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

Questo lavoro è stato finanziato dal National Institutes of Health di sovvenzione GM086822 e National Science Foundation importante ricerca strumentazione concessione DBI-0960313. Gli autori desiderano ringraziare Drs. Jon Hardy &amp; Donna Miyake (Bio-Rad) e Jennifer Loertscher (Seattle University) alla loro competenza tecnica. Selezionare reagenti cromatografia e strumentazione supplementare stati generosamente fornito da Bio-Rad.

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	<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>

Selezionare reagenti cromatografia e strumentazione supplementare sono stati forniti da Bio-Rad.

Tecniche di cromatografia liquida hanno dimostrato prezioso per la preparazione di proteine ​​altamente purificate necessari per lo svolgimento di 6 immunologica, biochimica 7 e 8 studi strutturali. Metodi di purificazione HIC più spesso richiedono determinazione empirica di un mezzo preferito, e la struttura ligando, densità di ligando, e matrice proprietà tallone tutti hanno dimostrato di impatto risultati cromatografici 2, 3. Colonna Automated scouting è un approccio efficace per la selezione di un mezzo di HIC per l&#39;ottimizzazione e la successiva purificazione di proteine ​​4. Il metodo automatizzato colonna esplorazione presentato può essere facilmente adattato alle strategie di varie proteine ​​HIC purificazione. Alterazioni concentrazione salina, scelta di sale, gradiente salino, e il pH può ulteriormente migliorare le condizioni di purificazione, e gli effetti delle variazioni di questi parametri essenziali sono stati precedentemente recensione 13,14. HIC eluato contenente una prote bersaglio parzialmente purificatoin può essere ulteriormente purificato utilizzando una tecnica cromatografica complementare, come cromatografia a scambio ionico (IEX) o gel filtrazione / cromatografia per esclusione dimensionale 9,10. A causa della sua assorbanza di luce unico e caratteristiche di emissione, il profilo di eluizione di GFP può essere identificato utilizzando in-linea e post-run approcci. A tal fine, questo protocollo può ulteriormente essere adattato per la purificazione di ricombinanti di fusione GFP-proteine, in cui una proteina bersaglio è correlato &quot;tag&quot; con GFP. Proteine ​​bersaglio GFP-etichetta può essere rilevato con luce UV 11 e purificato usando vari metodi cromatografici, compresa la strategia di purificazione HIC sopra descritto. Depurazione GFP è diventata anche un fiocco pedagogico nei laboratori di biochimica per l&#39;insegnamento di tecniche moderne della scienza delle proteine ​​12. Mentre base purificazione della proteina HIC può essere condotta utilizzando un sostanzialmente meno robusto cromatografia sygambo, la strumentazione qui presentata ha un numero di vantaggi distinti per aiutare a ottenere risultati favorevoli. Notevoli vantaggi di utilizzare un sistema altamente automatizzato includono il miglioramento delle run-to-run condizioni di riproducibilità, risparmio di tempo e diminuzione opportunità per l&#39;aria di essere introdotte nel sistema 10. Sistema di controllo miscelazione tampone, che viene facilitata dalla massimizzatore sistema, consente maggiore coerenza nella preparazione tampone e riproducibilità sperimentale. Disegno abbastanza carico campione nel ciclo del campione dinamico per tutti i scouting corre successiva garantisce l&#39;uniformità del campione che viene aggiunto ad ogni colonna e permette la replica in sequenza senza interruzioni o manuale ricarica. Iniezione del campione controllato dal circuito campione attraverso la colonna diminuisce variabilità che può verificarsi con caricamento manuale del campione. Una coppia di valvole di selezione di colonna per consentire corse campioni consecutivi, ciascuno utilizzando una colonna HIC differente, senza dover rinnovare gli impianti idraulici del sistema. Esecuzione di mul<html> ti-lunghezza d&#39;onda analisi è particolarmente vantaggioso quando saggiando una proteina con un unico profilo spettrofotometrico. Oltre a GFP, citocromi, flavoproteine ​​e altri eme contenenti proteine ​​possono beneficiare di questa tecnica. Dispositivi di controllo di conducibilità e pH per consentire la verifica delle condizioni sperimentali in tempo reale. Frazione collezione Split eluato consente una gestione migliore frazione e consente il trasferimento facile per Post-Run metodi di analisi che richiedono un volume minimo di campione (ad esempio ELISA, SDS-PAGE, western blot, e Experion elettroforesi microfluidica). Durante il funzionamento il secondo in linea collettore di frazioni richiede sincronizzazione manuale, permette la massima flessibilità nella scelta frazione collettore. Gli inconvenienti più significativi a utilizzare un sistema robusto cromatografia per la colonna HIC scouting e purificazione della proteina comprende il tempo iniziale e spese di bilancio connesse con l&#39;acquisizione dello strumento e la formazione degli operatori. t &quot;&gt; Il protocollo qui presentato utilizza un Bio-Rad DuoFlow sistema di cromatografia. tuttavia, strumentazione altrettanto robusto da altri produttori, come il Avant AKTA da GE Healthcare, possono anche essere utilizzati e sono in grado di produrre risultati equivalenti sistema di cromatografia Anche comparabile hanno caratteristiche uniche (ad esempio il metodo di programmazione, i limiti nomenclatura dei componenti, le preferenze dell&#39;operatore, e scalabilità) che devono essere considerati prima di avviare una procedura di purificazione o di acquisizione dello strumento.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">BioLogic DuoFlow Pathfinder 20 Sistema</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> Include il sistema di Maximizer, mixer, workstation F10, AVR7-3 valvole, QuadTec UV / Vis con rivelatore di cella di flusso, collettore di frazioni BioFrac, software BioLogic e starter kit </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 stream-selezionare valvola</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> Valvola di alta pressione, 9-port, 8 posizioni, 3.500 psi (233 bar) limite, per l&#39;utilizzo con sistema biologico DuoFlow </td></tr><tr><td> Valvola deviatrice SV3T-2 </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> 7600410 </td><td> Elettrovalvola, a 3 porte, 2-posizione, 30 psi (2 bar) limite, per l&#39;utilizzo con sistema biologico DuoFlow </td></tr><tr><td> Flusso splitter valvola </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td></td><td></td></tr><tr><td>verticale = LSR &amp; parentCategoryGUID = &quot;target =&quot; 22c495f1-36ff-44a3-9140-ee7e54d0b1ba _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> Sliding-pistone ciclo kit campione, include 25 ml DynaLoop scorrevole anello del pistone, DynaLoop parti kit (# 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">Raccoglitore di frazioni 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> Due frazione col<html> lettori usati </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 micropiastra goccia adattatore testa</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">BioFrac microtiter piastra di adattamento</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td>&amp; Country = US &amp; lang = en &amp; ProductID = &quot;target =&quot; _blank 741-0017 &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">UV Ottica 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">Lampada alogena</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 Gradient Pump</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> Pompa gradiente a bassa pressione purificazione della proteina, comprende tubi e raccordi kit </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224301" target="_blank">Experion sistema</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 = &quot;target =&quot; 224301 _blank &quot;&gt; 7.007.001 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">Experion Pro260 Analisi Kit</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 HIC colonna kit di selezione </td><td> GE Healthcare </td><td> 28-4110-07 </td><td> Include 7 x 1 ml preconfezionati colonne di mezzi di HIC per scouting </td></tr><tr><td> GE Healthcare-to-Bio-Rad colonna raccordi </td><td> GE Healthcare </td><td> 18111251 e 18111257 </td><td></td></tr></tbody></table>

automated hydrophobic interaction chromatography column selection for use in protein purification

A differenza di altri metodi cromatografici per proteine ​​purificante (gel ad esempio filtrazione, affinità, e scambio ionico), cromatografia di interazione idrofoba (HIC) richiede comunemente determinazione sperimentale (denominato screening o &quot;scouting&quot;) per selezionare il mezzo più adatto cromatografica per purificare una proteina dato 1. Il metodo qui presentato descrive un metodo automatizzato per scouting per un supporto ottimale HIC da utilizzare nella purificazione di proteine. HIC separa proteine ​​e di altre biomolecole da un lisato grezzo sulla base delle differenze di idrofobicità. Simile a cromatografia di affinità (AC) e cromatografia a scambio ionico (IEX), HIC è in grado di concentrare la proteina di interesse mentre avanza attraverso il processo cromatografico. Le proteine ​​più adatti per la purificazione da HIC sono quelli con le regioni superficie idrofoba e in grado di sopportare l&#39;esposizione a concentrazioni di sale in eccesso di 2 munizioni Mnio solfato ((NH 4) 2 SO 4). HIC è spesso scelta come metodo di purificazione di proteine ​​prive di una etichetta di affinità, e quindi inadatto per AC, e quando IEX non fornisce adeguata purificazione. Frazioni idrofobe sulla superficie della proteina temporaneamente legarsi ad un ligando non polare accoppiata ad un inerte, matrice immobile. L&#39;interazione tra proteina e legante sono altamente dipendenti dalla concentrazione salina del buffer che fluisce attraverso la colonna cromatografica, con alte concentrazioni ioniche rafforzamento della proteina-ligando e rendendo la proteina immobile (cioè vincolato all&#39;interno della colonna) 2. Come diminuzione delle concentrazioni di sale, la proteina-ligando dissipa, la proteina diventa nuovamente mobile e eluisce dalla colonna. Vari mezzi di HIC sono commercialmente disponibili in preconfezionati colonne, ciascuna contenente uno dei diversi ligandi idrofobi (ad esempio S-butile, butile, ottile, e fenil) reticolato a varie densità di perline di agarosio di una specificadiametro ific 3. Colonna di scouting automatizzato consente di adottare un approccio efficiente per determinare quali media HIC dovrebbe essere impiegato per i futuri, gli esperimenti di ottimizzazione più esaurienti e purificazione della proteina funziona 4. La proteina viene purificata specifico ecco ricombinante proteina verde fluorescente (GFP), tuttavia, il metodo può essere adattato per purificare altre proteine ​​con una o più regioni superficie idrofoba. GFP serve come una proteina modello utile, grazie alla sua stabilità, unico picco di assorbanza della luce a 397 nm, e la fluorescenza quando esposti a luce UV 5. Lisato batterico contenente GFP di tipo selvatico è stato preparato in un tampone ad alto sale, caricata in un Bio-Rad sistema mezzo cromatografia DuoFlow pressione del liquido, e adsorbiti colonne HiTrap HIC contenenti diversi supporti HIC. La proteina è stata eluita dalle colonne e analizzato in linea-e post-run metodi di rilevamento. Buffer blending, dinamica campione ansa di iniezione, col sequenzialeselezione UMN, multi-lunghezza d&#39;onda di analisi, e la frazione raccolta divisione eluato aumentato la funzionalità del sistema e la riproducibilità del metodo sperimentale.

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

Un metodo automatizzato per identificare adatti cromatografia interazione idrofoba (HIC) mezzi da utilizzare nel processo di purificazione di proteine ​​viene presentato. Il metodo utilizza un sistema di media pressione cromatografia liquida compreso il miscuglio tampone automatizzata, dinamico ciclo di iniezione del campione, selezione di colonna sequenziale, multi-lunghezza d&#39;onda di analisi, e la frazione raccolta divisione eluato.

Automated interazione idrofobica colonna di selezione cromatografia per l&#39;utilizzo in purificazione di proteine

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

automated-hydrophobic-interaction-chromatography-column-selection-for

Research

JoVE Journal

Biology

43.5K Views.  Seattle University. Un metodo automatizzato per identificare adatti cromatografia interazione idrofoba (HIC) mezzi da utilizzare nel processo di purificazione di proteine ​​viene presentato. Il metodo utilizza un sistema di media pressione cromatografia liquida compreso il miscuglio tampone automatizzata, dinamico ciclo di iniezione del campione, selezione di colonna sequenziale, multi-lunghezza d'onda di analisi, e la frazione raccolta divisione eluato.

Video: 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.

Espressione, purificazione, cristallizzazione e saggi enzimatici di Fumarylacetoacetate Hydrolase Domain-Containing Proteins

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

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Biochimica

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 sistema di purificazione delle proteine ad alta produttività economico e versatile che utilizza un adattatore a piastre multi-colonna

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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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

Metodi di purificazione di biomolecole basati sulla cromatografia

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

Didattica

JoVE Science Education

Chimica

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.

Purificazione delle proteine basata sulla cromatografia di affinità al nichel: una tecnica per purificare le proteine ricombinanti marcate con poliistidina dal lisato cellulare batterico

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

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.

Cromatografia liquida con interazione idrofobica butil-dipendente: una tecnica per caratterizzare i coniugati anticorpo-farmaco basati su interazioni idrofobiche

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.

Una tecnica di cromatografia di affinità per la purificazione di anticorpi monoclonali

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

Immunology

Antibody-Based Technologies

Production and Purification

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.

Purificazione di nanoparticelle proteiche autoassemblanti mediante cromatografia di affinità

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 ...

Immunotherapy

Immunogenics and Vaccine Development

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.

Tipi di cromatografia su colonna

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

JoVE Core

Cell Biology

Unit 1

Analyzing Cells and Proteins

TERMINI E CONCETTI CHIAVE

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.

Purificazione delle proteine basata sulla cromatografia a scambio anionico: una tecnica di separazione per isolare una proteina di interesse dal lisato batterico dializzato basato sulla carica netta

EoE Sub Articles

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.

Automated interazione idrofobica colonna di selezione cromatografia per l'utilizzo in purificazione di proteine

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