Serebriiskii Ilya
Cold Spring Harb Protoc. 2010 May;2010(5):pdb.prot5429. doi: 10.1101/pdb.prot5429.
An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the yeast two-hybrid system, uses transcriptional reporters in yeast to indirectly reflect the interaction between two proteins. The term two-hybrid derives from the two classes of chimeric, or "hybrid," proteins used in each screen. The first, commonly referred to as the "bait," is a fusion of a protein of interest "x" with a DNA-binding domain (DBD-x). The second, sometimes called the "prey," is a fusion of a cDNA library "y" to a transcriptional activation domain (AD-y). Together, DBD-x and AD-y provide the basis of the detection system. The two-hybrid approach has gained wide popularity because of the relative ease and speed with which it can be used to identify novel protein-protein interactions and to analyze known interactions. In stage 1 of the method, detailed in this protocol, characterization of a novel bait is described, with attention to controls that increase the chance of the bait functioning in a two-hybrid screen.
蛋白质功能表征的一个重要因素是鉴定与其相互作用的其他蛋白质。为此目的的一种强大的遗传策略，称为酵母双杂交系统，利用酵母中的转录报告基因来间接反映两种蛋白质之间的相互作用。双杂交一词源于每个筛选中使用的两类嵌合或“杂交”蛋白质。第一种，通常称为“诱饵”，是感兴趣的蛋白质“x”与DNA结合结构域（DBD-x）的融合体。第二种，有时称为“猎物”，是cDNA文库“y”与转录激活结构域（AD-y）的融合体。DBD-x和AD-y共同构成了检测系统的基础。双杂交方法因其相对容易且快速地用于鉴定新的蛋白质-蛋白质相互作用和分析已知相互作用而广受欢迎。在本方案详细描述的方法的第1阶段，描述了新型诱饵的表征，并关注增加诱饵在双杂交筛选中发挥作用机会的对照。
