Sortase‐Mediated Multi‐Fragment Assemblies by Ligation Site Switching

Abstract

Sortase‐mediated ligation (SML) is a powerful tool of protein chemistry allowing the ligation of peptides containing LPxTG sorting motifs and N‐terminal glycine nucleophiles. The installation of a sorting motif into the product prohibits the assembly of multiple fragments by SML. Here we report multi‐fragment SML based on switchable sortase substrates. Substitution of the Leu residue by disulfide‐containing Cys(StBu) results in active sorting motifs, which are inactivatable by reduction. In combination with a photo‐protected N‐Gly nucleophile, multi‐fragment SML is enabled by repetitive cycles of SML and ligation site switching. The feasibility of this approach was demonstrated by a proof‐of‐concept four‐fragment ligation, the assembly of peptide probes for bivalent chromatin binding proteins and oligomerization of peptide antigens. Biochemical and immuno‐assays demonstrated functionality of these probes rendering them promising tools for immunology and chromatin biochemistry.