Expression, purification, and characterization of thermolabile hemolysin (TLH) from Vibrio alginolyticus

A Jia, NYS Woo, XH Zhang
2010 Diseases of Aquatic Organisms  
Hemolysin is a putative pathogenicity factor in many bacterial pathogens. In this study, a DNA fragment containing the open reading frame (1254 bp) of the thermolabile hemolysin gene (tlh) from Vibrio alginolyticus V05 was amplified and cloned into the expression plasmid pET-24d(+). The deduced amino acid sequence of the thermolabile hemolysin (TLH) shared 94 and 83% identity with the lecithin-dependent hemolysin (LDH)/TLH of V. parahaemolyticus and V. harveyi thermolabile hemolysin (VHH),
more » ... molysin (VHH), respectively. The sequence analysis also indicated that it contained a GDSL lipase domain like VHH. The recombinant protein with a predicted molecular mass of 47.2 kDa was expressed in the Escherichia coli strain BL21 (DE3) as a His-tag fused protein. TLH purified by the nickel-nitrilotriacetic acid (Ni-NTA) His-Bind Resin method showed phospholipase activity on an egg yolk emulsion plate and hemolytic activity against flounder erythrocytes with a specific activity of 18 hemolytic units µg -1 . The addition of divalent cations at different concentrations decreased hemolytic activity of the purified TLH, but monavalent cations did not affect hemolytic activity. The hemolytic activity of TLH was also markedly inhibited by protein modification reagents, i.e. β-mercaptoethanol, phenylmethylsulfonyl fluoride, and 5, 5'-dithio-bis(2-nitrobenzoic acid). Moreover, TLH was toxic to zebrafish when injected intraperitoneally, with a median lethal dose (LD 50 ) of 0.8 µg protein g -1 fish. This work shows that TLH could potentially be developed as a vaccine and used as a diagnostic tool for vibriosis. KEY WORDS: Vibrio alginolyticus · Hemolysin · Expression · Purification · Pathogenicity Resale or republication not permitted without written consent of the publisher
doi:10.3354/dao02225 pmid:20662368 fatcat:w6rarzuo3bhjdfxa3gizcf3x3y