title: [Purification of recombinant H-NS protein of Yersinia pestis and characterization of its DNA-binding activity].
abstract: OBJECTIVE
  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.
  
  
  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 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China. zhangyiquang@163.com
authors.forename: Yiquan
authors.initials: Y
authors.lastname: Zhang
authors.forename: He
authors.initials: H
authors.lastname: Gao
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Zhang
authors.initials: Z
authors.lastname: Luo
authors.forename: Yafang
authors.initials: Y
authors.lastname: Tan
authors.forename: Zhaobiao
authors.initials: Z
authors.lastname: Guo
authors.forename: Ruifu
authors.initials: R
authors.lastname: Yang
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Zhou
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015010:Yersinia pestis / Q000235:genetics* / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D011994:Recombinant Proteins; C405626:RovA protein, Yersinia; D014157:Transcription Factors; C081814:pH 6 antigen, Yersinia