文献类型：期刊文献

英文题名：The renaturation of procarboxypeptidase B by urea gradient gel filtration and some properties of recombinant carboxypeptidase B

作者：Zhang, XY; Li, SX; Yuan, QS

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2005

卷号：12

期号：7

起止页码：671

外文期刊名：PROTEIN AND PEPTIDE LETTERS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000231958900012)】；

语种：英文

外文关键词：procarboxypeptidase B; recombinant carboxypeptidase B; urea gradient gel filtration renaturation; dilution refolding; properties

摘要：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.