Improving bioorthogonal protein ubiquitylation by click reaction

Bioorg Med Chem. 2013 Jun 15;21(12):3430-5. doi: 10.1016/j.bmc.2013.03.063. Epub 2013 Apr 3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Click Chemistry*
  • Humans
  • Models, Molecular
  • Proteins / chemistry
  • Proteins / metabolism*
  • Ubiquitin / chemistry
  • Ubiquitin / metabolism*
  • Ubiquitination

Substances

  • Proteins
  • Ubiquitin