A Bacterial Two-Hybrid System for In Vivo Assays of Protein-Protein Interactions and Drug Discovery.

Abstract

The bacterial adenylate cyclase-based two-hybrid (BACTH) system is a robust and simple genetic assay used to monitor protein-protein interactions in vivo. This system is based on functional complementation between two fragments from the catalytic domain of Bordetella pertussis adenylate cyclase (AC) to reconstitute a cyclic AMP (cAMP)-signaling cascade in Escherichia coli. Interactions between two chimeric proteins result in the synthesis of cAMP, which activates the transcription of various catabolite operons, leading to selectable phenotypes. One advantageous feature of this signaling cascade is that the physical association between the two interacting hybrid proteins is spatially separated from the transcriptional activation readout. Consequently, the BACTH system can detect protein-protein interactions occurring at various subcellular localizations. The system has been used to characterize interactions between soluble or membrane proteins of prokaryotic, eukaryotic, or viral origin. The BACTH assay can be used to uncover the region(s), domain(s), or amino acid residue(s) of a protein involved in an interaction with a specific partner. The BACTH system can also be adapted for the high-throughput screening of small molecules able to interfere with protein-protein interactions.