Name: automated hydrophobic interaction chromatography column selection for use in protein purification
Uploaded: 2011-09-21T13:00:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

Ce travail a été financé par les Instituts nationaux de la santé et de subvention GM086822 National Science Foundation majeure des instruments de recherche de subvention DBI-0960313. Les auteurs tiennent à remercier les Drs. Jon Miyake &amp; Donna Hardy (Bio-Rad) et Jennifer Loertscher (Seattle University) pour leur expertise technique. Sélectionnez réactifs de chromatographie et de l&#39;instrumentation supplémentaire a été généreusement fourni par la société Bio-Rad.

<ol>
	<li>Cummins, P. M., O'Connor, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography.">Hydrophobic interaction chromatography.</a> Methods Mol. Biol. 681, 431-437 (2011).</li><li>Nagrath, D., Xia, F., Cramer, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+modeling+of+nonlinear+hydrophobic+interaction+chromatographic+systems.">Characterization and modeling of nonlinear hydrophobic interaction chromatographic systems.</a> J. Chromatogr. A. 1218, 1219-1226 (2011).</li><li>To, B. C., Lenhoff, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography+of+proteins.+IV.+Protein+adsorption+capacity+and+transport+in+preparative+mode.">Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode.</a> J. Chromatogr. A. 1218, 427-440 (2011).</li><li>Huddleston, J. G., Wang, R., Lyddiatt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+use+of+mild+hydrophobic+interaction+chromatography+for+"method+scouting"+protein+purification+strategies+in+aqueous+two-phase+systems:+a+study+using+model+proteins.">On the use of mild hydrophobic interaction chromatography for "method scouting" protein purification strategies in aqueous two-phase systems: a study using model proteins.</a> Biotechnol Bioeng. 44, 626-635 (1994).</li><li>Arun, K. H., Kaul, C. L., Ramarao, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+proteins+in+receptor+research:+an+emerging+tool+for+drug+discovery.">Green fluorescent proteins in receptor research: an emerging tool for drug discovery.</a> J. Pharmacol. Toxicol. Methods. 51, 1-23 (2005).</li><li>Terrizzi, S. C., Banh, C., Brossay, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L.A+Protocol+for+the+Production+of+KLRG1+Tetramer.">L.A Protocol for the Production of KLRG1 Tetramer.</a> J. Vis. Exp. (35), e1701-e1701 (2010).</li><li>Murphy, P. J. M., Morishima, Y., Kovacs, J. J., Yao, T. P., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+dynamics+of+hsp90+action+on+the+glucocorticoid+receptor+by+acetylation/deacetylation+of+the+chaperone.">Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone.</a> J. Biol. Chem. 280, 33792-33799 (2005).</li><li>Zeytuni, N., Zarivach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+M.+magneticum+strain+AMB-1+Magnetosome+Associated+Protein+MamA&#916;41.">Purification of the M. magneticum strain AMB-1 Magnetosome Associated Protein MamA&#916;41.</a> J. Vis. Exp. (37), 10-3791 (2010).</li><li>Kong, Y., Li, X., Bai, G., Ma, G., Su, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automatic+system+for+multidimensional+integrated+protein+chromatography.">An automatic system for multidimensional integrated protein chromatography.</a> J. Chromatogr. A. 1217, 6898-6904 (2010).</li><li>Camper, D. V., Viola, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+protein+purification.">Fully automated protein purification.</a> Anal. Biochem. 393, 176-181 (2009).</li><li>Hammon, J., Palanivelu, D. V., Chen, J., Patel, C., Minor, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+green+fluorescent+protein+screen+for+identification+of+well-expressed+membrane+proteins+from+a+cohort+of+extremophilic+organisms.">A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms.</a> Protein Sci. 18, 121-133 (2009).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+green+and+red+fluorescent+proteins+to+teach+protein+expression,+purification,+and+crystallization.">Using green and red fluorescent proteins to teach protein expression, purification, and crystallization.</a> Biochem. Mol. Biol. Educ. 36, 43-54 (2008).</li><li>Queiroz, J. A., Tomaz, C. T., Cabral, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography+of+proteins.">Hydrophobic interaction chromatography of proteins.</a> J. Biotechnol. 87, 143-159 (2001).</li><li>McCue, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+and+use+of+hydrophobic+interaction+chromatography+in+protein+purification+applications.">Theory and use of hydrophobic interaction chromatography in protein purification applications.</a> Methods Enzymol. 463, 405-414 (2009).</li></ol>

Sélectionnez réactifs de chromatographie et de l&#39;instrumentation supplémentaire ont été offerts par Bio-Rad.

Les techniques de chromatographie liquide se sont avérés inestimables pour la préparation de protéines hautement purifiées nécessaires à la conduite 6 immunologiques, biochimiques 7, 8 et structurelles des études. Méthodes de purification HIC nécessitent le plus souvent la détermination empirique d&#39;un milieu privilégié, et la structure du ligand, la densité de ligand, et de la matrice des propriétés de perles ont toutes été montré à influer sur les résultats chromatographiques 2, 3. Colonne automatisé le scoutisme est une approche efficace pour la sélection d&#39;un support pour l&#39;optimisation ultérieure HIC et 4 de purification des protéines. La méthode de la colonne automatisé dépistage présenté peut être facilement adaptée à différents HIC stratégies de purification des protéines. Les modifications de la concentration en sel, le choix du sel, gradient de sel, et le pH peut en outre améliorer les conditions de purification, et les effets de la variation de ces paramètres essentiels ont déjà été examinés 13,14. Éluat contenant une HIC prote cible partiellement purifiéen peut encore être purifié en utilisant une technique de chromatographie en phase complémentaire, tels que chromatographie échangeuse d&#39;ions (IEX) ou filtration sur gel / chromatographie d&#39;exclusion de taille 9,10. En raison de son absorption de la lumière unique et des caractéristiques d&#39;émission, le profil d&#39;élution de la GFP peut être identifié à l&#39;aide en ligne et de post-terme des approches. À cette fin, ce protocole peut en outre être adaptée pour la purification de recombinants GFP-protéines de fusion, dans laquelle une protéine cible est sans rapport avec &quot;tag&quot; avec la GFP. Protéines cibles GFP-étiquette peut être détectée avec une lumière UV et 11 purifiée en utilisant diverses méthodes chromatographiques, y compris la stratégie de purification HIC décrit ci-dessus. Purification GFP est également devenu un aliment de base dans les laboratoires de biochimie pédagogique pour l&#39;enseignement des techniques modernes de la science des protéines 12. Alors que de base de purification des protéines HIC peut être effectuée en utilisant une chromatographie sensiblement moins robuste sytige, l&#39;instrumentation présentée ici possède un certain nombre d&#39;avantages distincts pour aider à l&#39;obtention de résultats favorables. Avantages notables de l&#39;utilisation d&#39;un système hautement automatisé comprennent l&#39;amélioration de fonctionner à fonctionner condition de reproductibilité, de gain de temps, et une diminution des possibilités de l&#39;air d&#39;être introduites dans le système 10. Buffer blending système contrôlé, qui est facilitée par le système Maximizer, permet une plus grande cohérence dans la préparation du tampon et de la reproductibilité expérimentale. Dessin de charge d&#39;échantillon suffisante dans la boucle d&#39;échantillonnage dynamique pour tous dépistage exécute en outre assure l&#39;uniformité de l&#39;échantillon étant ajouté à chaque colonne et permet pistes séquentielles sans interruption ou manuel de rechargement. Injection de l&#39;échantillon contrôlé à partir de la boucle d&#39;échantillonnage sur la colonne diminue la variabilité qui peut se produire avec un chargement manuelle de l&#39;échantillon. Une paire de soupapes de sélection de colonne pour permettre pistes consécutives, chacune échantillon en utilisant une colonne différente HIC, sans avoir à replumb le système. Exécution mul<html> ti-longueur d&#39;onde d&#39;analyse est particulièrement bénéfique lors du dosage d&#39;une protéine avec un profil unique spectrophotométrique. En plus de la GFP, les cytochromes, flavoprotéines et autres hème contenant des protéines peuvent bénéficier de cette technique. Dispositifs de surveillance de la conductivité et le pH permettent de vérifier en temps réel des conditions expérimentales. Collecte de l&#39;éluat de Split fraction permet une manipulation fraction améliorée et permet le transfert facile pour les méthodes d&#39;analyse post-terme qui nécessitent un volume d&#39;échantillon minime (par exemple ELISA, SDS-PAGE, western blot, et Experion microfluidique électrophorèse). Pendant le fonctionnement de la seconde fraction déconnecté collecteur nécessite une synchronisation manuelle, il permet une flexibilité maximale dans le choix collecteur de fractions. Les inconvénients les plus importants à l&#39;utilisation d&#39;un tel système de chromatographie robuste pour la colonne HIC le scoutisme et la purification des protéines incluent le temps initial et les dépenses budgétaires liées à l&#39;acquisition instrument et la formation des opérateurs. t &quot;&gt; Le protocole présenté ici utilise un Bio-Rad DuoFlow chromatographie système;. Cependant, l&#39;instrumentation aussi robuste d&#39;autres fabricants, tels que l&#39;Avant ÄKTA de GE Healthcare, peuvent également être utilisés et sont capables de produire des résultats équivalents système de chromatographie Même comparables ont des caractéristiques uniques de programmation (par exemple, la méthode, les limites nomenclature des composants, préférences de l&#39;opérateur, et l&#39;évolutivité) qui devraient être considérés avant d&#39;entamer une procédure de purification ou de l&#39;acquisition instrument.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">DuoFlow BioLogic Pathfinder 20 Système</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">7602257</a> </td><td> Comprend un système de Maximizer, mélangeur, poste de travail F10, AVR7-3 de soupape, UV Quadtec / détecteur de Vis avec cellule d&#39;écoulement, collecteur de fractions BIOFRAC, logiciels BioLogic, et kit de démarrage </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">AVR9-8-flux Sélectionnez la vanne</a><html></td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7600408</a> </td><td> Haut-pression à la valve, 9-port, en position 8, 3500 psi (233 bars) limite, pour une utilisation avec un système DuoFlow BioLogic </td></tr><tr><td> Vanne de dérivation SV3T-2 </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> 7600410 </td><td> Electrovanne, 3-port, en position 2, 30 psi (2 bars) limite, pour une utilisation avec un système DuoFlow BioLogic </td></tr><tr><td> Volet clapet de répartition </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td></td><td></td></tr><tr><td>tique = LSR &amp; parentCategoryGUID = 22c495f1-36ff-44a3-9140-ee7e54d0b1ba &quot;target =&quot; _blank &quot;&gt; DynaLoop 25 Kit </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0451&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7500451</a> </td><td> Glisse-piston kit boucle d&#39;échantillonnage, comprend 25 ml DynaLoop coulissant boucle piston, kit de pièces DynaLoop (# 750-0450) </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">Collecteur de fraction BIOFRAC</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">7410002</a> </td><td> Deux col fraction<html> lecteurs utilisés </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">BIOFRAC microplaque adaptateur de tête goutte</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">7410088</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=741-0017" target="_blank">Adaptateur BIOFRAC plaque de microtitration</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td>&amp; Country = Etats-Unis &amp; lang = fr &amp; ProductID = 741-0017 &quot;target =&quot; _blank &quot;&gt; 7410017 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">Optique UV Module</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">7500202</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-1331&vertical=LSR&parentCategoryGUID=c4095dc1-9260-4431-b066-823b050048c8" target="_blank">Lampe halogène</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> &lt;<html> un target = &quot;_blank&quot;&gt; 7601331 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">Econo dégradé la pompe</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">7319001</a> </td><td> une pompe à gradient pour la purification de protéines à basse pression, et un kit de tube comprend raccords </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224301" target="_blank">Experion système</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td>bio-rad.com/prd/en/US/adirect/biorad? cmd = BRCatgProductDetail &amp; ProductID = 224301 &quot;target =&quot; _blank &quot;&gt; 7007001 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">Kit Experion Analyse Pro260</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">7007102</a> </td><td></td></tr><tr><td> HiTrap kit HIC sélection de la colonne </td><td> GE Healthcare </td><td> 28-4110-07 </td><td> Comprend 7 x 1 ml pré-emballés colonnes des médias HIC pour le scoutisme </td></tr><tr><td> Raccords de GE Healthcare colonne à Bio-Rad </td><td> GE Healthcare </td><td> 18111251 18111257 et </td><td></td></tr></tbody></table>

automated hydrophobic interaction chromatography column selection for use in protein purification

Contrairement à d&#39;autres méthodes chromatographiques pour les protéines purification (filtration sur gel, par exemple, l&#39;affinité et l&#39;échange d&#39;ions), chromatographie d&#39;interaction hydrophobe (HIC) nécessite souvent la détermination expérimentale (dénommé dépistage ou &quot;scouting&quot;) afin de sélectionner le support le plus approprié par chromatographie en phase pour purifier une protéine donnée 1. La méthode présentée ici décrit une approche automatisée pour le scoutisme pour un média optimales HIC à être utilisés dans la purification des protéines. HIC sépare les protéines et autres biomolécules à partir d&#39;un lysat brut basée sur les différences de caractère hydrophobe. Semblable à la chromatographie d&#39;affinité (AC) et chromatographie échangeuse d&#39;ions (IEX), CIH est capable de concentrer la protéine d&#39;intérêt à mesure qu&#39;elle progresse dans le processus chromatographique. Protéines qui conviennent le mieux pour la purification par HIC comprennent ceux qui sont avec les régions de surface hydrophobes et capables de résister à l&#39;exposition au sel concentrations supérieures à 2 M munitionssulfate de titane ((NH 4) 2 SO 4). HIC est souvent choisi comme un procédé de purification des protéines qui n&#39;ont pas un marqueur d&#39;affinité, et donc impropres à AC, et quand IEX ne parvient pas à fournir de purification adéquate. Fractions hydrophobes sur la surface de la protéine temporairement se lier à un ligand non polaire couplé à une inerte, matrice immobile. L&#39;interaction entre la protéine et le ligand sont fortement tributaires de la concentration en sel de la mémoire tampon qui coule à travers la colonne de chromatographie, avec de fortes concentrations ioniques renforcement de l&#39;interaction protéine-ligand et de faire la protéine immobile (c.-à-lié à l&#39;intérieur de la colonne) 2. Sous forme de sel concentrations diminution, l&#39;interaction protéine-ligand se dissipe, la protéine est de nouveau mobile et est éluée de la colonne. Plusieurs médias HIC sont disponibles dans le commerce pré-emballés colonnes, chacune contenant plusieurs ligands hydrophobes (par exemple S-butyle, butyle, octyle, et phényl) réticulé à différentes densités de billes d&#39;agarose d&#39;un specdiamètre ific 3. Colonne automatisé dépistage permet une approche efficace pour déterminer quels médias HIC devrait être utilisée pour de futures expériences d&#39;optimisation, les plus exhaustives et purification des protéines fonctionne 4. La protéine spécifique se purifier ici est une protéine recombinante fluorescente verte (GFP), mais l&#39;approche peut être adaptée pour la purification d&#39;autres protéines avec une ou plusieurs régions de surface hydrophobes. GFP sert une protéine modèle utile, en raison de sa stabilité, unique pic d&#39;absorbance de lumière à 397 nm et la fluorescence lorsqu&#39;ils sont exposés à la lumière UV 5. Lysat bactérien contenant la GFP de type sauvage a été préparé dans un tampon riche en sel, chargé dans un Bio-Rad moyennes DuoFlow système de pression chromatographie en phase liquide, et adsorbés sur des colonnes HiTrap HIC contenant différents médias HIC. La protéine a été élue des colonnes et analysées par en ligne et les méthodes de détection post-terme. Mélange tampon, dynamique boucle d&#39;injection de l&#39;échantillon, col séquentiellelonne de sélection, multi-longueur d&#39;onde d&#39;analyse et de collecte de l&#39;éluat scission fraction augmenté la fonctionnalité du système et la reproductibilité de la démarche expérimentale.

Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Procédé automatisé pour identifier appropriés chromatographie d&#39;interaction hydrophobe (HIC) médias à être utilisés dans le procédé de purification de protéine est présenté. La méthode utilise un système de moyenne pression chromatographie en phase liquide, y compris buffer blending automatisé, dynamique boucle d&#39;injection de l&#39;échantillon, sélection de la colonne séquentielle, multi-longueur d&#39;onde d&#39;analyse et de collecte de l&#39;éluat scission fraction.

Automatisé d&#39;interaction hydrophobe Chromatographie sur colonne de sélection pour l&#39;utilisation dans la purification des protéines

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

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43.5K vues.  Seattle University. Procédé automatisé pour identifier appropriés chromatographie d'interaction hydrophobe (HIC) médias à être utilisés dans le procédé de purification de protéine est présenté. La méthode utilise un système de moyenne pression chromatographie en phase liquide, y compris buffer blending automatisé, dynamique boucle d'injection de l'échantillon, sélection de la colonne séquentielle, multi-longueur d'onde d'analyse et de collecte de l'...

Vidéo: Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Expression, Purification, Cristallisation et Essais enzymatiques des protéines contenant du domaine de l'hydrolase fumarylacetoacetate

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

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Biochimie

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

Un système de purification de protéines à haut débit économique et polyvalent à l’aide d’un adaptateur de plaque multicolonne

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
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&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
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Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
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To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
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Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

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In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
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<div class="chapter_text" id="script_5">
Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
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You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Size Exclusion and Affinity Chromatography

Méthodes de purification de biomolécules basées sur la chromatographie

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by ...

Enseignement

JoVE Science Education

Chimie

Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

Source: Pokhrel, A. et al. A <a href="https://www.jove.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018)
In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

Purification de protéines basée sur la chromatographie d’affinité du nickel : une technique pour purifier des protéines recombinantes marquées à la polyhistidine à partir d’un lysat de cellules bactériennes

Source: Pokhrel, A. et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018)
In this ...

JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

Source: Karch, C. P. et al., <a href="https://app.jove.com/v/60103">Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation.</a>&#160;J. Vis. Exp. (2019)
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.

1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)

	Mix 95 mL of ...

Immunology

Immunotherapy

Immunogenics and Vaccine Development

Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions

Source: Oller-Salvia, B. <a href="https://www.jove.com/v/58066">Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction</a>. J. Vis. Exp. (2018).
This video demonstrates the hydrophobic interaction liquid chromatography-based characterization of antibody-drug conjugates. Using a butyl ligand-based chromatography column, the antibody-drug conjugates are characterized via hydrophobic interactions with the ligand.

Chromatographie liquide à interaction hydrophobe dépendante du butyle : une technique pour caractériser les conjugués anticorps-médicament basée sur les interactions hydrophobes

Source: Oller-Salvia, B. Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal ...

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

Technique de chromatographie d’affinité pour la purification d’anticorps monoclonaux

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...

Antibody-Based Technologies

Production and Purification

Types Of Column Chromatography

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.
Gel Filtration Chromatography 
When the protein&#39;s chemical nature is unknown, gel filtration chromatography is used for purification based on size. Matrices such as agarose, polyacrylamide, dextran derivatives, or silica beads are used for low-pressure systems, whereas polymeric resins are used for higher flow rates. The varying degree of cross-linking of these polymeric materials determines the pore size of the matrix, which decides the size range of proteins that can be separated.
Ion Exchange Chromatography
Ion-exchange chromatography can purify proteins based on their net charge at a particular pH determined by their isoelectric point or pI. The net charge on matrix resin is used to purify oppositely charged proteins by replacing the cations or anions from the matrix resin with the charged proteins. The resins are categorized based on the type of ion that is exchanged &#8212; positively charged proteins replace cations from cation-exchange resins, and vice versa. Strong ion exchangers with stability over a wide range of buffer pH include quaternary ammonium, sulfonate, and sulfopropyl resins. In contrast, weak ion exchangers that are effective only in a narrow pH range include diethyl aminoethyl (DEAE) or carboxymethyl (CM) resins.
Affinity Chromatography
Affinity chromatography involves immobilizing protein-specific antibodies or enzyme-specific substrates to the column material, usually agarose beads. For example, a column with the immobilized antibody of choice is prepared to separate specific antigens. When the mixed antigen sample is passed through the&#160; column, the target antigens bind to the immobilized antibodies and separate from the mixture. To separate recombinant proteins tagged with enzymes such as beta-galactosidase, the corresponding substrate Isopropyl-&#946;-D-thiogalactopyranoside (IPTG) is immobilized on the column to purify the enzyme.
High-pressure liquid chromatography (HPLC) and gas chromatography (GC) are the most commonly used column chromatography techniques. In HPLC, the solvent flows at high pressure through a tightly packed column that improves efficiency. Gas chromatography analyzes volatile compounds by converting them to their gaseous forms upon heating. The volatilized sample is pushed through the column by inert gasses such as nitrogen and helium.

Types de chromatographie sur colonne

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating ...

JoVE Core

Biologie Cellulaire

Unité 1

Analyzing Cells and Proteins

TERMES ET CONCEPTS CLÉS

Calcium-Dependent Hydrophobic Interaction Chromatography: A Technique to Purify Calcium-Binding Proteins Based on Hydrophobic Interactions

To continue the protein purification, after the protein dialysis, add calcium chloride to the sample to a final concentration of 25 millimolar, which will facilitate binding of S100A12 to the resin in the next step. Then, filter the sample through a filter with a pour size of 0.45 microns.
Equilibrate HIC buffers and samples to 4 degrees Celsius. Next, connect column buffers HIC-A and B and the column, to the system and adjust the parameters.
Start the method to equilibrate the column with 1 to 2 column volumes of buffer HIC-A, and load the sample, and the &#39;wash unbound sample block&#39; when the UV signal reaches baseline level again.
Then, start elution with a calcium chelator-containing buffer EDTA. Collect 2 milliliters of peak fractions during elution.

Chromatographie d’interaction hydrophobe dépendante du calcium : une technique de purification des protéines de liaison au calcium basée sur des interactions hydrophobes

EoE Sub Articles

Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

Start the FPLC with general maintenance. Connect column buffers AIEX-A and AIEX-B to buffer valves, and the anion-exchange resin-containing column to the column ports of the FPLC. Adjust the general chromatographic parameters accordingly.
Start the AIEX program in the FPLC software and choose the equilibration volume for buffer A. Subsequently, load the sample onto the column, and elute the proteins with a linear gradient from 0% to 100% high-salt buffer. Collect 2-milliliter fractions during elution.
Analyze the eluted fractions after finishing the run.

Purification de protéines par chromatographie échangeuse d’anions : technique de séparation pour isoler une protéine d’intérêt d’un lysat bactérien dialysé sur la base de la charge nette

Automatisé d'interaction hydrophobe Chromatographie sur colonne de sélection pour l'utilisation dans la purification des protéines

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