BioID: A Screen for Protein-Protein Interactions

Abstract

BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species. © 2018 by John Wiley & Sons, Inc.

Keywords: BioID; biotinylation; protein-protein interaction; proximity-dependent labeling.

Figure 19.23.1

Outline of the BioID method. The following steps outline the BioID process. (1) Ligate bait cDNA and BioID in-frame and in an appropriate expression vector. (2) Express BioID fusion protein in cells to (3) generate stably expressing cells. (4) Induce biotinylation with excess biotin prior to (5) cell lysis and protein denaturation. (6) Perform biotin-affinity capture of biotinylated proteins. (7) Reserve 10% of the sample for streptavidin immunoblot analysis. Use the remainder of the sample for mass spectrometry either by (8A) SDS-PAGE separation with (8B) identification of proteins in isolated bands or (9) analysis of peptides released by on-bead digestion.

Figure 19.23.2

Analysis of cells stably expressing a BioID-fusion protein. U2OS cells stably expressing BioID-lamin A (LaA) were analyzed prior to large-scale pull-down experiments. (A) By fluorescence microscopy it can be observed that all of the cells express similar levels of the myc-tagged BioID-LaA protein (red) targeted to the nuclear envelope. Following 24 hr incubation with excess biotin, the vast majority of the biotin signal, detected with streptavidin–Alexa Fluor 488 (green), co-localizes with the fusion protein. DNA is detected with Hoechst dye (blue). Scale bar is 15 µm. (B) Following SDS-PAGE separation, the protein constituents of BioID-LaA and control U2OS cells were probed with both streptavidin-HRP (top) and anti-myc (bottom). As compared to the few naturally biotinylated proteins in the control sample, there is extensive biotinylation of endogenous proteins in the BioID-LaA sample.