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

Chemical methods for protein site-specific ubiquitination

“…2D ). 30 The authors named this method as lysine acylation using conjugating enzymes (LACE), which enables site-specific modifications including ubiquitination and ISGylation of recombinant proteins with just Ubc9 in the absence of E1 and E3 enzymes. According to this report, the authors found that Ubc9, a SUMO-conjugating enzyme, recognizes and modifies the lysine embedded in a consensus SUMOylation motif ΨKXD/E (Ψ is a hydrophobic residue) in a recombinant protein, yielding an isopeptide linkage.…”

“…[27][28][29] More recently, an E2 (Ubc9) mediated ubiquitination and ISGylation was reported. 30 While in vitro enzymatic generation of Ub dimers and polymers remains a very useful tool to study the different facets of ubiquitination and deubiquitination, the major limitation of the enzymatic approaches is the requirement of specific Ub ligases for a given chain linkage and a particular target protein.…”

Site-selective lysine conjugation methods and applications towards antibody–drug conjugates

“…Red star = rhodamine, blue star = coumarin. 56 the lysine-modified product was obtained within short timeframes (4-8 h). Further investigation occurred on a one-pot site-selective dual modification strategy on trastuzumab Fab.…”

Flavin-Tag: A Facile Method for Site-Specific Labeling of Proteins with a Flavin Fluorophore

“…Several enzymes have been discovered that can perform selective post-translational covalent modifications of proteins by attaching a molecular probe. Examples of such enzyme-based conjugation methods are biotin ligase (AP-tag) [5] , E2 small ubiquitin-like modifierconjugating enzyme Ubc9 (LACE-tag) [6], lipoic acid ligase (LAP-tag) [7] , AnkX [8] , transglutaminases (Qtag) [9,10] , sortase [11][12][13][14] and tubulin tyrosine ligase (Tub-tag) [15] . However, most of these methods rely on 2 (complex) synthetic fluorescent probes that are often not biocompatible.…”