Improved Sortase A-catalyzed transpeptidation by selective electrostatically-assisted aminolysis trapping.

Sortase A (SrtA) and some variants thereof have been used in a wide range of applications including fluorescent labeling, protein cyclization and immobilization due to their mild reaction conditions and high specificity. However, SrtA-catalyzed transpeptidation suffers the inherent limitation of being a reversible process which therefore requires an excessive amount of substrate to drive the reaction towards completion. Such an issue can prove prohibitive, especially in the case of high value-added substrate molecules. In this context, we disclose a novel substrate engineering strategy that enables to achieve high levels of SrtA-mediated protein modification with nearly stoichiometric amounts of substrate. Extension of the consensus sorting motif LPXTG with a positively charged peptidic module allows to achieve sequence-specific removal of by-products by applying the concept of electrostatic-assisted aminolysis reaction recently described by our group. The reaction equilibrium is driven to favor product formation, thereby greatly improving reaction yield.