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Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m) as a Tool for in Vivo Protein Labeling in Caenorhabditis elegans

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† Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, United States
‡ Department of Biology, Massachusetts Institute of Technology (MIT), 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
*E-mail: [email protected]
*E-mail: [email protected]
Cite this: ACS Chem. Biol. 2017, 12, 3, 664–673
Publication Date (Web):January 18, 2017
https://doi.org/10.1021/acschembio.6b00998
Copyright © 2017 American Chemical Society
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    Abstract

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    In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA7m) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca2+ environment. Feeding SrtA7m-expressing C. elegans with small peptide-based probes such as (Gly)3- biotin or (Gly)3-fluorophores enables in vivo target protein modification. SrtA7m also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00998.

    • Figures showing immunoblot analysis, SrtA7m-HA expression, protein labeling, small nucleophile reaction substrates, and inducible protein degradation. (PDF)

    • A table showing C. elegans proteins containing an LPXTG motif. (XLSX)

    • A table showing hypothetical fusion proteins. (XLSX)

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