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

Abstract

<i>In vivo</i> 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 <i>in vitro</i> sortase reactions in a low-Ca²⁺ environment. Feeding SrtA_7m-expressing C. elegans with small peptide-based probes such as (Gly)₃- biotin or (Gly)₃-fluorophores enables <i>in vivo</i> target protein modification. SrtA_7m also catalyzes the circularization of suitably modified linear target proteins <i>in vivo</i> 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 <i>in vivo</i> 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 <i>in vivo</i>