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

Abstract:

Abstract: [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.