Enhanced cell-free synthesis of active proteins containing disulfide bonds
View Patent ↗Compositions and methods are provided for the enhanced in vitro synthesis of active polypeptides containing disulfide bonds. In certain embodiments of the invention, the reaction mix includes a biological extract derived from a bacterial cell in which the glutathione reductase gene has been inactivated, which is pre-treated with a low concentration of a sulfhydryl inactivating agent.
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.