Universal procedure for refolding recombinant proteins
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C07K-014/00
C12P-021/06
출원번호
US-0420044
(2003-04-17)
발명자
/ 주소
Lin,Xinli
출원인 / 주소
Oklahoma Medical Research Foundation
대리인 / 주소
Morrison &
인용정보
피인용 횟수 :
7인용 특허 :
3
초록▼
A universal folding method that has been demonstrated to be effective in refolding a variety of very different proteins expressed in bacteria as inclusion bodies has been developed. Representative proteins that can be dissolved and refolded in biologically active form, with the native structure, are
A universal folding method that has been demonstrated to be effective in refolding a variety of very different proteins expressed in bacteria as inclusion bodies has been developed. Representative proteins that can be dissolved and refolded in biologically active form, with the native structure, are shown in Table I. The method has two key steps to unfold and then refold the proteins expressed in the inclusion bodies. The first step is to raise the pH of the protein solution in the presence of denaturing agents to pH greater than 9, preferably 10. The protein solution may be maintained at the elevated pH for a period of up to about 24 hours, or the pH immediately decreased slowly, in increments of about 0.2 pH units/24 hours, until the solution reaches a pH of about 8.0, or both steps used. In the preferred embodiment, purified inclusion bodies are dissolved in 8 M urea, 0.1 M Tris, 1 mM glycine, 1 mM EDTA, 10 mM beta-mercaptoethanol, 10 mM dithiothreitol (DTT), 1 mM reduced glutathione (GSH), 0.1 mM oxidized glutathione (GSSG), pH 10. The absorbance at 280 nm (OD280) of the protein solution is 5.0. This solution is rapidly diluted into 20 volumes of 20 mM Tris base. The resulting solution is adjusted to pH 9.0 with 1 M HCl and is kept at 4째 C. for 24 hr. The pH is adjusted to pH 8.8 and the solution is kept at 4째 C. for another 24 hrs. This process is repeated until the pH is adjusted to 8.0. After 24 hr at pH 8.0, the refolded proteins can be concentrated by ultrafiltration and applied to a gel filtration column for purification.
대표청구항▼
I claim: 1. A method for refolding of a recombinant protein comprising maintaining the protein at a pH of 9.0 or greater, in the presence of one or more chaotrophic and reducing agents, and decreasing the pH of the solution gradually over a period of at least about 24 hrs to pH about 8.0 to induce
I claim: 1. A method for refolding of a recombinant protein comprising maintaining the protein at a pH of 9.0 or greater, in the presence of one or more chaotrophic and reducing agents, and decreasing the pH of the solution gradually over a period of at least about 24 hrs to pH about 8.0 to induce renaturation of the protein so that it qualitatively exhibits a biological activity and structural characteristic of the protein. 2. The method of claim 1 wherein the pH is decreased in increments equivalent to 0.2 pH units per 24 hours. 3. The method of claim 1 wherein the protein is maintained at a pH of greater than 9.0 for a period of at least 24 hours. 4. The method of claim 1 wherein the pH is decreased by addition of acid. 5. The method of claim 1 wherein the pH is decreased by dilution or dialysis into a solution of a lower pH. 6. The method of claim 1 wherein the chaotrophic and reducing agents are selected from the group consisting of between 0.5 and 1.0 M urea, 0.1 mM to 100mM beta-mercaptoethanol, 0.1 mM to 100 mM DTT, 0.1 mM to 10 mM reduced glutathione, and 0.1 mM to 10 mM oxidized glutathione. 7. The method of claim 1 wherein the protein is first extracted from bacterial inclusion bodies. 8. The method of claim 7 wherein the bacteria is E. coli. 9. The method of claim 7 wherein the inclusion bodies are dissolved at a final pH of between 9 and 10. 10. The method of claim 1, wherein the protein is maintained at a pH above about 10.0. 11. The method of claim 1, wherein the protein is maintained at a pH above about 11.0. 12. The method of claim 1, wherein the protein is maintained at a pH above about 12.0. 13. The method of claim 1 wherein the pH is decreased over a period of at least about 36 hours. 14. The method of claim 1, wherein the pH of the solution is reduced more quickly at the higher pH and more gradually nearer the pH about 8.0. 15. The method of claim 1 comprising the additional step of separating protein species which exhibits biological activity from inactive, or wrongly folded, protein species. 16. The method of claim 1 wherein the chaotrophic agent is urea. 17. The method of claim 1 wherein the chaotrophic agent is guanidine hydrochloride. 18. The method of claim 1 wherein the chaotrophic agent is L-arginine. 19. The method of claim 1 wherein the reducing agent is dithiothreitol (DTT). 20. The method of claim 1 wherein the reducing agent is β-mercaptoethanol. 21. The method of claim 1 wherein the reducing agent is a combination of reduced glutathione (GSH) and oxidized glutathione (GSSG). 22. The method of claim 7 wherein the inclusion bodies are dissolved at a final pH of greater than 9.0. 23. The method of claim 7 wherein the inclusion bodies are dissolved at a final pH of greater than 10.0. 24. The method of claim 7 wherein the inclusion bodies are dissolved in a solution containing 8 M urea, 0.1 M Tris, 1 mM glycine, 1 mM EDTA, 10 mM beta-mercaptoethanol, 10 mM dithiothreitol, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, pH 10. 25. The method of claim 24 further comprising adjusting absorbance at 280 nm of the solution to 5.0; and rapidly diluting the solution into 20 volumes of 20 mM Tris base before the step of decreasing the pH of the solution.
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이 특허에 인용된 특허 (3)
Tang, Jordan J. N.; Lin, Xinli; Koelsch, Gerald; Hong, Lin, Catalytically active recombinant memapsin and methods of use thereof.
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