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).

İnsan kanı Ulusal Tsing Hua Üniversitesi Kurumsal Değerlendirme Kurulu, Hsinchu, Tayvan önceden yazılı aydınlatılmış onam ve onayı ile sağlıklı gönüllülerden alındı. E. Rekombinant HP NAP 1. İfade E. coli <ol><li> H. HP NAP-DNA dizisini içeren plazmid pET42a NAP-hazırlama pylori 26695 suşu olarak daha önce 16 nitelendirdi. tarif edildiği gibi arzu edilen nokta mutasyonu olan HP NAP DNA dizisini ihtiva eden plasmidler hazırlama (Protokol?...

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Burada sunulan DEAE anyon-değişim reçineleri ile negatif mod toplu kromatografisi E coli içinde aşırı eksprese rekombinant HP NAP saflaştırılması için uygundur. Hücre lizizi ve saflaştırma aşamasında kullanılan tamponların pH değerleri E. HP-NAP çözünürlüğünü sağlamak için çok önemlidir E. coli lizatları ve sırasıyla konak hücre yabancı maddelerden rekombinant HP NAP verimli ayrılması. Bakteri hücreleri, pH 9.0 lize olmalıdır ve negatif saflaştırma y...

Helikobakter pilori (H. pilori) gastrit ve peptik ülser bir ana sebebidir. Bu bakteri, aynı zamanda Kanser, Dünya Sağlık Örgütü adına Araştırmaları Uluslararası Ajansı tarafından insanlarda karsinojen olarak sınıflandırılmıştır, 1994 yılında tahmin edildiğini H. prevalansı pylori enfeksiyonu gelişmekte olan ülkelerde% 70 ve sanayileşmiş ülkelerde 1 30-40% &#39;dir. Olsa H. enfeksiyon oranı pylori, sanayileşmiş ülkelerde H. enfeksiyon oranını azaltmak olduğunu Gelişmekte olan ülkelerde pylori halen 2 yüksektir. Standart tedavi H. ortadan kaldırmak için pylori enfeksiyonu, bir proton pompası inhibitörü, PPI ve iki antibiyotik, klaritromisin artı amoksisilin veya metronidazol 3 uygulanmasından oluşur. H. antibiyotik direnci Ancak, yükselişi pylori ülser tedavisi o önlemek için yeni stratejilerin geliştirilmesini çağrısı lır enfeksiyonu tedavi. H. karşı önleyici ve / veya terapötik bir aşı geliştirilmesi H. pylori kontrol etmek için alternatif bir yaklaşım sağlayabilir pylori enfeksiyonunun. Helikobakter pilori nötrofil aktive edici protein (HP-NAP), H. pylori önemli bir virülans faktörü, birinci bir H. su ekstreleri tespit edilmiştir endotel hücrelerine ve reaktif oksijen türlerini (ROS) 4 üretmek için uygun nötrofillerin aktive etme kabiliyetine sahip pylori. H. bulunan mide mukozasının nötrofil infiltrasyonu pylori- aktif gastrit ile enfekte olan hastalarda mide enflamasyon ve doku hasarına yol açabilir. Bu nedenle, HP-NAP fazla ülser veya H. neden gastrik enflamasyon, indükleme nötrofillerin aktive ederek patolojik bir rol oynayabilir pylori mide hastalıkları -associated. Bununla birlikte, HP-NAP klinik uygulamalara 5,6 için potansiyel bir adaydır. Nedeniyle bağışıklık ve bağışıklık düzenleyici proHP UEP perties, bu proteinin aşılar, terapötik ajanlar ve diyagnostik araçlar geliştirmek için kullanılabilir. Bir klinik çalışma H. karşı bir protein aşı bileşenleri olarak yeniden birleştirici HP NAP kullanılarak gerçekleştirilmiştir pylori. Bu aşının, alüminyum hidroksit ile birlikte formüle rekombinant HP NAP, sitotoksin ilişkili gen A (CagA) ve vakuolasyon sitotoksin A (VacA) proteinlerden oluşur ve daha fazla güvenli ve insanlarda 7 imünojenik olduğu gösterilmiştir. Ayrıca HP NAP-kanser terapisi 8 immün yanıtları -polarized aşağı alerjik reaksiyonlar ve parazit enfeksiyonları 9,10 ile ortaya çıkarılan Th2 immün yanıtları düzenleyen (Th1) T yardımcı tip 1 tetiklemek için güçlü bir bağışıklık düzenleyici olarak işlev görür. Diagnostik olarak, yeniden birleştirici HP NAP-tabanlı ELISA H. HP-NAP karşı serum antikorları tespit etmek için uygulanmıştır pylori hasta 11 ile enfekte. Bir çalışma göstermiştir ki H. serumunda HP NAP-özgü antikorların seviyesi pylori mide kanserli hastaların kronik gastrit 12 olan hastalardan alınan göre anlamlı derecede yüksek olduğu ile enfekte. Bir başka çalışma, HP-NAP karşı serum antikorları olmayan kardiya gastrik adenokarsinoma 13 varlığı ile ilişkili olduğunu göstermiştir. Bu nedenle, yeniden birleştirici HP NAP-tabanlı ELISA H. mide kanseri prognoz için HP-NAP karşı serum antikorları tespit etmek için tatbik edilebilir pylori hastaları ile enfekte. Birlikte ele alındığında, arıtılmış, HP-NAP daha önleme, tedavi için kullanılan ve H. prognozu edilebilir pylori hastalıkları yanı sıra kanser tedavisi -associated. Rekombinant HP NAP saflaştırılması için kullanılan çeşitli yöntemler arasında doğal biçiminde Escherichia coli (E. Coli) &#39;de ifade edilen ikinci bir saflaştırma aşaması ile ilgili jel filtrasyon kromatografisi yüksek ölçüde saf, HP-NAP 14-16 elde etmek için gerekli olan şu ana kadar rapor . Burada, negatif mod toplu kromatografisi kullanılarak bir yöntemdietilaminoetil (DEAE) iyon-değiştirme reçineleri E. aşırı eksprese HP NAP saflaştırılması için açıklanan yüksek verim ve yüksek saflıkta coli. Bu saflaştırma tekniği konakçı hücre proteinleri ve / veya reçine HP-NAP dışındaki yabancı maddeler bağlanmasının dayanıyordu. pH 8.0, HP-NAP dışında neredeyse başka hiçbir proteinler bağlanmamış fraksiyonu geri kazanılır. negatif modda DEAE iyon değişimi kromatografisi kullanılarak bu saflaştırma yaklaşımı basit ve zaman bağlı olmayan fraksiyon toplama yoluyla tek adımlı kromatografisi ile yeniden birleştirici HP NAP saflaştırılmasını sağlayarak kazandıran. 17, immünoglobülin G (IgG) 18 hemoglobin 19, protein fosfataz 20 ve hastalık oluşturma faktörü flagellin 21 virüsleri gibi, aynı zamanda negatif iyon değişim kromatografisi ile saflaştırılabilir bildirilmiştir HP-NAP, birçok biyomoleküllerin ek olarak, modu. yabancı maddeler küçük ise negatif mod iyon değişimli kromatografi için tercih edilirtabi numunede mevcut olan bileşenlerin 22 arıtılacak. Doğal ya da yeniden birleştirici biyomoleküllerin saflaştırma negatif kromatografi uygulanması en son 23 gözden geçirilmiştir. Bu rapor E. rekombinant HP NAP ifadesi için adım protokolü tarafından bir adım sağlar E. coli, DEAE iyon değişim reçineleri ile negatif mod toplu kromatografisi kullanılarak HP-UEP hücrelerin parçalanması ve saflaştırılması. arıtma için arzu edilen bir protein negatif modda iyon değiştirme kromatografisi için uygun ise, açıklanan protokol, aynı zamanda bir saflaştırma işleminin geliştirilmesi için bir başlangıç ​​noktası olarak adapte edilebilir.

<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

Helikobakter pilori nötrofil aktive edici protein (HP-NAP) Helicobacter pylori (H. pylori) önemli bir virülans faktörü olduğu. Bu H. kritik bir rol oynar pylori nötrofiller, monositler ve mast hücreleri dahil olmak üzere birçok doğuştan lökositlerin aktive ederek mide iltihabı indüklenen. HP-NAP bağışıklık ve bağışıklık düzenleyici özellikleri onu H. için potansiyel bir tanı ve aşı adayı yapmak pylori ve kanser tedavisi için yeni bir ilaç adayı. klinik uygulamalarda kullanılan arıtılmış, HP-NAP önemli miktarlarda elde etmek için, etkili bir yöntem, yüksek verim ve saflıkta bu proteinin oluşturulmasına ihtiyaç saflaştırmak için. Bu protokol, dietilaminoetil (DEAE), iyon değiştirme reçineleri kullanılarak, Escherichia coli (E. Coli) &#39;de rekombinant aşırı eksprese HP UEP tek adımlı negatif kromatografik arıtma için bir yöntem tanımlanmıştır (örn., Sephadex i)n toplu modunda. Rekombinant HP-NAP E. toplam proteinin yaklaşık% 70 oluşturmaktadır E. coli ve hemen hemen tam olarak pH 9.0 hücre lizizi sonrasında çözünebilir fraksiyondan elde edilir. PH 8.0&#39;da iyi şartlar altında, HP-NAP çoğunluğu bağlanmamış fraksiyon geri kazanılır ise E. endojen proteinler E. coli etkin reçinesi ile giderilirler. DEAE iyon değişim reçineleri ile negatif mod toplu kromatografisi kullanılarak bu saflaştırma yöntemi E fonksiyonel HP NAP verir yüksek verim ve saflıkta doğal biçiminde coli. Saflaştınldı HP NAP daha önleme, tedavi için kullanılan ve H. prognozu edilebilir pylori hastalıkları yanı sıra kanser tedavisi -associated.

Rekombinant HP-NAP negatif arınma deneysel işlemin şematik E. ifade Toplu modunda DEAE iyon değişim reçineleri kullanılarak E. coli, Şekil 1 &#39;de gösterilmiştir. Bu saflaştırma tekniği konakçı hücre proteinleri ve / veya reçine HP-NAP dışındaki yabancı maddeler bağlanmasının dayanmaktadır. PH 8.0 &#39;de, kendi doğal formunda HP NAP dışında hemen hemen hiçbir proteinler, bağlanmamış fraksiyon (Şekil ...

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

Rekombinant Helicobacter tek-aşamalı negatif saflaştırılması için yüksek verimli bir yöntem tarif edilmektedir küme modunda dietilaminoetil reçineleri kullanılarak Escherichia coli içinde aşırı eksprese nötrofil aktive edici protein (HP-NAP) pylori. Bu yöntem ile arıtılmış, HP-NAP H. aşı, ilaç veya teşhis maddelerinin geliştirilmesi için faydalıdır pylori hastalıkları -associated.

Tek adımlı olumsuz Kromatografik arıtma<em&gt; Helicobacter pylori</em&gt; Nötrofil-aktive protein aşın<em&gt; Escherichia coli</em&gt; Toplu Modunda

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

Neuropathogenic olmayan İnvaziv Modeli<em&gt; Escherichia coli</em&gt; Yenidoğan Sıçan Enfeksiyon

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

İmmünoloji ve Enfeksiyon

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

Yüksek çözünürlüklü elektron mikroskobu<em&gt; Helikobakter pilori</emDemir Kullanılabilirlik Değişen Koşullarda Üretilen&gt; Çağ Tip IV Salgı Sistemi Pili

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

Enfeksiyon Hastalıkları Modeli Olarak Organoids: Helicobacter Pylori ve İnsan Kültür ve kemirgen Mide Organoids ve Mikroenjeksiyon

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

Non-invaziv Vivo içinde Bioluminescence sistemi tarafından fare konak Enterohemorrhagic Escherichia Coli kolonizasyon algılama

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

Bir yoğun Transposon ekleme kütüphane kullanarak bakteriyel konjugasyon Escherichia Coli Enterobacterial suşları gibi veya Shigella flexneri oluşturulması

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

Aşırı Ezme ve İnsanın Saflaştırılması<em&gt; Cis</em&gt; -prenıltransferaz in<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

Escherichia Coli proteinler Site-Specifically oluşturmak için Facile iletişim kuralı Acetylated

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

Bimolecular tamamlayıcı benzeşme arıtma (BiCAP) tarafından parçalanmış çok protein sinyal kompleksleri

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

Yeşil floresans Protein-ifade gelişmiş kullanma fare Peritoneal makrofaj fagositoz değerlendirmek için Escherichia Coli

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

Streptococcus mutans'larda Yüksek Moleküler Kütle Proteininin Saflaştırılması

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 ...