Zhang Yiquan, Gao He, Wang Li, Luo Zhang, Tan Yafang, Guo Zhaobiao, Yang Ruifu, Zhou Dongsheng
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.
Wei Sheng Wu Xue Bao. 2011 May;51(5):615-21.
The regulator protein H-NS of Yersinia pestis was expressed using the Escherichia coli BL21lambdaDE3 protein expression system, and its DNA-binding activity was characterized.
The entire coding region of the hns gene was amplified by PCR from Y. pestis strain 201, and then cloned into the BamHI and Sal sites of the vector pET28a. The recombinant plasmid pET28a-hns was transformed into BL21lambdaDE3. 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.
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.
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.
利用大肠杆菌BL21λDE3蛋白表达系统表达鼠疫耶尔森菌的调节蛋白H-NS，并对其DNA结合活性进行表征。
通过PCR从鼠疫耶尔森菌201株中扩增hns基因的整个编码区，然后克隆到载体pET28a的BamHI和Sal位点。将重组质粒pET28a-hns转化到BL21λDE3中。通过添加1 mM异丙基-β-D-硫代半乳糖苷（IPTG）在LB培养基中诱导His-H-NS的过表达。用镍负载的HiTrap螯合琼脂糖柱（Amersham）在天然条件下纯化过表达的蛋白。进行电泳迁移率变动分析和DNase I足迹实验以体外分析His-H-NS的DNA结合活性。
纯化的His-H-NS蛋白可与鼠疫耶尔森菌psaA、psaE和rovA的上游DNA区域结合，并在单核苷酸分辨率下确定了H-NS结合位点。
纯化的His-H-NS蛋白可与靶DNA片段结合，表明H-NS会调节鼠疫耶尔森菌中相关基因的转录。
