Protein-protein interactions (PPIs) are the backbone of signaling pathways, responsible for the basis of cell communication and, when deregulated, several diseases. Consequently, identifying and modulating PPIs can unravel the pathophysiological mechanisms of diseases. The two-hybrid system, particularly the mammalian two-hybrid system (MTH), is an efficient technique to validate PPIs ex vivo. Combining MTH with high-throughput screening has a huge advantage in biomedical research. In this review, we describe methodologies developed from MTH and the role of these adaptations in PPI discovery. We also highlight the powerful contribution of MTH to the identification of disease-related PPIs and its use in the development of potential new drug screens.
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