The yeast two-hybrid system was first established by Fields and Song to study eukaryotic gene transcription regulation. Typical eukaryotic transcription factors, such as GAL4, GCN4, etc., contain two different domains: DNA-binding domain and transcription-activating domain. The former can recognize specific sequences on DNA and position the transcription activation domain upstream of the gene it regulates. The transcription activation domain can interact with other components of the transcription complex to initiate the transcription of the gene it regulates. The two domains can not only be opened at the appropriate location in their connecting region, but also have their own functions. Moreover, the two different domains can be reconstituted to exert transcriptional activation effects.

Chinese name

Principle of yeast two-hybrid system

Type

System principle

Such as

GAL4

English

DNA-activating domain

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Advantage Application

Class II vector

The yeast two-hybrid system uses hybrid genes to detect protein-protein interactions by activating the expression of reporter genes. There are two main types of vectors: a. Vectors containing DNA-binding domain; b. Vectors containing DNA-activating domain. When constructing fusion genes with the above two types of vectors, the test protein gene and domain gene must be fused within the reading frame. The fusion gene is expressed in the reporter strain, and its expression product can only drive the transcription of the reporter gene if it is localized in the nucleus. For example, GAL4-bd has a nuclear-localization sequence, but GAL4-ad does not. Therefore, a sequence from the T-antigen of SV40 should be cloned at the amino terminus or carboxyl terminus of GAL4-ad as a nuclear localization sequence.

Advantages

Another important component of the two-hybrid system is the reporter strain. Reporter strain refers to a modified host cell containing a recombinant plasmid containing a reporter gene.

The most commonly used yeast cells are yeast cells. The yeast two-hybrid system of yeast cells as reporter strains has many advantages:

<1> It is easy to transform and recover the amplified plasmid.

<2>It has marker genes and characteristic reporter genes that can be directly selected.

<3>The endogenous proteins of yeast are not easy to combine with proteins derived from mammals.

Generally, a gene encoding a protein is fused to the DNA-binding domain of a clear transcriptional regulator (such as GAL4-bd, LexA-bd); another gene is fused to a transcriptional activation domain (such as GAL4- ad, VP16).

The activation domain fusion gene is transferred into a yeast cell line that expresses the binding domain fusion gene. The interaction between proteins causes the reconstruction of the transcription factor and leads to the expression of adjacent reporter genes (such as lacZ), so that the protein can be analyzed. the bonding effect between.

Application

The yeast two-hybrid system can measure protein binding in vivo and is highly sensitive.

Mainly due to:

① Using an expression vector with high copy and strong promoter to overexpress the hybrid protein.

② Signal measurement is carried out under natural equilibrium concentration conditions. However, physical methods such as immunoprecipitation require multiple washings to achieve this condition, which reduces the signal intensity.

③The stability of hybrid proteins can be enhanced by the combination of the activation domain and the binding domain to form a transcription initiation complex, which in turn binds to the promoter DNA. This ternary complex allows each group to The combination of points tends to be stable.

④A variety of stable enzymes are produced through 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.