Process for producing recombinant trypsin
View Patent ↗A process for producing recombinant trypsin from prokaryote host cells in high yield and high specific activity is described. In particular, a process for producing recombinant trypsin from E. coli is described.
1. A process for refolding recombinant trypsinogen produced from a prokaryote host cell comprising:
(a) providing the recombinant trypsinogen at a concentration of from 5 to 15 g/L in a solubilization solution comprising a chaotropic agent, a first buffer agent, and a first low molecular weight reducing agent, wherein the solubilization solution does not include a low molecular weight oxidizing agent that would provide a redox pair with the first low molecular weight reducing agent; and
(b) infusing the solubilization solution comprising the recombinant trypsinogen of step (a) over time into a diluent comprising a second buffer agent, a second low molecular weight reducing agent, the chaotropic agent, and arginine to provide a refold solution that comprises the recombinant trypsinogen at a concentration greater than 1 g/L, the chaotropic agent at from 20% to 30% of its concentration in the solubilization solution of step (a), the first and second buffer agents, arginine, and the first and second low molecular weight reducing agents and incubating the refold solution for a time sufficient for the recombinant trypsinogen to refold into a conformation characteristic of native trypsinogen and form the disulfide bonds characteristic of native trypsinogen, wherein the refold solution does not include a low molecular weight oxidizing agent that would provide a redox pair with either the first or second low molecular weight reducing agents.
2. The process of claim 1 , wherein in both steps (a) and (b) the chaotropic agent is urea.
3. The process of claim 1 , wherein the chaotropic agent is in the refold solution at a concentration of from 1.4 to 1.8 M.
4. The process of claim 1 , wherein the first and second low molecular weight reducing agents are cysteine or cysteine hydrochloride.
5. The process of claim 1 , wherein the first low molecular weight reducing agent is in the solubilization solution at a concentration sufficient to provide from 1 to 15 SH residues of the first low molecular weight reducing agent per cysteine residue of the recombinant trypsinogen.
6. The process of claim 1 , wherein the first and second low molecular weight reducing agents are in the refold solution at a concentration sufficient to provide from 1 to 30 SH residues of the low molecular weight reducing agents per cysteine residue of the recombinant trypsinogen.
7. The process of claim 1 , further comprising:
(c) diluting the refold solution and incubating the diluted refold solution for a time sufficient for the recombinant trypsinogen therein to auto-catalyze to provide the recombinant trypsin.
8. The process of claim 7 , wherein the recombinant trypsin is subjected to a chromatography step to provide a recombinant trypsin substantially free of tryptic peptides from the auto-catalysis of the recombinant trypsinogen and host cell residuals, including DNA, host cell proteins and endotoxin.
9. The process of claim 8 , wherein the recombinant trypsin substantially free of peptides from the auto-catalysis is concentrated to provide a composition comprising the recombinant trypsin at a concentration of from 20 to 80 mg/mL.
10. The process of claim 8 , wherein the chromatography step comprises affinity chromatography.
11. The process of claim 10 , wherein the affinity chromatography is performed on a matrix comprising benzamidine.
12. The process of claim 11 , wherein the affinity chromatography is performed on a matrix comprising benzamidine-Sepharose.
13. The process of claim 7 , wherein the first buffer agent is ethanolamine.
14. The process of claim 7 , wherein in both steps (a) and (b) the chaotropic agent is urea.
15. The process of claim 7 , wherein the chaotropic agent is in the refold solution at a concentration of from 1.4 to 1.8 M.
16. The process of claim 7 , wherein the first and second reducing agents are cysteine or cysteine hydrochloride.
17. The process of claim 7 , wherein the first low molecular weight reducing agent is in the solubilization solution at a concentration sufficient to provide from 1 to 15 SH residues of the first low molecular weight reducing agent per cysteine residue of the recombinant trypsinogen.
18. The process of claim 7 , wherein the first and second low molecular weight reducing agents are in the refold solution at a concentration sufficient to provide from 1 to 30 SH residues of the low molecular weight reducing agents per cysteine residue of the recombinant trypsinogen.
19. The process of claim 1 , wherein the recombinant trypsinogen is obtained from inclusion bodies isolated from the prokaryote host cells.
20. The process of claim 1 , wherein the first buffer agent is ethanolamine.
21. The process of claim 1 , wherein the second buffer agent is Tris.