The experiment of yeast two-hybrid mainly consists of three parts: basic principle, experimental operation and common problems.Let’s start with the principles section.
Yeast two-hybrid is one of the commonly used methods to detect protein interaction. The yeast two-hybrid system was first established by Fields and Song in studying the regulation of eukaryotic gene transcription. Typical eukaryotic transcription factors, such as GAL4 and GCN4, contain two distinct domains: DNA-binding domain and transcription-activating domain.The former can recognize the specific sequences on DNA and make the transcription activation domain locate upstream of the regulated gene. The transcription activation domain can interact with other components of the transcription complex to initiate the transcription of the regulated gene. DNA-BD and AD act separately and cannot activate the transcription reaction, but when the two are sufficiently close in space, they exhibit complete GAL4 transcription factor activity and can activate the UAS downstream promoter, so that the downstream genes of the promoter can be transcribed.
At present, two systems used in yeast two-hybrid experiment are LEXA system and GAL4 system.In the LEXA system, the DNA binding domain is composed of a complete prokaryotic protein LEXA, and the transcriptional activation domain is composed of an 88-amino acid acid Escherichia coli polypeptide B42, which can activate gene transcription in yeast. in the Gal4 system , BD and AD are respectively composed of two different domains (1-147aa and 768-881aa) on Gal4 protein.
The experiment used Takara’s Matchmarker gold system, which uses a two-hybrid system with 4 reporter genes/3 different bait recognition sequences. It uses a new and stable yeast two-hybrid antibiotic, Aureobasidin A (AbA), which can effectively kill non-resistant clones, thereby greatly reducing background clone growth. At the same time, it effectively reduces false positives. The 4 reporter genes are: 1 AbA antibiotic screening reporter gene, 2 nutrition screening reporter genes, and 1 blue-white spot screening reporter gene.
One gene is constructed into the vector containing the AD domain to act as Prey protein, and the other gene is constructed into the vector containing the BD domain to act as BaIT protein. When the two proteins interact with each other, the reporter gene can be activated, enabling yeast to grow in the nutrient deficient medium and make the yeast to turn blue.
Each experimental method has its advantages and disadvantages, and yeast two-hybrid is no exception.
Advantages of yeast two-hybrid:
(1) Eliminate the cumbersome steps of protein purification
(2) The test is carried out in living cells, which can represent the true situation in the cell to a certain extent
(3) It can detect weak or temporary interactions between proteins.
Limitations of yeast two-hybrid:
- Protein-protein interactions in two-hybrid system analysis are localized in the nucleus, while many protein-protein interactions rely on post-translational modifications, such as glycosylation, disulfide bond formation, etc., which cannot be carried out in the nucleus.
- An important problem of yeast two-hybrid system is “false positive”, that is, self-activation. Therefore, self-activation detection of target gene should be carried out before the experiment.