Bimolecular Fluorescence Complementation (BiFC) assay is a intuitive and fast method used to directly visualize protein-protein interaction and protein localization in vivo using live-cell imaging or fixed cells.
Proteins often do not perform their functions independently in cells, but usually interact with other proteins to form complexes to perform specific functions in a specific time and space.Understanding protein-protein interactions (PPI), especially large-scale high-throughput screening of PPI and thus mapping the network of PPI, is of irreplaceable importance for revealing the function of a protein and ultimately elucidating the molecular mechanisms of various life activities in cells.
There are many specific sites on the loop structure between the two β-sheet layers of fluorescent proteins (YFP, GFP, Luciferase, etc.) that can be inserted into exogenous proteins without affecting the fluorescent activity of the fluorescent protein. BiFC technology takes advantage of this property of the fluorescent protein family to split the fluorescent protein into two molecular fragments without fluorescence activity, and then fuse them with the target protein for expression respectively. If the two target proteins are brought into close proximity due to physical interaction, the two molecular fragments of the fluorescent protein are brought close to each other in space, thereby reforming the active fluorescent gene.
Split fluorescent protein (YFP):
This system is suitable for studying strong PPI. The YFP fluorescence intensity is controlled at a low level and is not disturbed by background and other factors. 2.
Split fluorescent protein (YFP) enhanced:
This system is suitable for studying weaker PPI, such as membrane protein interactions. YFPn and YFPc were edited for enhanced fluorescence, facilitating the observation of fluorescence signals of weak PPI. Meanwhile, amino acid A at position 206 is mutated to K, which reduces the interference of background and other factors, and its results are true and reliable.
The Split luciferase (LUC) system：
This system can quantify and compare the strength of the interactions, and is suitable for the study of protein interactions that are influenced by third-party substances. For example, the strength of protein interactions can be regulated by hormones and small molecule compounds, and the strength of interactions can be determined by exogenous addition of hormone compounds.
The PCA library of our BiFC protein system includes nearly 20,000 human proteins, allowing high-speed and convenient screening of protein interactions in human cells and also between pathogenic proteins and human host proteins. Our high-throughput screening of protein interactions based on protein fragment complementation technology has the following features and advantages.
1. Direct, in situ detection of protein interactions in living cells.
2. High sensitivity, can detect protein interactions at levels comparable to endogenous expression.
3. It can detect transient, indirect protein interactions.
4. Independently developed bioinformatics software for easy and fast analysis of results and mapping of protein interaction networks.
1. Clone the gene to be tested into an expression vector containing the N-terminal of the functional protein to construct a bait plasmid.
2. Transform the cells with the bait plasmid and select the transformed cells.
3. Then transform the library plasmid (containing 18,000 human genes, which has been cloned into the expression vector containing the C-terminal of the functional protein) into the cells expressing the bait plasmid.
4. High-throughput screening and identification of bait-interacting proteins by flow cytometry or luciferase reporter system.
5. Bioinformatics analysis, listing proteins that interact with bait proteins.
6. (Optional) Further validate the bait-interacting proteins by PCA or cellular immunofluorescence or co-immunoprecipitation.
High-throughput BiFC workflow