Lievens Sam, Van der Heyden José, Vertenten Els, Plum Jean, Vandekerckhove Joël, Tavernier Jan
Department of Medical Protien Research, Flanders Interuniversity Institute for Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, Belgium.
Methods Mol Biol. 2004;263:293-310. doi: 10.1385/1-59259-773-4:293.
The mammalian protein-protein interaction trap (MAPPIT) is a two-hybrid assay based on insights in type I cytokine signal transduction. Bait and prey polypeptides are tethered to mutant cytokine receptor chimeras which are impaired in signaling. On bait-prey interaction and after ligand stimulation, the JAK-STAT signaling cascade is initiated leading to transcription of a reporter or marker gene under the control of the STAT3-responsive rPAP1 promoter. In addition to a physiologically relevant context for mammalian protein-protein interactions this method provides separation of interactor and effector zones, and can be applied for both analytical and screening purposes. In the protocol described here, a cytokine receptor derived surface tag is used as a selectable marker. After an initial presort step using magnetic-activated cell sorting (MACS), "positive" cells are selected by fluorescence-activated cell sorting (FACS).
哺乳动物蛋白质-蛋白质相互作用陷阱（MAPPIT）是一种基于对I型细胞因子信号转导深入了解的双杂交检测方法。诱饵和猎物多肽与信号传导受损的突变细胞因子受体嵌合体相连。在诱饵-猎物相互作用以及配体刺激后，启动JAK-STAT信号级联反应，导致在STAT3反应性rPAP1启动子控制下的报告基因或标记基因转录。除了为哺乳动物蛋白质-蛋白质相互作用提供生理相关背景外，该方法还提供了相互作用体和效应区的分离，可用于分析和筛选目的。在此处描述的方案中，使用细胞因子受体衍生的表面标签作为选择标记。在使用磁激活细胞分选（MACS）进行初始预分选步骤后，通过荧光激活细胞分选（FACS）选择“阳性”细胞。
