One-Step Chromatographic Purification of <i>Helicobacter pylori</i> Neutrophil-Activating Protein Expressed in <i>Bacillus subtilis</i>

<div><p><i>Helicobacter pylori</i> neutrophil-activating protein (HP-NAP), a major virulence factor of <i>Helicobacter pylori</i> (<i>H. pylori</i>), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential <i>in vitro</i> diagnostic marker for <i>H. pylori</i> infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in <i>Bacillus subtilis</i> (<i>B. subtilis</i>). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from <i>B. subtilis</i> in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for <i>H. pylori</i> infection.</p>