Yeast Two-Hybrid (Y2H) Technology

Product Manager: Harrison Michael

Yeast Two-Hybrid (Y2H) technology is an important technique in the field of molecular biology, primarily used to reveal interactions between proteins. With the advancement of genomics and functional genomics, Y2H technology has become a core tool in the study of protein-protein interactions, cellular signal transduction, and the screening of new drug targets. This article will focus on the experimental aspects of Y2H, including the selection of reagents, experimental procedures, tips, and precautions, and will briefly review its principles, history, and applications.

I. Basic Principles of Yeast Two-Hybrid

The principle of Y2H technology is to detect interactions between two different proteins to reveal whether they collaborate in biological processes. This technique is based on the principle of splitting and recombining the functional domains of transcription factors, using yeast as the expression host and activating reporter genes to detect interactions between target proteins.

1.1 Working Principle

The Y2H system divides the interaction between two target proteins into two parts: the bait protein is fused with the DNA-binding domain (BD) of a transcription factor, and the prey protein is fused with the activation domain (AD). When the bait and prey proteins interact, the BD and AD are brought close together, activating the expression of the reporter gene. This allows yeast to grow on selective media containing specific substances or to produce color changes in blue-white screening. This method has high sensitivity and specificity, effectively screening for potential protein interaction pairs.

II. Experimental Procedures of Yeast Two-Hybrid

2.1 Reagents and Materials Required for the Experiment

The following reagents and materials are essential for Y2H experiments:

· Plasmid vectors: Used for cloning the genes of bait and prey proteins.

· Yeast strains: High-specificity yeast strains are commonly used, such as Y2HGold.

· Selective media: Such as SD/-Trp, SD/-Leu, SD/-Leu/-Trp, SD/-Leu/-Trp/-His/-Ade.

· Yeast transformation reagents: Such as PEG 3000 or PEG 4000, lithium acetate (LiAc), and salmon sperm DNA.

· X-α-Gal：Used for colorimetric detection of the LacZ reporter gene.

2.2 Vector Construction

1. Gene amplification:

· Use high-fidelity polymerase to amplify the coding genes of bait and prey proteins. The primers designed should include appropriate restriction sites for subsequent cloning.

2. Restriction and ligation:

· Digest the plasmid vectors and target genes with restriction enzymes, and then use DNA ligase to insert the target genes into the vectors. Commonly used vectors include pGBKT7 (for bait proteins) and pGADT7 (for prey proteins).

3. Plasmid verification:

· Verify the correct insertion of the target gene into the vector using restriction digestion or sequencing.

2.3 Yeast Transformation

1. Preparation of competent yeast:

· Cultivate yeast in YPDA medium to the logarithmic growth phase (OD600 ≈ 0.6). Take an appropriate volume of the culture, centrifuge to remove the supernatant, and resuspend the yeast in TE/LiAc solution.

2. Yeast transformation (LiAc/PEG method):

· Add the recombinant plasmid and salmon sperm DNA to the competent yeast. Add a mixture of PEG3350/LiAc, incubate at 30°C for 30 minutes, and then heat-shock at 42°C for 20 minutes to facilitate DNA uptake by yeast cells.

3. Selection of positive transformants:

· After transformation, spread the yeast onto selective media plates (e.g., SD/-Trp or SD/-Leu) and incubate at 30°C for 2-3 days. Pick single colonies for further analysis.

 

Experimental Tips and Precautions:

· Yeast culture density: Ensure the OD600 value is between 0.4 and 0.8 for optimal transformation efficiency.

· Fresh PEG solution: Use fresh PEG solution for the best results.

· Control experiments: For bait proteins, perform individual transformation experiments to confirm whether they can auto-activate the reporter gene.

2.4 Protein Interaction Screening

1. Co-transformation of bait and prey proteins:

· Mix yeast strains harboring bait and prey proteins at a 1:1 ratio and spread them onto SD/-Leu/-Trp selective media plates. Incubate at 30°C for 2-3 days and pick single colonies.

2. Selection of positive clones:

· Transfer the transformed yeast to more stringent selective media (SD/-Leu/-Trp/-His/-Ade) to screen for colonies that can grow. Further verify the interactions using X-α-Gal blue-white screening to observe whether the yeast colonies turn blue.

 

Experimental Tips and Precautions:

· Stringency of selection: Increase the stringency of the selective media to reduce false positives.

· Replicate verification: After each experiment, pick multiple independent colonies for verification to ensure the reliability of the results.

III. Applications of Yeast Two-Hybrid Technology

3.1 Elucidation of Cellular Signal Transduction Pathways

Y2H technology is widely used in the study of intracellular signaling pathways. By screening proteins that interact with key factors in signaling pathways (such as transcription factors and receptor proteins), researchers can reveal complex signal transduction processes. For example, Y2H technology has been used to elucidate the interactions between β-catenin and its interacting proteins in the Wnt signaling pathway.

3.2 Drug Target Screening

During drug development, Y2H can be used to screen for potential drug targets. By identifying molecules that interact with target proteins, key molecules regulating cellular functions can be discovered. In virology research, Y2H is also used to identify interactions between viral proteins and host cell proteins, providing clues for the development of antiviral drugs.

3.3 Study of Gene Interaction Networks

By constructing Y2H libraries, researchers can perform high-throughput protein interaction screening on a genomic scale, further revealing the connections between genes and their products. This method is widely used for gene function analysis and the discovery of new protein functions.

IV. Conclusion

Yeast Two-Hybrid technology provides a powerful tool for studying interactions between genes and proteins. With continuous development and optimization of the technology, Y2H will play an increasingly important role in functional genomics, drug screening, and disease research, offering deeper insights and new directions for biological research.

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