PT  - JOURNAL ARTICLE
AU  - Tess C. Branon
AU  - Justin A. Bosch
AU  - Ariana D. Sanchez
AU  - Namrata D. Udeshi
AU  - Tanya Svinkina
AU  - Steven A. Carr
AU  - Jessica L. Feldman
AU  - Norbert Perrimon
AU  - Alice Y. Ting
TI  - Directed evolution of TurboID for efficient proximity labeling in living cells and organisms
AID  - 10.1101/196980
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 196980
4099  - http://biorxiv.org/content/early/2017/10/02/196980.short
4100  - http://biorxiv.org/content/early/2017/10/02/196980.full
AB  - 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.