title: High yield purification of Helicobacter pylori neutrophil-activating protein overexpressed in Escherichia coli.
abstract: BACKGROUND
  Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-induced gastric inflammation. Due to its immunogenic and immunomodulatory properties, HP-NAP has been used for developing vaccines against H. pylori infection and new drugs for cancer therapy.
  
  
  RESULTS
  Here, we provide a simple process for high-yield production of HP-NAP by applying one-step negative chromatography to purify recombinant HP-NAP expressed in Escherichia coli (E. coli). In our E. coli expression system, recombinant HP-NAP constitutes nearly 70% of the total protein. Overexpressed recombinant HP-NAP is almost completely soluble upon cell lysis at pH 9.5. Under the optimal condition at pH 8.0, recombinant HP-NAP with purity higher than 95% can be obtained from E. coli by collecting the unbound fraction using diethylaminoethyl (DEAE) Sephadex resin in batch mode. The overall yield of HP-NAP from a 50-ml E. coli culture is ~19 mg. The purified HP-NAP folds into a multimer with a secondary structure of α-helix and is able to trigger the production of reactive oxygen species by neutrophils.
  
  
  CONCLUSIONS
  Purification of recombinant HP-NAP overexpressed in E. coli using DEAE Sephadex negative mode batch chromatography is an efficient method for high-yield production of highly pure HP-NAP in its native state. The purified HP-NAP is useful for various clinical applications including vaccine development, diagnosis, and new drug development.
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China. nevergone916@gmail.com.
authors.forename: Yu-Chi
authors.initials: YC
authors.lastname: Yang
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China. yoke12071015@hotmail.com.
authors.forename: Ting-Yu
authors.initials: TY
authors.lastname: Kuo
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China. a76062908@gmail.com.
authors.forename: Zhi-Wei
authors.initials: ZW
authors.lastname: Hong
authors.affiliation: Department of Internal Medicine, Division of Hepatology and Gastroenterology, Mackay Memorial Hospital, Hsinchu, 30055, Taiwan, Republic of China. 4074@mmh.org.tw.
authors.forename: Han-Wen
authors.initials: HW
authors.lastname: Chang
authors.affiliation: Department of Internal Medicine, Division of Hepatology and Gastroenterology, Mackay Memorial Hospital, Hsinchu, 30055, Taiwan, Republic of China. 4059@mmh.org.tw.
authors.forename: Chung-Chu
authors.initials: CC
authors.lastname: Chen
authors.affiliation: Department of Pathology and Laboratory Medicine, Mackay Memorial Hospital, Hsinchu, 30055, Taiwan, Republic of China. 5648@mmh.org.tw.
authors.forename: Te-Lung
authors.initials: TL
authors.lastname: Tsai
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China. hwfu@life.nthu.edu.tw.|Department of Life Science, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China. hwfu@life.nthu.edu.tw.
authors.forename: Hua-Wen
authors.initials: HW
authors.lastname: Fu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002845:Chromatography; D004926:Escherichia coli / Q000235:genetics*; D016480:Helicobacter pylori / Q000737:chemistry / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C092412:neutrophil-activating protein A, Helicobacter pylori