With the rapid development of molecular biology research, especially the human genome project, many new techniques have emerged in order to adapt to the development trend of functional research on many genes or proteins. Yeast two-hybrid is one of them. Yeast two-hybrid is one of them. So let’s briefly understand what is the yeast two-hybrid system:
The generation of yeast two-hybrid system is based on the study on the properties of eukaryotic transcription factors, especially the yeast transcription factor GAL4. Typical eukaryotic transcription factors, such as GAL4, GCN4, etc., contain two different domains:DNA-binding domain (DNA-BD) and transcriptional activating domain (AD). The former can recognize specific sequences on DNA and locate the transcription activation domain upstream of the regulated gene. The transcription activation domain can interact with other components of the transcription complex to initiate the transcription of the gene it regulates.
DNA-BD and AD alone could not activate the transcription response, but when the two were sufficiently close in space, the Gal4 transcription factor activity would be complete and the downstream promoter of UAS could be activated, so that the downstream gene of the promoter could be transcribed.
Fields first established a two-hybrid system in which BD fuses with the X protein and AD with the 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 is initiated.By using the interaction between SNF1 and SNF4, they fused SNF1 with BD and SNF4 with AD and constructed them on shuttle plasmids.Among them, SNF1 is a protein kinase dependent on serine and threonine, and SNF4 is one of its binding proteins. The researchers transformed the two shuttle plasmids into the yeast GGY:171 strain, which contains the LacZ reporter gene and the corresponding transcription factor gene has been removed. The results of this experiment show that the interaction between Snf1 and Snf4 makes AD and BD close in space , Activates the transcription of the reporter gene LacZ. Generally speaking, the fusion protein of BD-X is called bait,X is usually a known protein, Ad-Y is called prey, and the gene that can show the interaction between bait and prey is called reporter gene. Through the detection of reporter gene, it can in turn determine whether there is interaction between bait and 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.
Another important element of the two-hybrid system is the reporting plant.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:
It is easy to transform and to recover the amplified plasmid.
It has marker genes and characteristic reporter genes that can be directly selected.
Yeast’s endogenous proteins are not easy to bind to mammalian-derived proteins.
The activation domain fusion gene is transferred into the yeast cell line expressing the binding domain fusion gene, and the interaction between the proteins makes the transcription factor reconstruction lead to the expression of adjacent reporter genes (such as lacZ), so that the binding effect between the proteins can be analyzed. For example, the hybrid protein obtained by fusing the BD of the Gal4 protein of yeast cells with an acidic activation domain B42 of E. coli can still bind to the Gal4 binding site and activate transcription.
Screening genes using yeast two-hybrid
The yeast two-hybrid system can measure protein binding in vivo and is highly sensitive. It is mainly due to:
The expression vector with high copy and strong promoter is used to overexpress the hybrid protein.
The interstability of the hybrid proteins is enhanced by the binding of the activation and binding domains to form a transcription initiation complex, which in turn binds to the promoter DNA. The ternary complex stabilizes the binding of the components.
A variety of stable enzymes are generated by mRNA to amplify the signal. At the same time, detection methods such as yeast phenotype, X-Gal and HIS3 protein expression are all very sensitive.
The signal measurement is carried out under the condition of natural equilibrium concentration, while physical methods such as immunoprecipitation need to carry out multiple washings to achieve this condition, which reduces the signal strength.
The yeast two-hybrid system is highly sensitive for the determination of protein binding in vivo.Mainly due to:
The interaction between fusion proteins is carried out in eukaryotic yeast cells, the proteins keep the natural folded state, and the interaction between proteins will be closer to the real level in vivo.
The sensitivity of the two-hybrid system is very high, and it can be detected when the binding constant between proteins is as low as 1mmol/L.
When screening the library, the two-hybrid system can obtain the gene sequence encoding the interaction protein, omitting the cumbersome steps of protein extraction and purification necessary for other in vitro protein interaction detection methods.
Having said so much, in fact, yeast two-hybrid is not only this mode, through the continuous efforts and innovation of scientists, now commonly used yeast two-hybrid system and yeast two-hybrid system based on the isolated ubiquitin membrane protein yeast two-hybrid and RAS signaling pathway.The basic idea of this system is basically the same as the classical system introduced above, except that the site of interaction is moved to the cell membrane. Due to the directionality of membrane proteins, carrier selection and experimental design are more complicated than those of the classical model.This system uses a temperature-sensitive strain of yeast cells, using the idea that protein interactions activate the RAS signaling pathway to enable yeast cells to survive at low temperatures.
Yeast two-hybrid technology is simple, sensitive, efficient, and can reflect the interaction between different proteins in living cells. It will be more widely used in the study of protein interaction. In addition, on the basis of yeast two-hybrid, yeast one-hybrid, yeast three-hybrid and yeast reverse hybrid technology have now been developed. They are used for the study between nucleic acid and library protein, the interaction study between three different proteins, and the structure and site of the interaction between two proteins.