Protein–Protein Interactions: Yeast Two Hybrid

The yeast two-hybridYeast two-hybrid (Y2H) systemTwo-hybrid system is a powerful and commonly used genetic tool to investigate the interaction between artificial fusion proteins inside the nucleus of yeast. Here, we describe how to use the Matchmaker GAL4-based yeast two-hybridYeast two-hybrid (Y2H) systemTwo-hybrid system to detect the interaction of the Agrobacterium type VI secretion system (T6SS)Type VI secretion system (T6SS) sheath components TssB and TssC41. The baitBait and preyPrey gene are expressed as a fusion to the GAL4 DNA-binding domain (DNA-BDGAL4 DNA binding domain (DNA-BD)) and GAL4 activation domain (AD, preyPrey/library fusion protein), respectively. When baitBait and preyPrey fusion proteins interact in yeast nucleus, the DNA-BDGAL4 DNA binding domain (DNA-BD) and AD are brought into proximity, thus activating transcription of reporter genesReporter genes. This technology can be widely used to identify interacting partners, confirm suspected interactions, and define interacting domains.