Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions

Abstract

Methods for site-specific modification of proteins are in high demand. Reactions that yield bioconjugates should be quantitative, site-specific, and versatile with respect to nature and size of the biological/chemical targets involved, require minimal modification of the target, display acceptable kinetics under physiological conditions, and be orthogonal to other labeling methods. Sortase-mediated transpeptidation reactions meet these criteria. Here we describe the expression and purification conditions for two orthogonal sortase A enzymes and provide the protocol that allows functionalization of any given protein at its C-terminus or for select proteins at an internal site. Sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.

GPR107, a G-protein-coupled Receptor Essential for Intoxication by Pseudomonas aeruginosa Exotoxin A, Localizes to the Golgi and Is Cleaved by Furin

“…Sortase-mediated Labeling of PE-Sortase A was expressed and purified as described previously (19,20). Synthesis of the nucleophiles G 3 K(Atto647)-RDELK, G 3 K-(biotin)-RDELK, and G 3 K-biotin were done as described previously (21).…”

“…The supernatants that contain the toxin were centrifuged again at 10,000 rpm for 2 h, filtered to further remove any remaining debris, and concentrated using the 30K Amicon concentrator (Millipore). For the toxin that carries the sortase recognition motif (PE-LPETG-His 6 ) (19,20), the concentrate was applied to a nickel-nitrilotriacetic acid column equilibrated with 50 mM Tris-HCl, 150 mM NaCl, and 10 mM imidazole, pH 8.0. The column was washed with 10 column volumes of buffer, and the protein was eluted with 50 mM Tris-HCl, 150 mM NaCl, and 500 mM imidazole, pH 8.0.…”

“…To test whether PE reaches the ER, we installed a 3ϫGly-Lys-(biotin or Atto647) extended with the RDEL sequence or 3ϫGly-Lys-bi-otin without the RDEL sequence at the C terminus of PE via a sortase-mediated transacylation reaction (Fig. 2, A and B) (19,20). Both biotinylated and fluorescently labeled PE that carry the RDEL motif are toxic, whereas PE lacking the RDEL motif is not (Fig.…”

Noninvasive Imaging of Human Immune Responses in a Human Xenograft Model of Graft-Versus-Host Disease

“…To further characterize its specificity, VHH4 was conjugated to fluorophore tags in a sortase-mediated reaction ( Fig. 1A) (20). Sortase recognizes a LPXTG motif and cleaves the bond between the threonine and glycine, forming a thioester intermediate (21).…”

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

“…Sortase is a bacterial enzyme that functions to attach cell surface proteins bearing an "LPXTG" motif to the cell wall of Gram-positive bacteria via transacylation (21,22). Sortase-catalyzed transpeptidation allows for efficient site-specific modifications under physiological conditions, with excellent specificity and near-quantitative yields (23)(24)(25). To facilitate site-specific linking of the C termini of two antibodies, the fusion partners are labeled with either an azide or a cyclooctyne (DIBAC) functional group.…”