Yeast two-hybrid was proposed by Fields and Song in 1989, which was based on studies of the properties of eukaryotic transcription factors, particularly the yeast transcription factor GAL4. Yeast two-hybrid is the two domains of the transcription factor required for gene transcription, which are close to each other under the attraction of two interacting proteins, and induce gene expression.
The most important applications of the yeast two-hybrid system are rapid, direct analysis of interactions between known proteins, and the isolation of new ligands and known genes for their interaction with known proteins. The yeast two-hybrid system detects the interaction between proteins with the following advantages:
The action signal is given by the effect of reconstituting the transcription factor in the cell after expression of the fusion gene, eliminating the cumbersome steps of purifying the protein.
Detection is carried out in living cells and can represent the true state of the cells to a certain extent.
The result of the assay can be the cumulative effect of the gene expression product, thus detecting weak or transient interactions between proteins.
The yeast two-hybrid system can construct cDNA libraries from different tissues, organs, cell types and differentiation period materials, and can analyze various subcellular parts and functional proteins such as cytoplasm, nucleus and membrane-bound protein.
The yeast two-hybrid system is an efficient and rapid method for analyzing protein-protein interactions, but there are still some problems to be solved.
One of the problems often encountered in the application of the yeast two-hybrid system is that there are more false positives. The so-called false positive is that the reporter gene is activated in the absence of interaction between the two proteins to be studied. The main reason is that the BD fusion bait protein has a single activation effect, or the activation of this fusion protein is activated by a foreign protein. In addition, if the AD fusion target protein has specific binding of DNA, the expression of the reporter gene can also be activated separately. Therefore, a rigorous control trial is required to rule out false positives. The decoy and the target protein should be separately identified for the activation of the reporter gene. At present, the yeast double-hybrid system uses multiple reporter genes, and the upstream regulatory regions of each reporter gene are different, which can reduce a large number of false positives. In addition, the reporter gene is usually integrated into the chromosome, which can stabilize the gene expression level and eliminate false positives caused by fluctuations in gene expression levels due to changes in plasmid copy number.
Transformation efficiency is one of the keys to the success of yeast two-hybrid library screening. Especially when screening low-abundance cDNA libraries, the transformation efficiency must be improved. Co-transformation or sequential conversion can be used for conversion, which saves time and effort. More importantly, co-transformation can attenuate or eliminate this toxicity if the transformation alone produces toxicity to the yeast cells. A more effective method is to transfer the bait protein carrier and the target protein carrier into different ligated haploid yeasts respectively, and the bait protein and the target protein are brought into the same two by the hybridization of the two conjugated haploid cells and Ploid cells.
In recent years, many researchers have improved and developed the two-hybrid system. For example, false positive display assays and dual screening systems are employed to reduce the occurrence of "false positives"; development of mammalian two-hybrid systems to better study protein-protein interactions. Among them, the dual screening system uses two different reporter genes (usually lacZ and HIS3) to have the following advantages: using different promoters to express reporter genes located on two chromosomes of yeast can significantly reduce false positives. The screening ability is enhanced by nutrient screening, and is especially suitable for screening in the case of a large library capacity with less protein being selected.
The yeast two-hybrid system is carried out in eukaryotic model organism yeast. The study of protein interactions in living cells can be detected sensitively by the expression products of reporter genes. A large number of research literatures show that yeast two-hybrid technology is widely used in discovering new functions of new proteins and proteins, studying antigen and antibody interactions, screening drug sites and drug interactions with proteins, and establishing genomic protein linkage maps. With the deep and continuous optimization of yeast two-hybrid technology, yeast two-hybrid technology combined with other technologies will be more conducive to a more complete and accurate judgment of experimental results.