title: Improving bioorthogonal protein ubiquitylation by click reaction.
abstract: Posttranslational modification of proteins with ubiquitin (ubiquitylation) regulates numerous cellular processes. Besides functioning as a signal for proteasomal degradation, ubiquitylation has also non-proteolytic functions by altering the biochemical properties of the modified protein. To investigate the effect(s) of ubiquitylation on the properties of a protein, sufficient amounts of homogenously and well-defined ubiquitylated proteins are required. Here, we report on the elaboration of a method for the generation of high amounts of site-specifically mono-ubiquitylated proteins. Firstly, a one-step affinity purification scheme was developed for ubiquitin containing the unnatural amino acid azidohomoalanine at the C-terminal position. This ubiquitin was conjugated in a click reaction to recombinant DNA polymerase β, equipped with an alkyne function at a distinct position. Secondly, addition of defined amounts of SDS to the reaction significantly improved product formation. With these two technical improvements, we have developed a straight forward procedure for the efficient generation of site-specifically ubiquitylated proteins that can be used to study the effect(s) of ubiquitylation on the activities/properties of a protein.
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, 78457 Konstanz, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Schneider
authors.forename: Tatjana
authors.initials: T
authors.lastname: Schneider
authors.forename: Daniel
authors.initials: D
authors.lastname: Rösner
authors.forename: Martin
authors.initials: M
authors.lastname: Scheffner
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
mesh_terms: D057930:Click Chemistry; D006801:Humans; D008958:Models, Molecular; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D011506:Proteins; D025801:Ubiquitin