Abstract
Gram-positive bacteria display pili whose protein components (pilins) are covalently crosslinked by pilus-specific sortase enzymes. These cysteine transpeptidase enzymes catalyze a transpeptidation reaction that joins the pilins together via lysine isopeptide bonds. The crosslinking reaction that builds the SpaA pilus in Corynebacterium diphtheriae is mediated by the SrtA sortase ((Cd)SrtA) and has been reconstituted in vitro. Here, we present a protocol that can be used to measure the kinetics of (Cd)SrtA-catalyzed crosslinking using high-performance liquid chromatography (HPLC). In principle, this biochemical procedure can be used to measure the in vitro crosslinking activity of any pilus-specific sortase.