Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted ...Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, hetemlogous expression of the empA gene encoding metallopmtease and export of the recombinant metallopmtease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide （611 amino acids） consisting of four domains： a signal peptide, an Nterminal pmpoptide, a mature region and a C-terminal pmpoptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metallopmtease as the mature pmtease was further confirmed by SDS-PAGE and im- munoblotting. It was found that recombinant metallopmtease with the EmpA activity and antigenicity was exported into the poriplasm of Escherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. coli are similar to those in V. anguillarum.展开更多
基金Supported by the National Natural Science Foundation of China （ No. 30328021 ）.
文摘Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, hetemlogous expression of the empA gene encoding metallopmtease and export of the recombinant metallopmtease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide （611 amino acids） consisting of four domains： a signal peptide, an Nterminal pmpoptide, a mature region and a C-terminal pmpoptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metallopmtease as the mature pmtease was further confirmed by SDS-PAGE and im- munoblotting. It was found that recombinant metallopmtease with the EmpA activity and antigenicity was exported into the poriplasm of Escherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. coli are similar to those in V. anguillarum.
