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.

An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

“…Biotinylated and fluorescent probes were generated using Sortase A as described here 24 and here 25 . In brief, nanobodies were site-specifically biotinylated on the C-terminus using Sortase A 5M.…”

In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking

“…Importantly, we have shown here that the separated domains of the pilin, one containing the pilin motif and the other containing the sorting motif, could be ligated efficiently to produce a di-polypeptide conjugate containing the Lys-Thr isopeptide bond. This provides a powerful protein ligation platform for engineering designer proteins that is mechanistically different from the "sortagging" technology developed with the archetypal S. aureus sortase which normally functions to cross-link surface proteins to the bacterial cell wall but does not polymerize proteins (41,42). We envision that the surface display of protein polymers, protein labeling of living cells, and protein ligation are a few examples of many potential biotechnological and biological applications of this enzyme.…”

Synthetic Strategies for Artificial Lipidation of Functional Proteins

“…The resultant thioester intermediate is then subjected to nucleophilic attack by the amino terminus of an oligoglycine in the cell wall peptidoglycan, forming a new reversible amide bond. SrtA tolerantly recognizes the LPXTG motif at the C‐terminal of recombinant proteins, and therefore a protein fused with LPXTG at the C‐terminal can be labeled with synthetic nucleophiles containing a pentaglycine at the N‐terminal by SrtA catalysis in a site‐specific manner (Figure a) …”