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.

Scheme 1. C-terminal Labeling Using SrtA_staph

Figure 1

N-terminal labeling using SrtA_staph. (a) SrtA_staph catalyzes a transacylation reaction using labeled LPRT methyl esters as substrates. The labeled LPRT fragment is transferred to proteins containing N-terminal glycines in a site-specific fashion. (b) FITC (1) and biotin (2) LPRT methyl esters for N-terminal transacylation. (c) CtxB derivatives (50 μM) were treated with 500 μM 1 and 50 μM SrtA_staph for 2 h at 37 °C in 50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM CaCl₂. Reactions were analyzed by SDS-PAGE with visualization by coomassie staining and fluorescent gel scanning.

Figure 2

Site-specific N- and C-terminal labeling using multiple sortases. (a) Tetramethylrhodamine-labeled dialanine nucleophile (3) for SrtA_strep-mediated transpeptidation. (b) Strategy for the installation of discrete labels at both termini of the same protein using Δ59-SrtA_staph and SrtA_strep. (c, d) SDS-PAGE characterization with fluorescent gel scanning of dual-labeled (c) eGFP and (d) UCHL3.