AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions

• DOI: 10.7554/elife.54983
• Publication Date: 2020-05-11T12:00:22Z
• Authors: Kohki Kido; Satoshi Yamanaka; Shogo Nakano; Kou Motani; Souta Shinohara; Akira Nozawa; Hidetaka Kosako; Sohei Ito; Tatsuya Sawasaki
• Keywords: 0301 basic medicine; QH301-705.5; Cell Survival; Science; Recombinant Fusion Proteins; Biotin; Protein Engineering; 7. Clean energy; protein-protein interaction; 03 medical and health sciences; Biochemistry and Chemical Biology; Protein Interaction Mapping; Escherichia coli; enzymatic engineering; ancestral sequence reconstruction; Humans; cell-free; Biotinylation; Carbon-Nitrogen Ligases; Protein Interaction Maps; BioID; Biology (General); 2. Zero hunger; 0303 health sciences; Escherichia coli Proteins; Q; R; Repressor Proteins; HEK293 Cells; Mutation; Medicine; proximity labeling

Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with target protein in cell or organism. However, there have been some improvements the are used purpose. Here, we demonstrate novel enzyme, AirID (ancestral proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm metagenome data. AirID-fusion AirID-p53 AirID-IκBα indicated of MDM2 RelA, respectively, vitro cells, respectively. AirID-CRBN showed pomalidomide-dependent IKZF1 SALL4 vitro. biotinylated endogenous CUL4 RBX1 CRL4CRBN complex streptavidin pull-down assay. LC-MS/MS analysis cells were stably expressing top-level RelA proteins. These results indicate analyzing protein-protein interactions.Proteins need to each other perform many tasks required organisms thrive. A technique called proximity helps scientists pinpoint identity partner together. It relies attaching (either TurboID) interest; when comes close contact this construct, can attach chemical tag it. The tagged then be identified, revealing molecules interest. Although useful tools, they limitations. Experiments take more than 16 hours complete require high levels added cells. active able label within ten minutes. under certain conditions, it also likely toxic cell, make mistakes do not To address these issues, Kido et al. developed AirID, new biotinylation. conducted test how well would perform, interest whose partners already known. confirm human cells; compared TurboID, less mistakenly non-partners kill even over long periods. by suitable experiments Unlike may potential long-lasting living organisms, since time.