The renaturation of procarboxypeptidase B by urea gradient gel filtration and some properties of recombinant carboxypeptidase B

• Published in: Protein and peptide letters Vol. 12; no. 7; p. 671
• Main Authors: Xiao-Yan, Zhang, Su-Xia, Li, Qin-Sheng, Yuan
• Format: Journal Article
• Language: English
• Published: Netherlands 01.10.2005
• Subjects: Animals; Carboxypeptidase B - chemistry; Carboxypeptidase B - genetics; Carboxypeptidase B - isolation & purification; Carboxypeptidase B - metabolism; Cations, Divalent - chemistry; Chromatography, Gel; Drug Combinations; Electrophoresis, Gel, Two-Dimensional; Enzyme Stability; Indicator Dilution Techniques; Metals, Heavy - chemistry; Metals, Heavy - pharmacology; Oils; Phenols; Protein Folding; Protein Precursors - chemistry; Protein Precursors - genetics; Protein Precursors - isolation & purification; Protein Precursors - metabolism; Protein Renaturation - drug effects; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Spectrum Analysis; Temperature; Urea - pharmacology
• ISSN: 0929-8665
• DOI: 10.2174/0929866054696145

A new pro-carboxypeptidase (pCPB) gene was cloned by RT-PCR from SD rat pancreas and its overexpression in Escherichia coli resulted in the formation of inclusion bodies (IBs). The IBs of pCPB were solubilized in 8 M urea and successively refolded by urea gradient gel filtration. Subsequently, the renatured pCPB was digested by trypsin. Recombinant active CPB was obtained by passing through DEAE-FF ion exchange and Sephadex-G100 chromatographic column. Capillary electrophoresis assay showed that the purity of the recombinant CPB (rCPB) exceeded 90%. Further, some properties of rCPB were characterized. The optimum of activity was achieved at pH 7-9. The activity of rCPB was inhibited by typical metal chelating agents (EDTA) and Hg2+, and was activated by Co2+ and heat treatment at 40 degrees C. The two-dimension electrophoresis map of rCPB showed that the pI value of rCPB was 5.35. UV absorbance spectrum of the enzyme showed that an absorbance maximum was at 277 nm.