Kim Ji Young, Park Ok Gu, Lee Young Chul
School of Biological Sciences and Technology, Chonnam National University, Gwangju, Republic of Korea.
Methods Mol Biol. 2012;812:209-23. doi: 10.1007/978-1-61779-455-1_12.
In an effort to develop a method for the high-throughput analysis of protein interaction interfaces, we devised a novel yeast genetic screening method, termed the "one- plus two-hybrid system," which efficiently selects specific missense mutations that disrupt known protein-protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid screening system is used first to positively select intact prey proteins, harboring informative missense mutations, from a large library of randomly generated mutant alleles. Next, among the isolated missense mutants of the prey proteins, interaction-defective mutants for a given protein (bait) are selected using the two-hybrid screening system. As a validation of the feasibility of this method, we utilized this technique to rapidly characterize the molecular determinants of the interactions between vitamin D receptor and its transcriptional coactivator protein, thyroid hormone receptor-associated protein 220. This efficient and rapid method should prove useful in the systematic analysis of large numbers of interaction interfaces.
为了开发一种高通量分析蛋白质相互作用界面的方法，我们设计了一种新型酵母遗传筛选方法，称为“一加二杂交系统”，该方法能有效筛选出破坏已知蛋白质 - 蛋白质相互作用的特定错义突变。该系统对标准酵母双杂交系统进行了改进，以便在同一细胞内操作双报告系统。首先使用单杂交筛选系统从大量随机生成的突变等位基因文库中阳性筛选出携带信息性错义突变的完整猎物蛋白。接下来，在分离出的猎物蛋白错义突变体中，使用双杂交筛选系统选择针对给定蛋白质（诱饵）的相互作用缺陷突变体。作为该方法可行性的验证，我们利用此技术快速表征了维生素D受体与其转录共激活蛋白甲状腺激素受体相关蛋白220之间相互作用的分子决定因素。这种高效且快速的方法在大量相互作用界面的系统分析中应会证明是有用的。
