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

Summary

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.

Abstract

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.

Introduction

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 infection consists of the administration of a proton pump inhibitor, PPI, and two antibiotics, clarithromycin plus amoxicillin or metronidazole³. However, the rise of antibiotic resistance in H. pylori-related ulcer therapy urges the development of new strategies to prevent or cure the infection. Development of preventive and/or therapeutic vaccination against H. pylori could provide an alternative approach to control H. pylori infection.

Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of H pylori, was first identified in water extracts of H. pylori with the ability to activate neutrophils to adhere to endothelial cells and produce reactive oxygen species (ROS)⁴. Neutrophil infiltration of gastric mucosa found in H. pylori-infected patients with active gastritis may result in inflammation and tissue damage of the stomach. Thus, HP-NAP may play a pathological role by activating neutrophils to induce gastric inflammation, which further causes ulcer or H. pylori-associated gastric diseases. Nevertheless, HP-NAP is a potential candidate for clinical applications^5,6. Due to the immunogenic and immunomodulatory properties of HP-NAP, this protein could be used to develop vaccines, therapeutic agents, and diagnostic tools. A clinical trial has been conducted for using recombinant HP-NAP as one of the components of a protein vaccine against H. pylori. This vaccine consists of recombinant HP-NAP, cytotoxin-associated gene A (CagA), and vacuolating cytotoxin A (VacA) proteins formulated with aluminum hydroxide and has further been demonstrated to be safe and immunogenic in humans⁷. Also, HP-NAP acts as a potent immunomodulator to trigger T helper type 1 (Th1)-polarized immune responses for cancer therapy⁸ and to down regulate Th2-mediated immune responses elicited by allergic reactions and parasitic infections^9,10. As for diagnostics, recombinant HP-NAP-based ELISA has been applied to detect serum antibodies against HP-NAP in H. pylori-infected patients¹¹. One study showed that the level of HP-NAP-specific antibodies in sera from H. pylori-infected patients with gastric cancer was significantly higher than that from patients with chronic gastritis¹². Another study also showed that serum antibodies against HP-NAP are associated with the presence of non-cardia gastric adenocarcinoma¹³. Thus, recombinant HP-NAP-based ELISA may be applied to detect serum antibodies against HP-NAP for prognosis of gastric cancer in H. pylori-infected patients. Taken together, the purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

Among the several methods used for purification of recombinant HP-NAP expressed in Escherichia coli (E. coli) in its native form reported so far, a second purification step involving gel-filtration chromatography is needed to obtain highly pure HP-NAP^14-16. Here, a method using negative mode batch chromatography with diethylaminoethyl (DEAE) ion-exchange resins is described for purification of HP-NAP overexpressed in E. coli with high yield and high purity. This purification technique was 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 are recovered from the unbound fraction. This purification approach using DEAE ion-exchange chromatography in negative mode is simple and time saving by allowing purification of recombinant HP-NAP via one-step chromatography through the collection of the unbound fraction. In addition to HP-NAP, several other biomolecules, such as viruses¹⁷, Immunoglobulin G (IgG)¹⁸, hemoglobin¹⁹, protein phosphatase²⁰, and virulence factor flagellin²¹, have also been reported to be purified by ion-exchange chromatography in negative mode. The negative mode is preferred for ion-exchange chromatography if impurities are the minor components present in the sample subjected to be purified²². The application of negative chromatography in purification of natural or recombinant biomolecules has been recently reviewed²³.

The present report provides a step by step protocol for expression of recombinant HP-NAP in E. coli, lysis of the cells, and purification of HP-NAP using negative mode batch chromatography with DEAE ion-exchange resins. If a protein desired for purification is suitable for ion-exchange chromatography in negative mode, the described protocol could also be adapted as a starting point for development of a purification process.

Protocol

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 Protocol, step 8).
2. Transform 10 ng of the above DNA plasmids into E. coli BL21 (DE3) competent cells by heat shock for 45 seconds at 42 °C, streak the cells on lysogeny broth (LB) agar plates containing 50 µg/ml kanamycin, and incubate the plates at 37 °C for 16 hr.
3. Inoculate single colonies in individual tubes containing 5 ml of LB broth with 50 µg/ml kanamycin and grow them as precultures at 37 °C for 16 hr with shaking at 170 rpm.
4. Inoculate 2 ml of the above pre-culture cells into 200 ml of LB broth containing 50 µg/ml kanamycin in a 1 L flask. Incubate the inoculated culture flask at 37 °C for 2 hr with shaking at 170 rpm until the absorbance at 600 nm reaches approximately 0.4-0.5 detected by an UV/VIS spectrophotometer.
5. Add 80 µl of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) into the above culture to a final concentration of 0.4 mM to induce the expression of HP-NAP. Incubate the culture for 3 hr until the absorbance at 600 nm reaches approximately 1.6-1.7.
6. Centrifuge the cells at 6,000 x g at 4 °C for 15 min to remove the supernatant. Store the cell pellet at −70 °C until purification.

2. Preparation of the Soluble Protein Fraction Containing HP-NAP

Note: All of the following steps are carried out at 4 °C.

1. Resuspend 50 ml of the cell pellet prepared in Protocol step 1.6 in 20 ml of 20 mM Tris-HCl, pH 9.0, 50 mM NaCl containing 0.13 mM phenylmethylsulfonyl fluoride (PMSF), 0.03 mM N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK), and 0.03 mM N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) at 4 °C as previously described¹⁶.
2. Disrupt the bacteria cells by passing the bacterial suspension through a high pressure homogenizer operated at a range of 15,000 to 20,000 psi for 7 times at 4 °C.
3. Centrifuge the cell lysate at 30,000 × g at 4 °C for 1 hr to separate the soluble and insoluble protein fractions by using an ultracentrifuge. Transfer the supernatant as the soluble protein fraction to a beaker.
4. Measure the protein concentration of the soluble protein fraction by the Bradford method using a commercial kit with bovine serum albumin (BSA) as the standard according to the manufacturer's instructions.
5. Add 177.6 µl of 1 N HCl into 20 ml of the soluble protein fraction obtained from Protocol step 2.3 to adjust its pH value from pH 9.0 to pH 8.0.
6. Add 20 mM Tris-HCl, pH 8.0, 50 mM NaCl to the above protein solution to make the final protein concentration to be 0.5 mg/ml.

3. Purification of Recombinant HP-NAP From E. coli by Negative Mode Batch Chromatography with DEAE Ion-exchange Resins

1. Prepare 15 ml of DEAE resins.
   1. Weigh out 0.6 g dry powder of DEAE resins and suspend it in 30 ml of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl at room temperature for at least 1 day.
   2. Centrifuge the resin at 10,000 x g at 4 °C for 1 min.
   3. Remove and discard the supernatant.
   4. Add 15 ml of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl.
   5. Repeat Protocol steps 3.1.2 to 3.1.4 four more times.
   6. Store the 15 ml settled resin (50% slurry in 30 ml Tris buffer) at 4 °C for subsequent use.
      Note: All of the following steps are carried out at 4 °C.
2. Add 45 ml of the soluble proteins prepared in Protocol step 2.6 to 15 ml of the resin and stir the protein/resin slurry with a magnetic stirrer at 4 °C for 1 hr.
3. Pour the protein/resin slurry into a plastic or glass column fitted with a stopcock. Allow the resin to settle under gravity.
4. Open the stopcock to allow the protein solution to run through the column by gravity flow until the liquid level in the column is just above the resin. Collect the flow-through as the unbound fraction, which contains the purified HP-NAP.
5. Add 15 ml of ice-cold 20 mM Tris-HCl, pH 8.0, 50 mM NaCl into the column.
6. Open the stopcock to allow the wash buffer to run through the column by gravity flow until the liquid level in the column is just above the resin. Collect the flow-through as the wash fraction.
7. Repeat Protocol steps 3.5 to 3.6 four more times to collect the additional wash fractions.
8. Add 15 ml of ice-cold 20 mM Tris-HCl, pH 8.0, 1 M NaCl into a column.
9. Open the stopcock to allow the elution buffer to run through the column by gravity flow until the liquid level in the column is just above the resin. Collect the flow-through as the elution fraction.
10. Repeat Protocol steps 3.8 to 3.9 four more times to collect the additional elution fractions.

4. Buffer Exchange and Endotoxin Removal of HP-NAP Purified by Negative Mode Batch Chromatography with DEAE Resins

Note: The purified HP-NAP expressed in E. coli needs to be subjected to buffer exchange and endotoxin removal prior to stimulate neutrophils.

1. Perform dialysis to change the buffer of the purified HP-NAP to Dulbecco's phosphate-buffered saline (D-PBS), pH 7.2, at 4 °C by using dialysis tubing with molecular weight cutoff of 14 kDa as previously described²⁴.
2. Filter the dialyzed HP-NAP through a syringe filter with a positively-charged, hydrophilic membrane attached to a 20 ml disposable syringe at flow rates ranging from 2.5 to 4 ml/minute at 4 °C to remove endotoxin.

5. Characterization of the Molecular Properties of Purified Recombinant HP-NAP by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western Blotting, Native-PAGE, Gel Filtration Chromatography, and Circular Dichroism Spectroscopy

1. Analyze the purified recombinant HP-NAP by SDS-PAGE and western blotting with hybridoma culture supernatants containing mouse monoclonal antibody MAb 16F4²⁵ as previously described²⁴.
2. Analyze the purified HP-NAP by native-PAGE and gel filtration chromatography to examine its oligomeric status as previously described²⁶.
3. Analyze the purified recombinant HP-NAP by circular dichroism spectroscopy to examine its secondary structure as previously described²⁶.

6. Evaluation of the Purity of HP-NAP by Silver Staining of a SDS-PAGE Gel

1. Prepare the fixing solution, sensitizing solution, silver solution, developing solution, stop solution, and washing solution according to the manufacturer's instructions of a silver staining kit.
2. Perform silver staining of a SDS-PAGE gel according to the manufacturer's instructions of a silver staining kit.
3. Acquire the image of the gel with an imaging system.

7. Measurement of ROS Production From Neutrophils Induced by HP-NAP

1. Isolate human neutrophils from human heparinized blood by dextran sedimentation followed by density gradient centrifugation as previously described²⁴.
2. Measurement of ROS production from neutrophils stimulated with HP-NAP by using a luminol-dependent chemiluminescence assay.
   1. Turn on a plate reader and set the temperature at 37 °C. Place an empty flat bottom 96-well white plate inside the plate reader chamber, allowing it to warm to 37 °C.
   2. Prepare the stimulus mixtures in the D-PBS, pH 7.2, containing 0.9 mM CaCl₂, 0.5 mM MgCl₂, and 13.3 µM 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) with the presence and absence of 0.67 µM endotoxin-removed HP-NAP prepared from Protocol step 4.2.
   3. Adjust the concentration of human neutrophils prepared from Protocol step 7.1 to 2 x 10⁶ cells/ml in D-PBS, pH 7.2, containing 5 mM glucose.
   4. Add 50 µl of neutrophil suspension into each well of the 96-well while plate.
   5. Add 150 µl of the stimulus mixtures prepared from Protocol step 7.2.2 into the well containing neutrophils at a time interval of 5 seconds per well.
   6. Measure the emission of chemiluminescence for 5 sec per well over a period of three hours by using a plate reader.

8. Construction of DNA Plasmids Harboring HP-NAP Mutants by Polymerase Chain Reaction (PCR)-based Site-direct Mutagenesis

Note: PCR-based site-direct mutagenesis was generated basically as described previously²⁷ except that the "silent" restriction sites were introduced to the mutagenesis primers by site-directed mutagenesis (SDM)-assist software²⁸.

1. Perform PCR Reaction.
   1. Add 10 ng of plasmid pET42a-NAP, 1 µM of mutagenesis primer pairs listed in Table 1, 200 µM of deoxynucleoside triphosphates (dNTPs), and 3 units of high-fidelity PCR enzyme mix into a PCR tube containing deionized water, giving a final volume of 25 µl.
   2. Initiate the PCR cycles at 95 °C for 10 min to denature the template DNA, and follow with 12 amplification cycles at 95 °C for 1 minute, T_{m no}-5 °C for 1 minute, and 72 ^oC for 6 min.
   3. Finish the PCR cycles with an annealing step at T_{m pp}-5 ^oC for 1 minute and an extension step at 72 °C for 30 min.
2. Treat 15 µl of the PCR product with 0.4 µl of Dpn I restriction enzyme at 37 °C for 2 hr and then analyze 2 µl of the Dpn I-treated PCR product by agarose gel electrophoresis.
3. Screen mutants.
   1. Transform 2 µl of the PCR product into E. coli DH5α competent cells by heat shock for 45 sec at 42 °C.
   2. Spread the transformed cells on a LB plate containing 50 µg/ml kanamycin and incubate the plate at 37 °C for 16 hr.
   3. Isolate the plasmid DNA from the bacterial colonies using a commercial alkaline lysis kit according to the manufacturer's protocol.
   4. Treat the plasmid DNA isolated in Protocol step 8.3.3 with XhoI restriction enzyme to verify the presence of the desired silent mutations according to manufacturer's protocol.
   5. Sequence the plasmid DNA verified by Protocol step 8.3.4 with T7 promoter primer to confirm the correction of the coding sequences of HP-NAP mutants according to manufacturer's protocol.

Results

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

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
pET42a-NAP	N/A	N/A	prepared as described in Supplementary data of Refernce 15  http://www.sciencedirect.com/science/article/pii/S0006291X08018317
E. coli BL21 (DE3)	Thermo Fisher Scientific Inc	C6000-03	https://www.thermofisher.com/order/catalog/product/C600003
Kanamycin	Amresco	25389-94-0	http://www.amresco-inc.com/KANAMYCIN-SULFATE-0408.cmsx
Isopropyl β-D-1-thiogalactopyranoside (IPTG)	MD Biomedical Inc	101-367-93-1	http://www.antibody-antibodies.com/product_det.php?id=238064&supplier=search&name =IPTG%20
phenylmethylsulfonyl fluoride (PMSF)	Sigma-Aldrich	10837091001	protease inhibitor http://www.sigmaaldrich.com/catalog/product/roche/PMSFRO?lang=en®ion=TW
N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK)	Sigma-Aldrich	T7254	protease inhibitor http://www.sigmaaldrich.com/catalog/product/sigma/t7254?lang=en®ion=TW
N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK)	Sigma-Aldrich	T4376	protease inhibitor http://www.sigmaaldrich.com/catalog/product/sigma/t4376?lang=en®ion=TW
Protein standard (bovine serum albumin)	Sigma-Aldrich	P5619	a standard protein for Bio-Rad Protein Assay http://www.sigmaaldrich.com/catalog/product/fluka/p5619?lang=en®ion=TW
DEAE–Sephadex  A-25 chloride form	Sigma-Aldrich	A25120	http://www.sigmaaldrich.com/catalog/product/sigma/a25120?lang=en®ion=TW
Spectrum/Por dialysis tubing	Spectrum Laboratories	132720	with molecular weight cutoff of 14 kDa http://www.spectrumlabs.com/dialysis/RCtubing.html?Pn=132720;
Acrodisc® units with Mustang® E membrane	Pall	MSTG25E3	for endotoxin removal; operated at flow rates ranging from 1 to 4 ml/min http://www.pall.com/main/laboratory/product.page?id=19992
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 http://www.sciencedirect.com/science/article/pii/S0264410X10017585
HiLoad 16/600 Superdex 200 pg	GE Healthcare Life Sciences	28989335	for gel filtration chromatography http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/28989335
PlusOne silver staining kit, protein	GE Healthcare Life Sciences	17-1150-01	http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/17115001
Ficoll-Paque PLUS	GE Healthcare Life Sciences	17-1440-02	for density gradient centrifugation to purify human neutrophils http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-tw/products/AlternativeProductStructure _16963/17144002
Flat bottom 96-well white plate	Thermo Fisher Scientific Inc	236108	http://www.thermoscientific.com/en/product/nunc-f96-microwell-black-white-polystyrene-plate.html
Luminol	Sigma-Aldrich	A8511	protected from light http://www.sigmaaldrich.com/catalog/product/sigma/a8511?lang=en®ion=TW
Expand  long template PCR system	Sigma-Aldrich	11681834001	source of High Fidelity PCR enzyme mix http://www.sigmaaldrich.com/catalog/product/roche/elongro?lang=en®ion=TW
Dpn I	New England Biolabs	R0176S	https://www.neb.com/products/r0176-dpni
Xho I	New England Biolabs	R0146S	https://www.neb.com/products/r0146-xhoi
E. coli DH5α	Thermo Fisher Scientific Inc	18265-017	https://www.thermofisher.com/order/catalog/product/18265017
Name	Company	Product Number	Comments
Equipment
U-2800 double beam UV/VIS spectrophotometer	Hitachi	N/A	out of market and upgraded to a new model http://hitachi-hta.com/products/life-sciences-chemical-analysis/uvvisible-spectrophotometers
EmulsiFlex-C3 high pressure homogenizer	Avestin Inc	C315320	http://www.avestin.com/English/c3page.html
Hitachi Koki himac CP80WX general ultracentrifuge	Hitachi Koki Co	90106401	for separation of the soluble and insoluble protein fractions from E. coli lysates http://centrifuges.hitachi-koki.com/products/ultra/cp_wx/cp_wx.html
ÄKTA FPLC	GE Healthcare Life Sciences	18-1900-26	for gel filtration chromatography http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/18190026
Aviv model 62ADS CD spectrophotometer	Aviv Biomedical	N/A	out of market and upgraded to a new model http://www.avivbiomedical.com/circular.php
LAS-3000 imaging system	Fujifilm	N/A	discontinued and replaced http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/28955810
Wallac 1420 (Victor2) multilabel counter	Perkin-Elmer	1420-018	for chemiluminescence detection http://www.perkinelmer.com/catalog/product/id/1420-018