Hepta-Mutant <i>Staphylococcus aureus</i> Sortase A (SrtA_7m) as a Tool for <i>in Vivo</i> Protein Labeling in <i>Caenorhabditis elegans</i>

Abstract

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 (SrtA 7m ) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca 2+ environment. Feeding SrtA 7m -expressing C. elegans with small peptide-based probes such as (Gly) 3 - biotin or (Gly) 3 -fluorophores enables in vivo target protein modification. SrtA 7m 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 .