重组人谷氧还蛋白1在大肠杆菌中表达条件的优化、纯化及活性检测

The Optimization of Expression Condition,Purification and Activity Analysis of Recombination Human Glutaredoxin 1 in E.coli

为进一步研究谷氧还蛋白1(Grx1)的生物学功能及作用机制,将已构建的pRSETA-Grx1融合蛋白表达载体在大肠杆菌BL21(DE3)菌株中表达,并优化表达条件,用SDS-PAGE和凝胶影像分析.结果表明,当菌体密度OD600为1.0时开始诱导,加入终浓度0.5mmol/LIPTG,37℃,110±5r/min振荡培养5h,重组蛋白为可溶性,表达量达30%以上.用Ni-NTA树脂亲和层析纯化重组蛋白,获得蛋白质纯度达90%以上;W estern印迹结果表明,该蛋白具有免疫活性;MTT结果显示,终浓度为10mg/LGrx1重组蛋白具有保护细胞抵御500μmol/LH2O2作用(P<0.01).

In order to study the biological function and mechanism of human Grxl further, the recombinant plasmid pRSETA-Grxl（rhGrx1） constructed by our group previously were transformed into the host E. coli BL21 （DE3）, and the expression condition of rhGrxl were optimized in this test. When rhGrxl expression was induced at OD600 of 1.0 with 0.5 mmol/L IPTG for 5 h at 37 ℃ with the shaking speed of 110 ± 5 r/min, the maximum amounts of soluble proteins were obtained and the yield was more than 30% of the total mass of bacterial proteins. Then, the recombinant proteins were purified by Ni-NTA resin and protein amounts were more than 90%. The western blotting indicated that the rhGrxl protein expressed the immunological activity. In addition, MTT assay showed that rhGrx1 protein had antioxidant function.