New protein interaction imaging technology: BiFC technology

Recently, Professor Chen Kuangshi's research group from the Department of Biomedical Engineering of the Future Technology College has developed a new imaging technology that can study protein interactions in cells at the nanometer scale based on the Bimolecular Fluorescence Complementation (BiFC) technology. This technology overcomes the problem that traditional BiFC technology is prone to produce false positive signals, and is used to analyze the working mode of important host cell proteins involved in HIV-1 virus assembly at the nanometer scale. The research results have been published in the academic journal ACS Nano with the title "A Background Assessable and Correctable Bimolecular Fluorescence Complementation System for Nanoscopic Single-Molecule Imaging of Intracellular Protein-Protein Interactions".

BiFC technology is a method that can image protein interactions in cells, and its principle is to segment a complete fluorescent protein into two non-fluorescent fragments, and then connect these two non-fluorescent fragments with the two target proteins that may interact to form fusion proteins, respectively. When the target proteins interact in cells, these two non-fluorescent fragments will form complete fluorescent proteins due to the proximity of spatial distance. However, when the target protein does not interact, two non-fluorescent fragments may also self-assemble into a complete fluorescent protein due to random collisions, resulting in false positive signals, which limits the quantitative study of the target protein interaction with BiFC technology. In order to overcome this problem, Chen Kuangshi’s research group developed a method that can detect and eliminate this false positive signal generated by the self-assembly of non-fluorescent fragments, and named it BAC-BiFC (Background Assessable and Correctable-Bimolecular Fluorescence Complementation). Specifically, they connected a target protein in BiFC to a reference fluorescent protein, which can not only characterize the expression level and spatial distribution of the target protein in cells, but also allow researchers to determine the intensity of false positive signals at different protein expression levels by ratio imaging, so as to obtain the experimental conditions for false positive signal interference.

"By developing new super-resolved BiFC technologies, we found that the host cell AGO2 protein is involved in the whole process of HIV-1 viral particle assembly and is an indispensable original for viral proliferation. The findings are expected to provide a new perspective for the study of HIV and other retroviruses." Chen Kuangshi said.

During the proliferation of HIV-1 virus, its structural protein Gag needs to interact with many host cell proteins to complete the assembly and budding of virus particles. The researchers used the BAC-BiFC technology to study the interaction between the Gag protein and the host cell protein AGO2 at the nanometer scale by combining the super-resolution microscopy technology direct stochastic optical reconstruction microscopy (dSTORM). Unlike previous findings, the researchers found that Gag protein did not mediate only a fixed amount of AGO2 packaged by viral particles, but recruited more AGO2 at the late stage of viral assembly, suggesting that AGO2 is involved in the whole process of HIV-1 viral particle assembly and is an indispensable element for viral proliferation. The findings are expected to provide a new perspective for the study of HIV-1 virus and other retroviruses. It is worth mentioning that, in addition to the research objects involved in this work, BAC-BiFC technology also has the potential to visualize the interactions between various other types of proteins and even between other biomolecules during life.

Collected by Profacgen, an insect cell protein production provider.