NADH-细胞色素b5还原酶在大肠杆菌中的表达及其产物的纯化

Expression and Purification of Wild and Mutant Type NADH-ytochrome b5 Reductase in E.coli

为了探讨 NADH-细胞色素 b5还原酶基因突变引起遗传性高铁血红蛋白血症的分子病理机制 ,研究突变型 ( b5R)蛋白结构和功能的关系 ,用基因重组技术将野生型和突变型 ( C2 0 3Y) b5Rc DNA克隆于 p GEX- 2 T载体 ,在大肠杆菌 BL2 1中诱导表达 .Western印迹鉴定所表达的蛋白为GST- b5R融合蛋白 .应用谷胱甘肽 - Sepharose 4B亲和层析 ,还原型谷胱甘肽洗脱得到纯化的GST- b5R和 GST- b5RC2 0 3Y融合蛋白 .比较 GST- b5R和 GST- b5RC2 0 3Y酶活性及稳定性 ,发现野生型和突变型的酶活性基本相同 .但与野生型酶相比 ,突变型酶对热的稳定性较差 ,对胰蛋白酶更加敏感 .结果提示 ,C2 0 3Y突变可引起蛋白质二级结构改变而导致酶的稳定性下降 .

To characterize the effect of C203Y missense mutation on the enzyme function of NADH cytochrome b5 reductase,C203Y mutant b5R cDNA was isolated from plasmid pWR 450 b5RC203Y by RCR.The wild type and mutant b5R cDNAs were cloned into the expression vector pGEX 2T.The expressions of GST fused wild type and C203Y b5R mutant protein were analyzed by SDS PAGE and Western blotting after induced by IPTG for 4 hours.Then GST fused wild type b5R and GST fused C203Y b5R were purified with single step affinity chromatographic column of Glutathione Sepharose 4B.The purity of both b5R proteins was analyzed by electrophoresis on a PAGE and a single band with a molecular mass of 58 kD was obtained.Compared GST fused b5R with GST fused b5RC203Y expressing in E.coli ,it was found that mutant enzyme activity was the same as wild enzyme activity,but less thermostability and a greater susceptibility to trypsin than the normal counterpart.The results suggest that this amino acid substitution influences secondary structure of the enzyme,resulting in enzyme instability.