The yeast transcriptional activator GAL4 is composed of two separable and functionally independent domains. There is a DNA binding domain (BD) at the N terminal, which consists of 147 amino acids.There is a transcription activation domain (AD) at C terminal, which consists of 113 amino acids.BD can be combined with upstream activating sequence (UAS), while AD can activate downstream UAS genes for transcription.However, BD or AD alone cannot activate gene transcription, and only when they are combined in some way can they function fully as transcriptional activators. The yeast two-hybrid system mainly uses this feature of yeast GAL4 to study the interaction of proteins in living cells through the mutual binding of two hybrid proteins in yeast cells and the transcriptional activation of reporter genes. The effect can also be sensitively detected by the reporter gene. Yeast two-hybrid technology is not only used to study the interaction between mammalian proteins, but also can be used to study the interaction between higher plant proteins.
Vector plasmid: pLexA, pB42AD, p8op-LacZ, pB42AD-DNA library
1. Vector plasmids: Plexa, Pb42AD, P8OP-LacZ, Pb42AD -DNA library
2. Yeast strains: EGY48, EGY48(P8OP-LacZ), YM4271(the companion strain of EGY48)
3. E.coli strain: E.coli KC8 strain
4. The comparison:
pLexA-53, pB42AD-T positive control
pLexA-Pos (LexA/GAL4 AD fusion protein) positive control
pLexA-Lam (LaminC protein rarely interacts with other proteins) false positive detection plasmid
Primer:
pLexA sequencing primer and pB42AD sequencing primer.
The reporter gene p8OP-LacZ was transformed into yeast strain EGY48 and screened by medium SD/ -URA.
At the same time, DNA library was constructed and sufficient plasmids were purified to transform yeast cells.
DNA-BD/ target protein plasmid Plexa-X was constructed as bait.
Transform the above fishing bait plasmid pLexA-X into EGY48 (p8op-LacZ) cell line, screen with SD/-His/-Ura; and use solid induction medium SD/Gal/Raf/-His/-Ura to detect this DNA-BD / Whether the target protein has the activity of directly activating the reporter gene, and whether it has killing toxicity to yeast cells.
Transformation plasmid Selection medium Clonal growth description
(Contains Gal/Raf)
pLexA-Pos SD/-His, -Ura blue positive control
pLexA SD/-His, -Ura white negative control
PlexA-X SD/-His, -Ura white has no direct activation activity
PlexA-X SD/-His, -Ura blue has direct activation activity
PlexA-X SD/-His, -Ura colonies cannot grow Yeast cytotoxicity
Can automatically activate the reporter gene, try to remove its active site, or integrate the LacZ reporter gene into the genome,
1.4-1. If the signal effect of pLexA-X-galactosidase is reduced.
4-2. If Plexa-X is toxic to the yeast host cell, although not automatically activating the reporter gene, yeast transformation is required in conjunction with purified library DNA.
If Plexa-X does not automatically activate reporter genes and is not toxic, yeast cells can be transformed simultaneously and sequentially in the purified library DNA and plasmid transformation efficiency can be tested.
Transformed plasmid SD solid medium LacZ phenotype
Control 1 pLexA-Pos Gal/Raf/-His/-Ura blue
Control 2 pLexA-53 Gal/Raf/-His/-Trp/-Ura/-Leu blue
+pB42AD-T
Experiment pLexA-X Gal/Raf/-His/-Trp/-Ura/-Leu to be tested
+pB42AD-Library
5-1. Use SD/-His/-Trp/-Ura medium to select positive co-transformants and amplify them so that the plasmid in the host cell reaches the maximum copy number before induction.
5-2. The above recombinants were transferred to solid induction medium SD/ gal /Raf/-His/-Trp/-Ura/-Leu containing X-Gal, and the expression of LacZ and Leu reporter genes was observed. The blue clone was positive.The white clone was false positive, indicating that although Leu was expressed, B-galactosidase was not expressed.
5-3. The purpose of using LacZ and Leu reporter genes at the same time is to eliminate false positive errors in the experiment as much as possible. For example, AD fusion protein does not bind to the target protein, but directly binds to the promoter sequence binding domain.Because the promoters of the two reporter genes are different, the probability of these false positives is greatly reduced.
5-4. Seed the blue positive clone more than once, and isolate as many library plasmids as possible in the clone.
6.Screening of positive clones
6-1. Randomly select 50 positive clones, amplify and extract yeast plasmids, electrotransform E.coliKC8 host bacteria, extract plasmids in E.coli, and identify whether they have inserts and exclude the same library plasmids by restriction enzyme digestion.
6-2. If there are many repeated insertion sequences, another 50 positive clones can be taken for analysis.Finally, several insertion sequences of different fragment sizes were obtained, and then new host cells were transformed to detect whether they were still positive clones.
7.Use Plasmid Natural Segregation to screen out clones that only contain AD-library heterozygotes
7-1. The preliminary positive clones were inoculated into SD/-Trp/-Ura liquid medium and cultured for 1-2 days. The BD- target plasmid containing His3 coding sequence would be randomly lost at a frequency of about 10%-20% in the medium containing exogenous HIS.
7-2. Transfer the above clones to a solid medium SD/-Trp/-Ura and incubate at 30°C for 2-3 days.
7-3. Pick the grown single clones and transfer them into SD/-Trp/-Ura and SD/-His/-Trp/-Ura medium, and screen the clones with defects in His phenotype to obtain only AD- Recombinants that are heterozygous for the library.
7-4. Transform the clones with His phenotype defects into the solid induction medium SD/Gal/Raf/-Trp/-Ura to verify whether the AD-library can directly activate the expression of the reporter gene, discard the positive clones and retain the negative clones .
8.Yeast Mating test to determine true positive clones
As shown in the table below, the corresponding plasmid or library DNA was transferred into yeast EGY48 and its corresponding YM4271 host cells, and the true positive clones with interaction between Plexa – target DNA and PB42AD – library were screened by heterozygosity experiment.
Plasmid 1
(in YM4271) Plasmid 2
(in EGY48) LacZ phenotype Leu phenotype
pLexA pB42AD white can not grow
pLexA-target DNA pB42AD white cannot grow
pLexA pB42AD-library white cannot grow
pLexA-target DNA pB42AD-library blue true positive
pLexA-Lam pB42AD-library white cannot grow
9.Further screening and confirmation of positive clones
9-1. Amplify the preliminarily determined positive clones and extract yeast DNA. It contains both yeast genomic DNA and 3 types of transformed plasmid DNA.
9-2. Electrically transform the above DNA into E.coli KC8.In Escherichia coli, plasmids with different regulatory sequences of replication initiation are incompatible.Nutritional deficiency type was also used for screening.Therefore, on the M9/SD/-Trp medium, only the transformed bacteria containing the ad-library plasmid could grow, be amplified, and the plasmid DNA was extracted for enzymatic digestion and identification.
9-3. Plexa – target DNA was one-to-one corresponding to Pb42Ad – library DNA and co-transformed into yeast EGY48 containing only reporter genes. SD/-His/-Trp/-Ura plate was amplified first, and the comparison was made with the induction plate behind, indicating that the expression of reporter genes was related to the expression of induced AD fusion protein.The expression of LacZ and Leu reporter genes was confirmed.
9-4. Amplify the library DNA that interacts with the target DNA, perform sequence analysis and further structure and function research.
10.Further research on the positive results of the two-hybrid system
10-1. Verify with different two-hybrid systems
10-1-1. Perform a two-hybrid experiment after swapping the vector pLexA and pB42AD.
10-1-2. Choose different two-hybrid systems, such as the two-hybrid system based on the GAL4 transcription activator.
10-1-3. After the frameshift mutation of the library plasmid, and then interact with the target plasmid, whether the reporter gene can still be activated.
10-1-4. Remove or mutate specific binding sites, quantitatively detect the level of b-galactosidase, and compare changes in the intensity of action.
10-2. Determine the DNA sequence of the inserted fragment with the primer provided by the kit to prove its coding region.
10-3. Other assays, such as affinity chromatography or immunocoprecipitation, are used to demonstrate interactions between proteins screened by the two-hybrid system.
10-4. Further study the functional relationship between target protein and screening protein
It can study the interactions between known proteins, search for the key domains in protein-protein interactions, and search for new proteins that interact with target proteins.It is believed that with the development and popularization of molecular biology technology, yeast two-hybrid technology will play a greater role in proteomics research in the future.
Experience: yeast two-hybrid technology is mainly used to study the interaction between proteins, the core key step is the culture of yeast cells, it is related to the concentration and growth of yeast cells, yeast cell culture time must be strictly controlled.
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