Refolding and purification of yeast carboxypeptidase Y expressed as inclusion bodies in Escherichia coli.
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-His(6) was achieved but most of the expressed proteins were present in the form of inclusion bodies in the bacterial cytoplasm. The CPY and CPYPR-His(6) produced as inclusion bodies were separated from the cells and solubilized in 6 and 3 M guanidinium chloride, respectively. The denatured CPYPR-His(6) 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-His(6) 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-His(6) at various concentrations. Increasing the molar ratio of CPYPR-His(6) to CPY resulted in an increase in the CPY refolding yield, indicating that the CPYPR-His(6) plays a chaperone-like role in in vitro folding of CPY. The refolded CPY was purified to 92% purity by single-step p-aminobenzylsuccinic acid affinity chromatography. When refolding was carried out in the presence of 10 molar eq CPYPR-His(6), the specific activity, N-(2-furanacryloyl)-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.
Hahm
M S
MS
Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea.
Chung
B H
BH
D002268:Carboxypeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D043402:Cathepsin A; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013386:Succinates / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D010455:Peptides; D011993:Recombinant Fusion Proteins; D013386:Succinates; C033223:polyhistidine; D006639:Histidine; C027639:4-aminobenzylsuccinic acid; D002268:Carboxypeptidases; D043402:Cathepsin A