High pressure refolding of protein aggregates and inclusion bodies
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C07K-014/00
C12N-009/00
출원번호
UP-0341013
(2006-01-27)
등록번호
US-7767795
(2010-08-24)
발명자
/ 주소
Randolph, Theodore W.
Carpenter, John F.
St. John, Richard
출원인 / 주소
BaroFold Inc.
대리인 / 주소
Cooley LLP
인용정보
피인용 횟수 :
4인용 특허 :
23
초록▼
The present disclosure provides an effective method for the refolding of denatured proteins in solution so that properly folded, biologically active protein in solution is recovered in high yield. The refolding takes place at pressures between about 0.25 kbar to about 3.5 kbar, advantageously at abo
The present disclosure provides an effective method for the refolding of denatured proteins in solution so that properly folded, biologically active protein in solution is recovered in high yield. The refolding takes place at pressures between about 0.25 kbar to about 3.5 kbar, advantageously at about 1.5 kbar to about 3 kbar. Typically a chaotropic agent is present at a concentration which is not effective for denaturing protein at atmospheric pressure, and optionally, oxidation-reduction reagents can be incorporated in the refolding solution so that native intramolecular disulfide bonds can be formed where that is desired. The method is applicable to substantially all proteins, especially after solubilization and/or denaturation of insoluble protein aggregates, inclusion bodies, or abnormal oligomeric (soluble) aggregates.
대표청구항▼
What is claimed is: 1. A method for producing renatured, biologically active protein from a soluble denatured protein solution, said method comprising the steps of: (a) adjusting the concentration of denatured protein in solution to from about 0.01 mg/mL to about 500 mg/mL; (b) increasing pressure
What is claimed is: 1. A method for producing renatured, biologically active protein from a soluble denatured protein solution, said method comprising the steps of: (a) adjusting the concentration of denatured protein in solution to from about 0.01 mg/mL to about 500 mg/mL; (b) increasing pressure on the solution of denatured protein, which solution further comprises a chaotropic agent, to from about 0.25 kbar to about 3.5 kbar; and (c) incubating the solution of denatured protein under a pressure from about 0.25 kbar to about 3.3 kbar; then (d) reducing the chaotropic agent concentration to a level sufficient to permit biological activity of the protein at atmospheric pressure; then (e) after step (d), reducing the pressure to atmospheric pressure, whereby the protein has refolded to assume a native conformation and has biological activity of the native protein. 2. The method of claim 1, wherein during the incubation step (c), the solution or suspension further comprises an oxidizing agent, and a reducing agent wherein the oxidizing agent is oxidized glutathione and the reducing agent is dithiothreitol. 3. The method of claim 1, wherein the pressure in the incubation step (c) is from about 0.5 kbar to about 3.3 kbar. 4. The method of claim 3, wherein during the incubation step (c) the chaotropic agent is guanidine hydrochloride present at a concentration from about 0.1 to about 1M. 5. The method of claim 4, wherein during the incubation step (c) the protein concentration is from about 1 to about 100 mg/mL. 6. The method of claim 4, wherein during the incubation step (c) the protein concentration is from about 1 to about 20 mg/mL. 7. The method of claim 1, wherein at step (d), the concentration of the chaotropic agent is decreased to less than about 0.001M. 8. The method of claim 1, wherein, prior to step (a), the solubilized denatured protein is treated with a reducing agent. 9. The method of claim 1, wherein the solution of protein in step (a) comprises a detergent. 10. The method of claim 9, wherein the detergent is selected from the group consisting of sodium dodecyl sulfate, polyethoxysorbitan, deoxycholate, sodium octyl sulfate, sodium tetradecyl sulfate, polyoxyethylene ethers, sodium cholate, octylthioglucopyranoside, n-octylglucopyranoside, alkyltrimethylammonium bromides, alkyltrimethyl ammonium chlorides, and sodium bis (2-ethylhexyl) sulfosuccinate.
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이 특허에 인용된 특허 (23)
Randolph,Theodore W.; Carpenter,John F.; St. John,Richard, High pressure refolding of protein aggregates and inclusion bodies.
Chang Judy Y. (Hillsborough CA) McFarland Nancy C. (Hillsborough CA) Swartz James R. (Menlo Park CA), Method for refolding insoluble, misfolded insulin-like growth factor-1 into an active conformation.
Rudolph Rainer (Weilheim DEX) Buchner Johannes (Regensburg DEX) Lenz Helmut (Tutzing DEX), Process for activation of recombinant protein produced by prokaryotes.
Wolfe Sidney N. (El Cerrito CA) Dorin Glenn J. (San Rafael CA) Davis John T. (Berkeley CA) Smith Flint (Gardon Grove CA) Lim Amy (Hercules CA) Weissburg Robert (Oakland CA), Process for recovering purified, oxidized, renatured recombinant interleukin-2 from microorganisms.
Heilman ; Jr. Conrad J. (Chester NY), Process for solubilization, purification and characterization of protein from insoluble protein aggregates or complexes.
Rudolph Rainer (Regensburg DEX) Fischer Stephan (Weilheim DEX) Mattes Ralf (Oberhausen DEX), Process for the activating of gene-technologically produced, heterologous, disulphide bridge-containing eukaryotic prote.
Halenbeck Robert (San Rafael CA) Koths Kriston (El Cerrito CA) Cowgill Cynthia (Berkeley CA) Laird Walter J. (Pinole CA), Production of purified, biologically active, bacterially produced recombinant human CSF-1.
Fischer, Laurent Bernard; Carboulec, Nicolas Pierre Yves; Lux, Fabien, Expression of chimeric KSAC protein and method of producing soluble proteins by high pressure.
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