Gibson Assembly系统构建双标签原核类泛素蛋白表达载体

Constructing Prokaryotic ubiquitin protein expression vector with dual-tag by Gibson Assembly system

目的 体外构建表达原核类泛素蛋白 （prokaryoticubiquitin-likeprotein,Pup）的载体是研究Pup化靶蛋白的重要基础,为了提高蛋白纯化的效能和特异性需要构建带组氨酸 （his6）和链霉素 （strep）标签的Pup蛋白表达载体,为后续研究Pup-蛋白酶体在分枝杆菌中的应激调控奠定基础。方法 设计合成his6-strep片段,扩增富集his6-strep片段,从结核分枝杆菌 H37Rv基因组扩增Pup片段;应用GibsonAssembly系统采用同源片段重组方法将his6-strep片段和Pup片段依次连接到载体上,构建带双标签的原核表达载体 pET28a-his6-strep-Pup和大肠肝菌-结核杆菌穿梭质粒pMV261-his6-strep-Pup表达载体,经酶切和聚合酶链反应验证,分别导入大肠杆菌 （E.coli）和耻垢分枝杆菌 （M.sm）中,鉴定和测序获得正确单克隆菌株。培养富集菌株,超声破碎离心收集上清和沉淀,进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳 （SDS-PAGE）电泳,用免疫印迹法 （Westernblot）验证标签蛋白在E.coli和M.sm中的表达。结果 经聚合酶链反应鉴定及测序,证实成功构建了双标签Pup表达载体并导入目的菌株,诱导后蛋白表达,Westernblot结果表明标签蛋白在E.coli和M.sm中均可得到正确表达,在M.sm中可发现多种靶蛋白被Pup化结合。结论 快速高效地构建了表达his6-strep-Pup蛋白的双标签重组载体,可以应用于后续Pup的表达纯化及Pup化蛋白的功能分析,为进一步开展对Pup-蛋白酶体在分枝杆菌应激调控中的功能研究打下了良好的基础。

Objective Constructing Pup （prokaryotic ubiquitin-like protein） expression vector is key point for study pupylation in vitro. In order to improve effective and specificity of protein purification, we need to construct Pup expression with his6 and strep tag, which will establish basis for further study on regulation of Pup-proteasome system under stress in mycobacteria. Methods Designing and synthesizing his6-strep fragments, amplifying and enriching them. The Pup fragment was amplified from Mycobacterium tuberculosis H37Rv genome. His6-strep and Pup genes were inserted into pET28a and shuttle plasmid pMV261 based on homologous recombination by Gibson Assembly system, constructing dual-tags vector, named as pET28a-his6-strep-Pup and pMV261-his6-strep-Pup. The right vector checked by polymerase chain reaction （PCR） and sequencing was transformed into E. coil and M. sin. These strains containing right vector were cultured and enriched, separately collecting pellet and supernatant after lyse by ultra-sonic. After sodium dodecyl sulfate polyacrylamide gelelectrophoresis （ SDS-PAGE ）electrophoresis, the expression o{ dual-tags were checked by western blot. Results The vectors expressing dual-tags Pup were constructed successfully after clarified by PCR and sequencing analysis. The outcome of Western blot indicated positive expression of dual tags protein separately in E. coli and M. sm and many Pupylation protein were found in M. sm. Conclusions The dual-tag vectors expressing his6 and strep are constructed rapidly and efficiently. These vectors can be used to purifying Pupylation protein and study their functions, which will establish the good base for the further investigating regulation of Pup- proteasome system under the stress in mycobacteria.