NADH-细胞色素b5还原酶突变型蛋白的结构与功能研究  

Study on structure and function of mutant type NADH-cytochrome b5 reductase

作　　者：郑德柱[1] 兰风华[1] 黄粱浒[1] 吴玉水[1] 谢飞[1] 程烽[1] 朱忠勇[1] 

机构地区：[1]南京军区福州总医院全军医学检验中心,福州350025

出　　处：《临床检验杂志》2007年第1期13-16,共4页Chinese Journal of Clinical Laboratory Science

摘　　要：目的探讨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。方法用基因重组技术将野生型和突变型(L72P)b5RcDNA克隆于pGEX-2T载体,在大肠杆茵BL21中诱导表达。Western印迹鉴定表达的蛋白为GST-b5R融合蛋白。应用谷胱甘肽-Sepharose4B亲和层析,还原型谷胱甘肽洗脱得到纯化的GST-b5R和GST-b5RL72P融合蛋白,比较GST-b5R和GST-b5RL72P酶活性。结果突变型酶活性低,为野生型的3%。结论L72P突变可引起蛋白质二级结构改变从而导致酶的活性下降。Objective To characterize the effect of L72P missense mutation on the enzymic function of NADH-cytochrome b5 reductase. Methods L72P mutant bSR cDNA was isolated from a patient with mutant type NADH-cytochrome b5 reductase by RT-PCR. The wild type and mutant bSR cDNAs were cloned into the expression vector pGEX-2T. The expressions of GST-fused wild type and L72P bSR mutant protein were analyzed by SDS-PAGE and Western blotting after inducing with IPTG for 4 hours. GST-fused wild type bSR and GST-fused L72P bSR were then purified with single-step affinity chromatography column of glutathione Sepharose 4B. The purity of both bSR proteins was analyzed by electrophoresis on PAGE. Results A single band with molecular mass of 58 kD on PAGE gel was obtained. By comparing with GST-fused b5 R and GST-fused b5 R L72P expressed in E. coli, the mutant enzyme activity was only 3% of the wild type enzyme. Conclusion The findings suggest that the L72P missense mutation may influence the secondary structure of NADH- cytochromc b5 reductase,which consequently results in decrease of enzymic activity.

关 键 词：NADH-细胞色素B5还原酶 原核表达 GST融合蛋白 亲和层析 

分 类 号：Q754[生物学—分子生物学]