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Yeast two-hybrid technology can be used to screen LKB1 interacting proteins

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It is well known that proteins play a very key role in the process of cell life activities. The understanding of protein interactions can better understand the essence of life. The yeast two-hybrid system is an effective genetic method for analyzing protein interactions in vivo. In the yeast two-hybrid experiment service provided by some biomedical companies, customers only need to provide bait gene information, and they can screen the specified gene library according to the customer’s yeast two-hybrid screening. After the screening is completed, the customer will be provided with a detailed screening report and positive clones. In kind.

The operation of the yeast two-hybrid system is carried out at the nucleic acid level. It is to study the interaction between proteins in yeast cells, and it can also detect the weak interactions between proteins without the need to purify the proteins. Therefore, since the establishment of this technology, Has been quickly and widely used. Medicilon Biopharmaceuticals provides yeast two-hybrid experiment services. It has independent high-quality laboratories and professional experimenters, can issue authoritative test data reports, and provide detailed yeast two-hybrid experiment procedures, original data and pictures, and result analysis.

LKB1 is a tumor suppressor protein, its full name is liver kinase B1 (liver kinase B1), which can control cell growth and metabolism. In animal models, the researchers found that mice that lost LKB1 function had a fatal inflammatory response. Further pathological analysis confirmed that the spleens and lymph nodes of these mice were swollen, and there were infiltrating immune cells in multiple organs. These phenomena indicate that there is a problem with regulatory T cells that can suppress the immune response.

1.The relationship between LKB1 and T cells

To test this idea, the researchers further analyzed the regulatory T cells in these mice. They found that these cells exhibited the characteristics of functional failure no matter from the function or the molecular level. The researchers linked this finding to the function of LKB1. After knocking out LKB1, the metabolic pathways that are essential to the function of mitochondria were disturbed, which affected the activity and function of regulatory T cells.

LKB1 can not only affect the health of regulatory T cells itself, but also affect PD-1, a molecule that plays an important role in tumor immunotherapy. When analyzing the molecular differences in the expression of regulatory T cells before and after knocking out LKB1, the researchers unexpectedly found that the expression of PD-1 molecules increased in regulatory T cells lacking LKB1, which further inhibited the regulatory effect. The immunosuppressive ability of T cells caused a frantic immune response in mice. By inhibiting PD-1, the activity of regulatory T cells in mice was significantly restored, and the lethal immune response was also controlled. This shows that PD-1 can also inhibit the function of regulatory T cells

2. Using yeast two-hybrid technology to screen the protein interacting with LKB1

The yeast two-hybrid technology can be used to screen the proteins that interact with the tumor suppressor gene LKB1. The method is to use the yeast two-hybrid system and use human LKB1 as the bait to screen the human genome-wide open reading frame yeast two-hybrid library, and look for the interaction with it. The protein, and the interaction between them was confirmed by immunoprecipitation and GST-Pull down experiments.

Results: A total of 17 clones were obtained through yeast two-hybrid, and one non-repeated positive clone was finally confirmed after verification and analysis. Conclusion: The obtained protein encoded by 1 gene may reveal a new mechanism of action of LKB1.

3.Large-scale yeast two-hybrid screening method

(1) Library screening method

The library screening method generally uses relatively few known or unknown baits to screen a relatively large prey library. It can also screen the library with the prey as the bait after the positive prey is selected by the bait. This cycle continues to expand the spectrum. The bait can be one or several to thousands, and it can be ORF, full-length cDNA or their fragments (including domains); the prey library is generally large, which can be a library of many ORFs, or a full-length cDNA library Or cDNA fragment library, or random genomic fragment library. The advantage of the library screening method is that it can obtain the information of the interaction domain and reduce the false negative rate, but at the same time it may also increase the false positive rate.

(2) Array screening method

The array screening method can be divided into one-to-one array, high-throughput array method, and sub-library-to-sublibrary method. The one-to-one array method screens strains containing different BD-X and all strains containing different AD-Y in a one-to-one correspondence.

One-to-one cohort method: All strains containing different DBD-X and strains containing different AD-Y are screened in one-to-one correspondence. This screening method has a large workload, but positive clones can infer sequence information from the array position without sequencing, can be automated, and theoretically have a high possibility of detecting all protein interactions.

High-throughput array method: It is a screening method that combines all the different ORF-ADs into a library and then interacts with the different DBD-Xs arranged in an array. The essence is the screening of arrayed libraries in the case of a large number of decoys. The advantage is that it has a higher throughput, but it requires sequencing to identify positive clones.

Sub-library to sub-library method: Number the members of DBD-X and AD-Y with the larger total number and group them, each group contains a certain number of members, and then gather each group of members to form a sub-library, and then use the prey sub-library to decoy the bait The sub-library functions one by one to screen positive clones. This method is more high-throughput than the high-throughput array method in the ORF screening of yeast genome, but the prey and bait of positive clones need to be sequenced and identified.

(3) Two-step method

The two-step method combines library screening and array screening to give full play to the advantages of both. First, a single prey is used to screen the decoy sub-pool, and then the prey corresponding to the positive clone is used one-to-one with the decoy of the decoy sub-pool to select the positive clone. If researchers use this method for the study of fetal brain protein interaction, the advantage is equivalent to verifying the phenotype once, without sequencing, reducing workload, consumables and costs.

The yeast two-hybrid system is a sensitive and effective method for analyzing protein-protein interactions at the gene level in eukaryotic cells. This technology has been widely used in the study of protein interactions and revealed a large number of unknown proteins. The interaction between them.

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