Protein ubiquitination occurs through formation of an isopeptide bond between 
the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate 
lysine residue. This post-translational modification, which occurs through the 
attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin 
chains, is involved in crucial cellular events such as protein degradation, 
cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin 
pathways is also implicated in the pathogenesis of several human diseases 
ranging from cancer to neurodegeneration. However, despite the undoubted 
biological importance, understanding the molecular basis of how ubiquitination 
regulates different pathways has up to now been strongly limited by the 
difficulty of producing the amounts of highly homogeneous samples that are 
needed for a structural characterization by X-ray crystallography and/or NMR. 
Here, we report on the production of milligrams of highly pure Josephin 
mono-ubiquitinated on lysine 117 through large scale in vitro enzymatic 
ubiquitination. Josephin is the catalytic domain of ataxin-3, a protein 
responsible for spinocerebellar ataxia type 3. Ataxin-3 is the first 
deubiquitinating enzyme (DUB) reported to be activated by mono-ubiquitination. 
We demonstrate that the samples produced with the described method are correctly 
folded and suitable for structural studies. The protocol allows facile selective 
labelling of the components. Our results provide an important proof-of-concept 
that may pave the way to new approaches to the in vitro study of ubiquitinated 
proteins.