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1. One-step Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein.

　　HP‐NAP plays a crucial role in H. pylori‐induced gastric inflammation due to its ability to induce ROS production from human neutrophils and neutrophil adhesion to endothelial cells. HP‐NAP is highly immunogenic in humans and thus has become the vaccine candidate against H. pylori infection. The immunomodulatory property of HP‐NAP makes it a potential therapeutic agent for the treatment of allergic asthma and bladder cancer. HP‐NAP is a spherical dodecameric protein consisting of twelve identical monomers. Each monomer is a 17 kDa protein with a four‐helix bundle structure. Because of the large molecular size of this protein, the purification of HP‐NAP from the water extract of H. pylori was previously carried out by two consecutive gel‐filtration steps, followed by anion‐exchange chromatography. We had successfully purified recombinant HP‐NAP expressed in Escherichia coli in its native form with high purity by applying only two consecutive gel‐filtration steps (Wang et al., 2008, Biochem Biophys Res Commun. 377: 52-56). However, the purification of HP‐NAP using gel‐filtration chromatography is laborious and time‐consuming. We have developed one‐step negative chromatography using DEAE Sephadex resin for purification of HP‐NAP expressed in B. subtillis (Shih et al., 2013, PLoS One. 8(4):e60786). At pH 8.0, the majority of HP‐NAP was recovered in the flow‐through fraction while more than 99% of the endogenous proteins from B. subtilis were efficiently removed by DEAE Sephadex resin (Fig. 2). This negative purification method can also be applied to purify recombinant HP-NAP expressed in E. coli in one step. The US and ROC patents have been issued for this invention in 2014. The recombinant HP‐NAP purified by this one‐step chromatographic method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection or for other new therapeutic applications (Fig. 3). Currently, how surface charge influences the negative purification of HP-NAP is explored.


Fig. 2. Operation of a column showing one-step purification of recombinant HP-NAP using negative chromatography with DEAE anion-exchange resins in flow-through mode.	Fig. 3. Clinical applications of HP-NAP.

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2. Molecular and Functional Interaction between Helicobacter pylori Neutrophil-activating Protein and Its Receptors.

Helicobacter pylori is a microaerophilic gram-negative bacterium that colonizes the stomachs of an estimated half of all humans. Four diseases are now widely acknowledged to be caused by H. pylori: duodenal ulcer, gastric ulcer, adenocarcinoma of the distal stomach, and gastric mucosa-associated lymphoid tissue lymphoma. Helicobacter pylori neutrophil-activating protein (HP-NAP), a virulence factor of H. pylori, plays an important role in pathogenesis of H. pylori infection. HP-NAP was first found to be able to stimulate the production of reactive oxygen species (ROS) in neutrophils and promote adhesion of neutrophils to endothelial cells. Now, HP-NAP is known to play a role not only in innate immunity but also in adaptive immunity. HP-NAP has been shown as a ligand binding to Toll-like receptor 2 (TLR2) and an unidentified G protein-coupled receptor (GPCR). The engagement of TLR2 and GPCR seems to be related to HP-NAP-induced production of ROS and cytokines by leukocytes, respectively (Fig.4). However, the molecular mechanisms by which HP-NAP activates these two receptors are not clear. Molecular docking technique has been used to predict the binding site between HP-NAP and TLR2. Several possible docking conformations are shown in Fig. 5. The possible amino acid residues involved in the interaction of HP-NAP and TLR2 will be subjected to site-directed mutagenesis. The mutated HP-NAP will be expressed in the Escherichia coli expression system established in our lab (Wang et al., 2008, Biochem Biophys Res Commun. 377: 52-56). The identities of the amino acid residues responsible for the interaction of HP-NAP and TLR2 receptor will be further analyzed by examining the ability of the HP-NAP mutants to bind and to activate the receptor. Whether these amino acid residues are also involved in HP-NAP-induced GPCR activation will also be determined by examining if those HP-NAP mutants could stimulate ROS production in human neutrophils.

Fig. 4. The cellular responses of HP-NAP acting on its receptors. HP-NAP could induce leukocytes, such as neutrophils and monocytes, to produce cytokines through TLR2 activation or to release ROS through GPCR signaling pathway.

Fig. 5. The possible docking conformation of TLR2 paired with HP-NAP. The TLR2/HP-NAP docking complexes are sorted into five groups according to its different orientation and binding site on the complex. Molecular docking is predicted by docking servers of Patchdock and Gramm-X.

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