Protein-Protein Interaction: Bacterial Two Hybrid

Methods Mol Biol. 2024:2715:207-224. doi: 10.1007/978-1-0716-3445-5_13.


The bacterial two-hybrid (BACTH, for "Bacterial Adenylate Cyclase-based Two-Hybrid") system is a simple and fast genetic approach to detect and characterize protein-protein interactions in vivo. This system is based on the interaction-mediated reconstitution of a 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 from 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 the 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.

Keywords: Chimeric proteins; Membrane protein; Protein interaction assay; Two-hybrid system; cAMP signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclases*
  • Cytosol
  • DNA-Binding Proteins
  • Escherichia coli* / genetics
  • Genes, Bacterial


  • Adenylyl Cyclases
  • DNA-Binding Proteins