鼠疫菌H-NS蛋白的表达与纯化及其DNA结合活性分析

Purification of recombinant H-NS protein of Yersinia pestis and characterization of its DNA-binding activity

【目的】利用大肠杆菌BL21λDE3的表达系统,表达出有活性的鼠疫耶尔森氏菌(以下简称鼠疫菌)调控子蛋白H-NS,为进一步研究H-NS的转录调控奠定基础。【方法】PCR扩增鼠疫菌201株hns基因的编码区,将其直接克隆入pET28a质粒中,再将pET28a-hns重组质粒转入大肠杆菌BL21λDE3菌株中,所得菌株经IPTG诱导后能表达出鼠疫菌His-H-NS蛋白;通过体外的凝胶迁移实验(EMSA)和DNaseⅠ足迹实验对His-H-NS蛋白与DNA的结合活性进行分析。【结果】成功表达出有活性的鼠疫菌His-H-NS蛋白,该蛋白对鼠疫菌pH6抗原基因(psaA、psaE)及rovA基因均有结合活性。【结论】鼠疫菌His-H-NS具有DNA结合活性,说明H-NS能调控鼠疫菌基因的转录。

[Objective]The regulator protein H-NS of Yersinia pestis was expressed using the Escherichia coli BL21λDE3 protein expression system,and its DNA-binding activity was characterized.[Methods]The entire coding region of the hns gene was amplified by PCR from Y.pestis strain 201,and then cloned into the BamHI and SalI sites of the vector pET28a.The recombinant plasmid pET28a-hns was transformed into BL21λDE3.Over-expression of His-H-NS in the LB medium was induced by addition of 1 mM IPTG（isopropyl-b-D-thiogalactoside）.The over-expressed protein was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns（Amersham）.The electrophoretic mobility shift assay and DNase I footprinting experiments were carried out to analyze the DNA-binding activity of His-H-NS in vitro.[Results]The purified His-H-NS protein could bind to the upstream DNA regions of psaA,psaE and rovA of Y.pestis,and the H-NS-binding sites were determined at the single nucleotide resolution.[Conclusion]The purified His-H-NS protein could bind to target DNA fragments,suggesting that H-NS would regulate the transcription of relevant genes in Y.pests.