Site-specific protein labeling via sortase-mediated transpeptidation.
Creation of functional protein bioconjugates demands methods for attaching a diverse array of probes to target proteins with high specificity, under mild conditions. The sortase A transpeptidase enzyme from Staphylococcus aureus catalyzes the cleavage of a short 5-aa recognition sequence (LPXTG) with the concomitant formation of an amide linkage between an oligoglycine peptide and the target protein. By functionalizing the oligoglycine peptide, it is possible to incorporate reporters into target proteins in a site-specific fashion. This reaction is applicable to proteins in solution and on the living cell surface. The method described in this unit only requires incubation of the target protein, which has been engineered to contain a sortase recognition site either at the C terminus or within solvent-accessible loops, with a purified sortase enzyme and a suitably functionalized oligoglycine peptide.
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.|Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Maximilian Wei-Lin
MW
Popp
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.
John M
JM
Antos
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.|Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Hidde L
HL
Ploegh
D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D015202:Protein Engineering; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010455:Peptides; D011506:Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases