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

• Published in: eLife 2020-05, Vol.9
• Main Authors: Kido, Kohki, Yamanaka, Satoshi, Nakano, Shogo, Motani, Kou, Shinohara, Souta, Nozawa, Akira, Kosako, Hidetaka, Ito, Sohei, Sawasaki, Tatsuya
• Format: Article
• Language: English
• Subjects: Algorithms; ancestral sequence reconstruction; Antibodies; Biochemistry and Chemical Biology; BioID; Biotin; Biotin - chemistry; Biotin - metabolism; Biotinylation; Carbon-Nitrogen Ligases - chemistry; Carbon-Nitrogen Ligases - genetics; Cell Biology; Cell Survival; cell-free; Cells (Biology); Deoxyribonucleic acid; DNA; E coli; enzymatic engineering; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Genes; Genomes; HEK293 Cells; Humans; Identification; Labeling; Mass spectrometry; MDM2 protein; Mutation; p53 Protein; Pomalidomide; Protein Engineering; Protein interaction; Protein Interaction Mapping; Protein Interaction Maps; protein-protein interaction; Proteins; proximity labeling; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; RelA protein; Repressor Proteins - chemistry; Repressor Proteins - genetics; Scientific equipment industry; Scientific imaging; Software; Streptavidin; Technology application; Tools and Resources; Tumor proteins
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.54983

Proximity biotinylation based on BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4 complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.