Name: split-ubiquitin based membrane yeast two-hybrid (myth) system: a powerful tool for identifying protein-protein interactions
Uploaded: 2010-02-01T16:25:00
Description: Watch this Scientific Journal Video about Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions at JoVE.com

Wir möchten Morgenröte Edmonds für eine kritische Lektüre des Manuskripts danken. Die Stagljar Labor wird aus Mitteln der kanadischen Stiftung für Innovation (CFI), die Canadian Institute for Health Research (CIHR), der Heart and Stroke Foundation, die Canadian Cancer Society und Novartis unterstützt.

1. Hintergrundinformationen Protein-Protein-Interaktionen (PPI) sind die grundlegenden Bausteine ​​in über alle zellulären Prozesse beteiligt. Daher ist es wichtig, dass alle Interaktionen fest geregelt sind, um zelluläre Homöostase, wie eine Verschiebung in diesem biologische Gleichgewicht im Allgemeinen spielt eine Rolle bei Erkrankungen und Krebs Zelltransformation. Membran-assoziierten Proteine ​​gehören zu den biologisch wichtigen Klasse von Proteinen, wie sie komplexe Signal...

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	<li>Stagljar, I., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+membrane+protein+interactions+using+yeast-based+technologies.">Analysis of membrane protein interactions using yeast-based technologies.</a> Trends Biochem Sci. 27 (11), 559-563 (2002).</li><li>Iyer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+the+split-ubiquitin+membrane+yeast+two-hybrid+system+to+identify+protein-protein+interactions+of+integral+membrane+proteins.">Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins.</a> Sci STKE. 275, pl3-pl3 (2005).</li><li>Paumi, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+protein-protein+interactions+for+the+yeast+ABC+transporter+Ycf1p+by+integrated+split-ubiquitin+membrane+yeast+two-hybrid+analysis.">Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis.</a> Mol Cell. 26 (1), 15-25 (2007).</li><li>Stagljar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+system+based+on+split-ubiquitin+for+the+analysis+of+interactions+between+membrane+proteins+in+vivo.">A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo.</a> Proc Natl Acad Sci U S A. 95 (9), 5187-5192 (1998).</li><li>Gisler, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+protein-protein+interactions+between+the+mammalian+integral+membrane+transporters+and+PDZ-interacting+partners+using+a+modified+split-ubiquitin+membrane+yeast+two-hybrid+system.">Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system.</a> Mol Cell Proteomics. 7 (7), 1362-1377 (2008).</li><li>Scheper, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+N-glycosylation+and+protein+translocation+across+the+endoplasmic+reticulum+membrane+by+Sss1+protein.">Coordination of N-glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein.</a> J Biol Chem. 278 (39), 37998-38003 (2003).</li><li>Thaminy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+ErbB3-interacting+factors+using+the+split-ubiquitin+membrane+yeast+two-hybrid+system.">Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system.</a> Genome Res. 13 (7), 1744-1753 (2003).</li><li>Johnsson, N., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+ubiquitin+as+a+sensor+of+protein+interactions+in+vivo.">Split ubiquitin as a sensor of protein interactions in vivo.</a> Proc Natl Acad Sci U S A. 91 (22), 10340-10344 (1994).</li><li>Kelleher, D. J., Gilmore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Saccharomyces+cerevisiae+oligosaccharyltransferase+is+a+protein+complex+composed+of+Wbp1p,+Swp1p,+and+four+additional+polypeptides.">The Saccharomyces cerevisiae oligosaccharyltransferase is a protein complex composed of Wbp1p, Swp1p, and four additional polypeptides.</a> J Biol Chem. 269 (17), 12908-12917 (1994).</li><li>Chevallier, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+transcriptional+control+of+a+eucaryotic+permease+gene.">Cloning and transcriptional control of a eucaryotic permease gene.</a> Mol Cell Biol. 2 (8), 977-984 (1982).</li></ol>

MYTHOS ist das erste System mit hohem Durchsatz, die die Identifizierung von Wechselwirkungen zwischen full-length Membranproteinen und cytosolische oder membrangebundene Partnern ermöglicht. Es wurde verwendet, um Membranprotein aus einer Reihe von Organismen [3-7] zu studieren. Es gibt jedoch bestimmte Details, die müssen unter die Lupe, um sicherzustellen, das Protein-of-interest zugänglich ist mit MYTHOS Studie werden können. Viele membrangebundene Proteine ​​sind an die Plasmame...

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<td>&nbsp;</td>
<td>Bioshop</td>
<td>PEG335</td>
<td>&nbsp;</td>
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<td>Lithium Acetate Bihydrate</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>LIA001</td>
<td>&nbsp;</td>
<tr>
<td>X-Gal (5-Bromo-4-Chloro-3-Indolyl-b-D-galactopyranoside)</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>XGA001</td>
<td>&nbsp;</td>
<tr>
<td>N`,N-dimethyl formamide</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>DMF 451</td>
<td>&nbsp;</td>
<tr>
<td>3-amino-1,2,4-triazole (3-AT)</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>ATT124</td>
<td>&nbsp;</td>
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<td>Sodium phosphate dibasic</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>SPD307</td>
<td>&nbsp;</td>
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<td>Sodium phosphate monobasic</td>
<td>&nbsp;</td>
<td>FisherBiotech</td>
<td>BP329-500</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CA80601-120</td>
<td>&nbsp;</td>
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<td>D-Glucose</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>GLU501</td>
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<td>LB Broth LENOX</td>
<td>&nbsp;</td>
<td>Bioshop</td>
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<td>Bioshop</td>
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<td>&nbsp;</td>
<td>Bioshop</td>
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<td>Bioshop</td>
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<td>&nbsp;</td>
<td>Bioshop</td>
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<td>L-Histidine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>HIS200</td>
<td>&nbsp;</td>
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<td>L-Methionine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
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<td>Bioshop</td>
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<td>&nbsp;</td>
<td>Bioshop</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Bioshop</td>
<td>TYR333</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Bioshop</td>
<td>LEU222</td>
<td>&nbsp;</td>
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<td>L-Arginine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>ARG006</td>
<td>&nbsp;</td>
<tr>
<td>L-Tryptophane</td>
<td>&nbsp;</td>
<td>FisherBiotech</td>
<td>BP395-100</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Bioshop</td>
<td>LYS101</td>
<td>&nbsp;</td>
<tr>
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<td>&nbsp;</td>
<td>FisherBiotech</td>
<td>BP369-100</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Bioshop</td>
<td>AGR001</td>
<td>&nbsp;</td>
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<td>11079105</td>
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split-ubiquitin based membrane yeast two-hybrid (myth) system: a powerful tool for identifying protein-protein interactions

Die grundlegenden biologischen und klinischen Bedeutung von integralen Membranproteinen veranlasste die Entwicklung einer Hefe-basiertes System für die Hochdurchsatz-Identifizierung von Protein-Protein-Interaktionen (PPI) für full-length Transmembranproteine. Zu diesem Zweck entwickelt unser Labor das Split-Ubiquitin-basierten Membrane Yeast Two-Hybrid (Mythos) System. Diese Technologie ermöglicht den empfindlichen Nachweis von transienten und stabilen Protein-Wechselwirkungen mit<em&gt; Saccharomyces cerevisiae</em&gt; Als Wirtsorganismus. MYTHOS nutzt die Beobachtung, dass Ubiquitin in zwei stabile Einheiten getrennt werden können: Die C-terminale Hälfte von Hefe-Ubiquitin (C<sub&gt; Ub</sub&gt;) Und die N-terminale Hälfte des Ubiquitin-Einheit (N<sub&gt; Ub</sub&gt;). Im Mythos wird dieser Grundsatz für den Einsatz als &quot;Sensor&quot; von Protein-Protein-Wechselwirkungen angepasst. Kurz gesagt, ist die integrale Membranproteine ​​Köderprotein zu C verschmolzen<sub&gt; Ub</sub&gt; Was ist es, eine künstliche Transkriptionsfaktor gebunden. Prey Proteine, entweder in einzelnen oder Bibliothek-Format werden die N verschmolzen<sub&gt; Ub</sub&gt; Einheit. Protein-Interaktion zwischen den Köder und Beute führt zum Auflösen des Ubiquitin-Einheiten bilden eine full-length &quot;pseudo-Ubiquitin-Molekül. Dieses Molekül wiederum ist durch cytosolische deubiquitinating Enzyme erkannt, was zur Spaltung des Transkriptionsfaktors, und die anschließende Induktion der Expression des Reportergens. Das System ist sehr anpassungsfähig und eignet sich besonders für High-Throughput-Screening geeignet. Es wurde erfolgreich eingesetzt, um Interaktionen mit integralen Membranproteinen aus Hefe-und anderen Organismen zu untersuchen.

Watch this Scientific Journal Video about Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions at JoVE.com

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTHOS ermöglicht den empfindlichen Nachweis von transienten und stabilen Wechselwirkungen zwischen Proteinen, die im Modellorganismus Saccharomyces cerevisiae exprimiert werden. Es wurde erfolgreich angewendet, um exogene und Hefe integrale Membranproteine ​​zu studieren, um ihre Interaktionspartner in einem hohen Durchsatz Weise zu identifizieren.

Split-Ubiquitin Basierend Membrane Yeast Two-Hybrid (Mythos) System: Ein wirksames Instrument zur Ermittlung von Protein-Protein-Wechselwirkungen

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

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Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

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High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

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