Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity

John M Antos, Guo-Liang Chew, Carla P Guimaraes, Nicholas C Yoder, Gijsbert M Grotenbreg, Maximilian Wei-Lin Popp, Hidde L Ploegh
Journal of the American Chemical Society 2009 August 12, 131 (31): 10800-1
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.


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