单核细胞增生李斯特菌ladR基因在两种原核表达载体中的克隆表达及纯化分析

Expression and Purification of ladR Gene of Listeria monocytogenes in Two Prokaryotic Vectors

摘要:

利用特异性引物扩增ladR基因序列，分别构建单核细胞增生李斯特菌（Listeria monocytogenes）ladR基因的两种原核表达载体，即含有6个His标签的pET-28a-ladR表达载体和含有GST标签的pGEX-4T-ladR表达载体。通过诱导时间、温度和浓度优化两种表达载体的蛋白表达条件，比较分析两种表达载体的LadR蛋白可溶性表达水平，利用Western-Blot进行LadR蛋白表达验证。本研究成功构建了pET-28a-ladR和pGEX-4T-ladR表达载体，在大肠杆菌BL21中分别表达出His-LadR和GST-LadR融合蛋白。通过凝血酶切除GST标签，纯化后得到LadR蛋白。在最佳表达条件IPTG为0.5 mmol/L，22 ℃诱导过夜下，pGEX-4T-ladR表达载体中LadR的表达量比pET-28a-ladR（IPTG为1.0 mmol/L，22 ℃诱导过夜）中His-LadR蛋白的表达量高4.48倍。本研究结果表明，GST标签有利于ladR基因的可溶性表达，为后续的LadR蛋白的功能分析和转录调控机理研究奠定了良好基础。

Abstract:

The ladR genes was amplified using the specific primers, and then two prokaryotic expression vectors pET-28a-ladR (carried six His-tag) and pGEX-4T-ladR (carried a GST-tag) were constructed. The protein expression conditions of the two expression vectors were optimized, including induction time, temperature and concentration. The soluble expression levels of LadR protein in these two expression vectors were compared. The pET-28a-ladR and pGEX-4T-ladR expression vectors were successfully constructed and expressed the His-LadR and GST-LadR fusion proteins in E. coli BL21. The GST-LadR fusion protein was excised by thrombin and purified to obtain a LadR protein. Under the optimal expression conditions of 0.5 mmol/L isopropyl-β-D-thiogalactoside (IPTG) and induction at 22 ℃ for 12 h, the expression level of LadR protein in pGEX-4T-ladR expression vector was 4.48-fold higher than that of His-LadR protein in pET-28a-ladR (IPTG of 1.0 mmol/L, and induction at 22 ℃ for 12 h). The results of this study indicated that the GST-tag was beneficial for the soluble expression of the ladR gene, which could provide a good foundation for functional analysis and transcriptional regulation mechanism of the LadR protein in L. monocytogenes.