Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity.
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.
Antos
John M
JM
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Chew
Guo-Liang
GL
Guimaraes
Carla P
CP
Yoder
Nicholas C
NC
Grotenbreg
Gijsbert M
GM
Popp
Maximilian Wei-Lin
MW
Ploegh
Hidde L
HL
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
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D010455:Peptides; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D000409:Alanine