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Improving bioorthogonal protein ubiquitylation by click reaction

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SCHNEIDER, Daniel, Tatjana SCHNEIDER, Daniel RÖSNER, Martin SCHEFFNER, Andreas MARX, 2013. Improving bioorthogonal protein ubiquitylation by click reaction. In: Bioorganic & Medicinal Chemistry. 21(12), pp. 3430-3435. ISSN 0968-0896. eISSN 1464-3391. Available under: doi: 10.1016/j.bmc.2013.03.063

@article{Schneider2013-06-15Impro-23461, title={Improving bioorthogonal protein ubiquitylation by click reaction}, year={2013}, doi={10.1016/j.bmc.2013.03.063}, number={12}, volume={21}, issn={0968-0896}, journal={Bioorganic & Medicinal Chemistry}, pages={3430--3435}, author={Schneider, Daniel and Schneider, Tatjana and Rösner, Daniel and Scheffner, Martin and Marx, Andreas} }

Marx, Andreas Bioorganic & Medicinal Chemistry ; 21 (2013), 12. - S. 3430-3435 Schneider, Tatjana Rösner, Daniel Marx, Andreas 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 beta, 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. terms-of-use eng 2013-06-15 2014-06-30T22:25:05Z Rösner, Daniel Schneider, Daniel Improving bioorthogonal protein ubiquitylation by click reaction Scheffner, Martin Schneider, Daniel Schneider, Tatjana Scheffner, Martin 2013-06-21T09:14:51Z

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