Expression And Purification Of Important Regulatory Proteins Of Yersinia Pestis And Characterization Of Their DNA-binding Activity

Yersinia pestis is the causative agent of plague, a deadly zoonotic disease. During the cycles between fleas and host animals, Y. pestis will encounter multiple environmental adverse simulates (such as the change of pH, osmotic pressures, peroxides, and temperature). Y. pestis can sense and response to these diverse environment signals, then archive successful survive, and cause hosts fall ills, which is tightly controlled process for which Y. pestis multiply to produce virulence determinants. There are various transcriptional regulators, such as PhoP, RovA, H-NS, Fis, and CRP in Y. pestis. They play key roles in the above process through responding to stimulate signals and subsequently regulating the expression of their target genes.Objective: In order to set up a experiment platform for the study of the transcriptional regulation of virulence- or transmission-related genes, a total of eight regulatory proteins of Y. pestis (i.e., PhoP, OxyR, RcsB, RovA, H-NS, CRP, Fur, and OmpR) were expressed using the Escherichia coli BL21λDE3 protein expression system, and the DNA-binding activity of them was characterized.Methods: The entire coding regions of the crp, fur, phoP, oxyR, ompR, rcsB and rovA genes were amplified by PCR from Y. pestis strain 201, and then cloned into the vector pET28a directly, respectively. The recombinant plasmids were translocated into BL21λDE3, respectively, to generate the recombinant strains expressing the relevant proteins. Over-expression of His-tagged proteins were induced by addition of 1 mM IPTG (isopropyl-b-D-thiogalactoside). The over-expressed proteins were purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham), respectly. Based on the computational promoter analysis, the DNA-binding motifs of relavent regulatory proteins were predicted within the upstream regions of their potential target genes. The electrophoretic mobility shift assays (EMSA) were carried out to analyze the in vitro accosiation between regulatory proteins and their targeting promoter DNA regions. DNase I footprinting was further carried out for the His-H-NS protein to determine the H-NS-binding sites with targeting promoter DNA regions.Results: We successfully expressed eghit recombinant proteins of Y. pestis (i.e., PhoP, OxyR, RcsB, RovA, H-NS, CRP, Fur, and OmpR). As showed by EMSA, they were was able to bind to the promoter regions of YPO0736, hmsT, waaA, psaE, ompC, yfeA, and ompF, respectly, which suggested the regulatory proteins may regulate the transcription of relavent genes directly. The promoter regions of pla and pst were directly binded by the recombinant H-NS protein, as observed by both EMSA and DNase I footprinting. the GC contents of the H-NS sites within their promoter regions, revealed by DNase I footprinting experiments, were lower than 30%, confirming that H-NS trends to recognize the AT-rich sequences.Conclusion: We successful expressed eight recombinant proteins of Y. pestis, namely PhoP, OxyR, RcsB, RovA, H-NS, CRP, Fur, and OmpR. The purified His-tagged proteins were able to bind to the relevant target promoter regions, suggesting that they would regulate the transcription of relevant genes in Y. pestis. The GC contents of the H-NS sites were significantly lower than that of the whole genome.Significance: We succefully expressed eight important regulatory proteins of Y. pestis, and their DNA binding activities were further characterized by EMSA and DNase I footprinting, which provides a basis for the further gene regulation studies.