2C-BioID: An Advanced Two Component BioID System for Precision Mapping of Protein Interactomes

Abstract

Summary: The modulation of protein-protein interactions (PPIs) is an essential regulatory activity defining diverse cell functions in development and disease. BioID is an unbiased proximity-dependent biotinylation method making use of a biotin-protein ligase fused to a protein of interest and has become an important tool for mapping of PPIs within cellular contexts. We devised an advanced method, 2C-BioID, in which the biotin-protein ligase is kept separate from the protein of interest, until the two are induced to associate by the addition of a dimerizing agent. As proof of principle, we compared the interactomes of lamina-associated polypeptide 2β (LAP2β) with those of lamins A and C, using 2C- and conventional BioID. 2C-BioID greatly enhanced data robustness by facilitating the in silico elimination of non-specific interactors as well as overcoming the problems associated with aberrant protein localization. 2C-BioID therefore significantly strengthens the specificity and reliability of BioID-based interactome analysis, by the more stringent exclusion of false-positives and more efficient intracellular targeting. : Molecular Biology; Molecular Interaction; Methodology in Biological Sciences Subject Areas: Molecular Biology, Molecular Interaction, Methodology in Biological Sciences