Facile Generation of Protein-Protein Conjugates Using Enzymatic Oxidative Coupling Reactions - PROJECT SUMMARY/ABSTRACT Chimeric protein-protein conjugates provide a wide variety of successful platforms for immunotherapy, tar- geted drug delivery, cell biology studies, and vaccine development. However, many desirable constructs cannot be produced using genetic methods alone, and the targeted coupling of two or more proteins using chemical methods is still very challenging. In this program, a new approach will be explored for the rapid coupling of pro- teins using native amino acids. Tyrosinase enzymes will be used to oxidize solvent-exposed tyrosine residues on protein and peptide substrates to generate ortho-quinones that react rapidly with strategically placed cyste- ine residues in other proteins. Importantly, tyrosine residues that extend from the N- or C-terminal positions on proteins are oxidized readily, but internal tyrosine residues are unaffected during the reactions. The cysteine residues can be placed anywhere on the surface of the second protein target, allowing many different linkage locations to be accessed. Previous NIH-funded studies have shown that this approach can generate complex, multifunctional constructs from individual proteins in under 1 h at room temperature despite the high degree of steric interactions that are inherent in these reactions. The reaction strategy has been used to generate antibody drug conjugates, bispecific cell engagers with new geometric relationships, and multidomain protein chimeras. Due to these features, this method stands alone in its simplicity and flexibility for making complex protein con- structs, and thus it is greatly expanding the range of bioconjugates that can be accessed for biotechnology ap- plications. The first Specific Aim of the proposed research will capitalize on newly-available charge-sensitive tyrosi- nase mutants to make double- and triple-domain protein constructs. A central element of this work will be a systematically exploration the new protein-protein linkages that this chemistry can uniquely create, with the goal of developing heuristic models for linking proteins with desired geometries and flexibilities. This will be explored using a combination of experimental and computational approaches in the context of bispecific and trispecific cell engagers. The second Specific Aim will explore the ability of this chemistry to generate intramolecular crosslinks within a protein of interest, providing easy access to circular and knotted proteins that are envisioned to have greater stability and proteolysis resistance. We will do this in the context of the enzyme luciferase, with the goal of gen- erating imageable proteins that can be specifically activated in the presence of cancer-specific proteases. We will also explore this chemistry as a way to stabilize CRM197, which is a common platform for vaccine preparation. The third Specific Aim will focus on identifying new tyrosinase enzymes with desirable properties, and the engineering of these enzymes to achieve the activation of different tyrosine-containing sequences. This work will combine rational design with fitness landscaping methods to identify tyrosinase mutants with new specificities, higher stabilities, and other useful properties.
