Claims
- 1. A method for preparing tPA in host cells which method comprises:
- (a) transfecting said host cells with a plasmid effective in expressing DNA sequences encoding a DHFR protein and tPA protein contained therein;
- (b) growing the host cells under conditions such that said tPA protein is produced in amounts of at least 0.1 pg of tPA per cell per day; and
- (c) recovering said tPA from said cells.
- 2. A method of preparing tPA in mammalian host cells, which method comprises:
- (a) culturing Chinese hamster ovary cells deficient in DHFR;
- (b) transfecting said cells with an expression vector effective in expressing DNA sequences contained therein, said sequences encoding a DHFR protein and tPA protein;
- (c) selecting from said cells those cells capable of survival in HGT-deficient media:
- (d) growing said selected cells under culture conditions such that said tPA is produced; and
- (e) recovering from the culture at least 0.1 pg of tPA per cell per day.
- 3. A method of preparing tPA in mammalian host cells, which method comprises:
- (a) culturing Chinese hamster ovary cells deficient in DHFR;
- (b) transfecting said cells with an expression vector effective in expressing DNA sequences contained therein, said sequences encoding a DHFR protein and tPA protein;
- (c) selecting from said cells those cells capable of survival in HGT-deficient media;
- (d) growing said selected cells under culture conditions such that said tPA is produced; and
- (e) recovering from the culture at least 0.5 pg of tPA per cell per day.
- 4. A method of preparing tPA in mammalian host cells, which method comprises:
- (a) culturing Chinese hamster ovary cells deficient in DHFR;
- (b) transfecting said cells with an expression vector effective in expressing DNA sequences contained therein, said sequences encoding a DHFR protein and tPA protein;
- (c) selecting from said cells those cells capable of survival in HGT-deficient media;
- (d) growing said selected cells in the presence of a concentration of MTX effective to further select cells having amplified DNA sequences for tPA;
- (e) growing said amplified selected cells under culture conditions such that said tPA is produced; and
- (f) recovering from the culture at lest 20 pg of tPA per cell per day.
- 5. A method of preparing tPA in mammalian host cells, which method comprises:
- (a) culturing Chinese hamster ovary cells deficient in DHFR;
- (b) transfecting said cells with an expression vector effective in expressing DNA sequences contained therein, said sequences encoding a DHFR protein and tPA protein;
- (c) selecting from said cells those cells capable of survival in HGT-deficient media;
- (d) growing said selected cells in the presence of an effective amplifying concentration of a DHFR inhibitor;
- (e) growing these cells capable of survival in the presence of a DHFR inhibitor under culture conditions such that said tPA is produced; and
- (f) recovering from the culture at least 20 pg of tPA per cell per day.
- 6. The method of any one of claims 1, 2, 3, 4 or 5, wherein said DHFR protein is wild-type DHFR.
- 7. The method of any one of claims 1, 2, 3, 4 or 5 wherein said DHFR protein has a low binding affinity for MTX.
- 8. The method of any one of claims 1, 2, 3, 4 or 5 wherein the host cells prior to transfection are cells deficient in DHFR.
- 9. The method of any one of claims 1, 2, 3, 4 or 5 wherein the host cells are cells sensitive to MTX.
- 10. The method of claim 1 wherein the plasmid is pETPFR.
Parent Case Info
This is a continuation application of Ser. No. 07/149,032, filed Jan. 27, 1988, now abandoned, which is a continuating application under 35 U.S.C. 120/121 of U.S. Ser. No. 07/149,990, filed 27 January 1988, now abandoned, which is a continuating application under 35 U.S.C. 120/121 of U.S. Ser. No. 06/459,153, filed 19 Jan. 1983, now abandoned. Reference is hereby made under 35 U.S.C. 120/121 to U.S. Ser. No. 06/374,860 filed 5 May 1982, U.S. Ser. No. 06/398,003, filed 14 July 1982, both now abandoned, and U.S. Ser. No. 06/483,052, filed 7 April 1983, now U.S. Pat. No. 4,766,075.
US Referenced Citations (2)
Number |
Name |
Date |
Kind |
4399216 |
Axel et al. |
Aug 1983 |
|
4419446 |
Howley et al. |
Dec 1983 |
|
Foreign Referenced Citations (5)
Number |
Date |
Country |
37687 |
Oct 1981 |
EPX |
54223 |
Jun 1982 |
EPX |
93619 |
Nov 1983 |
EPX |
117060 |
Aug 1984 |
EPX |
8102425 |
Sep 1981 |
WOX |
Non-Patent Literature Citations (8)
Entry |
Blattner et al., Science, 202, 334 (1978). |
Chang et al., Nature, 275, 617 (1978). |
Christman et al., Proc. Natl. Acad. Sci., 79, 1815, (1982). |
Haber et al., Cell, 26, 355 (1981). |
Kaufman et al., J. Mol. Biol., 159, 601 (1982). |
Opdenakker et al., Eur. J. Biochem., 121, 269 (1982). |
Ringold et al., Journal of Molecular and Applied Genetics, 1, 165 (1981). |
Wigler et al., Proc. Natl. Acad. Sci. (USA), 77, 3567 (1980). |
Continuations (3)
|
Number |
Date |
Country |
Parent |
149032 |
Jan 1988 |
|
Parent |
149990 |
Jan 1988 |
|
Parent |
459153 |
Jan 1983 |
|