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