Sistema de dos hibridaciones en levadura: Detección in vivo de interacciones directas proteína-proteína

Yeast two-hybrid systems are one of the most popular and cost-effective in vivo genetic approaches for screening protein-protein interactions. The rationale behind the yeast two-hybrid (Y2H) system is the modular nature of transcription factors, which typically consist of a DNA-binding domain (BD) and an activation domain (AD), and the finding that both domains are not required to be covalently linked to activate transcription. By splitting a transcription factor into two separate domains, one fused to a "bait" protein (BD fusion) and the other to a "prey" protein (AD fusion), interaction between the bait and prey proteins brings the two domains together. This reconstitution of the transcription factor results in the activation of a reporter gene, signaling the protein-protein (bait-prey) interaction. Several variants of the Y2H system exist, but herein we will focus on the one based on the GAL4 transcription factor. In this chapter, we provide detailed protocols to efficiently co-transform and store competent yeast cells, test the interaction between two individual proteins, measure the relative strength of that interaction, and check the expression of the fusion proteins.