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).

La sangre humana se obtuvo de voluntarios sanos con consentimiento informado por escrito antes y aprobación de la Junta de Revisión Institucional de la Universidad Nacional Tsing Hua, en Hsinchu, Taiwán. 1. La expresión del recombinante HP-PAN en E. coli <ol><li> Preparar el plásmido pET42a-NAP que contiene la secuencia de ADN de HP-NAP de H. pylori cepa 26695 16 como se describió previamente. Preparar los plásmidos que contienen la secuencia de ADN de HP-NAP con las mutacio...

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La cromatografía por lotes modo negativo con resinas de intercambio aniónico DEAE que aquí se presenta es adecuado para la purificación de HP-NAP recombinante sobreexpresada en E coli. Los valores de pH de los tampones utilizados en las etapas de lisis celular y la purificación son muy críticos para asegurar la solubilidad de HP-NAP en E. Los lisados ​​de E. coli y la separación eficiente de HP-NAP recombinante a partir de las impurezas de la célula huésped, respectivamente. Las c...

Helicobacter pylori (H. pylori) es una causa importante de gastritis y úlcera péptica. Esta bacteria también ha sido clasificado como un carcinógeno en humanos por parte de la Agencia Internacional para la Investigación sobre el Cáncer, que forma parte de la Organización Mundial de la Salud, en el año 1994. Se ha estimado que la prevalencia de H. pylori es del 70% en los países en desarrollo y del 30-40% en los países industrializados 1. A pesar de que la tasa de infección de H. pylori está disminuyendo en los países industrializados, la tasa de infección de H. pylori en los países en desarrollo sigue siendo alta 2. El tratamiento estándar para erradicar H. pylori consiste en la administración de un inhibidor de la bomba de protones, IBP y dos antibióticos, claritromicina o metronidazol más amoxicilina 3. Sin embargo, el aumento de la resistencia a los antibióticos en H. pylori relacionados con la PI terapia de úlceras insta al desarrollo de nuevas estrategias para prevenir or curar la infección. Desarrollo de la vacunación preventiva y / o terapéutica contra H. pylori podría proporcionar un enfoque alternativo para el control de H. pylori. Helicobacter pylori proteína activadora de neutrófilos (NAP-HP), un importante factor de virulencia de H pylori, se identificó por primera vez en extractos de agua de H. pylori con la capacidad de activar los neutrófilos a adherirse a las células endoteliales y producir especies de oxígeno reactivas (ROS) 4. La infiltración de neutrófilos de la mucosa gástrica se encuentra en H. pylori pacientes infectados con gastritis activa pueden resultar en la inflamación y el daño tisular del estómago. Por lo tanto, HP-NAP puede jugar un papel patológico mediante la activación de los neutrófilos para inducir inflamación gástrica, lo que hace aún más úlcera o H. pylori -asociado enfermedades gástricas. Sin embargo, HP-PAN es un candidato potencial para aplicaciones clínicas 5,6. Debido a la pro inmunogénica e inmunomoduladorpiedades de HP-NAP, esta proteína se podrían utilizar para desarrollar vacunas, agentes terapéuticos, y herramientas de diagnóstico. Un ensayo clínico se ha realizado para el uso de HP-NAP recombinante como uno de los componentes de una vacuna de proteína contra H. pylori. Esta vacuna consiste en HP-NAP recombinante, asociado a la citotoxina gen A (CagA), y las proteínas vacuolizante citotoxina A (VacA) formulado con hidróxido de aluminio y además se ha demostrado que es segura e inmunogénica en humanos 7. Además, HP-PAN actúa como un potente inmunomodulador para desencadenar T auxiliares de tipo 1 (Th1) -polarized respuestas inmunes para la terapia del cáncer 8 y para regular hacia abajo las respuestas inmunitarias mediadas por Th2 provocadas por reacciones alérgicas e infecciones parasitarias 9,10. Como para el diagnóstico, recombinante ELISA basado en HP-PAN se ha aplicado a la detección de anticuerpos séricos contra HP-NAP en H. pylori pacientes infectados 11. Un estudio mostró que el nivel de anticuerpos HP-PAN-específica en sueros de H. pylori pacientes infectados con cáncer gástrico fue significativamente mayor que la de los pacientes con gastritis crónica 12. Otro estudio también demostró que los anticuerpos séricos contra HP-NAP se asocian con la presencia de no cardia adenocarcinoma gástrico 13. Por lo tanto, recombinante ELISA basado en HP-NAP se puede aplicar a la detección de anticuerpos séricos contra HP-NAP para el pronóstico del cáncer gástrico en H. pylori pacientes infectados. En su conjunto, el HP-PAN purificada podría ser utilizado más para la prevención, tratamiento y pronóstico de la H. pylori -asociado enfermedades, así como la terapia del cáncer. Entre los diversos métodos utilizados para la purificación de recombinante HP-NAP expresado en Escherichia coli (E. coli) en su forma nativa informado hasta ahora, se necesita una cromatografía de filtración en gel segunda etapa de purificación que implica obtener alta pureza HP-NAP 14-16 . Aquí, un método que utiliza cromatografía de modo por lotes negativa condietilaminoetilo (DEAE) resinas de intercambio iónico se describe para la purificación de HP-NAP sobreexpresa en E. coli con alto rendimiento y alta pureza. Esta técnica de purificación se basa en la unión de proteínas y / o impurezas que no sean HP-PAN a la resina de la célula huésped. A pH 8,0, casi no hay otras proteínas, excepto HP-NAP se recuperan de la fracción no unida. Este enfoque purificación utilizando cromatografía de intercambio iónico de DEAE en el modo negativo es simple y ahorrando al permitir la purificación de HP-PAN recombinante mediante cromatografía de un solo paso a través de la recogida de la fracción no unida tiempo. Además de HP-NAP, varias otras biomoléculas, tales como virus 17, la inmunoglobulina G (IgG) 18, la hemoglobina 19, la proteína fosfatasa 20, y el factor de virulencia flagelina 21, también se han informado de que se purificó por cromatografía de intercambio de iones en negativo modo. Se prefiere el modo negativo para la cromatografía de intercambio iónico, si las impurezas son el menorcomponentes presentes en la muestra sometida a purificarse 22. La aplicación de la cromatografía negativo en la purificación de biomoléculas naturales o recombinantes se ha revisado recientemente 23. El presente informe ofrece un paso a paso el protocolo para la expresión de HP-PAN recombinante en E. coli, lisis de las células, y la purificación de HP-NAP usando cromatografía por lotes modo negativo con resinas de intercambio iónico de DEAE. Si una proteína deseada para la purificación es adecuado para cromatografía de intercambio iónico en el modo negativo, el protocolo descrito también podría ser adaptado como punto de partida para el desarrollo de un proceso de purificación.

<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 activadora de neutrófilos (HP-PAN) es un importante factor de virulencia de Helicobacter pylori (H. pylori). Desempeña un papel crítico en H. pylori inducida por inflamación gástrica mediante la activación de varios leucocitos innatos incluyendo neutrófilos, monocitos y mastocitos. Las propiedades inmunogénicas e inmunomoduladoras de HP-NAP lo convierten en un candidato potencial de diagnóstico y de vacuna para H. pylori y un nuevo fármaco candidato para la terapia del cáncer. Con el fin de obtener cantidades sustanciales de purificado HP-NAP utilizado por sus aplicaciones clínicas, un método eficaz para purificar esta proteína con alto rendimiento y pureza necesita ser establecido. En este protocolo, hemos descrito un método para una sola etapa de purificación cromatográfica negativo de HP-NAP recombinante sobreexpresada en Escherichia coli (E. coli) mediante el uso de dietilaminoetilo resinas de intercambio iónico (DEAE) (por ejemplo., Sephadex) iel modo por lotes n. Recombinante HP-PAN constituye casi el 70% del total de proteínas en E. coli y está casi completamente recuperado en la fracción soluble tras la lisis celular a un pH de 9,0. Bajo la condición óptima a pH 8,0, la mayoría de HP-NAP se recupera en la fracción no unida, mientras que las proteínas endógenas de E. coli se eliminan de manera eficiente por la resina. Este método de purificación mediante cromatografía de lote modo negativo con resinas de intercambio iónico DEAE produce funcional HP-NAP de E. coli en su forma nativa con alto rendimiento y pureza. El HP-PAN purificada podría ser utilizado más para la prevención, tratamiento y pronóstico de la H. pylori -asociado enfermedades, así como la terapia del cáncer.

El diagrama esquemático del procedimiento experimental de la purificación negativa de recombinante HP-NAP expresa en E. coli mediante el uso de resinas de intercambio iónico de DEAE en modo por lotes se muestra en la Figura 1. Esta técnica de purificación se basa en la unión de proteínas y / o impurezas que no sean HP-PAN a la resina de la célula huésped. A pH 8,0, casi no hay otras proteínas, excepto HP-NAP en su forma nativa se recuperan de la fracci...

Watch this Scientific Journal Video about One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode at JoVE.co

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

Un método de alto rendimiento para la purificación negativa de un solo paso de Helicobacter pylori recombinante proteína activadora de neutrófilos (HP-PAN) sobreexpresado en Escherichia coli mediante el uso de resinas de dietilaminoetilo en modo batch se describe. HP-PAN purificado por este método es beneficioso para el desarrollo de vacunas, medicamentos o diagnósticos para H. pylori -asociado enfermedades.

De un solo paso negativo Purificación cromatográfica de<em&gt; Helicobacter pylori</em&gt; La proteína activadora de neutrófilos sobreexpresa en<em&gt; Escherichia coli</em&gt; En 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 no invasiva de neuropatogénico<em&gt; Escherichia coli</em&gt; La infección en Neonatología Rata

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

Investigación

Inmunología e Infección

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

Microscopía Electrónica de Alta Resolución de la<em&gt; Helicobacter pylori</em&gt; Cag secreción tipo IV Sistema Pili Producido en condiciones variables de Hierro disponibilidad

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

Organoides como Modelo para Enfermedades Infecciosas: Cultura de Derechos Humanos y organoides estomacales murinos y microinyección de 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

Detección de la colonización de Escherichia Coli enterohemorrágica en anfitrión murino no invasiva In Vivo el sistema de bioluminiscencia

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

Creación de un denso Transposon inserción biblioteca usando Conjugación bacteriana en cepas enterobacterianas tales como Escherichia Coli o 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

Sobreexpresión y Purificación de Humanos<em&gt; Cis</em&gt; -preniltransferasa en<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

Un protocolo fácil de generar proteínas Site-Specifically acetiladas en 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

Disección señalización complejos por purificación de la afinidad de complementación 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

Utilizando mayor expresión de proteína de fluorescencia verde Escherichia Coli evaluar fagocitosis de macrófagos peritoneales de ratón

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

Purificación de una proteína de masa molecular alta en 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 ...