Facile Generation of Protein-Protein Conjugates Using Enzymatic Oxidative Coupling Reactions

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PROJECT SUMMARY/ABSTRACT Chimeric protein-protein conjugates provide a wide variety of successful platforms for immunotherapy, targeted 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 proteins using chemical methods is still very challenging. In this program, a new approach will be explored for the rapid and site- specific coupling of proteins 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 cysteine residues in other proteins. Preliminary data have confirmed 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 tyrosinase enzymes are inexpensive, can be immobilized on solid supports to facilitate removal, and require only adventitious oxygen to function. 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. Due to these features, this method stands alone in its simplicity and flexibility for making complex, multidomain constructs, and thus it will greatly expand the range of bioconjugates that can be accessed for biotechnology applications. The first Specific Aim of the proposed research will explore the attachment of cell penetrating peptides to scFv constructs as useful model proteins, with the goal of increasing their ability to cross cell membranes and access the cytoplasm. Cysteines will be introduced into several locations on the scFv surfaces, and cell entry peptides bearing exposed tyrosine residues will be attached using the tyrosinase method. The uptake and intracellular localization of these constructs will be evaluated using 96-well plate assays, light microscopy and flow cytometry. Specific Aim 2 will explore the use of this approach for producing multifunctional proteins for immunotherapy applications. Tyrosine tags will be introduced in strategic locations on a panel of biomolecules, including scFv domains and IgG antibodies. Following tyrosinase activation, the ability of drug cargo molecules to couple to these sites will be evaluated, and the asymmetric nature of the protein-protein coupling chemistry will be explored for the production of bispecific and trispecific cell engagers. The success of this Aim would provide a highly efficient way to make and screen these therapeutically valuable constructs. 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.