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Immunology and Infection

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Published: June 18th, 2016



1Institute of Molecular and Cellular Biology, National Tsing Hua University, 2Department of Life Science, National Tsing Hua University

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.

In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.

This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

Helicobacter pylori (H. pylori) is a major cause of gastritis and peptic ulcer. This bacterium has also been classified as a carcinogen in humans by the International Agency for Research on Cancer, part of the World Health Organization, in 1994. It has been estimated that the prevalence of H. pylori infection is 70% in the developing countries and 30-40% in the industrialized countries1. Even though the infection rate of H. pylori is decreasing in the industrialized countries, the infection rate of H. pylori in the developing countries is still high2. The standard treatment to eradicate H. pylori

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Human blood was collected from healthy volunteers with prior written informed consent and approval from the Institutional Review Board of the National Tsing Hua University, Hsinchu, Taiwan.

1. Expression of Recombinant HP-NAP in E. coli

  1. Prepare the plasmid pET42a-NAP containing the DNA sequence of HP-NAP from H. pylori 26695 strain as previously described16. Prepare the plasmids containing the DNA sequence of HP-NAP with the desired point mutations as described (see Protoco.......

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The schematic diagram of the experimental procedure of negative purification of recombinant HP-NAP expressed in E. coli by using DEAE ion-exchange resins in batch mode is shown in Figure 1. This purification technique is based on the binding of host cell proteins and/or impurities other than HP-NAP to the resin. At pH 8.0, almost no other proteins except HP-NAP in its native form are recovered from the unbound fraction (Figure 2A and B

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The negative mode batch chromatography with DEAE anion-exchange resins presented here is suitable for purification of recombinant HP-NAP overexpressed in E coli. The pH values of the buffers used in the steps of cell lysis and purification are very critical to ensure the solubility of HP-NAP in E. coli lysates and efficient separation of recombinant HP-NAP from host cell impurities, respectively. Bacterial cells should be lysed at pH 9.0, and the negative purification should be performed at pH 8.0 to ob.......

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We thank Dr. Chao-Sheng Cheng at National Tsing Hua University, Taiwan, for performing the circular dichroism measurement. We also thank Drs. Evanthia Galanis and Ianko D. Iankov at Mayo Clinic, USA, for providing the anti-HP-NAP monoclonal antibody. We appreciate Drs. Han-Wen Chang and Chung-Chu Chen at Mackay Memorial Hospital, Hsinchu, Taiwan, for providing advice for IRB application, Mr. Te-Lung Tsai at the Mackay Memorial Hospital, Hsinchu, Taiwan, for supervising the analysis of isolated neutrophils, and Ms. Ju-Chen Weng at National Tsing Hua University, Taiwan, for her technical assistance. This work was supported by grants from the Ministry of Science and Tech....

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Name Company Catalog Number Comments
pET42a-NAP  N/A N/A prepared as described in Supplementary data of Refernce 15
E. coli BL21 (DE3) Thermo Fisher Scientific Inc C6000-03
Kanamycin Amresco 25389-94-0
Isopropyl β-D-1-thiogalactopyranoside (IPTG) MD Biomedical Inc 101-367-93-1
phenylmethylsulfonyl fluoride (PMSF) Sigma-Aldrich 10837091001 protease inhibitor
N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) Sigma-Aldrich T7254 protease inhibitor
N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) Sigma-Aldrich T4376 protease inhibitor
Bio-Rad protein assay dye reagent concentrate Bio‐Rad 500-0006 for protein quantitation
Protein standard (bovine serum albumin) Sigma-Aldrich P5619  a standard protein for Bio-Rad Protein Assay
DEAE–Sephadex  A-25 chloride form Sigma-Aldrich A25120
Spectrum/Por dialysis tubing Spectrum Laboratories 132720 with molecular weight cutoff of 14 kDa;
Acrodisc® units with Mustang® E membrane Pall MSTG25E3 for endotoxin removal; operated at flow rates ranging from 1 to 4 ml/min
mouse monoclonal antibody MAb 16F4 N/A N/A raised against the purified HP-NAP of H. pylori strain NCTC 11637 as described in Refernce 23;  A gift from Drs. Evanthia Galanis and Ianko D. Iankov at Mayo Clinic, USA
HiLoad 16/600 Superdex 200 pg GE Healthcare Life Sciences 28989335 for gel filtration chromatography
PlusOne silver staining kit, protein GE Healthcare Life Sciences 17-1150-01
Ficoll-Paque PLUS GE Healthcare Life Sciences 17-1440-02 for density gradient centrifugation to purify human neutrophils
Flat bottom 96-well white plate Thermo Fisher Scientific Inc 236108
Luminol Sigma-Aldrich A8511 protected from light
Expand  long template PCR system Sigma-Aldrich 11681834001 source of High Fidelity PCR enzyme mix
Dpn I New England Biolabs R0176S
Xho I New England Biolabs R0146S
E. coli DH5α Thermo Fisher Scientific Inc 18265-017
Name Company Product Number Comments
U-2800 double beam UV/VIS spectrophotometer Hitachi  N/A out of market and upgraded to a new model
EmulsiFlex-C3 high pressure homogenizer Avestin Inc C315320
Hitachi Koki himac CP80WX general ultracentrifuge Hitachi Koki Co 90106401 for separation of the soluble and insoluble protein fractions from E. coli lysates
ÄKTA FPLC GE Healthcare Life Sciences 18-1900-26 for gel filtration chromatography
Aviv model 62ADS CD spectrophotometer Aviv Biomedical N/A out of market and upgraded to a new model
LAS-3000 imaging system Fujifilm N/A discontinued and replaced
Wallac 1420 (Victor2) multilabel counter Perkin-Elmer 1420-018 for chemiluminescence detection

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