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In life activities, DNA replication, transcription, RNA splicing, protein translation and secretion, cell cycle regulation, signal transduction, and many metabolic processes are the result of the organic interaction of various related proteins. Proteins in cells or tissues are not a messy mixture. The interaction and coordination between proteins is the basis for all metabolic activities of cells. With the rapid development of molecular biology experimental technology, in the past 10 years, some methods for studying protein-protein interactions have been developed, of which the yeast two-hybrid technology created by Fields and Song is the best. This method utilizes the characteristics of fast growth of yeast and easy operation, and studies the protein-protein interaction of mammalian cells in its system. By screening the cDNA library, the protein that interacts with the protein of interest is found.
The establishment of the yeast two-hybrid system is due to the understanding of the eukaryotic cell regulation of transcription initiation process. The study found that many eukaryotic transcription activators are composed of two separable and functionally independent domains. For example, yeast’s transcriptional activation factor GAL4 has a 147-amino acid DNA binding domain (BD) at the N-terminus, and a 113-amino acid transcription activation domain (AD) at the C-terminus. ). The DNA binding domain of the GAL4 molecule can be combined with an upstream activating sequence (UAS), while the transcription activation domain can activate genes downstream of the UAS for transcription. However, a single DNA-binding domain cannot activate gene transcription, nor can a single transcription-activation domain activate downstream genes of UAS. Only when they are combined in some way can they have the function of a complete transcriptional activator.
The yeast two-hybrid system uses the functional characteristics of GAL4 to capture new proteins through the mutual binding of two hybrid proteins in yeast cells and transcriptional activation of reporter genes. The general steps are:
① Consider the cDNA sequence of known protein as bait, fuse it with DNA binding domain, and construct a bait plasmid.
② The cDNA sequence of the protein to be screened is fused with the transcription activation domain to construct a library plasmid.
③ Co-transform these two plasmids into yeast cells.
④In yeast cells, the isolated DNA binding domain and transcription activation domain will not interact, but if the bait protein can specifically interact with the unknown protein to be screened, it can activate the transcription of the reporter gene; otherwise, it cannot. Using the expression of four reporter genes, new proteins can be captured.
Advantages: Protein-protein interaction is the basis for all metabolic activities of the cell. The establishment of the yeast two-hybrid system provides an advantageous means and method for studying this problem.
Disadvantages: Although the system has proven to be a very effective method, it also has its own disadvantages and problems. 1. It is not suitable for all proteins, which is determined by its principle. The two-hybrid system requires that both hybrid proteins are fusion proteins and both must be able to enter the nucleus. Because the fusion protein interaction activates reporter gene transcription, it takes place in the nucleus. 2. False positives occur more frequently. The so-called false positive refers to a protein that fails to interact with the bait protein and is mistaken for a positive reaction. And some of the reasons for false positives are unclear, which may be related to the role of other proteins in yeast. 3. Extensive expression of foreign proteins in yeast strains will produce toxic effects, thereby affecting the growth of the strain and the expression of reporter genes.
Problems that should be paid attention to when using yeast two-hybrid technology
It is really clear that the main principle and screening method of yeast two-hybrid technology is the premise of yeast two-hybrid experiment. The successful construction of bait plasmids and a large amount of material preparation are the guarantee for yeast two-hybrid experiment. Only by understanding the principle of two-hybrid, it is possible to design the experimental process, to prepare materials for purpose, and to make predictions and analysis of the experimental results, especially for the purpose of using various selective pressure media in specific experiments. clear. A large amount of material preparation and a long experimental process are characteristics of yeast two-hybrid which are different from other experiments, and its operation technique itself is not very difficult. In particular, it should be mentioned that the number of a positive clone is often recorded repeatedly, so pay attention to the correctness of the number. In addition, if the yeast cDNA library to be screened is purchased from a company, it should be noted that different companies have different products, and the products of each company are continuously updated, and the experimental instruction manual should be carefully read to prevent mistakes.
Investigate known protein interactions, look for domains that play a key role in protein-protein interactions, and find new proteins that interact with target proteins. It is believed that with the development and promotion of molecular biology technology, yeast two-hybrid technology will play a greater role in future proteomics research.
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