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

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Title
Refolding and Purification of Yeast Carboxypeptidase Y Expressed as Inclusion Bodies in Escherichia coli
Author(s)
Moon Sun Hahm; Bong Hyun Chung
Bibliographic Citation
Protein Expression and Purification, vol. 22, no. 1, pp. 101-107
Publication Year
2001
Abstract
The 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.
ISSN
1046-5928
Publisher
Elsevier
DOI
http://dx.doi.org/10.1006/prep.2001.1418
Type
Article
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1. Journal Articles > Journal Articles
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