Enhanced cell-free synthesis of active proteins containing disulfide bonds

1. A method for cell-free synthesis of properly folded polypeptides comprising at least one disulfide bond, the improvement comprising:

synthesizing said polypeptide in a reaction mix comprising a biological extract derived from a bacterial cell that has been genetically modified to inactivate glutathione reductase and pre-treated with low concentrations of a sulfhydryl inactivating agent;

wherein the low concentration of the sulfhydryl inactivating agent is equivalent in sulfhydryl inactivating activity to iodoacetamide at a concentration of from 10 μM to about 100 μM.

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

3. The method of claim 2 , wherein the sulfhydryl inactivating agent is iodoacetamide.

4. The method according to claim 1 , wherein the biological extract is treated to remove thioredoxin reductase.

5. The method according to claim 4 , wherein the bacterial cell from which the biological extract is derived is genetically modified such that said thioredoxin reductase protein comprises an affinity tag; and the treatment to remove the thioredoxin reductase comprises contacting the biological extract with an affinity resin specific for the affinity tag.

6. The method according to claim 1 , wherein said reaction mix comprises an energy source lacking high energy phosphate bonds at a concentration of at least about 50 mM.

7. The method according to claim 6 , wherein said energy source is glucose or a glycolytic intermediate lacking high energy phosphate bonds.

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

9. The method according to claim 8 , wherein said redox buffer comprises one or more of glutathione, cysteine, and homocysteine.

10. The method according to claim 9 , wherein said redox buffer comprises a mixture of oxidized and reduced glutathione.

11. The method according to claim 1 , wherein said reaction mixture comprises one or more enzymes that enhance polypeptide folding or generation of disulfide bonds.

12. The method according to claim 11 , wherein said one or more enzymes that enhance polypeptide folding or generation of disulfide bonds are foldase enzymes.

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

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

15. A reaction mix for cell-free protein translation, comprising a biological extract derived from a bacterial cell that has been genetically modified to inactivate glutathione reductase and pre-treated with low concentrations of a sulfhydryl inactivating agent;

wherein the low concentration of the sulfhydryl inactivating agent is equivalent in sulfhydryl inactivating activity to iodoacetamide at a concentration of from 10 μM to about 100 μM.

16. The reaction mix according to claim 15 , wherein said sulfhydryl inactivating agent alkylates or acetylates free sulfhydryl groups.

17. The method of claim 16 , wherein the sulfhydryl inactivating agent is iodoacetamide.

18. The reaction mix according to claim 15 , wherein said biological extract is treated to remove thioredoxin reductase.

19. The reaction mix according to claim 18 , wherein the bacterial cell from which the biological extract is derived is genetically modified such that said thioredoxin reductase protein comprises an affinity tag; and the treatment to remove the thioredoxin reductase comprises contacting the biological extract with an affinity resin specific for the affinity tag.

20. The reaction mix according to claim 15 , wherein said reaction mix comprises an energy source lacking high energy phosphate bonds, at a concentration of at least about 50 mM.

21. The reaction mix according to claim 20 , wherein said energy source is glucose or a glycolytic intermediate lacking high energy phosphate bonds.