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Oxidative refolding chromatography for refolding of ribonuclease A and cyclohexanone monooxygenase expressed in recombinant escherichia coli : 재조합 대장균에서 발현된 cyclohexanone monooxygeanse[실은 monooxygenase]와 ribonuclease A의 재접힘을 위한 산화적 재접힘 크로마토그래피
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 서진호 | - |
dc.contributor.author | 이대희 | - |
dc.date.accessioned | 2010-07-08T23:06:16Z | - |
dc.date.available | 2010-07-08T23:06:16Z | - |
dc.date.copyright | 2003. | - |
dc.date.issued | 2003 | - |
dc.identifier.uri | http://dcollection.snu.ac.kr:80/jsp/common/DcLoOrgPer.jsp?sItemId=000000058217 | - |
dc.identifier.uri | https://hdl.handle.net/10371/68519 | - |
dc.description | Thesis (master`s)--서울대학교 대학원 :농생명공학부,2003. | en |
dc.description.abstract | Oxidative refolding of the denatured/reduced CHMO and RNase A was examined
through refolding chromatography. In order to construct the refolding matrices, E. coli dsbA gene, mini-chaperone (191 - 345 peptide fragment from E. coli GroEL) gene, and ppiA gene from human were cloned and overproduced in E. coli BL21(DE3). The folding machinery (DsbA, mini-chaperone and hPPIase) with the charged ten arginine stretch at the C-terminal was purified using a single ion-exchange chromatography with a high purity. Efficient immobilization of foldases and mini-chaperone on SP-Sepharose were achieved using a polyarginine stretch for preparative purposes. The maximum amounts of the immobilized folding machinery reached 5.2 £¿6.5 mg/mL. The immobilized foldases and mini-chaperone were fully functional when assayed in a batch mode. Two model proteins containing disulfide bonds were chosen to test the application of the refolding chromatography system. The first model protein is bovine pancreatic ribonuclease A (RNase A) containing four disulfide bonds. The refolding matrices (hPPIase, DsbA/mini-chaperone or DsbA/hPPIase/mini-chaperone) proved to be highly efficient (67 %, 68 % and 73 %, respectively) in restoring the native structure and biological properties of RNase A. The second model protein, cyclohexanone monooxygenase (CHMO) derived from Acinetobacter sp. NCIB 9871 was overproduced as inclusion bodies in recombinant Escherichia coli. After isolation and solubilization of inclusion bodies, active proteins could be generated through the refolding chromatography technique. A 53 % yield of protein with biological activity was recovered when treated with the ternary refolding matrix (DsbA/mini-chapereone/hPPIase-Sepharose). To examine the effects of coexpression of molecular chaperones or foldases on the production of foreign proteins in Escherichia coli, plasmids that permitted controlled expression of foldases were newly constructed. The model protein used to determine whether these systems were useful was cyclohexanone monooxygenase (CHMO), which are prone to aggregation when expressed in E. coli. The results reveal that chaperones (GroEL-GroES, DnaK-DnaJ-GrpE, and mini-chaperone) and foldases (DsbA, DsbC, and hPPIase) coexpression had marked effects on the production of soluble and active CHMO, presumably through facilitating correct folding. Whereas overexpression of the GroEL-GroES team alone was sufficient to prevent aggregation of CHMO, overexpression of the GroEL-GroES family together with DnaK-DnaJ-GrpE was more effective for CHMO, suggesting that GroEL-GroES and DnaK-DnaJ-GrpE play synergistic roles in vivo. Also, foldases were effective for improving soluble CHMO production in E. coli. DsbA coexpression was shown to have the most significant effect on the yield of correctly folded CHMO. These results demonstrate that coexpression of folding catalyst proteins could significantly enhance the production of active proteins in recombinant E. coli. | en |
dc.format.extent | xii, 123 leaves | en |
dc.language.iso | en | en |
dc.publisher | 서울대학교 대학원 | en |
dc.subject | 동시발현 | en |
dc.subject | Dsba | en |
dc.subject | 재접힘 | en |
dc.subject | Mini-chaperone | en |
dc.subject | 고정화 | en |
dc.subject | Peptidyl-prolyl cis/trans isomerase (ppiase) | en |
dc.subject | 리폴딩 크로마토그래피 | en |
dc.subject | Immobilization | en |
dc.subject | Foldases | en |
dc.subject | Renaturation | en |
dc.subject | Molecular chaperone | en |
dc.subject | Ribonuclease A (RNase A) | en |
dc.subject | CHMO | en |
dc.subject | Cyclohexanone monooxygenases (CHMO) | en |
dc.subject | RNase A | en |
dc.subject | Molecular chaperone | en |
dc.subject | Foldases | en |
dc.subject | Coexpression | en |
dc.title | Oxidative refolding chromatography for refolding of ribonuclease A and cyclohexanone monooxygenase expressed in recombinant escherichia coli | en |
dc.title.alternative | 재조합 대장균에서 발현된 cyclohexanone monooxygeanse[실은 monooxygenase]와 ribonuclease A의 재접힘을 위한 산화적 재접힘 크로마토그래피 | en |
dc.type | Thesis | en |
dc.contributor.department | 농생명공학부 | - |
dc.description.degree | Master | en |
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