Two-Hybrid System for Characterization of Protein-Protein Interactions in E. coli

Abstract

The yeast two-hybrid system has been used to characterize many protein-protein interactions. A two-hybrid system for E. coli was constructed in which one hybrid protein bound to a specific DNA site recruits another to an adjacent DNA binding site. The first hybrid comprises a test protein, the bait, fused to a chimeric protein containing the 434 repressor DNA binding domain. In the second hybrid, a second test protein, the prey, is fused downstream of a chimeric protein with the DNA binding specificity of the λ repressor. Reporters were designed to express cat and lacZ under the control of a low-affinity λ operator. At low expression levels, λ repressor hybrids weakly repress the reporter genes. A high-affinity operator recognized by 434 repressor was placed nearby, in a position that does not yield repression by 434 repressor alone. If the test proteins interact, the 434 hybrid bound to the 434 operator stabilizes the binding of the λ repressor hybrid to the λ operator, causing increased repression of the reporter genes. Reconstruction experiments with the fos and jun leucine zippers detected proteinprotein interactions between either homodimeric or heterodimeric leucine zippers.