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Protein‑protein interaction
1. Y2H (Yeast two‑hybrid)
1.1 Introduction
Yeast Two‑Hybrid (Y2H) is an in vivo protein‑protein interaction screening technology based on the modular structure of transcription factors. The system splits the GAL4 transcription factor into a DNA‑Binding Domain (BD) and a Transcription Activation Domain (AD), which are fused to a Bait protein and a Prey protein, respectively; when the Bait and Prey physically interact within the yeast nucleus, the BD and AD are brought into proximity and reconstitute a functional transcription factor, activating downstream reporter genes (e.g., HIS3, ADE2, LacZ). Protein interaction is thus determined through auxotrophic complementation or colorimetric assays. This technique requires no protein purification and enables high‑throughput screening of unknown interaction partners at the whole‑genome cDNA library scale, serving as a classical tool for discovering novel protein‑protein interactions.
1.2 Products
1.3 Applications
| Applications | Description |
|---|---|
| Interactome mapping | Y2H is the core technology for mapping proteome‑scale binary protein‑protein interaction networks. Through array‑based or pooled library screening strategies, it enables systematic identification of direct PPIs across the entire genome, constructing cellular "wiring diagrams." Integration with next‑generation sequencing dramatically increases throughput and coverage. |
| Drug target screening and validation | Y2H screens host proteins or endogenous regulators interacting with disease‑associated proteins to discover potential drug targets. Reverse Y2H enables screening of small‑molecule inhibitors that disrupt specific PPIs, providing a high‑throughput platform for PPI‑targeted drug development. |
| Host‑pathogen interaction studies | Y2H is one of the most widely used experimental methods for identifying interactions between pathogen proteins and host cellular proteins. By screening host cDNA libraries with pathogen proteins as bait, it reveals molecular mechanisms of pathogen invasion, replication, and immune evasion, providing novel targets for antiviral/antimicrobial drug development. |
| Protein function and Domain mapping | By constructing a series of deletion mutants or point mutations of bait proteins and performing pairwise Y2H assays with prey proteins, the key domains and critical amino acid residues mediating interactions can be precisely mapped. Combined with Deep Mutational Scanning, the effects of mutations on interactions can be systematically assessed at amino‑acid resolution. |
| Signal transduction pathway analysis | Y2H is widely applied to study intracellular signaling pathways. By screening proteins interacting with key signaling factors, complex signal transduction processes are revealed. Combined with Yeast Three‑Hybrid, ternary complexes or ligand‑dependent interactions can be studied to dissect hormone signaling and pathway crosstalk. |
References:
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