Kido, Kohki Proteo-Science Center
Yamanaka, Satoshi Proteo-Science Center
Nakano, Shogo University of Shizuoka
Motani, Kou Tokushima University Tokushima University Educator and Researcher Directory KAKEN Search Researchers
Shinohara, Souta Proteo-Science Center
Nozawa, Akira Proteo-Science Center
Kosako, Hidetaka Tokushima University Tokushima University Educator and Researcher Directory KAKEN Search Researchers
Ito, Sohei University of Shizuoka
Sawasaki, Tatsuya Proteo-Science Center
Proximity biotinylation based on Escherichia coli 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 CRL4CRBN 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.
eLife Sciences Publications
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elife_9_e54983.pdf 4.21 MB
Institute of Advanced Medical Sciences