RT Journal Article
SR Electronic
T1 Directed evolution of TurboID for efficient proximity labeling in living cells and organisms
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 196980
DO 10.1101/196980
A1 Tess C. Branon
A1 Justin A. Bosch
A1 Ariana D. Sanchez
A1 Namrata D. Udeshi
A1 Tanya Svinkina
A1 Steven A. Carr
A1 Jessica L. Feldman
A1 Norbert Perrimon
A1 Alice Y. Ting
YR 2017
UL http://biorxiv.org/content/early/2017/10/02/196980.abstract
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.