人重组核凋亡诱导因子1截短体在大肠杆菌中的表达及纯化

Expression and purification of truncated human recombinant nuclear apoptosis-inducing factor 1 in E.coli

目的核凋亡诱导因子1(Nuclearapoptosis-inducingfactor1,NAIF1)是本实验室首次克隆和鉴定的新凋亡基因,为了通过Polldown实验研究其结合蛋白,在大肠杆菌中表达和纯化人重组NAIF1(73-327)的截短体。方法通过PCR方法扩增出NAIF1(73-327)cDNA,并插入pGEX-KG载体,实现插入基因的融合表达,对表达产物进行SDS-PAGE、Westernblot和电喷雾电离-四极杆-飞行时间质谱(ESI-Q-TOF-MS/MS)检测分析。结果DNA测序结果证实成功构建了重组融合表达质粒pGEX-KG-NAIF1(73-327),并在大肠杆菌中稳定表达,表达产物分子量约为53kD,与预期一致,表达量约占菌体总蛋白的22%,纯化蛋白经Westernblot和二级质谱(MS/MS)分析证明表达蛋白为GST-NAIF1(73-327)融合蛋白。结论获得了重组GST-NAIF1(73-327)融合蛋白的高效表达,为下阶段NAIF1的结构与功能研究打下了基础。

Objective ： Nuclear apoptosis-inducing factor 1 （ NAIF1 ） is a novel apoptosis gene cloned in laboratory. To analyze the binding proteins of NAIF1 by pulldown method, the fusion expression vector of truncated human nuclear apoptosis-inducing factor 1 [ NAIF1 （73-327）] was constructed, were expressed and purified the recombinant GST-NAIF1 （73-327） fusion protein in E. coll. Methods ：The cDNA encoding human NAIF1 （73-327） was amplified by PCR and cloned into pGEX-KG vector. The GST-NA1F1 （73- 327 ） fusion protein was expressed in E. coli and purified by affinity chromatography. The purified protein was detected by SDS-PAGE, Western blot and ESI-Q-TOF-MS/MS. Results： A prokaryotic expression vector of GST-NAIF1 （73-327） was constructed and the GST- NAIF1 （73-327） fusion protein was expressed in E. coli at high level. SDS-PAGE analyses indicated that the purified protein was about 53 kD. Westem blot and MS/MS analyses verified the recombinant fusion protein. Conclusion：An eigcient method for obtaining recombinant GST-NAIF1 （73-327） fusion protein had been established and it could be used for further studies on the structure and function of NAIF1.