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Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions
Published on: December 23, 2022
Protein-Protein Interaction: Bacterial Two Hybrid
Gouzel Karimova1, Emilie Gauliard1,2, Marilyne Davi1
1Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Institut Pasteur, CNRS, UMR 3528, Paris, France.
Abstract:
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.
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