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      Efficient proximity labeling in living cells and organisms with TurboID

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          Abstract

          Protein interaction networks and protein compartmentalization underlie all signaling and regulatory processes in cells. Enzyme-catalyzed proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, current PL methods require over 18 hour labeling times or utilize chemicals with limited cell permeability or high toxicity. We used yeast display-based directed evolution to engineer two promiscuous mutants of biotin ligase, TurboID and miniTurbo, which catalyze PL with much greater efficiency than BioID or BioID2, and enable 10-minute PL in cells with non-toxic and easily deliverable biotin. Furthermore, TurboID extends biotin-based PL to flies and worms.

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          Most cited references45

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          Gene Ontology: tool for the unification of biology

          Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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            Directed evolution of APEX2 for electron microscopy and proteomics

            APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1.
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              An improved smaller biotin ligase for BioID proximity labeling

              A smaller promiscuous biotin ligase for proximity biotinylation called BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, exhibits enhanced labeling of proximate proteins, and demonstrates the use of a flexible linker to modulate the biotin-labeling radius.
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                Author and article information

                Journal
                9604648
                20305
                Nat Biotechnol
                Nat. Biotechnol.
                Nature biotechnology
                1087-0156
                1546-1696
                12 July 2018
                20 August 2018
                October 2018
                20 February 2019
                : 36
                : 9
                : 880-887
                Affiliations
                [1 ]Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                [2 ]Departments of Genetics, Stanford University, Stanford, California, USA
                [3 ]Departments of Chemistry, Stanford University, Stanford, California, USA
                [4 ]Department of Biology, Stanford University, Stanford, California, USA
                [5 ]Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
                [6 ]Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA
                [7 ]Howard Hughes Medical Institute, Boston, Massachusetts, USA
                [8 ]Chan Zuckerberg Biohub, San Francisco, California, USA
                Author notes
                [* ]Corresponding author, ayting@ 123456stanford.edu
                Article
                NIHMS979612
                10.1038/nbt.4201
                6126969
                30125270
                3fd0ab3f-082b-43b9-bdd5-99a2c751cde4

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                Biotechnology
                Biotechnology

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