PROTEIN INTERACTIONS: YEAST TWO HYBRID

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，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充分证明的方法之一（综述参见（布伦特和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，其中它们的相互作用导致报告基因的激活。 这两种蛋白质都用取自转录因子的氨基末端融合部分表达。 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。 如果两种蛋白质相互作用，AD激活包含DBD结合位点的报告基因的转录。 酵母双杂交系统提供了几个优势，比其他方法检测蛋白质-蛋白质相互作用。 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。 第二，双杂交测定是稳健的。 它已成功地用于各种不同的蛋白质类型，包括细胞质和核蛋白，并且令人惊讶地，甚至一些通常在膜中发现或在细胞外的蛋白质（例如参考文献（Li et al.，1996年;潘恩和斯尼德，1997年）。 第三，酵母双杂交试验简单，不需要昂贵的试剂或仪器。 第四，可以用来自缺乏稳健表达系统的生物体的蛋白质进行测定，这通常是替代蛋白质相互作用测定所必需的;例如，通常通过表达表位标记或亲和标记的融合蛋白来实现需要至少一种蛋白质的亲和标记可用性的测定。 最后，酵母双杂交系统是一种成熟的技术，具有众所周知的优点和缺陷。 包括我们在内的一些实验室已经使用该系统超过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，正确地解释数据，例如区分真阳性和可能的假阳性。 在过去的7年中，已经发表了2,800多篇论文，描述了最初使用双杂交方法检测到的新蛋白质或蛋白质相互作用的功能表征（未发表的分析）。 因此，使用酵母双杂交数据来产生假设和指导功能研究的价值是确定的。 这一成功启发了我们和其他一些研究人员开发方法来扩大系统的规模，以识别大量蛋白质之间的相互作用，包括整个蛋白质组。

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.