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

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).

Il sangue umano è stato raccolto da volontari sani con il previo consenso informato scritto e approvazione da parte del Institutional Review Board della National Tsing Hua University, Hsinchu, Taiwan. 1. Espressione ricombinante HP-NAP in E. coli <ol><li> Preparare il plasmide pET42a-NAP che contiene la sequenza di DNA di HP-NAP da H. pylori 26695 ceppo come descritto in precedenza 16. Preparare i plasmidi contenenti la sequenza di DNA di HP-NAP con le mutazioni puntiformi desider...

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La modalità di cromatografia lotto negativo con resine a scambio anionico DEAE qui presentata è adatto per la purificazione di ricombinante HP-NAP sovraespresso in E. coli. I valori di pH dei tamponi usati nelle fasi di lisi cellulare e purificazione sono molto critici per assicurare la solubilità di HP-NAP in E. lisati coli e efficiente separazione dei ricombinante HP-NAP dalle impurità cellula ospite, rispettivamente. Le cellule batteriche devono essere lisati a pH 9,0, e la purificazion...

Helicobacter pylori (H. pylori) è una delle principali cause di gastrite e ulcera peptica. Questo batterio è stato classificato come sostanza cancerogena per l&#39;uomo dall&#39;Agenzia internazionale per la ricerca sul cancro, che fa parte dell&#39;Organizzazione Mondiale della Sanità, nel 1994. E &#39;stato stimato che la prevalenza di H. pylori è del 70% nei paesi in via di sviluppo e il 30-40% nei paesi industrializzati 1. Anche se il tasso di infezione di H. pylori è in calo nei paesi industrializzati, il tasso di infezione di H. pylori nei paesi in via di sviluppo è ancora alto 2. Il trattamento standard per sradicare H. pylori consiste nella somministrazione di un inibitore della pompa protonica, PPI, e due antibiotici, claritromicina più amoxicillina o metronidazolo 3. Tuttavia, l&#39;aumento della resistenza agli antibiotici in H. pylori -related terapia dell&#39;ulcera sollecita lo sviluppo di nuove strategie per prevenire or curare l&#39;infezione. Sviluppo di vaccinazione preventiva e / o terapeutico contro H. pylori potrebbe fornire un approccio alternativo per controllare H. pylori. Helicobacter pylori neutrofili-attivazione di proteine ​​(HP-NAP), un importante fattore di virulenza di H. pylori, è stato identificato in estratti di acqua di H. pylori con la possibilità di attivare neutrofili di aderire alle cellule endoteliali e produrre specie reattive dell&#39;ossigeno (ROS) 4. Infiltrazione dei neutrofili della mucosa gastrica si trovano in H. pylori pazienti infettati con gastrite attiva possono provocare infiammazione e danni ai tessuti dello stomaco. Così, HP-NAP può giocare un ruolo patologico attivando neutrofili per indurre infiammazione gastrica, che provoca ulteriore ulcera o H. pylori -associated malattie gastriche. Tuttavia, HP-NAP è un potenziale candidato per le applicazioni cliniche 5,6. A causa della pro immunogenica e immunomodulanteprietà di HP-NAP, questa proteina potrebbero essere utilizzati per sviluppare vaccini, agenti terapeutici, e strumenti diagnostici. Uno studio clinico è stato condotto per l&#39;utilizzo ricombinante HP-NAP come uno dei componenti di un vaccino contro H. proteine pylori. Questo vaccino è costituito da ricombinante HP-NAP, cytotoxin associata gene A (CagA), e le proteine ​​vacuolizzante citotossina A (VacA) formulato con idrossido di alluminio ed è stato ulteriormente dimostrato di essere sicuro ed immunogenico negli esseri umani 7. Inoltre, HP-NAP agisce come un potente immunomodulatore per innescare T helper di tipo 1 (Th1) -polarized risposte immunitarie per la terapia del cancro 8 e per regolare le risposte immunitarie Th2-mediate indotte da reazioni allergiche e infezioni parassitarie 9,10. Per quanto riguarda la diagnostica, ricombinante ELISA HP-NAP-based è stata applicata per rilevare gli anticorpi sierici contro HP-NAP in H. pylori -infected pazienti 11. Uno studio ha mostrato che il livello di anticorpi specifici per HP-NAP nei sieri di H. pylori -infected pazienti con cancro gastrico è risultata significativamente più alta di quella di pazienti con gastrite cronica 12. Un altro studio ha anche dimostrato che gli anticorpi sierici contro HP-NAP sono associati con la presenza di non-cardias adenocarcinoma gastrico 13. Così, ricombinante ELISA HP-NAP-based può essere applicata per rilevare anticorpi contro HP-NAP per la prognosi del cancro gastrico in H. pylori -infected pazienti. Nel loro insieme, la HP-NAP purificata potrebbe essere ulteriormente utilizzato per la prevenzione, il trattamento e la prognosi di H. pylori -associated malattie e terapia del cancro. Tra i vari metodi utilizzati per la purificazione di ricombinante HP-NAP espresso in Escherichia coli (E. coli), nella sua forma nativa, presentate finora, una cromatografia di gel-filtrazione seconda fase di purificazione che coinvolge è necessario per ottenere elevata purezza HP-NAP 14-16 . Qui, un metodo che utilizza negativo cromatografia modalità batch condietilamminoetil (DEAE) resine a scambio ionico è descritto per la purificazione di HP-NAP sovraespresso in E. coli con alto rendimento e di elevata purezza. Questa tecnica di purificazione è stata basata sul legame di proteine ​​della cellula ospite e / o impurezze diverse HP-NAP alla resina. A pH 8,0, quasi senza altre proteine ​​tranne HP-NAP vengono recuperati dalla frazione non legata. Questo approccio purificazione mediante DEAE cromatografia a scambio ionico in modalità negativa è semplice e risparmio consentendo purificazione ricombinante HP-NAP tramite one-step cromatografia attraverso la raccolta della frazione libera tempo. Oltre ad HP-NAP, diverse altre biomolecole, come virus 17, Immunoglobulina G (IgG) 18, emoglobina 19, proteina fosfatasi 20, e fattore di virulenza flagellin 21, sono stati segnalati anche ad essere purificata dalla cromatografia a scambio ionico in negativo modalità. La modalità negativa è preferito per cromatografia a scambio ionico se impurità sono il minorecomponenti presenti nel campione sottoposto da depurare 22. L&#39;applicazione della cromatografia negativo purificazione di biomolecole naturali o ricombinanti è stato recentemente rivisto 23. Il presente rapporto fornisce un passo per passo il protocollo per l&#39;espressione ricombinante HP-NAP in E. coli, la lisi delle cellule, e purificazione di HP-NAP utilizzando modalità negativa cromatografia batch con resine a scambio ionico DEAE. Se una proteina desiderata per purificazione è adatto per cromatografia a scambio ionico in modo negativo, il protocollo descritto potrebbe anche essere adattato come punto di partenza per lo sviluppo di un processo di purificazione.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pET42a-NAP&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>prepared as described in Supplementary data of Refernce 15&#160; 
			http://www.sciencedirect.com/science/article/pii/S0006291X08018317</td>
		</tr>
		<tr>
			<td>E. coli 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 &#946;-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&#38;supplier=search&#38;name =IPTG%20</td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Sigma-Aldrich</td>
			<td>10837091001</td>
			<td>protease inhibitor 
			http://www.sigmaaldrich.com/catalog/product/roche/PMSFRO?lang=en&#38;region=TW</td>
		</tr>
		<tr>
			<td>N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK)</td>
			<td>Sigma-Aldrich</td>
			<td>T7254</td>
			<td>protease inhibitor 
			http://www.sigmaaldrich.com/catalog/product/sigma/t7254?lang=en&#38;region=TW</td>
		</tr>
		<tr>
			<td>N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK)</td>
			<td>Sigma-Aldrich</td>
			<td>T4376</td>
			<td>protease inhibitor 
			http://www.sigmaaldrich.com/catalog/product/sigma/t4376?lang=en&#38;region=TW</td>
		</tr>
		<tr>
			<td>Bio-Rad protein assay dye reagent concentrate</td>
			<td>Bio&#8208;Rad</td>
			<td>500-0006</td>
			<td>for protein quantitation 
			http://www.bio-rad.com/en-us/sku/5000006-bio-rad-protein-assay-dye-reagent-concentrate</td>
		</tr>
		<tr>
			<td>Protein standard (bovine serum albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>P5619&#160;</td>
			<td>a standard protein for Bio-Rad Protein Assay 
			http://www.sigmaaldrich.com/catalog/product/fluka/p5619?lang=en&#38;region=TW</td>
		</tr>
		<tr>
			<td>DEAE&#8211;Sephadex &#160;A-25 chloride form</td>
			<td>Sigma-Aldrich</td>
			<td>A25120</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/a25120?lang=en&#38;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 
			http://www.spectrumlabs.com/dialysis/RCtubing.html?Pn=132720;</td>
		</tr>
		<tr>
			<td>Acrodisc&#174; units with Mustang&#174; E membrane</td>
			<td>Pall</td>
			<td>MSTG25E3</td>
			<td>for endotoxin removal; operated at flow rates ranging from 1 to 4 ml/min 
			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;&#160; A gift from Drs. Evanthia Galanis and Ianko D. Iankov at Mayo Clinic, USA 
			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 
			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 
			http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-tw/products/AlternativeProductStructure _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 
			http://www.sigmaaldrich.com/catalog/product/sigma/a8511?lang=en&#38;region=TW</td>
		</tr>
		<tr>
			<td>Expand &#160;long template PCR system</td>
			<td>Sigma-Aldrich</td>
			<td>11681834001</td>
			<td>source of High Fidelity PCR enzyme mix 
			http://www.sigmaaldrich.com/catalog/product/roche/elongro?lang=en&#38;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>E. coli DH5&#945;</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td>18265-017</td>
			<td>https://www.thermofisher.com/order/catalog/product/18265017</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Product Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td> 
			&#160;</td>
		</tr>
		<tr>
			<td>U-2800 double beam UV/VIS spectrophotometer</td>
			<td>Hitachi&#160;</td>
			<td>N/A</td>
			<td>out of market and upgraded to a new model 
			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 E. coli lysates 
			http://centrifuges.hitachi-koki.com/products/ultra/cp_wx/cp_wx.html</td>
		</tr>
		<tr>
			<td>&#196;KTA FPLC</td>
			<td>GE Healthcare Life Sciences</td>
			<td>18-1900-26</td>
			<td>for gel filtration chromatography 
			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 
			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 
			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 neutrofili-attivazione di proteine ​​(HP-NAP) è un importante fattore di virulenza di Helicobacter pylori (H. pylori). Essa svolge un ruolo fondamentale in H. pylori indotta infiammazione gastrica attivando diversi leucociti innate tra neutrofili, monociti e mastociti. Le proprietà immunogeniche e immunomodulanti di HP-NAP lo rendono un candidato potenziale diagnostico e vaccino per H. pylori e un nuovo candidato farmaco per la terapia del cancro. Per ottenere quantità sostanziali di purificato HP-NAP utilizzato per le sue applicazioni cliniche, un metodo efficace per purificare questa proteina con alta resa e purezza deve essere stabilito. In questo protocollo, abbiamo descritto un metodo per un passo negativo purificazione cromatografica ricombinante HP-NAP sovraespresso in Escherichia coli (E. coli) utilizzando dietilamminoetil (DEAE) resine a scambio ionico (per es., Sephadex) imodalità batch n. Ricombinante HP-NAP costituisce quasi il 70% delle proteine ​​totali in E. coli ed è quasi completamente recuperato nella frazione solubile upon lisi cellulare a pH 9,0. A condizione ottimale a pH 8,0, la maggioranza di HP-NAP viene recuperato nella frazione non legata mentre le proteine ​​endogene da E. coli sono efficacemente rimossi dalla resina. Questo metodo di purificazione mediante cromatografia modalità negativa batch con resine a scambio ionico DEAE produce funzionale HP-NAP da E. coli nella sua forma nativa con alta resa e purezza. L&#39;HP-NAP purificata potrebbe essere ulteriormente utilizzato per la prevenzione, il trattamento e la prognosi di H. pylori -associated malattie e terapia del cancro.

Lo schema della procedura sperimentale di purificazione negativo ricombinante HP-NAP espressa in E. coli mediante resine a scambio ionico DEAE in modalità batch è mostrato in Figura 1. Questa tecnica di purificazione è basato sul legame di proteine ​​della cellula ospite e / o impurezze diverse HP-NAP alla resina. A pH 8,0, quasi senza altre proteine ​​tranne HP-NAP nella sua forma nativa vengono recuperati dalla frazione libera (Figura 2A</st...

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

Un metodo ad alto rendimento per un solo passaggio di purificazione negativo ricombinante Helicobacter pylori neutrofili-attivazione di proteine ​​(HP-NAP) sovraespresso in Escherichia coli utilizzando resine dietilamminoetil in modalità batch è descritta. HP-NAP purificata da questo metodo è utile per lo sviluppo di vaccini, farmaci, o diagnostica per H. pylori -associated malattie.

Un passo negativo cromatografica Purificazione di<em&gt; Helicobacter pylori</em&gt; Neutrofili-attivazione della proteina sovraespresso in<em&gt; Escherichia coli</em&gt; In modalità batch

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

The overall goal of this purification is to yield functional Helicobacter pylori Neutrophil-activating protein or HP-NAP in its native form with high purity and high yield using negative mode batch chromatography with di-ethyl amino-ethyl ion exchange resin. The main advantage of this technique is that HP-NAP can be purified in its native form in one step with high purity and high yield. Negative purification of HP-NAP starts with a batch binding procedure in which the soluble protein fraction from bacterial lysates is added to DEAE resin.The mixture is incubated to allow wholesale proteins to bind to the resin. HP-NAP present in the unbound fraction is then obtained by centrifugation of gravity flow. The implications of this technique is 10:1 prevention treatment and prognostic of Helicobacter pylori associated diseases as well as cancer therapy.Because of the immunogenic and immunomodulatory properties of HP-NAP. Generally, individuals new to this method will struggle. Because HP-NAP easily aggregates during the sterilization.Demonstrating the procedure will be Zhi-Wei Hong, a graduate student from our lab. Working at four degrees Celsius, we suspend 50 milliliters of the cell palate prepared as described in the text protocol in 20 milliliters of low salt, tris-buffer at pH 9.0 with protease inhibitor mixture. It's critical to keep sample at the four degrees Celsius to allow the procedure to prevent the protein aggregation.Disrupt the bacterial cells by passing the bacterial suspension through a high-pressure homogenizer operated at a range of 15, 000 to 20, 000 PSI for seven times at four degrees Celsius. After lysates baterializes, it should be centrifuged immediately. Transfer homogenized samples to a centrifuge tube.Immediately centrifuge the cell-lysate at 30, 000 times G at four degrees Celsius for one hour to separate the soluble and insoluble protein fractions by using an ultra-centrifuge. Following centrifugation, transfer the supernatant as the soluble protein fraction to a beaker. Measure the protein concentration of the soluble protein fraction by the Bradford method using a commercial kit with bovine serum albumin as the standard according to the manufacturer's instructions.Then add 177.6 microliters of one normal HCl into 20 milliliters of the soluble protein fraction to adjust its pH value from pH 9.0 to pH 8.0. Add 20 millimeter Tris-HCl, pH 8.0, 50 milliliter sodium chloride to the protein solution to result in a final protein concentration of 0.5 milligrams per milliliter. To prepare 15 milliliters of DEAE resin, suspend 0.6 grams of DEAE resin dry powder in 30 milliliters of Tris-HCl buffer at room temperature for at least one day.Centrifuge the resin at 10, 000 times G at four degrees Celsius for one minute and then remove and discard the supernatant. Then add 15 milliliters of Tris-HCl buffer. After repeating the centrifugation resuspension steps four more times, store the 15 milliliters of settled resin at four degrees Celsius for subsequent use.Working at four degrees Celsius, add 45 milliliters of the prepared soluble proteins to milliliters of the resin and transfer to a 100 milliliter beaker with stir bar. Stir the protein resin slurry using a stir plate at four degrees Celsius for one hour. Next, pour the protein resin slurry into a plastic or a glass column fitted with a stopcock and allow the resin to settle under gravity.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 neutrophil-activating protein. Add 15 milliliters of the ice-cold Tris-HCl buffer into the column.Open the stopcock again 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. Repeat washing with ice-cold buffer four more times to collect the additional wash fractions.Then add 15 milliliters of ice-cold 20 millimeter Tris-HCl pH 8.0, one molar sodium chloride into the column. Collect the flow through as the elution fractions as before. Repeat the elution steps four more times to collect the additional elution fractions.The effect of pH on solubility of HP-NAP in E.coli lysate are shown here. The amount of HP-NAP detected in this soluble fraction was markedly increased at pH 7.5 to 9.5. Recombinant HP-NAP was almost fully recovered in the soluble fraction upon cell lysis at pH 9.0.Here, the effect of pH on negative purification of recombinant HP-NAP expressed in E.coli by DEAE resins in batch mode are shown. At pH 8.0, only a little amount of HP-NAP was present in the elution fraction. The amount of HP-NAP present in the unbound fractions was the highest at pH 8.0.The optimized amount of proteins loaded onto the DEAE resin is 1.5 milligrams of protein per milliliter of resin for achieving maximum capacity of the resin to absorb the impurities from E.coli. At pH 8.0, very few proteins other than HP-NAP in its native form are recovered in the unbound fraction using DEAE resins in batch mode. The purity of HP-NAP was higher than 95 percent.The purified HP-NAP folds into a multimer with a secondary structure of alpha-helix as revealed by the gelifiltration chromotogram and the far UV circular dichroism spectrum. A luminal dependent chemiluminescence assay reveals that the purified HP-NAP is able to trigger the production of reactive oxygen species by neutrophiles. Once mastered, these techniques can be done within five hours if it is performed properly.After watching this video you should have a good understanding of how to purify functional HP-NAP in its native form with high yield and purity using negative mode batch chromotography with DEAE and exchange resin. While attempting this procedure, it's important to remember to use buffers at the appropriate pH in the step of sterilizes in the batch chromotography. We first had the idea for this method when we tried to apply other chromotographic approaches instead of filtration to purify HP-NAP in its native form.Though this method is designed to appeal by recombinant HP-NAP expressed in E.coli. It can also be applied to other bacterial expression systems such as per-sida sep-tidas. Visual demonstration of this measure is quick code.Plus the step of sterilizes and batch chromotography are difficult to learn. Because these steps must be carried out at the four degrees Celsius without any time delay. If a protein designed for purification is suitable for our exchange chromotography in negative mode, this procedure could also be adapted as a starting point for the development of purification process.After its development, this technique paved the way for researchers in the field of clinical research to explore the possibility of using HP-NAP in cancer therapy as well as the development of vaccines, drugs, and the diagnostics for Helicobacter pylori associated disease.

Research

JoVE Journal

Immunology and Infection

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

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non invasiva Modello di neuropatogeno<em&gt; Escherichia coli</em&gt; L&#39;infezione neonatale nel ratto

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

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Immunologia e infezioni

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

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Ad alta risoluzione microscopia elettronica del<em&gt; Helicobacter pylori</em&gt; Cag secrezione di tipo IV Sistema Pili Prodotto in diverse condizioni di Ferro Disponibilità

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

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Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

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Un protocollo Facile generare proteine site-specifically acetilati in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

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Dissezione multiproteici segnalazione di purificazione di affinità di complementazione bimolecolare (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

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Utilizzando Enhanced fluorescenza verde che esprimono proteine Escherichia Coli per valutare la fagocitosi dei macrofagi peritoneali del Mouse

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Purification of a High Molecular Mass Protein in Streptococcus mutans

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Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...