Yeast Three Hybrid Service

KMD Bioscience has an experienced research team that can provide customers with high-quality yeast three hybrid services to verify the interactions between proteins and between proteins and other biomolecules. At the same time, KMD Bioscience can also provide a variety of customized services, including protein interaction verification, construction of yeast three hybrid cDNA libraries for different samples (cDNA libraries for animal tissues, plant tissues, cells, and other species samples), construction of random peptide libraries, screening of targeted proteins/small molecule peptides, and other yeast three hybrid services. Based on the target protein sequence information provided by the customer, the types of libraries that need to be constructed, and sample information, KMD Bioscience can customize a reasonable experimental plan for the customer. KMD Bioscience, utilizing standardized experimental methodologies, offers robust support for subsequent research and development endeavors, encompassing the validation of established protein interactions, the creation and assessment of yeast three-hybrid libraries, the establishment and screening of peptide libraries adhering to yeast three-hybrid principles, the identification and confirmation of potential protein/small molecule peptide interactions, as well as the formulation of targeted small molecule peptide drugs.

KMD Bioscience has a well-established yeast three hybrid system and a protein interaction verification platform based on yeast hybrid system. With its in vivo interaction verification characteristics, the yeast three hybrid system allows interacting proteins to approach their natural conformation, resulting in more reliable results. The yeast three hybrid cDNA library and peptide library of KMD Bioscience have high library capacity, up to 10^6-10^7, and library diversity and insertion rate can reach over 90%, ensuring the quality of delivered libraries. In addition to library construction, KMD Bioscience can also provide library screening, as well as supporting expression validation and affinity determination services. Yeast hybrid system has become increasingly important in scientific research in fields such as biology. Through this service, we can assist clients in their research projects and provide new ideas.

Yeast Three Hybrid Service

By improving on the yeast two hybrid technology, yeast three hybrid technology has been developed, which is a specialized technique for verifying the interactions between proteins and between proteins and other biomolecules. Yeast three hybrid technology introduces a third protein as a regulatory factor to simulate and design the interactions of the three components, making it easier to explore complex biological processes such as signal transduction pathways and transfer complexes. The main advantage of this technology is its ability to provide intuitive data on the composition and dynamic changes of protein complexes, which can be used not only to identify unknown protein interactions, but also to validate the functional effects of known interactions, playing a role in intracellular nucleic acid level verification. Yeast three hybrid technology can be applied in biological experimental research and also in the food fermentation industry. The yeast three hybrid technology can be used to study protein interactions. By introducing a third molecule (such as protein or RNA) as a bridge, yeast three-hybrid technology possesses the capability to uncover more intricate protein-protein interactions, offering extensive potential for application in the fields of functional genomics and protein engineering. Additionally, this technique can be employed to investigate the interactions occurring between proteins and either RNA or DNA, thereby revealing the mechanisms of gene regulation and the regulation of gene expression. Yeast three hybrid technology can be used to identify potential targets of disease-related proteins and screen for potential drug candidate molecules. This is of great significance for target validation and drug screening stages in the drug development process. The use of yeast hybrid system for protein design and optimization can develop new biosynthetic pathways and improve the yield and efficiency of biological products. In addition, yeast three hybrid technology can also be used to improve yeast varieties. By hybridizing three different yeast strains together, yeast varieties with improved traits can be produced. These improved yeast varieties have higher fermentation efficiency. The improved yeast variety can also optimize the fermentation process, improve the fermentation efficiency of the product, and reduce production costs.

Fig 1: Principle of the yeast two-hybrid (A) and yeast three-hybrid (B) systems. Reference source: Pereira, Clara & Leão, et,al,. (2012). New Therapeutic Strategies for Cancer and Neurodegeneration Emerging from Yeast Cell-based Systems.

When conducting yeast three hybrid experiments, it is necessary to use methionine deficient culture medium, because in this system, the protein expressed at MCSII is controlled by the Pmet25 promoter, which is only expressed in media lacking methionine. In order to construct a yeast three hybrid system, a vector containing two different DNA binding domains (such as Gal4's BD binding domain and transcriptional activation domain) needs to be constructed and fused to two different proteins. At the same time, a third molecule (protein or RNA) needs to be introduced as a connecting bridge.

The yeast three hybrid technology adds a pBridge plasmid capable of simultaneously expressing two exogenous proteins on the basis of yeast two hybrid. The characteristic of the yeast three hybrid system is the fusion of the BD binding domain of Gal4 with the protein at MCSI. In the absence of a label, the Pmet25 promoter can control the protein of MCSII, allowing it to be expressed in a medium lacking methionine. It can be inferred that in the absence of labels, the yeast three hybrid system requires the use of methionine deficient culture medium for experiments, and the introduction of the third protein into the AH109 strain using the pGADT7 vector. If the strain can grow on a five deficiency medium lacking Met/Ade/His/Leu/Trp after binding, it indicates that the three proteins have interacted.

Yeast Three Hybrid Service Workflow

Advantages of Yeast Three Hybrid Service

FAQ-Yeast Three Hybrid

1. What is the yeast three-hybrid? What is the application of this technology?

A: The yeast three-hybrid system is a biotechnology based on yeast genetic analysis that analyzes the interaction of two proteins on another protein or RNA. The yeast three-hybrid system can utilize RNA binding proteins and fusion proteins required for DBD, and AD construction. For the two fusion proteins, we need to use additional proteins to bring them close to each other, thus triggering downstream transcription. Yeast three-hybrid experiments can help researchers to locate specific regions of action, and even more accurately locate individual genes. At the same time, if you want to understand the particular role of a protein, you can also use this technology to gain insight into the protein's function. Yeast three-hybrid technology can analyze the interaction between proteins and gene fragments. At the same time, this analysis can help us to complete the study of gene regulation mechanisms and gene expression regulation. This service can be used as a tool to reveal gene function and understand genetic diseases. The yeast three-hybrid system can also identify potential targets for proteins involved in disease and screen out drug candidates that could help researchers complete drug development. KMD Bioscience can provide yeast three-hybrid service to help customers better study the functional role of proteins and subsequent drug development.

2. In the yeast three-hybrid service, we often encounter the problem of self-activation phenomenon, low transformation efficiency, plasmid construction error, and so on. How do we solve it?

A: In the yeast three-hybrid service, the autoactivation phenomenon may be caused by the bait protein in some cases, activating the expression of its reporter gene, and eventually leading to false positive results in the experiment. If we encounter this situation in the experiment, we can perform background inhibition by using different concentrations of inhibitors (e.g., 3-amino-1,2,4-triazole, 3-AT or Aureobasidin A, AbA), thus reducing the experimental interference of false positives. Meanwhile, technicians also use adjusted truncation or mutation of bait proteins to reduce gene autoactivation. If none of the above methods can solve the autoactivation problem, we can also try to replace different yeast strains or use different reporter gene systems to validate the interacting proteins. In the yeast three-hybrid service, the transformation efficiency is low, such as the failure to transform successfully, and the poor state of the yeast strains after transformation. First, we need to ensure the correctness of our transformation steps, then our transformed plasmids need to be of high quality, and then we choose the appropriate transformation conditions, The optimal method is selected from various transformation methods such as PEG / LiAc method or electroconversion method and maintained the temperature and the time of heat shock during transformation, all of the above methods can help us to better complete the transformation.

Fig 2: Protein interactions in the yeast three-hybrid system. (Reference source：Glass F, Takenaka M. The Yeast Three-Hybrid System for Protein Interactions. Methods Mol Biol. 2018;1794:195-205.)

3. In the yeast three-hybrid service, we often encounter plasmid construction errors, non-specific interactions, and other problems. How do we solve it?

A: In the yeast three-hybrid service, there are many failures of our yeast three-hybrid experiments caused by plasmid construction errors, such as the deletion of genes, gene mutations, and insertion in the process of plasmid construction, which may lead to the failure of our subsequent experiments. What we can do is to sequence the plasmid by technology after the plasmid construction to ensure the accuracy of the plasmid we build. When we are in the yeast three-hybrid experiment, because the range of yeast screening conditions is too broad, resulting in the occurrence of non-specific interacting proteins, the final generation for our experimental results. In the face of the above problems, we should choose more stringent screening conditions according to the specific experimental requirements. For example, we can increase the concentration of AbA and other inhibitors, optimize the composition of the medium for cultivating yeast, change the PH value of the medium, and then reduce the non-specific interaction between proteins. At the same time, we can use a variety of biochemical methods for verification, such as co-immunoprecipitation, Western blot, etc., together to exclude non-specific interference, and ensure the credibility of the final experimental results. KMD Bioscience has rich project experience, a mature experimental system, and knowledgeable researchers, and can provide customers with high-quality yeast three-hybrid services.

4. In the yeast three-hybrid service, we often encounter media contamination, poor yeast cell growth, and other problems. How do we solve it?

A: In the yeast three-hybrid service, we may encounter contamination during the transformation or culture process, which will lead to interference in our experimental results or experimental failure. In this case, we must ensure the whole process of the experimental operation, and the experimental personnel should strictly abide by various aseptic requirements and standardize their operation. At the same time, all kinds of experimental instruments we use should also be strictly disinfected by the requirements. In the yeast three-hybrid service, if poor yeast cell growth, poor yeast cell growth may affect the vector transformation efficiency and the final experimental results. We can optimize the formulation of the medium and various nutrients, change the culture conditions of the strain, and ensure that the yeast grows at a normal growth rate. KMD Bioscience has its aseptic laboratory, the experimenters have received professional aseptic culture, strictly abide by the aseptic standards during the experiment, and years of rich project experience can help us quickly solve various problems encountered in the experimental process, provide a high-quality yeast three-hybrid service to our customers. In addition to the yeast three-hybrid service, KMD Bioscience can also provide yeast two-hybrid service and yeast single-hybrid service to help customers' scientific research and drug development.

5. The difference and the advantage between the yeast three-hybrid system and the yeast two-hybrid?

A: Compared with the yeast three-hybrid system, the yeast two-hybrid system is relatively simple. The system can be established in research groups with basic biological experimental equipment and molecular biology knowledge reserve. Therefore, in general, it appears as the first choice, helping specific proteins to select protein ligands that may interact. However, if there are now two proteins, "A" and "B", there is a direct interaction between them, but the interaction is not related to the other protein components. If this is the case, the use of the yeast three-hybrid system can help us better understand the role of the proteins. If the research group or the experimental platform already has the yeast two-hybrid system, then fewer experimental steps and experimental time can get the desired results. The yeast three-hybrid system is a process-convenient but more powerful application of the yeast two-hybrid system. The plasmid design for the bait protein in the yeast three-hybrid system has an additional expression cassette. This expression cassette encodes a third protein that can participate in and influence the interaction between the two proteins when co-expressed with the other two. Thus, we can monitor the effect of the third protein on the interaction between the first two proteins. KMD Bioscience provides yeast three-hybrid and two-hybrid services to help research and drug development.

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