N-terminally added tag selectively enhances heterologous expression of vaca cytotoxin variants from helicobacter pylori

Abstract

Background: Gastric pathogen Helicobacter pylori secretes VacA cytotoxin displaying a high degree of polymorphic variations of which the highest VacA pathogenicity correlates with m1-type variant followed by VacA-m2. Objective: To comparatively evaluate expression in Escherichia coli of the mature VacA variants (m1-and m2-types) and their 33-and 55/59-kDa domains fused with His(6) tag at N-or C-terminus. Methods: All VacA clones expressed in E. coli TOP10™ were analyzed by SDS-PAGE and West-ern blotting. VacA inclusions were solubilized under native conditions (~150-rpm shaking at 37°C for 2 h in 20 mM HEPES (pH7.4) and 150 mM NaCl). Membrane-perturbing and cytotoxic activities of solubilized VacA proteins were assessed via liposome-entrapped dye leakage and resazur-in-based cell viability assays, respectively. VacA binding to human gastric adenocarcinoma cells was assessed by immunofluorescence microscopy. Side-chain hydrophobicity of VacA was analyzed through modeled structures constructed by homology-and ab initio-based modeling. Results: Both full-length VacA-m1 and 33-kDa domain were efficiently expressed only in the presence of N-terminal extension while its 55-kDa domain was capably expressed with either N-or C-terminal extension. Selectively enhanced expression was also observed for VacA-m2. Protein expression profiles revealed a critical period in IPTG-induced production of the 55-kDa domain with N-terminal extension unlike its C-terminal extension showing relatively stable expression. Both Va-cA-m1 isolated domains were able to independently bind to cultured gastric cells similar to the ful-l-length toxin, albeit the 33-kDa domain exhibited significantly higher activity of membrane pertur-bation than others. Membrane-perturbing and cytotoxic activities observed for VacA-m1 appeared to be higher than those of VacA-m2. Homology-based modeling and sequence analysis suggested a potential structural impact of non-polar residues located at the N-terminus of the mature VacA toxin and its 33-kDa domain. Conclusion: Our data provide molecular insights into selective influence of the N-terminally added tag on efficient expression of recombinant VacA variants, signifying biochemical and biological implications of the hydrophobic stretch within the N-terminal domain.