223-R1979.sub.--2001,
F1401.sub.--1426-R1680.sub.--1705. Platinum Taq DNA Polymerase High
Fidelity (Life Technologies) was used for amplification. PCR
products, chromatographed in 1.5% agarose gel stained with Ethidium
Bromide, as shown in FIG. 9 (1.2 kbp for primers
F1201.sub.--1223-R1979.sub.--2001 in Lane 1, 800 bp for primers
F1201.sub.--1223-R1979.sub.--2001 in Lane 2, and 304 bp for primers
F1401.sub.--1426-R1680.sub.--1705 in Lane 3), were consistent with
an intact MS2 bacteriophage genome. An infectivity test was also
run on the same retentate as follows. 5 .mu.L of retentate were
used to infect 1 mL of bacterial culture as described in Example A
at the point it reached OD(600 nm)=0.22. OD(600 nm) was 0.621 hour
after of infection and dropped to 0.21 after 2 additional hours,
while during the same time a control sample attained OD(600 nm) of
0.82 1 hour after infection and 1.2 after 2 additional hours, as
shown in FIG. 7. This test showed a highly infectious phage in the
retentate and therefore demonstrated that the purification
processes used to isolate it did not compromise its integrity. In
conclusion, the product obtained contained MS2 bacteriophage with
purity higher than 99%.
Example G
Purification of MS2 Bacteriophage Using Different Exogenous
Proteases, and Ultrafiltration
[0115] Purification of MS2 bacteriophage using different exogenous
proteases was attempted substantially as described in Example E,
with the exception that proteases other than Proteinase K were
used. MS2 bacteriophage was successfully purified after proteolysis
promoted by Protease from Bacillus licheniformis (P5380, Sigma
Aldrich). However, a proteolysis reaction using Pepsin from porcine
gastric mucosa (P6887, Sigma Aldrich) at pH of 6 was found to
significantly degrade MS2 bacteriophage. On the other hand,
proteolysis reactions using Papain from papaya latex (P3125, Sigma
Aldrich) at pH 6 did not extensively degrade MS2 bacteriophage.
Example H
Production of MS2 Capsids Encapsidating RNA Coding for its Coat
Protein Attached to its Specific 19-Mer RNA Hairpin
[0116] Production of MS2 capsids was conducted as follows. Samples
were taken during the course of expression and SDS PAGE analysis
was run on the samples to monitor capsid production. Results
obtained are shown in FIG. 8. The following DNA sequence, encoding
MS2's coat protein and its specific RNA 19-mer PAC site was cloned
into pDEST14 A252 plasmid (Life Technologies):
TABLE-US-00003 (SEQ ID NO: 6)
ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTC
TAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGA
CTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCT
AACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTC
TGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAA
CCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC
TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGA
GCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTC
CCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAGACGCCGGCCATT
CAAACATGAGGATTACCCATGTACCCAGCT
One Shot BL21(DE3) Chemically Competent E. coli (Life Technologies)
cells were transformed using such plasmid. BL21(DE3) containing the
plasmid were grown in 750 mL of LB medium containing ampicillin at
37.degree. C., to OD(600 nm) equal to 0.8. A pre-induction sample
was then taken and analyzed: sample in Lane 1, FIG. 8. Isopropyl
.beta.-D-1-thiogalactopyranoside (Sigma-Aldrich) was then added to
a final concentration of 1 mM. Four hours post-induction cells were
harvested by centrifugation at 3,000 g and 4.degree. C. for 40 min.
A sample was then taken and analyzed: sample in Lane 2, FIG. 8.
Example I
Purification and Characterization of MS2 Capsids Encapsidating RNA
Coding For its Coat Protein Attached to its Specific 19-Mer RNA
Hairpin
[0117] Purification of MS2 capsids was conducted as follows.
Samples were taken during purification and SDS PAGE analysis was
run on the samples. Results obtained are shown in FIG. 8. A
fraction of the pellet from Example H equivalent to 115 mL of
culture was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM
MgCl2 and sonicated to lyse cells. Cell debris was removed by
centrifugation at 16,000 g. The cell lysate obtained was partially
purified by precipitation using ammonium sulfate and extraction
using trichlorofluoromethane (Freon 11) as described by Strauss
& Sinsheimer (1963) J. Mol. Biol. 7:43-54. To the partially
purified MS2 capsid solution (1.05 mL) 0.3 mg of Proteinase K
dissolved in 1.05 mL of 20 mM CaCl.sub.2 aqueous solution was
added. The mixture was incubated at 37.degree. C. and after 2.5
hours it was placed in an ice-water bath. A sample was then taken
and analyzed: sample in Lane 3, FIG. 8. Fifteen minutes afterwards,
0.14 mL of 1% phosphoric acid aqueous solution was slowly added
with vigorous agitation in an ice/water bath to bring the pH of the
liquid to 4.1. The liquid was kept at 0.degree. C. for 30 minutes
and centrifuged at 16,000 g at 4.degree. C. for 20 min. To the
supernatant, kept at 0.degree. C., 100 .mu.L of 1% NaOH was added
to bring the pH of the liquid to 7.9. 0.5 mL of ethanol at
0.degree. C. was then slowly added with vigorous agitation to bring
the ethanol concentration in the liquid to 20%. The liquid was kept
at 0.degree. C. for 30 minutes and centrifuged at 16,000 g at
4.degree. C. for 20 min. After adding 1% acetic acid to adjust the
pH of the solution to 7, the supernatant was filtered through a
Vivaspin 2 (Sartorius) 300 kDa membrane and the filtrate was
filtered through a 100 kDa membrane, from which 150 .mu.L of
retentate was obtained. The retentate was then diluted to 2 mL with
phosphate buffered saline and filtered through the same 100 kDa
membrane. Dilution and ultrafiltration of the retentate (150 .mu.L)
was repeated four more times. A sample of the retentate was then
taken and analyzed by SDS PAGE: sample in Lane 4, FIG. 8. MS2's
coat protein, of 14 kDa, retained by a membrane through which
proteins with less than 100 kDa molecular weight are able to
permeate, is clearly visible, consistent with the presence of
intact MS2 capsids. RNA was isolated from another sample of the
same retentate using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia,
Calif.) and a DNA-free kit (Life Technologies, Grand Island, N.Y.),
and reverse transcribed using a High Capacity cDNA Reverse
Transcription Kit (Life Technologies). The presence or absence of a
section of the MS2 capsid was then interrogated in PCR experiments.
The following pair of primers was used, each primer named for the
position of its first and last base in the MS2 genome, forward (F)
and reverse (R) respectively: F1401.sub.--1426-R1680.sub.--1705.
Platinum Taq DNA Polymerase High Fidelity (Life Technologies) was
used for amplification. The PCR product, chromatographed in 2%
agarose gel stained with Ethidium Bromide, as shown in FIG. 10 (304
by in Lane 1; the leftmost Lane corresponds to 1 kb plus ladder
from Life Technologies), was consistent with an intact MS2 coat
gene. In conclusion, the product obtained contained MS2 capsids
with purity higher than 99%.
Example J
Simple Precipitation with Ethanol for Purification of MS2
Virus-Like Particles (VLPs)
[0118] Purification of MS2 VLPs was conducted as follows. Samples
were taken during purification and SDS PAGE analysis was run on the
samples. Results obtained are shown in FIG. 11. One sixth of the
pellet obtained from an experiment identical to Example H was
resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mMMgCl.sub.2
and sonicated to lyse cells. Cell debris was removed by
centrifugation at 16,000 g. The cell lysate obtained was partially
purified by precipitation using ammonium sulfate and extraction
using trichlorofluoromethane (Freon 11) as described by Strauss
& Sinsheimer (1963) J. Mol. Biol. 7:43-54. A sample was taken
and analyzed: sample in Lane 1, FIG. 11. A strong band at about 14
kDa was found, consistent with the coat protein of MS2 phage. Other
bands--impurities--mostly of higher molecular weight, represent
about 27% of the sample weight. To the partially purified MS2 VLP
solution (1.35 mL) 1.36 mL of 20 mM CaCl2 aqueous solution was
added and placed in an ice-water bath. Fifteen minutes afterwards,
50 .mu.L of 10% acetic acid aqueous solution was added to bring the
pH of the liquid to 4.1. Then, at the same temperature and with
vigorous agitation, 1.44 mL of ethanol was slowly added. The liquid
was kept at 0.degree. C. for 30 minutes and centrifuged at 16,000 g
at 4.degree. C. for 20 min. The pellet was suspended in 2 mL of an
aqueous buffer consisting of 20 mM Tris-HCl and 10 mMMgCl.sub.2
adjusted to pH 7.5. A sample was taken and analyzed by SDS PAGE:
sample in Lane 2, FIG. 11. Impurities in this sample represented
about 24% of the sample weight. The diluted sample was filtered
through a Vivaspin 2 (Sartorius) 100 kDa membrane from which 200
.mu.L of retentate was obtained. The retentate was then diluted to
2 mL with the same buffer and filtered through the same 100 kDa
membrane. Dilution and ultrafiltration of the retentate (200 .mu.L)
was repeated four more times. A sample of the retentate was then
taken and analyzed by SDS PAGE: sample in Lane 3, FIG. 11.
Impurities in this sample represented about 9.7% of the sample
weight. In conclusion, the product obtained contained MS2 VLPs with
purity higher than 90%.
Example K
Use of Proteinase K (PK) and Simple Precipitation with Ethanol for
Purification of MS2 VLPs
[0119] Purification of MS2 VLPs was conducted as follows. Samples
were taken during purification and SDS PAGE analysis was run on the
samples. Results obtained are shown in FIG. 12. One sixth of the
pellet obtained from an experiment identical to Example H was
resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl.sub.2
and sonicated to lyse cells. Cell debris was removed by
centrifugation at 16,000 g. The cell lysate obtained was partially
purified by precipitation using ammonium sulfate and extraction
using trichlorofluoromethane (Freon 11) as described by Strauss
& Sinsheimer (1963) J. Mol. Biol. 7:43-54. A sample was taken
and analyzed: sample in Lane 1, FIG. 12. A strong band at about 14
kDa was found, consistent with the coat protein of MS2 phage. Other
bands--impurities--mostly of higher molecular weight represent
about 26% of the sample weight. To the partially purified MS2 VLP
solution (1.35 mL) 0.6 mg of Proteinase K dissolved in 1.36 mL of
20 mM CaCl.sub.2 aqueous solution was added. The mixture was
incubated at 37.degree. C. and after 2.5 hours placed in an
ice-water bath. A sample was taken and analyzed by SDS PAGE: sample
in Lane 2, FIG. 12. Impurities in this sample represented about 14%
of the sample weight. Fifteen minutes afterwards, about 50 .mu.L of
10% acetic acid aqueous solution was added in an ice/water bath to
bring the pH of the liquid to 4.1. Then, at the same temperature
and with vigorous agitation, 1.54 mL of ethanol was slowly added.
The liquid was kept at 0.degree. C. for 30 minutes and centrifuged
at 16,000 g at 4.degree. C. for 20 min. The pellet was suspended in
2 mL of an aqueous buffer consisting of 20 mM Tris-HCl and 10 mM
MgCl.sub.2 adjusted to pH 7.5. A sample was taken and analyzed by
SDS PAGE: sample in Lane 3, FIG. 12. Impurities in this sample
represented about 10% of the sample weight. The diluted sample was
filtered through a Vivaspin 2 (Sartorius) 100 kDa membrane from
which 200 .mu.L of retentate was obtained. The retentate was then
diluted to 2 mL with the same buffer and filtered through the same
100 kDa membrane. Dilution and ultrafiltration of the retentate
(200 .mu.L) was repeated four more times. A sample of the retentate
was then taken and analyzed by SDS PAGE: sample in Lane 4, FIG. 12.
Impurities in this sample represented about 5.1% of the sample
weight. In conclusion, the product obtained contained MS2 VLPs with
purity of about 95%.
Example L
Use of Constitutive Hydrolases (CH), Fractional Precipitation with
Ethanol, and Ultrafiltration for Purification of MS2 VLPs
[0120] Purification of MS2 VLPs was conducted as follows. Samples
were taken during purification and SDS PAGE analysis was run on the
samples. Results obtained are shown in FIG. 13. One sixth of the
pellet obtained from an experiment identical to Example H was
resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl.sub.2
and sonicated to lyse cells. Cell debris was removed by
centrifugation at 16,000 g. The cell lysate obtained was partially
purified by precipitation using ammonium sulfate and extraction
using trichlorofluoromethane (Freon 11) as described by Strauss
& Sinsheimer (1963) J. Mol. Biol. 7:43-54. To the partially
purified MS2 VLP solution (1.35 mL) 1.36 mL of 20 mM CaCl.sub.2
aqueous solution was added. The mixture was incubated at 37.degree.
C. during 2.5 hours (to allow constitutive hydrolases to act) and
afterwards was placed in an ice-water bath. A sample was taken and
analyzed by SDS PAGE: sample in Lane 1, FIG. 13. Impurities in this
sample represented about 12% of the sample weight. Fifteen minutes
afterwards, about 120 .mu.L of 1% sodium hydroxide aqueous solution
was added in an ice/water bath to bring the pH of the liquid to
7.86. Then, at the same temperature and with vigorous agitation,
0.81 mL of ethanol was slowly added. The liquid was kept at
0.degree. C. for 30 minutes and centrifuged at 16,000 g at
4.degree. C. for 20 min. About 100 .mu.L of 10% acetic acid aqueous
solution was slowly added to the supernatant with vigorous
agitation in an ice/water bath to bring the pH of the liquid to
4.01. Then, at the same temperature and with vigorous agitation,
1.3 mL of ethanol was slowly added. The liquid was kept at
0.degree. C. for 30 minutes and centrifuged at 16,000 g at
4.degree. C. for 20 min. The pellet was suspended in 2 mL of an
aqueous buffer consisting of 20 mM Tris-HCl and 10 mM MgCl.sub.2
adjusted to pH 7.5. The diluted sample was filtered through a
Vivaspin 2 (Sartorius) 100 kDa membrane from which 200 .mu.L of
retentate was obtained. The retentate was then diluted to 2 mL with
the same buffer and filtered through the same 100 kDa membrane.
Dilution and ultrafiltration of the retentate (200 .mu.L) was
repeated four more times. A sample of the retentate was then taken
and analyzed by SDS PAGE: sample in Lane 3, FIG. 13. Impurities in
this sample represented about 4.7% of the sample weight. In
conclusion, the product obtained contained MS2 VLPs with purity
higher than about 95%.
Example M
Use of Proteinase K (PK), Fractional Precipitation with Ethanol,
and Ultrafiltration for Purification of MS2 VLPs
[0121] Purification of MS2 VLPs was conducted as follows. Samples
were taken during purification and SDS PAGE analysis was run on the
samples. Results obtained are shown in FIG. 13. One sixth of the
pellet obtained from an experiment identical to Example H was
resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl.sub.2
and sonicated to lyse cells. Cell debris was removed by
centrifugation at 16,000 g. The cell lysate obtained was partially
purified by precipitation using ammonium sulfate and extraction
using trichlorofluoromethane (Freon 11) as described by Strauss
& Sinsheimer (1963) J. Mol. Biol. 7:43-54. To the partially
purified MS2 VLP solution (1.35 mL) 0.3 mg of Proteinase K
dissolved in 1.36 mL of 20 mM CaCl.sub.2 aqueous solution was
added. The mixture was incubated at 37.degree. C. during 2.5 hours
and afterwards was placed in an ice-water bath. A sample was taken
and analyzed by SDS PAGE: sample in Lane 2, FIG. 13. Impurities in
this sample represented about 8.1% of the sample weight. Fifteen
minutes afterwards, about 120 .mu.L of 1% sodium hydroxide aqueous
solution was added in an ice/water bath to bring the pH of the
liquid to 7.86. Then, at the same temperature and with vigorous
agitation, 0.81 mL of ethanol was slowly added. The liquid was kept
at 0.degree. C. for 30 minutes and centrifuged at 16,000 g at
4.degree. C. for 20 min. About 100 .mu.L of 10% acetic acid aqueous
solution was added to the supernatant in an ice/water bath to bring
the pH of the liquid to 4.01. Then, at the same temperature and
with vigorous agitation, 1.3 mL of ethanol was slowly added. The
liquid was kept at 0.degree. C. for 30 minutes and centrifuged at
16,000 g at 4.degree. C. for 20 min. The pellet was suspended in 2
mL of an aqueous buffer consisting of 20 mM Tris-HCl and 10 mM
MgCl.sub.2 adjusted to pH 7.5. The diluted sample was filtered
through a Vivaspin 2 (Sartorius) 100 kDa membrane from which 2004,
of retentate was obtained. The retentate was then diluted to 2 mL
with the same buffer and filtered through the same 100 kDa
membrane. Dilution and ultrafiltration of the retentate (200 .mu.L)
was repeated four more times. A sample of the retentate was then
taken and analyzed by SDS PAGE: sample in Lane 4, FIG. 13.
Impurities in this sample represented about 0.9% of the sample
weight. In conclusion, the product obtained contained MS2 VLPs with
purity higher than about 99%.
Example N
Use of Various Hydrolases, and Factional Precipitation with
Ammonium Sulfate for Purification of MS2 VLPs
[0122] Purification of MS2 VLPs was conducted as follows. Samples
were taken during purification and SDS PAGE analysis was run on the
samples. Results obtained are shown in FIG. 14. One sixth of the
pellet obtained from an experiment identical to Example H was
resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and
sonicated to lyse cells. Cell debris was removed by centrifugation
at 16,000 g. A sample of the supernatant was taken and analyzed by
SDS PAGE: sample in Lane 1, FIG. 14. Impurities in this sample
represented about 70% of the sample weight. Four other identical
fractions of the pellet obtained from such experiment identical to
Example H were processed in the same manner.
[0123] The five centrifuged cell lysates obtained, each 3.7 mL in
volume, were further processed in five different manners, as
follows. The first centrifuged cell lysate was placed in an
ice-water bath for 15 minutes and 0.1 grams of ammonium sulfate was
added. The mixture was vortexed until complete dissolution of
ammonium sulfate was achieved. The liquid was kept at 0.degree. C.
for 2 hours and centrifuged at 16,000 g at 4.degree. C. for 30 min.
0.4 grams of ammonium sulfate was added to the supernatant and
vortexed until complete dissolution of ammonium sulfate was
achieved. The liquid was kept at 0.degree. C. for 2 hours and
centrifuged at 16,000 g at 4.degree. C. for 30 min. The purified
MS2 VLPs pellet was suspended in 0.2 mL of an aqueous buffer
consisting of 20 mM Tris-HCl and 10 mM MgCl2 adjusted to pH 7.5.
The second centrifuged cell lysate was incubated at 37.degree. C.
for five hours, placed in an ice-water bath for the same amount of
time as the first centrifuged cell lysate and subsequently
processed in identical manner as the first centrifuged cell lysate.
0.15 mg of Proteinase K (Sigma Aldrich, St. Louis, Mo.) was added
to the third centrifuged cell lysate. It was incubated then at
37.degree. C. for five hours, placed in an ice-water bath for the
same amount of time as the first centrifuged cell lysate and
subsequently processed in identical manner as the first centrifuged
cell lysate. The fourth centrifuged cell lysate was incubated at
37.degree. C. for two hours. 0.15 mg of PK was then added. It was
incubated at 37.degree. C. for an additional three hours, placed in
an ice-water bath for the same amount of time as the first
centrifuged cell lysate and subsequently processed in identical
manner as the first centrifuged cell lysate.
[0124] 500 units of Benzonase.RTM. Nuclease (Sigma Aldrich, St.
Louis, Mo.) and 35 units of Lipase from Candida rugosa(Sigma
Aldrich, St. Louis, Mo.) was added to the fifth centrifuged cell
lysate and incubated at 37.degree. C. for one hour. 15 units of
.alpha.-Amylase from Bacillus sp. (Sigma Aldrich, St. Louis, Mo.)
was then added and incubated at 37.degree. C. for one additional
hour. 0.15 mg of PK was then added. The mixture was incubated at
37.degree. C. for an additional three hours, placed in an ice-water
bath for the same amount of time as the first centrifuged cell
lysate and subsequently processed in identical manner as the first
centrifuged cell lysate.
[0125] A sample was taken of the second centrifuged cell lysate
after its 5 hours incubation and analyzed by SDS PAGE: sample in
Lane 2, FIG. 14. A sample was taken of the third centrifuged cell
lysate after its 5 hours incubation and analyzed by SDS PAGE:
sample in Lane 3, FIG. 14. A sample was taken of the fourth
centrifuged cell lysate after its 5 hours incubation and analyzed
by SDS PAGE: sample in Lane 4, FIG. 14. A sample was taken of the
fifth centrifuged cell lysate after its 5 hours incubation and
analyzed by SDS PAGE: sample in Lane 5, FIG. 14.
[0126] A sample was taken of the purified MS2 VLPs suspension for
the first centrifuged cell lysate and analyzed by SDS PAGE: sample
in Lane 6, FIG. 14. The product obtained contained MS2 VLPs with
purity of about 88%. Protein concentration (Pierce.RTM. BCA Protein
Assay Kit, Thermo Fisher Scientific, Rockford, Ill.) of this sample
was 18.5 mg/mL. Optical density measured in a 1 cm cell at 260 nm
(OD-260 nm) of a 200:1 dilution of this sample was 0.553 and OD-280
nm was 0.303. These measurements are consistent with RNA yield of
about 9 mg per liter of culture.
[0127] A sample was taken of the purified MS2 VLPs suspension for
the second centrifuged cell lysate and analyzed by SDS PAGE: sample
in Lane 7, FIG. 14. The product obtained contained MS2 VLPs with
purity of about 75%. Protein concentration of this sample was 25.4
mg/mL. Optical density measured in a 1 cm cell at 260 nm (OD-260
nm) of a 200:1 dilution of this sample was 0.784 and OD-280 nm was
0.453. These measurements are consistent with RNA yield of about 11
mg per liter of culture.
[0128] A sample was taken of the purified MS2 VLPs suspension for
the third centrifuged cell lysate and analyzed by SDS PAGE: sample
in Lane 8, FIG. 14. The product obtained contained MS2 VLPs with
purity of about 94.3%. Protein concentration of this sample was
21.0 mg/mL. Optical density measured in a 1 cm cell at 260 nm
(OD-260 nm) of a 200:1 dilution of this sample was 0.632 and OD-280
nm was 0.321. These measurements are consistent with RNA yield of
about 10 mg per liter of culture.
[0129] A sample was taken of the purified MS2 VLPs suspension for
the fourth centrifuged cell lysate and analyzed by SDS PAGE: sample
in Lane 9, FIG. 14. The product obtained contained MS2 VLPs with
purity of about 95.6%. Protein concentration of this sample was
19.4 mg/mL. Optical density measured in a 1 cm cell at 260 nm
(OD-260 nm) of a 200:1 dilution of this sample was 0.666 and OD-280
nm was 0.353. These measurements are consistent with RNA yield of
about 11 mg per liter of culture.
[0130] A sample was taken of the purified MS2 VLPs suspension for
the fifth centrifuged cell lysate and analyzed by SDS PAGE: sample
in Lane 10, FIG. 14. The product obtained contained MS2 VLPs with
purity of about 96%. Protein concentration of this sample was 19.8
mg/mL. Optical density measured in a 1 cm cell at 260 nm (OD-260
nm) of a 200:1 dilution of this sample was 0.661 and OD-280 nm was
0.354. These measurements are consistent with RNA yield of about 11
mg per liter of culture.
Example O
Production of MS2 Capsids Encapsidating shRNA Targeting Green
Fluorescent Protein (GFP) and HDV Ribozyme Attached to MS2 19-Mer
RNA Hairpin
[0131] Production of MS2 capsids is conducted as follows. The
following DNA sequence Sequence A (SEQ ID NO: 7), encoding MS2's
coat protein is cloned into pDEST14 (Life Technologies)
plasmid:
TABLE-US-00004 (SEQ ID NO: 7)
ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTC
TAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGA
CTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCT
AACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTC
TGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAA
CCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC
TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGA
GCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTC
CCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG Sequence A
[0132] The following DNA sequence, Sequence B (SEQ ID NO: 8) was
cloned into plasmid pACYC184. A transcription terminator was also
cloned at the 3' end of Sequence B (SEQ ID NO: 8)(not shown).
Sequence B (SEQ ID NO: 8) encodes, shRNA hairpin, Hepatitis Delta
Virus (HDV) ribozyme designed to cleave the 3' end of the siRNA
hairpin, and MS2's specific RNA 19-mer, was cloned into plasmid
pACYC184:
TABLE-US-00005 Sequence T7-Rz6 (SEQ ID NO: 8)
GGATCCTAATACGACTCACTATAGGCAAGCTGACCCTGAAGTTCTCAAGA
GGAACTTCAGGGTCAGCTTGCCAAGGCCGGCATGGTCCCAGCCTCCTCGC
TGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACCACGCTTCAAACAT
GAGGATTACCCATGTCGAAGCGACCATGG.
[0133] One Shot BL21(DE3) Chemically Competent E. coli (Life
Technologies) cells were transformed with the 2 plasmids one
containing Sequence A (SEQ ID NO: 104) and Sequence B (SEQ ID NO:
105) selecting for chloramphenicol and ampicillin resistant
transformants. For capsid production these transformants were grown
at 37o in 32 mL LB medium containing both ampicillin and
chloramphenicol. When the culture density reached OD (600 nm)=0.8,
isopropyl .beta.-D-1-thiogalactopyranoside (Sigma-Aldrich) was then
added to a final concentration of 1 mM. Cells were harvested 4
hours post-induction by centrifugation at 3,000 g and 4.degree. C.
for 40 min. RNA was extracted from purified VLPs as described in
example Z, and analyzed as described in Example AA. A band of the
same molecular weight as expected for the encoded shRNA, as
observed in lane shRNA in FIG. 16, was observed.
Example P
Production of MS2 Capsids Using a Transcript Coding for siGFP
Flanked on its 5' End by a Long Hammerhead Ribozyme, and its 3' End
by an HDV Ribozyme Attached to MS2 19-Mer RNA Hairpin and a Second
HDV Ribozyme
[0134] Production of MS2 capsids is conducted as follows. The
following DNA sequence Sequence A (SEQ ID NO: 7), encoding MS2's
coat protein is cloned into pDEST14 (Life Technologies)
plasmid:
TABLE-US-00006 (SEQ ID NO: 7)
ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTC
TAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGA
CTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCT
AACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTC
TGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAA
CCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC
TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGA
GCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTC
CCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG Sequence A
[0135] The following DNA sequence, Sequence C (SEQ ID NO: 9) is
cloned into plasmid pACYC184. A transcription terminator is also
cloned at the 3' end of Sequence C (SEQ ID NO: 9)(not shown).
Sequence C (SEQ ID NO: 9) encodes T7 promoter, Hammerhead ribozyme
designed to cleave the 5' end of a siRNA hairpin, siRNA hairpin,
Hepatitis Delta Virus (HDV) ribozyme designed to cleave the 3' end
of the siRNA hairpin, and MS2's specific RNA 19-mer, and another
HDV ribozyme is cloned into plasmid pACYC184:
TABLE-US-00007 Sequence T7-Rz12 (SEQ ID NO: 9)
GGATCCTAATACGACTCACTATAGGGAGATAAATAAATAAATTTGAATGA
ACTTCAGGGTCAGCTTGCTGATGAGGCGCTTCGGCGCCGAAACACCCAGT
GGTGTCCAAGCTGACCCTGAAGTTCATTCAAGAGATGAACTTCAGGGTCA
GCTTGTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAA
CATTCGTGGCGAATGGGACCACGCTTCAAACATGAGGATTACCCATGTCG
AAGCGAATTTATTTATTTAATTATTATTATTATTATTGGCCGGCATGGTC
CCAGCCTCCTCGCTGGCGCCGGCTGGGCAACACCTTCGGGTGGCGAATGG
GACCAAAAAAAAATAATAATAATAATAATCCATGG
[0136] One Shot BL21(DE3) Chemically Competent E. coli (Life
Technologies) cells are transformed with the 2 plasmids one
containing Sequence A (SEQ ID NO: 104) and Sequence C (SEQ ID NO:
106) selecting for chloramphenicol and ampicillin resistant
transformants. For capsid production these transformants are grown
at 37o in 32 mL LB medium containing both ampicillin and
chloramphenicol. When the culture density reaches OD (600 nm)=0.8,
isopropyl .beta.-D-1-thiogalactopyranoside (Sigma-Aldrich) is then
added to a final concentration of 1 mM. Cells are harvested 4 hours
post-induction by centrifugation at 3,000 g and 4.degree. C. for 40
min.
Example Q
Production of MS2 Capsids Using Transcripts Coding for shRNA and
Slow HDV Ribozymes Attached to MS2 19-Mer RNA Hairpin
[0137] Several experiments producing MS2 capsids, each
encapsidating different cargoes are conducted as described in
Example O. However, instead of the HDV ribozyme sequence included
in Sequence B (SEQ ID NO: 8), a mutant cleaving the 3' end of the
siRNA with a slower reaction rate constant is used in each
experiment. The following sequences, containing slower HDV
ribozymes, are used instead of Sequence B, for each experiment:
TABLE-US-00008 Sequence G76U: (SEQ ID NO: 10) ##STR00001## Sequence
G40U: (SEQ ID NO: 11) ##STR00002## Sequence Al6G: (SEQ ID NO: 12)
TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAAC
TTCAGGGTCAGCTTGTCGGCCGGCATGGTCCCGGCCTCCTCGCTGGCGCC
GGCTGGGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGAT TACCCATGTCCATGG
Sequence G39U: (SEQ ID NO: 13)
TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAAC
TTCAGGGTCAGCTTGTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCC
GGCTGTGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGAT TACCCATGTCCATGG
Sequence A78G: (SEQ ID NO: 14)
TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAAC
TTCAGGGTCAGCTTGTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCC
GGCTGGGCAACATTCGTGGCGAGTGGGACCATATATATATACATGAGGAT TACCCATGTCCATGG
Sequence C21U: (SEQ ID NO: 15)
TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAAC
TTCAGGGTCAGCTTGTCGGCCGGCATGGTCCCAGCCTTCTCGCTGGCGCC
GGCTGGGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGAT TACCCATGTCCATGG
Sequence G25A: (SEQ ID NO: 16)
TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAAC
TTCAGGGTCAGCTTGTCGGCCGGCATGGTCCCAGCCTCCTCACTGGCGCC
GGCTGGGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGAT TACCCATGTCCATGG
Sequence A78U: (SEQ ID NO: 17)
TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAAC
TTCAGGGTCAGCTTGTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCC
GGCTGGGCAACATTCGTGGCGATTGGGACCATATATATATACATGAGGAT TACCCATGTCCATGG
Sequence G74C: (SEQ ID NO: 18)
TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAAC
TTCAGGGTCAGCTTGTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCC
GGCTGGGCAACATTCGTGCCGAATGGGACCATATATATATACATGAGGAT
TACCCATGTCCATGG
Example R
Production of MS2 Capsids Using a Transcript Coding for shGFP
Flanked on its 5' End by One Long Hammerhead Ribozyme, and its 3'
End by Another Long Hammerhead Ribozyme Attached to MS2 19-Mer RNA
Hairpin and an HDV Ribozyme
[0138] Production of MS2 capsids is conducted as follows. The
following DNA sequence, Sequence A (SEQ ID NO: 7), encoding MS2's
coat protein is cloned into pDEST14 (Life Technologies)
plasmid:
TABLE-US-00009 ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTC
TAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGA
CTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCT
AACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTC
TGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAA
CCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC
TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGA
GCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTC
CCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG
[0139] The following DNA sequence, Sequence D (SEQ ID NO: 19) is
cloned into plasmid pACYC184. A transcription terminator is also
cloned at the 3' end of Sequence D (SEQ ID NO: 19)(not shown).
Sequence D (SEQ ID NO: 19) encodes T7 promoter, Hammerhead ribozyme
designed to cleave the 5' end of a siRNA hairpin, siRNA hairpin,
Hammerhead ribozyme designed to cleave the 3' end of the siRNA
hairpin, MS2's specific RNA 19-mer and an HDV ribozyme:
TABLE-US-00010 Sequence T7-Rz15: (SEQ ID NO: 19)
GGATCCTAATACGACTCACTATAGGGAGACGTTCACGTTGAATGAACTTC
AGGGTCAGCTTGCTGATGAGGCGCTTCGGCGCCGAAACACCCAGTGGTGT
CCAAGCTGACCCTGAAGTTCATTCAAGAGATGAACTTCAGGGTCAGCTTG
TCACCGGATGTGCTCTCCGGTCTGATGAGTCCGTGAGGACGAAACAAGCT
GACCCTGAAGTTCATCCGTGAACGACGCTTCAAACATGAGGATTACCCAT
GTCGAAGCGAATATATATATATAGGCCGGCATGGTCCCAGCCTCCTCGCT
GGCGCCGGCTGGGCAACACCTTCGGGTGGCGAATGGGACCAAAAAAATAT
ATATATATACCATGG
One Shot BL21(DE3) Chemically Competent E. coli (Life Technologies)
cells are transformed with the 2 plasmids one containing Sequence A
(SEQ ID NO: 7) and the Sequence D (SEQ ID NO: 19) selecting for
chloramphenicol and ampicillin resistant transformants. For capsid
production these transformants are grown at 37.degree. in 750 mL LB
medium containing both ampicillin and chloramphenicol. When the
culture density reaches OD (600 nm)=0.8, isopropyl
.beta.-D-1-thiogalactopyranoside (Sigma-Aldrich) is then added to a
final concentration of 1 mM. Cells are harvested 4 hours
post-induction by centrifugation at 3,000 g and 4.degree. C. for 40
min. A sample is taken prior to induction and at the time of
harvest for analysis.
Example S
Production of MS2 Capsids Using Transcripts Coding for shRNA and
Long Hammerhead Ribozymes Attached to MS2 19-Mer RNA Hairpin
[0140] Several experiments producing MS2 capsids, each
encapsidating different cargoes, are conducted as described in
Example O. However, instead of the HDV ribozyme sequence included
in Sequence B (SEQ ID NO: 8), a long Hammerhead ribozyme sequence
hybridizing the 3' end of the siRNA, and designed to cleave it is
used in each experiment. The following sequences, containing long
Hammerhead ribozymes are used instead of Sequence B (SEQ ID NO: 8),
for each experiment:
TABLE-US-00011 Sequence 10MNT (doesn't cut well): (SEQ ID NO: 20)
##STR00003## Sequence 15MNT (doesn't cut well): (SEQ ID NO: 21)
##STR00004## Sequence 20MNT (cuts well): (SEQ ID NO: 22)
##STR00005## Sequence 22MNT (cuts well): (SEQ ID NO: 23)
##STR00006## Sequence 25MNT (cuts well): (SEQ ID NO: 24)
##STR00007## Sequence 27MNT (cuts well): (SEQ ID NO: 25)
##STR00008##
[0141] Experimental data set forth in the Examples supports the
conclusion that RNA strands which cut well according to the present
disclosure are those which include a Hammerhead ribozyme sequence
for which proper folding of the ribozyme sequence is
thermodynamically favored over the folding of the shRNA ("hairpin")
section of the whole strand. Thermodynamic parameters for each of
the above sequences were calculated to determine which sequences
cut well and which do not.
Example T
Production of MS2 Capsids Using a Transcript Coding for siRNA
Flanked by a Long Hammerhead Ribozyme at its 5' End, and
Trans-Acting HDV Ribozyme at its 3' End Attached to MS2 19-Mer RNA
Hairpin
[0142] Production of MS2 capsids is conducted as follows. The
following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's
coat protein is cloned into pDEST14 (Life Technologies)
plasmid:
TABLE-US-00012 (SEQ ID NO: 7)
ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTC
TAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGA
CTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCT
AACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTC
TGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAA
CCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC
TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGA
GCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTC
CCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG
[0143] The following DNA sequence, Sequence E (SEQ ID NO: 26) is
cloned into plasmid pACYC184. A transcription terminator was also
cloned at the 3' end of Sequence E (SEQ ID NO: 26)(not shown).
Sequence E (SEQ ID NO: 26) encodes BamHI restriction site, T7
promoter and start sequence, HDV ribozyme designed to cleave in
trans mode the 3' end of the siRNA against Enhanced Green
Fluorescent Protein (siEGPF), ATAT spacer, long Hammerhead ribozyme
designed to cleave the 5' end of the siEGFP hairpin, siEGFP
hairpin, complementary sequence to the HDV ribozyme cleaving in
trans mode the 3' end of the siEGFP hairpin, MS2's specific RNA
19-mer, and Pad restriction site.:
TABLE-US-00013 (SEQ ID NO: 26)
GGATCCTAATACGACTCACTATAGGGATCTCCCAGCCTCCTCGCTGGCGC
CGGCTGGGCAACATTCGTGGCGAATGGGGGATCATATCTTGATGAACTTC
AGGGTCAGCTTGCTGATGAGGCGCTTCGGCGCCGAAACACCGTGTCCAAG
CTGACCCTGAAGTTCATCAAGAATGAACTTCAGGGTCAGCTTGTCGGCCG
GCATGCATTCAAACATGAGGATTACCCATGTCGAAGTTAATTAA
[0144] BamHI and HindIII site are added at the 5' and 3' end
respectively to facilitate cloning into pACYC184 (not shown).
[0145] One Shot BL21(DE3) Chemically Competent E. coli (Life
Technologies) cells are transformed with the 2 plasmids one
containing Sequence A (SEQ ID NO: 104) and Sequence E (SEQ ID NO:
26) selecting for chloramphenicol and ampicillin resistant
transformants. For capsid production these transformants are grown
at 37.degree. in 750 mL LB medium containing both ampicillin and
chloramphenicol. When the culture density reaches OD (600 nm)=0.8,
isopropyl .beta.-D-1-thiogalactopyranoside (Sigma-Aldrich) is then
added to a final concentration of 1 mM. Cells are harvested 4 hours
post-induction by centrifugation at 3,000 g and 4.degree. C. for 40
min. A sample is taken prior to induction and at the time of
harvest for analysis.
Example U
Production of MS2 Capsids Using a Transcript Coding for 3 Different
siRNAs Targeting GFP Flanked by Long Hammerhead Ribozymes Attached
to MS2 19-Mer RNA Hairpin
[0146] Production of MS2 capsids is conducted as follows. The
following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's
coat protein is cloned into pDEST14 (Life Technologies)
plasmid:
TABLE-US-00014 (SEQ ID NO: 7)
ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTC
TAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGA
CTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCT
AACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTC
TGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAA
CCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC
TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGA
GCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTC
CCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG
[0147] The following DNA sequence, Sequence F (SEQ ID NO: 27) is
cloned into plasmid pACYC184. A transcription terminator was also
cloned at the 3' end of Sequence F (SEQ ID NO: 27)(not shown).
Sequence F (SEQ ID NO: 27) encodes T7 promoter and start sequence,
3 Hammerhead ribozymes designed to cleave the 5' ends of the siRNAs
against Enhanced Green Fluorescent Protein (siEGPF), 3 different
siEGFP hairpins, 3 long Hammerhead ribozymes designed to cleave the
3' ends of the siEGFP hairpins, MS2's specific RNA 19-mer:
TABLE-US-00015 (SEQ ID NO: 27)
GGATCCTAATACGACTCACTATAGGGAGACTTGATGAACTTCAGGGTCAG
CTTGCTGATGAGGCGCTTCGGCGCCGAAACACCCAGTGGTGTCCAAGCTG
ACCCTGAAGTTCATCAAGAATGAACTTCAGGGTCAGCTTGTCACCGGATG
TGCTCTCCGGTCTGATGAGTCCGTGAGGACGAAACAAGCTGACCCTGAAG
TTCATTATATCTTGGCAGATGAACTTCAGGGTCAGCTGATGAGACTCTTC
GGAGTCGAAACACCCAGTGGTGTCCTGACCCTGAAGTTCATCTGCCAAGA
GCAGATGAACTTCAGGGTCAGTCACCGGATGTGCTCTCCGGTCTGATGAG
TCCGTGAGGACGAAACTGACCCTGAAGTTCATCTGCTATATCTTGTGGTG
CAGATGAACTTCAGGGCTGATGAGGCTCTTCGGAGCCGAAACACCCAGTG
GTGTCCCCTGAAGTTCATCTGCACCACAAGATGGTGCAGATGAACTTCAG
GGTCACCGGATGTGCTCTCCGGTCTGATGAGTCCGTGAGGACGAAACCCT
GAAGTTCATCTGCACCATACGCCGGCCATTCAAACATGAGGATTACCCAT
GTCGAAGTTAATTAA
[0148] One Shot BL21(DE3) Chemically Competent E. coli (Life
Technologies) cells are transformed with the 2 plasmids one
containing Sequence A (SEQ ID NO: 7) and the other Sequence F (SEQ
ID NO: 27) selecting for chloramphenicol and ampicillin resistant
transformants. For capsid production these transformants are grown
at 37.degree. in 750 mL LB medium containing both ampicillin and
chloramphenicol. When the culture density reaches OD (600 nm)=0.8,
isopropyl .beta.-D-1-thiogalactopyranoside (Sigma-Aldrich) is then
added to a final concentration of 1 mM. Cells are harvested 4 hours
post-induction by centrifugation at 3,000 g and 4.degree. C. for 40
min. A sample is taken prior to induction and at the time of
harvest for analysis.
Example V
Production of MS2 Capsids Using a Transcript Coding for 3 Different
siRNAs Targeting GFP Flanked by Spacers Located at their 5' Ends
and HDV Ribozymes at their 3' Ends, Attached to MS2 19-Mer RNA
Hairpin
[0149] Production of MS2 capsids is conducted as follows. The
following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's
coat protein is cloned into pDEST14 (Life Technologies)
plasmid:
TABLE-US-00016 (SEQ ID NO 7)
ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTC
TAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGA
CTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCT
AACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTC
TGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAA
CCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC
TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGA
GCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTC
CCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG
[0150] The following DNA sequence, Sequence G (SEQ ID NO: 28) is
cloned into plasmid pACYC184. A transcription terminator was also
cloned at the 3' end of Sequence G (SEQ ID NO: 28) (not shown).
Sequence G (SEQ ID NO: 28) encodes BamHI restriction site, T7
promoter and start sequence, 3 spacers at the 5' ends of the siRNAs
against Enhanced Green Fluorescent Protein (siEGPF), 3 different
siEGFP hairpins, 3 HDV ribozymes designed to cleave the 3' ends of
the siEGFP hairpins, MS2's specific RNA 19-mer, and Pad restriction
site.:
TABLE-US-00017 (SEQ ID NO: 28)
GGATCCTAATACGACTCACTATAGGGAGAATATATATACAAGCTGACCCT
GAAGTTCATCAAGAATGAACTTCAGGGTCAGCTTGTCGGCCGGCATGGTC
CCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACC
AATTAATTACTGACCCTGAAGTTCATCTGCCAAGAGCAGATGAACTTCAG
GGTCAGTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCA
ACATTCGTGGCGAATGGGACCAATAATAATCCCTGAAGTTCATCTGCACC
ACAAGATGGTGCAGATGAACTTCAGGGTCGGCCGGCATGGTCCCAGCCTC
CTCGCTGGCGCCGGCTGGGCAACATTCGTGGCGAATGGGACCCATTCAAA
CATGAGGATTACCCATGTCGAAGTTAATTAA
[0151] One Shot BL21(DE3) Chemically Competent E. coli (Life
Technologies) cells are transformed with the 2 plasmids one
containing Sequence A (SEQ ID NO: 7) and Sequence G (SEQ ID NO: 28)
selecting for chloramphenicol and ampicillin resistant
transformants. For capsid production these transformants are grown
at 37.degree. in 750 mL LB medium containing both ampicillin and
chloramphenicol. When the culture density reaches OD (600 nm)=0.8,
isopropyl .beta.-D-1-thiogalactopyranoside (Sigma-Aldrich) is then
added to a final concentration of 1 mM. Cells are harvested 4 hours
post-induction by centrifugation at 3,000 g and 4.degree. C. for 40
min. A sample is taken prior to induction and at the time of
harvest for analysis.
Example W
Production of MS2 Capsids Using a Transcript Coding for the Two
Strands of An siRNA Targeting GFP Each Flanked by a Long Hammerhead
Ribozyme Located at their 3' Ends and HDV Ribozymes at their 5'
Ends, Attached to MS2 19-Mer RNA Hairpin
[0152] Production of MS2 capsids is conducted as follows. The
following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's
coat protein is cloned into pDEST14 (Life Technologies)
plasmid:
TABLE-US-00018 (SEQ ID NO: 7)
ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTC
TAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGA
CTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCT
AACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTC
TGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAA
CCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC
TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGA
GCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTC
CCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG
[0153] The following DNA sequence, Sequence H (SEQ ID NO: 29) was
cloned into plasmid pACYC184. A transcription terminator was also
cloned at the 3' end of sequence H (not shown).
TABLE-US-00019 Sequence T7-Rz8 (SEQ ID NO: 29)
GGATCCTAATACGACTCACTATAGGGAGAATGAACTTCAGGGTCAGCTTG
CTGATGAGGCGCTTCGGCGCCGAAACACCGTGTCCAAGCTGACCCTGAAG
TTCATGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACA
TTCGTGGCGAATGGGACCATTAGCCAAGCTGACCCTGAAGTTCATCTGAT
GAGACTCCGAATTCGGAGTCGAAACACGGTAACCGTGTCATGAACTTCAG
GGTCAGCTTGGCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTG
GGCAACATTCGTGGCGAATGGGACCCATTCAAACATGAGGATTACCCATG TCGAAGCCATGG
[0154] One Shot BL21(DE3) Chemically Competent E. coli (Life
Technologies) cells were transformed with the 2 plasmids one
containing Sequence A (SEQ ID NO: 104) and Sequence H (SEQ ID NO:
126) selecting for chloramphenicol and ampicillin resistant
transformants. For capsid production these transformants were grown
at 37o in 750 mL LB medium containing both ampicillin and
chloramphenicol. When the culture density reaches OD (600 nm)=0.8,
isopropyl .beta.-D-1-thiogalactopyranoside (Sigma-Aldrich) was then
added to a final concentration of 1 mM. Cells were harvested 4
hours post-induction by centrifugation at 3,000 g and 4.degree. C.
for 40 min.
Example X
Production of MS2 Capsids Using a Transcript Coding for 2 Different
siRNAs Targeting Green Fluorescent Protein (GFP) Flanked by Spacers
Located at their 3' Ends and 5' Ends, Attached to MS2 19-Mer RNA
Hairpin
[0155] Production of MS2 capsids is conducted as follows. The
following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's
coat protein was cloned into pDEST14 (Life Technologies)
plasmid:
TABLE-US-00020 (SEQ ID NO: 7)
ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTC
TAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGA
CTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCT
AACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTC
TGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAA
CCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC
TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGA
GCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTC
CCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG
[0156] The following DNA sequence, Sequence I (SEQ ID NO: 30) was
cloned into plasmid pACYC184. A transcription terminator was also
cloned at the 3' end of Sequence I (SEQ ID NO: 30)(not shown).
Sequence I (SEQ ID NO: 30) coded for T7 promoter and start
sequence, 3 spacers separating the ends of the siRNAs against Green
Fluorescent Protein (siGPF), 2 different siGFP hairpins, MS2's
specific RNA 19-mer:
TABLE-US-00021 (SEQ ID NO: 30)
GGATCCTAATACGACTCACTATAGGGAGAAATAATAATCAAGCTGACCCT
GAAGTTCATCAAGAATGAACTTCAGGGTCAGCTTGTCAATAATAATCCGC
TACCCCGACCACATGAACAAGATTCATGTGGTCGGGGTAGCGGTCAATAA
TAATACGCTTCAAACATGAGGATTACCCATGTCGAAGCGACCATGG
[0157] One Shot BL21(DE3) Chemically Competent E. coli (Life
Technologies) cells are transformed with the 2 plasmids one
containing Sequence A (SEQ ID NO: 7) and Sequence I (SEQ ID NO: 30)
selecting for chloramphenicol and ampicillin resistant
transformants. For capsid production these transformants are grown
at 37.degree. in 750 mL LB medium containing both ampicillin and
chloramphenicol. When the culture density reaches OD (600 nm)=0.8,
isopropyl .beta.-D-1-thiogalactopyranoside (Sigma-Aldrich) is then
added to a final concentration of 1 mM. Cells are harvested 4 hours
post-induction by centrifugation at 3,000 g and 4.degree. C. for 40
min. A sample is taken prior to induction and at the time of
harvest for analysis.
Example Y
Purification of MS2 VLPs Obtained in Examples O Through X
[0158] MS2 capsids obtained in Examples O through X are purified
using procedures outlined in Example N.
Example Z
Isolation of RNA Encapsidated in MS2 Capsids Obtained in Example
N
[0159] RNA encapsidated in MS2 capsids purified as described in
Example N was extracted from each experiment using TRIzol.RTM.
reagent according to the protocol supplied by the manufacturer
(Life Technologies, Grand Island, N.Y.). RNA obtained was denatured
by heating for 5 min at 95.degree. C. in formamide and analyzed by
electrophoresis in 17.6 cm.times.38 cm.times.0.04 cm (W, L, T) gels
composed of 8% polyacrylamide, 8 molar urea, 1.08% Tris base, 0.55%
Boric acid, and 0.093% EDTA. The running buffer had the same
concentrations of Tris base, Boric acid and EDTA as the gel. Power
was delivered at about 40 W. Gels were stained using a 0.025%
solution of Stains-All dye (Sigma-Aldrich, St. Louis, Mo.) in an
aqueous mixture containing 25% formamide, 19% isopropanol and 15 mM
Tris at pH 8. Results obtained are shown in FIG. 15. Lane numbers
for RNA electrophoresis in FIG. 15 refer to the same lane numbers
for protein electrophoresis in FIG. 14. A single RNA band can be
observed in each lane, consistent with high purity RNA recovered in
each case.
Example AA
HDV Ribozyme Produced shRNA During In-Vitro Transcriptions
[0160] Construct T7-Rz2 was used for in-vitro transcriptions. This
construct was cloned into pACYC184 plasmid (New England Biolabs).
One Shot BL21(DE3) Chemically Competent E. coli (Life Technologies)
cells were transformed using this plasmid. BL21(DE3) containing the
plasmid were grown in LB medium containing ampicillin at 37.degree.
C., to OD(600 nm) equal to 0.8. Plasmids were isolated using
QIAprep.RTM. Spin Miniprep Kit (Qiagen) following manufacturer's
instructions. NcoI (New England Biolabs) was used to cut isolated
plasmids at the restrictions sites introduced into this construct.
After digestion, templates were purified by electrophoresis on 1.5%
agarose gels and isolated using PureLink.TM. Quick Gel Extraction
Kit (Life Technologies) following manufacturer's instructions.
Reverse transcriptions were done using MAXIscript.RTM. T7 Kit
following manufacturer's instructions. RNA products were
electrophoresed in Novex.RTM. denaturing 15% polyacrylamide
TBE-Urea gels (Life Technologies) run at 70.degree. C. RNA bands
were visualized using ethidium bromide (Sigma-Aldrich). Gel imaging
was done using Image Lab 4.0.1 software (Bio-Rad).
[0161] Template T7-Rz2 encodes T7 promoter, shRNA hairpin, HDV
ribozyme designed to cleave the 3' end of the shRNA hairpin,
ATATATATAT spacer, and NcoI restriction site as follows:
TABLE-US-00022 (SEQ ID NO: 31)
TAATACGACTCACTATAGGCTTGTGATGCTTCAGCCAAATCAAGAGTTTG
GCTGAAGCATCACAAGCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCC
GGCTGGGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGAT
TACCCATGTCCATGG
[0162] The results show that the HDV ribozyme cut. The top band was
the uncut transcript and the two lower bands were the expected cut
pieces of RNA.
[0163] The gel in FIG. 16 shows a set of RNA markers in the
leftmost lane, a 49 nucleotide shRNA marker in the following lane,
in-vitro transcription and cutting of T7-Rz2 construct run for 1
hour at 37.degree. C. in the third lane and in-vitro transcription
and cutting of T7-Rz2 construct run for 1 hour at 37.degree. C. and
incubated one additional hour at 42.degree. C. in the fourth,
rightmost lane.
[0164] The slowest of the three most intense bands had a higher
intensity at 37 than at 37/42. In addition, the two faster bands
had higher intensity at 37/42 than at 37. The lowest molecular
weight band in the last lane run slightly slower than the 49 nt
shRNA standard, as expected for the resulting 51 nucleotide shRNA
in T7Rz2. In summary, the results as shown in FIG. 16 are
consistent with the hypothesis that HDV ribozyme cut in T7-Rz2
.
Example BB
Long Flanking Hammerhead Ribozymes Cut to a Significantly Higher
Extent During In-Vitro Transcriptions than Short Flanking
Hammerhead Ribozymes
[0165] Constructs T7-Rz1 and T7-Rz4 were used for in-vitro
transcriptions. T7-Rz1 comprises common ribozymes, i.e., with stems
hybridizing the siRNA being cut of less than 6 pairs of hybridizing
nucleotides. T7-Rz4 comprises flanking rybozymes with long-length
stems that hybridize the siRNA being cut, i.e. stems with more than
6 pairs of hybridizing nucleotides.
[0166] Construct T7-Rz1 encoded T7 promoter, Hammerhead (HH)
ribozyme designed to cleave the 5' end of a siRNA hairpin with 5
nucleotides complementary to siRNA, siRNA hairpin, HH ribozyme
designed to cleave the 3' end of the siRNA hairpin with 5
nucleotides complementary to siRNA, and NcoI restriction site:
TABLE-US-00023 (SEQ ID NO: 32)
TAATACGACTCACTATAGGCTCGAGCAAGCCTGATGAGGCGCTTCGGCGC
CGAAACACCGTGTCGCTTGTGATGCTTCAGCCAAATCAAGAGTTTGGCTG
AAGCATCACAAGCTCACCGGATGTGCTCTCCGGTCTGATGAGTCCGTGAG
GACGAAAGCTTGCCATGG
[0167] Construct T7-Rz4 encoded ansiRNA flanked by a HH ribozyme
designed to cut its 5' end having 12 nucleotides hybridizing to the
siRNA and a HH ribozyme designed to cut its 3' end having 23
nucleotides hybridizing to the siRNA:
TABLE-US-00024 (SEQ ID NO: 33)
TAATACGACTCACTATAGGGAGAACGCCGGCCATTCAAATAGTAAATAAT
AGAGGGTCAGCTTGCTGATGAGGCGCTTCGGCGCCGAAACACCGTGTCCA
AGCTGACCCTGAAGTTCATCAAGAGTGAACTTCAGGGTCAGCTTGTCACC
GGATGTGCTCTCCGGTCTGATGAGTCCGTGAGGACGAAACAAGCTGACCC
TGAAGTTCACTACGCCGGCCATTCAAACATGAGGATTACCCATGTCCATG G
In-vitro transcription experiments and analyses were run with these
constructs in a similar manner to those in Example Y. As shown in
FIG. 17, results obtained with construct T7-Rz1 were consistent
with a hypothesis that ribozymes cut to a very low extent. Results
obtained with construct T7-Rz4 shown in FIG. 17 were consistent
with a hypothesis that ribozymes cut to a significant extent.
Example CC
Production of MS2 Capsids Using a Transcript Coding for shRNA
Against EGFP Flanked by a Long Hammerhead Ribozyme at its 5' End
and Another Long Hammerhead Ribozyme Attached to MS2 19-Mer RNA
Hairpin at its 3 End
[0168] Production of MS2 capsids was conducted as follows. The
following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's
coat protein was cloned into pDEST14 (Life Technologies)
plasmid:
TABLE-US-00025 (SEQ ID NO 7)
ACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGAGATATACATATGGCTTC
TAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGA
CTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCT
AACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTC
TGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAA
CCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTAC
TTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGA
GCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTC
CCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAG
[0169] The following DNA sequence (construct T7-Rz4) was cloned
into plasmid pACYC184. A transcription terminator was also cloned
at the 3' end of construct T7-Rz4 (not shown).
TABLE-US-00026 (SEQ ID NO: 33)
TAATACGACTCACTATAGGGAGAACGCCGGCCATTCAAATAGTAAATAAT
AGAGGGTCAGCTTGCTGATGAGGCGCTTCGGCGCCGAAACACCGTGTCCA
AGCTGACCCTGAAGTTCATCAAGAGTGAACTTCAGGGTCAGCTTGTCACC
GGATGTGCTCTCCGGTCTGATGAGTCCGTGAGGACGAAACAAGCTGACCC
TGAAGTTCACTACGCCGGCCATTCAAACATGAGGATTACCCATGTCCATG G
[0170] One Shot BL21(DE3) Chemically Competent E. coli (Life
Technologies) cells were transformed with the 2 plasmids one
containing Sequence A (SEQ ID NO: 7) and construct T7-Rz4 (SEQ ID
NO: 33) selecting for chloramphenicol and ampicillin resistant
transformants. For capsid production these transformants were grown
at 37.degree. in 750 mL LB medium containing both ampicillin and
chloramphenicol. When the culture density reached OD (600 nm)=0.8,
isopropyl .beta.-D-1-thiogalactopyranoside (Sigma-Aldrich) was then
added to a final concentration of 1 mM. Cells were harvested 4
hours post-induction by centrifugation at 3,000 g and 4.degree. C.
for 40 min. A sample was taken prior to induction and at the time
of harvest for analysis.
Example DD
Purification of Virus Like Particles Obtained in Example CC
[0171] Purification of Virus Like Particles produced in Example CC.
was conducted as in Example N.
Example EE
Isolation of RNA in Virus Like Particles Obtained in Example DD
[0172] RNA encapsidated in Virus Like Particles purified as
described in Example DD were extracted using TRIzol.RTM. reagent
according to the protocol supplied by the manufacturer (Life
Technologies, Grand Island, N.Y.). RNA obtained was denatured by
heating for 5 min at 95.degree. C. in formamide and analyzed by
electrophoresis in Novex.RTM. denaturing 15% polyacrylamide
TBE-Urea gels (Life Technologies) run at 70.degree. C. RNA bands
were visualized using 0.5 .mu.g of Ethidium Bromide (Sigma-Aldrich,
St. Louis, Mo.) per mL of aqueous solution. Results obtained are
shown in lane 3, FIG. 18. Lane 1 shows a set of molecular
standards. Lane 2 shows a chemically synthesized shRNA 49
nucleotides long.
Example FF
Virus Like Particles Comprising MS2 Capsids Obtained in Example DD
are Resistant to Proteinase K from Engyodontium album,
licheniformis, Pepsin from Porcine Gastric Mucosa, and Papain from
Papaya Latex
[0173] Virus Like Particles comprising MS2 Capsids obtained from
250 mL of culture and purified as described in Example DD were
suspended in 400 .mu.L 20 mM CaCl.sub.2 aqueous solution at
pH=7.5.
[0174] A 66 .mu.L aliquote of this suspension was diluted to 0.25
mL with 20 mM CaCl.sub.2 aqueous solution at pH=7.5 and incubated
at 37.degree. C. Samples were taken for protein concentration
(Pierce.RTM. BCA Protein Assay Kit, Thermo Fisher Scientific,
Rockford, Ill.) and SDS PAGE analyses after 1 hour, and 4 hours of
incubation. Protein concentration in these 2 samples was 3086, and
4656 mg/L respectively. SDS PAGE analyses are shown in FIG. 19,
Lanes 1B, and 6 respectively. The same amount of protein was loaded
in each lane (4 .mu.g). This set of experiments was used as a
negative control. 2 .mu.g Protease from Streptomyces griseus (Sigma
Aldrich, St. Louis, Mo.) was diluted to 0.25 mL with 20 mM
CaCl.sub.2 aqueous solution at pH=7.5 and incubated at 37.degree.
C. Samples were taken for protein concentration and SDS PAGE
analyses after 1 hour, and 4 hours of incubation. Protein
concentration in these 2 samples was 361, and 324 mg/L
respectively. SDS PAGE analyses are shown in FIG. 19, Lanes 1, and
7 respectively. The same amount of protein was loaded in each lane
(4 .mu.g). This set of experiments was used as another negative
control.
[0175] 2 .mu.g of Protease from Streptomyces griseus was added to
another 66 .mu.L aliquote of the Virus Like Particles comprising
MS2 Capsids suspension, diluted to 0.25 mL with 20 mM CaCl.sub.2
aqueous solution at pH=7.5 and incubated at 37.degree. C. Samples
were taken for protein concentration and SDS PAGE analyses after 1
hour, and 4 hours of incubation. Protein concentration in these 2
samples was 2940, and 3012 mg/L respectively. SDS PAGE analyses are
shown in FIG. 19, Lanes 2, and 8 respectively. The same amount of
protein was loaded in each lane (4 .mu.g). This set of experiments
was used to test the proteolytic stability towards Protease from
Streptomyces griseus of MS2 capsids forming the Virus Like
Particles. Less than 10% degradation was observed.
[0176] Another 66 .mu.L aliquote of the Virus Like Particles
comprising MS2 Capsids suspension, diluted to 0.25 mL with 20 mM
CaCl.sub.2 aqueous solution at pH=7.5 was subjected to three cycles
of heating to 95.degree. C. for 10 minutes and cooling on wet ice
for 10 min to achieve the disassembly of the Virus Like Particles.
2 .mu.g of Protease from Streptomyces griseus was then added to
this suspension and was incubated at 37.degree. C. Samples were
taken for protein concentration and SDS PAGE analyses after 1 hour,
and 4 hours of incubation. Protein concentration in these 2 samples
was 2601, and 3033 mg/L respectively. SDS PAGE analyses are shown
in FIG. 19, Lanes 3, and 9 respectively. The same amount of protein
was loaded in each lane (4 .mu.g). Disassembled particles were
degraded to a significant extent by Protease from Streptomyces
griseus. This set of experiments was used as a positive
control.
[0177] 2 .mu.g of Protease from Streptomyces griseus dissolved in
0.002 mL of 20 mM CaCl.sub.2 aqueous solution at pH=7.5 was added
to 0.248 mL of bacterial cell lysate obtained from 41 mL of cell
culture from example CC and incubated at 37.degree. C. Samples were
taken for protein concentration and SDS PAGE analyses after 1 hour,
and 4 hours of incubation. Protein concentration in these 2 samples
was 3192, and 4837 mg/L respectively. SDS PAGE analyses are shown
in FIG. 19, Lanes 4, and 10 respectively. The last lane of FIG. 19,
labeled L, shows untreated bacterial cell lysate. The same amount
of protein was loaded in each lane (4 .mu.g). More than 90% of
proteins other than MS2 capsid protein were degraded by Protease
from Streptomyces griseus. This set of experiments was used as
another positive control.
[0178] This set of five experiments demonstrate that MS2 capsids
forming the Virus Like Particles of this disclosure are resistant
to proteolysis by Protease from Streptomyces griseus.
[0179] 2 .mu.g Protease from Bacillus licheniformis (Sigma Aldrich,
St. Louis, Mo.) was diluted to 0.25 mL with 10 mM Na acetate and 5
mM Ca acetate aqueous solution at pH=7.5 and incubated at
37.degree. C. Samples were taken for protein concentration and SDS
PAGE analyses after 1 hour, and 4 hours of incubation. Protein
concentration in these 2 samples was 976, and 1003 mg/L
respectively. SDS PAGE analyses are shown in FIG. 19, Lanes 2B, and
7B respectively.
[0180] The same amount of protein was loaded in each lane (4
.mu.g). This set of experiments was used as another negative
control.
[0181] 2 .mu.g of Protease from Bacillus licheniformis was added to
another 66 .mu.L aliquote of the Virus Like Particles comprising
MS2 Capsids suspension, diluted to 0.25 mL with 10 mM Na acetate
and 5 mM Ca acetate aqueous solution at pH=7.5 and incubated at
37.degree. C. Samples were taken for protein concentration and SDS
PAGE analyses after 1 hour, and 4 hours of incubation. Protein
concentration in these 2 samples was 3144, and 3727 mg/L
respectively. SDS PAGE analyses are shown in FIG. 19, Lanes 3B, and
8B respectively. The same amount of protein was loaded in each lane
(4 .mu.g). This set of experiments was used to test the proteolytic
stability towards Protease from Bacillus licheniformis of MS2
capsids forming the Virus Like Particles. Less than 10% degradation
was observed.
[0182] Another 66 .mu.L aliquote of the Virus Like Particles
comprising MS2 Capsids suspension, diluted to 0.25 mL with 10 mM Na
acetate and 5 mM Ca acetate aqueous solution at pH=7.5 was
subjected to three cycles of heating to 95.degree. C. for 10
minutes and cooling on wet ice for 10 min to achieve the
disassembly of the Virus Like Particles. 2 .mu.g of Protease from
Bacillus licheniformis was then added to this suspension and was
incubated at 37.degree. C. Samples were taken for protein
concentration and SDS PAGE analyses after 1 hour, and 4 hours of
incubation. Protein concentration in these 2 samples was 1769, and
1785 mg/L respectively. SDS PAGE analyses are shown in FIG. 19,
Lanes 4B, and 9B respectively. The same amount of protein was
loaded in each lane (4 .mu.g). Disassembled particles were degraded
by Protease from Bacillus licheniformis. This set of experiments
was used as a positive control.
[0183] 2 .mu.g of Protease from Bacillus licheniformis dissolved in
0.002 mL of 10 mM Na acetate and 5 mM Ca acetate aqueous solution
at pH=7.5 was added to 0.248 mL of bacterial cell lysate obtained
from 41 mL of cell culture from example CC and incubated at
37.degree. C. Samples were taken for protein concentration and SDS
PAGE analyses after 1 hour, and 4 hours of incubation. Protein
concentration in these 2 samples was 3696, and 4078 mg/L
respectively. SDS PAGE analyses are shown in FIG. 19, Lanes 6B, and
10B respectively. The last lane of FIG. 19, labeled L, shows
untreated bacterial cell lysate. The same amount of protein was
loaded in each lane (4 .mu.g). More than 90% of proteins other than
MS2 capsid protein were degraded by Protease from Bacillus
licheniformis. This set of experiments was used as another positive
control.
[0184] This set of four experiments demonstrated that MS2 capsids
forming the Virus Like Particles of this disclosure are resistant
to proteolysis by Protease from Bacillus licheniformis.
[0185] Three additional sets of equivalent experiments demonstrated
that MS2 capsids forming the Virus Like Particles of this
disclosure are resistant to proteolysis by any of the following
three proteases: Proteinase K from Engyodontium album, Pepsin from
porcine gastric mucosa (CAS Number 9001-75-6), and Papain from
papaya latex (CAS Number 9001-73-4) (Sigma-Aldrich, St. Louis,
Mo.). Each protease was used according the manufacturer's
instructions. Proteinase K was used at pH=7.5, Pepsin was used at
pH=1.6, and Papain was used at pH=6.6.
Example GG
Capsid Coat Protein Variants
[0186] The MS2 viral capsid protein (SEQ.ID No. 3) has a single
folding domain and belongs to fold family d.85.1 (RNA bacteriophage
capsid protein) of superfamily d.85, which includes leviviridae and
alloleviviridae capsid proteins. Each capsid monomer in this family
is made up of a 6-stranded beta sheet followed by the two helices
(sometimes described as a long helix with a kink) 180 monomers
assemble noncovalently to form an icosahedral (roughly spherical)
viral capsid with a continuous beta-sheet layer facing the capsid
interior and the alpha-helices on the capsid exterior. X-ray
crystal structures have been solved and placed in the public domain
for the enterobacteriophage MS2, GA (UniProt sequence identifier
P07234) and FR (UniProt sequence identifier P03614) viral capsids
and the capsid of MS2 formed from an MS2 dimer in which one
C-terminus of one MS2 has been fused to the N-terminus of another,
all d.85.1 family leviviridae coat proteins. The Protein Data Bank
identifiers for these structures are 1AQ3 (SEQ ID NO: 34), 1GAV
(SEQ ID NO: 35), 1FRS (SEQ ID NO: 36) and 2VTU (SEQ ID NO: 37),
respectively, and alignment of these is shown FIG. 20. In this an
all alignments described herein, the residue numbering is
sequential residue numbering, for example SEQ ID 3 starting with 0
for the lead Met (M) residue which is removed by the cell, as used
for most PDB structures.
The sequences of MS2 viral capsid protein vs the GA and FR viral
capsid proteins are 59% and 87% identical respectively. Only 56% of
the sequence positions have identical sequence and topologically
equivalent positions with respect to the backbone overlays when all
three sequences are considered together. The rms deviation of the
backbone conformations of MS2 viral capsid protein vs the GA and FR
viral capsid monomers are under 1 A. The backbone rms deviation of
1AQ3 monomer A vs 1GAV monomer 0 is 0.89 Angstroms. The backbone
rms deviation of 1AQ3 monomer A vs 1FRS monomer A is 0.37
Angstroms. Comparisons were made using the freeware utility jFATCAT
rigid (Prlic, et al, Bioinformatics 26, 2983-2985 (2010);
www.rcsb.org/pdb/workbench/workbench.do;
www.rcsb.org/pdb/workbench/workbench.do), a tool familiar to
practitioners of structure study protein available at the RCSB
Protein Data Bank site in their standard workbench of protein
structure tools. The overall fold of these proteins is identical.
There are no insertions or deletions. Each protein in the
crystallographic asymmetric unit is independently refined.
Different, compositionally identical proteins within an asymmetric
unit generally backbone rms deviations of 1 Angstrom or greater
although topologically equivalent Calpha atoms of the core tend to
differ by less, about 0.45 Anstroms (Cyrus Chothia & Arthur M
Lesk (1986) EMBO J. 5, 823-826). For example, 1AQ3 monomer A and
1AQ3 monomer B have rms deviation of 1.72 A (jFATCAT rigid)
primarily because of conformational differences in the Lys66-Trp82
region.
[0187] If sufficient members of a fold family have been identified,
a clear picture of conserved residues, topologically equivalent
residue positions within the sequences which seldom or never mutate
within the family, emerges. Nonconserved positions can be expected
to mutate from one sequence to another without disturbing the
family fold, perhaps in conjunction with the concerted mutation of
spatial neighbor(s) in the fold particularly if the sidechain packs
against the sidechain(s) of the spatial neighbors. Conserved
residues can be critical for fold stability, function or processing
of the protein, for example proteolytic digestion. Some can be
coincidentally conserved. GenBank (Dennis A. Benson, Ilene
Karsch-Mizrachi, David J. Lipman, James Ostell, and David L.
Wheeler (2005) Nucleic Acids Res 33, D34-D38) currently holds 353
leviviridae coat protein sequences. The alignment table shown in
FIG. 21 shows the multiple alignment of 40 complete leviviridae
coat protein sequences retrieved from the global protein sequence
database UniProt (Universal Protein Resource, (The UniProt
Consortium, Reorganizing the protein space at the Universal Protein
Resource (UniProt) Nucleic Acids Res. 40: D71-D75 (2012)),
http://www.uniprot.org) (See Table 1 below) and aligned with BLAST
(threshold=10, Auto weighting array selection, no filtering, gaps
allowed). All sequences except ef108465 were taken from UniProt.
ef108465 came from GenBank (www.ncbi.nlm.nih.gov/genbank). In the
alignment table, asterisk (*) indicates conserved residues, x is
calculated to be substitutable based on sidechain solvent
accessibility, hydrogen bonding requirements and backbone
conformational constraints. Fifty-seven (57) residues in the
sequences of these family members are conserved, or 45% of the
sequences are identical to on another. Some of these sequences have
an additional residue following the C-terminal Tyr129 residue of
SEQ ID NO: 3, others have 1-2 residues removed from the N-terminus
with respect to SEQ ID NO: 3. There are no insertions or deletions
within the fold.
TABLE-US-00027 TABLE 1 List of 40 complete leviviridae coat protein
sequences retrieved from the global protein sequence database
UniProt Accession Entry Name Organism Identifier G4WZU0
G4WZU0_BPMS2116 enterobacteria phage ms2 329852 D0U1D6
D0U1D6_BPMS2116 enterobacteria phage ms2 12022 C0M2U4
C0M2U4_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 C0M2S8
C0M2S8_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 C0M212
C0M212_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 C0M1M2
C0M1M2_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 C0M2L4
C0M2L4_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 C0M220
C0M220_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 Q2V0S8
Q2V0S8_BPB01116 enterobacteria phage bo1 12014 C0M216
C0M216_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 C0M1Y0
C0M1Y0_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 D0U1E4
D0U1E4_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 C0M309
C0M309_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 C0M325
C0M325_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 Q9T1C7
Q9T1C7_BPMS2116 enterobacteria phage ms12 110679 C0M2Z1
C0M2Z1_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 C0M1N8
C0M1N8_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 J9QBW2
J9QBW2_BPMS2116 enterobacteria phage ms2 12022 gene ms2g2 C8XPC9
C8XPC9_BPMS2113 enterobacteria phage ms2 329852 C0M2Y4
C0M2Y4_BPMS2115 enterobacteria phage ms2 12022 gene ms2g2 P69171
COAT_BPZR115 enterobacteria phage zr 332942 P69170 COAT_BPR17116
enterobacteria phage r17 12026 P03612 COAT_BPMS2 enterobacteria
phage ms2 329852 C0M1L4 C0M1L4_BPMS2116 enterobacteria phage ms2
12022 gene ms2g2 C8XPD7 C8XPD7_BPMS2116 enterobacteria phage ms2
329852 gene cp Q2VOT1 Q2V0T1_BPZR116 enterobacteria phage zr 332942
Q9MCD7 Q9MCD7_BPJP5115 enterobacteria phage jp501 12020 P03611
COAT_BPF2115 enterobacteria phage f2 12016 P34700 COAT_BPJP3115
enterobacteria phage jp34 12019 Q2V0U0 Q2V0U0_BPBZ1115
enterobacteria phage jp500 332939 Q2V0T7 Q2V0T7_BPBZ1115
enterobacteria phage sd 332940 Q9MBL2 Q9MBL2_BPKU1115
enterobacteria phage ku1 12021 P07234 COAT_BPGA115 enterobacteria
phage ga 12018 C8YJG7 C8YJG7_BPBZ1115 enterobacteria phage bz13
329853 C8YJH1 C8YJH1_BPBZ1115 enterobacteria phage bz13 329853
C8YJH5 C8YJH5_BPBZ1115 enterobacteria phage bz13 329853 Q2V0T4
Q2V0T4_BPTH1115 enterobacteria phage th13 12029 Q2V0U3
Q2V0U3_BPBZ1116 enterobacteria phage tl2 332938 P03614 COAT_BPFR116
enterobacteria phage fr 12017 ef108465 enterobacteria phage r17
329852 SEQ ID NO: 34 1AQ3 Enterobacteria phage MS2 coat protein
T59S ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYSI
KVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPS
AIAANSGIY SEQ ID NO: 35 1GAV Enterobacteria phage GA coat protein
A59T G79V
ATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIK
LEVPKIVTQVVNGVELPGSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIAEA
ISSQSGFYA SEQ ID NO: 36 1FRS Enterobacteria phage FR coat protei
>sp|P03614|COAT_BPFR Coat protein OS = Enterobacteria phage fr
PE = 1 SV = 4
ASNFEEFVLVDNGGTGDVKVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSANNRKYTV
KVEVPKVATQVQGGVELPVAAWRSYMNMELTIPVFATNDDCALIVKALQGTFKTGNPIAT
AIAANSGIY SEQ ID NO: 37 2VTU Enterobacteria phage MS2 coat protein
covalent dimer sp|P03612|COAT_BPMS2 Coat protein OS =
Enterobacteria phage MS2 PE = 1 SV = 2
ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTI
KVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPS
AIAANSGIYANFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSS
AQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLL
KDGNPIPSAIAANSGIY SEQ ID NO: 38 (SEQ ID NO: 38) G4WZU0
G4WZU0_BPMS2116 enterobacteria phage ms2 329852
>sp|P03612|COAT_BPMS2 Coat protein OS = Enterobacteria phage MS2
PE = 1 SV = 2
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYT
IKVEVPKVATQTVGGVELPVAAWRSYLNLELTIPIFATNSDCELIVKAMQGLLKDGNPIP
SAIAANSGIY SEQ ID NO: 39 D0U1D6 D0U1D6_BPMS2116 enterobacteria
phage ms2 12022 >tr|D0U1D6|D0U1D6 _BPMS2 Coat protein OS =
Enterobacterio phage MS2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNLELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 40 C0M2U4 C0M2U4 BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M2U4|C0M2U4_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVELPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 41 C0M2S8 C0M2S8_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M2S8|C0M2S8_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNVELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 42 C0M212 C0M212_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 tr|C0M212|C0M212_BPMS2 Coat protein OS = Enterobacterio phage
MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNPDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 43 C0M1M2 C0M1M2_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M1M2|C0M1M2_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVAVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 44 C0M2L4 C0M2L4_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M2L4|C0M2L4_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVXQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 45 C0M220 C0M220_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M220|C0M220_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTXFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 46 Q2V0S8 Q2V0S8_BPBO1116 enterobacteria phage bo1 12014
>tr|Q2V0S8|Q2V0S8_BPBO1 Coat protein OS = Enterobacteria phage
BO1 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGPLKDGNPIPSAIAANSGIY SEQ ID
NO: 47 C0M216 C0M216_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M216|C0M216_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNDGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 48 C0M1Y0 C0M1Y0_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M1Y0|C0M1Y0_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGXVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 49 D0U1E4 D0U1E4_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|D0U1E4|D0U1E4_BPMS2 Coat protein OS = Enterobacterio
phage MS2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNLELTIPIFATNPDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 50 C0M309 C0M309_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M309|C0M309_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPISSAIAANSGIY SEQ ID
NO: 51 C0M325 C0M325_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M325|C0M325_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNVELTIPIFATNSDCEXIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 52 Q9T1C7 Q9T1C7_BPMS2116 enterobacteria phage ms12 110679
>tr|Q9T1C7|Q9T1C7_BPMS2 Coat protein OS = Enterobacteria phage
M12 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVXPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCALIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 53 C0M2Z1 C0M2Z1_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M2Z1|C0M2Z1_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNVELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIX SEQ ID
NO: 54 C0M1N8 C0M1N8_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M2Z1|C0M2Z1_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNVELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIX SEQ ID
NO: 55 J9QBW2 J9QBW2_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|J9QBW2|J9QBW2_BPMS2 Capsid protein OS = Enterobacterio
phage MS2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVQLPVAAWRSYLNMELTIPIFATNDDCALIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 56 C8XPC9 C8XPC9_BPMS2113 enterobacteria phage ms2 329852
>tr|C8XPC9|C8XPC9_BPMS2 Coat protein OS = Enterobacteria phage
MS2 PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVQLPVAAWRSYLNMELTIPIFATNDDCALIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 57 C0M2Y4 C0M2Y4_BPMS2115 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M2Y4|C0M2Y4_BPMS2 Coat protein (Fragment) OS =
Enterobacterio phage MS2 GN = MS2g2 PE = 4 SV = 1
NFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTV
GGVELPVAAWRSYLNVELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 58 P69171 COAT_BPZR115 enterobacteria phage zr 332942
>sp|P69171|COAT_BPZR Coat protein OS = Enterobacteria phage ZR
PE = 1 SV = 1
ASNFTQFVLVNDGGTGNVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQ
TVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 59 P69170 COAT_BPR17116 enterobacteria phage r17 12026
>sp|P69170|COAT_BPR17 Coat protein OS = Enterobacteria phage R17
PE = 1 SV = 1
ASNFTQFVLVNDGGTGNVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQ
TVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 60 P03612 COAT_BPMS2 enterobacteria phage ms2 329852
>sp|P03612|COAT_BPMS2 Coat protein OS = Enterobacteria phage MS2
PE = 1 SV = 2
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 61 C0M1L4 C0M1L4_BPMS2116 enterobacteria phage ms2 12022 gene
ms2g2 >tr|C0M1L4|C0M1L4_BPMS2 Coat protein OS = Enterobacterio
phage MS2 GN = MS2g2 PE = 2 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 62 C8XPD7 C8XPD7_BPMS2116 enterobacteria phage ms2 329852 gene
cp >tr|C8XPD7|C8XPD7_BPMS2 Coat protein OS = Enterobacteria
phage MS2 GN = cp PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 63 Q2V0T1 Q2V0T1_BPZR116 enterobacteria phage zr 332942
>tr|Q2V0T1|Q2V0T1_BPZR Coat protein OS = Enterobacteria phage ZR
PE = 4 SV = 1
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 64 Q9MCD7 Q9MCD7_BPJP5115 enterobacteria phage jp501 12020
>tr|Q9MCD7|Q9MCD7_BPJP5 Coat protein OS = Enterobacteria phage
JP501 PE = 4 SV = 1
MASNFTEFVLVDNGETGNVTVAPSNFANGVAEWISSDSRSQAYKVTCSVRQSSAQNRKYTIKVAVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCALIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 65 P03611 COAT_BPF2115 enterobacteria phage f2 12016
>sp|P03611|COAT_BPF2 Coat protein OS = Enterobacteria phage f2
PE = 1 SV = 1
ASNFTQFVLVNDGGTGNVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQ
TVGGVELPVAAWRSYLNLELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 66 P34700 COAT_BPJP3115 enterobacteria phage jp34 12019
>sp|P34700|COAT_BPJP3 Coat protein OS = Enterobacteria phage
JP34 PE = 3 SV = 2
MATLRSFVLVDNGGTGDVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVTQ
VVNGVELPVSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIADAISSQSGFYA SEQ ID
NO: 67 Q2V0U0 Q2V0U0_BPBZ1115 enterobacteria phage jp500 332939
>tr|Q2V0U0|Q2V0U0_BPBZ1 Coat protein OS = Enterobacteria phage
JP500 PE = 4 SV = 1
MATLRSFVLVDNGGTGDVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVTQ
VVNGVELPVSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIADAISSQSGFYA SEQ ID
NO: 68 Q2V0T7 Q2V0T7_BPBZ1115 enterobacteria phage sd 332940
>tr|Q2V0T7|Q2V0T7_BPBZ1 Coat protein OS = Enterobacteria phage
SD PE = 4 SV = 1
MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVTQ
VVNGVELPISAWKAYASIDLTIPIFAATDDVTTISKSLAGLFKVGNPIADAISSQSGFYA SEQ ID
NO: 69 Q9MBL2 Q9MBL2_BPKU1115 enterobacteria phage ku1 12021
>tr|Q9MBL2|Q9MBL2_BPKU1 Coat protein OS = Enterobacteria phage
KU1 PE = 4 SV = 1
MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVTQ
SVNGVELPVSAWKAFASIDLTIPIFAATDDVTLISKSLAGLFKIGNPVADAISSQSGFYA SEQ ID
NO: 70 P07234 COAT_BPGA115 enterobacteria phage ga 12018
>sp|P07234|COAT_BPGA Coat protein OS = Enterobacteria phage GA
PE = 1 SV = 3
MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYAIKLEVPKIVTQ
VVNGVELPGSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIAEAISSQSGFYA SEQ ID
NO: 71 C8YJG7 C8YJG7_BPBZ1115 enterobacteria phage bz13 329853
>tr|C8YJG7|C8YJG7_BPBZ1 Capsid protein OS = Enterobacteria phage
BZ13 PE = 4 SV = 1
MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVTQ
VVNGVELPVSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIAEAISSQSGFYA SEQ ID
NO: 72 C8YJH1 C8YJH1_BPBZ1115 enterobacteria phage bz13 329853
>tr|C8YJH1|C8YJH1_BPBZ1 Capsid protein OS = Enterobacteria phage
BZ13 PE = 4 SV = 1
MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVTQ
TVNGVELPVSAWKAYASIDLTIPIFAATDDVTLISKSLAGLFKIGNPVADAISSQSGFYA SEQ ID
NO: 73 C8YJH5 C8YJH5_BPBZ1115 enterobacteria phage bz13 329853
>tr|C8YJH5|C8YJH5_BPBZ1 Capsid protein OS = Enterobacteria phage
BZ13 PE = 4 SV = 1
MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVTQ
VVNGVELPVSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGDPIADAISSQSGFYA SEQ ID
NO: 74 Q2V0T4 Q2V0T4_BPTH1115 enterobacteria phage th1 12029
>tr|Q2VOT4|Q2VOT4_BPTH1 Coat protein OS = Enterobacteria phage
TH1 PE = 4 SV = 1
MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVTQ
VVNGVELPVSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIADAISSQSGFYA SEQ ID
NO: 75 Q2V0U3 Q2V0U3_BPBZ1116 enterobacteria phage tl2 332938
>tr|Q2V0U3|Q2V0U3_BPBZ1 Coat protein OS = Enterobacteria phage
TL2 PE = 4 SV = 1
MATLRSFVLVDNGGTGNVTVVPVSNANGVAEWLSNNSRSQAYRVTASYRASGADKRKYTIKLEVPKIVTQ
VVNGVELPVSAWKAYASIDLTIPIFAATDDVTVISKSLAGLFKVGNPIADAISSQSGFYA SEQ ID
NO: 76 P03614 COAT_BPFR116 enterobacteria phage fr 12017
>sp|P03614|COAT_BPFR Coat protein OS = Enterobacteria phage fr
PE = 1 SV = 4
MASNFEEFVLVDNGGTGDVKVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSANNRKYTVKVEVPKVAT
QVQGGVELPVAAWRSYMNMELTIPVFATNDDCALIVKALQGTFKTGNPIATAIAANSGIY SEQ ID
NO: 77 ef108465 enterobacteria phage r17 329852
gi|132424616|gb|AB033465.1| coat protein [Enterobacteria phage MS2]
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVAT
QTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SEQ ID
NO: 78 1QBE >sp|P03615|COAT_BPQBE Coat protein OS =
Enterobacteria phage Qbeta PE = 1 SV = 2
AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKN
YKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLI
DAIDQLNPAY
Further, amino acid residues are distinguished by the identity of
their sidechains. They share a common backbone and a common set of
allowed backbone conformations (Kleywegt & Jones, Structure 4
1395-1400 (1996)), with two exceptions. Glycines can stably fold
into backbone conformations disallowed to other amino acids because
its sidechain consists of a single hydrogen atom. The proline
sidechain is cyclized into a stiff ring which is covalently bound
to its backbone nitrogen through elimination of its amide hydrogen,
constraining proline to a small subset of backbone conformations
with respect to the other amino acids and eliminating its ability
to be a hydrogen bond donor.
[0188] The domain fold and domain association for assembly into
capsids (for example of the amino sequence of SEQ ID NO: 3 is
stabilized by the backbone hydrogen bonding patterns that define
its secondary structural units, hydrogen bonds between sidechain
and backbone atoms that stabilize local structure or bind
neighboring secondary structure units (e.g. helices, strands, coil,
loops, turns and flexible termini) together, hydrogen bonds between
the atoms of different sidechains that stabilize local structure or
bind neighboring secondary structure units (e.g. helices, strands,
coil, loops, turns and flexible termini) together and the close
packing of hydrophobic sidechain atoms that serves to both
energetically stabilize the fold through van der Waals interactions
and to prevent solvent penetration into the fold which might lead
to destabilization and local unfolding. The sidechains of the
remaining residues do not participate in domain fold maintenance or
in domain-domain interactions. So long as their backbone
conformations do not have special requirements satisfied only by
Gly or cis-Pro in order to participate in the domain fold, these
residues can be mutated, singly or as a group, without
substantially affecting the final domain fold or the overall
topology of its surface, and can be identified as a class
unequivocally by surface accessibility calculations performed on
known structures (See, e.g., Fraczkiewicz & Braun, J M B; Meth
Enzym; J Comp Chem 19, 319 (1998)), followed by hydrogen bond
analysis of known structures, all conventional techniques in the
study of protein structure and function.
[0189] Using two MS2 capsid structures from the Protein Data Bank
for examination, 1AQ3 of an isosahedral capsid containing RNA &
2VTU of a stable octahedral capsid formed by 2 MS2 capsid protein
monomers fused C-terminus to N-terminus to form a single chain
protein 2 domain protein, 17 residues (Ala1, Ser2, Thr5, Gln6,
Ala21, Ala53, Val67, Thr69, Thr71, Val72, Val75, Ser99, Glu102,
Lys113, Asp114, Gly 115, Tyr129) were identified which have highly
solvated sidechain positions (Fraczkiewicz & Braun server
http://curie.utmb.edu/getarea with 1.4 A solvent probe, no
gradient, 2 area/energy per residue); do not participate in
hydrogen bonds with other parts of the capsid (hydrogen bonds
calculated in the widely used freeware software visualization
package Chimera (ERIC F. PETTERSEN, THOMAS D. GODDARD, CONRAD C.
HUANG, GREGORY S. COUCH, DANIEL M. GREENBLATT, ELAINE C. MENG,
THOMAS E. FERRIN (2004 J Comp Chem 25, 1605-1612) with hydrogen
bond criteria relaxed by 0.5 A and 30 deg); and which backbone
conformations allowed by all amino acid residues except proline.
When the subset of these 17 residues is compared to the structural
alignment of the enterobacteria phage MS2, wherein GA and FR capsid
sequences and residues which have mutated in the enterobacteria
phage GA or FR capsid sequences are disregarded, leaving 6
positions remaining which are putatively susceptible to mutation
without effecting the structure or function of the monomers or
their ability to assemble into stable capsids. This represents 52%
sequence identity to wild-type enterobacteria phage MS2 capsid
protein (SEQ ID NO:3).
[0190] The insertion and/or deletion of residues within secondary
structure elements (helices, strands, turns with defining hydrogen
bonding patterns and structured loops, e.g. omega loops) cause
those elements to lose their defining hydrogen bonding or
hydrophobic packing patterns or force a change in their hydrogen
bonding or hydrophobic packing patterns which can alter stability,
shape and/or function from the original protein sequence. This can
disrupt packing and effect the global stability of a fold. On the
other hand, unstructured loops, random coils and N- & C-termini
which have surface exposure but do not provide critical
stabilization to the rest of the protein fold (frequently via the
packing of sidechains against structured elements or the shielding
of interacting faces of adjacent structured elements from solvent
or in the case of capsids, cargo) are excellent candidates for (1)
residue deletion if significant repositioning of the joined
structured elements is not required, (2) insertion of amino acid
residues if the addition of residues will not significantly alter
the relative disposition of structured elements in the fold or
screen surface exposed residues from satisfying their hydrogen
bonding capacity with hydrogen bond donors or acceptors in the
protein's environment or (3) the incorporation of
naturally-occurring amino acid mutation(s) or mutation(s) to
normative residues which can be covalently linked to useful
moieties, e.g. fluorophores, phosphorescent groups, polyethylene
glycols, affinity tags, reporter groups, etc. Of course, such
insertions, deletions and mutations can occur within a single
suitable element concurrently or in any combination and their
incorporation may give rise to a protein with improved
characteristics. A straightforward way to distinguish optimal spots
for insertion and/or deletions is to scan the multiple alignments
of closely related sequences for insertions and/or deletions. Aside
from N- and C-terminal additions and deletions, the known
leviviridae coat protein sequences do not have insertion or
deletions with respect to each other. This does not mean insertion
and/or deletions cannot occur. One simply must examine more distant
members of the structure/function or fold family.
[0191] The simplest multiple alignment algorithms are usually
available to the general public at the public domain sequence and
structure data bases. These algorithms can correctly align
sequences that share a very low % identity if the sequence space is
populated by a continuous spectrum of sequences from a high %
identity, for example 90%, to a low % identity, for example 20%.
These algorithms tend to fail to correctly align clusters of
sequences with the same fold when those cluster share a low %
identity; however, such clusters can be successfully and
unequivocally aligned if the x-ray crystal structure of one or more
members of each cluster has been solved and well refined. By
optimally superimposing backbone atoms of the secondary structure
elements of the structures of proteins closely related by fold but
distantly related by sequence, a one-to-one correspondence between
their sequences is clearly defined and the high % identity clusters
successfully generated by straightforward sequence alignment
protocols can be anchored to the pairwise alignment resulting from
the backbone superposition and a correct global sequence alignment
for the fold family generated resulting in a topologically
meaningful alignment of the fold family members (Arthur M Lesk,
Michael Levitt, Cyrus Chothia (1986), Prot Eng 1, 77-78). By
examining the global sequence alignment, a comprehensive picture of
where the fold will tolerate insertion and/or deletion without
compromising its form or function can be viewed.
[0192] The alloleviviridae coat proteins belong to the same fold
family as the leviviridae coat proteins (fold family d.85.1) and
also assemble into isosahedral capsids comprised of 180 monomers.
The multiple alignments of the sequences of alloleviviridae coat
proteins deposited in UniProt are shown in the alignment table in
FIG. 27. Sixty percent (60%) of the alloleviviridae coat protein
sequence is conserved. The coat proteins of levi- and
alloleviviridae are both about 130 amino acid residues long but
because the percent of identical residues is low, about 20%,
multiple sequence alignment algorithms typically fail to correctly
alignment the allolevi- against the leviviridae sequences. A simple
way to recognize this is to reverse the sequences and then use the
same protocol to align the reversed sequences. The multiple
alignments of the sequences and reversed sequences will not agree.
This difficulty can be circumvented by examining representative
structures. An x-ray crystal structure of a capsid of allolevividae
Qbeta (PDB-ID:1QBE) (SEQ ID NO: 78. see below) has been deposited
in the public domain database, RCSB Protein Data Bank
(http://www.rcsb.org). The independently refined monomers of 1QBE
were fit to the independently refined monomers of 1AQ3 by
minimizing the rms deviation between {Calpha} atoms using the
jFATCAT comparison tool at the RCSB Protein Data Bank. The rms
deviation is in the range 2.33-2.76 Angstroms depending upon which
of the independently refined monomers is compared, primarily due to
differences in the backbone disposition of N-terminal residues 1-3
and segments 8-18, 26-28, 50-55 and 67-76 (numbering references the
topologically equivalent residues in the MS2 structure 1AQ3) which
connect secondary structure elements, as shown in FIGS. 22-25 and
described in the accompanying figure descriptions. The backbone rms
deviation measured by jFATCAT for independently refined monomers in
1AQ3 is 1.72 A due to conformational differences in the same
regions. The topological alignment is shown in the table, secondary
structure assignment by hydrogen bonding pattern (DSSP, W Wolfgang
Kabsch & Christian Sander (1983), Biopolymers 22, 2577-2636) is
indicated for 1AQ3 and segments that show the greatest deviation
either because the refined backbone conformations are substantially
different are because the segments were too mobile to be localized
in electron density during refinement are provided in lower case.
Regions which show backbone flexibility in the crystal environment
are also excellent candidates for insertion/and or deletion as if
the interactions between these residues and the rest of the fold
was important for fold stabilization, their electron density would
be localized. Appending the same information for 2VTU provides
further insight into segments best adapted to accommodate change.
These comparisons are captured symbolically in FIG. 26 which shows
alignment of 1AQ3 vs 2VTU vs 1QBE.
[0193] Examination of the 1AQ3 and 1QBE monomers provides the
following insights, as further illustrated by reference to FIGS.
28-31 and their respective descriptions. All residue numbers are
given with respect to the monomers in 1AQ3.
[0194] This also means that the fold of SEQ ID #3 Enterobacteria
phage MS2 coat protein is preserved down to 21% identity vs the
sequence of 1 QBE Enterobacteria phage coat protein Qbeta and 16%
identity with respect to the conserved residues for all of the
alloleviviridae coat protein sequences referenced here. Only one of
the highly solvated sidechain positions calculated earlier,
sidechains which do not participate in hydrogen bonds with other
parts of the capsid and whose backbone conformations are allowed by
all amino acid residues except proline, remains conserved, Y129 (in
SEQ NO 3 numbering). Its backbone position and sidechain packing is
substantially changed in the octahedral Enterobacteria phage MS2
capsid structure formed by the fused MS2 dimer (2VTU). Taking this
last change into account brings the threshold amino acid sequence
percent identity to 15%. See the alignment tables in FIG. 26 and
FIG. 27 (1AQ3 vs 2VTU vs 1QBE, & allolevi multiple sequence
alignment tables for clarification). All percent similarities in
this paragraph are valid only in the context of structure anchored
alignments.
[0195] N-terminal residues 1-3 can satisfy their hydrogen bonding
potential with the C-terminal residue 129 and water and vice versa;
therefore, it should be possible to delete some or all of these
residues and form stable VLPs with the truncated proteins. FIG. 32
shows backbone ribbon diagrams of 3 noncovalent Enterobacteria
phage MS2 noncovalent dimers packed around a symmetry point in the
assembled capsid. Three noncovalent MS2 coat protein dimers are
packed around a symmetry point in the assembled isosahedral capsid
(dimer one right, tan & brown chains; dimer two bottom, dark
and medium blue chains; dimer three upper right, medium gray and
dark blue chains). All chain N-termini are colored green, all
C-termini are colored red. The proximity of the termini mean that
that the sequences of the monomers can be fused into a single chain
to form a covalent dimer, either as done for 2VTU by appending one
monomer after the other, i.e., creating a single protein chain that
consists of (monomer residues 1-129-monomer residues 1-129) or by
adding additional linking residues between the monomer sequences
(monomer 1-129-linker residues-monomer 1-129) as long as the
relative chain directions (from N- to C-terminus) allow a
continuous peptide chain to be formed from the concatenated
monomers. A monomer-monomer concatenation without the addition of
linker residues was solved (PDB-ID:2VTU). In 2VTU each noncovalent
dimer has been engineered into a single protein; however, since the
Calpha's of residues 2 & 129 are around 6 Angstroms apart,
barely close enough to join with a linking segment without
disturbing the fold (The Calpha-Calpha distance is constrained to
about 3.8 Angstroms because of the resonance forms of the peptide
unit) and in some monomers their backbones hydrogen bond with each
other. The beta-sheet side of each dimer (covalent or noncovalent)
forms the interior wall of the capsid. The geometry of a beta sheet
can be defined by the curvature of the sheet (Cyrus Chothia, Jiri
Novotny, Robert Bruccoleri, Martin Karplus (1985) J Mol Biol 186,
651-663). The tight coupling in 2VTU constrains the beta sheet to a
lower curvature giving rise to an octahedral rather than an
isosahedral capsid. The incorporation of a linker between monomers
of 0-6 residues would provide enough flexibility to allow the
covalent dimer to relax into the same required for an isosahedral
capsid, with physical properties likely to be more closely related
to the isosahedral noncovalent capsid structure. Generally, the
linker will be 1-6 residues, however, for example, the covalent
dimer of 2VTU actually has Ser2 deleted in the second copy. In such
cases, the linker length can be 0.
[0196] Residues chosen for the linker should have small sidechains
to avoid steric strain which can be caused by a large number of
atoms packing into a relatively small volume. Strain can also be
minimized by avoiding the choice of amino acid residues with
smaller backbone conformational space, for example Pro. Avoiding
strain can translate into a protein which folds more quickly or
more efficiently. Bulkier and charged sidechains, particularly in
the middle section of longer loops tend to be binding targets for
proteases. Gly-containing linkers are preferred.
[0197] From FIG. 32 it is also clear that the C-terminus of one
monomer can be linked to the N-terminus of a monomer participating
in the neighboring noncovalent dimer and a stable isosahedral
capsid could still form as long as the linker was of appropriate
length and flexibility and did not contain a potential cleavage
site accessible by proteases in the capsid environment. In fact,
three monomers could be linked with appropriate linkers and still
form this section of capsid. Because the tan, gray and medium blue
monomers of FIG. 32, are also the asymmetric unit of the capsid.
Three monomers concatenated end to end with appropriate linking
segments should also be able to form a stable isosahedral
capsid.
[0198] N-terminal residues 1-3 can satisfy their hydrogen bonding
potential with the C-terminal residue 129 and water and vice versa;
therefore, it should be possible to delete some or all of these
residues and form stable VLPs with the truncated proteins or
alternatively with the corresponding potential linker lengths
extended by the number of deletions in concatenated proteins.
[0199] Accordingly, the present disclosure encompasses VLPs
comprising a capsid comprising a capsid protein which is a variant
of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) and is
resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. For example, a VLP may comprise a capsid protein
with the amino acid sequence of wild type Enterobacteria phage MS2
capsid (SEQ ID NO:3) except that the A residue at position 1 is
deleted. A VLP may comprise a capsid protein with the amino acid
sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3)
except that the A residue at position 1 is deleted and the S
residue at position 2 is deleted. A VLP may comprise a capsid
protein with the amino acid sequence of wild type Enterobacteria
phage MS2 capsid (SEQ ID NO:3) except that that the A residue at
position 1 is deleted, the S residue at position 2 is deleted and
the N residue at position 3 is deleted. A VLP may comprise a capsid
protein with the amino acid sequence of wild type Enterobacteria
phage MS2 capsid (SEQ ID NO:3) except that the Y reside at position
129 is deleted. A VLP may comprise a capsid protein with the amino
acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID
NO:3) but having a single (1) amino acid deletion in the 112-117
segment. A VLP may comprise a capsid protein with the amino acid
sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3)
but having a single (1) amino acid deletion in the 112-117 segment.
A VLP may comprise a capsid protein with the amino acid sequence of
wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having
a 1-2 residue insertion in the 65-83 segment and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. A
VLP may comprise a capsid protein with the amino acid sequence of
wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but NO:3)
having a 1-2 residue insertion in the 44-55 segment. A VLP may
comprise a capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a single
(1) residue insertion in the 33-43 segment and is resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. A
VLP may comprise a capsid protein with the amino acid sequence of
wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having
a 1-2 residue insertion in the 24-30 segment. A VLP may comprise a
capsid protein with the amino acid sequence of wild type
Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a single
(1) residue insertion in the 10-18 segment. A VLP may comprise a
capsid protein monomer sequence concatenated with a second capsid
monomer sequence which assembles into a capsid which resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. A
VLP may comprise a capsid protein monomer sequence whose C-terminus
is extended with a 0-6 residue linker segment whose C-terminus is
concatenated with a second capsid monomer sequence, all of which
assembles into a capsid which resistant to hydrolysis catalyzed by
a peptide bond hydrolase category EC 3.4. Suitable linker sequences
include but are not limited to -(Gly).sub.x-, where x is 0-6, or a
Gly-Ser linker such as but not limited to -Gly-Gly-Ser-Gly-Gly-,
-Gly-Gly-Ser and -Gly-Ser-Gly-. A VLP may further comprise a capsid
protein monomer sequence concatenated with a third capsid monomer
sequence which assembles into a capsid which resistant to
hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
Again, in the capsid protein, the C-terminus can be extended with a
0-6 residue linker segment whose C-terminus is concatenated with a
third capsid monomer sequence, all of which assembles into a capsid
which resistant to hydrolysis catalyzed by a peptide bond hydrolase
category EC 3.4. One or both linker sequences can be selected from
-(Gly).sub.x-, where x=0-6, or a Gly-Ser linker selected from
-Gly-Gly-Ser-Gly-Gly-, -Gly-Gly-Ser and -Gly-Ser-Gly-. For example,
in one or both linker sequences, the linker is -(Gly).sub.x-, and x
is 1, 2 or 3. A VLP may comprise one or more coat protein sequences
which are N-terminally truncated by 1-3 residues, wherein a linker
sequence is lengthened by the number of residues deleted, wherein
the linker sequence is -(Gly).sub.x-, wherein x=0-6. For example, a
VLP may comprise one or more coat protein sequences which is
C-terminally truncated by 1 residue and then a linker sequence is
lengthened by the 1 residue, wherein the linker sequence is
-(Gly).sub.x-, wherein x=0-6. A VLP may comprise two coat protein
sequences, wherein the first coat protein sequence in a
concatenated dimer is C-terminally truncated by 1 residue and a
linker sequence is lengthened by the one residue or wherein the
first and/or second coat protein sequence in the concatenated
trimer is C-terminally truncated by 1 residues, wherein the linker
sequence is -(Gly).sub.x-, wherein x=0-6.
[0200] All patents and publications cited herein are herein
incorporated by reference in their entireties, including those
listed below.
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Sequence CWU 1
1
7813569DNAEnterobacteria phage MS2 1gggtgggacc cctttcgggg
tcctgctcaa cttcctgtcg agctaatgcc atttttaatg 60tctttagcga gacgctacca
tggctatcgc tgtaggtagc cggaattcca ttcctaggag 120gtttgacctg
tgcgagcttt tagtaccctt gatagggaga acgagacctt cgtcccctcc
180gttcgcgttt acgcggacgg tgagactgaa gataactcat tctctttaaa
atatcgttcg 240aactggactc ccggtcgttt taactcgact ggggccaaaa
cgaaacagtg gcactacccc 300tctccgtatt cacggggggc gttaagtgtc
acatcgatag atcaaggtgc ctacaagcga 360agtgggtcat cgtggggtcg
cccgtacgag gagaaagccg gtttcggctt ctccctcgac 420gcacgctcct
gctacagcct cttccctgta agccaaaact tgacttacat cgaagtgccg
480cagaacgttg cgaaccgggc gtcgaccgaa gtcctgcaaa aggtcaccca
gggtaatttt 540aaccttggtg ttgctttagc agaggccagg tcgacagcct
cacaactcgc gacgcaaacc 600attgcgctcg tgaaggcgta cactgccgct
cgtcgcggta attggcgcca ggcgctccgc 660taccttgccc taaacgaaga
tcgaaagttt cgatcaaaac acgtggccgg caggtggttg 720gagttgcagt
tcggttggtt accactaatg agtgatatcc agggtgcata tgagatgctt
780acgaaggttc accttcaaga gtttcttcct atgagagccg tacgtcaggt
cggtactaac 840atcaagttag atggccgtct gtcgtatcca gctgcaaact
tccagacaac gtgcaacata 900tcgcgacgta tcgtgatatg gttttacata
aacgatgcac gtttggcatg gttgtcgtct 960ctaggtatct tgaacccact
aggtatagtg tgggaaaagg tgcctttctc attcgttgtc 1020gactggctcc
tacctgtagg taacatgctc gagggcctta cggcccccgt gggatgctcc
1080tacatgtcag gaacagttac tgacgtaata acgggtgagt ccatcataag
cgttgacgct 1140ccctacgggt ggactgtgga gagacagggc actgctaagg
cccaaatctc agccatgcat 1200cgaggggtac aatccgtatg gccaacaact
ggcgcgtacg taaagtctcc tttctcgatg 1260gtccatacct tagatgcgtt
agcattaatc aggcaacggc tctctagata gagccctcaa 1320ccggagtttg
aagcatggct tctaacttta ctcagttcgt tctcgtcgac aatggcggaa
1380ctggcgacgt gactgtcgcc ccaagcaact tcgctaacgg ggtcgctgaa
tggatcagct 1440ctaactcgcg ttcacaggct tacaaagtaa cctgtagcgt
tcgtcagagc tctgcgcaga 1500atcgcaaata caccatcaaa gtcgaggtgc
ctaaagtggc aacccagact gttggtggtg 1560tagagcttcc tgtagccgca
tggcgttcgt acttaaatat ggaactaacc attccaattt 1620tcgctacgaa
ttccgactgc gagcttattg ttaaggcaat gcaaggtctc ctaaaagatg
1680gaaacccgat tccctcagca atcgcagcaa actccggcat ctactaatag
acgccggcca 1740ttcaaacatg aggattaccc atgtcgaaga caacaaagaa
gttcaactct ttatgtattg 1800atcttcctcg cgatctttct ctcgaaattt
accaatcaat tgcttctgtc gctactggaa 1860gcggtgatcc gcacagtgac
gactttacag caattgctta cttaagggac gaattgctca 1920caaagcatcc
gaccttaggt tctggtaatg acgaggcgac ccgtcgtacc ttagctatcg
1980ctaagctacg ggaggcgaat ggtgatcgcg gtcagataaa tagagaaggt
ttcttacatg 2040acaaatcctt gtcatgggat ccggatgttt tacaaaccag
catccgtagc cttattggca 2100acctcctctc tggctaccga tcgtcgttgt
ttgggcaatg cacgttctcc aacggtgctc 2160ctatggggca caagttgcag
gatgcagcgc cttacaagaa gttcgctgaa caagcaaccg 2220ttaccccccg
cgctctgaga gcggctctat tggtccgaga ccaatgtgcg ccgtggatca
2280gacacgcggt ccgctataac gagtcatatg aatttaggct cgttgtaggg
aacggagtgt 2340ttacagttcc gaagaataat aaaatagatc gggctgcctg
taaggagcct gatatgaata 2400tgtacctcca gaaaggggtc ggtgctttca
tcagacgccg gctcaaatcc gttggtatag 2460acctgaatga tcaatcgatc
aaccagcgtc tggctcagca gggcagcgta gatggttcgc 2520ttgcgacgat
agacttatcg tctgcatccg attccatctc cgatcgcctg gtgtggagtt
2580ttctcccacc agagctatat tcatatctcg atcgtatccg ctcacactac
ggaatcgtag 2640atggcgagac gatacgatgg gaactatttt ccacaatggg
aaatgggttc acatttgagc 2700tagagtccat gatattctgg gcaatagtca
aagcgaccca aatccatttt ggtaacgccg 2760gaaccatagg catctacggg
gacgatatta tatgtcccag tgagattgca ccccgtgtgc 2820tagaggcact
tgcctactac ggttttaaac cgaatcttcg taaaacgttc gtgtccgggc
2880tctttcgcga gagctgcggc gcgcactttt accgtggtgt cgatgtcaaa
ccgttttaca 2940tcaagaaacc tgttgacaat ctcttcgccc tgatgctgat
attaaatcgg ctacggggtt 3000ggggagttgt cggaggtatg tcagatccac
gcctctataa ggtgtgggta cggctctcct 3060cccaggtgcc ttcgatgttc
ttcggtggga cggacctcgc tgccgactac tacgtagtca 3120gcccgcctac
ggcagtctcg gtatacacca agactccgta cgggcggctg ctcgcggata
3180cccgtacctc gggtttccgt cttgctcgta tcgctcgaga acgcaagttc
ttcagcgaaa 3240agcacgacag tggtcgctac atagcgtggt tccatactgg
aggtgaaatc accgacagca 3300tgaagtccgc cggcgtgcgc gttatacgca
cttcggagtg gctaacgccg gttcccacat 3360tccctcagga gtgtgggcca
gcgagctctc ctcggtagct gaccgaggga cccccgtaaa 3420cggggtgggt
gtgctcgaaa gagcacgggt gcgaaagcgg tccggctcca ccgaaaggtg
3480ggcgggcttc ggcccaggga cctcccccta aagagaggac ccgggattct
cccgatttgg 3540taactagctg cttggctagt taccaccca
35692396DNAEnterobacteria phage MS2 2atggcttcta actttactca
gttcgttctc gtcgacaatg gcggaactgg cgacgtgact 60gtcgccccaa gcaacttcgc
taacggggtc gctgaatgga tcagctctaa ctcgcgttca 120caggcttaca
aagtaacctg tagcgttcgt cagagctctg cgcagaatcg caaatacacc
180atcaaagtcg aggtgcctaa agtggcaacc cagactgttg gtggtgtaga
gcttcctgta 240gccgcatggc gttcgtactt aaatatggaa ctaaccattc
caattttcgc tacgaattcc 300gactgcgagc ttattgttaa ggcaatgcaa
ggtctcctaa aagatggaaa cccgattccc 360tcagcaatcg cagcaaactc
cggcatctac taatag 3963130PRTEnterobacteria phage MS2 3Met Ala Ser
Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly
Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25
30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser
35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys
Val Glu 50 55 60 Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val
Glu Leu Pro Val 65 70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu
Leu Thr Ile Pro Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu
Ile Val Lys Ala Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro
Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
436DNABacteriophage T7 4ctagcataac cccttggggc ctctaaacgg gtcttg
36547DNABacteriophage T7 5tagcataacc ccttggggcc tctaaacggg
tcttgagggg ttttttg 476480DNAArtificial SequenceSYNETHESIZED
6acaagtttgt acaaaaaagc aggctaagaa ggagatatac atatggcttc taactttact
60cagttcgttc tcgtcgacaa tggcggaact ggcgacgtga ctgtcgcccc aagcaacttc
120gctaacgggg tcgctgaatg gatcagctct aactcgcgtt cacaggctta
caaagtaacc 180tgtagcgttc gtcagagctc tgcgcagaat cgcaaataca
ccatcaaagt cgaggtgcct 240aaagtggcaa cccagactgt tggtggtgta
gagcttcctg tagccgcatg gcgttcgtac 300ttaaatatgg aactaaccat
tccaattttc gctacgaatt ccgactgcga gcttattgtt 360aaggcaatgc
aaggtctcct aaaagatgga aacccgattc cctcagcaat cgcagcaaac
420tccggcatct actaatagac gccggccatt caaacatgag gattacccat
gtacccagct 4807438DNAArtificial SequenceSYNTHESIZED 7acaagtttgt
acaaaaaagc aggctaagaa ggagatatac atatggcttc taactttact 60cagttcgttc
tcgtcgacaa tggcggaact ggcgacgtga ctgtcgcccc aagcaacttc
120gctaacgggg tcgctgaatg gatcagctct aactcgcgtt cacaggctta
caaagtaacc 180tgtagcgttc gtcagagctc tgcgcagaat cgcaaataca
ccatcaaagt cgaggtgcct 240aaagtggcaa cccagactgt tggtggtgta
gagcttcctg tagccgcatg gcgttcgtac 300ttaaatatgg aactaaccat
tccaattttc gctacgaatt ccgactgcga gcttattgtt 360aaggcaatgc
aaggtctcct aaaagatgga aacccgattc cctcagcaat cgcagcaaac
420tccggcatct actaatag 4388179DNAArtificial SequenceSYNTHESIZED
8ggatcctaat acgactcact ataggcaagc tgaccctgaa gttctcaaga ggaacttcag
60ggtcagcttg ccaaggccgg catggtccca gcctcctcgc tggcgccggc tgggcaacat
120tcgtggcgaa tgggaccacg cttcaaacat gaggattacc catgtcgaag cgaccatgg
1799385DNAArtificial SequenceSYNTHESIZED 9ggatcctaat acgactcact
atagggagat aaataaataa atttgaatga acttcagggt 60cagcttgctg atgaggcgct
tcggcgccga aacacccagt ggtgtccaag ctgaccctga 120agttcattca
agagatgaac ttcagggtca gcttgtcggc cggcatggtc ccagcctcct
180cgctggcgcc ggctgggcaa cattcgtggc gaatgggacc acgcttcaaa
catgaggatt 240acccatgtcg aagcgaattt atttatttaa ttattattat
tattattggc cggcatggtc 300ccagcctcct cgctggcgcc ggctgggcaa
caccttcggg tggcgaatgg gaccaaaaaa 360aaataataat aataataatc catgg
38510165DNAArtificial SequenceSYNTHESIZED 10taatacgact cactatagca
agctgaccct gaagttcatc aagagtgaac ttcagggtca 60gcttgtcggc cggcatggtc
ccagcctcct cgctggcgcc ggctgggcaa cattcgtggc 120taatgggacc
atatatatat acatgaggat tacccatgtc catgg 16511165DNAArtificial
SequenceSYNTHESIZED 11taatacgact cactatagca agctgaccct gaagttcatc
aagagtgaac ttcagggtca 60gcttgtcggc cggcatggtc ccagcctcct cgctggcgcc
ggctggtcaa cattcgtggc 120gaatgggacc atatatatat acatgaggat
tacccatgtc catgg 16512165DNAArtificial SequenceSYNTHESIZED
12taatacgact cactatagca agctgaccct gaagttcatc aagagtgaac ttcagggtca
60gcttgtcggc cggcatggtc ccggcctcct cgctggcgcc ggctgggcaa cattcgtggc
120gaatgggacc atatatatat acatgaggat tacccatgtc catgg
16513165DNAArtificial SequenceSYNTHESIZED 13taatacgact cactatagca
agctgaccct gaagttcatc aagagtgaac ttcagggtca 60gcttgtcggc cggcatggtc
ccagcctcct cgctggcgcc ggctgtgcaa cattcgtggc 120gaatgggacc
atatatatat acatgaggat tacccatgtc catgg 16514165DNAArtificial
SequenceSYNTHESIZED 14taatacgact cactatagca agctgaccct gaagttcatc
aagagtgaac ttcagggtca 60gcttgtcggc cggcatggtc ccagcctcct cgctggcgcc
ggctgggcaa cattcgtggc 120gagtgggacc atatatatat acatgaggat
tacccatgtc catgg 16515165DNAArtificial SequenceSYNTHESIZED
15taatacgact cactatagca agctgaccct gaagttcatc aagagtgaac ttcagggtca
60gcttgtcggc cggcatggtc ccagccttct cgctggcgcc ggctgggcaa cattcgtggc
120gaatgggacc atatatatat acatgaggat tacccatgtc catgg
16516165DNAArtificial SequenceSYNTHESIZED 16taatacgact cactatagca
agctgaccct gaagttcatc aagagtgaac ttcagggtca 60gcttgtcggc cggcatggtc
ccagcctcct cactggcgcc ggctgggcaa cattcgtggc 120gaatgggacc
atatatatat acatgaggat tacccatgtc catgg 16517165DNAArtificial
SequenceSYNTHESIZED 17taatacgact cactatagca agctgaccct gaagttcatc
aagagtgaac ttcagggtca 60gcttgtcggc cggcatggtc ccagcctcct cgctggcgcc
ggctgggcaa cattcgtggc 120gattgggacc atatatatat acatgaggat
tacccatgtc catgg 16518165DNAArtificial SequenceSYNTHESIZED
18taatacgact cactatagca agctgaccct gaagttcatc aagagtgaac ttcagggtca
60gcttgtcggc cggcatggtc ccagcctcct cgctggcgcc ggctgggcaa cattcgtgcc
120gaatgggacc atatatatat acatgaggat tacccatgtc catgg
16519365DNAArtificial SequenceSYNTHESIZED 19ggatcctaat acgactcact
atagggagac gttcacgttg aatgaacttc agggtcagct 60tgctgatgag gcgcttcggc
gccgaaacac ccagtggtgt ccaagctgac cctgaagttc 120attcaagaga
tgaacttcag ggtcagcttg tcaccggatg tgctctccgg tctgatgagt
180ccgtgaggac gaaacaagct gaccctgaag ttcatccgtg aacgacgctt
caaacatgag 240gattacccat gtcgaagcga atatatatat ataggccggc
atggtcccag cctcctcgct 300ggcgccggct gggcaacacc ttcgggtggc
gaatgggacc aaaaaaatat atatatatac 360catgg 36520154DNAArtificial
SequenceSYNTHESIZED 20taatacgact cactatagca agctgaccct gaagttcatc
aagagtgaac ttcagggtca 60gcttgtcacc ggatgtgctc tccggtctga tgagtccgtg
aggacgaaac aagctgacca 120tatatatata catgaggatt acccatgtcc atgg
15421159DNAArtificial SequenceSYNTHESIZED 21taatacgact cactatagca
agctgaccct gaagttcatc aagagtgaac ttcagggtca 60gcttgtcacc ggatgtgctc
tccggtctga tgagtccgtg aggacgaaac aagctgaccc 120tgaaatatat
atatacatga ggattaccca tgtccatgg 15922164DNAArtificial
SequenceSYNTHESIZED 22taatacgact cactatagca agctgaccct gaagttcatc
aagagtgaac ttcagggtca 60gcttgtcacc ggatgtgctc tccggtctga tgagtccgtg
aggacgaaac aagctgaccc 120tgaagttcaa tatatatata catgaggatt
acccatgtcc atgg 16423166DNAArtificial SequenceSYNTHESIZED
23taatacgact cactatagca agctgaccct gaagttcatc aagagtgaac ttcagggtca
60gcttgtcacc ggatgtgctc tccggtctga tgagtccgtg aggacgaaac aagctgaccc
120tgaagttcac tatatatata tacatgagga ttacccatgt ccatgg
16624169DNAArtificial SequenceSYNTHESIZED 24taatacgact cactatagca
agctgaccct gaagttcatc aagagtgaac ttcagggtca 60gcttgtcacc ggatgtgctc
tccggtctga tgagtccgtg aggacgaaac aagctgaccc 120tgaagttcac
tcttatatat atatacatga ggattaccca tgtccatgg 16925171DNAArtificial
SequenceSYNTHESIZED 25taatacgact cactatagca agctgaccct gaagttcatc
aagagtgaac ttcagggtca 60gcttgtcacc ggatgtgctc tccggtctga tgagtccgtg
aggacgaaac aagctgaccc 120tgaagttcac tcttgaatat atatatacat
gaggattacc catgtccatg g 17126244DNAArtificial SequenceSYNTHESIZED
26ggatcctaat acgactcact atagggatct cccagcctcc tcgctggcgc cggctgggca
60acattcgtgg cgaatggggg atcatatctt gatgaacttc agggtcagct tgctgatgag
120gcgcttcggc gccgaaacac cgtgtccaag ctgaccctga agttcatcaa
gaatgaactt 180cagggtcagc ttgtcggccg gcatgcattc aaacatgagg
attacccatg tcgaagttaa 240ttaa 24427615DNAArtificial
SequenceSYNTHESIZED 27ggatcctaat acgactcact atagggagac ttgatgaact
tcagggtcag cttgctgatg 60aggcgcttcg gcgccgaaac acccagtggt gtccaagctg
accctgaagt tcatcaagaa 120tgaacttcag ggtcagcttg tcaccggatg
tgctctccgg tctgatgagt ccgtgaggac 180gaaacaagct gaccctgaag
ttcattatat cttggcagat gaacttcagg gtcagctgat 240gagactcttc
ggagtcgaaa cacccagtgg tgtcctgacc ctgaagttca tctgccaaga
300gcagatgaac ttcagggtca gtcaccggat gtgctctccg gtctgatgag
tccgtgagga 360cgaaactgac cctgaagttc atctgctata tcttgtggtg
cagatgaact tcagggctga 420tgaggctctt cggagccgaa acacccagtg
gtgtcccctg aagttcatct gcaccacaag 480atggtgcaga tgaacttcag
ggtcaccgga tgtgctctcc ggtctgatga gtccgtgagg 540acgaaaccct
gaagttcatc tgcaccatac gccggccatt caaacatgag gattacccat
600gtcgaagtta attaa 61528431DNAArtificial SequenceSYNTHESIZED
28ggatcctaat acgactcact atagggagaa tatatataca agctgaccct gaagttcatc
60aagaatgaac ttcagggtca gcttgtcggc cggcatggtc ccagcctcct cgctggcgcc
120ggctgggcaa cattcgtggc gaatgggacc aattaattac tgaccctgaa
gttcatctgc 180caagagcaga tgaacttcag ggtcagtcgg ccggcatggt
cccagcctcc tcgctggcgc 240cggctgggca acattcgtgg cgaatgggac
caataataat ccctgaagtt catctgcacc 300acaagatggt gcagatgaac
ttcagggtcg gccggcatgg tcccagcctc ctcgctggcg 360ccggctgggc
aacattcgtg gcgaatggga cccattcaaa catgaggatt acccatgtcg
420aagttaatta a 43129362DNAArtificial SequenceSYNTHESIZED
29ggatcctaat acgactcact atagggagaa tgaacttcag ggtcagcttg ctgatgaggc
60gcttcggcgc cgaaacaccg tgtccaagct gaccctgaag ttcatggccg gcatggtccc
120agcctcctcg ctggcgccgg ctgggcaaca ttcgtggcga atgggaccat
tagccaagct 180gaccctgaag ttcatctgat gagactccga attcggagtc
gaaacacggt aaccgtgtca 240tgaacttcag ggtcagcttg gcggccggca
tggtcccagc ctcctcgctg gcgccggctg 300ggcaacattc gtggcgaatg
ggacccattc aaacatgagg attacccatg tcgaagccat 360gg
36230196DNAArtificial SequenceSYNTHESIZED 30ggatcctaat acgactcact
atagggagaa ataataatca agctgaccct gaagttcatc 60aagaatgaac ttcagggtca
gcttgtcaat aataatccgc taccccgacc acatgaacaa 120gattcatgtg
gtcggggtag cggtcaataa taatacgctt caaacatgag gattacccat
180gtcgaagcga ccatgg 19631165DNAArtificial SequenceSYNTHESIZED
31taatacgact cactataggc ttgtgatgct tcagccaaat caagagtttg gctgaagcat
60cacaagcggc cggcatggtc ccagcctcct cgctggcgcc ggctgggcaa cattcgtggc
120gaatgggacc atatatatat acatgaggat tacccatgtc catgg
16532168DNAArtificial SequenceSYNTHESIZED 32taatacgact cactataggc
tcgagcaagc ctgatgaggc gcttcggcgc cgaaacaccg 60tgtcgcttgt gatgcttcag
ccaaatcaag agtttggctg aagcatcaca agctcaccgg 120atgtgctctc
cggtctgatg agtccgtgag gacgaaagct tgccatgg 16833251DNAArtificial
SequenceSYNTHESIZED 33taatacgact cactataggg agaacgccgg ccattcaaat
agtaaataat agagggtcag 60cttgctgatg aggcgcttcg gcgccgaaac accgtgtcca
agctgaccct gaagttcatc 120aagagtgaac ttcagggtca gcttgtcacc
ggatgtgctc tccggtctga tgagtccgtg 180aggacgaaac aagctgaccc
tgaagttcac tacgccggcc attcaaacat gaggattacc 240catgtccatg g
25134129PRTEnterobacteria phage MS2 34Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu Trp 20 25 30 Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val 35 40 45 Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Ser Ile Lys Val Glu Val 50 55
60 Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val Ala
65 70 75 80 Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile
Phe Ala 85 90 95 Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met
Gln Gly Leu Leu 100 105 110 Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile
Ala Ala Asn Ser Gly Ile 115 120 125 Tyr 35129PRTEnterobacteria
phage GA 35Ala Thr Leu Arg Ser Phe Val Leu Val Asp Asn Gly Gly Thr
Gly Asn 1 5 10 15 Val Thr Val Val Pro Val Ser Asn Ala Asn Gly Val
Ala Glu Trp Leu 20 25 30 Ser Asn Asn Ser Arg Ser Gln Ala Tyr Arg
Val Thr Ala Ser Tyr Arg 35 40 45 Ala Ser Gly Ala Asp Lys Arg Lys
Tyr Thr Ile Lys Leu Glu Val Pro 50 55 60 Lys Ile Val Thr Gln Val
Val Asn Gly Val Glu
Leu Pro Gly Ser Ala 65 70 75 80 Trp Lys Ala Tyr Ala Ser Ile Asp Leu
Thr Ile Pro Ile Phe Ala Ala 85 90 95 Thr Asp Asp Val Thr Val Ile
Ser Lys Ser Leu Ala Gly Leu Phe Lys 100 105 110 Val Gly Asn Pro Ile
Ala Glu Ala Ile Ser Ser Gln Ser Gly Phe Tyr 115 120 125 Ala
36129PRTEnterobacteria phage FR 36Ala Ser Asn Phe Glu Glu Phe Val
Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asp Val Lys Val Ala Pro
Ser Asn Phe Ala Asn Gly Val Ala Glu Trp 20 25 30 Ile Ser Ser Asn
Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val 35 40 45 Arg Gln
Ser Ser Ala Asn Asn Arg Lys Tyr Thr Val Lys Val Glu Val 50 55 60
Pro Lys Val Ala Thr Gln Val Gln Gly Gly Val Glu Leu Pro Val Ala 65
70 75 80 Ala Trp Arg Ser Tyr Met Asn Met Glu Leu Thr Ile Pro Val
Phe Ala 85 90 95 Thr Asn Asp Asp Cys Ala Leu Ile Val Lys Ala Leu
Gln Gly Thr Phe 100 105 110 Lys Thr Gly Asn Pro Ile Ala Thr Ala Ile
Ala Ala Asn Ser Gly Ile 115 120 125 Tyr 37257PRTEnterobacteria
phage MS2 37Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly
Thr Gly 1 5 10 15 Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly
Val Ala Glu Trp 20 25 30 Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr
Lys Val Thr Cys Ser Val 35 40 45 Arg Gln Ser Ser Ala Gln Asn Arg
Lys Tyr Thr Ile Lys Val Glu Val 50 55 60 Pro Lys Val Ala Thr Gln
Thr Val Gly Gly Val Glu Leu Pro Val Ala 65 70 75 80 Ala Trp Arg Ser
Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe Ala 85 90 95 Thr Asn
Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu 100 105 110
Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly Ile 115
120 125 Tyr Ala Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr
Gly 130 135 140 Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val
Ala Glu Trp 145 150 155 160 Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr
Lys Val Thr Cys Ser Val 165 170 175 Arg Gln Ser Ser Ala Gln Asn Arg
Lys Tyr Thr Ile Lys Val Glu Val 180 185 190 Pro Lys Val Ala Thr Gln
Thr Val Gly Gly Val Glu Leu Pro Val Ala 195 200 205 Ala Trp Arg Ser
Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe Ala 210 215 220 Thr Asn
Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu 225 230 235
240 Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly Ile
245 250 255 Tyr 38130PRTEnterobacteria phage MS2 38Met Ala Ser Asn
Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp
Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30
Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35
40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val
Glu 50 55 60 Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu
Leu Pro Val 65 70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Leu Glu Leu
Thr Ile Pro Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile
Val Lys Ala Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile
Pro Ser Ala Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
39120PRTEnterobacteria phage MS2 39Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Leu Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro 115 120
40130PRTEnterobacteria phage MS2 40Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Leu Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
41130PRTEnterobacteria phage MS2 41Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Val Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
42130PRTEnterobacteria phage MS2 42Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Pro Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
43130PRTEnterobacteria phage MS2 43Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Ala Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
44130PRTEnterobacteria phage MS2misc_feature(50)..(50)Xaa can be
any naturally occurring amino acid 44Met Ala Ser Asn Phe Thr Gln
Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val
Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser
Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val
Xaa Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55
60 Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val
65 70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
45130PRTEnterobacteria phage MS2misc_feature(7)..(7)Xaa can be any
naturally occurring amino acid 45Met Ala Ser Asn Phe Thr Xaa Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
46130PRTEnterobacteria phage BO1 46Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Pro 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
47130PRTEnterobacteria phage MS2 47Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Asp Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
48130PRTEnterobacteria phage MS2misc_feature(18)..(18)Xaa can be
any naturally occurring amino acid 48Met Ala Ser Asn Phe Thr Gln
Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Xaa Val Thr Val
Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser
Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val
Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55
60 Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val
65 70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
49130PRTEnterobacteria phage MS2 49Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Leu Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Pro Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
50130PRTEnterobacteria phage MS2 50Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Ser Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
51130PRTEnterobacteria phage MS2misc_feature(104)..(104)Xaa can be
any naturally occurring amino acid 51Met Ala Ser Asn Phe Thr Gln
Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val
Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser
Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val
Arg Gln Ser Ser Ala
Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Val
Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65 70 75 80 Ala Ala
Trp Arg Ser Tyr Leu Asn Val Glu Leu Thr Ile Pro Ile Phe 85 90 95
Ala Thr Asn Ser Asp Cys Glu Xaa Ile Val Lys Ala Met Gln Gly Leu 100
105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser
Gly 115 120 125 Ile Tyr 130 52130PRTEnterobacteria phage
MS12misc_feature(22)..(22)Xaa can be any naturally occurring amino
acid 52Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly
Thr 1 5 10 15 Gly Asp Val Thr Val Xaa Pro Ser Asn Phe Ala Asn Gly
Val Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr
Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg
Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Val Ala Thr Gln
Thr Val Gly Gly Val Glu Leu Pro Val 65 70 75 80 Ala Ala Trp Arg Ser
Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe 85 90 95 Ala Thr Asn
Ser Asp Cys Ala Leu Ile Val Lys Ala Met Gln Gly Leu 100 105 110 Leu
Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly 115 120
125 Ile Tyr 130 53130PRTEnterobacteria phage
MS2misc_feature(130)..(130)Xaa can be any naturally occurring amino
acid 53Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly
Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly
Val Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr
Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg
Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Val Ala Thr Gln
Thr Val Gly Gly Val Glu Leu Pro Val 65 70 75 80 Ala Ala Trp Arg Ser
Tyr Leu Asn Val Glu Leu Thr Ile Pro Ile Phe 85 90 95 Ala Thr Asn
Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu 100 105 110 Leu
Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly 115 120
125 Ile Xaa 130 54130PRTEnterobacteria phage
MS2misc_feature(130)..(130)Xaa can be any naturally occurring amino
acid 54Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly
Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly
Val Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr
Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg
Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Val Ala Thr Gln
Thr Val Gly Gly Val Glu Leu Pro Val 65 70 75 80 Ala Ala Trp Arg Ser
Tyr Leu Asn Val Glu Leu Thr Ile Pro Ile Phe 85 90 95 Ala Thr Asn
Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu 100 105 110 Leu
Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly 115 120
125 Ile Xaa 130 55130PRTEnterobacteria phage MS2 55Met Ala Ser Asn
Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp
Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30
Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35
40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val
Glu 50 55 60 Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Gln
Leu Pro Val 65 70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu
Thr Ile Pro Ile Phe 85 90 95 Ala Thr Asn Asp Asp Cys Ala Leu Ile
Val Lys Ala Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile
Pro Ser Ala Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
56130PRTEnterobacteria phage MS2 56Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Gln Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Asp Asp Cys Ala Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
57127PRTEnterobacteria phage MS2 57Asn Phe Thr Gln Phe Val Leu Val
Asp Asn Gly Gly Thr Gly Asp Val 1 5 10 15 Thr Val Ala Pro Ser Asn
Phe Ala Asn Gly Val Ala Glu Trp Ile Ser 20 25 30 Ser Asn Ser Arg
Ser Gln Ala Tyr Lys Val Thr Cys Ser Val Arg Gln 35 40 45 Ser Ser
Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val Pro Lys 50 55 60
Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val Ala Ala Trp 65
70 75 80 Arg Ser Tyr Leu Asn Val Glu Leu Thr Ile Pro Ile Phe Ala
Thr Asn 85 90 95 Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly
Leu Leu Lys Asp 100 105 110 Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala
Asn Ser Gly Ile Tyr 115 120 125 58129PRTEnterobacteria phage ZR
58Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asn Asp Gly Gly Thr Gly 1
5 10 15 Asn Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu
Trp 20 25 30 Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr
Cys Ser Val 35 40 45 Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr
Ile Lys Val Glu Val 50 55 60 Pro Lys Val Ala Thr Gln Thr Val Gly
Gly Val Glu Leu Pro Val Ala 65 70 75 80 Ala Trp Arg Ser Tyr Leu Asn
Met Glu Leu Thr Ile Pro Ile Phe Ala 85 90 95 Thr Asn Ser Asp Cys
Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu 100 105 110 Lys Asp Gly
Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly Ile 115 120 125 Tyr
59129PRTEnterobacteria phage R17 59Ala Ser Asn Phe Thr Gln Phe Val
Leu Val Asn Asp Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val Ala Pro
Ser Asn Phe Ala Asn Gly Val Ala Glu Trp 20 25 30 Ile Ser Ser Asn
Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val 35 40 45 Arg Gln
Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val 50 55 60
Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val Ala 65
70 75 80 Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile
Phe Ala 85 90 95 Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met
Gln Gly Leu Leu 100 105 110 Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile
Ala Ala Asn Ser Gly Ile 115 120 125 Tyr 60130PRTEnterobacteria
phage MS2 60Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly
Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn
Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala
Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn
Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Val Ala Thr
Gln Thr Val Gly Gly Val Glu Leu Pro Val 65 70 75 80 Ala Ala Trp Arg
Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe 85 90 95 Ala Thr
Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu 100 105 110
Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly 115
120 125 Ile Tyr 130 61130PRTEnterobacteria phage MS2 61Met Ala Ser
Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly
Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25
30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser
35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys
Val Glu 50 55 60 Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val
Glu Leu Pro Val 65 70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu
Leu Thr Ile Pro Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu
Ile Val Lys Ala Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro
Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
62130PRTEnterobacteria phage MS2 62Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
63130PRTEnterobacteria phage ZR 63Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
64130PRTEnterobacteria phage JP501 64Met Ala Ser Asn Phe Thr Glu
Phe Val Leu Val Asp Asn Gly Glu Thr 1 5 10 15 Gly Asn Val Thr Val
Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser
Ser Asp Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val
Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Ala 50 55
60 Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val
65 70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Ala Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
65129PRTEnterobacteria phage F2 65Ala Ser Asn Phe Thr Gln Phe Val
Leu Val Asn Asp Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val Ala Pro
Ser Asn Phe Ala Asn Gly Val Ala Glu Trp 20 25 30 Ile Ser Ser Asn
Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val 35 40 45 Arg Gln
Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val 50 55 60
Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val Ala 65
70 75 80 Ala Trp Arg Ser Tyr Leu Asn Leu Glu Leu Thr Ile Pro Ile
Phe Ala 85 90 95 Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met
Gln Gly Leu Leu 100 105 110 Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile
Ala Ala Asn Ser Gly Ile 115 120 125 Tyr 66130PRTEnterobacteria
phage JP34 66Met Ala Thr Leu Arg Ser Phe Val Leu Val Asp Asn Gly
Gly Thr Gly 1 5 10 15 Asp Val Thr Val Val Pro Val Ser Asn Ala Asn
Gly Val Ala Glu Trp 20 25 30 Leu Ser Asn Asn Ser Arg Ser Gln Ala
Tyr Arg Val Thr Ala Ser Tyr 35 40 45 Arg Ala Ser Gly Ala Asp Lys
Arg Lys Tyr Thr Ile Lys Leu Glu Val 50 55 60 Pro Lys Ile Val Thr
Gln Val Val Asn Gly Val Glu Leu Pro Val Ser 65 70 75 80 Ala Trp Lys
Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro Ile Phe Ala 85 90 95 Ala
Thr Asp Asp Val Thr Val Ile Ser Lys Ser Leu Ala Gly Leu Phe 100 105
110 Lys Val Gly Asn Pro Ile Ala Asp Ala Ile Ser Ser Gln Ser Gly Phe
115 120 125 Tyr Ala 130 67130PRTEnterobacteria phage JP500 67Met
Ala Thr Leu Arg Ser Phe Val Leu Val Asp Asn Gly Gly Thr Gly 1 5 10
15 Asp Val Thr Val Val Pro Val Ser Asn Ala Asn Gly Val Ala Glu Trp
20 25 30 Leu Ser Asn Asn Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala
Ser Tyr 35 40 45 Arg Ala Ser Gly Ala Asp Lys Arg Lys Tyr Thr Ile
Lys Leu Glu Val 50 55 60 Pro Lys Ile Val Thr Gln Val Val Asn Gly
Val Glu Leu Pro Val Ser 65 70 75 80 Ala Trp Lys Ala Tyr Ala Ser Ile
Asp Leu Thr Ile Pro Ile Phe Ala 85 90 95 Ala Thr Asp Asp Val Thr
Val Ile Ser Lys Ser Leu Ala Gly Leu Phe 100 105 110 Lys Val Gly Asn
Pro Ile Ala Asp Ala Ile Ser Ser Gln Ser Gly Phe 115 120 125 Tyr Ala
130 68130PRTEnterobacteria phage SD 68Met Ala Thr Leu Arg Ser Phe
Val Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val Val
Pro Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30 Leu Ser Asn
Asn Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser
Tyr 35 40 45 Arg Ala Ser Gly Ala Asp Lys Arg Lys Tyr Thr Ile Lys
Leu Glu Val 50 55 60 Pro Lys Ile Val Thr Gln Val Val Asn Gly Val
Glu Leu Pro Ile Ser 65 70 75 80 Ala Trp Lys Ala Tyr Ala Ser Ile Asp
Leu Thr Ile Pro Ile Phe Ala 85 90 95 Ala Thr Asp Asp Val Thr Thr
Ile Ser Lys Ser Leu Ala Gly Leu Phe 100 105 110 Lys Val Gly Asn Pro
Ile Ala Asp Ala Ile Ser Ser Gln Ser Gly Phe 115 120 125 Tyr Ala 130
69130PRTEnterobacteria phage KU1 69Met Ala Thr Leu Arg Ser Phe Val
Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val Val Pro
Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30 Leu Ser Asn Asn
Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35 40 45 Arg Ala
Ser Gly Ala Asp Lys Arg Lys Tyr Thr Ile Lys Leu Glu Val 50 55 60
Pro Lys Ile Val Thr Gln Ser Val Asn Gly Val Glu Leu Pro Val Ser 65
70 75 80 Ala Trp Lys Ala Phe Ala Ser Ile Asp Leu Thr Ile Pro Ile
Phe Ala 85 90 95 Ala Thr Asp Asp Val Thr Leu Ile Ser Lys Ser Leu
Ala Gly Leu Phe 100 105 110 Lys Ile Gly Asn Pro Val Ala Asp Ala Ile
Ser Ser Gln Ser Gly Phe 115 120 125 Tyr Ala 130
70130PRTEnterobacteria phage GA 70Met Ala Thr Leu Arg Ser Phe Val
Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val Val Pro
Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30 Leu Ser Asn Asn
Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35 40 45 Arg Ala
Ser Gly Ala Asp Lys Arg Lys Tyr Ala Ile Lys Leu Glu Val 50 55 60
Pro Lys Ile Val Thr Gln Val Val Asn Gly Val Glu Leu Pro Gly Ser 65
70 75 80 Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro Ile
Phe Ala 85 90 95 Ala Thr Asp Asp Val Thr Val Ile Ser Lys Ser Leu
Ala Gly Leu Phe 100 105 110 Lys Val Gly Asn Pro Ile Ala Glu Ala Ile
Ser Ser Gln Ser Gly Phe 115 120 125 Tyr Ala 130
71130PRTEnterobacteria phage BZ13 71Met Ala Thr Leu Arg Ser Phe Val
Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val Val Pro
Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30 Leu Ser Asn Asn
Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35 40 45 Arg Ala
Ser Gly Ala Asp Lys Arg Lys Tyr Thr Ile Lys Leu Glu Val 50 55 60
Pro Lys Ile Val Thr Gln Val Val Asn Gly Val Glu Leu Pro Val Ser 65
70 75 80 Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro Ile
Phe Ala 85 90 95 Ala Thr Asp Asp Val Thr Val Ile Ser Lys Ser Leu
Ala Gly Leu Phe 100 105 110 Lys Val Gly Asn Pro Ile Ala Glu Ala Ile
Ser Ser Gln Ser Gly Phe 115 120 125 Tyr Ala 130
72130PRTEnterobacteria phage BZ13 72Met Ala Thr Leu Arg Ser Phe Val
Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val Val Pro
Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30 Leu Ser Asn Asn
Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35 40 45 Arg Ala
Ser Gly Ala Asp Lys Arg Lys Tyr Thr Ile Lys Leu Glu Val 50 55 60
Pro Lys Ile Val Thr Gln Thr Val Asn Gly Val Glu Leu Pro Val Ser 65
70 75 80 Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro Ile
Phe Ala 85 90 95 Ala Thr Asp Asp Val Thr Leu Ile Ser Lys Ser Leu
Ala Gly Leu Phe 100 105 110 Lys Ile Gly Asn Pro Val Ala Asp Ala Ile
Ser Ser Gln Ser Gly Phe 115 120 125 Tyr Ala 130
73130PRTEnterobacteria phage BZ13 73Met Ala Thr Leu Arg Ser Phe Val
Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val Val Pro
Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30 Leu Ser Asn Asn
Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35 40 45 Arg Ala
Ser Gly Ala Asp Lys Arg Lys Tyr Thr Ile Lys Leu Glu Val 50 55 60
Pro Lys Ile Val Thr Gln Val Val Asn Gly Val Glu Leu Pro Val Ser 65
70 75 80 Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro Ile
Phe Ala 85 90 95 Ala Thr Asp Asp Val Thr Val Ile Ser Lys Ser Leu
Ala Gly Leu Phe 100 105 110 Lys Val Gly Asp Pro Ile Ala Asp Ala Ile
Ser Ser Gln Ser Gly Phe 115 120 125 Tyr Ala 130
74130PRTEnterobacteria phage TH1 74Met Ala Thr Leu Arg Ser Phe Val
Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val Val Pro
Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30 Leu Ser Asn Asn
Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35 40 45 Arg Ala
Ser Gly Ala Asp Lys Arg Lys Tyr Thr Ile Lys Leu Glu Val 50 55 60
Pro Lys Ile Val Thr Gln Val Val Asn Gly Val Glu Leu Pro Val Ser 65
70 75 80 Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro Ile
Phe Ala 85 90 95 Ala Thr Asp Asp Val Thr Val Ile Ser Lys Ser Leu
Ala Gly Leu Phe 100 105 110 Lys Val Gly Asn Pro Ile Ala Asp Ala Ile
Ser Ser Gln Ser Gly Phe 115 120 125 Tyr Ala 130
75130PRTEnterobacteria phage T12 75Met Ala Thr Leu Arg Ser Phe Val
Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val Val Pro
Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30 Leu Ser Asn Asn
Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35 40 45 Arg Ala
Ser Gly Ala Asp Lys Arg Lys Tyr Thr Ile Lys Leu Glu Val 50 55 60
Pro Lys Ile Val Thr Gln Val Val Asn Gly Val Glu Leu Pro Val Ser 65
70 75 80 Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro Ile
Phe Ala 85 90 95 Ala Thr Asp Asp Val Thr Val Ile Ser Lys Ser Leu
Ala Gly Leu Phe 100 105 110 Lys Val Gly Asn Pro Ile Ala Asp Ala Ile
Ser Ser Gln Ser Gly Phe 115 120 125 Tyr Ala 130
76130PRTEnterobacteria phage FR 76Met Ala Ser Asn Phe Glu Glu Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Lys Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Asn Asn Arg Lys Tyr Thr Val Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Val Gln Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Met Asn Met Glu Leu Thr Ile Pro
Val Phe 85 90 95 Ala Thr Asn Asp Asp Cys Ala Leu Ile Val Lys Ala
Leu Gln Gly Thr 100 105 110 Phe Lys Thr Gly Asn Pro Ile Ala Thr Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
77130PRTEnterobacteria phage R17 77Met Ala Ser Asn Phe Thr Gln Phe
Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile Ser Ser
Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg
Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val 65
70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
78132PRTEnterobacteria phage Qbeta 78Ala Lys Leu Glu Thr Val Thr
Leu Gly Asn Ile Gly Lys Asp Gly Lys 1 5 10 15 Gln Thr Leu Val Leu
Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30 Ala Ser Leu
Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45 Thr
Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55
60 Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys
65 70 75 80 Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe
Ser Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val
Arg Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile
Asp Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala Tyr 130
* * * * *
References