Methods for Protein-Protein Interaction Research

Proteins are the most direct performers of biological functions. Although some proteins can accomplish their mission independently, most of them require the assistance of some chaperone molecules to complete the task together or form a complex before they can fully perform their functions. Therefore, understanding the interaction between protein and protein can help us better understand the vitality of cells and reveal the regulatory mechanism hidden under the surface. The classic protein-protein interaction research methods mainly include three: yeast two-hybrid, co-immunoprecipitation, and GST-pull down.

Yeast two hybrid is based on the study of the properties of eukaryotic transcription factors, especially yeast transcription factor GAL4. GAL4 consists of two separate but functionally necessary domains, the DNA binding domain (DNA-BD) at the N-terminal and the activation domain (AD) at the C-terminal. DNA-BD can recognize the upstream activating sequence (UAS) located in GAL4-responsive gene (GAL4-responsivegene) and bind to it. AD is through the combination with other components in the transcription machinery to initiate transcription of genes downstream of UAS. DNA-BD and AD alone cannot activate the transcription reaction. However, when the two are sufficiently close in space, they exhibit complete GAL4 transcription factor activity and can activate the UAS downstream promoter, so that the downstream genes of the promoter can be transcribed.

Based on this principle, the researchers established a two-hybrid system. BD is fused with protein X (X is often a known protein called bait), and AD is fused with protein Y (called prey). If a protein-protein complex is formed between X and Y to reconstitute the two domains of GAL4, the transcription of the reporter gene sequence will be initiated. Through the detection of the reporter gene, it can in turn determine whether there is an interaction between the bait and the prey.

This technology is mainly used for screening of interacting proteins. The disadvantage is that false positives are high, so result verification is required. Generally, immunoprecipitation or GST-pull down experiment is used for verification.

Co-immunoprecipitation is a classic method for studying protein interactions based on the specific interaction between antibodies and antigens. It is an effective method to determine the physiological interaction of two proteins in intact cells.

When cells are cleaved under nondenaturing conditions, many protein-protein interactions that exist within intact cells are preserved. When protein A is immunoprecipitated by the antibody of protein A pre-solidified on Argarose beads, the protein B that binds to protein A in the body can also be precipitated together. The interaction between the two is confirmed by denaturing protein isolation and Western blot analysis.

The GST pull-down experiment is an effective in vitro test technique to verify the yeast two-hybrid system. The basic principle is to construct the target protein-GST fusion protein vector first, and then perform in vitro expression and purification. The resulting target protein-GST (Glutathione-S-transferase) fusion protein is affinity immobilized on the glutathione affinity resin to act as a "bait protein". Pass the target protein solution through the column to capture the interacting protein from it. Then the target protein was eluted, and the interaction between the two proteins was confirmed by SDS-PAGE electrophoresis and western blot analysis.