细胞毒性T细胞抗原4胞外结构域的高效表达与纯化

目的:获得细胞毒性T淋巴细胞相关分子4胞外结构域(exCTLA4)在大肠杆菌中高效表达的条件,并纯化获得高纯度的重组蛋白.方法:构建原核表达质粒pET28a-exCTLA4并转入大肠杆菌Transetta(DE3)中,通过改变诱导物IPTG浓度、细菌培养温度和时间,获得了exCTLA4的最佳表达条件.收集最佳诱导条件下表达exCTLA4的菌体,将菌体超声破碎后,离心分离exCTLA4包涵体,用低浓度尿素(2mol·L~(-1))洗去大部分背景蛋白后,再用高浓度尿素(8mol·L~(-1))溶解包涵体,进一步用镍离子亲和层析方法得到高纯度exCTLA4.结果:构建了表达载体pET28a-exCTLA4,获得了exCTLA4在Transetta(DE3)中的最佳诱导条件,即在1mmol·L~(-1) IPTG、25℃下,诱导6h能得到最佳表达,但重组蛋白以包涵体形式存在.通过纯化exCTLA4包涵体及进一步的镍离子亲和层析获得了高纯度的目的蛋白,Western blot鉴定为CTLA4.结论:获得了exCTLA4重组蛋白在大肠杆菌中的最佳表达条件和纯化方法及步骤,为该蛋白的应用奠定了基础.

High Level Expression and Purification of Extracellular Domain of CTLA4

Objective:The work aims to obtain the conditions for efficient expression of extracellular domain of cytotoxic T-lymphocyte-associated protein 4(exCTLA4)in Escherichia coli and to acquire high purity recombinant protein.Methods:The sequence of exCTLA4 was constructed into prokaryotic expression vector of pET28 a,namely pET28a-exCTLA4,which was then transferred into Escherichia coli Transetta(DE3).The optimal expression conditions of exCTLA4 were obtained by changing the inducer concentration of IPTG,the temperature of bacterial growth and the time of recombinant protein induction.The bacteria were harvested under the best induction conditions.The inclusion body of exCTLA4 collected and purified from bacteria was dissolved in 8mol·L~(-1) urea,and then purified by nickel ion affinity chromatography.Results:The expression vector of pET28a-exCTLA4 was constructed and the optimal conditions for exCTLA4 expression in Transetta(DE3)were obtained,which was best expressed at 1mmol·L~(-1) IPTG at 25 ℃for 6h,but the recombinant protein was expressed in inclusion body forms.High purity protein was obtained by purification of exCTLA4 inclusion bodies and further by nickel ion affinity chromatography.The recombinant protein of exCTLA4 was identified by Western blot.Conclusion:The conditions of optimal expression for recombinant protein of exCTLA4 in Escherichia coli are obtained,and high purity recombinant protein is acquired by inclusion body purification and nickel ion affinity chromatography.