19 August 2021

All fundamental processes inside cells rely on protein-protein interactions. Understanding biological processes therefore requires a thorough and comprehensive understanding of protein-protein interactions. Proximity biotinylation recently emerged as a powerful interaction proteomics technology, that can be used to identify direct and indirect interactions between proteins in vivo.

Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells to fuse the proximity biotinylation enzyme to target genes of interest and can therefore not be applied to primary cells.

Irene Santos- Barriopedro, Guido van Mierlo and Michiel Vermeulen, theme cancer development and immune defense, overcame this bottleneck by fusing a proximity biotinylation enzyme, TurboID, to ProteinA (ProtA-Turbo). ProteinA binds with a high affinity to immunoglobulins. Thus, upon target cell permeabilization, the ProtA-TurboID enzyme can be targeted to proteins of interest using an endogenous antibody against the bait protein. Addition of exogenous biotin then triggers bait-proximal protein biotinylation.

These biotinylated bait-proximal proteins can subsequently be enriched from crude cell lysates using streptavidin conjugated beads and identified by quantitative mass spectrometry. They demonstrated this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, these experiments revealed that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells.

The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease. These results were published in Nature Communications.

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