Yeast Hybridization Platform

After years of development, Tek Biotech has accumulated rich experience in immunology research and testing, and established a perfect molecular base laboratory platform. Tek Biotech's Yeast two-hybrid system (Y2H) consists of libraries constructed based on the SMART method, yeast strains, rigorous reporter genes, and high expression vectors.

The yeast two-hybrid system is an immunological testing technique that uses genetic and molecular approaches to detect protein interactions at the gene level. This method requires only the construction of plasmids, does not involve protein purification or antibody immunization stages, and at the same time provides easy and rapid access to the genes coding for interacting proteins.Y2H can be used to screen for sequences of proteins interacting with target proteins, as well as to investigate the principles of interactions between known proteins.

Tek Biotech can provide protein interaction assay services, including Co-IP, Pull-down and other experiments for qualitative and quantitative analysis of protein, small molecule and antibody interactions, as well as commonly used immunological assay techniques such as ELISA and Western Blot.

█ Principles of the yeast two-hybrid system (Y2H):

Figure 1 Principle of yeast two-hybrid system

Bonding Domain (BD): BD is a structural domain of proteins that binds to specific DNA molecules to regulate gene expression and self-replication.DNA BDs are found in many gene regulatory elements, such as transcription factors, and confer the function of recognizing and binding to specific DNA sequences to regulate downstream genes.

Activation Domain (AD): AD is a functional protein sequence that activates gene transcription during transcription and is often found in transcription factor proteins.AD can be formed into protein complexes (transcription factors, co-activators, and gene regulatory complexes, etc.) by interacting with other regulatory factors, thus enabling transcription factors to acquire the ability to bind to specific sequences of DNA. can bind to specific sequences of DNA and then recruit other transcription factors and RNA polymerases to initiate transcription. Because each transcription factor can only bind to specific sequences, this process is highly specific.

In the yeast two-hybrid system, DNA BD and transcription factor AD are fused to Bait protein X and Prey protein Y, respectively, to form fusion proteins.The BD-Bait X fusion protein recognizes and binds to the Upstream Activation Sequence (UAS) of the reporter gene. At the same time, if there is an interaction between the Bait X protein and Prey Y protein, this will cause the DNA AD to recombine with the transcription factor BD to form a structural domain that has the function of initiating the reporter gene.

The yeast two-hybrid and system is a method performed in the nucleus to identify and detect interactions between nuclear and cytoplasmic proteins using gene-level techniques. Also, Y2H can be screened using a library of protein fragments prepared from desired cell types, tissues or whole organisms using proteins as bait, and then interactions can be identified by sequencing the corresponding plasmids in the selected yeast colonies.Y2H is performed in eukaryotic yeast, and is characterized by its high sensitivity, power, and applicability, and it is now used in several research The Y2H is performed in eukaryotic yeast and has been applied in several research fields.

█ Classification of Yeast Hybridization Assay Services:

To study protein interactions, scientists have developed several systems based on the traditional Y2H system to accommodate different subcellular localizations and biochemical characteristics of different proteins. Tek Biotech offers a wide range of systems to suit the different needs of different projects.

(1) Yeast one-hybrid system (Y1H): to study protein-DNA interactions.

Fig. 2 Principle of yeast single hybridization system

The yeast monohybrid system requires the integration of Bait DNA sequences into yeast gene sequences. The Prey protein is constructed into an AD vector and expressed in fusion with the AD structural domain of the transcription factor GAL4. If the Prey protein recognizes and binds to the Bait DNA sequence, the AD structural domain fusion-expressed by the Prey protein can recruit the transcription complex to activate reporter gene expression.

(1) Yeast three-hybrid system (Y3H): study of the interaction between two proteins and a third component (protein, RNA or small molecule).

Fig. 3 Principle of yeast three-hybrid system

In the yeast three-hybrid system, two RNA-binding proteins X, Y are conjugated to DNA BD and transcription factor AD, respectively, to form two fusion proteins. The third component can provide two specific RNA targets for these two RNA-binding proteins X, Y to connect the two fusion proteins.

(1) Membrane yeast two-hybrid system (Mb Y2H): for the study of membrane protein

Fig. 4 Principle of membrane yeast two-hybrid system

Ubiquitin (Ub) is a protein that can be experimentally separated into two motifs and whose functions can be reconfigured when they are in close proximity to each other.

In the Mb Y2H system, the target membrane protein Bait is fused to half a ubiquitin (Cub, the truncated C-terminal end of the ubiquitin molecule) and the Cub is also attached to an artificial transcription factor (TF) containing an artificial transcription factor (TF), which consists of the bacterial LexA-DNA BD and the Herpes Simplex Virus VP16 protein AD. Fusion of another target protein Prey (membrane protein or cytoplasmic protein) to another half of modified ubiquitin (NubG/NubA, which is the truncated N-terminal end of the ubiquitin molecule that is modified so as to reduce the affinity between the two, Cub and Nub, and to avoid the phenomenon of their spontaneous recombination). So only when there is an interaction between the protein bound to NubG/A and the protein bound to Cub can the two half ubiquitin molecules combine to form a complete ubiquitin molecule that can be recognized by ubiquitin-specific proteases (UBPs) for action. Then, in the presence of the enzyme, the regulatory factors attached to the Cub are released into the nucleus, where they bind to specific DNA sequences to activate transcriptional reporter genes.

█ Service Advantage:

-- Direct service from technical experts to meet different testing needs

-- Highly accurate analytical instruments, experienced operators and strict quality control ensure accurate and reliable results.

-- High efficiency of testing services and real-time process monitoring

-- High sensitivity and low sample consumption.

-- Short cycle time, saving waiting time

-- A true one-stop shop: from library construction to positive clone screening, which improves efficiency, avoids errors, and saves the client money objectively.

█ Service Content