sortaseassembled

Sortaseassembled refers to molecules, materials, or constructs that are produced through sortase-mediated ligation, a site-specific protein ligation technique based on the transpeptidase activity of sortase enzymes. In this approach, a donor substrate bearing a C-terminal motif such as LPXTG is joined to an acceptor substrate with an N-terminal glycine-rich sequence, yielding a covalent bond at a defined position.

Mechanism and scope: The most widely used enzyme is Staphylococcus aureus Sortase A (SrtA). SrtA cleaves between the threonine and glycine in the LPXTG sequence, forming a thioacyl-enzyme intermediate with a catalytic cysteine. A nucleophilic attack by an N-terminal glycine (or glycine-rich segment) on the second substrate resolves the intermediate and forms a new peptide bond, releasing the enzyme. Wild-type reactions often require calcium and proceed under mild aqueous conditions. Engineered sortases and alternative motifs have broadened substrate scope and reduced dependency on calcium.

Substrates and strategies: Typical applications involve LPXTG-tagged proteins (or peptides) and N-terminal oligoglycine motifs. This enables site-specific ligation of proteins, peptides, or small molecules, as well as cyclization of peptides or proteins by appropriately positioned donor and acceptor motifs. The method is also used for immobilization of proteins on surfaces or nanoparticles and for modular assembly of fusion constructs.

Applications and limitations: Sortaseassembled products are valued for site-specific modification, programmability, and mild reaction conditions and can be used in protein engineering, biomaterials, and biochemical labeling. Limitations include dependence on accessible motifs, potential reversibility in some contexts, variable efficiency with substrate choice, and the need for compatible partners. Engineered sortases continue to expand the range of usable substrates and reaction formats.