title: Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity.
abstract: The unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtA(strep) is used to label the protein's C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtA(staph) and LPXT derivatives. The generality of N-terminal labeling with SrtA(staph) is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.
authors.affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Antos
authors.forename: Guo-Liang
authors.initials: GL
authors.lastname: Chew
authors.forename: Carla P
authors.initials: CP
authors.lastname: Guimaraes
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Yoder
authors.forename: Gijsbert M
authors.initials: GM
authors.lastname: Grotenbreg
authors.forename: Maximilian Wei-Lin
authors.initials: MW
authors.lastname: Popp
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000201:enzymology; D013297:Streptococcus pyogenes / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D010455:Peptides; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D000409:Alanine