Site-Specific C-Terminal Labeling of Recombinant Proteins with Proximity-Based Sortase-Mediated Ligation (PBSL)

S. aureus sortase A (SrtA), a calcium-dependent transpeptidase, is frequently employed to site-specifically label the C-terminus of recombinant proteins bearing an LPXTG SrtA recognition motif. Unfortunately, SrtA suffers from low turnover rates, resulting in poor ligation efficiencies even with optimized reaction conditions. In this chapter, we describe proximity-based sortase-mediated ligation (PBSL), which uses the SpyTag-SpyCatcher peptide-protein pair to link SrtA to target proteins and dramatically improves reaction rate and ligation efficiency.