Journal
BACTERIAL PROTEIN SECRETION SYSTEMS: Methods and Protocols
Volume 1615, Issue -, Pages 159-176Publisher
HUMANA PRESS INC
DOI: 10.1007/978-1-4939-7033-9_13
Keywords
Two-hybrid system; Protein interaction assay; Membrane protein; cAMP signaling; Chimeric proteins
Funding
- Institut Pasteur
- Centre National de la Recherche Scientifique [CNRS UMR 3528]
- Universite Paris Diderot, Sorbonne Paris Cite, Cellule Pasteur, Paris, France
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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.
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