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Title: Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy
Abstract Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H2O2. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O2instead of toxic H2O2, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.  more » « less
Award ID(s):
2014862 1707356
PAR ID:
10623752
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
Publisher / Repository:
bioRxiv
Date Published:
Format(s):
Medium: X
Institution:
bioRxiv
Sponsoring Org:
National Science Foundation
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