Search

Although viruses have been successfully repurposed as vaccines, antibiotics, and anticancer therapeutics, they also raise concerns regarding genome integration and immunogenicity. Virus-like particles and non-viral protein cages represent a potentially safer alternative but often lack desired functionality. Here, we investigated the utility of a new enzymatic bioconjugation method, called lysine acylation using conjugating enzymes (LACE), to chemoenzymatically modify protein cages. We equipped two structurally distinct protein capsules with a LACE-reactive peptide tag and demonstrated their modification with diverse ligands. This modular approach combines the advantages of chemical conjugation and genetic fusion and allows for site-specific modification with recombinant proteins as well as synthetic peptides with facile control of the extent of labeling. This strategy has the potential to fine-tune protein containers of different shape and size by providing them with new properties that go beyond their biologically native functions.

Enzymes are powerful tools for protein labelling due to their specificity and mild reaction conditions. Many protocols, however, are restricted to modifications at protein termini, rely on non-peptidic metabolites or require large recognition domains. Here we report a chemoenzymatic method, which we call lysine acylation using conjugating enzymes (LACE), to site-specifically modify folded proteins at internal lysine residues. LACE relies on a minimal genetically encoded tag (four residues) recognized by the E2 small ubiquitin-like modifier-conjugating enzyme Ubc9, and peptide or protein thioesters. Together, this approach obviates the need for E1 and E3 enzymes, enabling isopeptide formation with just Ubc9 in a programmable manner. We demonstrate the utility of LACE by the site-specific attachment of biochemical probes, one-pot dual-labelling in combination with sortase, and the conjugation of wild-type ubiquitin and ISG15 to recombinant proteins. A chemoenzymatic method to site-specifically conjugate peptide and protein thioesters to folded proteins at lysine residues has been developed. The method uses a genetically encoded four-residue tag that is recognized by the E2 SUMO-conjugating enzyme Ubc9. This approach enables isopeptide formation with just Ubc9 in a programmable manner and obviates the need for E1 and E3 enzymes.