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 Western
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 resazurin-
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 Cterminal
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 VacA-
m1 isolated domains were able to independently bind to cultured gastric cells similar to the full-
length toxin, albeit the 33-kDa domain exhibited significantly higher activity of membrane perturbation
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.