Investigating enzymatic methods for preparation of protein conjugates

The ability to prepare well-defined protein conjugates is central to the industrial production of biopharmaceuticals including antibody-drug conjugates and PEGylated proteins. Sortases are transpeptidase enzymes that 'sort' and attach virulence factors to the cell wall of Gram-positive bacteria. They have been exploited for both N- and C-terminal labelling of proteins, including antibodies for construction of antibody-drug conjugates. Typically >10-fold excess of labelling reagent is required in conjunction with stoichiometric amounts of enzyme. Improvements in efficiency would make the method more useful for industrial biotechnology. We have reported very efficient methods for N-terminal labelling employing ester-linked depsipeptide substrates. In this project we test the hypothesis that methods developed for S. aureus Sortase A can be applied to orthogonal sortases with different substrate recognition sequences to enable specific multisite conjugation with different species at the N-and C-terminus of the target protein.