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

我们想感谢黎明埃德蒙兹关键读了这篇稿子。 Stagljar实验室是由从加拿大创新（CFI），加拿大卫生研究所（CIHR），心脏及中风基金会，加拿大癌症协会，和诺华基金会的资金支持。

1。背景资料蛋白质 - 蛋白质相互作用（生产者价格指数）是参与所有细胞过程的基本构建块。因此，它是必不可少的，所有交互严格管制，以维持细胞的动态平衡，在这个生物平衡的转变，通常扮演在疾病和癌症细胞转化的作用。膜相关蛋白是生物最重要的一类蛋白质之中，因为他们可以启动复杂的信号级联，并进行调解的进口和出口的各种分子，包括药物，被列为相关的问...

<ol>
	<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>

神话是第一个高通量确定全长膜蛋白和胞浆或膜结合的合作伙伴之间的的相互作用的系统，使。它已被用于研究膜蛋白的生物体[3-7]。有，但是，具体的细节，可能需要经过严格审核的程序，以确保蛋白质的利益服从研究神话。 许多膜结合蛋白裂解产生的成熟蛋白的信号序列，随后通过质膜。这个序列是生物体的特定的和有可能，这个原始信号序列在酵母中表达时，会导致定...

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<td>Bioshop</td>
<td>PEG335</td>
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<td>Lithium Acetate Bihydrate</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>LIA001</td>
<td>&nbsp;</td>
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<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>
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<td>N`,N-dimethyl formamide</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>DMF 451</td>
<td>&nbsp;</td>
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<td>3-amino-1,2,4-triazole (3-AT)</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>ATT124</td>
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<td>Sodium phosphate dibasic</td>
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<td>Bioshop</td>
<td>SPD307</td>
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<td>Sodium phosphate monobasic</td>
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<td>BP329-500</td>
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<td>Salmon Sperm DNA</td>
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<td>CA80601-120</td>
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<td>&nbsp;</td>
<td>Bioshop</td>
<td>TYR333</td>
<td>&nbsp;</td>
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<td>L-Leucine</td>
<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>L-Lysine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>LYS101</td>
<td>&nbsp;</td>
<tr>
<td>L-Alanine</td>
<td>&nbsp;</td>
<td>FisherBiotech</td>
<td>BP369-100</td>
<td>&nbsp;</td>
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<td>Agar</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>AGR001</td>
<td>&nbsp;</td>
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<td>Soda Lime Galss Beads</td>
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split-ubiquitin based membrane yeast two-hybrid (myth) system: a powerful tool for identifying protein-protein interactions

基本的生物和临床不可或缺的膜蛋白的重要性，提示为全长跨膜蛋白的蛋白质相互作用（PPI）的高通量鉴定酵母为基础的系统的发展。为此，我们的实验室开发的分裂泛基于膜酵母双杂交系统（神话）。这种技术允许的瞬时和稳定的蛋白质相互作用的敏感检测<em&gt;酿酒酵母</em作为东道主的有机体。神话的泛素可分隔成两个稳定的基团的观察优势：酵母C -末端的一半的泛素（彗星<sub&gt; UB</sub&gt;）和泛素基的N -末端的一半（N<sub&gt; UB</sub&gt;）。在神话中，这一原则适用于使用，作为一种“传感器”蛋白质 - 蛋白质相互作用。简言之，积分膜诱饵蛋白融合到C<sub&gt; UB</sub&gt;这是与人工转录因子。猎物的蛋白质，无论是在个人或库格式，融合到N<sub&gt; UB</sub&gt;基团。诱饵和猎物之间的蛋白质相互作用导致泛基团的重组，形成一个完整长度的“伪泛”分子。这种分子是由胞质deubiquitinating酶的认可，产生的转录因子，随后诱导报告基因的表达裂解。该系统适应性强，特别适合于高通量筛选。它已成功地调查使用从酵母和其他微生物的整合膜蛋白的相互作用。

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

神话允许的瞬时和稳定的互动模式生物酵母中表达的蛋白质之间的灵敏的检测。它已成功地应用于研究外源​​性和酵母的完整的膜蛋白，以确定其在高通量的方式相互作用的合作伙伴。

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

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

Research

JoVE Journal

Biology

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

Imaging Protein-protein Interactions in vivo

Imaging Protein-protein Interactions in vivo

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

科研

生物学

Associated Chromosome Trap for Identifying Long-range DNA Interactions

确定远距离的DNA相互作用的相关染色体陷阱

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

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

Identifying Protein-protein Interaction Sites Using Peptide Arrays

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...