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).

O sangue humano foi coletado de voluntários saudáveis ​​com o consentimento informado por escrito antes e aprovação do Conselho de Revisão Institucional da Universidade Nacional Tsing Hua, Hsinchu, Taiwan. 1. Expressão Recombinante de HP-PNA em E. coli <ol><li> Prepara-se o plasmídeo pET42a-PNA contendo a sequência de ADN de HP-NAP de H. pylori 26695 estirpe como descrito anteriormente 16. Prepare os plasmídeos contendo a sequência de ADN de HP-NAP com as mutações p...

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A cromatografia em lote modo negativo com resinas de permuta aniónica DEAE aqui apresentado é adequado para a purificação do recombinante HP-NAP sobre-expressos em E. coli. Os valores dos tampões utilizados nos passos de lise de células e purificação de pH são muito crítica para assegurar a solubilidade de HP-PNA em E. Os lisados ​​de E. coli e a separação eficiente de recombinante HP-NAP a partir de impurezas da célula hospedeira, respectivamente. As células bacterianas lisa...

Helicobacter pylori (H. pylori) é uma das principais causas de gastrite e úlcera péptica. Esta bactéria também foi classificado como cancerígeno em humanos pela Agência Internacional para Pesquisa sobre o Câncer, parte da Organização Mundial da Saúde, em 1994. Estima-se que a prevalência de H. pylori é de 70% nos países em desenvolvimento e 30-40% nos países industrializados 1. Mesmo que a taxa de infecção de H. pylori está diminuindo nos países industrializados, a taxa de infecção de H. pylori em países em desenvolvimento ainda é alto 2. O tratamento padrão para erradicar H. pylori consiste na administração de um inibidor da bomba de protões, IPP, e dois antibióticos, amoxicilina ou claritromicina mais metronidazole 3. No entanto, o aumento da resistência aos antibióticos em H. pylori relacionados com a terapia de úlcera insta o desenvolvimento de novas estratégias para prevenir oR curar a infecção. O desenvolvimento de vacina preventiva e / ou terapêutica contra o H. pylori pode proporcionar uma abordagem alternativa para controlar H. pylori. Helicobacter pylori proteína de activação de neutrófilos (HP-NAP), um importante fator de virulência do H. pylori, foi identificado pela primeira vez em extratos aquosos de H. pylori com a capacidade de activar os neutrófilos a aderir às células endoteliais e produzem espécies reactivas de oxigénio (ROS) 4. A infiltração de neutrófilos na mucosa gástrica encontrada em H. pylori pacientes infectados com gastrite ativa pode resultar em inflamação e danos nos tecidos do estômago. Assim, HP-PNA pode desempenhar um papel patológico através da activação de neutrófilos para induzir a inflamação gástrica, o que provoca ainda mais úlcera ou H. pylori -associated doenças gástricas. No entanto, HP-NAP é um candidato potencial para aplicações clínicas 5,6. Devido à pró imunogénica e imunomoduladorpriedades de HP-PNA, esta proteína pode ser utilizada para o desenvolvimento de vacinas, agentes terapêuticos, e ferramentas de diagnóstico. Um ensaio clínico foi conduzido para a utilização recombinante HP-NAP como um dos componentes de uma vacina contra a proteína H. pylori. Esta vacina consiste de recombinante HP-PNA, associado citotoxina-gene A (CagA), e proteínas citotoxina vacuolar A (VacA) formulado com hidróxido de alumínio e ainda tem sido demonstrado ser segura e imunogénica em humanos 7. Além disso, a HP-NAP actua como um potente imunomodulador para desencadear T auxiliar tipo 1 (Th1) -polarized respostas imunitárias para a terapia do cancro e 8 para regular negativamente as respostas imunitárias mediadas por Th2 provocadas por reacções alérgicas e infecções parasitárias 9,10. Como para o diagnóstico, com base ELISA-HP-NAP recombinante tem sido utilizada para a detecção de anticorpos séricos contra o HP-PNA em H. pylori pacientes infectados 11. Um estudo mostrou que o nível de anticorpos específicos de HP-NAP em soros de H. pyLori pacientes infectados com carcinoma gástrico foi significativamente mais elevada do que a partir de pacientes com gastrite crónica 12. Outro estudo também mostrou que os anticorpos séricos contra HP-PNA estão associados com a presença de não-cárdia adenocarcinoma gástrico 13. Assim, ELISA baseado em HP-NAP recombinante pode ser utilizada para a detecção de anticorpos séricos contra o HP-PNA para o prognóstico do cancro gástrico em H. pylori pacientes infectados. Tomados em conjunto, o HP-NAP purificada pode ser ainda utilizado para a prevenção, tratamento e prognóstico do H. pylori -associated doenças, bem como a terapia do cancro. Entre os vários métodos utilizados para a purificação do HP-NAP recombinante expressa em Escherichia coli (E. coli) na sua forma nativa relatados até agora, uma cromatograf ia de filtração em gel segundo passo de purificação envolvendo é necessária para se obter elevada pureza HP-NAP 14-16 . Aqui, um método utilizando a cromatografia modo descontínuo com negativodietilaminoetilo (DEAE) resinas de permuta iónica é descrito para a purificação do HP-PNA sobre-expresso em E. coli com elevado rendimento e elevada pureza. Esta técnica de purificação foi baseado na ligação de proteínas e / ou que não sejam HP-PNA para a resina de impurezas da célula hospedeira. A pH 8,0, quase sem outras proteínas, exceto HP-NAP são recuperados a partir da fracção livre. Esta abordagem purificação utilizando cromatografia de troca iônica DEAE no modo negativo é simples e economia de tempo, permitindo a purificação do recombinante HP-NAP através de cromatografia de uma etapa, através da recolha da fracção livre. Além de HP-PNA, várias outras biomoléculas, tais como vírus 17, Imunoglobulina G (IgG) 18, hemoglobina 19, fosfatase de proteína 20, e factor de virulência flagelina 21, também tem sido relatado para ser purificado por cromatografia de permuta iónica em negativo modo. O modo negativo é o preferido para a cromatografia de permuta iónica se as impurezas são o menorcomponentes presentes na amostra submetida a ser purificada 22. A aplicação de cromatografia negativa em purificação de biomoléculas naturais ou recombinantes foi recentemente revista 23. O presente relatório fornece um passo a passo de protocolo para a expressão de recombinante HP-NAP em E. coli, a lise das células, e a purificação do HP-PNA utilizando cromatografia lote modo negativo com resinas de permuta iónica em DEAE. Se uma proteína desejada para a purificação é adequada para cromatografia de permuta iónica em modo negativo, o protocolo descrito também pode ser adaptado como um ponto de partida para o desenvolvimento de um processo de purificação.

<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 proteína de activação de neutrófilos (HP-NAP) é um importante fator de virulência de Helicobacter pylori (H. pylori). Ela desempenha um papel crítico em H. pylori induzida por inflamação gástrica, activando vários leucócitos inatas, incluindo neutrófilos, monócitos, mastócitos e células. As propriedades imunogênicas e imunomoduladores da HP-NAP torná-lo um candidato potencial de diagnóstico e vacinas para H. pylori e uma nova droga candidatos para a terapia do cancro. A fim de obter quantidades substanciais de HP-NAP purificada utilizada para as suas aplicações clínicas, um método eficiente para purificar esta proteína com elevado rendimento e grau de pureza deve ser estabelecida. Neste protocolo, nós descrevemos um processo para um passo de purificação cromatográfica negativa recombinante de HP-PNA sobre-expresso em Escherichia coli (E. coli) usando dietilaminoetilo resinas de permuta iónica (DEAE) (por ex., Sephadex) iModo lote n. Recombinante HP-NAP constitui quase 70% do total de proteínas em E. coli e é quase totalmente recuperada na fracção solúvel após a lise celular, a pH 9,0. Sob a condição óptima a pH 8,0, a maioria dos HP-NAP é recuperada na fracção não ligada enquanto as proteínas endógenas da E. coli são eficientemente removida pela resina. Este método de purificação utilizando cromatograf ia em lotes modo negativo com resinas de permuta iónica em DEAE produz funcional HP-NAP a partir de E. coli, na sua forma nativa com elevado rendimento e pureza. A HP-NAP purificada pode ser ainda utilizado para a prevenção, tratamento e prognóstico do H. pylori -associated doenças, bem como a terapia do cancro.

O diagrama esquemático do processo experimental de purificação negativa recombinante de HP-NAP expressa em E. coli, usando resinas de permuta iónica em DEAE em modo de lote é mostrado na Figura 1. Esta técnica de purificação é baseado na ligação de proteínas e / ou que não sejam HP-PNA para a resina de impurezas da célula hospedeira. A pH 8,0, quase sem outras proteínas, exceto HP-NAP na sua forma nativa são recuperados a partir da fracção não...

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

Um método de alto rendimento para um passo de purificação negativa recombinante de Helicobacter pylori proteína activadora de neutrófilos (NAP-HP) sobre-expresso em Escherichia coli usando resinas dietilaminoetilo no modo de lote é descrito. HP-NAP purificada por este método é benéfico para o desenvolvimento de vacinas, medicamentos, ou diagnóstico de H. pylori -associated doenças.

Um passo de purificação cromatográfica de Negativo<em&gt; Helicobacter pylori</em&gt; Neutrófilos-proteína de activação sobre-expresso em<em&gt; Escherichia coli</em&gt; Em Modo 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

Modelo não-invasiva da neuropatogênicos<em&gt; Escherichia coli</em&gt; Infecção no Rato Neonatal

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

Imunologia e Infecção

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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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

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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

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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

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Criação de um denso Transposon inserção biblioteca usando bacteriana conjugação em entérobactérienne cepas tais como Escherichia Coli ou 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 ...

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Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

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

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

Um protocolo Facile para gerar proteínas Site-Specifically acetiladas em 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

Dissecando a proteínas multi sinalização complexos pela purificação da afinidade de complementação Bimolecular (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

Usando avançada proteína de fluorescência verde-expressando Escherichia Coli para avaliar a fagocitose de macrófagos peritoneais de Mouse

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

Purificação de uma proteína de massa molecular elevada em 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 ...