Yeast Two-Hybrid Assay for Studying Protein-Protein Interactions
Protein-protein interactions occur in a wide variety of biological processes and essentially control the cell fate from division to death. Today, the identification of proteins that interact with a protein of interest is a focus of intensive research and is an essential element of the rapidly growing field of proteomics. Yeast two-hybrid assays represent a versatile tool to study protein interactions in vivo. GAL4-based assay, for example, uses yeast transcription factor GAL4 for detection of protein interactions by transcriptional activation. Some transcription factors (such as GAL4) possess a characteristic phenomenon that the transactivation function can be restored when the factor’s DNA-binding domain (DBD) and its transcription-activation domain (AD) are brought together by two interacting, heterologous proteins. GAL4-yeast two-hybrid assay uses two expression vectors, one uses GAL4-DBD and the other uses GAL4-AD. DNA sequences encoding the two proteins of interest (or a protein and a complementary DNA library) can be cloned in the GAL4-DBD and GAL4-AD vectors to form the bait and the target of the interaction trap, respectively. A selection of host cells with different reporter genes and different growth selection markers provides a means to detect and confirm protein-protein interactions and highlight the flexibility of these assays to fit different applications. This chapter presents an outline for the GAL4-based yeast two-hybrid system with a detailed description of the vectors, host cells, and methods for detection and verifying protein interactions.
Key WordsADE2 Auxotrophic markers GAL4 GAL4-AD GAL4-BD HIS3 LacZ LacZ assays LEU2 MEL1 pADH TRP1 yeast plasmid preparation yeast transformation yeast two-hybrid
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