Protein–Protein Interaction: Bacterial Two-Hybrid

The bacterial two-hybrid (BACTH, for “Bacterial Adenylate Cyclase-Based Two-Hybrid”) system is a simple and fast genetic approach to detecting and characterizing protein–protein interactions in vivo. This system is based on the interaction-mediated reconstitution of a cyclic adenosine monophosphate (cAMP) signaling cascade in Escherichia coli. As BACTH uses a diffusible cAMP messenger molecule, the physical association between the two interacting chimeric proteins can be spatially separated from the transcription activation readout, and therefore it is possible to analyze protein–protein interactions that occur either in the cytosol or at the inner membrane level as well as those that involve DNA-binding proteins. Moreover, proteins of bacterial origin can be studied in an environment similar (or identical) to their native one. The BACTH system may thus permit a simultaneous functional analysis of proteins of interest—provided the hybrid proteins retain their activity and their association state. This chapter describes the principle of the BACTH genetic system and the general procedures to study protein–protein interactions in vivo in E. coli.