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.