Identifying Interacting Proteins in an Escherichia coli-Based Two-Hybrid System

The BacterioMatch™ two-hybrid system provides a fast, simple, and efficient method for in vivo detection of protein-protein interactions in Escherichia coli (1 ,2 ). This system is based on transcriptional activation of a reporter gene. In this system, a protein of interest (the bait) is fused to the full-length bacteriophage λ repressor protein (λcl), which consists of an amino-terminal DNA-binding domain and a carboxyl-terminal dimerization domain. Target proteins are fused to the N-terminal domain of the α-subunit of RNA polymerase (RNAP-α). When expressed in the bacterial reporter strain, the bait is tethered to the λ operator sequence upstream of the reporter promoter through the DNA-binding domain of λcl. When the bait and target proteins interact, they recruit and stabilize the binding of RNA polymerase at the promoter and activate transcription of the β-lactamase (Amp^r ) and β-galactosidase (lacZ ) reporter genes (see Fig. 1 ). Expression from the reporter promoter is correlated with the interaction affinity of the bait and target. A stronger interaction between bait and target proteins results in higher levels of gene expression of the reporter genes, as indicated by higher levels of carbenicillin-resistance (3 ) and β-galactosidase activity in the reporter strain. The BacterioMatch system detects an interaction between proteins with an equilibrium dissociation constant in the high nanomolar range.

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