Name: one-step negative chromatographic purification of helicobacter pylori neutrophil-activating protein overexpressed in escherichia coli in batch mode
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We thank Dr. Chao-Sheng Cheng at National Tsing Hua University, Taiwan, for performing the circular dichroism measurement. We also thank Drs. Evanthia Galanis and Ianko D. Iankov at Mayo Clinic, USA, for providing the anti-HP-NAP monoclonal antibody. We appreciate Drs. Han-Wen Chang and Chung-Chu Chen at Mackay Memorial Hospital, Hsinchu, Taiwan, for providing advice for IRB application, Mr. Te-Lung Tsai at the Mackay Memorial Hospital, Hsinchu, Taiwan, for supervising the analysis of isolated neutrophils, and Ms. Ju-Chen Weng at National Tsing Hua University, Taiwan, for her technical assistance. This work was supported by grants from the Ministry of Science and 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).

Humant blod samlades från friska frivilliga försökspersoner med skriftligt informerat samtycke och godkännande från Institutional Review Board of National Tsing Hua University, Hsinchu, Taiwan. 1. Expression av rekombinant HP-NAP i E. coli <ol><li> Förbereda plasmiden pET42a-NAP innehållande DNA-sekvensen för HP-NAP från H. pylori 26695 stammen som tidigare beskrivits 16. Framställa plasmider innehållande DNA-sekvensen av HP-NAP med de önskade punktmutationer såsom bes...

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Det negativa sättet sats-kromatografi med DEAE anjonbytarhartser som presenteras här är lämplig för rening av rekombinant HP-NAP överuttryckt i E. coli. PH-värdena för de buffertar som användes i stegen i cellys och rening är mycket avgörande för att säkerställa lösligheten av HP-NAP i E. coli-lysat och effektiv separation av rekombinant HP-NAP från värdcellföroreningar, respektive. Bakterieceller bör lyserades vid pH 9,0, och den negativa reningen bör utföras vid pH 8,0 för att e...

Helicobacter pylori (H. pylori) är en viktig orsak till gastrit och magsår. Denna bakterie har också klassificerats som cancerframkallande hos människor från International Agency for Research on Cancer, en del av Världshälsoorganisationen, 1994. Det har uppskattats att förekomsten av H. pylori-infektion är 70% i utvecklingsländerna och 30-40% i industriländerna 1. Även om infektionen hastigheten för H. pylori minskar i industriländerna, infektionsfrekvensen av H. pylori i utvecklingsländerna är fortfarande hög två. Standardbehandlingen för att utrota H. pylori-infektion består av administrering av en protonpumpshämmare, PPI, och två antibiotika, klaritromycin plus amoxicillin eller metronidazol 3. Men ökningen av antibiotikaresistens hos H. pylori -relaterade sår terapi uppmanar utvecklingen av nya strategier för att förebygga or bota infektionen. Utveckling av förebyggande och / eller terapeutisk vaccination mot H. pylori kan ge ett alternativt tillvägagångssätt för att styra H. pylori-infektion. Helicobacter pylori neutrofil-aktiverande protein (HP-NAP), en viktig virulensfaktor av H pylori, identifierades först i extrakt vatten från H. pylori med förmågan att aktivera neutrofiler att vidhäfta till endotelceller och producera reaktiva syreradikaler (ROS) 4. Neutrofil infiltration av magslemhinnan hittas i H. pylori-infekterade patienter med aktiv gastrit kan resultera i inflammation och vävnadsskada i magen. Således kan HP-NAP spela en patologisk roll genom att aktivera neutrofiler att inducera gastrisk inflammation, vilket ytterligare orsakar magsår eller H. pylori -associated gastric sjukdomar. Ändå är HP-NAP en potentiell kandidat för kliniska tillämpningar 5,6. På grund av den immunogena och immunmodulerande proegenskaper till HP-NAP, kunde detta protein användas för att utveckla vacciner, terapeutiska medel och diagnostiska verktyg. En klinisk prövning har genomförts för att använda rekombinant HP-NAP som en av komponenterna i ett proteinvaccin mot H. pylori. Detta vaccin består av rekombinant HP-NAP, cellgift associerad gen A (CagA) och vakuoliserande cytotoxin A (VacA) proteiner formuleras med aluminiumhydroxid och har dessutom visat sig vara säker och immunogen i människor 7. Också fungerar HP-NAP som en potent immunmodulator för att utlösa T-hjälpar typ 1 (Th1) -polarized immunsvar för cancerterapi 8 och att nedreglera Th2-medierade immunsvar som framkallas av allergiska reaktioner och parasitinfektioner 9,10. När det gäller diagnostik, har rekombinant HP-NAP-baserade ELISA använts för att påvisa serumantikroppar mot HP-NAP i H. pylori infekterade patienter 11. En studie visade att nivån av HP-NAP-specifika antikroppar i sera från H. pylori infekterade patienter med magcancer var betydligt högre än den från patienter med kronisk gastrit 12. En annan studie visade också att serumantikroppar mot HP-NAP är associerade med närvaron av icke-kardia magcancer 13. Således kan rekombinant HP-NAP-baserade ELISA appliceras för att detektera serumantikroppar mot HP-NAP för prognos av magcancer i H. pylori-infekterade patienter. Sammantaget kan det renade HP-NAP utnyttjas ytterligare för förebyggande, behandling och prognos av H. pylori -associated sjukdomar samt cancerterapi. Bland flera metoder som används för rening av rekombinant HP-NAP uttryckts i Escherichia coli (E. coli) i dess nativa form rapporterats hittills, behövs ett andra reningssteg inbegriper gelfiltreringskromatografi för att erhålla högren HP-NAP 14-16 . Här, en metod som använder negativ läge batch-kromatografi meddietylaminoetyl (DEAE) jonbytarhartser beskrivs för rening av HP-NAP överuttryckt i E. coli med högt utbyte och hög renhet. Denna reningsteknik baserades på den bindning av värdcellproteiner och / eller andra än HP-NAP till hartset föroreningar. Vid pH 8,0, är ​​nästan inga andra proteiner förutom HP-NAP utvinnas från den obundna fraktionen. Detta renings tillvägagångssätt och använda DEAE jonbyteskromatografi i negativt driftläge är enkel och tidsbesparande genom att tillåta rening av rekombinant HP-NAP via ett-steg-kromatografi genom insamling av den obundna fraktionen. Förutom HP-NAP, flera andra biomolekyler, såsom virus 17 immunglobulin G (IgG) 18, hemoglobin 19, proteinfosfatas 20, och virulensfaktor flagellin 21, har också rapporterats som skall renas genom jonbyteskromatografi i negativ läge. Det negativa sättet är att föredra för jonbyteskromatografi, om föroreningar är den mindrekomponenter som är närvarande i provet utsattes för skall renas 22. Tillämpningen av negativa kromatografi i rening av naturliga eller rekombinanta biomolekyler har nyligen över 23. Denna rapport ger en steg för steg-protokoll för uttryck av rekombinant HP-NAP i E. coli, lysering av cellerna, och rening av HP-NAP med användning av negativ läge batch-kromatografi med DEAE jonbyteshartser. Om ett protein som önskas för rening är lämplig för jonbyteskromatografi i negativ-läge, kan den beskrivna protokollet även anpassas som en utgångspunkt för utvecklingen av en reningsprocess.

<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 neutrofil-aktiverande protein (HP-NAP) är en viktig virulensfaktor av Helicobacter pylori (H. pylori). Den spelar en avgörande roll i H. pylori-inducerad gastrisk inflammation genom att aktivera flera medfödda leukocyter inklusive neutrofiler, monocyter och mastceller. De immunogena och immunmodulerande egenskaper hos HP-NAP gör det en potentiell diagnos och vaccinkandidat för H. pylori och en ny läkemedelskandidat för cancerbehandling. I syfte att erhålla väsentliga mängder av renat HP-NAP används för dess kliniska tillämpningar, till en effektiv metod att rena detta protein med högt utbyte och renhet behöver upprättas. I detta protokoll har vi beskrivit en metod för en-stegs negativ kromatografisk rening av rekombinant HP-NAP överuttryckt i Escherichia coli (E. coli) genom användning av dietylaminoetyl (DEAE) jonbytarmassa (eg., Sephadex) in satsvis. Rekombinant HP-NAP utgör nästan 70% av det totala proteinet i E. coli och är nästan helt återhämtat sig i den lösliga fraktionen efter cellys vid pH 9,0. Med optimal kondition vid pH 8,0, är majoriteten av HP-NAP utvanns i den obundna fraktionen medan de endogena proteiner från E. coli effektivt avlägsnas genom hartset. Denna reningsmetod med användning av negativ läge batch-kromatografi med DEAE jonbytarmassor ger funktionell HP-NAP från E. coli i dess nativa form med högt utbyte och renhet. Det renade HP-NAP skulle kunna utnyttjas ytterligare för förebyggande, behandling och prognos av H. pylori -associated sjukdomar samt cancerterapi.

Den schematiskt diagram över den experimentella proceduren av negativ rening av rekombinant HP-NAP uttryckt i E. coli genom att använda DEAE jonbytarmassor i batch-läge visas i figur 1. Denna reningsteknik baserad på bindning av värdcellproteiner och / eller andra än HP-NAP till hartset föroreningar. Vid pH 8,0, är nästan inga andra proteiner förutom HP-NAP i dess nativa form utvinnas från den obundna fraktionen (figur 2A och ...

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

En hög räntemetoden för ett steg negativ rening av rekombinant Helicobacter pylori neutrofilaktiverande protein (HP-NAP) överuttryckt i Escherichia coli genom att använda dietylaminoetyl hartser i satsvis beskrivs. HP-NAP renas genom denna metod är gynnsam för utvecklingen av vacciner, läkemedel eller diagnostik för H. pylori -associated sjukdomar.

Enstegs Negativ kromatografisk rening av<em&gt; Helicobacter pylori</em&gt; Neutrofilaktiverande Protein överuttryckt i<em&gt; Escherichia coli</em&gt; I batch-läge

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

Icke-invasiv Modell av Neuropathogenic<em&gt; Escherichia coli</em&gt; Infektion i neonatal Rat

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

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

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

Högupplöst elektronmikroskopi av<em&gt; Helicobacter pylori</em&gt; Cag Typ IV sekretionssystemet Pili Producerad i varierande förhållanden of Iron Tillgänglighet

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 ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Organoids som modell för Infektionssjukdomar: Culture of Human och Murina mage Organoids och mikroinjektion av Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Påvisande av entrohemoragiska Escherichia Coli koloniseringen i murina mottagande av icke-invasiva In Vivo Mareld System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

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

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

Skapandet av en tät Transposon införande bibliotek med bakteriell konjugation i Enterobacterial stammar som Escherichia Coli eller Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overuttryck och rening av människa<em&gt; Cis</em&gt; -prenyltransferas i<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Ett lättköpt protokoll att generera anläggningsvis acetylerade proteiner i 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)

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

Dissekera multi signalering proteinkomplex av Bimolecular komplettering affinitet rening (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

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

Använda förbättrade grön fluorescens Protein-uttryckande Escherichia Coli att bedöma mus Peritoneal makrofag fagocytos

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Purification of a High Molecular Mass Protein in Streptococcus mutans

Rening av ett högt molekylärt Mass protein i Streptococcus mutans

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 ...