Reprogramming the specificity of sortase enzymes

Significance The modification of proteins has proven to be crucial for many research and industrial applications. The bacterial transpeptidase Staphylococcus aureus sortase A (SrtA) is a powerful tool for conjugating proteins to a wide variety of molecules, but is limited to those proteins containing the five amino acid LPXTG sorting motif. Here we present a system for the directed evolution of reprogrammed SrtA variants that accept proteins with altered sorting motifs. We used this system to evolve two families of orthogonal sortases that recognize LAXTG and LPXSG motifs. These evolved sortases enabled the synthesis of triblock fluorophore–protein–PEG conjugates, the covalent and orthogonal functionalization of multiple proteins onto surfaces, and the manipulation of endogenous human proteins lacking a native LPXTG motif.