Protein-Protein Interactions: Yeast Two Hybrid

The yeast two-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-hybrid system to detect the interaction of the Agrobacterium type VI secretion system (T6SS) sheath components TssB and TssC₄₁. The bait and prey gene are expressed as a fusion to the GAL4 DNA-binding domain (DNA-BD) and GAL4 activation domain (AD, prey/library fusion protein), respectively. When bait and prey fusion proteins interact in yeast nucleus, the DNA-BD and AD are brought into proximity, thus activating transcription of reporter genes. This technology can be widely used to identify interacting partners, confirm suspected interactions, and define interacting domains.