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.