title: One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis.
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.
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
authors.forename: Kuo-Shun
authors.initials: KS
authors.lastname: Shih
authors.forename: Chih-Chang
authors.initials: CC
authors.lastname: Lin
authors.forename: Hsiao-Fang
authors.initials: HF
authors.lastname: Hung
authors.forename: Yu-Chi
authors.initials: YC
authors.lastname: Yang
authors.forename: Chung-An
authors.initials: CA
authors.lastname: Wang
authors.forename: Kee-Ching
authors.initials: KC
authors.lastname: Jeng
authors.forename: Hua-Wen
authors.initials: HW
authors.lastname: Fu
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D002852:Chromatography, Ion Exchange / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; C092412:neutrophil-activating protein A, Helicobacter pylori