Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation

Abstract

Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five-amino-acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. © 2017 by John Wiley & Sons, Inc.

Keywords: chemoenzymatic labeling; site-specific labeling; sortase; transpeptidation.

Figure 15.3.1

Sortase-catalyzed transpeptidation. Wild-type and engineered variants of sortase A from Staphylococcus aureus recognize substrates containing an LPXTG motif. The active site cysteine of sortase A cleaves between the threonine and glycine residues to generate a thioester-linked acyl enzyme intermediate. This intermediate is then attacked by an oligoglycine nucleophile, which releases the sortase enzyme and generates a site-specific ligation product linked via a native amide bond. This reaction can be harnessed to generate proteins site-specifically labeled at the N- or C-terminus.

Figure 15.3.2

Site-specific labeling of target proteins using sortase-catalyzed transpeptidation. (A) Reaction scheme for installing modifications at the protein C-terminus. Target proteins contain the requisite sortase substrate motif (e.g., LPETGG) separated from the body of the protein with an optional GGGGS linker. The sortase cleavage site is followed by an optional tag, such as His₆, to assist with reaction monitoring and labeled protein purification. For C-terminal labeling, the target protein is paired with an oligoglycine nucleophile tethered to the desired modification. (B) Reaction scheme for installing modifications at the protein N-terminus. Target proteins serve as the reaction nucleophile, and contain one or more N-terminal glycines. Protein targets are paired with synthetic peptides containing the sortase recognition site (e.g., LPETGG) and the modification of interest. (C) Representative examples of peptide probes compatible with sortase-mediated C-terminal (left) or N-terminal (right) labeling.