title: Chemo-enzymatic three-fragment assembly of semisynthetic proteins.
abstract: 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.
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tuebingen, Hoppe-Seyler-Str. 4, D-72076, Tuebingen, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Schmohl
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tuebingen, Hoppe-Seyler-Str. 4, D-72076, Tuebingen, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schwarzer
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D060326:Chemistry Techniques, Synthetic; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D011506:Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: FRET reporter; phosphopantetheine transferase; protein semisynthesis; sortase‐mediated ligation