Purification and DNA-binding of ToxR truncated protein of Vibrio parahaemolyticus

Abstract:

Abstract:［Objective］ The DNA-binding domain of ToxR protein of Vibrio parahaemolyticus was expressed using the Escherichia coli BL21λDE3 protein expression system，and its DNA-binding activity was characterized．［Methods］ The fragment of DNA-binding domain at N-terminal of ToxR (ToxR-N) was amplified by PCR from V．parahaemolyticus strain RIMD2210633，and then cloned into the BamHI and Hind III sites of the vector pET28a．The recombinant plasmid pET28a was transformed into BL21λDE3．Over-expression of His-ToxR-N in the LB medium was induced by adding 1 mmol/L IPTG (isopropyl-b-D-thiogalactoside)．The over-expressed protein was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham)，and then the His-tag was removed by using restricted thrombin．The electrophoretic mobility shift assay was used to analyze the DNA-binding activity of ToxR-N to the promoter-proximal DNA regions of calR and VP1687，respectively．The promoter-proximal regions of calR and VP1687 were separately cloned into the pHRP309 vector containing a promoterless lacZ gene．Then，each of the two recombinant LacZ reporter plasmids was transformed into the wide-type strain (WT) and the toxＲ null mutant strain (ΔtoxR)，respectively，to measure the promoter activity (the β-Galactosidase activity) of the target genes in WT and ΔtoxR by using the β-Galactosidase Enzyme Assay System．［Results］The purified ToxR-N protein had the ability to bind to the upstream DNA regions of calR but not VP1687．The LacZ fusion results showed that the transcription of calR and VP1687 was positively and negatively regulated by ToxR in V．parahaemolyticus，respectively． ［Conclusion］The recombinant ToxR-N protein could be used for studying the transcriptional regulation mechanism in V．parahaemolyticus．ToxR fulfills a mechanism of negative regulation of T3SS1 genes by activating the expression of calR through protein- proximal promoter DNA association.

Key words:Keywords: Vibrio parahaemolyticus，ToxR，DNA binding activity，transcriptional regulation