Updated project metadata. Protein ubiquitination orchestrates nearly all eukaryotic cellular events.1 It starts by attaching ubiquitin through isopeptide bonds to a single or multiple lysine residues of a target protein via a coordinated enzymatic reaction involving activating (E1), conjugating (E2), and ligating (E3) enzymes to form mono- or multi-mono-ubiquitinated products. There is a need for a technique that can facilitate the high-yield production of monoubiquitinated proteins. Here we present an efficient approach that fills this gap.