Directed evolution of sortase A mutants with altered substrate selectivity profiles

The ligation of two polypeptides in a chemoselective manner by the bacterial transpeptidase sortase A has become a versatile tool for protein engineering approaches. When sortase-mediated ligation is used for protein semisynthesis, up to four mutations resulting from the strict requirement of the LPxTG sorting motif are introduced into the target protein. Here we report the directed evolution of a mutant sortase A that possesses broad substrate selectivity. A phage-display screen of a mutant sortase library that was randomized in the substrate recognition loop was used to isolate this mutant. The altered substrate selectivity represents a gain-of-function that was exploited for the traceless semisynthesis of histone H3. Our report is a decisive step toward a platform of engineered sortases with distinct ligation properties that will conceivably allow for more versatile assemblies of modified proteins in biotechnological approaches.