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Omics techniques for protein-protein interactions

To probe the molecular mechanisms of biological processes, it is necessary to identify the protein-protein interactions that mediate this process. The main techniques for studying protein-protein interactions are summarized as follows:

1. The yeast two-hybrid system

Yeast two-hybrid system is an important method widely used in protein interactomics research. The principle is that when the target protein and the bait protein are specifically combined, the bait protein binds to the promoter of the reporter gene and starts the expression of the reporter gene in yeast cells. If the expression product of the reporter gene is detected, it means that there is an interaction between the two. effect, otherwise there is no interaction between the two. This technique can be used for large-scale protein interaction studies after micro-quantification and arraying. In practical work, one-hybrid system, three-hybrid system and reverse-hybrid system have been developed according to needs. Angermayr et al. designed an SOS protein-mediated two-hybrid system. The function of membrane proteins can be studied, which enriches the function of yeast two-hybrid system. In addition, the role of the yeast two-hybrid system has also been extended to the identification of proteins.

2. Phage display technology

The DNA sequence of a monoclonal antibody is connected to the gene encoding the phage coat protein. When the phage grows, the corresponding monoclonal antibody is expressed on the surface, and then the phage is passed through the column. If the column contains the target protein, it will be specific to the corresponding antibody. Combined, this is called phage display technology. This technology is also mainly used to study the interaction between proteins. It not only has the characteristics of high throughput and simplicity, but also has the advantages of directly obtaining genes, screening complex mixtures with high selectivity, and directly evaluating the interaction by appropriately changing the conditions during the screening process. specificity of binding. At present, using optimized phage display technology, cDNA libraries of two special cell lines of human and mouse have been displayed, and signaling molecules in the signaling pathway of human epithelial growth factor have been isolated.

3. Plasma Resonance Technology

Surface Plasmon Resonance (SPR) has become a new method in protein interaction research. Its principle is to use a nano-scale film to adsorb the “bait protein”. When the protein to be tested is combined with the bait protein, the resonance properties of the film will change, and the binding of the two proteins can be known through detection. The advantage of SPR technology is that no labels or dyes are required, and the reaction process can be monitored in real time. The assay is fast and safe, and can also be used to detect protein-nucleic acid and other biological macromolecules interactions.

4. Fluorescence Energy Transfer Technology

Fluorescence resonance energy transfer (FRET) is widely used to study intermolecular distances and their interactions; combined with fluorescence microscopy, it can quantitatively obtain spatiotemporal information about proteins, lipids, DNA, and RNA in living organisms. With the development of green fluorescent protein (GFP), FRET fluorescence microscopy has the potential to measure the dynamic properties of molecules in living cells in real time. A simple method for quantitatively measuring FRET efficiency and the distance between the donor and acceptor is presented, using only a set of filters and measuring a ratio, using the emission spectra of the donor and acceptor to eliminate spectral crosstalk. This method is simple and fast, and can quantitatively measure FRET efficiency and donor-acceptor distance in real time, especially for GFP-based donor-acceptor pairs.

5. Antibody and protein array technology

The emergence of protein chip technology brings new ideas to proteomics research. One of the main contents of proteomics research is to study the quantitative changes of protein levels in different physiological states. The miniaturized, integrated and high-throughput antibody chip is a very good research tool, and it is also the fastest growing chip among the chips. And the technology has become increasingly mature. Some of these antibody chips have been developed for clinical application, such as tumor marker antibody chips, and many others have been applied in various fields.

6. Co-immunoprecipitation technology

Co-immunoprecipitation is a technique mainly used to study the interaction of proteins and proteins. Staphylococcus aureus protein A (SPA), if there is a target protein that is binding to the protein of interest in the cell, it can form such a complex: “target protein-interest protein-anti-interest protein antibody-SPA\|Pansobin”, Because SPA\|Pansobin is relatively large, the complex is separated out during centrifugation. The four components of the complex were separated by denaturing polyacrylamide gel electrophoresis. Then, by Western blotting method, the antibody is used to detect what the target protein is and whether it is a predicted protein. The target protein obtained by this method is naturally bound to the protein of interest in the cell, which is in line with the actual situation in vivo, and the obtained protein is highly reliable. But this method has two drawbacks: one is that the binding of the two proteins may not be direct, but a third party may act as a bridge in the middle; the other is that the target protein must be predicted before the experiment to select the final detection Antibodies, so if the prediction is incorrect, the experiment will not yield results, and the method itself is risky.

7. Pull-down technology

There are two types of protein interactions: firm interactions and transient interactions. Robust interactions are common in multi-subunit protein complexes and are best studied by co-immunoprecipitation (Co-IP), Pull-down techniques, or Far-western methods. Pull-down technology uses immobilized, labeled bait or tag proteins (Biotin-, PolyHis- or GST-) to fish out interacting proteins from cell lysates. The pull-down technique can determine the interaction between the known protein and the extracted protein or purified related protein, and detect the protein interaction from the in vitro pathway or translation system.