Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

We report a strategy for chemical protein modification by using tyrosinase enzymes to oxidize exposed tyrosine residues on protein N or C-termini. We explore the chemical space for coupling partners in this reaction and find combinations that can proceed in near quantitative conversion. This strategy is used to conjugate a dye onto a Trastuzumab antibody fragment and a Protein L fragment and demonstrate that these constructs can be used as immunostaining reagents

Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues

Site-selective protein-protein coupling has long been a goal of chemical biology research. In recent years, that goal has been realized to varying degrees through a number of techniques, including the use of tyrosinase-based coupling strategies. Early publications utilizing tyrosinase from Agaricus bisporus showed the potential to convert tyrosine residues into ortho-quinone functional groups, but this enzyme is challenging to produce recombinantly and suffers from some limitations in substrate scope. Initial screens of several tyrosinase candidates revealed that the tyrosinase from Bacillus megaterium (megaTYR) as an enzyme that possesses a broad substrate tolerance. We use the expanded substrate preference as a starting point for protein design experiments and show that single point mutants of megaTYR are capable of activating tyrosine residues in various sequence contexts. We leverage this new tool to enable the construction of protein trimers via a charge-directed sequential activation of tyrosine residues (CDSAT)

Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues

Site-selective protein-protein coupling has long been a goal of chemical biology research. In recent years, that goal has been realized to varying degrees through a number of techniques, including the use of tyrosinase-based coupling strategies. Early publications utilizing tyrosinase from Agaricus bisporus(abTYR) showed the potential to convert tyrosine residues into ortho-quinone functional groups, but this enzyme is challenging to produce recombinantly and suffers from some limitations in substrate scope. Initial screens of several tyrosinase candidates revealed that the tyrosinase from Bacillus megaterium (megaTYR) is an enzyme that possesses a broad substrate tolerance. We use the expanded substrate preference as a starting point for protein design experiments and show that single point mutants of megaTYR are capable of activating tyrosine residues in various sequence contexts. We leverage this new tool to enable the construction of protein trimers via a charge-directed sequential activation of tyrosine residues (CDSAT)

Site-Specific Generation of Protein-Protein Conjugates Using Native Amino Acids

Chimeric protein-protein conjugates provide platforms for immunotherapy, targeted drug delivery, and vaccine development. However, many desirable constructs cannot be produced through direct expression, and the targeted coupling of two proteins is chemically challenging. Here we present a new approach for the rapid and site-specific coupling of proteins using native amino acids. Tyrosinase oxidizes exposed tyrosine residues on polypeptides, generating ortho-quinones that react rapidly with strategically placed cysteine residues in other proteins. This approach was used to modify CRISPR-Cas9 and other substrates with small molecules, peptides and even intact proteins. The conjugation of cell penetrating peptides to CRISPR-Cas9 was shown to increase cellular genome editing efficiency by 20-fold relative to unmodified Cas9. This technology represents a new paradigm for biomolecular coupling, and paves the way to an unprecedented range of multifunctional bioconjugates.</p

Site-Specific Bioconjugation through Enzyme-Catalyzed Tyrosine-Cysteine Bond Formation.

The synthesis of protein-protein and protein-peptide conjugates is an important capability for producing vaccines, immunotherapeutics, and targeted delivery agents. Herein we show that the enzyme tyrosinase is capable of oxidizing exposed tyrosine residues into o-quinones that react rapidly with cysteine residues on target proteins. This coupling reaction occurs under mild aerobic conditions and has the rare ability to join full-size proteins in under 2 h. The utility of the approach is demonstrated for the attachment of cationic peptides to enhance the cellular delivery of CRISPR-Cas9 20-fold and for the coupling of reporter proteins to a cancer-targeting antibody fragment without loss of its cell-specific binding ability. The broad applicability of this technique provides a new building block approach for the synthesis of protein chimeras

Site-Specific Bioconjugation through Enzyme-Catalyzed Tyrosine-Cysteine Bond Formation.

The synthesis of protein-protein and protein-peptide conjugates is an important capability for producing vaccines, immunotherapeutics, and targeted delivery agents. Herein we show that the enzyme tyrosinase is capable of oxidizing exposed tyrosine residues into o-quinones that react rapidly with cysteine residues on target proteins. This coupling reaction occurs under mild aerobic conditions and has the rare ability to join full-size proteins in under 2 h. The utility of the approach is demonstrated for the attachment of cationic peptides to enhance the cellular delivery of CRISPR-Cas9 20-fold and for the coupling of reporter proteins to a cancer-targeting antibody fragment without loss of its cell-specific binding ability. The broad applicability of this technique provides a new building block approach for the synthesis of protein chimeras