BioID as a Tool for Protein-Proximity Labeling in Living Cells

Abstract

BioID has become an increasingly utilized tool for identifying candidate protein-protein interactions (PPIs) in living cells. This method utilizes a promiscuous biotin ligase, called BioID, fused to a protein of interest that when expressed in cells can be induced to biotinylate interacting and proximate proteins over a period of hours, thus generating a history of protein associations. These biotinylated proteins are subsequently purified and identified via mass spectrometry. Compared to other conventional methods typically used to screen strong PPIs, BioID allows for the detection of weak and transient interactions within a relevant biological setting over a defined period of time. Here we briefly review the scientific progress enabled by the BioID technology, detail an updated protocol for applying the method to proteins in living cells, and offer insights for troubleshooting commonly encountered setbacks.

Keywords: BioID; Biotin ligase; Biotinylation; Protein–protein interactions; Proximity-dependent labeling.

Figure 1

Overview of the BioID method. The BioID fusion protein induces biotinylation of proteins in a proximity-dependent manner. Biotinylated proteins are denatured followed by affinity-isolation using streptavidin-coupled beads. After subsequent tryptic digest, the isolated proteins are identified by MS analysis.

Figure 2

Representation of stable BioID-fusion protein expression in cells. NIH3T3 cells stably expressing BioID2-only or a BioID-fusion protein of interest (BioID2-BAF) were analyzed for fusion protein expression, localization and biotinylation by fluorescence microscopy and immunoblot. A) Using florescent microscopy, fusion proteins were detected by anti-BioID2 antibody (red) and biotinylation was detected by streptavidin-488 Alexa Fluor (green). Scale bar = 10 µm. B) Biotinylation efficiency and fusion protein expression in whole cell lysate were also assessed by western blot, using streptavidin-HRP and anti-BioID2 antibody.