Design of a fluorescence-activated cell sorting-based Mammalian protein-protein interaction trap.

ABSTRACT

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).