Overview of Yeast Two Hybrid Technology-Yeast Display Technology Special Topic-Tekbiotech-Yeast Display Service,Phage display technology

Overview of Yeast Two Hybrid Technology

In 1989, Fields et al. initially established the yeast two-hybrid system when conducting research on eukaryotic gene transcription regulation. This is a molecular biology technique that uses yeast genetics to analyze protein interactions. This technique is based on the structural and functional characteristics of transcription factors (such as GAL4) in eukaryotic cells. GAL4 has two non-interfering domains that perform their respective functions when separated. The DNA binding domain (BD) has the function of binding to DNA, and the transcription activation domain (AD) has the function of activating transcription. When the two are connected together, they will have transcriptional activity. The two proteins to be studied are fused with the BD and AD of GAL4 to form two fusion proteins. When the two proteins are transferred into yeast cells at the same time and can interact with each other in the yeast cells, they will cause the BD and AD to be close in space, thereby activating specific reporter genes, such as LacZ, HIS3, etc. If the two proteins do not interact, the reporter gene will not be expressed. By detecting whether the reporter gene is expressed, it can be indirectly determined whether there is an interaction between the two proteins.

Yeast two-hybrid technology principle diagram - Tekbiotech.png

Fig.1 Yeast two-hybrid technology principle diagram

Application of Yeast Two-hybrid Technology:

Yeast two-hybrid technology has high sensitivity and can simulate the real protein interaction environment in cells. It can not only be used to verify the interaction between known proteins, but also to screen genes encoding unknown proteins. It has been widely used in many fields such as proteomics, cell signal transduction and drug screening. Yeast two-hybrid technology can be used to quickly and high-throughput screen the interaction between proteins, providing important data support for the construction of protein interaction networks in organisms, which is conducive to providing new targets for disease diagnosis and treatment. Through yeast two-hybrid technology, unknown proteins that interact with known proteins can be found, and then new genes can be discovered. Yeast two-hybrid technology can be used to screen drug action sites and study the effects of drugs on protein interactions, which is helpful for drug optimization and modification. With the emergence of new technologies such as the DUALmembrane system, yeast two-hybrid technology can be used to study membrane proteins, which helps to reveal the role of membrane proteins in cell signal transduction.

Yeast Two-hybrid Library Construction:

An important part of yeast two-hybrid technology is yeast two-hybrid library construction, which is used to screen unknown proteins that interact with specific proteins. First, extract total RNA from the target tissue and separate and purify mRNA. Reverse transcribe mRNA into cDNA and amplify it by PCR. Connect cDNA with AD vector to construct AD library. There are two library construction methods: SMART amplification technology and Gateway recombination technology. Choose the appropriate method according to the amount of starting RNA. SMART technology can continuously synthesize the sequence of the 5' untranslated region, while Gateway technology uses proprietary recombination sequences to enable DNA fragments to be more effectively transferred into plasmids, which is suitable for large-fragment gene cloning. The constructed library is tested for quality control, including library capacity identification, insert size identification, etc., to ensure the quality and reliability of the library. Finally, the plasmid containing BD-X fusion protein and AD library are co-transformed into yeast cells, and the unknown protein that interacts with the protein is identified by screening the expression of the reporter gene.

TekBiotech is committed to providing customers with high-quality yeast display services. Based on yeast display technology, TekBiotech can provide customers with high-quality antibody yeast bi-hybrid library construction services, including but not limited to single-chain antibody yeast display technologies such as VHH and scFv. The yeast library capacity can reach 10^8, and the library diversity, insertion rate, and positive rate can reach more than 90%, meeting the quality requirements of various customers for antibody yeast display libraries. At the same time, TekBiotech can also provide downstream antibody in vitro validation (including but not limited to affinity validation, antibody blocking validation, cross-reaction validation and other downstream validation experiments), antibody humanization, antibody affinity maturation, CAR-T/CAR-NK lead sequence design and cell killing validation and other one-stop technical services for antibody yeast display. Customers only need to provide specific experimental requirements and target information. We can design and customize reasonable solutions according to customer needs to help customers' scientific research projects and drug antibody development.

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