Talking About Protein Interaction Detection Method I


Proteins are indispensable in the mechanism of organisms, and they are responsible for initiating and performing tasks in biological systems. Every biological process in an organism, from the replication of genetic material to cell senescence and death, depends on the functional coordination between several and even hundreds of proteins. Changes in the function and structure of proteins can disrupt this balance and lead to disease, usually due to protein-protein interactions. The current methods used to study protein interactions include two-dimensional electrophoresis, phage display technology, GST pull-down, yeast two-hybrid, tandem affinity purification, bi-molecular fluorescence complementary technology, protein chips, Far western blot, and co-immunoprecipitation. Below we will divide into two parts to introduce the characteristics of these related technologies.

Figure 1. Maps of protein–protein interactions.

Two-Dimensional Electrophoresis

Two-dimensional electrophoresis (2-DE) was first proposed in 1975 as a technique for the separation and analysis of proteins. It was not originally designed to verify protein-protein interactions, but after continuous development and improvement, the technology can cooperate with technologies such as TAP To verify protein interaction, the results can also reflect the modified state of the protein after translation. The principle of two-dimensional electrophoresis is to perform isoelectric focusing electrophoresis according to the isoelectric point of the predicted protein, and then perform SDS-PAGE electrophoresis for secondary separation based on the molecular mass. After staining, a two-dimensionally distributed protein electrophoresis diagram is obtained, followed by image analysis software To analyze the properties of the protein. The advantage of this technology is that it can detect differences in properties between proteins with high resolution and sensitivity. The disadvantage is that late gel staining such as fluorescent dyes or silver stains require expensive equipment or cost, so most laboratories use low-cost Coomassie blue staining methods.

Phage Display Technology

The technology was first established by Smith GPS of the University of Missouri in the United States. In the past few years, the technology has developed rapidly and has shined in various fields, such as verification of protein-protein interactions, clinical applications, drug discovery, vaccine development, antibody engineering separation technology, epitope mapping, and so on. The principle of this technology is to clone the complete reading frame of the target gene to the gene position of the phage coat protein structure, so that the foreign gene and the phage coat protein are fused and expressed, and the fusion protein is displayed on the phage surface with the recombination of the progeny phage. The displayed molecule or protein maintains a relatively independent structure and activity. After incubating for a certain period of time, the peptide library and the target protein molecule on the solid phase are washed away from the unbound free phage, and then the target molecule is eluted with a competitive receptor or acid. Combined with the adsorbed phage, the eluted phage infects the host cell and then multiplies and expands. The next round of elution is performed. After three to five rounds of “adsorption-elution-amplification”, the phage that specifically binds to the target molecule Get highly enriched. The resulting phage preparation can be used to further enrich the target phage with desired binding properties. The advantages of this technology are efficient screening, simple operation and low cost. The disadvantage is that the copy number is low, the expression is small, and the phage display system depends on the expression of genes in the cell. Therefore, some molecules that are toxic to the cell, such as biotoxin molecules, are difficult to effectively express and display, limiting the diversity of the molecule.

GST Pull-Down Technology

The purpose of this technology is to verify the interaction between proteins in vitro, and it has been widely used in the field of molecular biology. The principle of this technology is to first construct a fusion expression vector with a GST tag, then purify the protein through a GST affinity purification column, and finally the test protein is passed through the column. If there is an interaction between the tag protein and the test protein, the test protein can be bound to the column, and then the interaction protein can be obtained by elution. The disadvantage is that large-scale protein interactions cannot be screened, and some endogenous proteins can interfere with the test and lead to false positive results.

Yeast two-hybrid technology

In 1989, Filds established yeast two-hybrid through the results of the study of the transcription factor GAL4. It is a classic method to verify protein-protein interactions in vivo. The principle is that there are two special domains in yeast: specific binding domain (BD) and transcription activation domain (AD). These two structure domains are functional even when separated, and do not affect each other. When BD binds to a certain gene and needs to be expressed, it must recognize AD to bind, so that transcription and translation can form a complete eukaryotic expression system. So as long as the two protein genes are connected to AD and BD respectively, if there is an interaction between the target proteins, AD and BD can complete the transcription and activate the downstream reporter gene, and the interaction result can be judged based on the reporter gene . At the same time, it is recommended to use two or more types of reporter genes (such as ADE2 and URA3) to make the test results more accurate. The advantage of yeast two-hybrid is that it can establish a cDNA library, select a large number of proteins that interact with the target protein on a large scale, and establish a gene map (GPLM). The advantage of yeast two-hybrid is that it is highly efficient. The transient and stable binding between proteins has good sensitivity. The results are easy to observe. The most important thing is that yeast two-hybrid is performed in vivo. It can effectively eliminate the interference of in vitro factors to make the result more Credible. The disadvantage is that the cell membrane, cytoplasm, and proteins secreted outside the cell cannot be detected. In addition, the test period is slightly longer, and the results are prone to false positives. Multiple screening and rotary yeast tests are required to rule out.

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