Protein–Protein Interactions: Yeast Two-Hybrid System
The yeast two-hybrid system is a powerful and commonly used genetic tool to investigate interactions 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, thereby activating the transcription of reporter genes. This technology can be widely used to identify interacting partners, confirm suspected interactions, and define interacting domains.
Key wordsProtein–protein interaction Yeast two-hybrid Gal4 transcriptional activation domain (AD) Gal4 DNA-binding domain (BD) Saccharomyces cerevisiae AH109 Type VI secretion system TssB TssC
This work was supported by a research grant from the Ministry of Science and Technology (MOST 104-2311-B-001-025 -MY3) to E.M. Lai. J. S. Lin is the recipient of postdoctoral fellowships from Academia Sinica.
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