Improving bioorthogonal protein ubiquitylation by click reaction

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2013
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Bioorganic & Medicinal Chemistry. 2013, 21(12), pp. 3430-3435. ISSN 0968-0896. eISSN 1464-3391. Available under: doi: 10.1016/j.bmc.2013.03.063
Zusammenfassung

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.

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540 Chemie
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Ubiquitin, Unnatural amino acids, Click reaction, DNA polymerase beta, Protein conjugation
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ISO 690SCHNEIDER, Daniel, Tatjana SCHNEIDER, Daniel RÖSNER, Martin SCHEFFNER, Andreas MARX, 2013. Improving bioorthogonal protein ubiquitylation by click reaction. In: Bioorganic & Medicinal Chemistry. 2013, 21(12), pp. 3430-3435. ISSN 0968-0896. eISSN 1464-3391. Available under: doi: 10.1016/j.bmc.2013.03.063
BibTex
@article{Schneider2013-06-15Impro-23461,
  year={2013},
  doi={10.1016/j.bmc.2013.03.063},
  title={Improving bioorthogonal protein ubiquitylation by click reaction},
  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}
}
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