His-AMPKα1^312截短缺失融合蛋白原核表达载体的构建与纯化

Prokaryotic expression and purification of rat c-terminal truncation mutant of the AMPK fusion protein

目的构建His-AMPKα1312截短缺失融合蛋白原核表达载体,表达重组AMPKα1312为深入研究各种病理生理条件对AMPK活化的影响奠定基础。方法经RT-PCR获得AMPKα1截短基因片段,定向插入原核表达载体PET28a(+),构建重组表达质粒YH2/PET28a(+),转化DH5αE.coli,经IPTG诱导表达及镍琼脂糖凝胶亲和层析纯化后SDS-PAGE分析及Western blot检测。结果经测序和PCR证实重组表达质粒YH2/PET28a(+)构建正确。表达的截短缺失蛋白相对分子量约为38 kD,Westernblot分析表明,表达蛋白具有良好的抗原性。结论已成功构建并纯化了具有生物学活性的AMPKα1312截短缺失蛋白,为深入研究AMPK奠定了基础。

Objective To express rattus His-AMPKal^312 truncated fusion protein in E. coli, the expressed product for its bioactivity were purified and analyzed. Methods The truncated AMPKoL1 gene was amplified by RT-PCR and inserted into prokaryotic expression vector PET28a（ ＋ ）. The constructed recombinant plasmid YH2/PET28a（ ＋ ） was transformed to DH5a E. coli for expression under induction of IPTG. The expressed His-AMPKcd 312 fusion protein was purified by Ni2＋ -NTA Agarose affinity chromatography and identified for reactogenieity. Results The DNA sequence of amplified truncated AMPKcd gene was consistent with that reported in GenBank. Recombinant plasmid YH2/PET28a（ ＋ ） was constructed correctly. The expressed recombinant protein ,with a relative molecular mass of about 38kd, showed good reactogenicity. Conclusion The His-AMPKal^312truncated fusion protein was successfully constructed and purified with bioactivity, which laid a foundation of further study on the function of AMPK.