Prokaryotic expression and biochemical characteristics of glutamyl endopeptidase

Objective To express the glutamyl endopeptidase in prokaryocytes and evaluate its biochemical characteristics.Methods The plasmid PQE60Glu△Cmut5 was transferred into E.coli M15,and was induced to express the recombinant glutamyl endopeptidase with IPTG.The recombinant protein was purified by Ni-NTA metal affinity resins,and its activity was determined with azocasein as substrate.The effects of concentrations of substrate,different temperature,pH,metal ions and EDTA on the recombinant glutamyl endopeptidase were investigated.The specific enzymolysis of the proteinase to hemoglobin was also analyzed with SDS-PAGE and mass spectrometry.Results The recombinant glutamyl endopeptidase was highly expressed in E.coli M15 and showed high enzymatic activity.Its relative molecular mass was about 33 000 as shown on SDS-PAGE.Analysis of the glutamyl endopeptidase dynamics revealed that the Km was 0.26 mmol/L,the optimal reaction temperature was 55 ℃ and the optimal pH was 8.0.The activity of the recombinant proteinase was enhanced by Ca2+and Mg2+,but inhibited by Fe2+,Zn2+,Mn2+and EDTA.Hemoglobin could be specifically hydrolyzed by the recombinant proteinase.Conclusion Glutamyl endopeptidase could be highly expressed in E.coli M15,and the recombinant proteinase showed some specific biochemical characteristics and strict specificity of hydrolyzing substrate.