SUMO基因的内在原核启动子活性及其在大肠杆菌蛋白表达系统中的应用

Intrinsic prokaryotic promoter activity of SUMO gene and its applications in the protein expression system of Escherichia coli

SUMO融合系统已成为目前大肠杆菌重组蛋白生产的重要手段,但在载体构建效率和蛋白可溶性等方面仍有待改进。本研究在PCR克隆酿酒酵母SUMO基因Smt3(Sm)时意外发现Sm具有组成型原核启动子活性;而且经软莓BPROM程序预测发现大多数物种SUMO基因编码区都具有依赖σ70的原核启动子。进一步通过整合Sm启动子和Sm 3′末端StuⅠ位点特性以及引入His标签和超酸增溶标签,构建了基于Sm’-LacZα融合基因的一系列通用克隆表达载体,并通过蓝白斑筛选和SDS-PAGE分析进行了多个靶蛋白基因的克隆和表达,结果表明实现了基因表达载体的快速构建、蛋白高可溶性表达和关联蛋白的共表达等目标。因此,集成诸多特性而改良的SUMO融合技术可以成为大肠杆菌蛋白表达系统的有力工具,建立的共表达载体系统还可以用作研究细胞内蛋白间的相互作用。

Nowadays,SUMO fusion system is important for recombinant protein production in Escherichia coli,yet a few aspects remain to be improved,including the efficacy for vector construction and protein solubility.In this study,we found the SUMO gene Smt3（Sm） of Saccharomyces cerevisiae conferred an unexpected activity of constitutive prokaryotic promoter during its PCR cloning,and the gene coding regions of SUMOs in most species had a ？70-dependent prokaryotic promoter embedded,through the prediction via the BPROM program developed by Softberry.By combining the characters of Sm promoter activity and the Stu I site（added at the 3＇-terminal of Sm）,and introducing a His-tag and a hyper-acidic solubility-enhancing tag,we further constructed a set of versatile vectors for gene cloning and expression on the basis of Sm＇-LacZα？fusion gene.Experimentally started from these vectors,several target genes were subcloned and expressed through blue-white screening and SDS-PAGE analysis.The results manifest a few of expectable advantages such as rapid vector construction,highly soluble protein expression and feasible co-expression of correlated proteins.Conclusively,our optimized SUMO fusion technology herein could confer a large potential in E.coli protein expression system,and the simultaneously established co-expression vector systems could also be very useful in studying the protein-protein interactions in vivo.