Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificity

Abstract

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.