Active yeast ribosome preparation using monolithic anion exchange chromatography

Antonio M. Munoz, Paul Yourik, Vaishnavi Rajagopal, Jagpreet S. Nanda, Jon R. Lorsch, Sarah E. Walker

Research output: Contribution to journalArticle

Abstract

In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes.

Original languageEnglish (US)
Pages (from-to)188-196
Number of pages9
JournalRNA Biology
Volume14
Issue number2
DOIs
StatePublished - Feb 1 2017
Externally publishedYes

Keywords

  • Eukaryotic translation
  • in vitro translation
  • ribosome
  • ribosome purification
  • yeast

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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  • Cite this

    Munoz, A. M., Yourik, P., Rajagopal, V., Nanda, J. S., Lorsch, J. R., & Walker, S. E. (2017). Active yeast ribosome preparation using monolithic anion exchange chromatography. RNA Biology, 14(2), 188-196. https://doi.org/10.1080/15476286.2016.1270004