title: Site-specific protein modification on living cells catalyzed by Sortase.
abstract: The use of enzymes is a promising approach for site-specific protein modification on living cells owing to their substrate specificity. Herein we describe a general strategy for the site-specific modification of cell surface proteins with synthetic molecules by using Sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of Sortase and an N-terminal triglycine-containing probe results in the site-specific labeling of the tagged protein. We were successful in the C-terminal-specific labeling of osteoclast differentiation factor (ODF) with a biotin- or fluorophore-containing short peptide on the living cell surface. The labeling reaction occurred efficiently in serum-containing medium, as well as serum-free medium or PBS. The labeled products were detected after incubation for 5 min. In addition, site-specific protein-protein conjugation was successfully demonstrated on a living cell surface by the Sortase-catalyzed reaction. This strategy provides a powerful tool for cell biology and cell surface engineering.
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1, Hongo, Tokyo, 113-8656, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tanaka
authors.forename: Teruyasu
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Shinya
authors.initials: S
authors.lastname: Tsukiji
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry; D002384:Catalysis; D002470:Cell Survival; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D053245:RANK Ligand / Q000737:chemistry; D013211:Staphylococcus aureus / Q000201:enzymology; D013379:Substrate Specificity; D013499:Surface Properties; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010455:Peptides; D053245:RANK Ligand; D001710:Biotin; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases