PROTEIN INTERACTIONS: YEAST TWO HYBRID

蛋白质相互作用：酵母二杂交体

• 批准号: 7602883
• 负责人: RUSSELL L FINLEY
• 金额: $ 23.26万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602883
• 关键词: Affinity; Binding Sites; Biological Assay; Cell Nucleus; Cells; Chimeric Proteins; Computer Retrieval of Information on Scientific Projects Database; Condition; DNA Binding Domain; Data; Epitopes; Funding; Genetic Transcription; Grant; Institution; Laboratories; Membrane; Methods; Nuclear Protein; Nuclear Proteins; Organism; Paper; Personal Satisfaction; Physiological; Proteins; Proteome; Publishing; Reagent; Reporter Genes; Research; Research Personnel; Resources; Set protein; Source; System; Technology; Testing; Transcriptional Activation; Two-Hybrid System Techniques; United States National Institutes of Health; Work; Yeasts; experience; extracellular; in vitro Assay; instrumentation; protein protein interaction; scale up; success; transcription factor; yeast two hybrid system

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The yeast two-hybrid system (Fields and Song, 1989) is one of the most widely used and well-proven methods to detect biologically important protein-protein interactions (for review see (Brent and Finley, 1997; Fields and Sternglanz, 1994). In these systems the two proteins to be tested for interaction are expressed in the nucleus of a yeast cell, where their interaction leads to activation of reporter genes. Both proteins are expressed with amino-terminal fusion moieties taken from a transcription factor. One protein is fused to the DNA-binding domain (DBD) and the other is fused to a transcription activation domain (AD). If the two proteins interact, the AD activates transcription of reporter genes that contains binding sites for the DBD. Yeast two-hybrid systems offer several advantages over other approaches for detecting protein-protein interactions. First, the assay is conducted inside cells, which may mimic the appropriate physiological conditions for some interactions better than in vitro assays. Second, the two-hybrid assay is robust. It has been used successfully with a variety of different protein types including cytoplasmic and nuclear proteins, and surprisingly, even some proteins that are normally found in membranes or that are extracellular (e.g. ref. (Li et al., 1996; Paine and Snead, 1997). Third, the yeast two-hybrid assay is simple and does not require expensive reagents or instrumentation. Fourth, the assay can be conducted with proteins from organisms lacking robust expression systems, which are often necessary for alternative protein interaction assays; e.g., assays that require the availability of an affinity tag for at least one of the proteins, are often achieved by expressing an epitope- or affinity-tagged fusion protein. Finally, the yeast two-hybrid system is a mature technology with well-understood advantages and pitfalls. Some laboratories, including ours, have been working with the system for over 10 years. We and others have gained the necessary experience to understand the experimental nuances and, most importantly, to properly interpret the data, distinguishing, for example, true positives from likely false positives. In the last 7 years, over 2,800 papers have been published that describe the functional characterization of new proteins or protein interactions that were initially detected using a two-hybrid approach (unpublished analysis). Thus, the value of using yeast two-hybrid data to generate hypotheses and to guide functional studies is well established. This success inspired us and a handful of other researchers to develop approaches to scale-up the system to identify the interactions among large sets of proteins, including entire proteomes.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 酵母双杂交系统（Fields 和 Song，1989）是检测生物学上重要的蛋白质-蛋白质相互作用的最广泛使用和经过充分验证的方法之一（综述参见（Brent 和 Finley，1997；Fields 和 Sternglanz，1994）。在这些系统中，待测试相互作用的两种蛋白质在酵母细胞的细胞核中表达，它们的相互作用导致报告基因的激活 基因。 两种蛋白质均表达有取自转录因子的氨基末端融合部分。 一种蛋白质与 DNA 结合结构域 (DBD) 融合，另一种蛋白质与转录激活结构域 (AD) 融合。 如果这两种蛋白质相互作用，AD 就会激活含有 DBD 结合位点的报告基因的转录。 与其他检测蛋白质-蛋白质相互作用的方法相比，酵母双杂交系统具有多种优势。 首先，该测定在细胞内进行，这可以比体外测定更好地模拟某些相互作用的适当生理条件。 其次，双杂交测定是稳健的。 它已成功地用于多种不同的蛋白质类型，包括细胞质和核蛋白，令人惊讶的是，甚至一些通常在膜中或细胞外发现的蛋白质（例如参考文献（Li et al., 1996; Paine and Snead, 1997））。第三，酵母双杂交测定很简单，不需要昂贵的试剂或仪器。第四，该测定可以用 来自缺乏强大表达系统的生物体的蛋白质，这通常是替代蛋白质相互作用测定所必需的；例如，需要至少一种蛋白质的亲和标签的可用性的测定通常通过表达表位或亲和标签的融合蛋白来实现。 最后，酵母双杂交系统是一项成熟的技术，其优点和缺点众所周知。 一些实验室，包括我们的实验室，一直在努力 使用该系统已超过 10 年。 我们和其他人已经获得了必要的经验来理解实验的细微差别，最重要的是，正确解释数据，区分真阳性和可能的假阳性等。 在过去 7 年中，已经发表了 2,800 多篇论文，描述了最初使用两种混合方法（未发表的分析）检测到的新蛋白质或蛋白质相互作用的功能特征。 因此，使用酵母的价值 用于生成假设和指导功能研究的两种混合数据已经很成熟。 这一成功激励我们和其他一些研究人员开发扩大系统规模的方法，以识别大量蛋白质（包括整个蛋白质组）之间的相互作用。