Enhancedin vitrosynthesis of active proteins containing disulfide bonds

• US 7,041,479 B2
• Filed: 03/31/2003
• Issued: 05/09/2006
• Est. Priority Date: 09/06/2000
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Swartz, James Robert, Kim, Dong-Myung
• Primary Examiner(s): KAM, CHIH MIN
• Application Number:
  US10/404,599
• Publication Number:
  US 20040038332A1
• Time in Patent Office: 1,135 Days
• Field of Search: 435/70.1, 435/69.1, 435/25, 435/189, 435/215, 435/243, 530/350
• US Class Current: 435/70.1
• CPC Class Codes: C07K 1/1133 by redox-reactions involving cystein/cystin side chains

Abstract

Compositions and methods are provided for the enhanced in vitro synthesis of polypeptides containing disulfide bonds. In order to improve the performance of in vitro protein synthesis reactions, pre-treatment and redox buffering of the reaction mix is performed in order to optimize the redox potential. Exogenous enzymes that enhance protein folding and disulfide bond formation may also be added to the reaction.

Claims

1. A method for in vitro synthesis of properly folded polypeptides comprising at least one disulfide bond, in a reaction mix comprising a biological extract comprising components of polypeptide synthesis machinery, wherein such components are capable of expressing a nucleic acid encoding a desired polypeptide, the method comprising:

synthesizing said polypeptide in a reaction mix wherein the biological extract has been pre-treated with a sulfhydryl inactivating agent that irreversibly inactivates free sulfhydryl groups;

isolating said polypeptide from said reaction mixture, wherein the amount of said polypeptide that is properly folded is enhanced relative to polypeptide synthesized in the absence of pretreatment with said sulfhydryl inactivating agent.

2. The method according to claim 1, wherein said sulfhydryl inactivating agent alkylates free sulfhydryl groups.

3. The method according to claim 1, wherein said sulthydryl inactivating agent is selected from the group consisting of iodoacetamide, N-ethyl maleimides, and iodoacetate.

4. The method according to claim 1, wherein said reaction mix further comprises a redox buffer.

5. The method according to claim 4, wherein said redox buffer comprises one or more of glutathione, dithiothreitol, dithioerythritol, β

-mercaptoethanol, thioglycolate and cysteine.

6. The method according to claim 5, wherein said redox buffer comprises a mixture of oxidized and reduced glutathione.

7. The method according to claim 1, wherein said biological extract is a bacterial cell extract from a bacterial cell that has been genetically modified to inactivate one or more reducing enzymes selected from thioredoxin reductase and glutathione reductase.

8. The method according to claim 1, wherein said biological extract is treated to inactivate one or more reducing enzymes selected from thioredoxin reductase and glutathione reductase.

9. The method according to claim 1, wherein said reaction mixture is further modified by the addition of one or more enzymes that enhance polypeptide folding or generation of disulfide bonds.

10. The method according to claim 9, wherein said one or more enzymes that enhance polypeptide folding or generation of disulfide bonds are foldase enzymes and/or chaperonins.

11. The method according to claim 10, wherein said foldase enzyme or chaperonin is selected from the group consisting of RotA (PpiA), FkpA, Skp, SurA, PpiD, DsbA, DsbB, DsbC, DsbD, PDI (protein disulfide isomerase), GroEL/ES, DnaK, DnaJ, GrpE, BIP (immunoglobulin heavy chain binding protein), PPI (peptidylprolyl isomerase) and cyclophilin.

12. The method according to claim 1, wherein said reaction mixture is substantially free of polyethylene glycol.

13. The method according to claim 12, wherein said reaction mixture further comprises one or more of spermine, spermidine and putrescine.

14. The method according to claim 1, wherein said sulfhydryl inactivating agent acetylates free sulfhydryl groups.

15. The method according to claim 1, wherein said sulfhydryl inactivating agent is N-iodoacetyl-N′

-(5-sulfo-1-naphthyl)ethylenediamine.

16. A method for optimizing the in vitro synthesis of a properly folded polypeptide comprising at least one disulfide bond, in a plurality of reaction mixtures comprising a biological extract comprising components of polypeptide synthesis machinery, wherein such components are capable of expressing a nucleic acid encoding a desired polypeptide the method comprising:

varying at least one of;

the concentration of a foldase or chaperonin, a redox buffer, and temperature, in a plurality of reaction mixtures, wherein the biological extract has been pre-treated with a sulfhydryl inactivating agent that irreversibly inactivates free sulfhydryl groups;

synthesizing said polypeptide in said plurality of reaction mixtures;

quantitating the amount of properly folded polypeptide synthesized in each of said reaction mixtures;

determining which of said plurality of reaction mixtures provides for an optimized yield of properly folded polypeptide.

17. The method according to claim 16, wherein said sulfhydryl inactivating agent alkylates or acetylates free sulfhydryl groups.

18. The method according to claim 17, wherein said redox buffer comprises a mixture of oxidized and reduced glutathione, and said plurality of reaction mixtures varies the ratio of said mixture of oxidized and reduced glutathione.

19. The method according to claim 16, wherein said sulfhydryl inactivating agent is selected from the group consisting of iodoacetamide, N-ethyl maleimides, and iodoacetate, and N-iodoacetyl-N′

-(5-sulfo-1-naphthyl)ethylenediamine.

20. The method according to claim 16, wherein said redox buffer comprises one or more of glutathione, dithiothreitol, dithioerythritol, β

-mercaptoethanol, thioglycolate and cysteine.

21. The method according to claim 16, wherein said foldase enzyme or chaperonin is selected from the group consisting of RotA (PpiA), FkpA, Skp, SurA, PpiD, DsbA, DsbB, DsbC, DsbD, PDI (protein disulfide isomerase), GroEL/ES, DnaK, DnaJ, GrpE, BIP (immunoglobulin heavy chain binding protein), PPI (peptidylprolyl isomerase) and cyclophilin.

22. The method according to claim 21, wherein said reaction mixture further comprises one or more of spermine, spermidine and putrescine.

23. The method according to claim 16, wherein said reaction mixture is substantially free of polyethylene glycol.