重组羧肽酶G2的原核表达、纯化及活性分析

摘要

在原核表达系统中实现羧肽酶G2（Carboxypeptidase G2，CPG2）的表达，并对CPG2进行了纯化和活性测定。通过PCR扩增得到编码CPG2的基因片段，利用基因重组技术构建原核表达质粒pET-30a-CPG2，在Escherichia coli中进行诱导表达，菌体经超声破碎后利用金属螯合亲和柱和阴离子交换层析柱对目标蛋白进行纯化。目的蛋白能够以可溶形式表达，SDS-PAGE和Western blotting检测有特异的蛋白表达条带。纯化的CPG2有降解氨甲喋吟（MTX）活性，酶活力达到400 U/mg。%This work is to express carboxypeptidase G2(CPG2)in prokaryotic expression system,purify it and detect its activityin vitro. The gene fragment encoding CPG2 was amplified by PCR,and the prokaryotic expressed plasmid pET-30a-CPG2 was constructed by gene recombinant technology,then it was transformed intoEscherichia coli BL21(DE3)for the expression with induction. The target protein from grounded cells was purified by metals chelating affinity chromatogram and anion-exchange chromatography. Majority of the fusion protein was expressed in soluble form,and its specificity was confirmed via SDS-PAGE and Western blotting. The preliminary experimental result showed that the recombinant protein degraded MTX,and the specific activity reached 400 U/mg.