PROTEIN INTERACTIONS: YEAST TWO HYBRID

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 酵母双杂交系统(Fields和Song，1989)是检测生物上重要的蛋白质相互作用的最广泛和最有效的方法之一(见Brent和Finley，1997；Fields和Sternglanz，1994)。在这些系统中，被测试相互作用的两种蛋白质在酵母细胞的细胞核中表达，在那里它们的相互作用导致报告基因的激活。这两种蛋白都是用取自转录因子的氨基末端融合部分表达的。一个蛋白质融合到DNA结合结构域(DBD)，另一个融合到转录激活结构域(AD)。如果这两种蛋白质相互作用，AD就会激活包含DBD结合位点的报告基因的转录。 酵母双杂交系统在检测蛋白质-蛋白质相互作用方面具有其他方法无法比拟的优势。首先，化验是在细胞内进行的，这可能比体外化验更好地模拟某些相互作用的适当生理条件。其次，双杂交试验是稳健的。它已经成功地用于各种不同的蛋白质类型，包括细胞质和核蛋白，令人惊讶的是，甚至一些正常存在于细胞膜或细胞外的蛋白质(例如REF)。(Li等人，1996；Paine和Snead，1997)。第三，酵母双杂交试验简单，不需要昂贵的试剂或仪器。第四，可以用缺乏强健表达系统的生物体的蛋白质进行分析，这通常是替代蛋白质相互作用分析所必需的；例如，要求至少一种蛋白质有亲和标记的分析通常是通过表达表位或亲和标记的融合蛋白来实现的。最后，酵母双杂交系统是一项成熟的技术，具有众所周知的优势和缺陷。一些实验室，包括我们的实验室，已经使用该系统超过10年了。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年里，已经发表了2800多篇论文，描述了最初使用双杂交方法(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，使用酵母双杂交数据来产生假说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几位研究人员开发方法来扩大系统，以识别包括整个蛋白质组在内的大量蛋白质之间的相互作用。

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.