The Yeast Two-Hybrid Screen

The yeast two-hybrid screen for detection of protein-protein interactions was originally developed by Stanley Fields (Fields and Song, 1989). Improvements to the initial procedure included use of multiple reporter genes (Bartel et al., 1993; Durfee et al., 1993; Gyuris et al., 1993) and screening by mating (Bendixen et al., 1994; Finley and Brent, 1994). In addition, multiple variations have been developed, such as one-hybrid for detection of DNA-binding proteins (Li and Herskowitz, 1993; Wang and Reed, 1993, three-hybrid for use with RNA-binding proteins (SenGupta et al., 1996), reverse two-hybrid to identify molecules that may disrupt protein interactions (Vidal et al., 1996), and tethered catalysis two-hybrid, for protein interactions dependent on post-translational modifications (Guo et al., 2004).

The basic two-hybrid screen is reviewed in detail in numerous review articles (see Phizicky and Fields, 1999; Vidal and Legrain, 1999; Toby and Golemis, 2002; Miller and Stagljar, 2004 as examples.) In brief, a "bait" protein to be tested for interaction is fused to a DNA binding domain (BD) of a transcription factor (usually Gal4 or LexA), and placed in a yeast expression vector that contains a selectable marker. In parallel, a library of proteins (the "prey") are fused to an activation domain (AD) of the same or another transcription factor, and placed in a separate yeast expression vector. When the bait and prey are transformed into yeast, the binding domain binds to a DNA-binding site on a promoter sequence that drives a reporter protein, such as His3, Leu2, Ade2, or LacZ. An interaction between bait and prey brings the activation domain in the vicinity of the DNA-binding domain, and initiates transcription.