谷氨酰内切酶的原核表达及生物化学特性分析

Prokaryotic expression and biochemical characteristics of glutamyl endopeptidase

目的原核表达谷氨酰内切酶,评价其生物化学特性。方法 PQE60Glu△Cmut5质粒转化大肠埃希菌M15,经异丙基β-D-硫代半乳糖(IPTG)诱导、Ni-NTA亲和层析等步骤获得重组谷氨酰内切酶。以偶氮酪蛋白为底物测定其酶活性,分析底物浓度、温度、pH、金属离子和EDTA对酶活性的影响。采用SDS-PAGE电泳和质谱研究谷氨酰内切酶对Hb的酶解作用。结果重组谷氨酰内切酶可以在M15菌中高效表达,并具有较高的酶活性。SDS-PAGE分析显示其相对分子质量约33 000。谷氨酰内切酶的Km值为0.26 mmol/L,最适反应温度为55℃,最适pH为8.0,Ca2+和Mg2+能激活酶活性,Fe2+、Zn2+、Mn2+和EDTA则对酶活性有抑制作用。SDS-PAGE和质谱分析显示重组谷氨酰内切酶对Hb有特异性水解作用。结论谷氨酰内切酶在大肠埃希菌M15中能获得高效表达,具有特有的生物化学特性和严格的底物水解特异性。

Objective To express the glutamyl endopeptidase in prokaryocytes and evaluate its biochemical characteristics. Methods The plasmid PQE60GluACmut5 was transferred into E. coli M15, and was induced to express the recombinant glutamyl endopepti- dase 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 activ- ity. 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 ~C 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 hydro- lyzed by the recombinant proteinase. Conclusion Glutamyl endopeptidase could be highly expressed in E. coli M15, and the recombi- nant proteinase showed some specific biochemical characteristics and strict specificity of hydrolyzing substrate.