Abstract
Background: VacA is the only exocrine toxin of H. pylori, which plays a very important role in the customization of H. pylori in the gastric mucosa, contributing to the pathogenesis of gastritis-cancer. Studies about vacA structure and function was hindered by the lack of efficient production system. In this study, we developed methodology for expression, purification and stable storage of a functionally active vacA toxin in E.coli.Results: A 2502-bp fragment and vacA gene were identified. An 89.7-kDa VacA34-854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay.Conclusions: A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).
Acetic acid buffer as extraction medium for free and bound phenolics from dried blackcurrant (
Ribes nigrum
L.) skins
