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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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&amp;nbsp;</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&amp;nbsp;</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-his-6-tagged-recombinant

Universidad de Cartagena UDEC

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

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Metodo per ripiegamento efficiente e purificazione del chemocettore Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Ricerca

Biochimica

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Purificazione e aggregazione della proteina precursore dell&#39;amiloide dominio intracellulare

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Medicina

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

Sintesi di una proteina artificiale mediata da inteina Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Bioingegneria

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Espressione, purificazione, cristallizzazione e saggi enzimatici di Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Targeting dei tioli cisteina per in Vitro site-specific glicosilazione delle proteine ricombinanti

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Immunologia e infezioni

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 ...

Biologia

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Produzione di Nanoparticelle proteiche auto-assemblanti E. coliper vaccini che richiedono la presentazione dell'epitoscopio trimerico

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Una purificazione e l'analisi di attività In Vitro per un ppGpp (p) sintetasi da Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Purificazione biochimica e caratterizzazione proteomica dei nuclei di fibrille amiloidi dal cervello

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Purificazione e ripiegando a amiloide fibrille di (sua)

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Capitoli in questo video

Metodo per ripiegamento efficiente e purificazione del chemocettore Ligand Binding Domain

Purificazione e aggregazione della proteina precursore dell'amiloide dominio intracellulare

Sintesi di una proteina artificiale mediata da inteina Hydrogel

Espressione, purificazione, cristallizzazione e saggi enzimatici di Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Targeting dei tioli cisteina per in Vitro site-specific glicosilazione delle proteine ricombinanti

Purificazione di Hsp104, una proteina Disaggregase

Produzione di Nanoparticelle proteiche auto-assemblanti E. coliper vaccini che richiedono la presentazione dell'epitoscopio trimerico

Una purificazione e l'analisi di attività In Vitro per un ppGpp (p) sintetasi da Clostridium difficile

Rapid Generation of Amyloid from Native Proteins In vitro

Purificazione biochimica e caratterizzazione proteomica dei nuclei di fibrille amiloidi dal cervello