A bidirectional fluorescent two-hybrid system for monitoring protein–protein interactions

Abstract

Two-hybrid systems have been the cornerstone of research into protein–protein interactions, but these systems typically rely on life/death reporters that put additional selective pressure on the host organism, and potentially lead to false positives. Here we report a bidirectional fluorescence-based bacterial two-hybrid system that enables both the association and dissociation of a given protein–protein interaction to be monitored. The functionality of this system and its compatibility with FACS screening are demonstrated in the forward and reverse direction using known interacting protein-partners and their cyclic peptide inhibitors. The reported fluorescent two-hybrid system may be used in the forward direction for the identification of interacting protein partners, or as a reverse two-hybrid system for the high-throughput identification of protein–protein interaction inhibitors.