Methods and reagents for the detection and selection of two interacting-polypeptides, especially integral membrane proteins and transcription factors, by monitoring the reassembly of ubiquitin amino-terminal and carboxy-terminal chimeric polypeptide fragments are disclosed. Negative selection against an N-end rule-labilized marker released following ubiquitin reassembly allows direct selection of the interacting polypeptide pair. Methods to identify agonists and antagonists for certain protein-protein interactions; methods and reagents/kits for identifying proteins that binds a target protein are also provided. The dynamic and adaptable nature of the assay allows adaptation to a number of applications—such as probing the molecular environment of cellular membrane proteins in vivo.
揭示了一种用于检测和选择两个相互作用的
多肽的方法和试剂,特别是通过监测泛素
氨基末端和羧基末端嵌合
多肽片段的
重组来选择整合膜蛋白和转录因子。针对随泛素
重组释放的N-末端规则稳定标记物的负选择允许直接选择相互作用的
多肽对。还提供了用于识别某些蛋白质-蛋白质相互作用的激动剂和拮抗剂的方法,以及用于识别结合目标蛋白的蛋白质的方法和试剂/试剂盒。该检测方法的动态和适应性特性使其能够适用于多种应用,例如在体内探究细胞膜蛋白的分子环境。