title: Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions.
abstract: Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Carla P
authors.initials: CP
authors.lastname: Guimaraes
authors.forename: Martin D
authors.initials: MD
authors.lastname: Witte
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Theile
authors.forename: Gunes
authors.initials: G
authors.lastname: Bozkurt
authors.forename: Lenka
authors.initials: L
authors.lastname: Kundrat
authors.forename: Annet E M
authors.initials: AE
authors.lastname: Blom
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002473:Cell Wall / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D013058:Mass Spectrometry; D015202:Protein Engineering / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases