Diverse approaches to efficient sortase-mediated protein modification

The site-specific modification of proteins is a widely used biochemical tool. However, there are many challenges associated with the development of protein modification techniques, in particular, maintaining site-specificity, reaction efficiency and versatility. Peptide ligases, chiefly the sortase enzyme, have been used to address these challenges through exploitation of their inherent specificity and engineering of both enzymes and substrates. Sortases are a class of transpeptidase enzymes that covalently attach an array of proteins to the surface of Gram-positive bacteria. Sortase A does this by recognising an LPXTG sequence within a protein and attaching it to an oligoglycine substrate on the cell surface. Despite being the subject of extensive engineering, the modification reaction, mediated by Staphylococcus aureus sortase A, remains limited by the rigid specificity of the enzyme.
In this project, variants of S. aureus sortase A with altered specificity were investigated. Those identified to show specificity to defined target sequences and low cross-reactivity towards other sortase sequences were used, alongside depsipeptide substrates, to efficiently and orthogonally N-terminally dual label a protein. A strategy to increase the efficiency of the C-terminal labelling strategy was also investigated and used in combination with the N-terminal approach for dual protein modification. As well as the orthogonal dual labelling strategies, further applications for sortase-mediated ligation such as protein polymerisation and ligation of an internal loop in a protein were explored. The project resulted in the development of novel sortase-mediated conjugation strategies, increasing the versatility of the technique and expanding the biochemical toolkit.