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).

Menneskeblod blev indsamlet fra raske frivillige med forudgående skriftlig informeret samtykke og godkendelse fra Institutional Review Board for National Tsing Hua University, Hsinchu, Taiwan. 1. Ekspression af rekombinant HP-NAP i E. coli <ol><li> Forbered plasmid pET42a-NAP indeholdende DNA-sekvensen af HP-NAP fra H. pylori 26.695 stamme som tidligere beskrevet 16. Forbered plasmider indeholdende DNA-sekvensen af ​​HP-NAP med de ønskede punktmutationer som beskrevet (se pro...

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Den negative tilstand batch-kromatografi med DEAE anionbytterharpikser præsenteret her er egnet til oprensning af rekombinant HP-NAP overudtrykkes i E. coli. PH-værdierne af de anvendte buffere i trinnene cellelyse og oprensning er meget afgørende for at sikre opløseligheden af HP-NAP i E. coli lysater og effektiv separering af rekombinant HP-NAP fra værtscelleurenheder hhv. Bakterieceller bør lyseres ved pH 9,0, og den negative oprensning bør udføres ved pH 8,0 til opnåelse af HP-NAP i højt ...

Helicobacter pylori (H. pylori) er en væsentlig årsag til gastritis og mavesår. Denne bakterie er også blevet klassificeret som kræftfremkaldende i mennesker ved Det Internationale Agentur for Kræftforskning, en del af World Health Organization, i 1994. Det er blevet anslået, at forekomsten af H. pylori-infektion er 70% i udviklingslandene og 30-40% i de industrialiserede lande 1. Selvom infektion på H. pylori er faldende i de industrialiserede lande, infektion på H. pylori i udviklingslandene er stadig høj 2. Standarden behandling for at udrydde H. pylori infektion består af administrationen af en protonpumpehæmmer, PPI, og to antibiotika, clarithromycin plus amoxicillin eller metronidazol 3. Men fremkomsten af antibiotikaresistens i H. pylori-relaterede sår terapi tilskynder udviklingen af nye strategier til forebyggelse or helbrede infektionen. Udvikling af forebyggende og / eller terapeutisk vaccination mod H. pylori kunne give en alternativ tilgang til at styre H. pylori-infektion. Helicobacter pylori neutrofil-aktiverende protein (HP-NAP), en væsentlig virulensfaktor af H pylori, blev først identificeret i vand ekstrakter af H. pylori med evnen til at aktivere neutrofiler til at adhærere til endotelceller og producere reaktive oxygenarter (ROS) 4. Neutrofil infiltration af maveslimhinden fundet i H. pylori-inficerede patienter med aktiv gastritis kan resultere i inflammation og vævsskade af maven. Således kan HP-NAP spille en patologisk rolle ved at aktivere neutrofiler at inducere gastrisk inflammation, hvilket yderligere bevirker ulcus eller H. pylori-associeret gastrisk sygdomme. Ikke desto mindre, HP-NAP er en potentiel kandidat til kliniske anvendelser 5,6. På grund af den immunogene og immunmodulerende proejendommene af HP-NAP, kan dette protein anvendes til at udvikle vacciner, terapeutiske midler og diagnostiske værktøjer. Et klinisk forsøg er gennemført for anvendelse af rekombinant HP-NAP som en af komponenterne i et protein vaccine mod H. pylori. Denne vaccine består af rekombinant HP-NAP, cytotoksin-associeret gen A (CagA), og vakuolerende cytotoksin A (VacA) proteiner formuleret med aluminiumhydroxid og er yderligere blevet demonstreret at være sikkert og immunogent i mennesker 7. Også, HP-NAP virker som en potent immunmodulator til at udløse T-hjælper type 1 (Th1) -polarized immunresponser til cancerterapi 8 og at nedregulere Th2-medierede immunresponser fremkaldt af allergiske reaktioner og parasitære infektioner 9,10. Som for diagnostik, har rekombinant HP-NAP-baserede ELISA blevet anvendt til påvisning af serumantistoffer mod HP-NAP i H. pylori inficerede patienter 11. En undersøgelse viste, at niveauet af HP-NAP-specifikke antistoffer i sera fra H. pylori inficerede patienter med mavekræft var signifikant højere end fra patienter med kronisk gastritis 12. En anden undersøgelse viste også, at serum antistoffer mod HP-NAP er forbundet med tilstedeværelsen af ikke-mavemunden gastrisk adenocarcinom 13. Således kan rekombinant HP-NAP-baserede ELISA anvendes til at måle serum antistoffer mod HP-NAP til prognose af gastrisk cancer hos H. pylori-inficerede patienter. Tilsammen kan det oprensede HP-NAP yderligere anvendes til forebyggelse, behandling og prognose af H. pylori-associeret sygdomme og cancer terapi. Blandt de mange metoder, der anvendes til oprensning af rekombinant HP-NAP udtrykt i Escherichia coli (E. coli) i sin native form rapporteret hidtil, et andet oprensningstrin involverer gelfiltreringskromatografi er nødvendig for at opnå særdeles rene HP-NAP 14-16 . Her, en fremgangsmåde under anvendelse negativ modus batch kromatografi meddiethylaminoethyl (DEAE) ionbytterharpikser er beskrevet for oprensning af HP-NAP overudtrykt i E. coli med højt udbytte og høj renhed. Denne rensning teknik var baseret på bindingen af ​​værtscelleproteiner og / eller andre end HP-NAP til harpiksen urenheder. Ved pH 8,0, er næsten ingen andre proteiner undtagen HP-NAP genvundet fra den ubundne fraktion. Denne rensning tilgang med DEAE ionbytningskromatografi som negativ er enkel og tidsbesparende ved at tillade oprensning af rekombinant HP-NAP via et-trins kromatografi gennem indsamling af den ubundne fraktion. Ud over HP-NAP, flere andre biomolekyler, såsom vira 17, immunoglobulin G (IgG) 18, hæmoglobin 19, proteinphosphatase 20, og virulensfaktor flagellin 21, også er blevet rapporteret at renses ved ionbytningskromatografi i negativ mode. Den negative modus foretrækkes til ionbytningskromatografi hvis urenheder er den mindreårigekomponenter til stede i prøven underkastes oprenses 22. Anvendelsen af negative chromatografi i rensning af naturlige eller rekombinante biomolekyler er for nylig blevet revideret 23. Denne rapport giver en trinvis protokol for ekspression af rekombinant HP-NAP i E. coli, lysis af cellerne, og oprensning af HP-luven negativ modus batch-kromatografi med DEAE ionbytterharpikser. Hvis et ønsket til oprensning protein er egnet til ionbytningskromatografi i negativ modus, kan den beskrevne protokol også tilpasses som et udgangspunkt for udvikling af en rensningsproces.

<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-aktiverende protein (HP-NAP) er en væsentlig virulensfaktor af Helicobacter pylori (H. pylori). Det spiller en kritisk rolle i H. pylori-induceret gastrisk inflammation ved at aktivere flere medfødte leukocytter, herunder neutrofiler, monocytter og mastceller. De immunogene og immunmodulerende egenskaber af HP-NAP gør det til en potentiel diagnostisk og vaccine kandidat til H. pylori og en ny lægemiddelkandidat til kræftbehandling. For at opnå betydelige mængder oprenset HP-NAP anvendes til dets kliniske anvendelser, til en effektiv fremgangsmåde oprense dette protein med højt udbytte og renhed skal etableres. I denne protokol, har vi beskrevet en fremgangsmåde til ettrins negativ kromatografisk oprensning af rekombinant HP-NAP overudtrykkes i Escherichia coli (E. coli) ved anvendelse diethylaminoethyl (DEAE) ionbytterharpikser (f.eks. Sephadex) in batch-mode. Rekombinant HP-NAP udgør næsten 70% af det totale protein i E. coli og er næsten fuldt tilbagebetalt i den opløselige fraktion efter cellelysis ved pH 9,0. Under den optimale tilstand ved pH 8,0, er størstedelen af HP-NAP udvundet i den ubundne fraktion, mens de endogene proteiner fra E. coli effektivt fjernet ved harpiksen. Denne rensning metode bruger negativ tilstand batch kromatografi med DEAE ionbytterharpikser giver funktionel HP-NAP fra E. coli i sin native form med højt udbytte og renhed. Det oprensede HP-NAP kunne yderligere anvendes til forebyggelse, behandling og prognose af H. pylori-associeret sygdomme og cancer terapi.

Det skematiske diagram af den eksperimentelle procedure for negativ oprensning af rekombinant HP-NAP udtrykt i E. coli ved anvendelse af DEAE ionbytterharpikser i batch-tilstand er vist i figur 1. Denne rensning teknik er baseret på bindingen af værtscelleproteiner og / eller andre end HP-NAP til harpiksen urenheder. Ved pH 8,0, er næsten ingen andre proteiner undtagen HP-NAP i sin naturlige form genvundet fra den ubundne fraktion (figur 2A o...

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

Et højt udbytte fremgangsmåde til ettrins negativ oprensning af rekombinant Helicobacter pylori neutrofil-aktiverende protein (HP-NAP) overudtrykkes i Escherichia coli ved hjælp af diethylaminoethyl harpikser i batchvis, er beskrevet. HP-NAP renset ved denne metode er til gavn for udviklingen af vacciner, lægemidler, eller diagnostik for H. pylori-associeret sygdomme.

Et-trins Negativ kromatografisk oprensning af<em&gt; Helicobacter pylori</em&gt; Neutrofilaktiverende protein overudtrykt i<em&gt; Escherichia coli</em&gt; I 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. ...

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-invasive Model of 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

High Resolution Electron Microscopy af<em&gt; Helicobacter pylori</em&gt; Cag Type IV sekretionssystem Pili Produceret i varierende forhold Iron Tilgængelighed

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 model for infektionssygdomme: Kultur i humane og murine mave Organoids og Mikroinjektion af 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

Påvisning af Enterohemorrhagic Escherichia Coli kolonisering i Murine vært ved Non-invasiv In Vivo bioluminescens 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

Oprettelsen af en tætte Transposon indsættelse bibliotek ved hjælp af bakteriel konjugation i Enterobacterial stammer 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

Overekspression og oprensning af mennesket<em&gt; Cis</em&gt; -prenyltransferase 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

En letkøbt protokol til at generere Site-Specifically acetyleret 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

Dissekere multi protein Signaling komplekser af Bimolecular komplementering affinitet rensning (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

Ved hjælp af forbedret grønne fluorescens Protein-udtryk for Escherichia Coli at vurdere musen Peritoneal makrofag fagocytose

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

Oprensning af et Højmolekylært masse 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 ...