一种用于识别功能上重要的蛋白质-蛋白质相互作用的两步双杂交系统。

A two-step two-hybrid system to identify functionally significant protein-protein interactions.

作者信息

Poustovoitov Maxim, Serebriiskii Ilya, Adams Peter D

机构信息

Fox Chase Cancer Center, 333 Cotman Ave, Philadelphia, PA 19111, USA.

出版信息

Methods. 2004 Apr;32(4):371-80. doi: 10.1016/j.ymeth.2003.10.012.

DOI:10.1016/j.ymeth.2003.10.012

PMID:15003599

Abstract

The two-step two-hybrid approach described here is an adaptation of the classic two-hybrid system. Its purpose is to identify proteins that interact with a relatively small, defined, functionally significant domain of a protein of interest. In this method, a first round of screening is performed to identify proteins that interact with bait comprised of the wild type protein. Next, each of the prey identified in this first round is tested for its ability to interact with functionally impaired, mutant bait. Any proteins that interact with the wild type bait, but not the mutant bait, are candidate effectors or regulators of the protein of interest.

摘要

这里描述的两步双杂交方法是经典双杂交系统的一种改良。其目的是鉴定与感兴趣蛋白质中相对较小、明确且具有功能意义的结构域相互作用的蛋白质。在该方法中，首先进行一轮筛选，以鉴定与由野生型蛋白质组成的诱饵相互作用的蛋白质。接下来，测试在第一轮中鉴定出的每个猎物与功能受损的突变型诱饵相互作用的能力。任何与野生型诱饵相互作用但不与突变型诱饵相互作用的蛋白质，都是感兴趣蛋白质的候选效应物或调节剂。