鼠伤寒沙门菌AvrA蛋白的原核表达及纯化

Expression and purification of Salmonella typhimurium AvrA protein

目的克隆,表达和纯化沙门菌AvrA蛋白,为AvrA蛋白的结构和功能研究奠定基础。方法采用生物信息学方法对AvrA蛋白的性质进行预测。使用PCR的方法从沙门菌基因组上获得AvrA36-277基因,并克隆到原核表达载体pGl01上;使用大肠埃希菌BL21(DE3)菌株进行目的蛋白的表达,表达产物分别经镍离子亲和柱、阴离子交换柱以及凝胶层析柱层析纯化。结果成功获得重组质粒AvrA36-277/pGl01,转化BL21后经IPTG诱导获得分子质量单位约为27ku的重组AvrA36-277蛋白。该蛋白以可溶形式表达,层析纯化后蛋白浓度达5mg/ml,蛋白纯度95%。结论使用大肠原核表达系统可稳定表达沙门菌的AvrA36-277蛋白,该蛋白可溶性良好,获得蛋白纯度较高,可用于蛋白底物的筛选、晶体生长及三维结构解析。

Objective To clone,express,and purify the AvrA protein in order to lay the foundation for study of the structure and function of that protein.Methods Characteristics of the AvrA protein were predicted hioinformatically. The AvrA36-277 gene was obtained from Salmonella typhimurium via amplification with PCR,it was inserted into the prokaryotic expression vector pG101,and it was then transformed into BL21(DE3).The AvrA protein was purified using an Ni-NTA column,anion exchange,and gel filtration chromatography.Results The recombinant plasmid AvrA36-277/ pG101was successfully constructed and transformed into BL21(DE3).The AvrA protein was expressed in a soluble state.The concentration of the purified AvrA protein reached 5mg/ml with 95%purity.Conclusion The AvrA36-z77 protein of S.typhimurium was stably expressed in an E.coli prokaryotic expression system .The protein is soluble, highly pure,and is suitable for use in substrate screening and X-ray diffraction experiments.