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

Abstract

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.

Parallel Automated Flow Synthesis of Covalent Protein Complexes That Can Inhibit MYC-Driven Transcription

“…Preparing homogeneous, stable, well-defined protein–protein conjugates can be a challenge. 28 − 30 Chemical synthesis approaches to generate covalently linked multimeric proteins have been mainly focused on preparing ubiquitinylated or sumoylated proteins. 31 − 38 These strategies relied on chemical ligation or chemoenzymatic workflows, requiring the incorporation of unnatural amino acids or an engineered recognition sequence, respectively.…”

Tyrosine bioconjugation – an emergent alternative

“…Although sometimes a reason for side reactions, the attack of nucleophilic amino acids on o -quinones can be exploited to prepare site-specific protein-peptide or protein–protein conjugates. This was demonstrated recently by Lobba et al 55 with the addition of surface-exposed cysteine thiols of one protein to o -quinones obtained by tyrosinase catalysed oxidation of tyrosine on another protein or peptide. The attack occurs at the 5-position of the o -quinone (numbered from the amino acid branching point) and the resulting product is predominantly in its catechol form.…”

“… 51 The cysteine/ o -quinone linkage formed was shown to be more stable compared to an analogous thiosuccinimide linkage after incubation in human blood serum for 7 days. 55 The method was used to attach a variety of proteins and peptides to other proteins. For example, GFP bearing a tyrosine tag was conjugated successfully to Cas9.…”

Integrating Biomaterials and Genome Editing Approaches to Advance Biomedical Science

“…In addition, some CPPs help particles cross the blood-brain barrier ( 52), including delivery of CRISPR components for genome editing across the blood-brain barrier (53). CPPs have been conjugated to the Cas9 protein and gRNA to modulate cellular delivery (54), and the Cas9 protein has been directly modified with peptides or small molecules (36,55) to aid in targeting, delivery, or stability. Other peptides can be used to control intercellular trafficking, such as the microtubule-associated sequence and nuclear localization signal, which supports transport along the cellular microtubule network for intracellular trafficking to the nucleus (56).…”