Yeast two-hybrid (Y2H) screening is an extension of the Y2H assay, used to systematically identify potential protein-protein interactions in a large-scale or high-throughput manner. It allows researchers to screen a "library" of potential interacting proteins (prey) against a "bait" protein to discover new binding partners or interactions that may have biological significance.

 Steps in Yeast Two-Hybrid Screening

1. Preparation of Bait and Prey Libraries

   Bait Protein: A protein of interest is fused to the DNA-binding domain (DBD) of a yeast transcription factor.

   Prey Library: A large collection of proteins or peptides (often the entire proteome of an organism or a specific subset of proteins) is fused to the activation domain (AD) of the same yeast transcription factor.

2. Transformation into Yeast:

   The yeast cells are co-transformed with the bait construct and a large library of prey constructs. Each yeast cell receives one bait construct and one prey construct.

3. Selection of Positive Interactions:

   If a prey protein interacts with the bait protein, the DBD and AD are brought together, reconstituting a functional transcription factor. This leads to the expression of a reporter gene (such as HIS3 or lacZ), allowing yeast to grow on selective media or show a detectable signal (e.g., blue color from lacZ activity).

   Yeast cells that survive or show a color change are considered "positives," indicating a potential interaction between the bait and a prey protein.

4. Isolation and Identification of Prey Proteins:

   Positive yeast colonies are isolated, and the interacting prey proteins are identified by sequencing the DNA of the prey constructs. This reveals the identity of the protein that interacts with the bait.

5. Verification of Interactions:

   After identifying potential interacting partners, follow-up assays such as independent Y2H tests, co-immunoprecipitation (Co-IP), or pull-down assays are performed to validate the interaction.

Applications of Yeast Two-Hybrid Screening

Protein Interaction Networks: Y2H screening can map complex interaction networks, revealing how proteins communicate in cellular pathways.

Drug Target Discovery: Screening with a specific disease-related protein as the bait can help identify drug targets or pathways involved in disease progression.

Functional Genomics: This technique helps assign function to unknown proteins by identifying their interaction partners.

Mutation Analysis: Mutant forms of a bait protein can be used in screening to see how mutations affect its interaction network.

Advantages

Can detect novel and unknown protein-protein interactions.

Can screen a large library of proteins in a relatively short time.

Inexpensive compared to other interaction-mapping techniques.

 Limitations

False Positives: Some interactions may be non-specific or due to artifacts of the assay.

False Negatives: Weak or transient interactions may not be detected, and membrane-bound or insoluble proteins may not function correctly in yeast cells.

Yeast-Specific Conditions: Proteins that require post-translational modifications not present in yeast may not be properly tested.

Y2H screening remains one of the most widely used methods for mapping protein-protein interactions and discovering new biological insights.