耐热、高产重组人Cu,Zn-SOD改构体的构建及纯化方法的研究

目的通过对天然人Cu,Zn-SOD基因的改造,得到热稳定性、活性产量提高的重组人Cu,Zn-SOD(rhCu,Zn-SOD)改构体,并进行蛋白纯化方法的研究。方法重叠PCR技术将天然hCu,Zn-SOD的Cys6突变为A la6,Cys111突变为Ser111,构建重组质粒pET22b-rmhCu,Zn-SOD6A la,111Ser,转化大肠杆菌BL21(DE3),IPTG诱导后SDS-PAGE分析改构体蛋白的表达,并进行W estern blot鉴定;研究改构体蛋白的纯化方法,HPLC检测蛋白纯度,比较rhCu,Zn-SOD及其改构体的活性和热稳定性。结果成功构建了rmhCu,Zn-SOD6A la,111Ser表达载体;SDS-PAGE显示rmhCu,Zn-SOD6A la,111Ser蛋白在BL21(DE3)宿主菌中得到了有效表达,表达量占菌体蛋白总量的64.6%;Western blot结果显示表达产物可以特异性地与小鼠抗人SOD多克隆抗体结合;纯化得到纯度大于95%的rmhCu,Zn-SOD6A la,111Se改构体蛋白,从1g湿菌体中获得的活性蛋白总量高于未改构体的2倍;改构体的热稳定性较未改构体获得大幅提高。结论构建的rmhCu,Zn-SOD6A la,111Ser改构体具有较好的热稳定性和较高的活性产量,摸索出一种比较简易的蛋白纯化工艺,为Cu,Zn-SOD的广泛应用奠定基础。

Construction,expression and purification of a thermostable,high yield recombined hCu,Zn-SOD mutant

Aim To obtain the rhCu,Zn-SOD mutant with high thermostability and yield,rhCu,Zn-SOD cDNA was modified by overlap PCR;and to establish the purification method of the rhCu,Zn-SOD mutant protein.Methods The Cys6 and Cys111 of hCu,Zn-SOD gene were mutated into Ala6 and Ser111 by overlap PCR.Then,the gene was cloned into vector pET22b(+),and transfered to BL21(DE3).After induced with IPTG,the expression of the mutant protein was detected by SDS-PAGE and western blot.The purification method of the mutant protein...