Technologies

Proximity labeling has emerged as a powerful alternative to immunoprecipitation and biochemical fractionation for the proteomic analysis of macromolecular complexes, organelles, and protein interaction networks. However, current proximity labeling techniques either require toxic reagents or long labeling times that prevent the study of dynamic structures or processes. Here, we disclose two promiscuous biotin ligases that we engineered for non-toxic and efficient proximity labeling in living cells and organisms.

Researchers at Stanford have engineered two promiscuous biotin ligases for non-toxic, efficient proximity labeling (PL) in living cells and organisms. PL is a powerful technique for the proteomic analysis of macromolecular complexes, organelles or protein interaction networks. In PL, a promiscuous labeling enzyme is fused to a protein of interest or targeted to a subcellular region and upon the addition of biotin, the enzyme begins labeling nearby (within a few nanometers) endogenous proteins. Then the biotin labelled proteins are harvested and identified by mass spectrometry. Existing PL techniques are not optimal as they require either toxic regents or long labelling times that prevent the study of dynamic structures or processes. To overcome these limitations the inventors have engineered two mutants of biotin ligase, called TurboID and miniTurbo, which are both more active and efficient than existing variants. TurboID and miniTurbo enable PL with greater signal in shorter time windows under physiologically relevant conditions in live cells. This technology will expand the scope and utility of PL by enabling the study of dynamic processes in vivo and thus can provide insight into proteomic changes to aid general research and therapeutic development.

Proximity-dependent biotinylation by promiscuous biotin ligases. Ligases catalyze the formation of biotin-5’-AMP anhydride, which diffuses out of the active site to biotinylate proximal endogenous proteins.Stage of researchIn addition to shortening PL time by 100-fold and increasing PL yield in cell culture, the inventors have shown that TurboID enabled biotin-based PL in new settings including yeast, Drosophila and C. elegans .Related technologiesResearchers in the Ting lab have also developed additional technologies to detect protein-protein interactions.