苏云金芽胞杆菌Sigma K在大肠杆菌中的表达与纯化

Expression and purification of Bacillus thuringiensis Sigma K in Escherichia coli

摘要:

【目的】在大肠杆菌中表达纯化苏云金芽胞杆菌HD73的转录调控因子Sigma K (σK)。【方法】PCR扩增出苏云金芽胞杆菌HD73中sigK基因的ORF (open reading frame)装载到带有His标签的表达载体pET21b上，转入到表达菌株BL21(DE3)中获得重组菌株BL21(pETsigK)，通过SDS-PAGE、镍柱亲和纯化、阴离子交换纯化和凝胶迁移实验(EMSA)等方法对Sigma K蛋白进行提取、纯化和生物活性分析。【结果】正确表达出大小约为27 kD的His-Sigma K蛋白，并获得了纯化的蛋白。EMSA结果表明纯化的His-Sigma K蛋白可以与受其控制的cry1Ac基因启动子结合。【结论】表达和纯化了His-Sigma K蛋白，His-Sigma K具有与受其控制的启动子结合的功能。

Abstract:

[Objective] To express and purify the regulator protein Sigma K (σK) from Bacillus thuringiensis HD73 in Escherichia coli. [Methods] The ORF (open reading frame) of the sigK gene was amplified by PCR from Bt strain HD73, and then cloned into the vector pET21b to generate pETsigK. The pETsigK was transformed into BL21(DE3). The SDS-PAGE, nickel column affinity purification, Q-Sepharose fast flow column purification and electrophoretic mobility shift assay (EMSA) experiments were carried out to analyze the purity and binding activity of His-Sigma K with cry1Ac gene controlled by Sigma K in vitro. [Results] The 27 kD His-Sigma K was expressed. EMSA results showed that the His-Sigma K could bind to the promoter of cry1Ac gene, which is controlled by Sigma K. [Conclusion] The His-Sigma K protein was successfully expressed and purified. The purified His-Sigma K could bind to Sigma K-controlled promoter.