Name: purification and aggregation of the amyloid precursor protein intracellular domain
Uploaded: 2012-08-28T12:00:00
Description: Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

De auteurs willen graag Dr Hui Zheng (Baylor College of Medicine) bedanken voor de APP cDNA. Dit werk werd gefinancierd door NIH subsidie ​​R21AG031948 (DB, JMB), F30AG030878 (ESS), R01DK073394 (AFO), de John Sealy Memorial Endowment Fonds voor biomedisch onderzoek (AFO), en de Jean C. en William D. Willis Neuroscience Research Endowment (ESS). JMB is een geleerde in de Translationeel Onderzoek Scholar Program en een lid van de Universiteit van Texas Medical Branch Claude E. Pepper Oudere Amerikanen Independence Center (ondersteund door NIH subsidies UL1RR029876 en P30-AG-024832, respectievelijk).

<html> 1. Expressie van recombinant APP intracellulaire domein (AICD) <ol><li> Transform E. coli-stam BL21 met menselijke AICD (resten 649 tot 695 van APP, neuronale isovorm nummering) gekloneerd in vector pGEX-4T-1 (GE Healthcare). Deze vector uitdrukkelijke AICD het C-terminale deel van een fusie-eiwit van glutathion-S-transferase (GST). Deze vector codeert ook trombine splitsingssequentie om loskomen van de GST eenheid. Details van het klonen AICD in pGEX-4T-1 kan worden gevonden in onze vorige publicatie <...

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In dit protocol hebben we geschetst van een procedure voor het verkrijgen van zeer zuivere AICD om structurele, biofysische en biochemische analyses. Deze procedure vereist geen geavanceerde chromatografie apparatuur en is daarom toegankelijk voor de meeste laboratoria. Andere groepen hebben gezuiverd AICD 5-7,16, met inbegrip van GST-AICD 17-19, voor de biochemische / structurele analyses. Nadelen naar de vorige protocollen zijn: een slechte oplosbaarheid van AICD 16, minder dan ideaal ...

<table border="1"><tbody><tr><td> Naam van het reagens </td><td> Vennootschap </td><td> Catalogusnummer </td><td> Reacties </td></tr><tr><td> pGEX-4T-1 </td><td> GE Healthcare </td><td> 28-9545-49 </td><td></td></tr><tr><td> Trombine </td><td> GE Healthcare </td><td> 27-0846-01 </td><td></td></tr><tr><td> Ampicilline </td><td> Fisher Scientific </td><td> BP1760 </td><td></td></tr><tr><td> Bradford eiwittest reagens </td><td> Bio-Rad </td><td> 500-0002 </td><td></td></tr><tr><td> Coomassie blauw </td><td> Bio-Rad </td><td> 161-0786 </td><td></td></tr><tr><td> IPTG (isopropyl-beta-D thiogalactopyranoside) </td><td> Sigma-Aldrich </td><td> I6758 </td><td></td></tr><tr><td> Glutathion-agarose </td><td> Sigma-Aldrich </td><td> G4510 </td><td></td></tr><tr&gt; <td> p-aminobenzamidine-agarose </td><td> Sigma-Aldrich </td><td> A7155 </td><td></td></tr><tr><td> Volledige protease inhibitor cocktail </td><td> Roche </td><td> 11836170001 </td><td></td></tr><tr><td> Slide-A-Lyzer dialyse cassettes </td><td> Thermo Scientific </td><td> 66380 </td><td></td></tr><tr><td> Chromatografiekolommen </td><td> Evergreen Wetenschappelijke </td><td> 208-3367-050 </td><td></td></tr><tr><td> Emulgator </td><td> Avestin, Inc </td><td> EmulsiFlex-C3 </td><td> Zeer aan te bevelen </td></tr><tr><td> Eppendorf Thermomixer </td><td> Eppendorf </td><td> 022670107 </td><td></td></tr><tr><td> Mica schijven </td><td> Ted Pella </td><td> 50 tot 12 </td><td></td></tr><tr><td> AFM uitkragingen </td><td> Bruker </td><td> MSNL-10 </td><td></td></tr><tr><td> WSxM software </td><td> Nanotec </td><td> N / A </td><td> Free downloaden </td></tr></tbody></table>

purification and aggregation of the amyloid precursor protein intracellular domain

Amyloïde voorlopereiwit (APP) is een type I transmembraan eiwit geassocieerd met de pathogenese van de ziekte van Alzheimer (AD). APP wordt gekenmerkt door een groot extracellulair domein en een korte cytosolische domein zogenaamde APP intracellulaire domein (AICD). Tijdens rijping door de secretieroute kan APP worden gesplitst door proteasen genoemd α, β, γ-en secretasen 1. Sequentiële proteolytische splitsing van APP met β en γ-secretasen leidt tot de productie van een kleine proteolytische peptide, aangeduid Aß, dat amyloidogene en de kern bestanddeel van seniele plaques. De AICD is bevrijd van het membraan na secretase verwerking en via interacties met Fe65 en Tip60 kan naar de kern om aan transcriptie regulatie van meerdere doelgenen 2,3. Eiwit-eiwit interacties waarbij AICD kunnen beïnvloeden handel, verwerking en cellulaire functies van holo-APP en C-terminal fragmenten. We hebben onlangs aangetoond dat AICD kan totaal in vitro, en dit proces wordt geremd door de AD-betrokken moleculaire chaperonne ubiquilin-1 4. Consistent met deze bevindingen de AICD hydrofobe domeinen heeft belicht en intrinsiek ongeordend in vitro 5,6, maar het verkrijgt stabiele secundaire structuur wanneer gebonden aan Fe65 7. We hebben voorgesteld ubiquilin-1 ongepast inter-en intramoleculaire interacties van AICD voorkomt, waardoor aggregatie in vitro en in intacte cellen 4. Terwijl de meeste studies richten zich op de rol van APP in de pathogenese van AD, de rol van de AICD in dit proces is niet duidelijk. Expressie van AICD is aangetoond dat apoptose induceren 8, te moduleren signaalwegen 9, en calcium reguleren signalering 10. Over-expressie van AICD en Fe65 in een transgeen muismodel induceert Alzheimer pathologie zoals 11 en onlangs AICD is gedetecteerd in brain lysaten door western blotting, wanneer er gebruik geschikte antigeen retrieval technieken 12. Structurele, biochemische en biofysische studies van AICD vergemakkelijken, hebben we een procedure om recombinant grote hoeveelheden zeer zuivere eiwit AICD. We beschrijven verder een werkwijze voor het induceren van de in vitro aggregatie van thermische analyse door AICD en atomic force microscopie. De beschreven methoden zijn nuttig voor biochemische, biofysische en structurele karakterisering van de AICD en de effecten van moleculaire chaperones op AICD aggregatie.

Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Werkwijze voor grootschalige zuivering van het APP intracellulaire domein (AICD) beschreven. We beschrijven ook de methodologie voor het induceren<em&gt; In vitro</em&gt; AICD aggregatie en visualisatie van atomaire kracht microscopie. De beschreven methoden zijn nuttig voor biochemische / structurele karakterisering van de AICD en de effecten van moleculaire chaperones op de aggregatie.

Zuivering en Aggregatie van het amyloïde voorlopereiwit intracellulaire domein

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

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