Lysine acylation using conjugating enzymes for site-specific modification and ubiquitination of recombinant proteins

Raphael Hofmann, Gaku Akimoto, Thomas G. Wucherpfennig, Cathleen Zeymer, Jeffrey W. Bode

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

Abstract

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. [Figure not available: see fulltext.].

Original languageEnglish
Pages (from-to)1008-1015
Number of pages8
JournalNature Chemistry
Volume12
Issue number11
DOIs
StatePublished - 1 Nov 2020

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