重组人MBD4蛋白在大肠杆菌中的表达、纯化及活性分析

Purification and Characterization of Recombinant Human MBD4 Protein Expressed in E.coli

为获得重组人MBD4蛋白,将编码MBD4的开放式阅读框(ORF)插入原核表达载体pGEX-6P1GST基因下游的多克隆位点(MCS).将获得的表达质粒转化入大肠杆菌BL21(DE3)菌株扩大培养并用IPTG诱导融合蛋白的表达.用谷胱甘肽琼脂糖凝胶4B亲和介质从菌体裂解液中纯化了GST-MBD4融合蛋白.经过Prescisionprotease专一性裂解成功去除了融合蛋白上的GST标签.通过MonoQ阴离子交换层析获得了纯度达94%以上的MBD4蛋白,该蛋白具有甲基化DNA结合和糖苷酶生物活性.

To produce recombinant human MBD4 protein, the ORF of MBD4 cDNA was inserted into pGEX- 6P1 expression vector fused in frame with an upstream GST gene. The expression plasmid was transformed into E. coli BL21（DE3） strain and protein expression was induced by IPTG. The GST-MBD4 fusion protein was purified from cell lysates using glutathione Sepharose 4B affinity chromatography. The GST tag was removed from fusion protein by site-specific Prescision protease cleavage. MBD4 protein was purified through Mono Q anion exchange to more than 94 % homogeneity. The purified protein has both methylated DNA binding and DNA glycosylase activity.