GST-SRY融合蛋白原核表达和纯化

Prokaryotic Expression and Purification of GST-SRY Fusion Protein

目的构建GST-SRY融合蛋白原核表达载体,在E.coli中诱导其表达,并进行纯化.方法 PCR扩增SRY基因,将SRY基因插入原核表达载体p GEX-6P-1,经酶切和测序验证后,p GEX-6p-1-SRY转化到原核表达菌株E.coli Rosetta DE3,IPTG诱导表达GST-SRY融合蛋白,用GST标签纯化树脂对其进行纯化.结果经双酶切和测序证实p GEX-6P-1-SRY构建正确.重组质粒转化到Rosetta DE3经IPTG诱导后表达了分子质量单位约为49 KD的重组蛋白.结论 p GEX-6P-1-SRY原核表达载体构建成功,SRY蛋白成功诱导表达并纯化,为今后深入研究SRY蛋白的功能奠定了实验基础.

Objective To construct GST-SRY fusion protein expression vector and induce its expression in E.coli, then purificate so as to obtain GST-SRY fusion protein. Me thods SRY gene was amplified by PCR and cloned into prokaryotic expression vector p GEX-6 P-1. p GEX-6 P-1-SRY plasmid was verified by restriction enzyme digestion and DNA sequencing. After that, the recombinant plasmid was transformed into E.coli Rosetta DE3.Expression of GST-SRY fusion protein was induced by IPTG,which was purified by Glutathione Sepharose later on.Re s ults Restriction enzyme digestion and sequencing demonstrated that p GEX-6 P-1-SRY was successfully obtained. The result of SDS-PAGE assay indicated that SRY was successfully expressed in E.coli Rosetta DE3 and expressed a 49 KD SRY protein.Conclus ion The successful construction of SRY gene recombinant prokaryotic expression vector and expression of SRY protein will be helpful for further studies of SRY gene function.