Enhancing Sortase mediated transpeptidation reaction conversion via metal-peptide complexation

Sortase A from Staphylococcus aureus has grown in popularity as a robust and reliable enzyme for site-specifically modifying proteins. Recognizing and attacking the five amino acid recognition sequence (LPXTG), Sortase A forms a thioester intermediate which is susceptible to nucleophilic attack by the N-terminal amine of a glycine. During formation of the intermediate, sortase A excises the region of the peptide C-terminal to threonine. The excised fragment thus has an N-terminal glycine, and can attack the enzyme-protein intermediate, leading to reversal of the reaction. To attenuate reaction reversal, the recognition motif has been extended to LPETGGH, as GGH peptides are known to be strong chelators of metal ions. Sortase-catalyzed reactions performed using this modified recognition sequence in the presence of Ni2+ have been found to have a higher conversion to product; although little to no effect has been seen in the presence of other metal ions. To demonstrate the increase of efficiency and the potential for decreased cost, here we present the results of applying this metal chelation strategy to a range of model proteins and peptides