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Analysis on the Method of Yeast Two-hybrid Library Construction

2021-08-30
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Yeast two-hybrid technology is an important experimental method in the field of molecular biology research. It uses yeast genetics to analyze the interactions between proteins. It has now been widely used in proteomics, cell signal transduction, and functional genomics. . Yeast two-hybrid library construction generally includes two methods: nuclear system and membrane system.

The nuclear system includes the construction of the library by homologous recombination, and the construction of the library according to the technology of Gateway, which uses BP recombination and LR recombination to obtain the primary library and the secondary library respectively. The membrane system is based on the method of restriction enzyme digestion and connection, which is mainly based on the recognition of separated ubiquitin-mediated membrane protein interaction signals. Medicilon Biopharma specializes in yeast two-hybrid, and issues authoritative test data reports. It has independent high-quality laboratories and professional experimenters to provide detailed yeast two-hybrid experiment procedures, original data and pictures, and result analysis.

The difference between the membrane system and the nuclear system yeast double-mixed screen library

The strains used in the screening library are different. When screening the library by co-transformation, the nuclear system uses Y2HGold yeast and the membrane system uses NMY51 strain. In addition to the choice of co-rotating sieve library for nuclear system, it can also be carried out by mating method. Mating naturally uses two genders of strains, Y187 containing the AD library plasmid and Y2HGold containing the BD plasmid. The following table is the basic information of the three strains.

StrainsMating typeReporter geneConversion marker
Y2HGoldMATaAbAr,HIS3,
 
ADE2,   MEL1
trp1,   leu2
Y187MATaMEL1,LacZtrp1,   leu2
NMY51MATaHIS3, ADE2,LacZtrp1,   leu2

The difference between nuclear system and membrane system in the whole experimental process.

It is worth noting that if the BD gene itself is a transcription factor in the nuclear system, there may be self-activation. Therefore, the first thing to do after the bait vector is constructed is the self-activation test of the bait gene. The yeast double hybrid membrane system, theoretically, because the bait protein is “hanged” on the cytoplasmic membrane, when there is no interacting protein close to each other, the two parts of ubiquitin are separated from each other, and the transcription factor will not be cleaved, which will not cause the report. gene expression. For the membrane system yeast double hybrid, because the bait protein has different transmembrane methods on the cell membrane, the plasmids used when constructing the BD vector are different. Based on this, after the completion of the BD vector construction, the first thing to do is functional verification, that is, whether the constructed BD plasmid is suitable for the system. The following are the main experimental procedures of the sieve library of the two systems.

The two methods are also different in the selection of positive clones.

In the nuclear system, because of the MEL1 in the reporter gene, α-galactosidase is expressed when there is protein interaction, that is, the positive colonies on the selective medium containing X-α-Gal are blue. In the membrane system, positive clones are screened mainly by the expression level of the reporter gene ADE2. When there is no interacting protein, the synthesis of adenine is blocked, the intermediate product accumulates, the cells turn red, and the clones show a change from light pink to dark red from the beginning of growth to the prolonged time. When there is protein interaction, the color of the clone is different due to the different intensity of action, and the expression of ADE is different, and the clone appears pinkish white (weak interaction) to white (strong interaction).

How to determine whether to choose to construct a nuclear system library or a membrane system library

If the bait gene is a protein located on the membrane, select a membrane library, if the bait gene is located in the nucleus, select a nuclear library. If the bait gene has a transmembrane region, you can also consider removing the transmembrane region and screen it with a nuclear library. Library, but this operation has certain risks. The yeast two-hybrid system has the functions of discovering new proteins and proteins, studying the interaction of antigens and antibodies in the cell body, screening the action sites of drugs, and establishing genomic protein linkage maps. Its application will become more and more important.

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