Name: purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli
Uploaded: 2015-12-19T14:00:00
Description: 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

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. Generering af plasmid og Shadoo Expression Bemærk: Genet, der koder murint Shadoo protein (Shadoo 25-122), blev subklonet ind i ekspressionsvektoren pET-28 11. Denne klon blev transformeret ind i E. coli SoluBL21 bakteriestamme, at udtrykke Shadoo protein med en N-terminal hexahistidinmærke, (HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG) som forklaret tidligere 11.</su...

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En nem og effektiv protokol til både ekspression og oprensning af en stor mængde af rekombinant muse Shadoo protein præsenteres. Den beskrevne fremgangsmåde tillader vellykket solubilisering og oprensning af (His 6) -mærket rekombinant Shadoo protein. Det er vigtigt, som påpeget i titlen, at når det udtrykkes i en prokaryot bakterie E. coli Shadoo akkumuleres i inklusionslegemer. Før Shadoo oprensning bakterierne skal lyseres ved lydbehandling i en puffer indeholdende Triton X-100. Efterfølg...

Prion neurodegenerative sygdomme, som omfatter BSE hos kvæg, scrapie hos får og Creutzfeldt-Jakobs sygdom hos mennesker, er dødelige og uhelbredelige. Prionsygdomme er kendetegnet ved konformationelle ændringer af cellulære prionprotein hovedsagelig sammensat af a-helixer, ind på tværs af β-sheet beriget amyloid konformer 1,2. Cross-p-strukturer indeholder tæt pakkede og yderst ordnede krystallinske-lignende p-sheets stabiliseret med hydrogenbindinger 3,4. Prion amyloider kan selv replikere på grund af p-strenge på den voksende kant, der giver en skabelon for at rekruttere og konvertering en monomer protein enhed. Ifølge &quot;eneste protein&quot; hypotesen, det amyloid konformer af prion protein er det eneste smitstoffet. Imidlertid har nogle andre biomolekyler blevet foreslået at være afgørende for prionlidelser. For eksempel er cellemembraner menes at være et sted, hvor omstilling;på finder sted, og nogle negativt ladede lipider er blevet vist at forbedre konverteringsprocessen 5,6. Desuden kan nogle proteiner også være involveret i prion sygdomme. Konkret er der to andre medlemmer af prionproteinet familien: Doppel og Shadoo 7,8. Doppel har en relativt stiv struktur stabiliseret med di-sulfid broer og undlader at selvpolymerisere og aggregat til amyloide fibriller 9. I modsætning hertil svarer til prionproteinet, kan Shadoo vedtage en β-ark-struktur og beriget selvassociere i amyloidfibrildannelse strukturer. Det blev vist, at Shadoo kan fibrillerer under native betingelser eller efter binding negativt ladede membraner 10,11. Omdannelse af Shadoo i amyloid-lignende fibre kan være forbundet med patologier. Faktisk er de mekanismer, hvormed amyloid-lignende strukturer forårsage sygdom dårligt forstået. Shadoo bærer et hydrofobt domæne (HD) og en række tandem Arg / Gly gentager tilsvarende to N-terminale del af prionprotein (figur 1A). Som den N-terminale del af prionprotein, er Shadoo stærkt positivt ladet og synes at være en indbygget ustruktureret protein 11,12. Shadoo synes at være funktionelt relateret til prionlidelser eftersom den direkte kan binde prionprotein. Desuden er dets ekspression nedreguleres under prion patologi 13,14. Imidlertid er endnu ikke etableret rolle Shadoo i prionsygdom. Vi udviklede et plasmid, der bærer den kodende sekvens af muse Shadoo genet. Plasmidet blev anvendt til at transformere E. coli til fremstilling af N-terminale His-6 fusion Shadoo protein. Dette ekspressionssystem er veletableret i vores laboratorium og er almindeligt anvendt i vores igangværende projekter 6,15,16. Et vigtigt spørgsmål for ekspression af Shadoo er valget af E. coli-stamme. Mens kompetente BL21 bakteriestamme er almindeligt anvendt til ekspression af rekombinant proproteiner Shadoo blev med succes udtrykt og oprenset kun fra den transformerede SoluBL21 bakteriestamme. SoluBL21 kompetent E. coli er en forbedret mutant af BL21 værtsstamme udviklet til produktion af proteiner, hvis ekspression i moder- BL21 gav intet påviseligt opløselige produkt. Højniveauekspression af Shadoo i SoluBL21 E. coli fører til akkumulering af proteinet i inklusionslegemer. Som et generelt træk, når en ikke-nativt protein er overudtrykt i E. coli, dette protein har tendens til at ophobe sig i uopløselige inklusionslegemer. Shadoo aggregater sandsynligvis gennem ikke-kovalente hydrofobe eller ioniske interaktioner (eller en kombination af begge) til højt beriget dynamiske strukturer dannet af proteinet foldes til forskellige grader. Som følge heraf oprensningen omfatter mindst to trin: (i) en selektiv separation af proteinet fra andre biomolekyler i et denatureringstrin medium og (ii) en renaturering af det oprensede protein under anvendelse af in vitro foldningteknikker. Den selektive adskillelse af Shadoo blev opnået i en pufferopløsning indeholdende 8M urinstof (eller alternativt 6M guanidin-HCI). Urinstoffet fjernelse og renaturering af proteinet kan ske ved at anvende forskellige protokoller: (i) renaturering i et surt pH opløsning for at opnå en ustruktureret monomere Shadoo protein eller (ii) renaturering i en opløsning med pH ≥ 7 til opnåelse Shadoo polymeriseret til stabile amyloide fibre med karakteristiske cross-β-sheet motiver.

<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

Escherichia coli-ekspressionssystemet er et kraftfuldt værktøj til fremstilling af rekombinante eukaryote proteiner. Vi bruger den til at producere Shadoo, et protein, der tilhører prion familien. En kromatografisk fremgangsmåde til oprensning af (His) 6-mærkede rekombinante Shadoo udtrykt som inklusionslegemer er beskrevet. Inklusionslegemerne solubiliseres i 8M urinstof og bundet til en Ni 2+ -charged kolonne for at udføre ion-affinitetskromatografi. Bundne proteiner elueres med en gradient af imidazol. Fraktioner indeholdende Shadoo protein underkastes gelpermeationskromatografi til opnåelse af et højt oprensede protein. I det sidste trin oprenses Shadoo afsaltes til fjernelse af salte, urinstof og imidazol. Rekombinant Shadoo protein er en vigtig reagens til biofysiske og biokemiske studier af proteinkonformation sygdomme, der opstår i prionsygdomme. Mange rapporter viste, at prion neurodegenerative sygdomme stammer fra aflejring af stable, beordrede amyloid fibriller. Sample protokoller, der beskriver, hvordan man fibrillere Shadoo til amyloide fibriller på sure og neutrale / basale pH-værdier præsenteres. Fremgangsmåderne om, hvordan man producere og fibrillere Shadoo kan lette forskning i laboratorier, der arbejder i prionsygdomme, da det giver mulighed for produktion af store mængder protein i en hurtig og billig måde.

Ca. 5-10 mg oprenset protein Shadoo opnås per liter bakteriekultur. Er behov for en to-trins rensning for at opnå rekombinant Shadoo af høj renhed. Det første trin udføres ved affinitetskromatografi med en Ni2 + -charged kolonne, der bevarer sin-mærkede proteiner. Eluerede fraktioner underkastes SDS-PAGE og farvet med Coomassie Brilliant Blue (figur 5A). Fraktioner indeholdende Shadoo protein samles og renses yderligere ved gelpermeationskromatografi som adskiller proteinerne ifølge de...

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.

Oprensning og genfoldning til amyloidfibriller af (His)<sub&gt; 6</sub&gt; -mærket rekombinant Shadoo protein udtrykt som inklusionslegemer i<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 ...

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

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