Yeast two-hybrid, proposed by Fields in 1989, is primarily used to analyze the gene system for protein-protein interactions in yeast and is a genetic method for the module structure of transcription factors.
The yeast two-hybrid system was developed in eukaryotic model yeast to study protein interactions in living cells, and weak, transient interactions between proteins can also be observed sensitively through the expression products of reporter genes.
This technique can be used to study the interactions between proteins encoded in mammalian and plant genomes.
The yeast two-hybrid system can detect interactions between proteins, and more importantly, discover new unknown proteins that interact with known proteins.
In a two-hybrid system, BD fuses with X protein, AD fuses with Y protein. If a protein-protein complex is formed between X and Y, and the two domains of Gal4 are reconstituted, the transcription of specific gene sequences will be initiated.
Generally, the fusion protein of BD-X is called bait, X is often a known protein, AD-Y is called prey, and genes that can show the interaction between bait and prey are called reporter genes. Genetic testing, in turn, can determine whether there is an interaction between the bait and the prey.
Therefore, yeast two-hybridization can be briefly summarized as: two domains of transcription factors required for gene transcription are located close together by the attraction of two interacting proteins to induce gene expression.
When we use a known gene as a bait and screen the selected cDNA library for proteins that interact with the bait protein, the AD-LIBRARY plasmid can be isolated from the positive yeast strains screened, and the cDNA fragments in the plasmid are sequenced. The coding sequence of this fragment was compared in GENEBANK to study the biological function connection with known genes.
Another important element of the two-hybrid system is the reporter strain.
Reporter strain refers to the host cell which has been modified and contains the recombinant plasmid of reporter gene.The most commonly used are yeast cells, which have many advantages as the yeast two-hybrid system of the report strain:
▲ Easy to transform, easy to recover and expand plasmid.
▲ It has directly selectable marker genes and characteristic reporter genes.
▲ The endogenous proteins of yeast do not easily bind to mammalian proteins.
The common yeast two-hybrid systems on the market are nuclear system and membrane system hybrid libraries. Nuclear system yeast two-hybrid libraries include Clontech system and Invitrogen system, and membrane system is Dual systerm system.
Picture note: It is better to choose Invitrogen system instead of Clontech system for rice and other high GC species. The Clontech system requires a PCR process, while the Invitrogen system can directly reverse transcription from mRNA. High GC will affect the amplification efficiency of two-strand PCR, so in general, high GC species will choose the Invitrogen system when building a library, but the Invitrogen system requires a higher sample volume.
The so-called false positive means that the reporter gene is activated when there is no interaction between the two proteins to be studied. In order to eliminate false positives, strict control tests are required. The bait and target protein should be separately activated for the identification of the reporter gene.
At present, the yeast two-hybrid system launched by many technical service companies uses multiple reporter genes, and the upstream regulatory region of each reporter gene is different, which can reduce a large number of false positives.
Transformation efficiency is one of the key factors in the screening of yeast two-hybrid libraries, especially in the screening of low abundance cDNA libraries, so it must be improved.Co-transformation or sequential transformation can be used during transformation, which saves time and effort.More importantly, co-transformation can attenuate or eliminate virulence of the fusion-expressed protein to yeast cells if transformation alone would occur.
A more effective method is to transfer the bait protein carrier and target protein carrier into different conjugated haploid yeast, and then hybridize the two conjugated haploid cells to bring the bait protein and target protein into the same diploid cell.
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Yeast two-hybrid technology and common problems
Yeast Two-hybrid: Basic Principles of Experimental Technical Posts
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