Abstract
Helicobacter pylori (H. pylori) is a pathogen that causes the gastric mucosal diseases. H. pylori neutrophil-activating protein (HP-NAP) is a virulent factor of H. pylori and has the ability to activate various immune cells such as neutrophils. Recombinant DNA technology for expressing HP-NAP is a useful tool to study the function of HP-NAP. Bacillus subtilis (B. subtilis) is a bacterium commonly used to express recombinant proteins for avoiding contamination of endotoxin. Though B. subtilis was served as a host for the expression of recombinant HP-NAP, only one strategy for purification of recombinant HP-NAP expressed in B. subtilis has been reported. In this study, B. subtilis was served as a host for the expression of recombinant HP-NAP and DEAE ion-exchange chromatography was utilized for purification of recombinant HP-NAP. The optimized culture time for expression of HP-NAP in my B. subtilis expression system was around 12 to 15 hr. To determine the optimal purification condition for obtaining HP-NAP with high purity, DEAE Sephadex resin and DEAE Sepharose resin at pH 7.0, 7.5, and 8.0 were utilized to determine the optimal purification condition. HP-NAP was remained in the unbound fraction by DEAE Sephadex resin at pH 8.0. HP-NAP was then purified by a single DEAE Sephadex ion-exchange column. The flow-through fractions containing HP-NAP with purity exceeding 92 % was obtained. The purified HP-NAP assembled into a multimeric protein with secondary structure of α-helix and stimulated the production of reactive oxygen species from neutrophils. Thus, HP-NAP can be efficiently purified from B. subtilis expression system by collection of flow-through fraction using DEAE Sephadex resin at pH 8.0.