One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis

Kuo-Shun Shih,Yu-Chi Yang,Hua-Wen Fu,Chung-An Wang,Hsiao-Fang Hung,Chih-Chang Lin,Kee-Ching Jeng,William R Abrams

doi:10.1371/journal.pone.0060786

Abstract

Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), 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 in vitro diagnostic marker for H. pylori 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 Bacillus subtilis (B. subtilis). 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 B. subtilis 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 H. pylori infection.

Highlights

Helicobacter pylori (H. pylori), a microaerophilic Gram-negative bacterium, infects about half of the entire human population [1,2]
We have developed a new strategy to allow onestep chromatographic purification of the recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) expressed in B. subtilis
At pH 7.5 and 8.0, HP-NAP did not bind to the resin, whereas the majority of the other proteins from B. subtilis did bind to the resin

Summary

Introduction

Helicobacter pylori (H. pylori), a microaerophilic Gram-negative bacterium, infects about half of the entire human population [1,2]. Infection of H. pylori is associated with chronic gastritis, gastric ulcer, and gastric cancer. In H. pylori-infected patients with chronic gastritis, a strong infiltration of neutrophils was detected in their gastric mucosa, and the extent of gastric neutrophil infiltration was correlated to the degree of mucosa damage [3,4]. H. pylori neutrophil-activating protein (HP-NAP) is a virulence factor that recruits neutrophils to inflamed mucosal tissue during H. pylori infection. It was first characterized by its ability to promote neutrophil adherence to endothelial cells and induce the production of reactive oxygen species (ROS) by neutrophils [5]. In addition to acting as a chemoattractant to induce neutrophil migration, HP-NAP can cross the endothelium to promote neutrophil-endothelial cell adhesion [6,7]. HP-NAP could play a critical role in recruiting neutrophils towards the infected area to trigger the gastric inflammatory response during H. pylori infection

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Results

Conclusion