Study on protein binding and expression

Posted September 20, 2019 by Bonnibelle

The interaction between white matter and proteins constitutes one of the main components of the cellular biochemical response network.

The interaction between white matter and proteins constitutes one of the main components of the cellular biochemical response network. The types of protein interactions are firm and temporary. Firm interactions are common in multisubunit protein complexes, and proteins interacting with them are extracted from cell lysates. Pull-down technique can determine the Protein Conjugation relationship between the known Protein and the fished Protein or the purified related Protein, and detect the Protein interaction relationship from in vitro transmission or translation system.

Yeast two-hybrid system is an important method for studying Protein interaction and Protein Expression. The principle is that when the target protein and the decoy protein are specifically combined, the decoy protein binds to the promoter of the reporter gene and initiates the expression of the reporter gene in yeast cells. If the expression product of the reporter gene is detected, it indicates that there is an interaction between the two, and vice versa. This technique can be used to study the interaction between proteins on a large scale. In practice, people have developed single-hybrid system, three-hybrid system and reverse hybrid system. An SOS protein-mediated two-hybrid system already exists. This system can study the function of membrane protein and enrich the function of yeast two-hybrid system. In addition, the role of yeast two-hybrid system has been extended to identify proteins.

E. coli is a prokaryote with only ribosomes that can only form polypeptide chains. Insulin is a protein, and its synthesis requires the processing of endoplasmic reticulum and golgi body. Therefore, in the process of Protein co-expression in e. coli, expression products are often formed into inclusion bodies rather than secreted. Active human insulin is obtained through the process of denaturation and refolding. Due to the fact that escherichia coli can only express insulin gene in cells without specific spatial structure of polypeptide chain, and its lack of protein processing system of eukaryotic cells, escherichia coli cannot express insulin with biological activity.

When the phage grows, the corresponding monoclonal antibody is expressed on the surface. Then the phage is placed on a column. If the target protein is contained on the column, it will be specifically bound to the corresponding antibody. This technique is also mainly used to study protein-protein interactions. It not only has the characteristics of high throughput and simplicity, but also has the advantages of obtaining genes directly, screening complex mixtures with high selectivity, and evaluating the specificity of binding directly by changing the conditions in the screening process. At present, the cDNA libraries of two special cell lines of human and mouse have been demonstrated by using the optimized phage display technology, and the signal molecules in the signaling pathway of human epithelial growth factor have been isolated.

Recombinant proteins are proteins produced by using techniques such as recombinant DNA or recombinant RNA. Currently, in vitro production of recombinant proteins mainly includes four systems: prokaryotic Protein expression, Protein co-expression in Mammalian Cells, yeast Protein expression and insect cell Protein expression. The proteins produced differ in their activity and application. Select the appropriate protein expression system according to their own downstream application, improve the success rate of expression. Hemoglobin extraction and separation procedures can be divided into four major steps: sample processing, crude separation, purification and purity identification.

First, the hemoglobin solution is collected by washing red blood cells, releasing hemoglobin, centrifugation and other operations, i.e. sample treatment. After dialysis, impurities with small molecular weight are removed, that is, coarse separation of samples. Then, the impurity proteins with high molecular weight are removed by gel chromatography, that is, the sample is purified. Finally, haemoglobin is a colored protein identified by polyacrylamide gel electrophoresis, so the color can be used to determine when the eluent should be collected.

In the application of yeast two-hybrid system, there are many false positives and low conversion efficiency. False positives are when the reporter gene is activated without any interaction between the two proteins being studied. The main reason is that BD fusion decoy protein has a separate activation effect, or the activation effect of this fusion protein is activated by foreign proteins. In addition, AD fusion target protein can independently activate reporter gene expression if it has specific DNA binding. Therefore, in order to eliminate false positive results need to be strict Yeast Two - Hybrid Screening test. The reporter gene activation should be independently identified for bait and target proteins. In addition, reporter genes are usually integrated into chromosomes to stabilize gene expression and eliminate false positives caused by gene expression fluctuations caused by plasmid copy number changes. Even if the protein-protein interactions do occur based on rigorous controlled experiments, the following aspects should be analyzed:

(1) whether such interaction will occur naturally in the cell, that is, whether the protein pair will be expressed at the same time and located in the same region in the normal life activities of the cell.

(2) some proteins are dependent on members of the proteolytic pathway that pervades the protein, and they have a universal ability to interact with one another.

(3) interactions can occur between two a-heliophilic proteins that do not actually interact with each other but have the same pattern. In the past ten years, yeast two-hybrid technology has been improving in eliminating false positives and has achieved good results.
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Country United States
Categories Biotech
Last Updated September 20, 2019