Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH under award number R01AI114362 and Welch Foundation grant I-1782 to Iv&#225;n D'Orso.

1. Cellule placcatura <ol><li> Un giorno prima della trasfezione, piastra di 3,5 x 10 6 cellule HEK293T in 10 mL di Dulbecco Modified Eagle Medium (DMEM) supplementato con 10% (v / v) di siero fetale bovino (FBS) e 1% (v / v) di penicillina / streptomicina (10.000 U / ml magazzino) per ogni piatto a 100 mm. Tavola 3 - 5 100 piatti mm per esperimento / condizione per garantire il recupero di proteine ​​sufficiente. NOTA: Il numero di lastre deve essere determinato per ogni esperimento / condizione, che si baserà sui livelli di espressione e la solubilità del complesso proteina di interesse. In alternativa, se è necessaria la purificazione scala ingrandita, linee cellulari possono essere adattati a crescere in sospensione in fiaschi filatore 2, ma questo metodo non funziona per trasfezioni transitorie. </li></ol> 2. transfecting cellule o indurre linee cellulari stabili <ol><li> Il giorno seguente, controllare le cellule sotto il microscopio. Le cellule devono essere 70 - 80% confluenti prima proceEding. </li><li> Trasfezione delle cellule con una miscela totale di 7 mg di DNA plasmidico e 21 ml di reagente per trasfezione piatto a 100 mm. Cellule trasfezione con un vettore che esprime GFP (Tabella 1) come controllo per l&#39;efficienza di trasfezione. NOTA: Se si utilizza un HEK293-T-Rex o simile linea cellulare stabile che induce l&#39;espressione di due o più complessi subunità in risposta alla doxiciclina 14, 15, l&#39;induttore dovrà essere aggiunto alle cellule e incubate per 48 ore. Analogamente, una linea cellulare stabile che induce l&#39;espressione di GFP dopo l&#39;aggiunta del induttore sarebbe necessaria anche per scopi di convalida. Passare al punto 2.7. </li><li> Preparare la miscela di trasfezione per ogni piatto a 100 mm. In una provetta da 1,5 ml microcentrifuga, miscelare 500 ml di DMEM senza supplementi con 7 mg di DNA plasmidico totale (corrispondente a due o più plasmidi). In una provetta separata, mescolare 500 ml di DMEM senza supplementi with 21 ml di reagente di trasfezione. Ripetere l&#39;operazione per ogni reazione trasfezione. </li><li> Pipetta la soluzione di trasfezione del reagente nella soluzione plasmide DNA (non mescolare soluzioni in ordine inverso). Mescolare pipettando almeno 4 volte. </li><li> Incubare la miscela a temperatura ambiente per 10 - 15 min, ma non più di 15 min. </li><li> La piastra per creare un serbatoio media e accuratamente pipetta la miscela di trasfezione goccia a goccia sul lato interno della piastra senza disturbare le cellule. Riportare il piatto nella posizione piatta originale e miscelare delicatamente per distribuire il composto. </li><li> Incubare le cellule a 37 ° C in un incubatore umidificato coltura cellulare con 5% di CO 2 (questa condizione è seguito indicato come &quot;coltura tissutale incubatore&quot;). Sostituire i media 5 ore dopo la trasfezione (opzionale). </li></ol> 3. Controllo Transfection o induzione efficienza <ol><li> A 24 ore dopo la trasfezione, visualizzare le cellule in una influenzaMicroscopio orescent per verificare l&#39;efficienza di trasfezione (o induzione di controllo HEK293T-Rex linea cellulare stabile che esprimono GFP). Incubare per altre 24 ore. </li></ol> 4. STREP Affinity Purification (STREP AP) <ol><li> Raccogliere le cellule <ol><li> A 48 ore dopo la trasfezione, rimuovere il supporto e aggiungere 8 ml di soluzione fisiologica fredda 1x tampone fosfato (PBS). Pipettare su e giù per staccare le cellule dalla piastra. </li><li> Raccogliere e trasferire la sospensione cellulare in una provetta da 15 ml e girare le celle in basso a 800 xg per 4 minuti a 4 ° C. </li><li> Rimuovere il surnatante PBS. Ripetere la rotazione verso il basso (800 xg per 1 min a 4 ° C) per eliminare eventuali residui di PBS. Conservare il pellet a -80 ° C per un uso futuro o seguire la procedura qui sotto per continuare purificazione della proteina. </li></ol></li><li> Lisi delle cellule <ol><li> Risospendere il pellet di cellule utilizzando 5 × volume pellet cellulare (~ 250 microlitri per piastra) di Passive Lysis Buffer(PLB: 20 mM Tris-HCl pH 7,5, 150 mM NaCl, 1,5 mM MgCl 2, 1 mM DTT, proteasi tablet inibitore cocktail, 5% v / v di glicerolo, e 1,0% NP-40) e trasferire la sospensione cellulare in un 1,5 ml provetta. </li><li> Brevemente vortice sospensione e nutate per 30 minuti a 4 ° C. </li><li> Centrifugare la sospensione cellulare a 10.000 xg per 10 min a 4 ° C. </li><li> Trasferire i lisati solubili in una nuova provetta da 1,5 ml e scartare il pellet di cellule. Risparmi 10% del volume finale del lisato solubile &quot;STREP AP Input.&quot; Conservare a 4 ° C. </li></ol></li><li> Equilibrando le perline STREP NOTA: Utilizzare STREP perline per il primo passo di AP! Complessi tirato giù con perline STREP recuperare significativamente più proteine ​​di quelle abbattute con perline FLAG. <ol><li> Estrarre (n × 40 ml) + n ml di STREP tallone slurry 50% (n = numero di campioni) e spin down a 1.500 xg per 2 minuti a 4 ° C. NOTA: utilizzare in tutto il mpunte outhed a perline pipetta. </li><li> Rimuovere il surnatante e aggiungere 500 ml di PLB ai talloni. Nutate la miscela perline per 5 minuti a 4 ° C. Spin down a 1.500 xg per 2 minuti a 4 ° C. <ol><li> Ripetere per un totale di 3 lavaggi. Risospendere le sfere in PLB ad un volume finale di n × 40 microlitri. </li></ol></li><li> Aggiungere 40 ml di 50% STREP tallone slurry per ogni campione lisato cellulare e nutate per 2 ore a 4 ° C. </li></ol></li><li> STREP AP <ol><li> Spin down soluzione a 1.500 xg per 2 minuti a 4 ° C. </li><li> Rimuovere il surnatante e conservare come &quot;STREP AP flusso-through (FT)&quot; a 4 ° C. </li><li> Aggiungere 500 pl di STREP AP Wash Buffer I (20 mM Tris-HCl pH 7,5, 250 mM NaCl, 1,5 mM MgCl 2, 1 mM DTT, 5% glicerolo e 0,2% NP-40) ai talloni. Nutate la miscela perline per 5 minuti a 4 ° C e centrifugare a 1500 xg per 2 minuti a 4 ° C. Eliminare il surnatante. <ol><li> Ripetere per un totale di 4 lavaggi. <br/&gt; NOTA: Prima quarto lavaggio, trasferire la soluzione perline in una nuova provetta da 1,5 ml ridurre lo sfondo proteine. Questo passaggio è fondamentale. </li></ol></li><li> Aggiungere 500 microlitri di tampone di lavaggio II (100 mM Tris-HCl pH 8,0, 150 mM NaCl, e 1 mM EDTA) ed equilibrare le perline invertendo i tubi. Spin down a 1.500 xg per 2 minuti a 4 ° C e scartare il surnatante. </li><li> Eluire la proteina di interesse con 50 ml di tampone di eluizione STREP (100 mM Tris-HCl pH 8,0, 150 mM NaCl, 1 mM EDTA e 2,5 mM Desthiobiotin). Vortex a 800 rpm per 15 min a 4 ° C. </li><li> Spin giù le perline a 1.500 xg per 1 min a 4 ° C. Raccogliere il surnatante. Tale quota corrisponde al campione &quot;STREP AP Elution&quot;. </li><li> Salvare la frazione perline a 4 ° C per una seconda eluizione, che potrebbe produrre fino a metà della quantità di proteina ottenuta con la prima eluizione. Aliquotare 10% del STREP AP Elution per la colorazione argento e / o western blotting16. </li></ol></li><li> Equilibrando le perline FLAG <ol><li> Estrarre (nx 16 ml) + n ml di soluzione di FLAG perline (n = numero di campioni) e spin down a 1.500 xg per 2 minuti a 4 ° C. NOTA: utilizzare punte bocca larga per perline pipetta. </li><li> Rimuovere il surnatante e aggiungere 500 ml di tampone di lavaggio FLAG (100 mM Tris-HCl pH 8,0, 150 mM NaCl, 1 mM EDTA e 0,1% NP-40) ai talloni. Spin down a 1.500 xg per 2 minuti a 4 ° C. Ripetere per un totale di 3 lavaggi. </li><li> Risospendere le sfere in tampone di lavaggio FLAG ad un volume finale di nx 16 ml. </li><li> Aggiungere 16 ml di liquame FLAG tallone per il restante eluizione STREP AP e pernottamento nutate a 4 ° C. </li></ol></li></ol> 5. FLAG IP <ol><li> Spin giù la sospensione perline FLAG a 1.500 xg per 2 minuti a 4 ° C. Salvare il surnatante e conservare come la &quot;FLAG IP Flow-through&quot; (FT) a 4 ° C. </li><li> Aggiungere 50081; l di tampone di lavaggio FLAG alla soluzione. Nutate per 5 minuti a 4 ° C e centrifugare a 1500 xg per 2 minuti a 4 ° C. Ripetere per un totale di 4 lavaggi e rimuovere il surnatante dopo ogni lavaggio. NOTA: prima il quarto lavaggio, trasferire la soluzione proteica-perline in una nuova provetta da 1,5 ml per ridurre sfondo. Questo passaggio è fondamentale. </li><li> Rimuovere il surnatante dalla centrifuga finale e aggiungere 30 ml di tampone di lavaggio FLAG contenente 200 ng / ml di FLAG peptide nelle perline. Vortex a 800 rpm per 2 ore a 4 ° C. </li><li> Centrifugare la sospensione a 1.500 xg per 2 minuti a 4 ° C. Raccogliere il surnatante e conservare a 4 ° C come la &quot;FLAG IP eluizione.&quot; NOTA: I campioni di eluizione possono essere confermate con macchia d&#39;argento e / o Western Blot utilizzando protocolli standard di 16, e sono pronti per ulteriori analisi di proteine. Quando si imposta un Western Blot, eseguire tutti i seguenti campioni: (1) STREP AP Ingresso, (2) STREP AP FT, (3) STREP AP Elutisu, (4) FLAG IP FT, e (5) bandiera IP eluizione. I volumi caricati dovranno essere determinati per ogni esperimento. Tutti i campioni raccolti e perline possono essere conservati a 4 ° C per una conservazione a breve termine e a -80 ° C per una conservazione a lungo termine. Campioni TAP possono essere analizzati mediante spettrometria di massa per verificare l&#39;identità dei loro componenti, per identificare nuove modificazioni post-traduzionali (PTM), e / o per scoprire ogni interazione proteina romanzo con il complesso purificato 2, 17. A tale scopo, dopo l&#39;esecuzione di un gel Coomassie o argento macchia, bande possono essere tagliate dal gel utilizzando un bisturi pulito. Diversi ottime recensioni che discutono i metodi di spettrometria di massa sono stati pubblicati in precedenza 18, 19. </li></ol>

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The authors have nothing to disclose.

Il protocollo qui descritto per l&#39;espressione e l&#39;isolamento di complessi proteici da cellule eucariotiche non è limitata alla caratterizzazione biochimica di tali complessi molecolari, ma può anche essere utilizzato per l&#39;identificazione di nuovi interattori e modificazioni post-traduzionali che potrebbe regolare la loro funzione. L&#39;utilizzo di tag di affinità non si limita a ciò che è menzionato in questo protocollo; ma la nostra esperienza suggerisce che l&#39;utilizzo STREP come primo passo AP m...

<html> Interazioni proteina-proteina (PPI) sono fondamentali per la precisa regolazione dei processi biologici 1, e ulteriori studi su questi inibitori della pompa protonica può dare informazioni relative la loro funzione 2. Diversi approcci sono stati ideati per lo studio e la caratterizzazione di IPP e per l&#39;identificazione de novo di nuove componenti proteici normativi. Nel 1989, Stanley Campi e colleghi hanno riportato il lievito doppio ibrido (Y2H) test 3. Questo approccio consente l&#39;identificazione imparziale e completa di interattori (prede) per una proteina definito di interesse (esche) in Saccharomyces cerevisiae. Oltre alla sua notevole utilità per scoprire IPP, il saggio Y2H può essere utilizzato per caratterizzare coppie di proteine ​​in cellule di lievito, definendo domini interagenti minime, e identificare mutazioni che aboliscono tali interazioni. Modificando il test Y2H, inibitori della pompa protonica può anche essere studiata in cellule di mammiferoclass = &quot;xref&quot;&gt; 4. Variazioni del dosaggio Y2H (ad esempio, il sistema di lievito tre ibrido) possono essere applicate anche a studiare proteina-RNA e proteina-piccola interazioni legante organico nelle cellule. Un altro strumento comunemente utilizzato per studiare PPI in un sistema omologo è il test 5 co-immunoprecipitazione (co-IP). Utilizzando un anticorpo per immunoprecipitare una proteina di interesse, il test di co-IP permette ai ricercatori di monitorare PPI nelle celle per diverse condizioni ambientali e situazioni sperimentali. L&#39;uso di proteine epitopi-tag (ad esempio, la bandiera, Myc, STREP, e HA, tra gli altri) in purificazione di affinità (AP) metodi ha facilitato l&#39;isolamento di proteine da miscele complesse di proteine per diversi saggi a valle, tra cui Western Blot, macchia d&#39;argento , e l&#39;analisi enzimatica. Tuttavia, nessuno di questi approcci precedenti consentire l&#39;isolamento di grandi quantità di complessi proteici per un&#39;ulteriore caratterizzazione compreso in vitro unssays, scoperta di subunità regolatorie mediante spettrometria di massa, e l&#39;identificazione di modificazioni post-traslazionali. Una versione migliorata del metodo AP viene chiamato Tandem AP (TAP), che è una tecnica di purificazione per studiare IPP creando una proteina di fusione con due epitopi che viene purificato mediante due AP successive 6, 7. In questo articolo, vi presentiamo una variante del metodo TAP per purificare complessi proteici in cui due subunità sono contrassegnati con diversi epitopi e poi purificato attraverso due punti di accesso sequenziali (STREP AP seguiti da FLAG IP). In primo luogo abbiamo a disposizione una panoramica minimalista di TAP (figura 1) e poi una descrizione dettagliata di tutte le fasi sperimentali (figura 2), in modo che i ricercatori li possono applicare alla loro complesso proteina di interesse. Per dimostrare l&#39;applicabilità del metodo di TAP, abbiamo scelto un ben caratterizzato ciclina-CDK complesso (di seguito PTEFB chinasi), che è composto della ciclina T1 subunità regolatrice (CycT1) e una chinasi (CDK9), ed è coinvolto nella regolazione della trascrizione di RNA polimerasi II (Pol II) 8, 9, 10. P-TEFb fosforila il dominio C-terminale di Pol II e dei suoi fattori di allungamento negativi associati, che allevia la pausa trascrizionale al promotore e facilita in tal modo la trascrizione allungamento 11, 12, 13. Con questa interazione conosciuta in mente, STREP-tag CycT1 e CDK9 FLAG-tag sono stati oltre espresso in cellule HEK293T. Un esperimento TAP reciproca è stata eseguita con CDK9 STREP-tag e FLAG-tagged CycT1 per convalidare ulteriormente che l&#39;interazione proteina è indipendente dalle epitopi utilizzati. Le cellule sono state raccolte e lisate 48 ore dopo la trasfezione. Il lisato solubile è stato purificato dalla TAP (STREP AP seguito da FLAGIP). Input e proteine purificate sono state analizzate mediante western blot e macchia d&#39;argento (Figura 3).

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH3002.2FS</td><td></td></tr><tr><td>Fetal bovine serum</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH30071</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>MP Biomedicals</td><td>MP091670049</td><td></td></tr><tr><td>PolyJet</td><td>SignaGen Laboratories</td><td>SL100688</td><td></td></tr><tr><td>Protease inhibitor cocktail&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>11836153001</td><td></td></tr><tr><td>STREP-Tactin Superflow</td><td>IBA Lifesciences</td><td>2-1208-010</td><td></td></tr><tr><td>STREP-tag elution buffer</td><td>IBA Lifesciences</td><td>2-1000-025</td><td></td></tr><tr><td>EZview Red ANTI-FLAG M2 Affinity Gel</td><td>Sigma-Aldrich</td><td>F2426</td><td></td></tr><tr><td>Corning 100 mm &amp;times; 20 mm style dish cell culture treated nonpyrogenic polystyrene 20/sleeve</td><td>Corning&amp;nbsp;</td><td>430167</td><td></td></tr><tr><td>Protein Lo-Bind Eppendorf</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific&amp;nbsp;</td><td>02-215-370</td><td></td></tr><tr><td>48 hole micro tube foam rack</td><td>Fisher Scientific</td><td>02-215-386</td><td></td></tr><tr><td>Labquake shaker rotisserie&amp;nbsp;</td><td>Thermo&amp;nbsp;</td><td>415110</td><td></td></tr></tbody></table>

tandem affinity purification of protein complexes from eukaryotic cells

La purificazione della proteina-proteina attiva e complessi acido nucleico-proteina è cruciale per la caratterizzazione delle attività enzimatiche e de novo identificazione di nuovi subunità e modificazioni post-traduzionali. sistemi batterici permettono l&#39;espressione e la purificazione di una grande varietà di singoli polipeptidi e proteine ​​complessi. Tuttavia, questo sistema non consente la purificazione di subunità proteiche che contengono modificazioni post-traduzionali (ad esempio, fosforilazione e acetilazione), e l&#39;identificazione di nuovi subunità regolatorie che sono presenti solo / espressi nel sistema eucariotico. Qui, forniamo una descrizione dettagliata di un romanzo, robusto, ed efficiente metodo di purificazione tandem affinità (TAP) utilizzando STREP- e le proteine ​​FLAG-tag che facilita la purificazione di complessi proteici con transitoriamente o stabilmente espresso proteine ​​epitopi-tag da cellule eucariotiche. Questo protocollo può essere applicata per caratterizzare protEin funzionalità complesse, alla scoperta di modificazioni post-traduzionali su subunità complesse, e per identificare nuovi componenti complessi normativi mediante spettrometria di massa. In particolare, questo metodo TAP può essere applicato per studiare complessi proteici formati da componenti eucariotiche o patogeni (virali e batteriche), ottenendo così una vasta gamma di possibilità sperimentali valle. Proponiamo che i ricercatori che lavorano con i complessi proteici potrebbero utilizzare questo approccio in molti modi diversi.

In questo articolo, abbiamo dimostrato l&#39;applicabilità del metodo TAP per il ben caratterizzato CycT1-CDK9 complesso (noto anche come P-TEFb chinasi). I plasmidi codificanti ciclina T1-STREP (CycT1: S) e CDK9-FLAG (CDK9: F), o CDK9-STREP (CDK9: S) e ciclina T1-FLAG (CycT1: F) (Tabella 1), sono state trasfettate in cellule HEK293T . I controlli negativi inclusi trasfezioni con un vettore vuoto e il CDK9: F...

Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Tandem Affinity Purification di complessi proteici da cellule eucariotiche

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

tandem-affinity-purification-protein-complexes-from-eukaryotic

Research

JoVE Journal

Biochemistry

14.9K Views.  The University of Texas Southwestern Medical Center. We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Video: Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Proteine ​​Complesso affinità Acquisizione da cellule di mammifero Cryomilled

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Ricerca

Biochimica

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Risoluzione purificato per affinità complessi proteici di Blue Native PAGE e proteine ​​correlazione Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 in 1: purificazione di affinità per l'analisi parallela di proteina-proteina e proteina-metabolita complessi in un unico passaggio

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Purificazione di affinità del cloroplasto Translocon proteina complessi usando il Tag di rubinetto

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

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 ...

Biologia

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 ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Il MultiBac Protein Complex Piattaforma di produzione al EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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. ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Purificazione di Native Complessi di studio strutturale utilizzando un metodo Tag Tandem Affinity

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://app.jove.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

Saggio di purificazione di affinità tandem per studiare le interazioni proteina-proteina

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...

Tandem Affinity Purification di complessi proteici da cellule eucariotiche

Riepilogo

Esplora Altri Video

Capitoli in questo video

Proteine Complesso affinità Acquisizione da cellule di mammifero Cryomilled

Risoluzione purificato per affinità complessi proteici di Blue Native PAGE e proteine correlazione Profiling

2 in 1: purificazione di affinità per l'analisi parallela di proteina-proteina e proteina-metabolita complessi in un unico passaggio

Purificazione di affinità del cloroplasto Translocon proteina complessi usando il Tag di rubinetto

Individuazione di partner proteine interagenti mediante Tandem Affinity Purification

Identificazione di complessi proteici in

Il MultiBac Protein Complex Piattaforma di produzione al EMBL

Identificare proteina-proteina interazione in

Purificazione di Native Complessi di studio strutturale utilizzando un metodo Tag Tandem Affinity

Saggio di purificazione di affinità tandem per studiare le interazioni proteina-proteina

Tandem Affinity Purification di complessi proteici da cellule eucariotiche

Riepilogo

Esplora Altri Video

Capitoli in questo video

Proteine ​​Complesso affinità Acquisizione da cellule di mammifero Cryomilled

Risoluzione purificato per affinità complessi proteici di Blue Native PAGE e proteine ​​correlazione Profiling

2 in 1: purificazione di affinità per l'analisi parallela di proteina-proteina e proteina-metabolita complessi in un unico passaggio

Purificazione di affinità del cloroplasto Translocon proteina complessi usando il Tag di rubinetto

Individuazione di partner proteine ​​interagenti mediante Tandem Affinity Purification

Identificazione di complessi proteici in

Il MultiBac Protein Complex Piattaforma di produzione al EMBL

Identificare proteina-proteina interazione in

Purificazione di Native Complessi di studio strutturale utilizzando un metodo Tag Tandem Affinity

Saggio di purificazione di affinità tandem per studiare le interazioni proteina-proteina

Proteine Complesso affinità Acquisizione da cellule di mammifero Cryomilled

Risoluzione purificato per affinità complessi proteici di Blue Native PAGE e proteine correlazione Profiling

Individuazione di partner proteine interagenti mediante Tandem Affinity Purification