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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
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- Authors
- Advisor
- 서진호
- Issue Date
- 2003
- Publisher
- 서울대학교 대학원
- Keywords
- 동시발현 ; Dsba ; 재접힘 ; Mini-chaperone ; 고정화 ; Peptidyl-prolyl cis/trans isomerase (ppiase) ; 리폴딩 크로마토그래피 ; Immobilization ; Foldases ; Renaturation ; Molecular chaperone ; Ribonuclease A (RNase A) ; CHMO ; Cyclohexanone monooxygenases (CHMO) ; RNase A ; Molecular chaperone ; Foldases ; Coexpression
- Description
- Thesis (master`s)--서울대학교 대학원 :농생명공학부,2003.
- 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.
- Language
- English
- URI
- http://dcollection.snu.ac.kr:80/jsp/common/DcLoOrgPer.jsp?sItemId=000000058217
https://hdl.handle.net/10371/68519
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