sortasemediated

Sortase-mediated ligation is a bioconjugation technique that utilizes bacterial transpeptidase enzymes to join biomolecules in a site-selective manner under mild, aqueous conditions. The most widely used enzyme is Sortase A from Staphylococcus aureus. The method enables covalent linkage of a substrate bearing an LPXTG recognition motif at its C-terminus to a second substrate bearing an N-terminal oligoglycine sequence, producing a defined product.

Mechanism: Sortase A cleaves between the threonine and glycine in LPXTG, forming a thioacyl enzyme intermediate. The N-terminal amino group of the donor substrate (often G5 or another glycine-containing peptide) acts as a nucleophile, attacking the intermediate and forming a new amide bond. The reaction can be performed in vitro or in living cells, and it generally requires calcium ions for the wild-type enzyme, though calcium-independent variants exist.

Variants and scope: Engineered sortases with altered substrate specificities extend the range of ligatable motifs beyond LPXTG and improve kinetics. Other sortases and mutant forms enable alternative ligation schemes and faster turnover.

Applications: SML is used for protein labeling, construction of fusion proteins, site-specific antibody–drug conjugates, immobilization on surfaces or nanoparticles, and assembly of multi-domain protein architectures. It offers site specificity, mild conditions, and compatibility with aqueous media, though it relies on accessible recognition motifs and can be limited by competing hydrolysis or reversibility under certain conditions.

Terminology: In some literature, the approach is referred to as sortase-mediated ligation or simply sortase-mediated conjugation, and the term sortasemediated may be used as a shorthand for this technique.