TwoHybrid

The two-hybrid system, also called the two-hybrid assay, is a molecular biology method for detecting protein-protein interactions by reconstituting a functional transcription factor from two modular fusion proteins inside a host cell. When the proteins of interest interact, a DNA-binding domain and a transcriptional activation domain are brought together, driving expression of a reporter gene.

The best known form is the yeast two-hybrid (Y2H), developed in 1989 by Fields and Song. In Y2H, a bait protein is fused to a DNA-binding domain and a prey protein to an activation domain; interaction activates transcription of selectable reporters such as HIS3, lacZ, or ADE2, enabling growth or screening. Variants include bacterial two-hybrid and mammalian two-hybrid systems, as well as adaptations for membrane proteins (split-ubiquitin, membrane yeast two-hybrid).

Principle and components: a bait-BD fusion and a prey-AD fusion are expressed in a host; their interaction reconstitutes a transcription factor that binds a promoter controlling a reporter gene. Readouts can be growth selection, colorimetric, or luminescent signals. The approach is widely used to map binary protein interactions and to screen libraries for novel interactors.

Limitations: false positives from autoactivation or nonspecific interactions; false negatives due to improper folding, lack of post-translational modifications, or mislocalization in the host; and effects of fusion orientation or linker sequences. Results are typically validated with orthogonal methods such as co-immunoprecipitation, pull-downs, or fluorescence-based assays.

Applications and scope: used to generate interaction networks, study domain requirements, and identify compounds that disrupt interactions. Advances include quantitative readouts, alternative host systems, and high-throughput sequencing approaches that identify interactors from screened libraries.