Ringraziamo Anne-Marie Dion-Costa per le discussioni che portano allo sviluppo del metodo. JK e RB sono FQNRT studiosi di dottorato, M.-MG è uno studioso Vanier CIHR, e J.-YM è un ricercatore senior FRSQ. Questo lavoro è stato sostenuto da fondi dalle scienze naturali e ingegneria Research Council di JY. M.

1. Produzione di ricombinante Baculovirus Baculoviruses vengono generati utilizzando il Baculovirus Expression System Bac-to-Bac di Invitrogen prevalentemente secondo il protocollo del produttore con solo lievi modifiche: <ol><li> Un pFastBac1 vettore modificato (Gibco, vita) è stato creato, contenente la GST e suo-tag (Figura 2). Il MultiCloning Site (MCS) di una PET-52b (+) vettore (Novagen), contenente un 10xHis-tag, è stato inserito nel MCS di un vettore pFastBac1 per generare pFastBac1-Strep-10xHis. La sequenza MCS pET-52b (+) è stato amplificato mediante PCR tra i siti di restrizione XbaI e BlpI, l&#39;aggiunta di un sito di restrizione BglII all&#39;estremità 5 &#39;estremità e un sito di restrizione HindIII al 3&#39; estremità. Il prodotto di PCR è stato poi clonato tra BamHI e HindIII sito di restrizione di pFastBac1, utilizzando la compatibilità di BamHI e BglII sequenze di restrizione. Poiché l&#39;obiettivo era di generare una pFastBac1 permettendo l&#39;espressione diproteina ricombinante con streptavidina-e 10xHis-tag, il sito di restrizione BamHI della PET-52b (+) la sequenza MCS non è stato utilizzato. Inoltre, il sito di restrizione XbaI non è stato utilizzato per evitare la ridondanza dei siti di restrizione che si verificano tra i siti già presenti nel MCS di pFastBac1 e questi inserite con la MCS di PET-52b (+). La sequenza codificante GST con il sito prescissione proteasi di riconoscimento (LeuGluValLeuPheGln / GlyPro) è stato poi inserito nel pFastBac1-Strep-10xHis. La sequenza di cDNA da GST-pGEX 6P-2 (GE Healthcare) è stato amplificato mediante PCR con primer contenenti NcoI e KpnI siti di restrizione al 5 &#39;e 3&#39; estremità, rispettivamente. Il sito NcoI permette l&#39;aggiunta di un codone di inizio, ma anche una alanina al GST. Il prodotto di PCR è stato poi clonato in pFastBac1-Strep-10xHis tra i siti di restrizione NcoI e KpnI, con la conseguente soppressione della streptavidina-tag. Il nuovo vettore di fusione così ottenuto è stato nominato pFastBac1-GST-10xHis. </li><li> Clonare la codifica cDNA per il protein di interesse nel vettore pFastBac-GST-10xHis tra la GST (N-terminale) e la His-tag (C-terminale). Fate attenzione a rimuovere il fermo cDNA codone per consentire la fusione con la C-terminale His-tag. </li><li> Trasforma E. coli DH10Bac con il ricombinante pFastBac (ottenuta nella fase 1.2) per generare il bacmid. Consentire colonie di crescere durante la notte a 37 ° C e diverse ore a 30 ° C su piastre di selezione contenenti Bluo-gal e IPTG (10 mcg / ml tetraciclina, 50 pg / ml kanamicina, 7 mg / ml di gentamicina, 100 pg / ml Bluo- Gal, 40 mcg / ml IPTG). </li><li> Pick and restreak 2 colonie bianche (a partire dal punto 1.3) su piastre di selezione per confermare il fenotipo bianco. </li><li> Inoculare 2 ml di LB contenenti 10 ug / ml tetraciclina, 50 pg / ml kanamicina, 10 mg / ml di gentamicina con le due colonie bianche dal punto 1.4 e farli crescere per una notte sotto agitazione (250 rpm) a 37 ° C. </li><li> Al fine di purificare il DNA bacmid, pellet i batteri dal punto 1.5,sospendere nuovamente le cellule in 300 ml di soluzione di I e aggiungere 300 ml di soluzione II (kit Maxiprep da Qiagen). Incubare per 5 min a temperatura ambiente, aggiungere 300 ml di 3 M KOAc pH 5.5 e incubare per 10 min in ghiaccio. Centrifugare i campioni a 4 ° C, velocità massima per 10 min. Aggiungere 800 ml di isopropanolo per surnatanti e incubare per 10 min in ghiaccio. Centrifugare i campioni per 15 min a 4 ° C e lavare il precipitato con 500 ml di etanolo al 70%. Lasciare il pellet asciutto e risospendere in condizioni sterili in 40 ml di Tris-EDTA pH 8,0. Il DNA bacmid deve essere conservato a 4 ° C. </li><li> Generare baculovirus come descritto nel protocollo del produttore. Baculoviruses possono essere conservati a 4 ° C al riparo dalla luce. </li></ol> 2. Ricombinante Protein Expression <ol><li> Per scegliere il baculovirus più efficiente per la purificazione e di monitorare ciò che il tempo si raggiunge il picco di espressione della proteina, eseguire un mini-infezione test uns segue: Infect 2 x 10 7 cellule infette Sf9 coltivate a 27 ° C in mezzi di Grace (integrato con 10% FBS e 1% P / S) con 133 microlitri (1/150) di baculovirus. Raccogliere aliquote di 1,5 ml a 0, 24, 48, e 72 ore dopo l&#39;infezione. Agglomerare le cellule e analizzare il livello di espressione della proteina di interesse mediante western blotting utilizzando anticorpi anti-tag. </li><li> Utilizzare il baculovirus più efficiente dal punto 2.1 per infettare un filatore contenente 1,0 x 10 6 cellule Sf9 per ml in 500 ml di media con un rapporto di 1/150 di tra virus e media. Lasciare che le cellule infettate crescere in sospensione a 27 ° C e raccogliere le cellule quando viene raggiunta la massima espressione della proteina (come determinato nella fase precedente punto 2.1). Cellule pellet possono essere conservati a -80 ° C sino al successivo utilizzo. </li></ol> 3. Preparazione del lisato cellulare Solubile Tutte le seguenti fasi di incubazione vengono effettuati a 4 ° C sotto lieve rotazione. <ol><li> Lyse SF9 cellule esprimenti la vostra proteina di interesse in ~ 20 ml di tampone di legame GST (NaCl 150 mM, EDTA 1 mM, 0,05% Triton-X-100, 1 mM DTT in PBS1X per una concentrazione finale di NaCl di 250 mM, e inibitore della proteasi cocktail (Roche). In un bagno di acqua ghiacciata, Dounce la soluzione 20x con un omogeneizzatore Dounce, ultrasuoni 3x 30 secondi ciascuno (in uscita il 70%), e Dounce nuovo 20x. </li><li> (Facoltativo). Incubare il lisato cellulare totale con 1 mM MgCl 2 e benzonasi 7 nucleasi (2,5 U / ml) per 30 min a 4 ° C per rimuovere il DNA o RNA contaminazione. </li><li> Centrifugare a 18.000 rpm per 30 min a 4 ° C. Conservare il surnatante. </li><li> (Facoltativo). Centrifugare il surnatante nuovamente per 30 min a 18.000 rpm a 4 ° C per ottenere un lisato solubile chiaro. Passare il surnatante con un filtro da 0,2 o 0,45 micron per evitare intasamenti. </li></ol> 4. Il legame di proteine ​​su Beads GST <ol><li> Incubare il lisato cellulare solubile con 1 ml di perline GST (lavata due volte con10 ml di tampone GST) vincolante per 1 ora a 4 ° C sotto lieve rotazione. </li></ol> Commento: Il tempo di incubazione deve essere ottimizzato secondo la stabilità della proteina e l&#39;efficienza di legame. <ol start="2"><li> Giro veloce a 700 rpm e rimuovere il surnatante contenente proteine ​​non legate (questo può essere conservato per ulteriori analisi). Lavare le proteine ​​GST-bound (Figura 3B, corsia 1) con tampone di lavaggio GST (GST binding buffer con 350 mM NaCl finale). </li><li> Ripetere il punto 4.2 2x. All&#39;ultimo lavaggio, rimuovere il più possibile surnatante. </li><li> Incubare le perline con 5 mM ATP e 15 mM MgCl 2 (in 10 ml di tampone di legame GST) per 1 ora a 4 ° C per evitare legami aspecifici di proteine ​​da shock termico 8. Lavare le perline tre volte con tampone di lavaggio GST. </li></ol> 5. Prescissione scissione del GST <ol><li> Centrifugare le perline GST, rimuovere il surnatante e lavare le perline GST con P5 buffer (50 NaHPO 4 mM pH 7,0, NaCl 500 mM, glicerolo 10%, 0,05% Triton-X-100, imidazolo 5 mM). </li><li> Incubare le GST-perline con prescissione enzima nel buffer P5 da 3 ore a notte a 4 ° C. (Dividete le perle GST in frazioni di 100 ml e aggiungere 4-8 unità (2-4 microlitri) di enzima diluito in 100 ml di tampone P5). </li></ol> Commento: Il tempo di incubazione del passo prescissione deve essere ottimizzato in funzione del peso molecolare e la stabilità della proteina. Se la degradazione viene osservata, questo tempo di incubazione può essere ridotto fino ad un minimo di 2 ore anziché overnight. <ol start="3"><li> Giro veloce e raccogliere il surnatante. Ripetere questa operazione per due volte dopo l&#39;aggiunta di 100 ml di P5 tampone ai talloni e piscina tutte le frazioni (Figura 3B, corsia 2). </li></ol> Commento: I restanti sfere possono essere utilizzate per l&#39;analisi di efficienza clivaggio (Figura 3B </strong&gt;, corsia 3). Solitamente, il 70-80% delle proteine ​​viene aperto. 6. -Proteina di legame per TALON metallo Affinity Resin <ol><li> Dividere l&#39;eluizione dall&#39;alto in 3 frazioni da 1 ml e incubare ciascuno con 100 microlitri Talon resina di affinità metallo perline secco (lavata due volte con tampone P5) per 1 ora in rotazione. </li></ol> Commento: Dividendo il eluizione in diverse frazioni aumenta l&#39;efficienza di legame / lavaggi. <ol start="2"><li> Lavare la resina 5 min con tampone P30 (tampone P5 con una concentrazione finale di 30 mM imidazolo). </li><li> Ripetere passo 6.2 2x e in comune le perline in un unico tubo. Prima l&#39;ultimo lavaggio, rimuovere il più possibile surnatante. </li></ol> Commento: Questo corrisponde al TALON vincolato campione (figura 3B, corsia 4). 7. Eluizione della proteina purificata <ol><li> Incubare la proteina legata resina TALON per 5 min in rotazione con P500 tampone (buffer P5 con una concentrazione finale di 500 mM imidazolo). Utilizzare un rapporto tra buffer e perle di 1:01 v / v (Ad esempio, se tu avessi preso 200 ml di perline secco, dovrete aggiungere 200 ml di P500). Ripetere questa operazione due volte e mantenere ogni frazione eluita separatamente (denominato eluizione 1 (E1), eluizione 2 (E2) e eluizione 3 (E3), figura 3B, corsie 5-7). </li></ol> Commento: I restanti sfere possono essere utilizzate per l&#39;analisi di efficienza di eluizione (Figura 3B, corsia 8). Tipicamente, 60-80% della proteina viene eluita in totale. <ol start="2"><li> Analizzare la quantità e la qualità della proteina purificata caricando circa 20-40 microlitri di ogni frazione con una concentrazione standard BSA su SDS-PAGE e colorazione con Coomassie blue (Figura 3B) o SYPRO macchia proteina per la visualizzazione. </li></ol> Commento: La proteina di interesse può essere rilevata anche throughout la procedura di purificazione utilizzando anti-GST e anti-His anticorpi (Figura 3C). 8. Conservazione della proteina purificata <ol><li> Dializzare la proteina purificata contro un tampone di conservazione appropriata (ad esempio 20 mM Tris acetato pH 8,0, KAC 200 mM, glicerolo 10%, EDTA 1 mM, DTT 0,5 mM) a 4 ° C. È importante controllare la precipitazione della proteina purificata durante la dialisi. Di solito Dializzare 2x per 1 ora a 4 ° C sotto agitazione rotazione. </li><li> Effettuare piccole aliquote della proteina purificata (10-20 ml) e congelare in ghiaccio secco per 30 min. Conservare le aliquote a -80 ° C ed evitare molte disgelo / cicli di congelamento. </li></ol>

<ol>
	<li>Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J., Tan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+affinity+tags+for+protein+purification.">Comparison of affinity tags for protein purification.</a> Protein Expr. Purif. 41, 98-105 (2005).</li><li>Thain, A., Gaston, K., Jenkins, O., Clarke, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+separation+of+GST+fusion+proteins+from+co-purifying+GroEL.">A method for the separation of GST fusion proteins from co-purifying GroEL.</a> Trends Genet. 12, 209-210 (1996).</li><li>Carr, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+recombinant+form+of+the+V+antigen+of+Yersinia+pestis,+using+three+different+expression+systems.">Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems.</a> Vaccine. 18, 153-159 (1999).</li><li>Armisen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+adsorption+of+poly-His+tagged+glutaryl+acylase+on+tailor-made+metal+chelate+supports.">Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.</a> J. Chromatogr. A. 848, 61-70 (1999).</li><li>Kuhn, S., Zipfel, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+baculovirus+expression+vector+pBSV-8His+directs+secretion+of+histidine-tagged+proteins.">The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins.</a> Gene. 162, 225-229 (1995).</li><li>Buisson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+of+breast+cancer+proteins+PALB2+and+piccolo+BRCA2+in+stimulating+homologous+recombination.">Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.</a> Nat. Struct. Mol. Biol. 17, 1247-1254 (2010).</li><li>Filimonova, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoforms+of+Serratia+marcescens+nuclease.+The+role+of+Mg2++in+the+hydrolysis+mechanism.">Isoforms of Serratia marcescens nuclease. The role of Mg2+ in the hydrolysis mechanism.</a> Biochemistry. 62, 983-988 (1997).</li><li>Thorslund, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+breast+cancer+tumor+suppressor+BRCA2+promotes+the+specific+targeting+of+RAD51+to+single-stranded+DNA.">The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA.</a> Nat. Struct. Mol. Biol. 17, 1263-1265 (2010).</li><li>Genois, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+BRCA2+and+RAD51+for+promoting+homologous+recombination+in+Leishmania+infantum.">Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.</a> Nucleic Acids Res. 40, 6570-6584 (2012).</li><li>Shrestha, B., Smee, C., Gileadi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+vector+system:+an+emerging+host+for+high-throughput+eukaryotic+protein+expression.">Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression.</a> Methods Mol. Biol. 439, 269-289 (2008).</li></ol>

Il GST-His protocollo di purificazione qui presentato è adatto per la purificazione di una vasta gamma di dimensioni di proteine ​​ricombinanti: Abbiamo purificati successo Li RAD51 (41kDa), piBRCA2 (120kDa), PALB2 (130kDa), e un alto contenuto di proteine ​​di peso molecolare instabile Leishmania infantum: Li BRCA2 (125kDa) 6,9. Inoltre, le proteine ​​purificate con questo protocollo erano biochimicamente attiva 6. Il successo di questo metodo dipende unicamente dall...

La purificazione di proteine ​​ricombinanti è fondamentale per affrontare le questioni fondamentali in biochimica. Modi convenzionali di purificazione delle proteine ​​come cromatografia a scambio ionico e cromatografia ad esclusione dimensionale basano sulle proprietà fisiche della proteina bersaglio come suo punto isoelettrico e carica o dimensione, rispettivamente. Le caratteristiche proteiche ultimi sono condivisi da una varietà di proteine, che aumenta notevolmente la possibilità di proteine ​​contaminanti in strategie di purificazione delle proteine ​​convenzionali. Questo problema può essere aggirato con l&#39;utilizzo di più colonne di purificazione, che richiede molto tempo. Allo stesso tempo, questi ultimi metodi cromatografici richiedono costosi setup sperimentale. Affinità-tag purificazione aumenta fortemente bersaglio specificità, come nella maggior parte dei casi il tag sarà unico per la proteina di interesse. In studi recenti, purificazione-Flag o HA-affinità è stato ampiamente utilizzato. In contrasto con rec esistenteprotocolli di purificazione di proteine ​​ombinant in cui vengono utilizzati singoli tag, abbiamo stabilito la combinazione unica di due tag. Il nostro metodo prevede la fusione di GST-tag al N-terminale e un His-tag al C-terminale della proteina di interesse, per un rapporto ottimale tra quantità e la purezza della proteina desiderata. Il GST è molto variabile (29 kDa), che è molto efficace per la purificazione su glutatione Sepharose perline. Inoltre, utilizzando GST garantisce costo-efficacia del nostro metodo 1. La possibilità di fendere largo della GST con l&#39;enzima prescissione (con sequenza di riconoscimento LeuGluValLeuPheGln / GlyPro, conseguente aggiunta di soli due amminoacidi) ha molti vantaggi, per esempio, questa strategia evita alterazioni delle funzioni fisiologiche proteina causa ostacolo allosterico. Il piccolo His-tag fusa alla proteina altra estremità serve in una seconda fase di purificazione per aumentare proteina purezza lavando via il GST spaccati, così come proteine ​​degradate e altre<html> contaminanti. Inoltre, il protocollo non richiede una fase di dialisi quando si passa dalla GST alla colonna passo His-purificazione (resina TALON). Contaminanti comuni in tali processi di purificazione sono proteine ​​da shock termico (HSP). L&#39;aggiunta di una fase di incubazione con MgCl 2 e ATP permette la rimozione di questi contaminanti (Figura 1). Proteina rapidi Liquid Chromatography (FPLC) e High Performance Liquid Chromatography (HPLC) sono tecniche comuni che dipendono strumentazione costosa per generare alta resa della proteina purificata di interesse. Il protocollo di purificazione lotto presentiamo qui, al contrario, è manuale e non richiede una configurazione strumentale costoso. Un elevato rendimento di proteina può essere raggiunto scalando il protocollo. Allo stesso tempo, con il protocollo di purificazione batch, il volume di eluizione può essere regolato in modo da aumentare la concentrazione di proteine, che non è dato con FPLC o HPLC. S copi &quot;&gt; Inoltre, con la GST-His protocollo di purificazione batch, proteine ​​con un intervallo di dimensioni di circa 10-300 kDa possono essere purificati. Uno dei maggiori vantaggi è dato dal fatto che si può purificare diverse proteine ​​allo stesso tempo , per esempio, sia di tipo selvatico e la sua proteina mutante. Il successo del protocollo presentato dipende unicamente il livello di espressione e la solubilità della proteina di interesse.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>E.Coli DH10Bac (competent)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bac-to-Bac Baculovirus Expression System</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Maxi Prep Kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Solution I and II</td>
		</tr>
		<tr>
			<td>Ultra Pure Bluo-Gal</td>
			<td>Gibco (Life)</td>
			<td>15519028</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Gibco (Life)</td>
			<td>15529019</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 infected cells</td>
			<td>ATCC</td>
			<td>CRL-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>PreScission enzyme</td>
			<td>GE Healthcare</td>
			<td>27084301</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#39;s infected medium supplemented</td>
			<td>Gibco (Life)</td>
			<td>11605-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum characterized</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>P/S (Penicillin-Streptomycin)</td>
			<td>Gibco (Life)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose resin</td>
			<td>GE bioscience</td>
			<td>17075605</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Talon resin</td>
			<td>Clontech</td>
			<td>635504</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>BioShop</td>
			<td>288324</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco (Life)</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal GST-antibody</td>
			<td>Production in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal 6X-His-antibody</td>
			<td>Clontech</td>
			<td>631212</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dounce homogenizer (tight)</td>
			<td>Wheaton</td>
			<td>357546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator (Fisher dismembrator)</td>
			<td>Fisher</td>
			<td>Model 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis bag (50 mm)</td>
			<td>Fisher Scientific</td>
			<td>2115217</td>
			<td></td>
		</tr>
	</tbody>
</table>

gst-his purification: a two-step affinity purification protocol yielding full-length purified proteins

Saggi chiave enzimologia per la caratterizzazione biochimica delle proteine ​​in vitro richiedono alte concentrazioni di proteina purificata di interesse. Protocolli di purificazione di proteine ​​dovrebbero coniugare efficienza, semplicità ed economicità 1. Qui, descriviamo la GST-His metodo come un nuovo sistema di purificazione per affinità su piccola scala per le proteine ​​ricombinanti, basato su un N-terminale Tag glutatione Sepharose (GST) 2,3 e un tag 10xHis C-terminale 4, che sono entrambi fusa per la proteina di interesse. Quest&#39;ultimo costrutto viene utilizzato per generare baculovirus, per infezione di Sf9 cellule infette per l&#39;espressione proteica 5. GST è piuttosto lunga tag (29 kDa) che serve a garantire l&#39;efficienza di depurazione. Tuttavia, potrebbe influenzare le proprietà fisiologiche della proteina. Quindi, viene successivamente tagliato fuori la proteina usando l&#39;enzima prescissione 6. Al fine di garantire la massima purezza e per rimuovere la GST spaccati, abbiamoaggiunta una seconda fase di purificazione di affinità basata sul relativamente piccolo His-tag. È importante sottolineare che la nostra tecnica si basa su due diversi tag che fiancheggiano le due estremità della proteina, che è uno strumento efficace per rimuovere le proteine ​​degradate e, quindi, arricchisce proteine ​​integrali. Il metodo qui presentato non richiede una configurazione strumentale costoso, come FPLC. Inoltre, abbiamo incorporato MgCl 2 e lava ATP per rimuovere le impurità di calore proteina da shock e il trattamento nucleasi per abolire contaminanti acidi nucleici. In sintesi, la combinazione di due diversi tag che fiancheggiano la N-e C-terminale e la capacità di scindere fuori uno dei tag, garantisce il recupero di una proteina altamente purificata e full-length di interesse.

Al fine di illustrare l&#39;efficacia del-His GST protocollo di purificazione, si purificati Rec14, un S. proteine ​​pombe del 32,9 kDa. La Rec14 cDNA è stato clonato nel nostro vettore pFastBac1 modificata permettendo le aggiunte di GST-e le Sue-tag presso la N-e C-terminali, rispettivamente (Figura 1A). Baculovirus ricombinanti sono stati poi preparati e usati per infettare le cellule infettate SF9 per l&#39;espressione della proteina. I lisati cellulari solubili sono stati incu...

Watch this Scientific Journal Video about GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins at JoVE.com

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Nel presente protocollo, si dimostra un metodo altamente efficiente e conveniente purificazione di proteine ​​su piccola scala, che permette la purificazione di proteine ​​ricombinanti, combinando in modo univoco un scindibile GST-tag e un piccolo His-tag.

GST-la sua purificazione: A Two-step Affinity Purification protocollo Cedendo Full-length proteine ​​purificate

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

gst-his-purification-two-step-affinity-purification-protocol-yielding

Research

JoVE Journal

Biology

41.1K Views.  Hôtel-Dieu de Québec.  Nel presente protocollo, si dimostra un metodo altamente efficiente e conveniente purificazione di proteine ​​su piccola scala, che permette la purificazione di proteine ​​ricombinanti, combinando in modo univoco un scindibile GST-tag e un piccolo His-tag. 

Video: GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Ricerca

Biologia

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Purificazione di Hsp104, una proteina Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificazione cromatografica dei ribosomi di lievito Highly Active

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Purificazione delle proteine ​​ortogonali Facilitato da una piastrina Affinity Bispecific

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Individuazione di partner proteine ​​interagenti mediante Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

High-throughput Purificazione di affinità-tagged proteine ​​ricombinanti

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Protein Purification-libera Metodo di Binding Affinity Determinazione Thermophoresis microscala

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Un protocollo per Phage Display e affinità selezione con esche proteina ricombinante

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Identificare proteina-proteina interazione in<em&gt; Drosophila</em&gt; Adult capi di Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Purificazione delle proteine ​​tecnica che permette la rilevazione di sumoilazione e Ubiquitinazione di lievito in erba cinetocore proteine ​​Ndc10 e Ndc80

GST-la sua purificazione: A Two-step Affinity Purification protocollo Cedendo Full-length proteine ​​purificate

GST-la sua purificazione: A Two-step Affinity Purification protocollo Cedendo Full-length proteine purificate