The world-famous genome project has led to the discovery of a large number of new genes, but simple base-group DNA sequences are still unable to answer many life questions. Genes are relatively static, and the products encoded by genes - proteins are dynamic, spatial and temporal, and regulatory, and are the main executors and performers of biological functions. Protein expression levels, patterns of presence, and interactions are directly related to biological function. The interaction between proteins is essential in all life activities, and it is the basis for all metabolic activities of cells. Cells receive exogenous or endogenous signals and regulate their gene expression through their unique signaling pathways to maintain their biological properties. In this process, protein plays an important role in regulating and mediating many biological activities of cells. Although some proteins can function in monomeric form, most proteins work with a partner molecule or form a complex with other proteins. Therefore, in order to better understand the biological activity of cells, the function of protein monomers and complexes must be well understood, which involves the study of protein interactions. In modern molecular biology, the study of protein interactions plays a very important role. Therefore, revealing the interaction between proteins and establishing a network map of interactions has become a hot topic in proteomics research. First, the biophysical method 1. Fusion protein pull-down experiment The fusion protein pull-down technology is based on the principle of immobilizing a protein on a substrate (such as Sepharose), when the cell extract passes through the matrix, The ligand protein interacting with the immobilized protein is adsorbed, while the "impurities" that are not adsorbed elute with the eluent. The adsorbed protein can be recovered by changing the eluent or elution conditions. In order to utilize the pull-down technique more efficiently, the protein to be purified can be expressed as a fusion protein, that is, the "bait" protein is fused with a ligand protein that is easy to purify. In 1988, Smith et al. used a glutathione-S-transferase (GST) fusion tag to purify GST fusion protein from bacteria. Since then, GST fusion proteins have been greatly promoted in the field of protein interaction research. The GST fusion protein can be adsorbed by the interaction of GST and GSH when it is passed through a GST (glutathione) column. When the cell extract is passed through the column, a protein of interest capable of interacting with the "bait" protein can be obtained. In general, the GST fusion protein pull-down method is used in two ways: one is to identify unknown proteins that interact with known fusion proteins; the other is to identify the presence of interactions between two known proteins. The method is relatively simple, avoids the use of dangerous substances such as isotopes, and has a wide range of applications in protein interaction research. There are many similar fusion proteins, such as the "bait" protein fused to staphylococcal protein A can be purified by IgG-immobilized column; the "bait" protein fused with oligohistidine peptide can be combined with Ni2+ column Purification; the "bait" protein fused with dihydrofolate reductase can be purified by a column immobilized with methotrexate, and the like. 2. Affinity Blot Affinity Imprinting is the transfer of a protein sample separated by polyacrylamide gel electrophoresis onto a nitrocellulose membrane and then detecting which protein interacts with the labeled "bait" protein. The method to be considered is how to maintain the biological activity of the protein on the membrane, how to obtain a purified "bait" protein and the like. 3. Immune coprecipitation If the cells are lysed under non-denaturing conditions, the interaction between proteins can be maintained, so the co-immunoprecipitation method can be used to find the interacting proteins. Examples of this include Harlow et al. When an EIA protein of an adenovirus was precipitated by an antibody, a protein which specifically binds to the EIA protein was found in eukaryotic cells; McMahon et al. used this method to identify a protein in which a transcription domain-related protein can interact with E2F1 and c-Myc transcription factors. Co-immunoprecipitation can be used to test the interaction of two known proteins in vivo, and to find unknown protein interactions, no matter which of the two, the principle is the same, all need to use specific The antibody binds to one of the proteins, and then the complex is precipitated by Protein A or Protein G-Sepharose microbeads and then identified by SDS-PAGE. It is important to set the correct control in co-immunoprecipitation because the probability of false positives in this method is relatively high. The control set includes: using a control antibody in the control group, a cell line lacking the protein of interest as a negative control, and the like. 4. Fluorescence Resonance Energy Transfer Technology The principle of fluorescence resonance energy transfer technology is that an active donor can deliver its own fluorescence to a receptor that is very close to it (usually within 10 nm). Therefore, if a protein is labeled with a genetically-mutated green fluorescent protein (GFP) or a synthetic fluorescent dye, the interaction of the two proteins can be determined by energy transfer between the phosphors. This method has two advantages over the above method: (1) The interaction between proteins occurs in intact cells and relatively completely reflects the activity of the protein in physiological state. (2) Using this method, the localization of the protein in the cell can be observed. Second, molecular biology methods 1. Phage display technology Phage display technology is based on a profound understanding of phage genetic and physiological characteristics. The principle is to integrate a protein library into a simple phage particle, and use the affinity of the protein of interest to a specific ligand to screen for a protein that specifically binds to the ligand. Phage display technology generally involves multiple rounds of screening so that a very low concentration of ligand-specific proteins in the library can be obtained. 2. Yeast two-hybrid In 1989, Field et al. invented the yeast two-hybrid system. The system utilizes two domains contained in the yeast growth transcription factor GAL4, a DNA binding domain (BD) and a DNA activating domain (AD), and a known gene (bait gene) and a target gene. Or the cDNA containing the target gene is separately constructed on the BD-containing and AD plasmid vectors, and when the two plasmids co-transform yeast competent cells, if the BD-binding bait protein can bind to AD, the target protein or certain cDNA encoded in the library Protein interactions, when combined with each other, result in spatially close proximity of BD and AD, presenting the full activity of the GAL4 transcription factor, and initiating the expression of downstream reporter genes such as His and LacZ, thus causing specific defects Growing on the medium. Therefore, the yeast two-hybrid system can be used to screen proteins that interact with bait proteins, and the interaction between known proteins can also be studied. The yeast two-hybrid technique is used to screen cDNA libraries to separate proteins interacting with bait proteins or to study protein interactions. The first consideration is generally the construction of bait vectors. After the gene encoding the bait protein is digested, it is ligated to the bait vector such as pGBTK7 according to the correct reading frame, and then further confirmed by enzyme digestion or sequencing. The bait vector was constructed and the bait protein yeast strain such as Y187 and the target protein yeast strain such as AH109 were simultaneously transformed to test for toxicity and self-activation. It was proved that the bait protein expression was not toxic to the normal growth of the yeast, and the yeast reporter gene could not be activated by itself. Expression can be used to screen libraries or to study interactions between proteins. The second issue to consider is the construction of a target protein vector or a yeast two-hybrid cDNA library. A homologous recombination sequence is generally added to the gene or cDNA encoding the target protein, and then the target protein vector such as pGADT72Rec is co-transformed with a target protein yeast strain such as AH109, ​​and the target protein gene or cDNA sequence is under the action of the yeast homologous recombinase. Integration into a vector completes the construction of a target protein vector or cDNA library. The final step is to screen the library or directly study the interaction between the two proteins. After hybridization of the yeast containing the bait protein vector and the target protein carrier, the BD bait and the AD target protein are expressed in yeast. The interaction between the bait protein and the target protein results in the spatial proximity of BD and AD, showing the full activity of the GAL4 transcription factor, and the initiation of downstream reporter genes such as His and LacZ, depending on the expression of the reporter gene. Thereby achieving the purpose of separating proteins or studying protein interactions. Third, genetics technology synthesis lethal screening synthetic lethal effect refers to the simultaneous death of two genes will produce a lethal effect, and when each gene alone mutation, there is no lethal effect. This total genetic approach can be used to analyze the interaction between two proteins that share the same important functions. Since the synthetic lethal mutant is not viable and the lethal strain cannot be directly obtained, the conditional mutant strain is often used in the laboratory. For example, temperature-sensitive strains are mutants that can be lethal at certain temperature conditions. The two interacting proteins obtained by synthetic lethal screening may be two components of the same complex or all proteins that regulate one another. 4. Prospects The development of proteomics has promoted the development and improvement of a variety of research technologies, and more and more technologies tend to develop in a large-scale, high-throughput direction. At the same time, the development of basic science research in physics, chemistry, and informatics has greatly promoted the improvement of these technologies. A newly developed computer analysis method that predicts protein interactions based on gene and genomic information and involves prediction of surface residues involved in protein interactions. Analytical methods that rely on gene sequences to analyze protein interactions are emerging. The advancement of these advanced, simple, and large-scale analysis of protein interactions has driven advances in proteomics. It is foreseeable that in the near future, with the deepening of genomic research and proteomics, various problems in the life sciences will be broken one by one.
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