The renaturation of procarboxypeptidase B by urea gradient gel filtration and some properties of recombinant carboxypeptidase B.
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.
Xiao-Yan
Zhang
Z
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Su-Xia
Li
L
Qin-Sheng
Yuan
Y
D000818:Animals; D043424:Carboxypeptidase B / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000737:chemistry; D002850:Chromatography, Gel; D004338:Drug Combinations; D015180:Electrophoresis, Gel, Two-Dimensional; D004795:Enzyme Stability; D007201:Indicator Dilution Techniques; D019216:Metals, Heavy / Q000737:chemistry / Q000494:pharmacology; D009821:Oils; D010636:Phenols; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020673:Protein Renaturation / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis; D013696:Temperature; D014508:Urea / Q000494:pharmacology*
D002413:Cations, Divalent; D004338:Drug Combinations; D019216:Metals, Heavy; D009821:Oils; C027222:P & S Liquid; D010636:Phenols; D011498:Protein Precursors; D011994:Recombinant Proteins; D014508:Urea; D043424:Carboxypeptidase B