title: Site-specific N-terminal labeling of proteins using sortase-mediated reactions.
abstract: This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPXTG (for Streptococcus aureus sortase A) or LPXTA (for Staphylococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Theile
authors.forename: Martin D
authors.initials: MD
authors.lastname: Witte
authors.forename: Annet E M
authors.initials: AE
authors.lastname: Blom
authors.forename: Lenka
authors.initials: L
authors.lastname: Kundrat
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
authors.forename: Carla P
authors.initials: CP
authors.lastname: Guimaraes
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D007700:Kinetics; D015202:Protein Engineering / Q000379:methods*; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases