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Directed evolution of TurboID for efficient proximity labeling in living cells and organisms

Tess C Branon, Justin A Bosch, Ariana D Sanchez, Namrata D Udeshi, Tanya Svinkina, Steven A Carr, Jessica L Feldman, Norbert Perrimon, Alice Y Ting
doi: https://doi.org/10.1101/196980
Tess C Branon
Massachusetts Institute of Technology;
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Justin A Bosch
Harvard Medical School;
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Ariana D Sanchez
Stanford University;
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Namrata D Udeshi
Broad Institute of MIT and Harvard
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Tanya Svinkina
Broad Institute of MIT and Harvard
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Steven A Carr
Broad Institute of MIT and Harvard
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Jessica L Feldman
Stanford University;
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Norbert Perrimon
Harvard Medical School;
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Alice Y Ting
Stanford University;
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  • For correspondence: ayting@stanford.edu
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Abstract

Protein interaction networks and protein compartmentation underlie every signaling process and regulatory mechanism in cells. Recently, proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, the two enzymes commonly used for PL come with tradeoffs: BioID is slow, requiring tagging times of 18-24 hours, while APEX peroxidase uses substrates that have limited cell permeability and high toxicity. To address these problems, we used yeast display-based directed evolution to engineer two mutants of biotin ligase, TurboID and miniTurbo, with much greater catalytic efficiency than BioID, and the ability to carry out PL in cells in much shorter time windows (as little as 10 minutes) with non-toxic and easily deliverable biotin. In addition to shortening PL time by 100-fold and increasing PL yield in cell culture, TurboID enabled biotin-based PL in new settings, including yeast, Drosophila, and C. elegans.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted October 02, 2017.
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Directed evolution of TurboID for efficient proximity labeling in living cells and organisms
Tess C Branon, Justin A Bosch, Ariana D Sanchez, Namrata D Udeshi, Tanya Svinkina, Steven A Carr, Jessica L Feldman, Norbert Perrimon, Alice Y Ting
bioRxiv 196980; doi: https://doi.org/10.1101/196980
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Directed evolution of TurboID for efficient proximity labeling in living cells and organisms
Tess C Branon, Justin A Bosch, Ariana D Sanchez, Namrata D Udeshi, Tanya Svinkina, Steven A Carr, Jessica L Feldman, Norbert Perrimon, Alice Y Ting
bioRxiv 196980; doi: https://doi.org/10.1101/196980

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