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 Technology of Taiwan (MOST 104-2311-B-007-003, NSC101-2311-B-007-007 and NSC98-2311-B-007-006-MY3), the Joint Research Program of National Tsing Hua University and Mackay Memorial Hospital (100N7727E1, 101N2727E1, 103N2773E1), and the research program of National Tsing Hua University (104N2052E1).

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
<ol>
	<li>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).</li>
	<li>Transform 10 ng of the above DNA plasmids into E. coli BL21 (DE3) competent cells by heat shock for 45 seconds at 42 &#176;C, streak the cells on lysogeny broth (LB) agar plates containing 50 &#181;g/ml&#160;kanamycin, and incubate the plates at 37 &#176;C for 16 hr.</li>
	<li>Inoculate single colonies in individual tubes containing 5 ml&#160;of LB broth with 50 &#181;g/ml kanamycin and grow them as precultures at 37 &#176;C for 16 hr with shaking at 170 rpm.</li>
	<li>Inoculate 2 ml of the above pre-culture cells into 200 ml of LB broth containing 50 &#181;g/ml kanamycin in a 1 L flask. Incubate the inoculated culture flask at 37 &#176;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.</li>
	<li>Add 80 &#181;l of 1 M isopropyl &#946;-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.</li>
	<li>Centrifuge the cells at 6,000 x g at 4 &#176;C for 15 min to remove the supernatant. Store the cell pellet at &#8722;70 &#176;C until purification.</li>
</ol>
2. Preparation of the Soluble Protein Fraction Containing HP-NAP
Note: All of the following steps are carried out at 4 &#176;C.
<ol>
	<li>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 &#176;C as previously described16.</li>
	<li>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 &#176;C.</li>
	<li>Centrifuge the cell lysate at 30,000 &#215; g at 4 &#176;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.</li>
	<li>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&#39;s instructions.</li>
	<li>Add 177.6 &#181;l 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.</li>
	<li>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.</li>
</ol>
3. Purification of Recombinant HP-NAP From E. coli by Negative Mode Batch Chromatography with DEAE Ion-exchange Resins
<ol>
	<li>Prepare 15 ml of DEAE resins.
	<ol>
		<li>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.</li>
		<li>Centrifuge the resin at 10,000 x g at 4 &#176;C for 1 min.</li>
		<li>Remove and discard the supernatant.</li>
		<li>Add 15 ml of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl.</li>
		<li>Repeat Protocol steps 3.1.2 to 3.1.4 four more times.</li>
		<li>Store the 15 ml settled resin (50% slurry in 30 ml Tris buffer) at 4 &#176;C for subsequent use. 
		Note: All of the following steps are carried out at 4 &#176;C.</li>
	</ol>
	</li>
	<li>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 &#176;C for 1 hr.</li>
	<li>Pour the protein/resin slurry into a plastic or glass column fitted with a stopcock. Allow the resin to settle under gravity.</li>
	<li>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.</li>
	<li>Add 15 ml of ice-cold 20 mM Tris-HCl, pH 8.0, 50 mM NaCl into the column.</li>
	<li>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.</li>
	<li>Repeat Protocol steps 3.5 to 3.6 four more times to collect the additional wash fractions.</li>
	<li>Add 15 ml of ice-cold 20 mM Tris-HCl, pH 8.0, 1 M NaCl into a column.</li>
	<li>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.</li>
	<li>Repeat Protocol steps 3.8 to 3.9 four more times to collect the additional elution fractions.</li>
</ol>
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.
<ol>
	<li>Perform dialysis to change the buffer of the purified HP-NAP to Dulbecco&#39;s phosphate-buffered saline (D-PBS), pH 7.2, at 4 &#176;C by using dialysis tubing with molecular weight cutoff of 14 kDa as previously described24.</li>
	<li>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 &#176;C to remove endotoxin.</li>
</ol>
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
<ol>
	<li>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.</li>
	<li>Analyze the purified HP-NAP by native-PAGE and gel filtration chromatography to examine its oligomeric status as previously described26.</li>
	<li>Analyze the purified recombinant HP-NAP by circular dichroism spectroscopy to examine its secondary structure as previously described26.</li>
</ol>
6. Evaluation of the Purity of HP-NAP by Silver Staining of a SDS-PAGE Gel
<ol>
	<li>Prepare the fixing solution, sensitizing solution, silver solution, developing solution, stop solution, and washing solution according to the manufacturer&#39;s instructions of a silver staining kit.</li>
	<li>Perform silver staining of a SDS-PAGE gel according to the manufacturer&#39;s instructions of a silver staining kit.</li>
	<li>Acquire the image of the gel with an imaging system.</li>
</ol>
7. Measurement of ROS Production From Neutrophils Induced by HP-NAP
<ol>
	<li>Isolate human neutrophils from human heparinized blood by dextran sedimentation followed by density gradient centrifugation as previously described24.</li>
	<li>Measurement of ROS production from neutrophils stimulated with HP-NAP by using a luminol-dependent chemiluminescence assay.
	<ol>
		<li>Turn on a plate reader and set the temperature at 37 &#176;C. Place an empty flat bottom 96-well white plate inside the plate reader chamber, allowing it to warm to 37 &#176;C.</li>
		<li>Prepare the stimulus mixtures in the D-PBS, pH 7.2, containing 0.9 mM CaCl2, 0.5 mM MgCl2, and 13.3 &#181;M 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) with the presence and absence of 0.67 &#181;M endotoxin-removed HP-NAP prepared from Protocol step 4.2.</li>
		<li>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.</li>
		<li>Add 50 &#181;l of neutrophil suspension into each well of the 96-well while plate.</li>
		<li>Add 150 &#181;l 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.</li>
		<li>Measure the emission of chemiluminescence for 5 sec per well over a period of three hours by using a plate reader.</li>
	</ol>
	</li>
</ol>
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 &#34;silent&#34; restriction sites were introduced to the mutagenesis primers by site-directed mutagenesis (SDM)-assist software28.
<ol>
	<li>Perform PCR Reaction.
	<ol>
		<li>Add 10 ng of plasmid pET42a-NAP, 1 &#181;M of mutagenesis primer pairs listed in Table 1, 200 &#181;M 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 &#181;l.</li>
		<li>Initiate the PCR cycles at 95 &#176;C for 10 min to denature the template DNA, and follow with 12 amplification cycles at 95 &#176;C for 1 minute, Tm no-5 &#176;C for 1 minute, and 72 oC for 6 min.</li>
		<li>Finish the PCR cycles with an annealing step at Tm pp-5 oC for 1 minute and an extension step at 72 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Treat 15 &#181;l of the PCR product with 0.4 &#181;l of Dpn I restriction enzyme at 37 &#176;C for 2 hr and then analyze 2 &#181;l of the Dpn I-treated PCR product by agarose gel electrophoresis.</li>
	<li>Screen mutants.
	<ol>
		<li>Transform 2 &#181;l of the PCR product into E. coli DH5&#945; competent cells by heat shock for 45 sec at 42 &#176;C.</li>
		<li>Spread the transformed cells on a LB plate containing 50 &#181;g/ml kanamycin and incubate the plate at 37 &#176;C for 16 hr.</li>
		<li>Isolate the plasmid DNA from the bacterial colonies using a commercial alkaline lysis kit according to the manufacturer&#39;s protocol.</li>
		<li>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&#39;s protocol.</li>
		<li>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&#39;s protocol.</li>
	</ol>
	</li>
</ol>

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HWF and YCY are inventors of patents TW I 432579 and US 8,673,312 for the method of one-step purification of Helicobacter pylori neutrophil-activating protein. All materials described in the manuscript will be available for research purposes. The authors confirm that this does not alter their adherence to all of the JoVE's policies on sharing data and materials.

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...

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 applications5,6. 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 infections9,10. 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-NAP14-16. 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.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pET42a-NAP&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>prepared as described in Supplementary data of Refernce 15&amp;nbsp;&lt;br/&gt; http://www.sciencedirect.com/science/article/pii/S0006291X08018317</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21 (DE3)</td><td>Thermo Fisher Scientific Inc</td><td>C6000-03</td><td>https://www.thermofisher.com/order/catalog/product/C600003</td></tr><tr><td>Kanamycin</td><td>Amresco</td><td>25389-94-0</td><td>http://www.amresco-inc.com/KANAMYCIN-SULFATE-0408.cmsx</td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>MD Biomedical Inc</td><td>101-367-93-1</td><td>http://www.antibody-antibodies.com/product_det.php?id=238064&amp;amp;supplier=search&amp;amp;name&lt;br/&gt;=IPTG%20</td></tr><tr><td>phenylmethylsulfonyl fluoride (PMSF)</td><td>Sigma-Aldrich</td><td>10837091001</td><td>protease inhibitor&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/roche/PMSFRO?lang=en&amp;amp;region=TW</td></tr><tr><td>N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK)</td><td>Sigma-Aldrich</td><td>T7254</td><td>protease inhibitor&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/t7254?lang=en&amp;amp;region=TW</td></tr><tr><td>N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK)</td><td>Sigma-Aldrich</td><td>T4376</td><td>protease inhibitor&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/t4376?lang=en&amp;amp;region=TW</td></tr><tr><td>Protein standard (bovine serum albumin)</td><td>Sigma-Aldrich</td><td>P5619&amp;nbsp;</td><td>a standard protein for Bio-Rad Protein Assay&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/fluka/p5619?lang=en&amp;amp;region=TW</td></tr><tr><td>DEAE&amp;ndash;Sephadex &amp;nbsp;A-25 chloride form</td><td>Sigma-Aldrich</td><td>A25120</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/a25120?lang=en&amp;amp;region=TW</td></tr><tr><td>Spectrum/Por dialysis tubing</td><td>Spectrum Laboratories</td><td>132720</td><td>with molecular weight cutoff of 14 kDa&lt;br/&gt; http://www.spectrumlabs.com/dialysis/RCtubing.html?Pn=132720;</td></tr><tr><td>Acrodisc&amp;reg; units with Mustang&amp;reg; E membrane</td><td>Pall</td><td>MSTG25E3</td><td>for endotoxin removal; operated at flow rates ranging from 1 to 4 ml/min&lt;br/&gt; http://www.pall.com/main/laboratory/product.page?id=19992</td></tr><tr><td>mouse monoclonal antibody MAb 16F4</td><td>N/A</td><td>N/A</td><td>raised against the purified HP-NAP of H. pylori strain NCTC 11637 as described in Refernce 23;&amp;nbsp; A gift from Drs. Evanthia Galanis and Ianko D. Iankov at Mayo Clinic, USA&lt;br/&gt; http://www.sciencedirect.com/science/article/pii/S0264410X10017585</td></tr><tr><td>HiLoad 16/600 Superdex 200 pg</td><td>GE Healthcare Life Sciences</td><td>28989335</td><td>for gel filtration chromatography&lt;br/&gt; http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/28989335</td></tr><tr><td>PlusOne silver staining kit, protein</td><td>GE Healthcare Life Sciences</td><td>17-1150-01</td><td>http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/17115001</td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare Life Sciences</td><td>17-1440-02</td><td>for density gradient centrifugation to purify human neutrophils&lt;br/&gt; http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-tw/products/AlternativeProductStructure&lt;br/&gt;_16963/17144002</td></tr><tr><td>Flat bottom 96-well white plate</td><td>Thermo Fisher Scientific Inc</td><td>236108</td><td>http://www.thermoscientific.com/en/product/nunc-f96-microwell-black-white-polystyrene-plate.html</td></tr><tr><td>Luminol</td><td>Sigma-Aldrich</td><td>A8511</td><td>protected from light&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/a8511?lang=en&amp;amp;region=TW</td></tr><tr><td>Expand &amp;nbsp;long template PCR system</td><td>Sigma-Aldrich</td><td>11681834001</td><td>source of High Fidelity PCR enzyme mix&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/roche/elongro?lang=en&amp;amp;region=TW</td></tr><tr><td>Dpn I</td><td>New England Biolabs</td><td>R0176S</td><td>https://www.neb.com/products/r0176-dpni</td></tr><tr><td>Xho I</td><td>New England Biolabs</td><td>R0146S</td><td>https://www.neb.com/products/r0146-xhoi</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; DH5&amp;alpha;</td><td>Thermo Fisher Scientific Inc</td><td>18265-017</td><td>https://www.thermofisher.com/order/catalog/product/18265017</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Product Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td>&lt;br/&gt; &amp;nbsp;</td></tr><tr><td>U-2800 double beam UV/VIS spectrophotometer</td><td>Hitachi&amp;nbsp;</td><td>N/A</td><td>out of market and upgraded to a new model&lt;br/&gt; http://hitachi-hta.com/products/life-sciences-chemical-analysis/uvvisible-spectrophotometers</td></tr><tr><td>EmulsiFlex-C3 high pressure homogenizer</td><td>Avestin Inc</td><td>C315320</td><td>http://www.avestin.com/English/c3page.html</td></tr><tr><td>Hitachi Koki himac CP80WX general ultracentrifuge</td><td>Hitachi Koki Co</td><td>90106401</td><td>for separation of the soluble and insoluble protein fractions from &lt;em&gt;E. coli&lt;/em&gt; lysates&lt;br/&gt; http://centrifuges.hitachi-koki.com/products/ultra/cp_wx/cp_wx.html</td></tr><tr><td>&amp;Auml;KTA FPLC</td><td>GE Healthcare Life Sciences</td><td>18-1900-26</td><td>for gel filtration chromatography&lt;br/&gt; http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/18190026</td></tr><tr><td>Aviv model 62ADS CD spectrophotometer</td><td>Aviv Biomedical</td><td>N/A</td><td>out of market and upgraded to a new model http://www.avivbiomedical.com/circular.php</td></tr><tr><td>LAS-3000 imaging system</td><td>Fujifilm</td><td>N/A</td><td>discontinued and replaced&lt;br/&gt; http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/28955810</td></tr><tr><td>Wallac 1420 (Victor2) multilabel counter</td><td>Perkin-Elmer</td><td>1420-018</td><td>for chemiluminescence detection&lt;br/&gt; http://www.perkinelmer.com/catalog/product/id/1420-018</td></tr></tbody></table>

one-step negative chromatographic purification of helicobacter pylori neutrophil-activating protein overexpressed in escherichia coli in batch mode

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.

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</s...

Watch this Scientific Journal Video about One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode at JoVE.co

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

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.

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

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

one-step-negative-chromatographic-purification-helicobacter-pylori

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.6K Views.  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.

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

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

Biochemistry

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Biology

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

qPCR-Based Rapid, Noninvasive Diagnosis of Helicobacter pylori Infection and Antibiotic Resistance

Quantitative Polymerase Chain Reaction (qPCR)-Based Rapid Diagnosis of Helicobacter pylori Infection and Antibiotic Resistance

Helicobacter pylori is a major human pathogen that&#160;infects approximately half of the global population and is becoming a serious health threat due to its increasing antibiotic resistance. It is the causative agent of chronic active gastritis, peptic ulcer disease, and gastric cancer and has been classified as a Group I Carcinogen by the International Agency for Research on Cancer. Therefore, the rapid and accurate diagnosis of H. pylori and the determination of its antibiotic resistance are important for the efficient eradication of this bacterial pathogen. Currently, H. pylori diagnosis methods mainly include the urea breath test (UBT), the antigen test, the serum antibody test, gastroscopy, the rapid urease test (RUT), and bacterial culture. Among them, the first three detection methods are noninvasive, meaning they are easy tests to conduct. However, bacteria cannot be retrieved through these techniques; thus, drug resistance testing cannot be performed. The last three are invasive examinations, but they are costly, require high skills, and have the potential to cause damage to patients. Therefore, a noninvasive, rapid, and simultaneous method for H. pylori detection and drug resistance testing is very important for efficiently eradicating H. pylori in clinical practice. This protocol aims to present a specific procedure involving the string test in combination with quantitative polymerase chain reaction (qPCR) for the rapid detection of H. pylori infection and antibiotic resistance. Unlike bacterial cultures, this method allows for easy, rapid, noninvasive diagnosis of H. pylori infection status and drug resistance. Specifically, we used qPCR to detect rea for H. pylori infection and mutations in the 23S rRNA and gyrA genes, which encode resistance against clarithromycin and levofloxacin, respectively. Compared to routinely used culturing techniques, this protocol provides a noninvasive, low-cost, and time-saving technique to detect H. pylori infection and determine its antibiotic resistance using qPCR.

Author Spotlight: Detection and Treatment of Helicobacter pylori Infection 

The protocol presents a noninvasive method for the rapid diagnosis of Helicobacter pylori stomach infections through the string test and determines its antibiotic resistance to clarithromycin and levofloxacin using quantitative polymerase chain reaction (qPCR).

Helicobacter pylori is a major human pathogen that&#160;infects approximately half of the global population and is becoming a serious health threat due to ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

Source: Pokhrel, A. et al. A <a href="https://jove.unicartagenaproxy.elogim.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018)
In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

Source: Pokhrel, A. et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018)
In this ...

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

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A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

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High-throughput Purification of Affinity-tagged Recombinant Proteins

Quantitative Polymerase Chain Reaction (qPCR)-Based Rapid Diagnosis of Helicobacter pylori Infection and Antibiotic Resistance

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate