Isopeptide Ligations Catalyzed by Streptococcus Suis Sortase A

Chemically modified proteins are critical components of modern therapeutics and basic research. To generate non-natural protein derivatives, bacterial sortase enzymes have been effective due to their ability to catalyze selective ligations between protein targets and functional groups that are uncommon in nature. Thus far, the enzymatic approach using sortase has been limited to modifications at the termini of peptide chains. Here we describe efforts to develop a sortase-mediated strategy for the formation of isopeptide bonds at the side chains of internal lysine residues. To this end, we have identified a sortase A homolog from Streptococcus suis (SrtAsuis) that is capable of generating isopeptide linkages at levels far superior to sortases that are typically used for protein modification. As confirmed by RP-HPLC, ESI-MS, and MS/MS analysis, we have succeeded in generating isopeptide linkages with model peptide substrates and larger biological targets by utilizing SrtAsuis. Additionally, we have optimized these reactions by using Ni(II) ions to sequester reaction by-products in order to drive the equilibrium toward the desired products. Overall, SrtAsuisis uniquely effective at catalyzing isopeptide bond formation and shows promise for future applications such as generating protein oligomers linked together by isopeptide bond, and the formation of protein therapeutics modified at lysine residues.