title: Active yeast ribosome preparation using monolithic anion exchange chromatography.
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.
authors.affiliation: a Laboratory on the Mechanism and Regulation of Protein Synthesis, The Eunice Kennedy Shriver National Institute of Child Health and Human Development , Bethesda , MD , USA.
authors.forename: Antonio M
authors.initials: AM
authors.lastname: Munoz
authors.affiliation: a Laboratory on the Mechanism and Regulation of Protein Synthesis, The Eunice Kennedy Shriver National Institute of Child Health and Human Development , Bethesda , MD , USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Yourik
authors.affiliation: b Ra Pharmaceuticals , Cambridge , MA , USA.
authors.forename: Vaishnavi
authors.initials: V
authors.lastname: Rajagopal
authors.affiliation: a Laboratory on the Mechanism and Regulation of Protein Synthesis, The Eunice Kennedy Shriver National Institute of Child Health and Human Development , Bethesda , MD , USA.
authors.forename: Jagpreet S
authors.initials: JS
authors.lastname: Nanda
authors.affiliation: a Laboratory on the Mechanism and Regulation of Protein Synthesis, The Eunice Kennedy Shriver National Institute of Child Health and Human Development , Bethesda , MD , USA.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Lorsch
authors.affiliation: a Laboratory on the Mechanism and Regulation of Protein Synthesis, The Eunice Kennedy Shriver National Institute of Child Health and Human Development , Bethesda , MD , USA.|c Department of Biological Sciences , The State University of New York at Buffalo , Buffalo , NY , USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Walker
mesh_terms: D000837:Anion Exchange Resins / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D066298:In Vitro Techniques; D011189:Potassium Chloride / Q000737:chemistry; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism*
chemical_list: D000837:Anion Exchange Resins; D011189:Potassium Chloride
keywords: Eukaryotic translation; in vitro translation; ribosome; ribosome purification; yeast