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

Overview of Yeast Two Hybrid System Principle and Application

The yeast two-hybrid system is based on the discovery that many eukaryotic transcription factors have discrete, separable DNA binding and transcriptional activation domains. In their system, protein-protein interactions are tested by fusing one test protein to the DNA binding domain of the yeast GAL4 transcription factor and another protein to the GAL4 activation domain. The fusion proteins are expressed in a suitable yeast strain and their interaction is detected by detecting the expression of a GAL4-responsive reporter gene.

I. Classical Yeast Two-hybrid System

The early yeast two-hybrid system was based on the discovery that many eukaryotic transcription factors have separable DNA binding and transcriptional activation domains. The test protein is fused to each separate host to reconstitute an active transcription factor, thereby promoting the interaction of the test protein. The expression of a reporter gene can be monitored to detect the interaction, and the reporter gene contains an upstream element that binds to the DNA binding domain (BD). The most commonly used systems are the GAL4 system (in which the DNA binding and activation hosts of the yeast GAL4 protein are used) and the LexA system (the DNA binding host of the bacterial inhibitor protein LexA is combined with the Escherichia coli B42 activation domain).

The yeast strains used for two-hybrid experiments have mutations in many genes required for amino acid biosynthesis, such as TRP1, LEU2, HIS3, and URA3. If these amino acids are not present in the growth medium, the yeast strain will not grow. Many of the two-hybrid plasmids carry genes that accomplish these mutations and allow selection of transformed yeast.

The classical system has two main applications. It can be used to determine whether two known proteins interact or not, and it can also be used to identify unknown proteins encoded by a cDNA library that interact with a protein of interest. In another case, the yeast two-hybrid system has become a powerful tool for studying the interaction networks formed between proteins involved in specific disease processes.

Three disadvantages of the classical yeast two-hybrid system are potential difficulties with transcriptional repressors, the auto-activation of transporter genes by bait constructs, and the problem that some proteins do not localize to the yeast nucleus.

II. Other Yeast Two-hybrid Technology

Technical Name	Basic Introduction
hSos/Ras Recruitment System	This system relies on the activation of the Ras signaling pathway to rescue yeast cdc25 mutants that exhibit a temperature-sensitive growth phenotype. Cdc25en encodes a Ras guanosine nucleotide exchange factor (GEF). If the cdc25 phenotype is recruited to the yeast plasma membrane activating yRas, it can be rescued by human mRas or the human Ras GEF hSos. hSos is fused to one test protein. A second test protein is fused to the v-Src myristoylation signal, which targets it to the yeast plasma membrane. The interaction between the two test proteins leads to the recruitment of hSos to the plasma membrane and activation of the Ras signaling pathway by exchange of GDP or GTP.
Split-ubiquitin System	In the split-ubiquitin system, the proteins of interest are fused to separate domains of the ubiquitin polypeptide. The interaction between the proteins of interest reconstitutes an active ubiquitin molecule, resulting in proteolytic cleavage of the reporter protein from one of the ubiquitin domains. In a variant of this system, designed primarily for studying interactions between membrane proteins, the transcription factor a-LexA-VP16 is used as the reporter protein. For the classic two-hybrid yeast strain, protein-protein interactions can be detected by activating the LacZandHIS3 reporter genes.
Three-protein System	If the interaction between two proteins requires the presence of a third protein, or if one protein interacts with a domain formed by the interaction of two other proteins, the interaction may be missed in the two-hybrid analysis. Yeast protein-protein interaction systems have been developed to study the formation of these ternary complexes. They are based on the same principle as the classic two-hybrid system. Two of the proteins are fused to the DNA binding and activation domains of the GAL4 protein, while the third is expressed with a nuclear localization signal. The system is designed so that the activity of the reporter gene can only be detected when the three proteins bind together to form a complex when activated upstream of GAL4.

III. Application of the Yeast Two-hybrid System

The yeast two-hybrid system has been widely used in plant research to analyze known interactions and isolate new interaction partners. It has been used to study protein-protein interactions involved in many different processes, such as flower development, self-incompatibility mechanisms, circadian clocks, plant disease resistance, and plant hormone signaling.

After years of development, Tek Biotech has accumulated rich experience in immunological research and detection and established a complete molecular basis laboratory platform. Tek Biotech's yeast two-hybrid system (Y2H) is composed of a library constructed based on the SMART method, yeast strains, rigorous reporter genes, and high-expression vectors.

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