Overexpression, Purification, Characterization And Pathogenicity Of Vibrio Harveyi VHH Hemolysin

Vibrio harveyi is a Gram-negative, luminous marine bacterium, which is widely distributed in the marine environment. The organism is a major pathogen of cultured penaeid shrimp, and has also been associated with diseases in fish. However, little is known about the pathogenicity mechanisms of V. harveyi. Previous studies revealed that the hemolysin activity in the extracellular product (ECP) of V. harveyi was involved in the pathogenesis in salmonids. The most pathogenic isolate, VIB 645, contained two closely related hemolysin genes (designated vhhA and vhhB), which were cloned and sequenced.In this study, vhhA was cloned into an expression plasmid pET-24d(+). The recombinant protein, VHH, was expressed in Escherichia coli strain BL21(DE3) as His-tag fused protein. The recombinant E.coli and its culture supernatant had hemolytic activity on blood agar plate. The expression conditions of the recombinant E. coli were optimized. It was found that the production of the VHH hemolysin was maximum when the transformed E. coli was induced with 1 mM IPTG at 25°C. VHH hemolysin expressed in recombinant E.coli cells and its culture supernatant were purified by metal chelating affinity chromatography (Ni-NTA His-Bind Resin). Estimated molecular weights of VHH hemolysin purified from E. coli cells and its culture supernatant by SDS-PAGE were 46.2 kDa and 45.6 kDa, respectively. The molecular weight of the mature VHH was determined to be ~ 45.9 kDa by Sephadex G-200 gel filtration. It is concluded that the mature VHH was a monomer polypeptide when expressed in E. coli. The N-terminal amino acid sequence of the mature VHH was determined to be Ala-Glu-Pro-Thr-Leu-Ser (A E P T L S). From the N-terminal amino acid sequence of the mature VHH and the deduced amino acid sequence of vhhA, it was revealed that the preprotein and the mature protein of VHH hemolysin consisted of 418 and 398 amino acids, respectively. Therefore,the former 20 N-terminal amino acids may well be the signal peptide of VHH hemolysin.