复苏促进因子Rpf结构域突变体蛋白的原核表达和纯化

Expression in E. coli of Micrococcus luteus Rpf domain mutants and its purification

【目的】构建藤黄微球菌复苏促进因子Rpf结构域突变体基因E54K、E54A的原核表达质粒,在大肠杆菌中表达和纯化。【方法】采用PCR方法从藤黄微球菌基因组中扩增出Rpf结构域E54A、E54K突变体基因,连接进入pMD-18T载体中,测序正确的基因插入到pGEX-4T-1表达载体中,转化大肠杆菌DH5α,经IPTG诱导,SDS-PAGE鉴定表达蛋白,利用Rpf结构域单克隆抗体进行Western-blotting分析。用GS-4B亲和层析柱纯化目的蛋白。【结果】获得藤黄微球菌Rpf结构域E54K和E54A突变体基因,测序结果与GenBank公布的序列相同,突变体位点与设定一致;表达蛋白相对分子质量与文献报道一致;Western-blotting结果显示,在相对分子量约32 KD处有与Rpf结构域单克隆抗体特异性结合带。通过GS-4B系统,得到纯化的GST融合蛋白。【结论】成功表达、纯化了Rpf结构域突变体E54K、E54A融合蛋白,为上述蛋白在结核病中的应用奠定基础。

[Objective] To express Micrococcus luteus resuscitation-promoting factor （Rpf） domain mutants in prokaryotic cells. [Methods] The gene of Micrococcus luteus Rpf domain mutants （E54K, E54A） were amplified by polymerase chain reaction （PCR） from genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain mutant gene were subcloned into expression vector PGEX-4T-1 and transfected into E. coli DH5a. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE and analyszed by Western-blotting with monoclonal antibodies against Rpf domain. [Resuits] It was showed that the sequences of PCR products were identical with those in GenBank. Relative molecular mass identified by SDS-PAGE was consistent with what had been reported, which was also confirmed by Western-blotting analysis that there were specific bindings at 32 KD with Rpf domain mAb. [Conclusions] Two kinds of Rpf domain mutant protein have obtained.