Refolding and Purification of Yeast Carboxypeptidase Y Expressed as Inclusion Bodies in Escherichia coli

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dc.contributor.authorMoon Sun Hahm-
dc.contributor.authorBong Hyun Chung-
dc.date.accessioned2017-04-19T08:58:17Z-
dc.date.available2017-04-19T08:58:17Z-
dc.date.issued2001-
dc.identifier.issn1046-5928-
dc.identifier.uri10.1006/prep.2001.1418ko
dc.identifier.urihttps://oak.kribb.re.kr/handle/201005/5538-
dc.description.abstractThe genes encoding carboxypeptidase Y (CPY) and CPY propeptide (CPYPR) from Saccharomyces cerevisiae were cloned and expressed in Escherichia coli. Six consecutive histidine residues were fused to the C-terminus of the CPYPR for facilitated purification. High-level expression of CPY and CPYPR-His6 was achieved but most of the expressed proteins were present in the form of inclusion bodies in the bacterial cytoplasm. The CPY and CPYPR-His6 produced as inclusion bodies were separated from the cells and solubilized in 6 and 3 M guanidinium chloride, respectively. The denatured CPYPR-His6 was refolded by dilution 1:30 into the renaturation buffer (50 mM Tris-HCl containing 0.5 M NaCl and 3 mM EDTA, pH 8.0), and the refolded CPYPR-His6 was further purified to 90% purity by single-step immobilized metal ion affinity chromatography. The denatured CPY was refolded by dilution 1:60 into the renaturation buffer containing CPYPR-His6 at various concentrations. Increasing the molar ratio of CPYPR-His6 to CPY resulted in an increase in the CPY refolding yield, indicating that the CPYPR-His6 plays a chaperone-like role in in vitro folding of CPY. The refolded CPY was purified to 92% purity by single-step p-amino-benzylsuccinic acid affinity chromatography. When refolding was carried out in the presence of 10 molar eq CPYPR-His6, the specific activity, N-(2-furanacryl-oyl)-L-phenylalanyl-L-phenylalanine hydrolysis activity per milligram of protein, of purified recombinant CPY was found to be about 63% of that of native S. cerevisiae CPY.-
dc.publisherElsevier-
dc.titleRefolding and Purification of Yeast Carboxypeptidase Y Expressed as Inclusion Bodies in Escherichia coli-
dc.title.alternativeRefolding and Purification of Yeast Carboxypeptidase Y Expressed as Inclusion Bodies in Escherichia coli-
dc.typeArticle-
dc.citation.titleProtein Expression and Purification-
dc.citation.number1-
dc.citation.endPage107-
dc.citation.startPage101-
dc.citation.volume22-
dc.contributor.affiliatedAuthorMoon Sun Hahm-
dc.contributor.affiliatedAuthorBong Hyun Chung-
dc.contributor.alternativeName함문선-
dc.contributor.alternativeName정봉현-
dc.identifier.bibliographicCitationProtein Expression and Purification, vol. 22, no. 1, pp. 101-107-
dc.identifier.doi10.1006/prep.2001.1418-
dc.description.journalClassY-
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