Improving bioorthogonal protein ubiquitylation by click reaction

Schneider, Daniel; Schneider, Tatjana; Rösner, Daniel; Scheffner, Martin; Marx, Andreas

Improving bioorthogonal protein ubiquitylation by click reaction

Dateien

Schneider_234611.pdfGröße: 4.24 MBDownloads: 779

Datum

2013

Open Access-Veröffentlichung

Open Access Green

Sammlungen

Chemie
Biologie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen 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

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.

Fachgebiet (DDC)

540 Chemie

Schlagwörter

Ubiquitin, Unnatural amino acids, Click reaction, DNA polymerase beta, Protein conjugation

Zitieren

ISO 690

SCHNEIDER, 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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Universitätsbibliographie

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