MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions

Abstract

MAPPIT (MAmmalian Protein-Protein Interaction Trap) is a two-hybrid technology that facilitates the detection and analysis of interactions between proteins in living mammalian cells. The system is based on type 1 cytokine receptor signaling. The bait protein of interest is fused to a chimeric signaling-deficient cytokine receptor, the signaling competence of which is restored upon recruitment of a prey protein that is coupled to a functional cytokine receptor domain. MAPPIT exhibits an excellent signal-to-noise ratio, detects a wide variety of protein-protein interactions (PPIs) including transient and indirect interactions, and has been shown to be highly complementary to other two-hybrid methods with respect to the interactions it can detect. Variants of the method were developed to allow large-scale PPI screening, mapping of protein interaction interfaces, PPI inhibitor screening and drug profiling. This chapter describes a basic 4-day MAPPIT protocol for the analysis of interaction between two designated proteins.