U.S. patent application number 09/769396 was filed with the patent office on 2001-11-15 for gene coded for interleukin-2 polypeptide, recombinant dna carrying the said gene, a living cell line possessing the recombinant dna, and method for producing interleukin-2 using the said cell.
This patent application is currently assigned to AJINOMOTO CO., INC.. Invention is credited to Hamuro, Junji, Kashima, Nobukazu, Matsui, Hiroshi, Muramatsu, Masami, Sugano, Haruo, Taniguchi, Tadatsugu.
Application Number | 20010041362 09/769396 |
Document ID | / |
Family ID | 27550424 |
Filed Date | 2001-11-15 |
United States Patent
Application |
20010041362 |
Kind Code |
A1 |
Taniguchi, Tadatsugu ; et
al. |
November 15, 2001 |
Gene coded for interleukin-2 polypeptide, recombinant DNA carrying
the said gene, a living cell line possessing the recombinant DNA,
and method for producing interleukin-2 using the said cell
Abstract
A gene coded for a polypeptide which possesses interleukin-2 is
isolated, and connected with a vector DNA which is capable of
replicating in a procaryotic or eucaryotic cell at a position
downstream of a promoter gene in the vector obtaining a recombant
DNA, with which the cell is transformed to produce
interleukin-2.
Inventors: |
Taniguchi, Tadatsugu;
(Tokyo, JP) ; Muramatsu, Masami; (Tokorozawa-shi,
JP) ; Sugano, Haruo; (Tokyo, JP) ; Matsui,
Hiroshi; (Yokohama-shi, JP) ; Kashima, Nobukazu;
(Yokohama-shi, JP) ; Hamuro, Junji; (Yokohama-shi,
JP) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
AJINOMOTO CO., INC.
|
Family ID: |
27550424 |
Appl. No.: |
09/769396 |
Filed: |
January 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09769396 |
Jan 26, 2001 |
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09046786 |
Mar 24, 1998 |
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09046786 |
Mar 24, 1998 |
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08621097 |
Mar 22, 1996 |
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5795777 |
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08621097 |
Mar 22, 1996 |
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08516563 |
Aug 18, 1995 |
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5795769 |
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08516563 |
Aug 18, 1995 |
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07814049 |
Dec 26, 1991 |
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5620868 |
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07814049 |
Dec 26, 1991 |
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07332364 |
Apr 3, 1989 |
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07332364 |
Apr 3, 1989 |
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07036309 |
Apr 7, 1987 |
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07036309 |
Apr 7, 1987 |
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06463496 |
Feb 3, 1983 |
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4738927 |
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Current U.S.
Class: |
435/325 ;
435/69.52; 536/23.5 |
Current CPC
Class: |
C12N 15/81 20130101;
Y10S 930/141 20130101; C07K 14/55 20130101; Y10S 435/849 20130101;
C12N 15/70 20130101 |
Class at
Publication: |
435/325 ;
435/69.52; 536/23.5 |
International
Class: |
C12P 021/04; C07H
021/04; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 1982 |
JP |
51122/1982 |
May 18, 1982 |
JP |
82509/1982 |
Dec 15, 1982 |
JP |
219518/1982 |
Dec 24, 1982 |
JP |
229619/1982 |
Dec 27, 1982 |
JP |
234607/1982 |
Dec 29, 1982 |
JP |
230371/1982 |
Claims
What is claimed as new and desired to be secured by letters patent
of the united states is:
1. A cloned gene coded for a polypeptide possessing the activity of
interleukin-2.
2. The gene of claim 1, which is prepared from a messenger RNA
produced by an interleukin-2 producing mammalian cell line.
3. The gene of claim 2, wherein said messenger RNA is obtainable as
a sediment of 11 to 12S of sucrose density gradient
centrifugation.
4. The gene of claim 2, wherein said mammalian cell line is a human
T-lymphocyte, a transformed human T-lymphocyte or a human T-cell
hybridoma.
5. The gene of claim 1, which has sites cleaved with restriction
endonuclease in the order of Bst NI, Xba I and Bst NI from
5'-terminus of the coding sequence.
6. The gene of claim 1, which has sites cleaved with restriction
endonuclease in the order of Dde I, Hinf I, Bst NI, Xba I, Bst NI
and Sau 3A from 5'-terminus of the coding sequence.
7. The gene of claim 1, which has the base sequence shown in FIG. 2
(a).
8. The gene of claim 1, of which the base sequence initiates from
ATG sequence at position 48 to 50 and has the sequential bases
following the ATG sequence up to at least ACT sequence at position
504 to 506 in FIG. 2 (a).
9. The gene of claim 1, of which the base sequence initiates from
GCA sequence at position 108 to 110 and has the sequential bases
following the GCA sequence up to at least ACT sequence at position
504 to 506 in FIG. 2 (a).
10. The gene of claim 1, of which the base sequence initiates from
CCT sequence at position 111 to 113 and has the sequential bases
following the CCT sequence up to at least ACT sequence at position
504 to 506 in FIG. 2 (a).
11. The gene of claim 1, of which the base sequence initiates from
A at position 1 and has the sequential bases following the A ending
at ACT sequence at position 504 to 506 in FIG. 2 (a).
12. The gene of claim 8, of which the base sequence ends at ACT
sequence at position 504 to 506 in FIG. 2 (a).
13. The gene of claim 9, of which the base sequence ends at ACT
sequence at position 504 to 506 in FIG. 2 (a).
14. The gene of claim 10, of which the base sequence ends at ACT
sequence at position 504 to 506 in FIG. 2 (a).
15. The gene of claim 1, of which the base sequence initiates from
A at position 1 and has the sequential bases following the A ending
at TGA sequence at position 507 to 509 in FIG. 2 (a).
16. The gene of claim 8, of which the base sequence ends at TGA
sequence at position 507 to 509 in FIG. 2 (a).
17. The gene of claim 9, of which the base sequence ends at TGA
sequence at position 507 to 509 in FIG. 2 (a).
18. The gene of claim 10, of which the base sequence ends at TGA
sequence at position 507 to 509 in FIG. 2 (a).
19. The gene of claim 1, of which the base sequence initiates from
A at position 1 and has the sequential bases following the A ending
at C at position 801 in FIG. 2 (a).
20. The gene of claim 8, of which the base sequence ends at C at
position 801 in FIG. 2 (a).
21. The gene of claim 9, of which the base sequence ends at C at
position 801 in FIG. 2 (a).
22. The gene of claim 10, of which the base sequence ends at C at
position 801 in FIG. 2 (a).
23. The gene of claim 8, of which the base sequence ends at poly
(A) in FIG. 2 (a).
24. The gene of claim 9, of which the base sequence ends at poly
(A) in FIG. 2 (a).
25. The gene of claim 10, of which the base sequence ends at poly
(A) in FIG. 2 (a).
26. The gene of claim 1, which has the base sequence corresponding
to Amino Acid Sequence I in FIG. 2 (b).
27. The gene of claim 1, which has the base sequence corresponding
to Amino Acid Sequence II in FIG. 2 (b).
28. The gene of claim 1, which has the base sequence corresponding
to Amino Acid Sequence III in FIG. 2 (b).
29. A DNA, prepared recombinantly which comprises a gene coded for
a polypeptide which possesses the activity, of inter-leukin-2, and
a vector DNA capable of propagating in a procaryotic or eucaryotic
the coding sequence of said gene being located at a position
downstream of a promoter sequence.
30. The recombinant DNA of claim 29, wherein said gene is prepared
with a messenger RNA-produced by an interleukin-2 producing
mammalian cell line.
31. The recombinant DNA of claim 30, wherein said mammalian cell is
a human T-lymphocyte, a transformed human T-lymphocyte or a human
T-cell hybridoma.
32. The recombinant DNA of claim 30, wherein said messenger RNA is
obtainable as a sediment of 11 to 12S of sucrose density gradient
centrifugation.
33. The recombinant DNA of claim 29, wherein said gene has sites
cleaved with restriction endonuclease in the order of Bst NI, Xba I
and Bst NI from 5'-terminus of the coding sequence.
34. The recombinant DNA of claim 29, wherein said gene has sites
cleaved with restriction endonuclease in the order of Dde I, Hinf
I, Bst NI, Xba I, Bst NI and Sau 3A from 5'-terminus of the coding
sequence.
35. The recombinant DNA of claim 29, wherein said gene has the base
sequence shown in FIG. 2 (a).
36. The recombinant DNA of claim 29, wherein the base sequence of
said gene initiates from ATG sequence at position 48 to 50 and has
the sequential bases following the ATG sequence up to at least ACT
sequence at position 504 to 506 in FIG. 2 (a).
37. The recombinant DNA of claim 29, wherein the base sequence of
said gene initiates from GCA sequence at position 108 to 110 and
has the sequential bases following the GCA sequence up to at least
ACT sequence at position 504 to 506 in FIG. 2 (a).
38. The recombinant DNA of claim 29, wherein the base sequence of
said gene initiates from CCT sequence at position 111 to 113 and
has the sequential bases following the CCT sequence up to at least
ACT sequence at position 504 to 506 in FIG. 2 (a).
39. The recombinant DNA of claim 29, wherein the base sequence of
said gene initiates from A at position 1 and has the sequential
bases following the A ending at ACT sequence at position 504 to 506
in FIG. 2 (a).
40. The recombinant DNA of claim 36, wherein the base sequence of
said gene ends at ACT sequence at position 504 to 506 in FIG. 2
(a).
41. The recombinant DNA of claim 37, wherein the base sequence of
said gene ends at ACT sequence at position 504 to 506 in FIG. 2
(a).
42. The recombinant DNA of claim 38, wherein the base sequence of
said gene ends at ACT sequence at position 504 to 506 in FIG. 2
(a).
43. The recombinant DNA of claim 29, wherein the base sequence of
said gene initiates from A at position 1 and has the sequential
bases following the A ending at TGA sequence at position 507 to 509
in FIG. 2.
44. The recombinant DNA of claim 36, wherein the base sequence of
said gene ends at TGA sequence at position 507 to 509 in FIG. 2
(a).
45. The recombinant DNA of claim 37, wherein the base sequence of
said gene ends at TGA sequence at position 507 to 509 in FIG. 2
(a).
46. The recombinant DNA of claim 38, wherein the base sequence of
said gene ends at TGA sequence at position 507 to 509 in FIG. 2
(a).
47. The recombinant DNA of claim 29, wherein the base sequence of
said gene initiates from A at position 1 and has the sequential
bases following the A ending at C at position 801 in FIG. 2
(a).
48. The recombinant DNA of claim 36, wherein the base sequence of
said gene ends at C at position 801 in FIG. 2 (a).
49. The recombinant DNA of claim 37, wherein the base sequence of
said gene ends at C at position 801 in FIG. 2 (a).
50. The recombinant DNA of claim 38, wherein the base sequence of
said gene ends at C at position 501 in FIG. 2 (a).
51. The recombinant DNA of clam 36, wherein the base sequence of
said gene ends at poly (A) in FIG. 2 (a).
52. The recombinant DNA of claim 37, wherein the base sequence of
said gene ends at poly (A) in FIG. 2 (a).
53. The recombinant DNA of claim 38, wherein the base sequence of
said gene ends at poly (A) in FIG. 2 (a).
54. The recombinant DNA of claim 29, wherein the base sequence of
said gene corresponds to Amino Acid Sequence I in FIG. 2 (b).
55. The recombinant DNA of claim 29, wherein the base sequence of
said gene corresponds to Amino Acid Sequence II in FIG. 2 (b).
56. The recombinant DNA of claim 29, wherein the base sequence of
said gene corresponds to Amino Acid Sequence III in FIG. 2 (b).
57. The recombinant DNA of claim 29, wherein said procaryotic cell
line belongs to the genus Escherichia.
58. The recombinant DNA of claim 29, wherein said procaryotic cell
line belongs to Escherichia coli.
59. The recombinant DNA of claim 29, wherein said eucaryotic cell
line belongs to the genus Saccharomyces.
60. The recombinant DNA of claim 29, wherein said eucaryotic cell
line belongs to the genus Saccharomyces cerevicea.
61. The recombinant DNA of claim 29, wherein said eucaryotic cell
line is a monkey cell transformed with SV-40 constitutively
expressing large T antigen.
62. A cell of eucarlote or procaryote transformed with a
recombinant DNA comprising a gene coding for a polypeptide
possessing the activity of interleukin-2 and a vector DNA capable
of propagating in said cell, and the coding sequence of said gene
being located at a position downstream of a promoter sequence.
63. The cell of claim 62, wherein said gene is prepared with a
messenger RNA produced by an interleukin-2 producing mammalian
cell.
64. The cell of claim 63, wherein said mammalian cell is a human
T-lymphocyte, a transformed human T-lymphocyte or a T-cell
hybridoma.
65. The cell of claim 63, wherein said messenger RNA is obtainable
as a sediment of 11 to 12S of sucrose density gradient
centrifugation.
66. The cell of claim 63, wherein said gene has sites cleaved with
restriction endonuclease in the order of Bst NI, Xba I and Bst NI
from 5'-terminus of the coding sequence.
67. The cell of claim 62, wherein said gene has sites cleaved with
restriction endonuclease in the order of Dde I, Hinf I, Bst NI, Xba
I, Bst NI and Sau 3A from 5'-terminus of the coding sequence.
68. The cell of claim 62, wherein said gene has the base sequence
shown in FIG. 2 (a).
69. The cell of claim 62, wherein the base sequence of said gene
initiates from ATG sequence at position 48 to 50 and has the
sequential bases following the ATG codon up to at least ACT
sequence at position 504 to 506 in FIG. 2 (a).
70. The cell of claim 62, wherein the base sequence of said gene
initiates from GCA sequence at position 108 to 110 and has the
sequential bases following the CCA sequence up to at least ACT
sequence at position 504 to 506 in FIG. 2 (a).
71. The cell of claim 62, wherein the base sequence of said gene
initiates from CCT sequence at position 111 to 113 and has the
sequential bases following the CCT sequence up to at least ACT
sequence at position 504 to 506 in FIG. 2 (a).
72. The cell of claim 62, wherein the base sequence of said gene
initiates from A at position 1 and has the sequential bases
following the A ending at ACT sequence at position 504 to 506 in
FIG. 2 (a).
73. The cell of claim 69, wherein the base sequence of said gene
ends at ACT sequence at position 504 to 506 in FIG. 2 (a).
74. The cell of claim 70, wherein the base sequence of said gene
ends at ACT sequence at position 504 to 506 in FIG. 2 (a).
75. The cell of claim 71, wherein the base sequence of said gene
ends at ACT sequence at position 504 to 506 in FIG. 2 (a).
76. The cell of claim 62, wherein the base sequence of said gene
initiates from A at position 1 and has the sequential bases
following the A ending at TGA sequence at position 507 to 509 in
FIG. 2 (a).
77. The cell of claim 69, wherein the base sequence of said gene
ends at TGA sequence at position 507 to 509 in FIG. 2 (a).
78. The cell of claim 70, wherein the base sequence of said gene
ends at TGA sequence at position 507 to 509 in FIG. 2 (a).
79. The cell of claim 71, wherein the base sequence of said gene
ends at TGA sequence at position 507 to 509 in FIG. 2 (a).
80. The cell of claim 62, wherein the base sequence of said gene
initiates from A at position I and has the sequential bases
following the A ending at C at position 801 in FIG. 2 (a).
81. The cell of claim 69, wherein the base sequence of said gene
ends at C at position 801 in FIG. 2 (a).
82. The cell of claim 70, wherein the base sequence of said gene
ends at C at position 801 in FIG. 2 (a).
83. The cell of claim 71, wherein the base sequence of said gene
ends at C at position 801 in FIG. 2 (a).
84. The cell of claim 69, wherein the base sequence of said gene
ends at poly (A) in FIG. 2 (a).
85. The cell of claim 70, wherein the base sequence of said gene
ends at poly (A) in FIG. 2 (a).
86. The cell of claim 71, wherein the base sequence of said gene
ends at poly (A) in FIG. 2 (a).
87. The cell of claim 65, wherein the base sequence of said gene
corresponds to Amino Acid Sequence I in FIG. 2 (b).
88. The cell of claim 65, wherein the base sequence of said gene
corresponds to Amino Acid-Sequence II in FIG. 2 (b).
89. The cell of claim 65, wherein the base sequence of said gene
corresponds to Amino Acid Sequence III in FIG. 2 (b).
90. The cell of claim 62, wherein said procaryotic cell belongs to
the genus Escherichia.
91. The cell of claim 62, wherein said procaryotic cell belongs to
Escherichia Coli.
92. The cell of claim 62, wherein said eucaryotic cell belongs to
the genus Saccharomyces.
93. The cell of claim 62, wherein said eucaryotic cell belongs to
the genus Saccharomyces cerevicea.
94. The cell of claim 62, wherein said eucaryotic cell is a monkey
cell transformed with SV-40 constitutively expressing large T
antigen.
95. A method for producing interleukin-2 which comprises culturing
aleucaryotic or procaryotic cell culture medium, transformed with a
recombinant DNA to produce interleukin-2 and recovering the
produced interleukin-2; said recombinant DNA comprising a gene
coded for a polypeptide which posesses the activity of
interleukin-2 and a vector DNA which is capable of replicating in
said cell, and the coding sequence of said gene being located at a
position downstream of a promoter sequence.
96. The method of claim 95, wherein said gene is prepared with a
messenger RNA produced by an interleukin-2 producing mammalian cell
line.
97. The method of claim 96, wherein said mammalian cell is a human
T-lymphocyte, a transformed human lymphocyte or a T-cell
hybridoma.
98. The method of claim 96, wherein said messenger RNA is
obtainable as a sediment of 11 to 12S of sucrose density gradient
centrifugation.
99. The cell of claim 95, wherein said gene has sites cleaved with
restriction endonuclease in the order of Bst NI, Xba I and Bst NI
from 5'-terminus of the coding sequence.
100. The method of claim 95, wherein said gene has sites cleaved
with restriction endonuclease in the order of Dde I, Hinf I, Bst
NI, Xba I, Bst NI and Sau 3A from 5'-terminus of the coding
sequence.
101. The method of claim 95, wherein said gene has the base
sequence shown in FIG. 2 (a).
102. The method of claim 95, wherein the base sequence of said gene
initiates from ATG sequence at position 48 to 50 and has the
sequential bases following the ATG sequence up to at least ACT
sequence at position 504 to 506 in FIG. 2 (a).
103. The method of claim 95, wherein the base sequence of said gene
initiates from GCA sequence at position 103 to 110 and has the
sequential bases following the GCA sequence up to at least ACT
sequence at position 504 to 506 in FIG. 2 (a).
104. The method of claim 95, wherein the base sequence of said gene
initiates from CCT sequence at position 111 to 113 and has the
sequential bases following the CCT sequence up to at least ACT
sequence at position 504 to 506 in FIG. 2 (a).
105. The method of claim 95, wherein the base sequence of said gene
initiates from A at position 1 and has the sequential bases
following the A ending at ACT sequence at position 504 to 506 in
FIG. 2 (a).
106. The method of claim 102, wherein the base sequence of said
gene ends at ACT sequence at position 504 to 506 in FIG. 2 (a).
107. The method of claim 103, wherein the base sequence of said
gene ends at ACT sequence at position 504 to 506 in FIG. 2 (a).
108. The method of claim 104, wherein the base sequence of said
gene ends at ACT sequence at position 504 to 506 in FIG. 2 (a).
109. The method of claim 95, wherein the base sequence of said gene
initiates from A at position 1 and has the sequential bases
following the A ending at TGA sequence at position 507 to 509 in
FIG. 2 (a).
110. The method of claim 102, wherein the base sequence of said
gene ends at TGA sequence at position 507 to 509 in FIG. 2 (a).
111. The method of claim 103, wherein the base sequence of said
gene ends at TGA sequence at position 507 to 509 in FIG. 2 (a).
112. The method of claim 104, wherein the base sequence of said
gene ends at TGA sequence at position 507 to 509 in FIG. 2 (a).
113. The method of claim 95, wherein the base sequence of said gene
initiates from A at position 1 and has the sequential bases
following the A ending at C at position 801 in FIG. 2 (a).
114. The method of claim 102, wherein the base sequence of said
gene ends at C at position 801 in FIG. 2 (a).
115. The method of claim 103, wherein the base sequence of said
gene ends at C at position 801 in FIG. 2 (a).
116. The method of claim 104, wherein the base sequence of said
gene ends at C at position 801 in FIG. 2 (a).
117. The method of claim 102, wherein the base sequence of said
gene ends at poly (A) in FIG. 2 (a).
118. The method of claim 103, wherein the base sequence of said
gene ends at poly (A) in FIG. 2 (a).
119. The method of claim 104, wherein the base sequence of said
gene ends at poly (A) in FIG. 2 (a).
120. The method of claim 101, wherein the base sequence said gene
corresponds to Amino Acid Sequence I in FIG. 2 (b).
121. The method of claim 101, wherein the base sequence of said
gene corresponds to Amino Acid Sequence II in FIG. 2 (b).
122. The method of claim 101, wherein the base sequence of said
gene corresponds to Amino Acid Sequence III in FIG. 2 (b).
123. The method of claim 95, wherein said procaryotic cell belongs
to the genus Escherichia.
124. The method of claim 95, wherein said procaryotic belongs to
Escherichia coli.
125. The method of claim 95, wherein said eucaryotic cell belongs
to the genus Saccharomyces.
126. The method of claim 95, wherein said eucaryotic cell belongs
to the genus Saccharomyces cerevicea.
127. The method of claim 95, wherein said eucaryotic cell is a
monkey cell transformed with SV-40 constitutively expressing large
T antigen.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a gene, especially a cloned
gene coding for a interleukin-2 polypeptide, recombinant DNA
carrying the gene, a living cell line possessing the recombinant
DNA and a method for producing interleukin-2 using the cell
line.
[0003] 2. Brief Description of the Prior Art
[0004] Interleukin 2 (hereinafter referred to as "IL-2"), formerly
referred to as T cell growth factor, is a soluble protein
(generally known as "lymphokine"), and is produced from T cells
activated with a lectin or an antigen (Morgan. D. A., et al.,
Science, 193, 1007-1008 (1976), Gillis, S. et al., J. Immunol.,
120, 2027-2033 (1978). Interleukin 2 (IL-2) is capable of
modulating lymphocyte reactivity and promoting the in vitro
long-term culture of antigen specific effector T-lymphocytes
(Gillis. S. et al., Nature 268, 154-156 (1977)). IL-2 is also known
to manifest other relevant biological activities such as
enhancement of thymocyte mitogenesis (Chen, B. M. et al., Cell.
Immunol., 22, 211-224, (1977), Shaw, J. et al., J. Immunol. 120,
1967-1973, (1978)), induction of cytotoxic T cell reactivity
(Wagner, H. et al., Nature, 284, 278-280, (1980)) and anti-SRBC
plaque forming cell responses (Gillis, S. et al., J. Exp. Med.,
149, 1960-1968, (1979)) in cultures of nude mouse spleen cells.
Accordingly, this lymphocyte regulatory substance is useful in
potentiating humoral and cellular immune responses and in restoring
immune deficient state to a normal humoral and cellular immune
state. These identified immunological activities of IL-2 strongly
indicate that IL-2 is useful for medical immunotherapy against
immunological disorders including neoplastic diseases, bacterial or
viral infections, immune deficient diseases, autoimmune diseases
etc. (Papermaster, B. et al., Adv. Immunopharm., 507, (1980)). Like
inteferons, IL-2 has been shown to augment natural killer cell
activity, suggesting a potential use in the treatment of neoplastic
diseases. Furthermore, IL-2 enables the maintenance of cultures of
functional monoclonal T cells and hence appears to play a key role
in the studying of the molecular nature of T cell differentiation,
and of the mechanism of differentiated T cell functions as well as
the mechanism of T cell antigen receptors. It is also useful for
producing, by long term culturing of monoclonal T cell,,many other
T cell derived lymphokines, which are useful in a wide range of
fields. In addition, IL-2 production and the response c lymphocytes
to IL-2 can be important parameters of immunological functions
which are useful in the clinical diagnosis of aberrant immunity.
IL-2 has been produced in the prior art by stimulating mouse, rat
or human lymphocytes with a mitogen (Gillis. S. et al., Nature,
268, 154-156, 1977, Farrar, J. et al., J. Immunol., 121,
1353-1360,(1978) Gillis, S. et al., J. Immunol., 120, 2027-2033,
1978,)). By stimulating human peripheral blood mononuclear
lymphocytes with a mitogen (Gillis. S. et al., J. Immunol., 124,
1954-1962, (1980)). Gillis et al. reported the preparation of
murine IL-2 from murine T cell lymphoma cell line (Gillis. S. et
al, J. Immunol., 125, 2570-2578 (1980)) and the preparation of
human IL-2 from a human leukemia cell line (Gillis, S. et al., J.
Exp. Med., 152, 1709-1719, (1980)).
[0005] The above noted articles by Gillis et. al. discuss the
method of producing human IL-2 from mitogen-stimulated human T cell
leukemia cell line by cell culture methods. However, such a
technique results in undesirably low concentrations of human IL-2,
and necessiates complex purification procedures to obtain even
small amounts of IL-2 from a huge volumes of culture media.
Moreover, since the human T cell leukemia cell lines produce trace
amounts of many other biologically active substances which are
analogous to human IL-2, significant difficulties are encountered
in isolating IL-2 from these other immunologically active
molecules, or in isolating IL-2 from the occasionally present toxic
lectins.
[0006] As an alternative approach it would seem to be desirable to
use recombinant DNA (DNA is an abbreviation for deoxyribonucleic
acid) techniques as are used in the production of other
biologically active human proteins, such as interferons, (Gray, P.
W. et al., Nature, 295, 503-508, (1981), Nagata, S., et. al.,
Nature, 284, 316-320, (1980), Taniguchi, T. et. al., Gene, 10,
11-15, (1980)) to produce IL-2. However to date, attempts at the
production of IL-2, by recombinant DNA techniques have not been
successful. For instance, it was reported in "NIKKEI BIOTECHNOLOGY
(Japan), No. 19, Jul. 5, 1982 that attempts to construct
IL-2-producing organisms by recombinant DNA were unsuccessful,
probably due to the fact that the gene coding for IL-2 polypeptide
had not yet been cloned.
[0007] A need therefore, continues to exist for a cloned gene,
coded for interleukin-2, and for DNA produced recombinantly which
carries the gene. A need also continues to exist for a living cell
line which possesses the recombinantly produced DNA, and for a
method of producing interleukin-2 using the cell line.
SUMMARY OF THE INVENTION
[0008] These and other objects of the present invention which will
hereinafter become more readily apparent from the following
description have been attained by providing:
[0009] A cloned gene coded for a polypeptide which possesses the
activity of IL-2, and by providing:
[0010] A DNA, produced recombinantly which comprises a gene coded
for a polypeptide possesses the activity of IL-2, and a vector DNA
capable of replicating in a procaryotic or eucaryotic cell, the
coding sequence of the said gene being located at a position
down-stream of a promoter sequence.
[0011] Further in accordance with the present invention,
procaryotic or eucaryotic cell lines are provided which have been
transformed to produce IL-2 with the above said DNA, vector DNA and
coded gene. DNA capable of replicating in the cell; the coding
sequence of said the gene being located at a position downstream of
a promoter sequence.
[0012] In accordance with the present invention, IL-2 is produced
by aerobically culturing a medium containing a eucaryotic or
procaryotic cell line which has been transformed to produce IL-2
with a DNA which has been recombinantly modified by invention of a
gene coded to produce which possesses the activity of IL-2, and, by
insertion of a vector DNA, which is capable of replicating in the
cell; the coding sequence of said gene being located at a position
downstream of a promoter sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily attained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0014] FIG. 1 shows a restriction endonuclease cleavage map of a
cloned gene coded to produce a polypeptide which possesses the
activity of IL-2 (hereinafter referred to as "IL-2
polypeptide").
[0015] FIG. 2(a) shows the base sequence of the cloned gene.
[0016] FIG. 2(b) shows Amino Acid Sequence I, and Amino Acid
Sequences II and III, of the polypeptides which possess IL-2
activity.
[0017] FIG. 3 is a flow chart showing the construction of a
recombinant DNA (pCEIL-2), in which the coded gene is inserted.
[0018] FIG. 4 shows the plasmid vector pTrS-3.
[0019] FIGS. 5(a), 5(b) and 5(c) are flow charts showing the
construction of recombinant DNAs (pTIL 2-22, pTIL2-21, pTIL2-14 and
pTIL2-15) using pTrS-3 as a vector.
[0020] FIGS. 6 is a flow chart showing the construction of a
recombinant DNA (pKIL2-21) using pKT218 as a vector.
[0021] FIG. 7 is a flow chart showing the construction of a
recombinant DNA (pTuIL2-22) using pTUB1P-5 as a vector.
[0022] FIG. 8 is vector DNAs which are capable of replicating in a
cell of Saccaromyces cerevicea.
[0023] In the Figures, "A", "G", "C" and "T" represent
deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid and
thymidylic acid, respectively.
BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENT
[0024] The cloned gene, coded for an IL-2 polypeptide, may be
obtained by transcription of messenger RNA (mRNA; "RNA" is an
abbreviation for ribonucleic acid) corresponding to IL-2
(hereinafter referred to as "IL-2 mRNA"), originating from a
mammalian cell which is characterized by the capability of
producing a polypeptide which possesses IL-2 activity, to a
complementary DNA (cDNA). The single stranded cDNA (ss-cDNA)
obtained can be converted into a double stranded cDNA (ds-cNA).
[0025] The mRNA used as a template for the preparation of cDNA can
be conventionally separated from a mammalian cell capable of
producing IL-2 polypeptide. The separated RNA is polyadenylated
(Gills. et al., Immunological Rew., 63, 167-209 (1982)), and the
polyadenylated RNA can be fractionated by, for example,
centrifugation on a sucrose density gradient as a sediment of 11 to
12S. Occasionally mRNA of 13S will demonstrate IL-2 mRNA activity,
and in those instances, it is presumed that the mRNA is in an
aggregated form of 11 to 12 S mRNA.
[0026] The mammalian cells capable of producing IL-2 which are the
source of mRNA of the present invention, may be T-lymphocytes, such
as peripheral blood mononuclear cells, tonsil cells, spleen cells
or the like, which are operationally obtainable from mammals. The
cells may be conventionally pretreated such as with a nylon column,
antiserum-complement, density gradient fractionation, multiple
enzyme treatment such as a combination of neuraminidase and
galactose oxidase, by x-ray irradiation or with trypsin to confer
the cells with the IL-2 productivity or to increase the IL-2
activity. Also cloned T lymphocytes, obtained from the said
mammalian cells after cultivation in the presence of T cell growth
factor, may be also used as a source of mRNA and is the preferred
T-lymphocytes. Transformed lymphocyte cell lines such as T
lymphocytes derived from leukemia or lymphoma cell line per se or
from their derivatives obtained by pretreatment or mutation by the
methods mentioned above, or the cloned transformed cell lines are
preferred as sources of the mRNA. Evidently, cloned cells line
usually contain larger amounts of IL-2 mRNA as compared to parental
bulk cell lines. T cell hybridomas, obtained by fusion of the
lymphocyte derived cells mentioned above and tumor cell lines, such
as CEM, Molt 4F, and BW5147, are also preferred mammalian cell
lines for use in this invention. In such instance the lymphocyte
derived cell lines include (1) constitutive producers of IL-2 and
(2) those which are producers of IL-2 only in the presence of a
mitogen introduced into the culture, either in the absence or
presence of other IL-2 production co-stimulatory cells.
[0027] In order to generate IL-2 mRNA in constitutive IL-2 producer
cells, the constitutive IL-2 producer cells are cultured under
conditions commonly known in the field of cell culture. For the
generation of the mRNA in cells producing IL-2 only in the presence
of mitogen, cultured cells are washed extensively with culture
medium and resuspended in a culture medium, such as Rosewell Park
Memorial Institute 1640 (hereinafter "RPMI 1640"), Dulbecco
Modified Eagle Medium (hereinafter "DMEM") or in Click's medium,
which may or may not contain serum. These culture media may be
supplemented with various additives, such as penicillin,
streptomycin or other antibiotics, or with fresh L-glutamine, Hepes
buffer and sodium bicarbonate in a concentrations as are generally
used in the field of cell culture. The preferred cell density may
be from 0.5 to 4.times.10.sup.6 cells/ml. To induce activation of
the mRNA and production of IL-2, appropriate stimulants are added.
Suitable such stimulants include mitogens, neuraminidase, galactose
oxidase, zinc derivatives such as zinc chloride, or lymphocyte
activating substances originated from microorganisms, such as
protein A, streptolysin-O. The stimulated cells are recovered and
washed. The co-presence of macrophages or dendritic cells during
the mitogen stimulation may also activate the mRNA, or may increase
the amount of the activated mRNA. Likewise the co-presence of cell
lines derived from B lymphocytes or B lymphocyte lines, such as
Raji, Daudi, K562, and BALL-1 may activate the mRNA or increase the
amount of activated mRNA.
[0028] To propagate the mammalian cells, they are maintained in an
in vitro cell culture or in histocompatibility matched animals,
under normal conditions. When in vitro culture maintenance is used
to prepare the source of mRNA, the cells may be grown in any of the
culturing media as were previously found to foster growth of T
cells. These culture media may or may not be supplemented with
mammal serum, serum component or serum albumin. The culture period
for the activation of the mRNA will correspond to the period
necessary for the activation of cells to generate the mRNA. This
period usually coincides with the time needed to start the
excretion of IL-2 into the culture medium. The preferred period may
be from 3 to 12 hours after addition of a stimulant, such as a
mitogen. Undue prolongation of the culture period may occasionally
result in the decomposition of the generated IL-2 mRNA. During the
course of the activation of IL-2 producing cells, phorbol esters,
such as PMA or TPA may preferably utilized in a concentration from
10 to 50 ng/ml to boost the level of activation.
[0029] The above described process for activation of IL-2 mRNA may
be carried out at temperatures ranging from 32 to 38.degree. C. in
a humidified atmosphere and in a pH of approximately 7.0 to
7.4.
[0030] The procedures to obtain and culture mammalian cells capable
of producing IL-2 will now be explained.
[0031] (1) Acquisition of Constitutively IL-2 Producing Cell
Line.
[0032] Jurkat cell line of human leukemic T cell (freely available
from Fred Hutchinson Cancer Institute, Seattle, United States, Salk
Institute, San Diego, United States, German Cancer Center,
Heidelberg, West Germany) is suspended in Click's medium at a cell
density of 1.times.10.sup.6 cells/ml and 8.times.10.sup.3 x-ray is
irradiated at irradiation rate of 150 R/min. Thereafter 0.1 cells
of the thus irradiated cells per 200 .mu.l of medium are inoculated
into Click's medium containing 5% FCS in 96 well flat-bottom
microplates (Falcon 3072) and cultured for 3 weeks at 37.degree. C.
in 5% CO.sub.2 incubator (cloning by limiting dilution method). The
grown viable cells are transferred into 24 well culture plate
(Nunc) before the cell layer becomes confluent and is further
cultured for 5 days. The grown cells are further cultured in serum
and serum albumin free synthetic culture medium for about two days
at a initial cell density of between 1-2.times.10.sup.6/ml. The
culture supernatant is harvested by centrifugation and filtered
with 0.22 millipore filter paper to clear off debris and to
sterilize the supernatant, and then x-ray treated mutants capable
of producing IL-2 constitutively are selected and cloned by
measuring the IL-2 activity present in the supernatant.
[0033] (2) Acquisition of IL-2 Producer Cell from Human Peripheral
Blood Mononuclear Cells.
[0034] Human peripheral blood is harvested and peripheral blood
lymphocytes (hereinafter "PBL") are isolated by density gradient
centrifugation on Ficoll-Hypaque. The PBL is inoculated in 2 ml of
Click's medium containing 5% FCS at a cell density of 1.times.10
.sup.6 cells/ml in 24 well Nunc culture plate together with 100
.mu.l of 5 .mu.g/ml of phytohemmaglutinin-M(Gibco)(pHA), and
cultured for 48 hrs under the conditions described above. The cells
are washed and inoculated again in 1 ml of Click's medium at a cell
density of 1.times.10.sup.5 cells/ml together with 1 ml of a
conditioned medium which has been prepared from human splenocytes
stimulated by 2.5 .mu.g/ml of concanavalin A (hereinafter "Con A")
for 48 hrs, and the culture medium containing 50% conditioned
medium is exchanged in every three days to get long term culture of
human T lymphocytes from PBL. The thus prepared long term cultured
human T lymphocytes are cloned by the limiting dilution method as
described above, in the presence of human splenocytes derived
conditioned medium and the cell clones are propagated similarly.
Thereafter cloned human T lymphocytes are inoculated in 1 ml of
RPMI 1640 at a cell density of 1.times.10 .sup.6 cells/ml in 24
well Nunc culture plate in the presence of 10 .mu.g/ml of PHA and
cultured for 24 hrs at 37.degree. C. in 7.5% CO.sub.2 incubator.
The supernatants of the culture liquid are harvested, centrifuged,
filtered through a 0.22 .mu. millipore filter and assayed for IL-2
activity to specify the IL-2 producing human normal T lymphocytes
clones.
[0035] (3) Acquisition of Malignant Cell Line Derived from Human
Lymphocytes Capable of Producing IL-2 in the Presence of
Mitogen.
[0036] Jurkat cell line or cloned cell lines such as Jurkat 111
obtained by the limiting dilution method described above are able
to produce from 10 to 4,000 units/ml of IL-2 when cultured for 24
hours in a serum free synthetic medium described prebiously or in
RPMI 1640 containing 1-2% mammalian serum in the or presence of a
mitogen such as 10 .mu.g/ml Con A or 2.5 .mu.g/ml PHA. These
malignant human cell lines also produce IL-2 when cultured in the
presence of zinc chloride, protein A or picibanil.
[0037] (4) Acquisition of Cells Capable of Producing IL-2 in the
Co-Presence of a Mitogen and Other Co-Stimulatory Cells or
Co-Stimulatory Soluble Factors.
[0038] Human malignant cell line Molt 4F and some cloned cell lines
such as Jurkat J99, obtained according to the limiting dilution
method, do not produce IL-2 even when cultured for 24 to 72 hours
in the presence of lectins or mitogens in any concentration.
However, these cells become able to produce IL-2 in significant
amount (10-100 .mu./ml) during culture period of 24 hours at
37.degree. C., when cocultured with 5-10 .mu./ml interleukin 1, one
of monokines, or with 50% number of K562 or Raji cells.
[0039] The extraction of IL-2 mRNA from cells activated by the
manner as mentioned above is carried out according to the
conventional well known procedures, irrespective of the difference
of cell sources. For instance, cells are partially or completely
disrupted by addition of a detergent such as NP-40, SDS, Triton-X
and deoxycholic acid or by mechanical homogenization or
freeze-thawing. To prevent degradation of RNA by ribonuclease
during extraction of mRNA, it is preferred to add RNase inhibitors
such as heparin, polyvinylsulfate, bentonite, macaroid,
diethylpyrocarbonate or vanadyl complex. IL-2 mRNA can be obtained
from precipitated polysome in the IL-2 biosyntheis, which is
precipitated with anti-IL-2 antibody by extracting with a
detergent.
[0040] The poly A-containing mRNA can be fractionated or
concentrated by any conventional manner, such as by affinity
chromatography or batch absorption on oligo dT-cellulose, poly
U-sepharose of sepharose 2B, sucrose density gradient
centrifugation or by agarose gel electrophresis.
[0041] The mRNA fractions are then assayed for IL-2 mRNA activity
by testing biological activities of proteins translated from the
mRNA fractions or by identifying the translated protein using
monoclonal antibody against the IL-2 peptide. For instance mRNA is
usually translated into the corresponding protein by microinjection
into frog (Xenoous lauvis) egg (Gurdon, J. B., et al., Nature, 233,
177-182 (1972)) or by employing the mRNA dependent reticulolysate
or wheat germ translation cell free systems.
[0042] The activity of IL-2 may be ascertained by the microassay
procedure principally discussed by Gillis et. al (Gillis. S., et
al., J. Immunol., 120, 2027-2033 (1978)). The assay monitors the
IL-2 dependent cellular proliferation of a cytotoxic T lymphocyte
cell lines (hereinafter "CTLL") generated according to the methods
described by Gillis et al., That is, 4.times.10.sup.3 CTLL cells
are inoculated into 100 .mu.l of RPMl 1640 medium containing 2% FCS
in 96 well flat-bottomed microplates together with 100 .mu.l of the
serially diluted translation products . After 20 hours incubation
at 37.degree. C. in 5% CO.sub.2 incubator, cells are pulsed for 4
hours with 0.5 .mu.Ci of .sup.3H-TdR, harvested onto glass fibre
strips with the aid of an automated cell harvester and then the
incorporated radioactivity is measured by liquid scintillation
counting. By these assay procedures, the CTLL cells cultured in the
presence of IL-2 were found to incorporate .sup.3H-TdR in a dose
dependent manner resulting in the definite calculation of the
amount of IL-2 contained in test samples.
[0043] IL-2 possesses the activity to promote the proliferation of
T lymphocytes, which enables the measurement of IL-2 activity using
an index of T cell growth activity. That is, five CTLL cells are
transferred into 100 .mu.l of DMEM containing 2% FCS in 96 well
flat-bottomed microplates together with 100 .mu.l of the serially
diluted translation products. After 72 to 96 hours incubation at
37.degree. C. in a 5% CO.sub.2 incubator, the number of cells grown
and activated is counted under microscopy. As an positive external
control group, 100 units/ml, 10 units/ml of IL-2 are added and the
IL-2 activity of the test sample is calculated in comparison with
the number of grown viable cells in these control groups.
[0044] The thus obtained IL-2 mRNA from the most active fraction is
used as a template to synthesize ds-cDNA and the ds-cDNA is
connected with a vector DNA.Synthesis of cDNA is carried out by
conventional procedures.
[0045] At first ss-cDNA which is complementary to mRNA is prepared
in the presence of dATP, dGTP, dCTP, d-TTP employing reverse
transcriptase and using mRNA as a template and oligo-dT as a
primer. The template mRNA is then removed by a alkaline treatment
and ds-cDNA is achieved by employing reverse transcriptase or DNA
polymerase and using the above synthesizes ss-cDNA as a
[0046] DNA produced recombinantly is prepared from the ds-cDNA thus
obtained and a vector DNA containing replicon capable of
replicating in eucaryotic or procaryotic cells. The recombinant DNA
is thereafter incorporated into the host cells.
[0047] The ds-cDNA and a vector DNA capable of propagating in
eucaryotic or procaryotic cells are, prior to ligation, modified by
various procedures such as exonuclease treatment, addition of
chemically synthesied DNA pieces and G, C-tailing to give ligatable
termini to the ends of the ds-cDNA and the vector DNA. Ligation of
the ligatable DNAs is performed by, for example, T.sub.4-phage DNA
ligase in the presence of ATP and dithiothreitol.
[0048] With the recombinant DNA thus obtained, living cells are
transformed to amplify the cloned cDNA or to produce IL-2
polypeptide.
[0049] Suitable eucaryotic host organisms, which are usually used
for production of IL-2, include vertebrates, yeasts and so on. For
instance, monkey cells, e.g. CV-1 cells, transformed by an origin
defective mutant of SV-40 and expressing the SV-40 large T antigen
(COS cells) as discussed by Y. Gluzman (Cell, 33, 175-182, 1981),
mouse derived cells discussed by Ohne, S and Taniguchi, T (Nucleic
Acids Research, 10, 967-977,(1982)), and yeast host-vector systems
applied for the expression of IFN gene, discussed by R. Hitzeman et
al. (Nature, 293, 717-722,(1981)) may be used. Suitable procaryotic
host organisms include Escherichia coli, Bacillus subtilis and so
on. For the amplification of DNA in host organisms, it may be
preferred to use E. coli as a host, however other hosts can also be
employed.
[0050] Suitable vectors used for E. coli include EK type plasmid
vector (stringent type): pSC101, pRK353, pRK646, pRK248, pDF4l
etc., EK type plasmid vector (relaxed type): Co1E1, pVH51, pAC105,
RSF2124, pCR1, pMB9, pBR313, pBR322, pBR324, pBR325, pBR327,
pBR328, pKY2289, pKY2700, pKN180, pKC7, pKB158, pMK2004, pACYC1,
pACYC184, dul etc. .lambda.gt type phage vector: .lambda.gt.
.lambda.c, .lambda.gt. .lambda.B, .lambda.WES, .lambda.C,
.lambda.WES. .lambda.B, .lambda.ZJvir., .lambda.B', .lambda.ALO,
.lambda.B, .lambda.WES. Ts622, .lambda.Dam etc.. In general pBR322
has been frequently used as a vector for E. coli and in that
instance the best cloning sites are the Pst I and EcoRI sites.
[0051] Transformation of the host cell with the recombinant. DNA
may be carried out by conventionally used manner as follows:
[0052] Where the host is of procaryote such as E. coli, competent
cells which are capable of DNA uptake are prepared from cells
harvested after exponential growth phase and subsquently treated by
the CaCl.sub.2-method well known procedures. When MgCl.sub.2 or
RbCl exists in the transformation reaction medium, the
transformation efficiency increases. Transformation can be also
performed after forming a protoplast of the host cell.
[0053] Where the host used is an eucaryote, transfection method of
DNA as calcium phosphate-precipitates, conventional mechanical
procedures such as microinjection, insertion of a plasmid
encapsulated in red blood cell hosts or in liposomes, treatment of
cells with agents such as lysophosphatidylcholine, or use of virus
vectors, or the like may be used.
[0054] Cells possessing IL-2 gene can be isolated after the
transformation, by either of the following two ways.
[0055] (1) In the plus-minus method, partially purified IL-2 mRNA
is obtained by sucrose density gradient centrifugation of mRNAs
extracted from mitogen activated mammalian cells as 11 to 12s
sediment and then 32p-radiolabelled ss-cDNA is synthesized using
the partially purified mRNA as a template. After removal of the
template mRNA by alkaline treatment, isolated cDNA is hybridized
with partially purified 11 to 12s mRNA extracted from mitogen non
activated mammalian cells. Thereafter nonhybridized and hybrid
forming cDNA are fractionated on hydroxylapatite column
chromatography. The non hybridized cDNA and hybridized cDNA are
tentatively called probe A and probe B, respectively. Transformants
are grown on two nitrocellulose filters in quite the same way: and
the DNA of the cells is fixed on the filter paper by alkaline
treatment. Probe A and Probe B are respectively hybridized with the
DNA on two different filter papers and thereafter autoradiography
assay is carried out to select the transformants which react
positively to probe A (plus), but react weakly or do not at all to
probe B (minus)(Taniguchi et al., Proc. Jpn. Acad., v 155B 464-469,
1979).
[0056] (2) The second method consists of dividing, for example,
1,000 to 10,000 transformant clones into several tens or several
hundreds of clone groups. The divided clone groups are respectively
cultured by conventional means to obtain plasmid DNAs. Thereafter
these plasmid DNAs are converted into ss-cDNAs, for example, by
heat denaturation, and the ss-cDNAs obtained are fixed onto
nitrocellulose filter papers to achieve the hybridization of mRNA
complementary to the fixed DNAs and prepared from mammalian cells
including activated IL-2 mRNA. Alternatively, mRNAs containing IL-2
mRNA are hybridized with heat denaturated plasmid DNAs and then
DNA-mRNA hybrid is fixed onto nitrocellulose filter papers. These
filter papers are then washed with low salt concentration buffer,
such as 1 mM HEPES, or with 10 mM NaCl, and mRNA adsorbed on filter
paper is extracted by treatment with a solution containing 0.5 mM
EDTA and 0.1% SDS solution for e.g. 1 min. at 95.degree. C.
Purified mRNA is recovered by elution through oligo dT-cellulose
column chromatography. Thereafter, the mRNA is translated into
protein by microinjection into Xenopus laevis egg to ascertain IL-2
activity, or the mRNA is translated into a protein using the mRNA
dependent reticulocyte or wheat germ in vitro cell free translation
system, to analyse IL-2 activity using ant i-IL-2 antibody.
According to these procedures, the group in which the presence of
IL-2 activity was detected was further divided repeatedly into
groups consisting of smaller number of transformant clones until a
single clone possessing IL-2 DNA is specified.
[0057] To obtain cDNA coding for IL-2 polypeptide from the IL-2
producing transformant, the recombinant DNA in the transformant is
separated and cleaved with a restriction endonuclease. From the DNA
fragments formed by the cleaving, the insert cDNA fraction is
separated.
[0058] The complete nucleotide sequence of the PstI DNA inset
coding for IL-2 polypeptides from the recombinant DNA of pIL2-50A
was determined by the procedure of Maxam and Gilbert (Meth. Enzym.
65. 499-560, (1980)) and by the dideoxynucleotide chain termination
method (Smith, A. J. M. Meth. Enzym. 65, 560-580 (1980)).
[0059] The restriction endonuclease cleavage map of the cDNA insert
and base sequence of the insert are shown in FIG. 1, and FIG. 2(a)
in which the cDNA has sites cleaved with restriction endonuclease
of BstNI, XbaI and BstNI in this order, respectively.
[0060] The DNA sequence of the insert contains a single large open
reading frame. The first ATG sequence, which usually serves as the
initiation sequence in eukaryotes (Kozak, M. Cell, 15, 1109-1123
(1973)), is found at nucleotides 48-50 from the 5' end This ATG is
followed 152 codons before the termination TGA is encountered at
nucleotides 507 to 509. A stretch of A residues corresponding to
the 3'-poly (A) terminus of the mRNA is found at the end of the
cDNA and this is preceeded by the hexanucleotide AATAAA (position
771-776) which is usually found in most eukaryotic mRNA (Proudfoot,
N. J. and Brownlee, C. G., Nature 263, 211-214, (1976)).
[0061] The amino acid sequence, for which the cDNA codes, could be
deduced as shown in FIG. 2(b) (Amino Acid sequence I), and the
polypeptide of the amino acid sequence I consists of 153 amino
acids and its molecular weight is calculated to be 17631.7 daltons.
As has been reported as a common feature in most of the secretion
proteins known to date (Blobel, G. et al., Sym. Soc. exp. Med., 33,
9-36 (1979)), the N-terminal region of the deduced IL-2 polypeptide
is also quite hydrophobic and this region probably serves as a
signal peptide which is cleaved during the secretion process of the
mature IL-2. Such cleavage occurs either between Ser and Ala at
position 20 and 21 or between Ala and Pro at position 21 and 22
respectively, forming the polypeptide having amino acid sequences
II and III, since similar cleavage sites have often been found in
other secretion proteins (Blobel, G. et. al., Symp. Soc, exp. Med.
33, 9-36, (1979)). The mature IL-2 polypeptide would then contain
133 or 132 amino acids with the calculated molecular weight being
15420.5 daltons or 15349.4 daltons. This value is then compared
with the reported value for human IL-2 protein from Jurkat cells
(15,000 daltons) (Gillis, S. et al., Immunological Rev., 63,
67-209, (1982)). Additionally, the DNA fragment initiating from CCT
codon at position 111 to 113 in base sequence, which, therefore,
codes for a polypeptide initiating from Pro at position 22 (Amino
Acid Sequence III in FIG. 2(b)), was confirmed to express a
polypeptide possessing IL-2 activity as shown in Example 5. It is
also confirmed that the DNA fragment initiating from GCA sequence
at position 107 to 110 in the base sequence, which therefore codes
for a polypeptide initiating from Ala at position 21 (Amino Acid
Sequence II in FIG. 2(b)) expresses a polypeptide possessing IL-2
activity as shown in Example 8.
[0062] It has been known that genes of eukaryotes often show
polymorphysm for example in human interferon genes. (Taniguchi et
al. Gene 10. 11-15 (1980), Ohno & Taniguchi, Proc. Natl. Acad.
Sci USA, 77, 5305-5309, (1986); Gray et al., Nature, 295 501-508
(1981)). In some cases, polymorphysm is accompanied with
replacement of certain amino acids of the protein products and in
other cases, the structure of the protein product remains
unchanged. In the case of human IL-2 cDNA, another cDNA clone
(pIL2-503) in which the A residue at position 503 of pIL2-50A cDNA
(FIG. 2) is replaced by a G residue can be detected. Other cDNA
clones with some base substitution compared to pIL 2-50A cDNA can
also be expected.
[0063] As can be understood from the above the genes of present
invention include DNA having the base sequence shown in FIG. 2(a)
DNAs initiating from ATG sequence at position 48 to 50 and having
the sequencial bases following the ATG sequence up to at least ATC
sequence at position 504-506, DNAs initiating from GCA sequence at
position 108-110 and having the sequencial bases following the GCA
sequence up to at least the ATC codon and DNAs initiating from CCT
sequence at position 111-113 and having the sequencial bases
following the CCT sequence up to at least the ACT sequence. The
genes of the present invention also include DNAs ending at the ACT
sequence at position 504 to 506 and initiating from A at position
1, ATG sequence at position 48 to 50, GCA sequence at position 108
to 110 or CCT sequence at position 111 to 113. The genes of the
present invention further include DNAs ending at TGA sequence at
position 507 to 509 and initiating from A at position 1, ATG
sequence at position 48 to 50, GCA sequence at position 108 to 110
or CCT sequence at position 111 to 113. The genes of the present
invention further include DNAs ending at C at position 801 and
initiating from A at position 1, ATG sequence at position 48 to 50,
GCA sequence at position 108 to 110 or CCT sequence at position 111
to 113. The genes of the present invention additionally include
DNAs ending with poly (A) and initiating from ATG codon at position
48-50, GCA at position 108-110 or CCT sequence at position 111 to
113. The genes of the present invention also include those of Faith
base sequences correspond to Amino Acid Sequence I, II and III.
Furthermore, polyeptides deficient in one or more amino acids in
Amino Acid Sequence I, or polypeptides in which one or more amino
acids in Amino Acid Sequence I are replaced with one or more amino
acids may have IL-2 activity. Therefore genes coded for such
polypeptides are suitable genes for the present invention.
Similarly, genes having additive connection of one or more base
sequences, capable of expressing one or more amino acids to Amino
Aid Sequences I, II or III, are suitable in this invention so far
as the additively connected amino acids do not interfere with the
action of the polypeptides in expressing IL-2 activity. Modified
additively connected amino acid region which interfere with the
polypeptide function as IL-2, can be used in this invention so far
as the additively connected region can be easily eliminated. This
situation is quite the same for the additive connection of DNA to
the 3'-terminus of genes corresponding to Amino Acid Sequence I, II
and III coding additional amino acids at C-terminal of the I, II
and III having Amino Acid Sequence I, II and III respectively.
Therefore use of genes coded for such polypeptides are to be
considered to be included in the present invention.
[0064] Recombinant DNAs which direct the production of IL-2 in
living cells can be constructed by various methods. For example,
the coding sequence of IL-2 cDNA can be inserted in an expression
vechole downstream of the promoter sequence. Alternatively, a cDNA
piece carrying a promoter sequence can be inserted upstream of the
IL-2 coding sequence, after or prior to, the insertion of cDNA in
the expression vechole.
[0065] Procedures to construct cells procaryotic or euaryotic which
express the IL-2 cDNA and produce IL-2 polypeptide are explained
more precisely below:
[0066] (1) Expression of the IL-2 cDNA in E. coli
[0067] In order to express the IL-2 cDNA in E. coli the cDNA is
fused with various bacterial promoters and hybrid plasmids
containing the cDNA downstream of the promoters are obtained. The
plasmids are transfected into, for example. E. coli HB101 strain
and bacteria synthesizing a protein product with human IL-2
activity are cloned. Essentially any kind of bacterial promoter
should direct the expression of IL-2 cDNA when they are abutted
appropriately to the cDNA. Examples of this cDNA expression are
described here.
[0068] The cloned cDNA for IL-2 encodes a polypeptide consisting of
153 amino acids as illustrated in FIG. 2. The N-terminal region
corresponding to about 20 amino acids of this polypeptide is quite
hydrophobic and this is characteristic of most of the secretion
proteins. Such a hydrophobic sequence, so-called signal sequence,
is cleaved during the secretion process. Therefore, mature IL-2
polypeptide should contain less than 153 amino acids. It is
therefore desirable to express the cDNA portion encoding the mature
IL-2 polypeptide but not the portion corresponding to the IL-2
signal sequence.
[0069] (i) Construction of an expression plasmid vechole, pTrS-3,
which includes E. coli trp promoter, its ribosome binding site (SD
sequence) for the leader peptide was previously reported (G.
Miozzari and Yanofsky, C. J. Bacteriol. 133, 1457-1466,(1978)) and
an ATG codon situated 13 bp downstream of the SD sequence (Nishi
et. al., SEIKAGAKU, 54, No. 8, 676 (1982)). The plasmid vehicle
also contains a single SphI site just downstream of the ATG
intiation sequence (FIG. 4).
[0070] To express IL-2 cDNA, the plasmid is first digested by SphI
and treated either with E. coli DNA polymerase I (Klenow Fragment)
or with bacteriophage T4 DNA polymerase I to remove the 3'
protruding ends (Fig. (a)). Plasmid pIL2-50A is double digested by
PstI and HgiAI, and a larger cDNA fragment is isolated. The DNA is
then treated either with E. coli DNA polymerase I (Klenow Fragment)
or with bacteriophage T4 DNA polymerase so that the 3' protruding
ends are rendered flush. The above treated cDNA encodes IL-2
polypeptide of 132 amino acids as shown in FIG. 5(a). This cDNA is
then ligated to the pTrS-3 plasmid DNA pre-treated as above, such
that the ATG initiation codon is abutted to the CCT (Pro) sequence
of the IL-2 cDNA. Thus, a plasmid pTIL2-22 is obtained. The
junction between trp promoter sequence and IL-2 cDNA sequence of
pTIL2-22 is also illustrated in FIG. 5(a).
[0071] The plasmid pTIL2-22 should :direct the synthesis in E. coli
of an IL-2 polypeptide consisting 132 amino acids starting with
proline.
[0072] (ii) Since it is also possible that the mature IL-2 contains
alanine (position 21) as the N-terminal amino acid instead of
proline, the following plasmid which direct the synthesis of IL-2
polypeptide, consisting of 133 amino acids, is discussed,
[0073] Plasmid pTrS-3 contains a single ClaI site between SD
sequence and ATG sequence (FIG. 4). This plasmid is digested by Cla
I and Sal I. Plasmid pIL2-50A is partially digested by PstI,
treated with E. coli DNA polymerase I and the largest linear DNA is
isolated. The DNA was then ligazed with a synthetic DNA linker
containing a restriction XhoI cleavage site and a clone containing
the plasmid pIL2-50A (Xho) in which the linker DNA is introduced at
the 3' downstream of IL-2 coding sequence is isolated. The plasmid
pIL2-50A(Xho) is first digested by HgiAI, treated either with E.
coli Klenow Fragment or with T4 DNA polymerase, digested by XhoI
and the cDNA fragment is isolated. This cDNA fragment is then
ligated with pTrS-3 DNA pretreated with ClaI and SalI and with a
synthetic DNA shown in FIG. 5(b). Thus, a plasmid pTIL2-21 which
should direct the synthesis in E. coli of an IL-2 polypeptide
consisting 133 amino acids starting from alanine can be obtained as
illustrated in FIG. 5(b). Similar construction can also be made
without using XhoI linker.
[0074] (iii) IL-2 polypeptides with different size with different
N-terminal amino acid can be produced by using the pTrS-3
expression plasmid vechole by the following procedure. The cloned
IL-2 cDNA in pIL2-50A contains a sole DdeI site at nucleotide
position 81-85. Plasmid pIL2-50A(Xho) is digested by DdeI and the
DNA fragment containing he larger portion of the cDNA is isolated.
The fragment should also contain DNA of base about 3,000 pairs from
pBR322 (FIG. 5(c)). The DNA fragment is treated by exonuclcease Bal
31 and then digested by XhoI. The above treated DNA is then ligated
with pTrS-3 which is digested by SphI, treated either with Klenow
Fragment or with T4 DNA polymerase and then digested by SalI as
illustrated in FIG. 5(c). The ligated DNA is transfected into E.
coli HB101 and bacterial clones expressing human IL-2 are screened.
Those clones should express human IL-2 of various sizes since the
DNA corresponding to the N-terminal region of human IL-2 is
variably chewed away. Thus pTIL2-14 and pTIL2-15 carrying IL-2 cDNA
could be obtained.
[0075] (iv) The IL-2 cDNA can also be expressed by the use of
pKT218 (provided by K. Talmage)(Proc. Natl, Acad, Sci, USA 77,
p3369-3373, (1980)). Plasmid pKT213 is digested by PstI and ligated
with an IL-2 cDNA insert obtained by digesting pIL2-50A DNA by
HgiAI and PstI (FIG. 6). The resulting plasmid pKIL2-21 has the
sequence at the beginning of the protein synthesis initiation as
shown in FIG. 6. Thus, the plasmid pKIL2-21 should direct the
synthesis in E. coli of a fused polypeptide consisting of 133 amino
acids of IL-2 and amino acid of .beta.-lactamase (The first
methionine is cleaved off in E. coli).
[0076] (v) An expression plasmid pTuB1P-5 in which the promoter
sequence for tuf B is inserted into pBR322 was previously
constructed (Taniguchi etal.,SEIKAGAKU,53, 966,(1981)). The plasmid
contains a single ClaI site and this is located 2 bp downstream of
the SD sequence as shown in FIG. 7.
[0077] Since pTrS-3 also contains a ClaI site between the SD
sequence and ATG initiation sequence, and since this ClaI site is
not destroyed during the construction of expression plasmid by
using pTrS-3 and IL-2 cDNA as described above, it is very simple to
replace the bacterial Trp promoter with that of tufB so that the
human IL-2 cDNA is expressed under the control of tufB promoter.
For example, pTIL2-22 is digested by ClaI and pvuII and the DNA
fragment containing the IL-2 cDNA is isolated. This fragment is
then ligated with pTUB1P-5 DNA, pre-digested by ClaI and PvuII and
a plasmid pTuIL2-22 is constructed as illustrated in FIG. 7. The
IL-2 activity could be detected in the extract of E. coli HB101
harboring the plasmid pTuIL2-22.
[0078] (vi) Similar construction can also be made by using, for
example, pTL2-21 and essentially all expression plasmid which are
constructed with the use of pTrS-3. It is also possible to optimize
the distance between SD and ATG sequence by digesting, for example,
pTuIL2-22 with ClaI, removing (or adding) a few base pairs of DNA
by Bal 31 or S1 or DNA polymerase I (E. coli) and then re-ligating
the plasmid.
[0079] (2) Expression of the IL-2 cDNA in Yeast
[0080] IL-2 cDNA can be also expressed in yeast by inserting the
cDNA into appropriate expression vectors and introducing the
product into the host cells. Various shuttle vectors for the
expression of foreign genes in yeast have been reported (Heitzman.
R. et al., Nature 293, 717-722 (1981); Valenzuela, P. et al.,
Nature 298, 347-350 (1982); Miyanoshita et al., Proc. Natl, Acad,
Sci. USA 80, 1-5 (1983)). The vectors are capable of replicating
both in E. coli and in yeast hosts and they contain promoter
sequences of genes of yeast, Essentially all such expression
vectors can be used for the expression of IL-2 cDNA. It may be
possible to achieve higher levels of IL-2 production by using yeast
as compared to use of animal cells or bacteria. An example of human
IL-2 cDNA expression in yeast is now described.
[0081] A yeast E. coli shuttle vectors pAT77 and pAM82 have been
described by Miyanoshita et al. (Proc. Natl. Acad. Sci. USA 80,
1-5. (1983)). The vector pAM82 is a derivative of pAT77 and both
carrying markers of ars 1 (Stinchcomb, D. T. et al, Nature 292,
39-43, (1979), 2 .mu.m ori (Broach, J. R et al. Gene 8, 121-133
(1979)) and leu 2 (Ratzkin, B. et al. Proc. Natl. Acad. Sci. USA
74, 474-491 (1979)) and the promoter for the yeast acid phosphatase
(APase) gene. They also carry a 3.7 kb DNA segment of pBR322 which
contains an ampicillin resistance marker (Ap.sup.r) and the origin
of replication (FIG. 8). The APase promoter is inducible by
shifting a high concentration of phosphate into a low concentration
in the culture media. In order to express human IL-2 cDNA, pIL2-50A
is digested by PstI after treating either by the E. coli Klenow
Fragment of by T4 DNA polymerase, the cDNA is ligated with pAM82
previously digested by XhoI and incubated with the E. coli Klenow
Fragment to fill in the ends. Hybrid plasmids in which the cDNA
coding sequence are downstream of the yeast APase promoter sequence
are selected by cloning them in E. coli. The obtained plasmid,
pYIL-2a, is introduced into yeast and, after induction of the APase
promoter, IL-2 activity in the yeast extract is measured. The
plasmid pYIL-2a contains a stretch of GC residues between the yeast
promoter and the IL-2 cDNA. It is possible that such a sequence
inhibits the expression of IL-2 cDNA. In order to overcome this
problem, following construction of a plasmid can be made: Plasmid
pIL2-50A is digested by PstI and the cDNA insert is isolated. This
cDNA is then treated by T4 DNA polymerase in the presence of dATP,
dGTP and dTTP so that streches of C-residues at the both ends of
the cDNA are chewed off and subsequently treated by Nuclease S1, to
remove stretches of G-residues. This DNA is ligated with XhoI DNA
linker and plasmid pBR322 whose EcoRI site is cleaved and rendered
flush by EcoRI and the Klenow Fragment, and the resulting plasmid,
pIL2-Xho, is digested by XhoI and the cDNA insert is isolated. The
cDNA is then introduced into the single XhoI site of pAM82 and
a-plasmid containing the IL-2 coding sequence correctly oriented
with respect to the yeast APase promoter is cloned in E. coli. The
plasmid, pYIL-2b, is introduced into yeast and, after induction of
the APase promoter, IL-2 activity in the yeast extract is
measured.
[0082] (3) Expression of the cDNA in mammalian cell
[0083] A plasmid which should direct the synthesis of human IL-2 in
mammalian cells can be constructed as follows. A plasmid pCE-1 is
constructed from pKCR (O'Hare, K., et al., Proc. Natl. Acad. Sci.
USA., 78, 1527-1531, (1981)) and pSR328 (Soberon, X., et al., Gene,
9, 287-305, 1980) by a series of modification procedures as
illustrated in FIG. 2, and initiation sequence ATG of IL-2 gene is
connected to the downstream of the SV40 early gene. This plasmid
contains a single PstI site just downstream of the SV40 early
promoter and upstream of the part of the rabbit .beta.-globin
chromosomal gene containing one intron. The plasmid also contains
the replication origin of SV40 as well as the polyadenylation site
for the early gene. Thus a plasmid pCEIL-2, in which the IL-2
structural gene should be transcribed from the early promoter of
SV40 in appropriate host cells, is obtained (FIG. 2).
[0084] This plasmid is digested by HhaI and then introduced by DNA
transfection into the transformed monkey cell line COS-7 which
allows replication of DNA containing SV40 origin sequences. It
appears to be important to digest the plasmid by HhaI before
transfection for the efficient expression of cDNA since sequences
which could hamper replication of the transfected DNA in COS cells
can be removed from the essential part of the plasmid for cDNA
expression by this procedure. One to three days culture under
conventional culture conditions after transfection of this vector
to monkey cultured cell COS-7 (Gluzman, Y. Cell, vol. 23, 175-182,
(1981)), IL-2 is usually secreted and produced in cultured cell
medium. In order to insert amplified DNA into other eucaryotic
cells, similarly a vector appropriate to host organisms is
connected to cDNA insert cleaved and isolated from procaryotic
cells and the eucaryotic cell may be transfected with thus
synthesized vector and cultured.
[0085] Cells incorporating the recombinant DNA are cultured to
amplify the recombinant DNA or to produce IL-2 polypeptide. The
cultivation is carried out by conventional means. For instance,
transformed yeast may be cultured in a medium containing source of
carbon, a nitrogen source, inorganic salts and, when required,
organic nutrients such as vitamin and amino acid at a temperature
in the range from 20.degree. to37.degree. C., and a pH ringing from
4 to 7 under aerobic condition. Transformed procaryotic organisms,
such as E. coli or B. subtilis may also be cultured under
conventional conditions.
[0086] The IL-2 produced intracellularly or extracellular, is
recovered by any known method, such as precipition with ammonium
sulfate, dialysis to remove salts (under normal or vacuum
pressure), gel filtration, chromatography, preparative flat-bed
iso-electric focusing, gel electropheresis, high performance liquid
chromatography (hereinafter "HPLO"), (ion exchange, gel filtration
and reverse phase chromatography), and affinity chromatography on
dye bound carrier, on activated Sepharose 4B coupled with
monoclonal antibody against said IL-2 or on lectin bound Sepharose
4B and the like. Methods of recovery, and purification of IL-2, are
described in Watson et. al., J. Exp. Med., 150, 849-861 (1979),
Gillis et. al., J. Immunol., 124, 1954-1962, (1980), Mochizuki et.
al., J. Immunol Methods 39, 185-201, (1980), and Welte, K. et. al.,
J. Exp. Med., 156, 454-464 (1982).
[0087] The polypeptide thus obtained shows the same biochemical and
biological behavior as has been known for IL-2 produced by
mammalian cells by mitogen stimulation, and has IL-2 activity. The
molecular weight is around 15,000 dalton and IL-2 activity was
completely neutralized or precipitated with monoclonal anti-IL-2
antibody in the presence or absence of immunoabsorbents, such as
Igsorb (Enzyme Center). In immunoelectrophoresis, the IL-2
polypeptide shows only a single precipitate against the
corresponding anti-IL-2 antibody. The IL-2 activity remains stable
after reduction with 2-mercaptoethanol, and is resistant to
treatment with DNAse and RNAse as well as to heat treatment at
56.degree. C. for 30 min.. The activity is stable at a pH between
pH 2 to 9. The IL-2 produced could promote the growth of monoclonal
functional T cells (cytotoxic T lymphocyte), enhance the thymocyte
mitogenesis, give rise to the generation of anti-tumor specific
cytotoxic T lymphocytes from memory state in the absence of the
antigen, and could be used to augment natural killer cell activity
against YAC-1 and RL.male.1 cells.
[0088] Having now generally described this invention, the same will
become better understood by reference to certain specific examples
which are included herein for purpose of illustration only and are
not intended to be limiting unless otherwise specified.
EXAMPLE 1
[0089] (1) Human T leukemia cell line, Jurkat cells (freely
available in Japan, W. Germany and United States) were suspended in
RPMI 1640 medium containing 10 vol/vol % FCS and were irradiated
with X-ray till 10,000 roentgen at a room temperature for 50
seconds using X-ray irradiation apparatus Exs 150/300-4 (Toshiba,
Japan), and thereafter the irradiated cell was cultured for 5 days
at 37.degree. C. in 5% CO.sub.2 incubator at a initial cell density
of 1.times.10.sup.5 cells/ml in the culture medium mentioned above.
The mutated cells (0.2 cells/well) were placed in wells 10 pieces
of flat-bottomed microplates having 96 wells, and cultured at
37.degree. C. in 5% CO.sub.2 incubator for 21 days.
[0090] Clones obtained from the wells showing growth were
repeatedly transferred into fresh culture medium to propagate the
clone sizes, and the propagated clones were cultured for 24 hrs at
a initial cell density of 1.times.10.sup.6 cells/ml in the presence
of 50 .mu.g/ml of Con A and IL-2 activity was measured according to
the methods described before. Consequently a human T cell line
designated as Jurkat-111 (hereinafter "J-111") (ATCC CRL8129),
cloned from parent Jurkat, was selected, of which productivity of
IL-2 was increased 40 times as much as that of the parent strain.
The cloned cell line J-111 could grow under conventional conditions
and the growth rate shows almost the same with ordinary Jurkat
cells.
[0091] (2) Cells (1.times.10.sup.5/ml) of J-111 were inoculated in
1,000 ml of serum free synthetic culture medium, RITC 55-9 (Sato,
T. et al., Exp. Cell Res., 138, 127-134, (1932)) in roller culture
bottles (Falcon 3027) and cultured for 4 days at 37.degree. C., and
cells propagated were harvested by centrifugation. The harvested
cells were again inoculated in the medium mentioned above which had
been added with 25 .mu.g/ml of Con A to contain 4.times.10.sup.6
cells/ml. In four batches of roller culture bottles (Falcon), 1,000
ml of the inoculated culture medium was placed into each batch. The
cultivation was continued for 6 hours with rotating.
[0092] (3) Jurkat cells (1.2.times.10.sup.6) thus stimulated with
25 .mu.g/ml of Con A for 6 hrs were suspended in 8,000 ml of
phosphate buffer balanced with saline (hereinafter "PBS"). The
cells were washed twice by centrifugation and were resuspended in
800 ml of RSB solution (10 ml Tris-HCl, pH 7.5, 10 mM NaCl, 1.5 mM
MgCl.sub.2) containing Ribonucleosides-Vanadyl Complex (10 mM), an
inhibitor of nuclease. Then a detergent NP-40 was added to contain
0.05% as final concentration, followed by gentle mixing and the
cell nuclei were removed by centrifugation for five minutes at
3,000 rpm at 4.degree. C. SDS (0.5%) and EDTA (5 mM) were added to
the supernatant and cytoplasmic RNA was extracted by addition of
equal volume of phenol. After three times extraction with phenol,
RNA was precipitated with two times volume of ethanol and
precipitates were collected by centrifugation, which were
solubilized in 10 mM Tris-HCl of pH 7.5. The amount of RNA obtained
was 196 mg.
[0093] Fractionation of mRNA was carried out using affinity
chromatography on oligo (dT) -Cellulose (P. L. Biochemicals, Type
7). An adsorption solution was a solution or pH 7.5 containing 20
mM Tris-HCl, 0.5 Ml NaCl, 1 mM EDTA and 0.5%. SDS and elution was
carried out with water and 10 mM Tris-HCl (pH 7.5) by turns after
washing the column with the buffer (20 mM Tris-HCl, pH 7.5, 0.5M
NaCl, 1 mM EDTA). The resultant mRNA eluted was 3.6 mg. Next, 2.4
mg of the mRNA obtained was fractionated by sucrose density
gradient centrifugation (5 to 2.5% sucrose density gradient in a
solution of pH 7.5 containing 50 mM Tris-HCl, 1 mM EDTA and 0.2 M
NaCl, centrifuged at 26,000 rpm for 24 hrs at 4.degree. C., and 11
to 12S fraction of mRNA was fractionated into fractions No. 12, 13,
14 in the amount of 59 .mu.g, 46 .mu.g and 60 .mu.g,
respectively.
[0094] (4) The mRNA obtained in fraction No. 13 was microinjected
into the oocyte of Xenopus laevis (50 ng mRNA/egg) and the culture
supernatant was served for the assay of IL-2 activity. As shown in
Table 1, the increase of the incorporation of .sup.3H-TdR and the
increase of number of activated T lymphocytes were confirmed,
clearly verifying that mRNA in this fraction contains human IL-2
mRNA.
1TABLE 1 (a) Uptake of .sup.3H-TdR Amount of IL-2* Sample Dilution
(cpm) (unit/ml) Control I -- 553 0 (Medium for assay) Control II
.times.2 590 0 (Supernatant of egg .times.32 572 culture
non-treated) Translation product .times.8 14,683 32 of fraction 13
.times.32 10,165 (b) Cell number of T-lymphocyte Amount of IL-2*
Dilution (No./well) (unit/ml) Control I .times.2 0 0 (Medium for
assay) .times.16 0 Control II .times.2 0 0 (Supernatant of egg
.times.16 0 culture non-treated) Translation product .times.2 115
40 of fraction 13 .times.16 55 *The unit was calculated by
comparing the amount of incorporated .sup.3H-TdR with that of
standard IL-2 (10 unit/ml) according to probit analysis.
[0095] (5) Thereafter cDNA was synthesized in vitro from No. 13
fraction of 11 to 12S mRNA containing IL-2 mRNA and recombinant DNA
was constructed with the plasmid vector PBR 322. With the
recombinant DNA, Escherichia coli was transformed, and clone
acquired IL-2 cDNA clones was selected, as follows:
[0096] (5-1) Fifty mM Tris-HCl buffer (pH 7.5) , 30 mM NaCl, 6 mM
MgCl.sub.2, 5 mM dithiothreitol (hereinafter "DTT"), 0.5 mM of each
dATP, dGTP, dCTP, dTTP (dCTP contained .sup.32p radiolabelled one),
0.7 .mu.g oligo (dT).sub.10 10 .mu.g mRNA and 15 unit AMV reverse
transcriptase (J. W. Beard) were mixed and maintained for 90 min.
at 41.degree. C.
[0097] After termination of the reaction, DNA was recovered as
ethanol precipitates after the phenol treatment, and DNA was
solubilized in a solution of pH 7.5 containing 20 mM Tris and 1 mM
EDTA.
[0098] Two point five .mu.g of ss-cDNA was synthesized. To remove
mRNA present in this solution, the solution was made 0.33 N-NaOH by
addition of NaOH, allowed to stand for 15 hrs at a room
temperature, then the solution was neutralized with equal volume of
1 M-Tris-HCl of pH 7.5 and passed through "Sephadex G-50"
column.
[0099] The recovered cDNA was 1.8 .mu.g.
[0100] (5-2) Fifty mM phosphate buffer (pH 7.5), 10 mM MgCl.sub.2,
10 mM DTT, 0.75 mM of each dATP, dGTP, dCTP, dTTP (dCTP contains
.sup.3H radiolabelled one), 1.8 .mu.g ss-cDNA, and 8 unit of
polymerase I (BRL, United States) were mixed and were allowed to
react for 15 hrs at 15.degree. C. After the termination of the
reaction, DNA was recovered as ethanol precipitate, after
treatments with phenol and with chloroform. 1.10 .mu.g of ds-cDNA
was generated. A mixture of 50 mM sodium acetate (pH 4.5), 0.2M
NaCl, 1 mM ZnCl.sub.2 and 1.10 .mu.g of ds-cDNA was incubated for
20 min. at 37.degree. C., added with 0.25 unit of nuclease S.sub.1
(Sankyo, Japan), and incubated further for 15 min.
[0101] After the termination of the reaction, the reaction product
treated twice with phenol was applied onto sephadex G-50 to get
0.55 .mu.g of ds-cDNA.
[0102] (5-3) A mixture of 0.14M potassium cacodylate, 30 mM Tris
base, 0.1 mM DTT, 1 mM COCl.sub.2, 0.64 mM .sup.32p-dCTP (spc. act.
2.7.times.10.sup.6 cpm/n mol), 0.55 .mu.m of ds-cDNA and 5 unit of
terminal transferase (BRL) were incubated for 7 min. at 37.degree.
C., then applied onto sephadex G-50 column after phenol treatment
to get 0.50 .mu.m DNA as ethanol precipitates. The recovered DNA
was found to be extended with around 50 dCMP residues at the both
3' terminus.
[0103] Ten .mu.g of pBR 322 DNA was cleaved with restriction enzyme
PstI, and 3'-termini of the cleaved DNA were added with dGMP chain,
by the same method as that used in the addition of dCMP to ds-cDNA
mentioned above, except dGTP was used in place of dCTP.
[0104] (5-4) A mixture of 50 mM Tris-HCl (pH 7.5), 0.1M NaCl, 5 mM
EDTA, 0.05 .mu.g of pBR 322 elongated with dGMP residues and 0.01
.mu.g of cDNA extended with dCMP was incubated firstly for 2 min.
at 65.degree. C., then for 120 min. at 46.degree. C., for 60 min.
at 37.degree. C. and finally for 60 min. at a room temperature. E.
coli.times.1776 (Curtiss III, R. et al., in Molecular Cloning
Recombinant DNA, (W. A. Scott & R. Werner ed.) Academic Press,
(1977)) was inoculated in 50 ml of L broth containing 100 .mu.g/ml
of diaminopimelic acid, 50 .mu.g/ml of thymidine, 1% tryptophan,
0.5% yeast extract, 0.5% NaCl and 0.1% glucose and cultured in
shaking at 37.degree. C. until the absorbance of culture liquid at
562 nm became around O. D 0.3. After the termination of the
culture, the culture liquid was left at 0.degree. C. for 30 min.,
then the bacterial cells were collected by centrifugation followed
by twice washing with 25 ml of a solution containing 5 mM Tris-HCl
(pH 7.6), 0.1M NaCl, 5 mM MgCl.sub.2 and 10 mM RbCl.
[0105] Thus obtained cells were suspended in 20 ml of a solution
containing 5 mM Tris-HCl (pH 7.6), 0.25M KCl, 5 mM MgCl.sub.2, 0.1M
CaCl.sub.2 and 10 mM RbCl and Keys left at 0.degree. C. for 25
min., then cells were collected to resuspend them into 1 ml of the
same solution, the recombinant DNA described above was added into
0.2 ml of the cell suspension and the suspension was left at
0.degree. C. for 60 min. Then 0.7 ml of L broth was added to
culture in shaking for 30 min. at 37.degree. C. Thus obtained
culture medium (0.1 ml) was thoroughly spread on the surface of
1.5% agarose medium composed of L broth containing 100 .mu.g/ml
diaminopimelic acid, 50 .mu.g/ml thymidine and 15 .mu.g/ml
tetracycline, and incubated at 37.degree. C. for two days.
[0106] (5-5) Four hundred and thirty two colonies appeared were
divided into 18 groups, each containing 24 different bacterial
clones, inoculated in 200 ml of L-broth containing 100 .mu.g/ml of
diaminopimelic acid 50 .mu.g/ml of thymidine and 10 .mu.g/ml of
tetracycline and cultured in shaking at 37.degree. C. for 5 to 7
hrs. Then, 200 ml of fresh L-broth containing chloramphenicol at a
final concentration of 170 .mu.g/ml was added to culture further
for an overnight. Thus amplified plasmid DNA was purified according
to a conventional mean. Clones possessing IL-2 cDNA were screened
by a mRNA hybridization-translation assay (hereinafter "H-T"
assay"). H-T assay here employed was carried out as follows:
[0107] Purified DNA (25 .mu.g) was cleaved with restriction enzyme
Hind III, treated with phenol three times, treated with
phenol-choroform and with chloroform, respectively, precipitated
with ethanol, washed with 80% ethanol and dissolved in 40 .mu.l of
80% formamide.
[0108] The reaction mixture was heated for denaturation at
90.degree. C. for 5 min., then diluted to 1.3 ml with 10.times.SSC
(1.5M NaCl, 0.15M sodium citrate). The DNA was thereafter fixed
onto nitrocellulose filters, which filters were dried up at
80.degree. C. for 3 hrs, and incubated for 18 hrs at 37.degree. C.
in the solution containing 50% formamide, 20 mM Pepes of pH 6.5,
0.75M NaCl, 5 mM EDTA, 0.2% SDS and 250 .mu.g of poly (A) mRNA from
induced J-111 cells to hybridize the DNA fixed on filters with IL-2
mRNA. Then the filters were washed at 65.degree. C. three times
with solution consisting of 10 mM Pipes of pH 6.5, 0.15M NaCl, 1 mM
Pipes, 10 ml NaCl solution and treated with 0.5 mM EDTA, 0.1% SDS
solution at 95.degree. C. for 1 min. to recover the hybridized mRNA
from the filters. Thus extracted mRNA was purified on oligo
dT-Cellulose column according to the conventional methods and
injected into Xenonus oocates to determine the IL-2 activity of
translated proteins. One out of 18 groups, each consisting of 24
clones, gave positive 48 unit/ml IL-2 activity in .sup.3H-TdR
incorporation assay described previously, while others being
clearly negative. Then 24 single colonies belonging to the positive
group were inoculated in 200 ml of L-broth possessing the same
composition described, cultured aerobically for 5 to 7 hrs. at
37.degree. C. and similarly chloramchenicol containing fresh
L-broth was further added. After amplification of plasmid DNA by an
overnight culture, plasmid DNA was similarly purified according to
the standard procedures. After cleavage of about 5 .mu.g of each
plasmid DNA with Hind III, each plasmid DNA was bound to
nitrocellulose filters similarly. The filters were hybridized with
IL-2 mRNA and hybridized mRNA was recovered to inject into Xenoaus
oocyte to determine the IL-2 activity of translated proteins.
[0109] As shown in Table 2, only plasmid DNA purified from a single
colony, designated as p3-16, gave the positive IL-2 activity.
Therefore this clone was identified as the clone possessing IL-2
cDNA (E. coli.times.1776/p3-16 AJ 11995 (FERM-BP-225)) Thus plasmid
DNA, p3-16, was confirmed to share exactly the DNA (Il-2 gene)
capable of forming the specific hybrid with IL-2 mRNA.
2TABLE 2 (a) Uptake of .sup.3H-TdR Amount of IL-2 Sample Dilution
(cpm) (unit/ml) Control I -- 2,010 0 (Medium for assay) Control II
.times.2 2,120 (Supernatant of .times.32 2,482 0 culture liquid of
non-treated egg) Translation product .times.2 20,453 58 of mRNA
.times.32 20,961 (b) Cell number of T-lymphocyte Amount of IL-2
Sample Dilution (cells/well) (unit/ml) Control I -- 0 0 (Medium for
assay) Control II .times.2 0 0 (Supernatant of .times.32 culture
liquid of non-treated egg) Translation product .times.2 88 32 of
mRNA* .times.32 42 *mRNA hybridized with cDNA from plasmid
p3-16.
[0110] The cDNA insert of plasmid p3-16 showed characteristics to
be cleaved by restriction enzyme XbaI at a single site and by BstN
at two sites, (at upstream and downstream of XbaI cleavage site).
However the plasmid p3-16 contained a cDNA insert consisting of
about 650 base pairs, which apparently corresponds to a part of
IL-2 mRNA of 11 to 12S size.
[0111] Therefore another cDNA library were prepared according to
the procedure of Land et al. (Land et al., Nucleic Acids Res., vol
9, p2551, (1981)) using IL-2 mRNA as a template. Single stranded
cDNA (1.6 .mu.g) was synthesized by using 4 .mu.m of IL-2 mRNA
elongated by dCMP residues, and ds-cDNA was synthesized by using
oligo (dG) 12-18 as the primer for DNA polymerase I (Klenow
fragment). The cDNA (0.6 .mu.g) longer than 680-base pair DNA size
marker was obtained by a sucrose gradient centrifugation and
inserted into the PstI site of pBR322 by the standard G-C tailing
method. After transformation of E. coli.times.1776 by the
recombinant DNA, approximately 2,000 colonies were screened by in
situ hybridization method of Grunstein-Hogness with nick-translated
p3-16 cDNA insert as the probe and the colony containing plasmid
pIL 2-50A containing around 850 base pairs and the transformed
clone (E. coli.times.1776/pIL 2-50A, AJ 11996 (FERM-BP-226)) were
identified. A restriction endonuclease cleavage maps of the cDNA
insert of pIL 2-50A are shown in FIG. 1.
[0112] To isolate a gene coding IL-2 peptide from transformed E.
coli..times.1776 pIL 2-50A, plasmid DNA was digested with
restriction enzyme pstI after isolation of DNA region from the
cells according to the conventional means. Thus produced smaller
fragment among generated two DNA fragments was DNA gene coding for
IL-2 peptide. The complete nucleotide sequence of the PstI insert
from pIL 2-50A was determined by the procedure of Maxam and Gilbert
(,Maxam, A. W. et al., Enzym. 65, 499-560, 1980), and the whole
structure is shown in FIG. 2.
EXAMPLE 2
[0113] The plasmid pKCR (O'Hare et al. Proc. Natl. Acad, Sci., USA,
vol 78, No. 3, 1527-1531, (1981)) consists of (i) segments of SV40
DNA (shown as hatched blocks in FIG. 3) containing an early gene
promoter and an origin of replication (0.725-0.648 m.u.) and a
polyadenylation site from the early gene (0.169-0.144 m.u.) (ii) a
part of the rabbit 3-globin gene (shown as open blocks) (BamHI-PvuI
fragment) (iii) a seament from pBR322 (EcoRI-BamHI fragment)
containing an origin of replication and ampicillin resistance gene.
This plasmid was cleaved by BamHI, and, after filling both ends of
the cleaved DNA by DNA polymerase I (Klenow fragment), a synthetic
PstI linker DNA was introduced to construct pKCR (PstI). Plasmid
pKCR (PstI) was cleaved by SalI, treated by the Klenow fragment to
fill the ends and then partially cleaved by EcoRI to obtain
EcoRI-SalI fragment which contains the whole DNA derived from SV40
and the globin gene. This fragment was then ligated to a piece of
pBR323 DNA which contains tetracycline resistance gene and an
origin of replication as outlined in the FIG. 3. The resulting
plasmid pCE-1 contains a single PstI site just downstream of the
SV40 early promoter.
[0114] The cDNA insert of pIL 2-50A was excised by PstI cleavage
and ligated to PstI-cleaved pCE-1 to construct pCEIL-2 in which
expression of the IL-2 structural gene should be under control of
SV40 early promoter. Plasmid pCE-1 was originally constructed for
the cDNA cloning by G-C tailing method (Chang, A. C. Y. et al.
Nature, 275, 617-624, 1978) in bacteria and direct expression in
mammalina cells.
[0115] This plasmid was digested by HhaI and then introduced by DNA
transfection (.AcCutc.-an et al., J. Natl. Cancer Inst. 41,
351-357, 1968) into the transformed monkey cell line Cos-7 which
allows replication of DNA containing SV40 origin sequences and is
available from Gluzman, Y.(Cell, 23, 175-182, 1981). It appears to
be important to digest the plasmid by HhaI before transfection for
the efficient expression of cDNA since sequences which could hamper
replication of the transected DNA in COS cells can be removed from
the essential part of the plasmid for cDNA expression by this
procedure. COS-7 cells (6.times.10.sup.4/ml) were suspended in 0.5
ml of DMEM containing 5% FCS in 24 well culture plate (Nunc) and
incubated for 4 hrs. at 37.degree. C. Then mixture of 1 .mu.g of
the above described vector, 17.6 .mu.l of 1 mM Tris-HCl containing
0.1 mM EDTA, 2.4 .mu.l of 2M CaCl.sub.2 and 120 .mu.l of
2.times.HBS containing 50 mM Pipes, 280 mM NaCl, and 1.5 mM
Na.sub.2HPO.sub.4.12H.sub.2O (pH 7.10) were added to the cultured
cells. The cells were further incubated for 4 hrs. at 37.degree. C.
and the culture medium was aspirated off, washed with 1 mL of PBS,
then 0.5 ml of PBS containing 20% glycerol was added to leave at a
room temperature for 3 min. Again the medium was aspirated off and
the cells were washed with 1 ml of PBS and cultured in 1 ml of DMEM
containing 5% FCS. Every 24 hrs., 500 .mu.l of medium was exchanged
with fresh medium. Each media, collected at appropriate interval
was kept at 4.degree. C. until use. Two to three days after the
transfection, the cultured cell medium was assayed for the human
IL-2 activity. As shown in Table 3 the resulting culture
supernatant of COS-7 cell transfected with PCEIL-2 contained IL-2
activity. No IL-2 activity was detectable in the culture media of
cells transfected with pCE-1.
3 TABLE 3 IL-2 activity measured DNA with which by .sup.3H-TdR
uptake Growth of transfected (.mu./ml) T-lymphocyte PCEIL-2 12 ++++
pCE-1 1 -
[0116] The IL-2 activity found in culture cell medium after
transfection of COS-7 with pCEIL-2 was neutralized from 12 unit/ml
to below 1 unit/ml by mouse (BALB/c) anti-human IL-2 monoclonal
actibody. The result that COS-7 cell transfected with pCEIL-2
secreted human IL-2 cleary shows that cells of eukaryote
transformed with a recombinant DNA comprising a gene coding for
IL-2 polypeptide and a vector DNA capable of replicating in said
cells can exactly useful for the production of IL-2.
[0117] The plasmid PCEIL-2 incorporated in E. coli. HB101 has been
deposited in the accession number of FERM-BP 244.
EXAMPLE 3
[0118] Escherichia coli.times.1776/pIL 2-50A (AJ 11996
(FERM-BP-226)) prepared in Example 1 was inoculated in 250 ml of L
broth, containing 100 .mu.g/ml diaminopimelic acid, 50 .mu.g/ml
thymidine, 1% tryptophan, 0.5% yeast extracts, 0.5% NaCl and 0.1%
glucose, and cultured with shaking at 37.degree. C. till optical
density at 562 nm of the cultured medium became 0.5. After the
termination of the culture, cultured medium was allowed to stand at
0.degree. C. for 30 min. and the cells were harvested by
centrifugation, washed once with 20 mM Tris-HCl containing 30 my
NaCl and wee resuspended in 1.8 ml of the same buffer. A solution
containing 0.2 .mu.l of lysozyme (10 mg/ml) and 20 .mu.l of 0.5M
EDTA was then added to the calls and the mixture was allowed to
stand at 0.degree. C. for 20 min., followed by freeze-thawing three
times successively. Then extracts of cells (1.5 ml) were obtained
after centrifugation 40,000 rpm for 30 min. The extract was
subjected to salting out with 85% ammonium sulfates, applied on
sephadex G15 to remove salts, then applied on DEAE cellulose column
chromatography and the fraction eluted with 0.06M Tris-HCl buffer
(pH 7.6) was pooled. Thus pooled fraction was freeze-dried and was
applied on controlled pore glass beads (250 .ANG., Funakoshi
pharmaceuticals, Japan) chromatography to get IL-2 activity in
eluant with 0.3M glycine-HCl buffer, where IL-2 containing fraction
exerted 12 unit/ml of IL-2 activity. The results clearly indicate
that E. coli..times.1776/ pIL 2-50A, AJ 11996 actually produces
IL-2.
EXAMPLE 4
[0119] Constitutive IL-2 producer cell line J-A1886 (ATCC CRL3130),
cloned from Jurkat cells according to the means described in
Example 1, was similarly grown in roller culture bottle. The grown
cells were resuspended in fresh synthetic medium RITC-55-9 at a
initial cell density of 1.times.10.sup.6 cells/ml and 8 hrs. after
the start of the culture, the cells were served for the extraction
of IL-2 mRNA as 11 to 12S fraction, from 3.times.10.sup.9 cells,
accord4ng to the steps detailed in Example 1.
[0120] Double stranded cDNA was synthesized similarly as Example 1
and the cDNA longer than 600 base pairs (2.4 .mu.g) was obtained
after fractionation on a sucrose density gradient. The cDNA was
then extended with dCMP residues using terminal deoxynucleotidyl
transferase and an aliquot (50 ng) was annealed with 250 ng of
dGMP-elongated, PstI-cleaved pBR322. The resulting hybrid plasmids
were used to transform E. coli..times.1776 and the transformants of
around 4,000 clones were obtained. According to the
Gvunstein-Hogness method, three clones complementary with plasmid
3-16 cDNA used as a probe were selected. Namely thus selected
clones are transformed clones possessing human IL-2 gene.
EXAMPLE 5
[0121] A plasmid which should direct the synthesis of Human IL-2 in
E. coli. cells was constructed as follows. A plasmid pTIL2-22 was
constructed from pTrS-3 (Nishi T., Taniguchi T. et al., SEIKAGGAKU
53, 967, (1981)), and pIL 2-50A containing the IL-Z cDNA by a
series of modification procedures as illustrated in FIG. 5(a). A
plasmid pTrS-3 include insertion of the region of Trp promoter and
Shine Dalgarno (hereinafter "SD") between EcoRI site and ClaI site
of pBR322. The plasmid also contains an ATG initiation codon 13 bp
downstream of the SD sequence as well as a single SphI site as
illustrated in FIG. 4. The vector is very efficient to produce the
said protein when DNA sequence corresponding to the said protein is
inserted in phase just downstream of the ATG codon, which is
generated by SphI digestion and by subsequent treatment by T4 DNA
polymerase of pTrS-3. Therefore the plasmid pTrS-3 (30 .mu.g) was
cleaved with a restriction enzyme SphI in a conventional manner and
after successive treatment with phenol and chloroform, ethanol
precipitates were recovered, then both ends were rendered flush by
the treatment of T4 DNA polymerase. Then the DNA (21.4 .mu.g) was
recovered by similar successive phenol, chloroform treatment and
ethanol precipitation. On the other side, 380 .mu.g of pIL 2-50A
containing an IL-2 cDNA was cleaved by PstI and the IL-2 cDNA
insert was isolated by agarose gel electrophoresis. cDNA insert (11
.mu.g) was cleaved by HgiAI, treated by T4 DNA polymerase and 10
.mu.m of the DNA of larger site was isolated by agarose gel
electrophoresis. According to the procedures a cDNA (7.2 .mu.g)
coding for 132 amino acids was obtained and this DNA fragment had
blunt ends (FIG. 5(a)). Then the thus obtained cDNA ligated to a
pTrS-3 vector, previously digested by SphI and treated by T4 DNA
polymerase just downstream of ATG sequence. Thus ligated plasmid
was then used to transform into E. coli. HB101 according to the
conventional procedures. Ligation was carried out as follows. IL-2
cDNA (0.4 .mu.g) larger fragment and 0.2 .mu.g of pTrS-3 vector DNA
were mixed with 0.8 unit of T4 DNA ligase in 66 mM Tris-HCl of pH
7.5 containing 6.6 mM MgCl.sub.2, 1 mM ATP and 10 mM DTT, and the
mixture was allowed to react at 4.degree. C. overnight. Among the
transformants appeared on L broth agar plate containing ampicillin,
colonies containing the IL-2 cDNA portion, which encodes 132 amino
acids were selected by in situ colony hybridization assay. Thus
selected colonies were cultured (10 ml) again to prepare plasmid
DNA by lysozyme treatment and by freeze-thawing. The plasmid DNAs
were cleaved with PstI and XbaI, and the resulting products were
analysed by agarose gel electrophoresis in order to identify pTIL
2-22 in which the cDNA was linked to the ATG sequence of pTrS-3 in
correct orientation. The E. coli. HB101 containing pTIL 2-22 was
cultured under the conventional conditions known for the
propagation of micro-organisms. The cells were grown in 10 ml of X
broth (2.5% Bactotrypton, 1.0% yeast extracts, 0.1% glucose, 20 mM
MgSO.sub.4, 50 mM Tris-HCl, pH 7.5) containing 25 .mu.g/ml
streptomycin and 25 .mu.g of ampicillin at 37.degree. C. for an
overnight. One ml of the culture suspension was inoculated into the
same X broth (100 ml) and cultured at 37.degree. C. When O.D at 650
m.mu. arrived around 1.5-2.0, 3-indole acrylic acid (IAA) was
added. Three hours after the addition of inducer, the cells were
collected, washed with 20 mM Tris-HCl (pH 7.5, 30 mM NaCl) and
resuspended into 8 ml of the same buffer. For the efficient
functioning of Trp promoter inducers such as IAA was added at a
final concentration of 50 .mu.g/ml. Thus produced proteins in
bacterial cells were extracted by sonication (0+ C. 2 min.) or
lysozyme (8 .mu.g) digestion 0.degree. C., 20 min.) followed with
three successive freeze-thawing. According to this procedures IL-2
was usually extracted from organisms. The extracted IL-2 activity
ranged from 10,000 to 120,000 units/ml.
[0122] E. coli. HB101 containing pTIL 2-22 has been deposited in
accession number of FERM-BP 245.
EXAMPLE 6
[0123] A plasmid pTuIL 2-22, carrying IL-2 cDNA, was constructed
from pTuB1P-5 (Taniguchi, T. et al., Seikagaku, 53, 966, 1931) and
pTIL 2-22 shown in Example 5, by the procedures as illustrated in
FIG. 7. A plasmid pTuB1P-3 include; insertion of the promoter
sequence for tufB in PBR322. The plasmid also contains a single
ClaI site and this is located 2bP downstream of the SD sequence as
shown in FIG. 7. Since pTrS-3 also contains a ClaI site between the
SD sequence and ATG initiation codon, and since this ClaI site is
not destroyed during the construction of expression plasmid by
using pTrS-3 IL-2 cDNA as described in Example 5, it is very simple
to replace the bacterial trp promoter with that of tufB so that the
IL-2 cDNA is expressed under the control of tuft promoter.
[0124] Therefore the plasmid pTIL 2-22 (30 .mu.g) was cleaved with
a restriction enzyme ClaI and PvuII in a conventional manner. The
fragment (ca 2.2 kb) containing IL-2 cDNA was isolated and purified
by agarose gel electrophoresis to recover 3 .mu.g of DNA. On the
other side, 20 .mu.g of pTuB1P-5 vector was cleaved similarly by
ClaI and PvuII, and the larger fragment (ca. 3.4 kb) containing
ampicillin resistant gene was isolated and purified by agarose gel
electrophoresis to recover 3.5 .mu.g of DNA. Then thus obtained two
fragments, one (ca. 3.4 kb) containing tufB promoter, the other
(ca. 2.2 kb) containing IL-2 cDNA, were ligated as follows. The
fragment containing IL-2 cDNA (1.2 .mu.g) and 0.3 .mu.g of the
fragment containing tufB promoter were mixed with 0.8 unit of T4
DNA ligase in 66 mM Tris-HCl of pH 7.5 containing 6.6 mM
MgCl.sub.2, 1 mM ATP and 10 mM DTT, and the mixture was allowed to
react at 4.degree. C. overnight. Thus ligated plasmid was then used
to transform into E. coli HB101 according to the conventional
procedures. Among the transformants appeared on Lbroth agar plate
containing ampicillin, eight colonies containing the IL-2 cDNA
portion such as pTuIL 2-22 in FIG. 7 were selected and plasmid DNA
was prepared as described in Example 5. The E. coli HB101
containing pTuIL 2-22 were cultured in L broth (100 ml) at
37.degree. C. When O.D at 650 mu arrived around 0.5-1.0, the
bacterial cells were collected, washed with 20 my Tris-HCl (pH 7.5,
30 mM NaCl) and resuspended into 2 ml of the same buffer. Thus
produced proteins were extracted similarly as Example 5. The
extracted IL-2 activity ranged from 6,0 00 to 56,0 00 units/ml.
[0125] Escherichia coli HB101 containing pTuIL 2-22 has been
deposited as in the accession number of FERM-BP 246.
EXAMPLE 7
[0126] A plasmid PGIL 2-22, carrying IL-2 cDNA was constructed from
pGL 101 (Roberts, T. M. and Laucer G. D., Meth. Enzym., 68,
473-483, 1979) and pTIL 2-22 shown in Example 5.
[0127] The plasmid pGL 101 (20 .mu.g) containing a lac promoter was
cleaved with a restriction enzyme PvuII in a conventional manner to
recover 17 .mu.g of DNA by successive treatment with phenol,
chloroform and ethanol precipitation. On the other side, pTIL 2-22
(75 .mu.g) was cleaved with ClaI and SalI to recover 2.2 .mu.g of a
DNA fragment containing IL-2 cDNA by agarose gel electrophoresis.
The fragment was rendered flush by the treatment with DNA
polymerase I (Klenow fragment), then thus obtained two fragments
(0.25 .mu.m and 0.66 .mu.g) were ligated with 1.0 unit of T4 DNA
ligase in the same manner as Example 6. Thus ligated plasmid was
then used to transform E. coli HB101 according to the conventional
manner. Among the transformants, the transformants possessing the
insertion of the ClaI-SalI fragment containing IL-2 cDNA as a
probe. These transformants were then cultured in X broth (10 ml)
containing 25 .mu.g/ml of ampicillin and the plasmid DNA was
prepared by the manner as described in Example 5. Thus the plasmid
DNA possessing the initiation sequence ATG of IL-2 cDNA just
downstream of a lac promoter was obtained by cleavage with PstI and
Xba.
[0128] Thus prepared pGIL 2-22 was inoculated in 100 ml of L-broth
containing 25 .mu.g/ml of ampicillin and 25 .mu.g/ml of
streptomycin and were cultured. When optical density at 650 m.mu.
arrived around 0.5, isopropyl-.beta.-D-thiogalactopyranoside (IPTG)
was added in the concentration of 1 mM and one hour later the
bacterial cells were collected and the cell extracts prepared in
the manner as described in Example 6. The extracted IL-2 activity
ranged from 6,0 00 to 80,0 00 units/ml.
[0129] Escherichia coli HB101 containing pGIL 2-22 as been
deposited in the accession number of FERM-BP 247.
EXAMPLE 8
[0130] Plasmid pTrS-3 (10 .mu.g) seas at first cleaved with the
restriction enzyme SalI and the SalI site was rendered flush by the
treatment with DNA polymerase (Klenow fragment) or with T4 DNA
polymerase. After cleavage with ClaI, a larger fragment, containing
the trp promoter region, was isolated bay agarose gel
electrophoresis in a conventional manner to recover 3 .mu.g of
DNA.
[0131] On the other side, 11 .mu.g of cleaved with HgiAI, treated
with T4 DNA polymerase and a larger fragment was isolated and
purified by agarose gel electrophoresis. Thus cDNA fragment coding
for 132 amino acids of IL-2 was obtained in an amount of 7.2 .mu.g.
Then 0.45 .mu.m of the fragment containing a trp promoter
(described above), 0.5 .mu.g of HgiAI-PstI fragment containing IL-2
cDNA and synthetic oligonucleotides (5') CGATAAGC TATGGCA (3'), and
(3') TATTCGATACCGT (5') (each 20 pmole), both of which were
phosphorylated at 5'-terminus, were ligated with 1 unit of T4 DNA
ligase in the same manner as described in Example 5.
[0132] Thus ligated plasmid was then used to transform E. coli
HB101. Among the transformants appeared, the target transformants
were selected as follows. The candidate transformants able to
hybridize which both of IL-2 cDNA and synthetic oligonucleotides
were firstly selected by colony hybridization method, then the
transformants possessing the insertion of DNA fragment initiating
from CCT sequence at position III to 113 in FIG. 2(a) (CCTACT-----)
just downstream of ATG GCA sequence were selected by PstI, XbaI
cleavage.
[0133] The above transformant, which contains pTIL2-21a or
pTIL2-21b, is cultured in L broth by the manner shown in Example 5,
and high activities of IL-2 can be found in cell extracts of the
transformants when assayed by the manner shown in Example 5.
[0134] Escherichia coli HB101 possessing pTIL2-2.a (AJ 12013) and
Escherichia coli HB101 possessing pTIL2-21b (AJ 12014) have been
deposited in the assession numbers of FERM-BP and FERM-BP
respectively.
[0135] The hosts, E. coli 1776 and HB101 (Boyer H. W. et al., J.
Mol. Biol. 41, 459, (1969)) used in the above Examples are known
and available for any public. Additionally, the hosts can be
obtained from the deposited transformants by culturing the
transformants in L-broth at 37.degree. C. to make release the
respective recombinant DNAs in the transformants and separating
strains which become sensitive to tetracycline and ampicillin as
the hosts.
[0136] The plasmid vectors pBR322 (Which is commercially sold by,
for example, Bethesda Research Laboratory), pCE-1, pTrS-3 and
pGL101 are known and available for any public. In addition, the
plasmid vectors can be obtained from the deposited transformants by
separating the recombinant plasmid DNAs in the transformants by a
conventional manner and by separating the plasmid vectors by the
manners which are naturally obvious from the disclosures in the
respective examples. For example, pCE-1 can be obtained by
digesting pCEIL-2 by PstI and separating larger DNA fragment
formed. Additionally, pTrS-3 and pTuB1P-5 have been deposited as E.
coli FERM-P 6735 and E. coli ATCC 31871 respectively.
[0137] Having now fully described this invention, it will be
understood by those of skill in the art that the same can be
performed within a wide and equivalent range of conditions,
parameters and the like without affecting the spirit or scope of
the invention or of any embodiment thereof.
* * * * *