Protein–Protein Interaction: Bacterial Two-Hybrid

  • Gouzel Karimova
  • Emilie Gauliard
  • Marilyne Davi
  • Scot P. Ouellette
  • Daniel LadantEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)


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.

Key words

Two-hybrid system Protein interaction assay Membrane protein cAMP signaling Chimeric proteins 



This work was supported by Institut Pasteur and the Centre National de la Recherche Scientifique (CNRS UMR 3528, Biologie Structurale et Agents Infectieux). E.G. was supported by Ph.D. funding from the Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France.


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Gouzel Karimova
    • 1
  • Emilie Gauliard
    • 1
    • 2
  • Marilyne Davi
    • 1
  • Scot P. Ouellette
    • 3
  • Daniel Ladant
    • 1
    Email author
  1. 1.Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et ChimieInstitut Pasteur, CNRS, UMR 3528ParisFrance
  2. 2.Université Paris Diderot, Sorbonne Paris Cité, Cellule PasteurParisFrance
  3. 3.Division of Basic Biomedical Sciences, Sanford School of MedicineUniversity of South DakotaVermillionUSA

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