克隆、表达及纯化核糖体蛋白S6用于体外激酶活性检测

目的:构建40S核糖体蛋白S6的原核表达载体,表达并纯化S6蛋白,将其作为底物用于S6激酶(S6K)的体外活性测定。方法:采用RT-PCR方法从人胚肾细胞HEK293中获取S6 cDNA,将扩增产物克隆至大肠杆菌表达载体中,进行酶切及测序鉴定;IPTG诱导GST-S6融合蛋白在大肠杆菌中表达,用谷胱甘肽亲和层析纯化GST-S6,免疫沉淀法检测该蛋白是否可作为底物用于S6K的体外激酶活性测定。结果:酶切及测序鉴定表明构建了S6原核表达载体,并表达及纯化出GST-S6融合蛋白,相对分子质量为55×103。该蛋白可用于S6K的体外激酶活性测定,特异性强。结论:S6蛋白的克隆、表达与纯化成功,可用于S6K的体外激酶活性测定,为研究S6K的功能奠定了基础。

Cloning, Expression, and Purification of Ribosomal Protein S6 for In Vitro Kinase Assay

Objective：To obtain purified recombinant 40S ribosomal protein S6 for p70 S6 kinase（S6K） in vitro kinase assay.Methods：The ribosomal protein S6 cDNA was cloned from human embryonic kidney 293（HEK293） cells by RT-PCR and ligated to prokaryotic expression plasmid pGEX-6P-1 for construction of the recombinant vector pGEX-6P-1-S6.After sequencing,the recombinant vector was transformed into E.coli BL21 and GST-tagged S6 was induced by IPTG.The S6 was purified by glutathione affinity chromatography column and characterized by SDS-PAGE.Overexpressed S6K was immunoprecipitated from HEK293 cells and the purified S6 was used as sub-strate for S6K in vitro kinase assay.Results：The S6 gene was successfully cloned from HEK293 cells,which was identical to that reported in GenBank.After IPTG induction,the E.coli transformed with pGEX6P-1-S6 plasmid expressed a about 55 kD GST-tagged protein,which was successfully purified and identified.S6K in vitro kinase assay demonstrated that the purified S6 protein could be used as specific substrate for S6K in vitro kinase assay.Conclusion：We successfully cloned the S6 gene,expressed and purified S6 protein which is a suitable substrate for S6K in vitro kinase assay.It will be used in further investigation of functions and of S6K and S6 signaling and its regulatory mechanisms