The authors would like to acknowledge Natalie Doude and David Westerway (University of Alberta) for providing us Sho cDNA, Christophe Chevalier (INRA) for plasmid construction, Michel Br&#233;mont (INRA) for providing us BL21Sol bacteria; Christine Longin (INRA) for TEM assistance and Edith Pajot-Augy (INRA) for comments and proofreading.

1. Generazione di plasmidi e Shadoo Expression Nota: Il gene che codifica la proteina murino Shadoo (Shadoo 25-122), è stato subclonato nel vettore di espressione pET-28 11. Questo clone è stata trasformata in E. coli SoluBL21 ceppo batterico, per esprimere la proteina Shadoo con un tag hexahistidine N-terminale, (HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG), come spiegato in precedenza 11. Il plasmide può essere richiesto da parte degli autori. Il peso molecolare di Shadoo calcolata dalla sua sequenza aminoacidica è 12,243.2 Da. Nota: Eseguire tutte le manipolazioni con i batteri sotto una cappa a flusso laminare o in prossimità di Bunsen fiamma. <ol><li> Trasforma batteri SoluBL21 competenti con la PET-28-Il suo 6 -Shadoo plasmide, utilizzando un protocollo shock termico standard. Per questo mix 1 a 5 ml di plasmide (tipicamente 10 a 100 pgng) in 50 ml di batteri in un tubo. Eseguire lo shock termico a 42 ° C per 45 sec. </li><li> Cultura batteri trasformati a 37 ° C fino a 600 di 0,5-0,7 in Luria Bertani-mezzo supplementato con 40 mg / ml kanamicina (Figura 1B). </li><li> Indurre Shadoo espressione O / N con l&#39;aggiunta di 240 mg / ml isopropilico-β-D-thiogalactopyranoside, ITPG, alla cultura. Batteri Cultura sotto agitazione a 150 rpm a 37 ° C. Nota: In genere, la coltura batterica raggiunge A 600 3 O / N. Nota: Trasformato batteri possono essere conservati a -80 ° C per la futura produzione di proteine. Per questo, aggiungere 10% -50% (v / v) di glicerolo sterile in aliquote coltura batterica e congelarli a -80 ° C. </li></ol><img alt="Figura 1" src="/files/ftp_upload/53432/53432fig1.jpg" /> Figura 1. Schema di Shadoo espressione costrutto, batteri trasformazione e l&#39;induzione dell&#39;espressione Shadoo (come descritto al punto 1). </strong&gt; (A) Il costrutto di espressione per Shadoo codifica un tag hexahistidine fusa all&#39;N-terminale della proteina, Nt. Lo schema mostra che le proteine ​​Shadoo contiene ripetizioni base arginina / glicina (cilindro verde), un dominio idrofobico (HD), e un singolo sito N-glicosilazione (CHO), seguiti da un C-terminale (Ct). (B) pET-28-Il suo 6 -Shadoo plasmide si trasforma in batteri SoluBL21 da shock termico per 45 secondi a 42 ° C. Batteri trasformati sono placcati su agar selettivo contenente 40 mg / ml kanamicina. Una singola colonia dalla piastra è usata per inoculare una coltura in una beuta agitatore 1000-3000 ml. L&#39;espressione della proteina ricombinante è indotta con 1 mM IPTG. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig1highres.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> 2. Purificazione di Shadoo proteine ​​da bassa pressione cromatografia liquida (LPLC) <ol><li> Lisi cellulare e inconclusione corpi preparazione <ol><li> Raccogliere la sospensione batterica e le trasferisce in tubi centrifugazione (Figura 2). </li><li> Spin a 2.500 xg per 20 min a 4 ° C. Rimuovere il surnatante per decantazione e risospendere pellet in 15 ml di tampone (50 mM Tris, EDTA 10 mM, pH 8 con anti-proteasi cocktail) per ogni pastiglia derivata da 500 ml di coltura cellulare. Nota: precipitato risospeso possono essere congelati e conservati a -20 ° C per diversi giorni. Congelamento facilita cellule batteriche lisi. </li><li> Controllare la sovra-espressione della proteina ricombinante utilizzando batteri greggio pellet mediante analisi SDS-PAGE e colorazione Coomassie. <ol><li> Per questo, centrifuga 1 ml di coltura batterica a pellet cellule. Aggiungere 100 microlitri soluzione Laemmli denaturazione (277,8 mM Tris-HCl, pH 6,8, 4,4% SDS, 44,4% w / v glicerolo, 0,02% blu di bromofenolo) a pellet e riscaldare a 100 ° C per 5 min. </li><li> Carico 10 ml di campione ad un gel di acrilammide 12%, eseguire SDS-PAGE e visuAlize proteine ​​separate da Coomassie colorazione. </li></ol></li><li> Aggiungere Triton X-100 a concentrazione finale 0,5% al ​​pellet risospeso completamente dalla 2.1.2). </li><li> Incubare la sospensione in bagno d&#39;acqua a 37 ° C per 30 min. </li><li> Sonicare la sospensione per 5 minuti a 6-12 micron ampiezza picco-picco in acqua ghiacciata per lisare le cellule batteriche. </li><li> Trasferimento sonicata sospensione nella pulite provette da 50 ml di centrifugazione. </li><li> Tubi centrifugare a 15.000 xg per 15 min a 4 ° C. </li><li> Rimuovere il surnatante per decantazione e aggiungere 5 ml di tampone di legame (20 mM tampone Tris-HCl, pH 7,4, 0,5 M NaCl, 8 M Urea, imidazolo 5 mM) per provetta. </li><li> Tubi immettono sul rotante volante O / N di proteine ​​da corpi di inclusione risospendere completamente. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53432/53432fig2.jpg" /> Figura 2. Schema di purificazione di inclusioneorgani (come descritto al punto 2.1). trasformate batteri esprimenti Shadoo vengono raccolte per centrifugazione e risospese in tampone di lisi. La sospensione viene incubata a 37 ° C per 30 minuti, e quindi sonicato per rompere meccanicamente cellule batteriche. Sonicazione è fatto su ghiaccio per evitare il riscaldamento del campione. Celle rotte sono raccolte per centrifugazione e risospese in un tampone contenente 8 M di urea per solubilizzare proteine ​​da corpi inclusi. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig2highres.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> </li><li> Cromatografia Ni 2+ -affinity Nota: Cromatografia viene eseguita utilizzando un sistema FPLC Ni 2+ -charged, 5 ml Sepharose colonna chelante (Figura 3A). Tutte le soluzioni vengono caricati sulla colonna con una portata di 1 ml / min. <ol><li> Iniettare 10 ml di 0,2 M Niso soluzione 4 acqua nella colonna Sepharose per caricarlacon ioni Ni 2+. </li><li> Equilibrare la colonna caricata con Ni 2+ ioni iniettando 30-50 ml di tampone di legame contenente una bassa concentrazione di imidazolo (20 mM tampone Tris-HCl, pH 7,4, 0,5 M NaCl, 8 M Urea, imidazolo 5 mM). </li><li> Caricare le proteine ​​urea-solubilizzato con un sistema di iniezione 50 ml. </li><li> Recupero della frazione flow-through lavando a fondo la colonna con 10 ml bassa imidazolo tampone di legame (20 tampone mM Tris-HCl, pH 7,4, 0,5 M NaCl, 8 M Urea, 5 Imidazole mM) al fine di rimuovere un massimo di proteine ​​non legate dalla colonna. </li><li> Lavare la colonna con 50-100 ml di tampone di lavaggio (20 mM Tris-HCl, pH 7,4, 0,5 M NaCl, 8 M urea, 80 mM imidazolo) per eluire le proteine ​​non specificamente legato alla colonna. </li><li> Applicare 15 min gradiente lineare di 80-800 mM imidazolo in tampone contenente 20 mM Tris-HCl, pH 7,4, 0,5 M NaCl, 8 M Urea. Raccogliere 1 ml frazioni durante l&#39;applicazione del gradiente. Nota: proteine ​​His-tag vengono eluiti ingegnoha pendenza di tampone contenente imidazolo-(utilizzata come concorrente). </li><li> Prendete una un&#39;aliquota 8 microlitri di ogni frazione e aggiungere 4 ml soluzione denaturazione 4x Laemmli (vedi 2.1.3.1). Soluzioni di calore a 100 ° C per 5 min. </li><li> Carico 10 ml di dimensioni proteine ​​marcatore e 10 ml di ogni campione al gel di acrilammide al 12%, eseguire SDS-PAGE e visualizzare le proteine ​​separate da Coomassie colorazione. Nota: Il peso molecolare di Shadoo calcolato formano la sua sequenza di aminoacidi è 12,243.2 Da. </li><li> Piscina tutte le frazioni contenenti Shadoo. </li></ol><img alt="Figura 3" src="/files/ftp_upload/53432/53432fig3.jpg" /> Figura 3. procedura di purificazione (come descritto nei punti 2.2-2.4). (A) Ni 2+ ioni possono essere coordinati da residui di istidina, che permette la purificazione selettiva di proteine ​​polyhistidine-tag. Imidazolo è usato per eluire la proteina, attraversola sua capacità di coordinare con il 2+ -ion Ni e spostare la proteina legato. (B) Proteine ​​di varia raggio idrodinamico che sono stati presenti a Ni 2+ -column sono separati su una colonna esclusione dimensioni. Le proteine ​​vengono eluite dal più grande al quelle più piccole. (C) Prima dell&#39;uso, frazioni contenenti Shadoo sono raggruppati e dissalate per rimuovere l&#39;urea, imidazolo e sali. Saggi di controllo qualità includono SDS-PAGE / Coomassie colorazione, Western blotting. Purificata Shadoo può essere liofilizzato. Liofilizzato Shadoo è stabile per mesi se conservato a -20 ° C. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig3highres.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> </li><li> Cromatografia di esclusione Nota: Una seconda fase di purificazione consiste nella cromatografia di esclusione dimensionale che separa proteine ​​Shadoo dalle altre proteine ​​co-eluite durante la prima fase di purificazione(Figura 3B). <ol><li> Equilibrare Superdex colonna 120 ml di volume totale letto iniettando 250 ml di tampone 20 mM Tris-HCl, pH 7,4, 0,5 M NaCl, 8 M Urea alla portata di 1,5 ml / min. </li><li> Carica frazioni pool contenenti Shadoo dalla 2.2.9 alla colonna equilibrata. Raccogliere 1 ml frazioni dopo il volume vuoto della colonna 40 ml al flusso costante del buffer sopra. </li><li> Prelevare un&#39;aliquota 10 microlitri di ogni frazione ed effettuare un&#39;analisi SDS-PAGE e colorazione Coomassie come descritto sopra per scoprire quale frazioni contengono Shadoo. </li><li> Piscina frazioni contenenti Shadoo. </li></ol></li><li> Dissalazione Nota: Procedura Desalting rimuove sali, imidazolo e l&#39;urea per ottenere proteine ​​Shadoo ricombinante in un buffer adatto per ulteriori studi biofisici e biochimici (Figura 3C). Lo stesso risultato può essere ottenuto campioni dialisi contro tampone. <ol><li> Equilibrare una colonna dissalazione di 53 ml con 150 ml10 mM di tampone di acetato di ammonio, pH 5, a velocità di flusso di 5 ml / min. </li><li> Carico pool frazioni Shadoo sulla colonna. Dopo iniezione del tampone acetato di ammonio raccogliere manualmente frazioni che mostrano un aumento del segnale a 280 nm in uscita del rivelatore UV. </li><li> Liofilizzare il Shadoo dissalato e conservarlo a -20 ° C. </li><li> Sciogliere Shadoo liofilizzata in un buffer adeguata, ad esempio 10 mM di tampone sodio acetato, pH 5, prima dell&#39;uso. Nota: Il tag polyhistidine può essere rimosso con enterokinase. </li></ol></li></ol> 3. In Vitro Assemblaggio di Shadoo in amiloide fibrille a pH fisiologico Nota: I aggregati proteici Shadoo e spontaneamente forme fibrille a pH ≥ 7 11. <img alt="Figura 4" src="/files/ftp_upload/53432/53432fig4.jpg" /> Figura 4. Schema di Shadoo ripiegamento di fibrille amiloidi (come descriLetto al punto 3). purificato Shadoo converte spontaneamente di fibrille amiloidi quando disciolto in tampone a pH ≥ 7. In una soluzione acida, Shadoo converte in fibrille amiloidi su incubazione con 1 M Gdn-HCl agitando. Test di controllo di qualità sono la microscopia elettronica e ThT colorazione. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig4highres.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <ol><li> Diluire Shadoo ad una concentrazione finale di 2 mM in tampone 20 mM Tris-HCl, pH 7.4. Misurare la concentrazione misurando la densità ottica della soluzione a 280 nm ed utilizzando il coefficiente di estinzione dedotta dalla composizione aminoacidica Shadoo di 20.970. </li><li> Incubare 500 microlitri della soluzione in un tubo di plastica conica a 4 ° C per due settimane, per consentire la conversione spontanea proteine ​​per strutture amiloidi (Figura 4). </li><li> Aggiungere appena preparato thioflavin T (ThT) per una conce finalentration di 10 mM di un&#39;aliquota di 100 microlitri della soluzione Shadoo. Incubare la soluzione per 5 minuti a temperatura ambiente. Misurare emissione di fluorescenza da 460 a 520 nm con una lunghezza d&#39;onda di eccitazione di 435 nm per verificare la presenza di strutture amiloidi 17. </li></ol> 4. In Vitro Assemblaggio di Shadoo di amiloide fibrille a pH acido <ol><li> Diluire Shadoo in 1 M GdnHCl, tampone 20 mM Na-acetato, pH 5 per una concentrazione finale di 2 mM. </li><li> Incubare 500 ml di questa soluzione in un tubo conico con agitazione continua a 37 ° C, per 3-7 giorni. </li><li> Controllare la presenza di fibrille amiloidi aggiungendo ThT ad un&#39;aliquota della soluzione, come in 3.3. </li><li> Dializzare sospensione ottenuta contro 10 mM tampone Na-acetato, pH 5,0, per purificare Shadoo fibrille amiloidi. </li><li> Conservare fibrille purificati a +4 ° C. </li></ol><img alt="Figura 5" src="/files/ftp_upload/53432/53432fig5.jpg"/&gt; Figura 5. Risultati rappresentativi. Frazioni proteiche eluite da Ni 2 + -charged colonna chelante (A) e della colonna esclusione dimensione (B) sono stati analizzati utilizzando SDS-PAGE e colorazione Coomassie. Identificazione di Shadoo purificato è stata valutata utilizzando SPRN anticorpo anti-Shadoo (C). <a href="https://www.jove.com/files/ftp_upload/53432/53432fig5large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

<ol>
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The authors have nothing to disclose.

Un protocollo semplice ed efficace sia per l&#39;espressione e la purificazione di una grande quantità di topo ricombinante proteina Shadoo è presentato. Il metodo descritto consente solubilizzazione successo e purificazione (His 6) -tagged proteina Shadoo ricombinante. È importante, come indicato nel titolo, che quando espresso in un batterio E. procariotico coli Shadoo accumula nei corpi di inclusione. Prima Shadoo purificazione, i batteri devono essere lisate per sonicazione in un tamp...

Prion malattie neurodegenerative, tra cui l&#39;encefalopatia spongiforme bovina nel bestiame, la scrapie ovina, malattia di Creutzfeldt-Jakob negli esseri umani, sono fatali e incurabile. Le malattie da prioni sono caratterizzate da cambiamenti conformazionali di proteina prionica cellulare principalmente composti da alfa-eliche, in cross-β-sheet arricchito amiloide conformer 1,2. Cross-beta-strutture contengono densamente imballati e beta-sheets cristallini come altamente ordinati stabilizzati con legami idrogeno 3,4. Amiloidi da prioni grado di autoreplicarsi causa delle beta-filoni sul bordo crescente che forniscono un modello per il reclutamento e la conversione di un&#39;unità proteina monomerica. Secondo l&#39;ipotesi &quot;unica proteina&quot;, conformero amiloide della proteina prionica è l&#39;unico agente infettivo. Tuttavia, alcune altre biomolecole sono state proposte per essere essenziali per disturbi prioni. Ad esempio, le membrane cellulari si pensa di essere un luogo in cui conversiin si svolge e alcuni lipidi caricati negativamente hanno dimostrato di migliorare il processo di conversione 5,6. Inoltre, alcune proteine ​​possono anche essere coinvolti in patologie da prioni. In particolare, ci sono altri due membri della famiglia proteina prionica: Doppel e Shadoo 7,8. Doppel ha una struttura relativamente rigida stabilizzata con ponti di-solfuro e non riesce ad auto-polimerizzarli e aggregata per fibrille amiloidi 9. In contrasto, simile alla proteina prionica, Shadoo può adottare una struttura β-sheet arricchito e di auto-associate in strutture fibrille amiloidi. È stato dimostrato che Shadoo può fibrillazione in condizioni native o dal legame membrane con carica negativa 10,11. Conversione di Shadoo in fibre amiloidi simili può essere associato a patologie. Infatti, i meccanismi con cui le strutture amiloidi simili causano la malattia sono poco conosciuti. Shadoo porta un dominio idrofobico (HD) e una serie di tandem Arg / Gly ripete simile to la parte N-terminale della proteina prionica (Figura 1A). Poiché la parte N-terminale della proteina prionica, Shadoo è altamente caricato positivamente e sembra essere una proteina nativa strutturati 11,12. Shadoo sembra essere funzionalmente correlati a disturbi da prioni dal momento che può legarsi direttamente proteina prionica. Inoltre, la sua espressione è regolata giù durante prioni patologia 13,14. Tuttavia, il ruolo di Shadoo in malattie da prioni non è ancora stabilita. Abbiamo sviluppato un plasmide che porta la sequenza codificante del gene murino Shadoo. Il plasmide è stato usato per trasformare E. coli per la produzione di N-terminale suo 6 proteina di fusione Shadoo. Questo sistema di espressione è ben consolidata nel nostro laboratorio ed è comunemente usato nei nostri progetti in corso 6,15,16. Una questione importante per l&#39;espressione del Shadoo è la scelta del E. coli. Mentre BL21 competente ceppo batterico è comunemente utilizzato per l&#39;espressione di pro ricombinanteproteine ​​Shadoo è stato espresso con successo e purificato solo dal ceppo batterico trasformato SoluBL21. SoluBL21 competente E. coli è una migliore mutante di ceppo ospite BL21 sviluppato per la produzione di proteine ​​la cui espressione nel BL21 genitore non dava prodotto solubile rilevabile. Espressione ad alto livello di Shadoo in SoluBL21 E. coli porta all&#39;accumulo della proteina in corpi di inclusione. Come caratteristica generale, quando una proteina non nativo è altamente espresso in E. coli, questa proteina si accumula nei corpi di inclusione insolubili. Shadoo probabilmente aggrega attraverso interazioni non covalenti idrofobiche o ionici (o una combinazione di entrambi) strutture dinamiche altamente arricchito formata dalla proteina ripiegata a vari gradi. Di conseguenza, la purificazione comprende almeno due fasi: (i) una separazione selettiva della proteina da altre biomolecole in un mezzo denaturazione, e (ii) un rinaturazione della proteina purificata mediante piegatura in vitrotecniche. La separazione selettiva di Shadoo è stato ottenuto in una soluzione tampone contenente urea 8M (o in alternativa 6M guanidina-HCl). La rimozione di urea e rinaturazione della proteina può essere fatto mediante diversi protocolli: (i) rinaturazione in una soluzione a pH acido per ottenere una proteina non strutturata Shadoo monomerica, o (ii) rinaturazione in una soluzione di pH ≥ 7 per ottenere Shadoo polimerizzato in fibre amiloidi stabili con caratteristici motivi cross-β-sheet.

<table>
	<tbody>
		<tr>
			<td>AKTA FPLC&#160;</td>
			<td>GE, Amersham</td>
			<td># 1363</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 16/60 Superdex</td>
			<td>GE Healthcare</td>
			<td>17-1069-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap IMAC</td>
			<td>GE Healthcare</td>
			<td>GE17-0920-05</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep26/10 Desalting column</td>
			<td>GE Healthcare</td>
			<td>GE17-5087-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SoluBL21 Competent E. coli</td>
			<td>Genlantis</td>
			<td>C700200</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-28b+</td>
			<td>Novogen</td>
			<td>69865-3</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Broth medium</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Biophotometar</td>
			<td>Eppendorf</td>
			<td>550507804</td>
			<td></td>
		</tr>
		<tr>
			<td>Trito X-100</td>
			<td>Life Technologies</td>
			<td>85111</td>
			<td></td>
		</tr>
		<tr>
			<td>NiSO4</td>
			<td>Sigma-Aldrich</td>
			<td>656895</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Gdn-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr>
			<td>protein size marker&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>26620</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli

Il sistema di espressione coli Escherichia è uno strumento potente per la produzione di proteine ​​eucariotiche ricombinanti. Lo usiamo per produrre Shadoo, una proteina appartenente alla famiglia prione. Un metodo cromatografico per la purificazione di (suo) 6 -tagged Shadoo ricombinante espressa come corpi di inclusione è descritto. I corpi di inclusione vengono solubilizzati in 8 M urea e legati ad un 2+ colonna -charged Ni eseguire cromatografia di affinità ionico. Proteine ​​legate sono eluite da un gradiente di imidazolo. Le frazioni contenenti proteine ​​Shadoo mediante cromatografia di esclusione dimensionale per ottenere una proteina altamente purificata. Nella fase finale è purificato Shadoo dissalati per eliminare i sali, urea e imidazolo. Proteina ricombinante Shadoo è un reagente importante per gli studi biofisici e biochimici di disturbi conformazione della proteina che si verificano nelle malattie da prioni. Molti rapporti hanno dimostrato che le malattie neurodegenerative da prioni provengono dalla deposizione di pugnalatale, ordinò fibrille amiloidi. Protocolli di esempio descrivono come alla fibrillazione Shadoo in fibrille amiloidi in acido e neutro / pHs di base sono presentati. I metodi su come produrre e fibrillazione Shadoo può facilitare la ricerca in laboratori che lavorano su malattie da prioni, in quanto consente per la produzione di grandi quantità di proteine ​​in maniera rapida e basso costo.

Circa 5-10 mg di proteine ​​Shadoo purificato viene ottenuto per litro di coltura batterica. Una purificazione a due fasi è necessario per ottenere Shadoo ricombinante di elevata purezza. Il primo passo è effettuata mediante cromatografia di affinità con un Ni 2 + colonna -charged che conserva i suoi-proteine ​​taggati. Frazioni eluite sono sottoposti a SDS-PAGE e colorazione con Coomassie Brilliant Blue (Figura 5A). Le frazioni contenenti proteine ​​Shadoo sono raggruppati e u...

Watch this Scientific Journal Video about Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli at JoVE.com

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Purificazione e ripiegando a amiloide fibrille di (sua)<sub&gt; 6</sub&gt; -tagged proteina ricombinante Shadoo Espresso come corpi di inclusione in<em&gt; E. coli</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

purification-refolding-to-amyloid-fibrils-his6-tagged-recombinant

Research

JoVE Journal

Biology

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

14.1K Views.  INRA. A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures. 

Video: Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Patch di registrazione pinza dei canali ionici Espressa in ovociti di Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Ricerca

Biologia

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 ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Protocolli di selezione mutagenesi e funzionale per l&#39;evoluzione Regia di proteine ​​in E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Purification of Hsp104, a Protein Disaggregase

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

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

Purificazione di Hsp104, una proteina Disaggregase

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

Identification of Protein 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 ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Saggi accoppiati per il monitoraggio delle proteine ​​in ripiegamento<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Purificazione della fibrosi cistica regolatore della conduttanza transmembrana proteina espressa in<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-base Espressione Proiezione di proteine ​​di membrana in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...