Questo lavoro è stato sostenuto da un progetto Wellcome Grant (078043/Z/05/Z) a ROJW

A PARTE: High-throughput crescita di colture batteriche. 1. Crescere batteri Pernottamento in 2 ml di terreno autoinduzione Utilizzando piastre da 24 pozzetti <ol><li> Sterilizzare le piastre da 24 pozzetti da microonde. </li><li> Inoculare 1,5 ml di mezzo di autoinduzione (Overnight Medio Express) con appena cresciute colonie batteriche o stock glicerolo congelati. Di solito inoculare tre pozzi per mutante con tre singole colonie clonati. Riserva sei pozzetti per i controlli positivi e negativi. Per i controlli positivi si cresce tre cloni di tipo selvatico e per i controlli negativi si cresce 2 cloni che sono state trasformate con un plasmide che esprime non. Controllare per la sterilizzazione sufficiente della piastra lasciando un pozzetto vuoto per il medio-solo il controllo. </li><li> Crescere le cellule per 18 ore a 37 ° C e agitazione a 250 rpm. Usiamo lac-inducibile BL21 (DE3) Rosetta 2 celle. Medie autoinduzione contiene una miscela di glucosio e lattosio. I batteri inizialmentenutrono di glucosio e quindi iniziare ad utilizzare il lattosio, che induce anche l&#39;espressione delle proteine ​​ricombinanti. </li><li> Togliere il coperchio e posizionare la piastra sulla piattaforma robotica. </li></ol> PARTE B: Robotic purificazione di proteine ​​ricombinanti. 2. Preparare la piattaforma robotica <ol><li> Compongono il tampone costituito da 20 mM imidazolo, 0,1% Triton X-100, 0,5 M NaCl, 20 mM Tris-acetato, pH 7,9, 10 mM MgOAc 2, 0,7 mM ZnOAc 2, 10% glicerolo, e il tampone di eluizione costituito di 0,5 M imidazolo, 0,1% Triton X-100, 0,5 M NaCl, 20 mM Tris-acetato, pH 7,9, 10 mM MgOAc 2, 0,7 mM ZnOAc 2, 10% glicerolo. Questi buffer verrà utilizzato per lavare le perle dopo le proteine ​​marcate sono stati legati a loro, o per eluire le proteine ​​dalle perline, rispettivamente. </li><li> Portare il diluente batterico (100 ml di acqua distillata con 15 microlitri di reagente antischiuma). Questa soluzione sarà utilizzataeffettuare delle diluizioni delle colture batteriche durante la notte per la densità ottica (A600) misurazioni. La presenza di antischiuma nel diluente previene la formazione di bolle d&#39;aria che potrebbero interferire con le misurazioni lettore a piastra. </li><li> Portare la soluzione di lisi composto da reagente contropiede 10x, 2 lysonase microlitri per ogni campione e 15 mM MgOAc 2. Fastbreak contiene una miscela di detergenti e sali che rompono le pareti cellulari batteriche e facilitare il rilascio di proteine ​​intracellulari. Lysonase è una miscela brevettata di lisozima e una nucleasi. Lisozima aiuta a distruggere la parete cellulare e la nucleasi digerisce le Liberatoria acidi nucleici batterici. </li><li> Opzionale: Fare una M 6 soluzione cloridrato di guanidina. Alcune proteine ​​ricombinanti tendono ad attaccarsi agli aghi di pipettaggio rivestiti in teflon. Il cloridrato di guanidina soluzione denatura le proteine ​​e si lava di dosso più efficacemente di acqua che viene solitamente usata per sciacquare i lavabili suggerimenti robotici pipettaggio dopo ogni step. </li><li> Preparare la (acido bicinconinico) BCA reagente quantificazione delle proteine ​​miscelando una soluzione di acido bicinconinico (reagente A) e la soluzione di solfato di rame (reagente B): legami peptidici ridurre Cu 2 + dal componente 4 CuSO presente nel reagente BCA di Cu 1 + cui la quantità di Cu 1 + è proporzionale al numero di legami peptidici presenti nella soluzione. In una seconda fase, due molecole di acido bicinconinico chelato Cu 1 + conseguente cambiamento di assorbanza a 562 nm, risultante in un colore viola. La reazione del colore è dipendente dal tempo e ha bisogno di solito diverse ore per essere definitivo. Dopo che il colore è stabile per diverse ore. </li><li> Riempire abbeveratoi o piatti della giusta misura e metterli nelle loro posizioni pre-assegnati sulla piattaforma robotica. </li><li> Posizionare una piastra a 24 pozzetti per i rifiuti guanidina cloridrato, due lastre trasparenti a 96 pozzetti per OD 600 e misurazioni di assorbanza e una blu piastra a 96 pozzetti per the proteine ​​purificate sulle loro posizioni sulla piattaforma. Posizionare un 96-Deepwell piastra sul supporto magnetico. </li><li> Diluire MagneHis Ni-particelle che si legano le proteine ​​formando chelati con i loro suo-tag 5 volte in acqua distillata e riempirli nel tallone-agitazione unità. Accendere l&#39;agitatore a mantenere le microsfere in sospensione. </li><li> Accendere la piattaforma robotica e lavare gli aghi di dispensazione per alcuni minuti per eliminare le bolle d&#39;aria che altrimenti interferire con la precisione di pipettaggio. Gli aghi riutilizzabili pipettaggio sono lavata tra le fasi di pipettamento individuali. In alternativa, puntali monouso potrebbe essere utilizzato. Tutte le fasi successive sono eseguite robot. Il protocollo robot è disponibile su richiesta. </li></ol> 3. La crescita cellulare è verificato misurando la OD600 <ol><li> 10 pl di coltura durante la notte è diluito in 90 ml di soluzione diluente. </li><li> Misurare la OD 600 per garantire che i batteri sono cresciuti a densità simili(Figura 1). </li></ol> 4. Le cellule sono rotto fino a rilasciare le proteine ​​e per consentire Bead-binding <ol><li> 100 pl di sospensione magnetica Ni-bead è distribuito in ciascun pozzetto di una seconda piastra a 24 pozzetti. </li><li> 900 microlitri di ogni piccola coltura batterica è trasferito nella piastra a 24 pozzetti contenenti le perline. Aggiungere 100 microlitri della 10x contropiede / lysonase mix. </li><li> Il 24-pozzetti viene trasferito alla piattaforma agitazione per scuotimento rapido (800 rpm) per 30 minuti a temperatura ambiente. Pareti cellulari batteriche sono interrotti da una combinazione di forze meccaniche e agli agenti chimici e le proteine ​​ricombinanti prodotte vengono rilasciati in soluzione. Con i loro tag di affinità che legano immediatamente le perline chelanti nichel paramagnetiche. </li></ol> 5. Le perline vengono lavati <ol><li> Gli lisati cellulari contenenti le microsfere in sospensione magnetici sono trasferiti ad un 96-Deepwell Plate che è posizionato su un supporto con maste agnetica che scorrono tra i pozzetti. Le piastre a 96 pozzetti hanno ottenuto un quadrato forma conica del fondo, che facilita la rimozione del supernatante. I pozzetti possono contenere volumi fino a 2,1 ml, ma sono pieni di volumi molto piccoli. Questo ci permette di effettuare misure vigorose stringono senza campione di cross-contaminazione da spruzzi. </li><li> I puntali vengono lavati tra le singole fasi di pipettamento con 6 M guanidina-HCl. Questo passaggio è fondamentale per la purificazione di TFIIB e altri &quot;sticky&quot; proteine ​​per evitare la contaminazione incrociata. Con altre proteine ​​può essere sufficiente per risciacquare gli aghi estensivamente con acqua tra le fasi di pipettamento. </li><li> Le barre magnetiche del supporto magnetico attirare le perline paramagnetiche, strappandoli i centri della pozzetti e lasciare gli aghi pipettaggio libero accesso al supernatante. Il supernatante viene scartato. </li><li> 500 pl di tampone di lavaggio prima aggiunta al 24-pozzetti e successivamente trasferito alla piastra a 96 pozzetti a eNsure il completo trasferimento delle perle. Il 24-pozzetti viene rimosso dal shaker. </li><li> Altri 500 pl di tampone di lavaggio viene aggiunto direttamente alla piastra a 96 pozzetti, la piastra viene trasferito shaker e agitare vigorosamente per 1 min. La piastra viene riportato al supporto magnetico e il tampone di lavaggio scartato. </li><li> Questo passaggio viene ripetuto due volte e la procedura di lavaggio è finito rimuovendo eventuali residui tampone dalla piastra. </li></ol> 6. Eluire le proteine ​​in tampone di eluizione <ol><li> 100 pl di tampone di eluizione viene aggiunto ai talloni, la piastra viene spostato shaker e agitare vigorosamente per 30 min a temperatura ambiente. </li><li> La piastra viene riportato l&#39;agitatore e l&#39;eluato, contenente la proteina ricombinante purificata, viene trasferito in una nuova piastra. </li></ol> 7. Misurare le concentrazioni di proteine ​​purificate <ol><li> 190 pl di miscela di reagente BCA viene trasferito ad una chiara piastra da 96 pozzetti e 10 pl della proproteina soluzione aggiunta. </li><li> Dopo diverse ore di incubazione (tipicamente 5-6 o durante la notte), misurare l&#39;assorbanza e confrontarlo con standard BSA per determinare le concentrazioni di ciascuna delle preparazioni proteina purificata (Figura 2). </li><li> Le proteine ​​purificate possono essere utilizzati per applicazioni a valle, alle concentrazioni appropriate (Figura 3). </li></ol> 8. Risultati rappresentativi Il protocollo di purificazione offre due fasi di controllo di qualità, esempi delle quali sono mostrate. Siamo in grado di identificare e documentare potenziali problemi in fase di crescita batterica (Figura 1) e successivamente su di valutare il tenore di proteine ​​purificate (Figura 2). Noi di solito purificare proteine ​​e testarli in triplice copia. Questo, in combinazione con le due fasi di controllo di qualità, ci dà fiducia che qualsiasi variazione abbiamo osservato nei nostri saggi funzionali sono dovute al phenotyp mutantee (Figura 3) e non causata da variazioni sperimentali o purificazioni falliti. Le rese ottenute variano tipicamente 50-200 mcg e sono più che sufficienti per vari saggi funzionali. <img alt="Figura 1" src="/files/ftp_upload/4110/4110fig1.jpg" /> Figura 1. Istogramma delle misurazioni di OD di una piastra a 24 pozzetti con culture overnight. Tre cloni di sei varianti TFIIB mutanti nonché del TFIIB tipo selvatico sono state coltivate. Due cloni non trasportano un plasmide esprimente e un pozzo con mezzo servono solo come controlli negativi. Le misurazioni OD mostrano che ci sono piccole variazioni nei tassi di crescita tra le culture individuali. <img alt="Figura 2" src="/files/ftp_upload/4110/4110fig2.jpg" /> Figura 2. Rese proteici ottenuti da queste colture come determinato mediante un saggio BCA e confermata mediante SDS PAGE. Una delle varianti non è espressa a livelli elevati. In comcombinazione con la figura 1, possiamo concludere che ciò non è dovuto alla crescita delle cellule differenziale ma a causa di espressione della proteina non essere indotti in modo corretto. <img alt="Figura 3" src="/files/ftp_upload/4110/4110fig3.jpg" /> Figura 3. Risultato rappresentativo di un saggio di trascrizione. Abbiamo misurato l&#39;attività di stimolazione varianti TFIIB sulla produzione di piccoli trascritti da RNAP abortivi. Qui, gli effetti di stimolazione di una libreria piena di singole sostituzioni aminoacidiche di residui TFIIB K87 sono mostrati. L&#39;alto grado di riproducibilità è confermata dal piccolo errore. Un gel di esempio che mostra le prestazioni di tre mutanti rispetto al tipo selvatico (in peso), controlli negativi (NC) e tampone di eluizione controlla solo è raffigurato sotto.

<ol>
	<li>Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleotide+addition+cycle+of+RNA+polymerase+is+controlled+by+two+molecular+hinges+in+the+Bridge+Helix+domain.">The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain.</a> BMC Biol. 8, 1741-7007 (2010).</li><li>Nottebaum, S., Tan, L., Trzaska, D., Carney, H. C., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+polymerase+factory:+a+robotic+in+vitro+assembly+platform+for+high-throughput+production+of+recombinant+protein+complexes.">The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant protein complexes.</a> Nucleic Acids Res. 36, 245-252 (2008).</li><li>Tan, L., Wiesler, S., Trzaska, D., Carney, H. C., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridge+helix+and+trigger+loop+perturbations+generate+superactive+RNA+polymerases.">Bridge helix and trigger loop perturbations generate superactive RNA polymerases.</a> J. Biol. 7, 40 (2008).</li><li>Wiesler, S. C., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+linker+domain+of+basal+transcription+factor+TFIIB+controls+distinct+recruitment+and+transcription+stimulation+functions.">The linker domain of basal transcription factor TFIIB controls distinct recruitment and transcription stimulation functions.</a> Nucleic Acids Res. 39, 464-474 (2011).</li><li>Weinzierl, R. O., Wiesler, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+Function+of+TFIIB.">Revealing the Function of TFIIB.</a> Transcription. , (2011).</li><li>Bell, S. D., Magill, C. P., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+and+regulated+transcription+in+Archaea.">Basal and regulated transcription in Archaea.</a> Biochemical Society transactions. 29, 392-395 (2001).</li><li>Parvin, J. D., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+topology+and+a+minimal+set+of+basal+factors+for+transcription+by+RNA+polymerase+II.">DNA topology and a minimal set of basal factors for transcription by RNA polymerase II.</a> Cell. 73, 533-540 (1993).</li><li>Tyree, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+minimal+set+of+proteins+that+is+sufficient+for+accurate+initiation+of+transcription+by+RNA+polymerase+II.">Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II.</a> Genes. 7, 1254-1265 (1993).</li><li>Batada, N. N., Westover, K. D., Bushnell, D. A., Levitt, M., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+nucleoside+triphosphates+and+role+of+the+entry+site+to+the+RNA+polymerase+II+active+center.">Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active center.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 17361-17364 (2004).</li><li>Liu, X., Bushnell, D. A., Wang, D., Calero, G., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+an+RNA+Polymerase+II-TFIIB+Complex+and+the+Transcription+Initiation+Mechanism.">Structure of an RNA Polymerase II-TFIIB Complex and the Transcription Initiation Mechanism.</a> Science (New York, N.Y). 327, 206-209 (2010).</li><li>Kostrewa, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+II-TFIIB+structure+and+mechanism+of+transcription+initiation.">RNA polymerase II-TFIIB structure and mechanism of transcription initiation.</a> Nature. 462, 323-330 (2010).</li><li>Werner, F., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+modulation+of+RNA+polymerase+core+functions+by+basal+transcription+factors.">Direct modulation of RNA polymerase core functions by basal transcription factors.</a> Molecular and cellular biology. 25, 8344-8355 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

Il metodo automatizzato purificazione della proteina ricombinante qui descritto consente la produzione e purificazione di un gran numero di proteine ​​mutanti in una piccola formato in condizioni altamente riproducibili con minimo intervento umano. Figure 1 e 2 mostrano i risultati di sistematiche qualità-controlli ed esempi del proteine ​​purificate. Figura 3 mostra che i fattori di trascrizione purificati utilizzati in questo esempio effettuare in un modo altamente riproducibile in saggi funzionali. Anche se la procedura è stata sviluppata per la purificazione di archaeal TFIIB, è ampiamente applicabile per la purificazione di proteine ​​affinità-etichetta. L&#39;uso di tali protocolli di purificazione automatizzati così notevolmente facilitare l&#39;analisi biochimica delle proteine ​​ricombinanti e, quindi, ulteriormente la nostra comprensione di interazioni proteina-proteina su una scala che è difficile da ottenere manualmente.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Overnight Express Media TB istantanea </td><td> VWR International srl </td><td> 71491-4 </td><td></td></tr><tr><td> Contropiede </td><td> Promega Ltd. </td><td> V8573 </td><td></td></tr><tr><td> Lysonase Reagente Bioprocessing / 1 ml </td><td> VWR International srl </td><td> 71230 </td><td></td></tr><tr><td> Antischiuma 204 </td><td> Sigma-Aldrich Company Ltd </td><td> A6426 </td><td></td></tr><tr><td> MagneHis Ni-particelle </td><td> Promega </td><td> V8565 </td><td></td></tr><tr><td> Imidazolo </td><td> Sigma-Aldrich Company Ltd </td><td> 56750 </td><td></td></tr><tr><td> Trizma di base </td><td> Sigma-Aldrich Compqualsiasi Ltd. </td><td> 93362 </td><td></td></tr><tr><td> NaCl </td><td> VWR </td><td> 27810.295 </td><td></td></tr><tr><td> Bicinconinico Acid determinazione delle proteine </td><td> Sigma-Aldrich Company Ltd </td><td> BCA1-1KT </td><td></td></tr><tr><td> Deep Well Tavola 2.2 ml Piazza Wells PP PK10 </td><td> Anachem Ltd </td><td> 1810-1800 </td><td></td></tr><tr><td> Polistirene fondo Micropiastra MicroWell 96 pozzetti piatto non trattata 12 bussole su 5 piastre chiaro il volume di 0.4 ml e 128 millimetri x 86 mm Thermo Scientific Nunc </td><td> Fisher Scientific Ltd. </td><td> DIS-984-090M </td><td></td></tr><tr><td> Micropiastre Blu </td><td> VWR </td><td> NUNC367001 </td><td></td></tr><tr><td> 24 Well Blocchi RB (24) </td><td> Qiagen </td><td> 19583 </td><td></td></tr><tr><td> Guanidina cloridrato </td><td> VWR </td><td> ALFAA13543.0B </td><td></td></tr><tr><td> Ago lavabile per TheOnyx </td><td> Aviso GmbH </td><td> 8152-317001 </td><td></td></tr><tr><td> Rack Reagente per sfere magnetiche </td><td> Aviso GmbH </td><td> 8152-035003 </td><td></td></tr><tr><td> Piatto Reader Synergy HT </td><td> BioTek </td><td> 4200-000043 </td><td></td></tr><tr><td> Robotic Platform TheOnyx 44OH/150/100 </td><td> Aviso GmbH </td><td> 8145-050046 </td><td></td></tr><tr><td> Shaker per micropiastre Variomag Teleshaker </td><td> Inheco </td><td> 3800047 </td><td></td></tr><tr><td> 96 pozzetti Tipo di magnete A </td><td> Qiagen </td><td> 36915 </td><td></td></tr></tbody></table>

high-throughput purification of affinity-tagged recombinant proteins

Cristallografia a raggi X è il metodo di scelta per ottenere una vista dettagliata della struttura delle proteine. Tali studi devono essere integrate da ulteriori analisi biochimiche di acquisire le conoscenze dettagliate in relazioni struttura / funzione. Advances in oligonucleotide-gene e tecnologia di sintesi rendono grandi strategie mutagenesi sempre possibili, compresa la sostituzione dei residui bersaglio da tutti e 19 altri amminoacidi. Guadagno o perdita di funzione fenotipi quindi consentire sistematici conclusioni da trarre, come il contributo di residui particolari per l&#39;attività catalitica, la stabilità della proteina e / o proteina-proteina specificità dell&#39;interazione. Al fine di assegnare diversi fenotipi alla natura della mutazione - anziché fluttuanti condizioni sperimentali - è vitale per purificare e analizzare le proteine ​​in modo controllato e riproducibile. High-throughput strategie e l&#39;automazione di protocolli manuali surobotici liquido di gestione piattaforme hanno creato opportunità di eseguire tali complesse procedure di biologia molecolare, con poco intervento umano ei tassi di errore minimo 1-5. Qui, presentiamo un metodo generale per la purificazione di His-tag proteine ​​ricombinanti in un modo ad alta produttività. In un recente studio, abbiamo applicato questo metodo per una dettagliata struttura-funzione di indagine TFIIB, un componente della macchina trascrizione basale. TFIIB è indispensabile per promotore-diretta trascrizione in vitro ed è essenziale per l&#39;assunzione di RNA polimerasi in un complesso preinitiation 6-8. TFIIB contiene un dominio linker flessibile che penetra il sito attivo fessura di RNA polimerasi 9-11. Questo dominio linker conferisce due attività biochimicamente quantificabili TFIIB, vale a dire (i) la stimolazione dell&#39;attività catalitica durante la fase di &#39;fallita&#39; di inizio trascrizione, e (ii) un ulteriore contributo allaassunzione specifica di RNA polimerasi nella preinitiation complesso 4,5,12. Abbiamo sfruttato l&#39;alta produttività metodo di purificazione per generare singola, doppia sostituzione e triple mutazioni e delezioni nel linker TFIIB e successivamente analizzarli in saggi funzionali per il loro effetto sulla stimolazione dell&#39;attività catalitica della polimerasi RNA 4. Complessivamente, abbiamo generato, purificati ed analizzati 381 mutanti - compito che sarebbe stato lungo e laborioso effettuare manualmente. Abbiamo prodotto e analizzato le proteine ​​in multiplicates che ci ha permesso di apprezzare le variazioni sperimentali e ci ha dato una chiara idea della riproducibilità dei nostri risultati. Questo metodo serve da protocollo generico per la purificazione di His-tag proteine ​​ed è stato usato con successo per purificare altre proteine ​​ricombinanti. Attualmente è ottimizzato per la purificazione di proteine ​​di 24, ma può essere adattato per purificare fino a 96 proteine.

Watch this Scientific Journal Video about High-throughput Purification of Affinity-tagged Recombinant Proteins at JoVE.com

High-throughput Purification of Affinity-tagged Recombinant Proteins

Si descrive un metodo per la purificazione per affinità-tag di proteine ​​ricombinanti utilizzando liquido movimentazione robotica. Questo metodo è generalmente applicabile alla piccola scala purificazione di solubili His-tagged proteine ​​in una high-throughput formato.

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 ...

high-throughput-purification-of-affinity-tagged-recombinant-proteins

Research

JoVE Journal

Biology

14.1K Views.  Imperial College London. Si descrive un metodo per la purificazione per affinità-tag di proteine ​​ricombinanti utilizzando liquido movimentazione robotica. Questo metodo è generalmente applicabile alla piccola scala purificazione di solubili His-tagged proteine ​​in una high-throughput formato.

Video: High-throughput Purification of Affinity-tagged Recombinant Proteins

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

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

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

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

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

Ricerca

Biologia

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Espressione di proteine ​​ricombinanti nel lievito Methylotrophic Pichia pastoris</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

High-throughput iperespressione schermo lievito plasmidi

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Cristallizzazione high-throughput di proteine ​​di membrana con il metodo lipidica Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

L&#39;utilizzo di un robot per High-throughput cristallizzazione delle proteine ​​di membrana in mesofasi lipidici

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Agroinfiltration efficiente di impianti di alto livello espressione transiente di proteine ​​ricombinanti

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

High Throughput microiniezioni di Sea Urchin zigoti

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening espressione quantitativa e purificazione Applicata al ricombinante Disulfide ricchi di Venom proteine ​​prodotte in<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Purificazione non-cromatografico di polipeptidi di elastina ricombinante simili e le loro fusioni con Peptidi e Proteine ​​da<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

High-throughput Titolazione della luciferasi che esprimono ricombinanti virus

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

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

High-throughput Purificazione di affinità-tagged proteine ricombinanti