Chemo-enzymatic three-fragment assembly of semisynthetic proteins

Here, we report the development of a method for three-fragment assemblies of semisynthetic proteins by combining sortase-mediated ligation with site-specific bioconjugation catalyzed by the 4'-phosphopantetheine transferase Sfp. This method enables the introduction of synthetic peptides into central regions of proteins without the need to purify intermediates. The assembled proteins are linked at the N-terminal junction with a 4'-phosphopantetheine moiety and with a peptide bond at the C-terminal ligation site. We have demonstrated the applicability of this method by assembling a semisynthetic model protein derived from fluorescence resonance energy transfer-based reporters from three fragments in a one-pot reaction.