Two-hybrid screening originally known as yeast two-hybrid system or Y2H, is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.
The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS). For two-hybrid screening, the transcription factor is split into two separate fragments, called the DNA-binding domain and activating domain (AD). The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for the activation of transcription. The Y2H is thus a protein-fragment complementation assay.
Y2H is based on the reconstitution of a functional transcription factor (TF) when two proteins or polypeptides of interest interact. This takes place in genetically modified yeast strains, in which the transcription of a reporter gene leads to a specific phenotype, usually growth on a selective medium or change in the color of the yeast colonies. The most popular reporter genes are HIS3 to select yeast on a medium lacking histidine, and LacZ to screen yeast in a colorimetric assay.
Two fusions (‘hybrids’) are constructed between each protein of interest and either the DNA Binding Domain (DBD) or the Activation Domain (AD) of the TF. The protein fused to the DBD is referred to as the ‘bait’, and the protein fused to the AD as the ‘prey’.
Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter gene. The most popular fusions use the DBD and AD of the yeast TF Gal4. The bacterial protein LexA is also frequently used as a DBD in combination with Gal4 AD.
As a genetic technique, a yeast two-hybrid screen offers a sensitive and cost-effective means to test the direct interaction between two targeted proteins, or to use one’s favorite protein as a bait to screen libraries of protein fragments prepared from the desired cell types, tissues or entire organisms. The identity of the interacting partners is then obtained by sequencing the corresponding plasmids in the selected yeast colonies. Collections of full-length proteins (‘ORF omes’) are also becoming available for several species, but they do not cover the entire proteome yet.
For all the benefits of Y2H screening, this method has varieties of applications:
• Find new protein and new function of the protein
• Determination of sequences crucial for interaction
• Study the interaction between antigen and antibody in vivo
• Screening the functional site of new drugs and the affection of the drug to the interaction of target protein
• Establish genomic protein linkage map
The term “two-hybrid” is coined from the two classes of hybrid or chimeric proteins. Protein of interest “x” and a DNA binding domain (DBD-x) fuse to form “bait.” Fusion of transcriptional activation domain (AD-y) and a cDNA library “y” results in “prey.” These two classes form the basis of the protein–protein interaction detection system. Without bait–prey interaction, the activation domain cannot restrict to the reporter gene-to-gene expression drive.
This Y2H system has been successfully implemented for analysis of interaction between Calnexin and protein disulfide isomerase ERp57, and interactions between antibodies and antigens.