One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis.

PLoS One. 2013;8(4):e60786.

Shih KS, Lin CC, Hung HF, Yang YC, Wang CA, Jeng KC, Fu HW.

Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan, Republic of China; Department of Medical Technology, Jen‐Teh Junior College of Medicine, Nursing and Management, Miaoli 35665, Taiwan, Republic of China; Departments of Research, Taichung Veterans General Hospital, Taichung 40705, Taiwan, Republic of China.

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 inflammatory 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.

PMID: 23577158

 

Supplement:

HP‐NAP was first identified from the water extract of H. pylori. This protein might play 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 (1). However, the purification of HP‐NAP using gel‐filtration chromatography is laborious and time‐consuming. An alternative strategy for efficient purification of HP‐NAP with high purity needs to be developed.

In this study, one‐step negative chromatography using DEAE Sephadex resin has been developed for purification of HP‐NAP from B. subtillis. 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 1). We expect that if a higher level of recombinant HP‐NAP were expressed in B. subtilis, then a higher recovery and higher purity of recombinant HP‐NAP could be achieved by using this purification method. 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.

Hua-Wen Fu-1Figure 1

Operation of a column showing one‐step purification of recombinant HP‐NAP using negative chromatography with DEAE anion‐exchange resins in flow‐through mode.

 

 

 

 

 

 

 

 

 

 

Reference:

1. Wang C‐A, Liu Y‐C, Du S‐Y, Lin C‐W, Fu H‐W (2008) Helicobacter pylori neutrophil‐activating protein promotes myeloperoxidase release from human neutrophils. Biochem Biophys Res Commun 377: 52‐56.

Acknowledgments:

This work was supported by grants from the National Research Program for Genomic Medicine, National Science Council, Taiwan (NSC95‐3112‐B‐007‐003 and NSC96‐3112‐B‐007‐003) and partially supported by grants from the National Science Council of Taiwan (NSC98‐2311‐B‐007‐006‐MY3 and NSC101‐2311‐B‐007‐007) and the Joint Research Program of National Tsing Hua University and Mackay Memorial Hospital (100N7727E1 and 101N2727E1).

Contact:

Hua‐Wen Fu, Ph.D.
Associate Professor
Institute of Molecular and Cellular Biology & Department of Life Science
College of Life Science
National Tsing Hua University
101, Sec 2, Kuang‐Fu Rd.
Hsinchu 30013, Taiwan
E‐mail:hwfu@life.nthu.edu.tw
http://life.nthu.edu.tw/~labfhw/

 

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