AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions.
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-I?B? 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-I?B? showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.
Mesh Terms:
Biotin, Biotinylation, Carbon-Nitrogen Ligases, Cell Survival, Escherichia coli, Escherichia coli Proteins, HEK293 Cells, Humans, Mutation, Protein Engineering, Protein Interaction Mapping, Protein Interaction Maps, Recombinant Fusion Proteins, Repressor Proteins
Biotin, Biotinylation, Carbon-Nitrogen Ligases, Cell Survival, Escherichia coli, Escherichia coli Proteins, HEK293 Cells, Humans, Mutation, Protein Engineering, Protein Interaction Mapping, Protein Interaction Maps, Recombinant Fusion Proteins, Repressor Proteins
Elife
Date: May. 11, 2020
PubMed ID: 32391793
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