Questo progetto è stato finanziato dalla Knut e Alice Wallenberg Foundation e il Consiglio svedese della ricerca.

1. Clonazione della fusione obiettivo gene/ABDz1-tagged costruire <ol><li> Preparare frammenti PCR del gene ABDz1 affiancato da siti di restrizione adatti per N-terminale legatura del gene target nel plasmide di espressione (un plasmide contenente ABDz1 è disponibile gratuitamente attraverso un accordo di trasferimento di materiale). </li><li> Cleave purificata espressione vettoriale e frammenti di PCR con enzimi di restrizione scelto in un tampone di reazione adeguato. Purificare i prodotti prima di legatura. </li><li> Legare il limitato ABDz1 frammento nel vettore di espressione contenente il gene di interesse e di trasformare il prodotto legatura a E. coli (nel metodo originale RR1ΔM15 ceppo è stato utilizzato 8). Diffondere cellule trasformate su piastre di agar integrato con gli antibiotici adatti per la selezione. </li><li> PCR schermo alcune colonie e la sequenza verificare la cassetta espressione risultante dal sequenziamento del DNA. </li><li> Preparare plasmide da un giorno all&#39;altro la cultura di una sequenza VERIFcolonia IED e trasformare al ceppo espressione preferita (in originale metodo di E. coli Rosetta (DE3), ospita un pRARE plasmide per migliorare la produzione delle proteine ​​codificate da geni umani, sono stati utilizzati). </li></ol> 2. Espressione della proteina <ol><li> Inoculare una singola colonia batterica in 10 ml di brodo di soia trittico completato con 5 g / l estratto di lievito e antibiotici appropriati. Incubare a 150 rpm a 37 ° C durante la notte. </li><li> Seminare 1 ml di cultura durante la notte in 100 ml di mezzo fresco e indurre l&#39;espressione della proteina (originariamente 1 mM isopropil-β-D-tiogalattoside quando un operone lac è stato utilizzato) quando le cellule raggiungono la fase logaritmica di crescita. </li><li> Incubare a 150 rpm a 25 ° C le cellule durante la notte e messe per centrifugazione. </li></ol> 3. Affinità purificazione ortogonali <ol><li> Risospendere il pellet in 25 ml di tampone in esecuzione (25 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0,05% (w / v) di Tween 20, pH 8,0) e disturbare °cellule e mediante ultrasuoni al 60% di ampiezza e 1.0/1.0 impulsi per 3 min. Centrifugare il campione e filtrare la proteina bersaglio contenente surnatante (0,45 micron) prima di un&#39;ulteriore purificazione. </li><li> Equilibrare un 1 ml di HSA colonna Sepharose o un NHS-attivato colonna, precedentemente accoppiato con HSA secondo le raccomandazioni del fornitore, con 10 volumi di colonna (CV) di correre tampone a 1 ml / min su un adeguato sistema di purificazione delle proteine. Caricare il lisato batterico a 0,5 ml / min e poi ripristinare il flusso di 1 ml / min. </li><li> Lavare la colonna con 5 CV di tampone di corsa seguiti da 5 CV di tampone di lavaggio (5 mM NH 4 Ac, pH 5,5). </li><li> Eluire il campione con tampone di eluizione (0,5 M HAC, pH 2.5) a 1 ml / min e raccogliere frazioni, monitorare l&#39;assorbimento a 280 nm per selezionare frazioni dalla vetta eluiti per un&#39;ulteriore purificazione. </li><li> Piscina le frazioni con la più alta concentrazione di proteine, diluire due volte in certi tampone di corsa (20 mM fosfato, 150 mM NaCl, pH 7,2) eassicurarsi che il pH è intorno neutro. Aggiungere 1 M Tris-HCl pH 8 per aumentare il pH, se necessario. </li><li> Caricare il campione a 0,5 ml / min su una colonna 1 MabSelect HITRAP ml Certo che è stata equilibrata con 10 CV di sicuro tampone di corsa a 1 ml / min. Ripristina il flusso di 1 ml / min dopo il caricamento. </li><li> Lavare la colonna con 5 CV di tampone di corsa sicuro (punto 5) ed eluire le proteine ​​con 0,2 M HAC, pH 2.7. Per le proteine ​​bersaglio sensibile l&#39;eluato può essere neutralizzato direttamente al momento della raccolta con l&#39;aggiunta di Tris-HCl. </li></ol> Se i passaggi cromatografici sono invertiti l&#39;eluato dal MabSelect colonna Certo possono essere diluiti con tampone (passo 3.1) e il pH è aumentato a circa 8 mediante aggiunta di 1 M Tris-HCl. In generale, più stretto picchi si osservano quando la matrice Certo è impiegato nella seconda fase. 4. Valutazione della purezza di sodio dodecil solfato elettroforesi su gel di poliacrilammide (SDS-PAGE) <ol><li> Caricare le frazioni purificata su unariducendo SDS-PAGE e, se necessario, analizzare il peso molecolare del prodotto purificato mediante spettrometria di massa. E &#39;importante ridurre completamente il campione in quanto una cisteina libera in ABDz1 può causare dimerizzazione del prodotto in condizioni non riducenti. </li></ol> 5. Rappresentante dei risultati: Come prova di principio, purificazione di affinità ortogonali facilitato dal tag ABDz1 è stato valutato per tre proteine ​​bersaglio umano che rappresentano classi di diversa solubilità, pesi molecolari e punti isoelettrico (Tabella 1). Il gene ABDz1 era geneticamente fuso con i geni bersaglio ed i costrutti sono stati espressi in E. coli, la Figura 2 mostra un diagramma di flusso per tutte le fasi del metodo consecutivamente. A seguito di purificazione dal protocollo ortogonali, campioni raccolti in diversi punti sono stati analizzati mediante SDS-PAGE (Figura 3). I risultati mostrano chiaramente l&#39;utilità di un tag a doppia e due fasi di purificazione altamente specifiche. Anche se è ragionevole purezza achieved dopo la fase di purificazione iniziale visto dal gel, il passo successivo si ottiene un prodotto molto puro adatto per applicazioni altamente esigenti. <img alt="Figura 1" src="/files/ftp_upload/3370/3370fig1.jpg" /> Figura 1. Progettazione della biblioteca combinatoria utilizzata per la selezione del bispecific ABDz1 tag. I 46 aminoacidi albumina vincolante pieghe di dominio in una stalla tre fascio elica e contiene un sito di legame per l&#39;albumina sierica umana principalmente trova a elica secondo. Con geneticamente randomizing undici superficie esposta amino acidi situato nel elica di prima e terza, una superficie romanzo vincolante è stato progettato. Gli undici posizioni sono indicate nella figura e numerati secondo Kraulis et al. 9. A seguito di biopanning contro un dimero di Z-dominio della proteina A stafilococcica con la libreria combinatoria espressa fagi, il bispecific ABDz1 molecola è stata identificata. <img alt = &quot;Figura 2&quot; src = &quot;/ files/ftp_upload/3370/3370fig2.jpg&quot; /&gt; Figura 2. Un diagramma di flusso semplificato per il protocollo di purificazione di affinità ortogonale. Una PCR-frammento contenente la sequenza ABDz1 è legatura (N-terminale) con un gene di interesse in un vettore di espressione adatta. Il prodotto legatura si trasforma in E. coli per la verifica di sequenza e la preparazione plasmide. A seguito di trasformazione di un host espressione, una grande cultura di espressione della proteina ricombinante è costituito da una cultura iniziale durante la notte. I batteri sono raccolte per centrifugazione e lisate mediante ultrasuoni per la produzione di lisato batterico contenente la proteina bersaglio. Dopo una fase di filtrazione per rimuovere residue particelle solide, il lisato è sottoposto a purificazione di affinità ortogonale su un sistema di gestione dei liquidi, subendo due passaggi di purificazione in successione. Picchi eluiti da entrambe le fasi di purificazione sono campionati e valutate da SDS-PAGE per valutare la purezza e rispetto al tha lisato prima di purificazione. <img alt="Figura 3" src="/files/ftp_upload/3370/3370fig3.jpg" /> Figura 3. SDS-PAGE analisi delle proteine ​​bersaglio espresse e purificate in fusione con ABDz1. I campioni prelevati dal lisato batterico di tre proteine ​​espresse in fusione di ABDz1, che rappresentano diverse proprietà, e il ABDz1 tag stesso sono stati raccolti insieme con i campioni dai picchi corrispondenti dalla purificazione su una colonna HSA-seguita da un MabSelect sicuro colonna (A) . Corsie 1-4 rappresentano campioni di lisati prima purificazione, corsie 5-8 dalla HSA-purificazione (prima fase) e corsie 9-12 dalla purificazione MabSelect sicuro (seconda fase). I campioni vengono caricati nel seguente ordine: ABDz1-141377, ABDz1-HT875, ABDz1-HT2375 e ABDz1. Inoltre, i campioni acquisiti dai lisati stesso purificato in ordine inverso sulle colonne stesse sono state analizzate (B). Corsie 1-3 rappresentano campioni di lisati prima purificazione, corsie 4-6 from MabSelect Sure-purificazione (prima fase) e corsie 7-9 da HSA-purificazione (seconda fase). I campioni sono stati caricati nello stesso ordine come in (A), ma il tag non è incluso. Da questi risultati è chiaro che questo metodo in due fasi produce proteine ​​altamente purificate indipendentemente dall&#39;ordine in cui vengono applicati i gradini. <table border="1"><tbody><tr><td> Nome b </td><td> Proteina bersaglio UniProt c </td><td> Molecolare peso (kDa) </td><td> Solubilità Classe D </td><td> Peso molecolare di prodotto di fusione (kDa) </td><td> Punto isoelettrico di prodotto di fusione </td></tr><tr><td> 141377 </td><td> B7Z315 </td><td> 17,4 </td><td> 4 </td><td> 23,7 </td><td> 8,5 </td></tr><tr><td> HT875 </td><td> P01040 </td><td> 10,8 </td><td> 3 </td><td> 17,1 </td><td> 4,9 </td></tr><tr><td> HT2375 </td><td> P00740 </td><td> 8,9 </td><td> 5 </td><td> 15,2 </td><td> 6,8 </td></tr></tbody></table><ol class="lalpha"><li> Peso molecolare di ABDz1 tag da solo è di 6,3 kDa, punto isoelettrico 6.7. </li><li> Proteina bersaglio rappresenta una parte della proteina UniProt. </li><li> <a href="http://www.uniprot.org">http://www.uniprot.org</a> . </li><li> Classe di solubilità di frammenti proteici con N-terminale del suo 6 SOA 10. </li></ol> Tabella 1. Proteine ​​bersaglio e prodotti di fusione con ABDz1 contrassegnare una valutazione in una prova di studio principio. Tre proteine ​​umano unico con diverso peso molecolare, solubilità e punto isoelettrico sono stati scelti per l&#39;espressione e la purificazione dai approccio affinità ortogonale.

<ol>
	<li>Waugh, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+the+most+of+affinity+tags.">Making the most of affinity tags.</a> Trends Biotechnol. 23, 316-320 (2005).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+engineering+strategies+for+selective+protein+purification.">Protein engineering strategies for selective protein purification.</a> CET. 28, 1315-1325 (2005).</li><li>Porath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+chelate+affinity+chromatography,+a+new+approach+to+protein+fractionation.">Metal chelate affinity chromatography, a new approach to protein fractionation.</a> Nature. 258, 206-213 (1975).</li><li>Linhult, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+the+interaction+between+albumin-binding+domain+from+streptococcal+protein+G+and+human+serum+albumin.">Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin.</a> Protein Sci. 11, 206-213 (2002).</li><li>Nilsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+IgG-binding+domain+based+on+staphylococcal+protein.">A synthetic IgG-binding domain based on staphylococcal protein.</a> A. Protein Eng. 1, 107-113 (1987).</li><li>Alm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+bispecific+protein+selected+for+orthogonal+affinity+purification.">A small bispecific protein selected for orthogonal affinity purification.</a> Biotechnol. J. 5, 605-617 (2010).</li><li>Nilsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+affinity+domains+for+the+detection,+purification+and+immobilization+of+recombinant+proteins.">Multiple affinity domains for the detection, purification and immobilization of recombinant proteins.</a> J. Mol. Recognit. 9, 585-594 (1996).</li><li>R&#252;ther, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pUR+250+allows+rapid+chemical+sequencing+of+both+DNA+strands+of+its+inserts.">pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts.</a> Nucleic Acids Res. 10, 5765-5772 (1982).</li><li>Kraulis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serum+albumin-binding+domain+of+streptococcal+protein+G+is+a+three-helical+bundle:+a+heteronuclear+NMR+study.">The serum albumin-binding domain of streptococcal protein G is a three-helical bundle: a heteronuclear NMR study.</a> FEBS Lett. 378, 190-194 (1996).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+flow+cytometry-based+method+for+analysis+of+expression+levels+in+E+coli+giving+information+about+precipitated+and+soluble+protein.">Novel flow cytometry-based method for analysis of expression levels in E coli giving information about precipitated and soluble protein.</a> J. Biotechnol. 119, 133-146 (2005).</li><li>Carter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+proteolysis+of+fusion+proteins.">Site-specific proteolysis of fusion proteins.</a> ACS Sympos. Ser. 427, 181-193 (1990).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negatively+charged+purification+tags+for+selective+anion-exchange+recovery.">Negatively charged purification tags for selective anion-exchange recovery.</a> PEDS. 17, 779-786 (2004).</li><li>Gr&#228;slund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+engineering+of+a+protein+domain+to+allow+efficient+ion-exchange+recovery.">Charge engineering of a protein domain to allow efficient ion-exchange recovery.</a> Protein Eng. 13, 703-709 (2000).</li></ol>

Nessun conflitto di interessi dichiarati.

Il protocollo di purificazione di affinità ortogonale qui presentata permette la purificazione efficiente di una vasta gamma di proteine ​​bersaglio. Combinando il sito inerente vincolante del albumina dominio di legame con una superficie romanzo vincolante, un tag piccola proteina bispecific è stato sviluppato. E &#39;molto semplice da utilizzare il tag dato che può semplicemente essere clonato ed espresso in fusione a qualsiasi proteina di interesse in qualsiasi vettore di espressione preferito. Equipaggiamento di serie disponibile nella maggior parte dei laboratori può essere impiegato per le due fasi di purificazione delle proteine. Poiché la funzione del tag ABDz1 dipende corretto ripiegamento del dominio per esporre in modo efficiente le sue due superfici vincolanti, il metodo è limitato alle proteine ​​espresse in forma solubile e non adatto per le purificazioni in condizioni di denaturazione. Agenti riduttori dovrebbero essere evitati in quanto interferiscono con il legame di ABDz1 alla Z-dominio sulla matrice MabSelect Certo. Ortogonali affinità depurazionezione fornisce una valida alternativa ai metodi tradizionali al fine di soddisfare le esigenze di proteine ​​altamente purificate in molte applicazioni esigenti. Allo stesso tempo, questo metodo elimina la necessità di tagging combinatorio quando le strategie di purificazione vengono utilizzati diversi in successione. Per applicazioni che richiedono una proteina nativa bersaglio, un sito di clivaggio della proteasi possono essere introdotti per rendere la rimozione enzimatica del tag ABDz1 possibili 11. Inoltre, il tag ABDz1 è il primo dominio bispecific piccola proteina e piegato descritto fino ad oggi. E &#39;unico in quanto fornisce una strategia di purificazione che dipende da due interazioni affinità target specifici. In futuro sarebbe interessante estendere questo concetto attraverso lo sviluppo di nuovi tag bispecific che portano nuove superfici vincolanti che sono compatibili con resine cromatografiche prontamente disponibili e poco costoso. Per esempio, sostituendo gli aminoacidi coinvolti nel legame con l&#39;albumina con residui di base, un tag compatibile con isulla cromatografia a scambio può essere realizzabile. Un concetto simile è stato dimostrato efficace l&#39;ingegneria incaricato del Z-dominio per generare tag conosciuto come acido Z 12 e Z 13 di base compatibile con anioni e cationi di scambio, rispettivamente.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> E. coli RR1ΔM15 </td><td> Tipo americano Culture Collection </td><td> 35102 </td></tr><tr><td> E. coli Rosetta (DE3) </td><td> Novagen </td><td> 70954 </td></tr><tr><td> Trittico di soia brodo </td><td> Difco </td><td> 211822 </td></tr><tr><td> Estratto di lievito </td><td> Difco </td><td> 212720 </td></tr><tr><td> Vibra cellula ultrasuoni </td><td> Sonics e dei materiali </td><td> - </td></tr><tr><td> HSA Sepharose </td><td> Pharmacia Biotech </td><td> Prodotto ex </td></tr><tr><td> HITRAP Certo MabSelect </td><td> GE Healthcare </td><td> 11-0034-93 </td></tr><tr><td> HITRAP NHS-attivato HP colonna </td><td> GE Healthcare </td><td> 17-0716-01 </td></tr></tbody></table>

orthogonal protein purification facilitated by a small bispecific affinity tag

A causa dei costi elevati associati alla purificazione di proteine ​​ricombinanti i protocolli devono essere razionalizzate. Per high-throughput sforzi vi è una domanda generale per i metodi che non richiedono l&#39;ottimizzazione specifica proteina bersaglio 1. Per raggiungere questo obiettivo, i tag di depurazione che possono essere geneticamente fuso con il gene di interesse sono comunemente usati 2. La maniglia affinità più usato è il esa-istidina tag, che è adatto per la depurazione sia in condizioni native e denaturanti 3. L&#39;onere metabolici per produrre il tag è bassa, ma non fornisce come alta specificità come concorrenti cromatografia basata 1,2 strategie di affinità. Qui, un tag purificazione bispecific con due diversi siti di legame su un acido 46 aminoacidi, proteine ​​piccolo dominio è stato sviluppato. L&#39;albumina dominio di legame deriva dalla proteina G streptococco e ha una forte affinità inerente alla albumina sierica umana(HSA). Undici superficie esposta aminoacidi, non coinvolti nel legame albumina-4, sono stati randomizzati geneticamente per produrre una libreria combinatoria. La biblioteca di proteine ​​con il romanzo casualmente disposti superficie vincolante (Figura 1) è stato espresso sulle particelle dei fagi per facilitare la selezione dei leganti dalla tecnologia phage display. Attraverso varie tornate di biopanning contro un dimerica Z-dominio indicato da proteine ​​Staphylococcal A 5, una piccola molecola bispecific con affinità per i HSA e il nuovo bersaglio è stato identificato 6. Il dominio nuova proteina, denominata ABDz1, è stata valutata come un tag di depurazione per una selezione di proteine ​​bersaglio con diverso peso molecolare, solubilità e punto isoelettrico. Tre proteine ​​bersaglio sono stati espressi in Escherichia Escherishia con il tag romanzo fusi alla loro N-termini e successivamente purificato per affinità. Purificazione iniziale su una colonna con immobilizzato HSA o Z-dominio ha portato in relazionvamente prodotti puri. In due fasi purificazione di affinità con il tag bispecific determinato un sostanziale miglioramento della purezza delle proteine. Mezzi di comunicazione cromatografica con la Z-dominio immobilizzato, per esempio MabSelect Certo, sono facilmente disponibili per la purificazione degli anticorpi e HSA può essere facilmente accoppiato chimicamente ai media per fornire la matrice secondo. Questo metodo è particolarmente vantaggioso quando c&#39;è una forte domanda sulla purezza della proteina bersaglio recuperato. Il bifunctionality del tag consente due diverse fasi cromatografiche da utilizzare, mentre il peso metabolico sull&#39;host espressione è limitata a causa delle ridotte dimensioni del tag. Fornisce un&#39;alternativa competitiva ai cosiddetti tag combinatoria dove più tag vengono utilizzati in combinazione 1,7.

Watch this Scientific Journal Video about Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag at JoVE.com

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Un romanzo e altamente efficiente in due fasi cromatografia di affinità protocollo è stato sviluppato ed è descritto in dettaglio. Il metodo si basa su un tag purificazione con due affinità intrinseca ed è applicabile ad una vasta gamma di proteine ​​bersaglio con proprietà diverse.

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 ...

orthogonal-protein-purification-facilitated-small-bispecific-affinity

Research

JoVE Journal

Biology

16.9K Views.  Royal Institute of Technology. Un romanzo e altamente efficiente in due fasi cromatografia di affinità protocollo è stato sviluppato ed è descritto in dettaglio. Il metodo si basa su un tag purificazione con due affinità intrinseca ed è applicabile ad una vasta gamma di proteine ​​bersaglio con proprietà diverse.

Video: Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

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

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolamento di Labile Multi-proteina complessi da In vivo Controllato Cellular Cross-Linking e Immuno-magnetica Cromatografia di affinità

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

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 ...

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificazione di complessi proteici in<em&gt; Escherichia coli</em&gt; Con sequenziale purificazione per affinità Peptide in combinazione con Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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 ...

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

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small 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. ...

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. ...

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

Purificazione delle proteine ortogonali Facilitato da una piastrina Affinity Bispecific