Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method.
One of the major challenges facing researchers working with eukaryotic ribosomes lies in their lability relative to their eubacterial and archael counterparts. In particular, lysis of cells and purification of eukaryotic ribosomes by conventional differential ultracentrifugation methods exposes them for long periods of time to a wide range of co-purifying proteases and nucleases, negatively impacting their structural integrity and functionality. A chromatographic method using a cysteine charged Sulfolink resin was adapted to address these problems. This fast and simple method significantly reduces co-purifying proteolytic and nucleolytic activities, producing good yields of highly biochemically active yeast ribosomes with fewer nicks in their rRNAs. In particular, the chromatographic purification protocol significantly improved the quality of ribosomes isolated from mutant cells. This method is likely applicable to mammalian ribosomes as well. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.
Leshin
Jonathan A
JA
Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, MD, USA.
Rakauskaitė
Rasa
R
Dinman
Jonathan D
JD
Meskauskas
Arturas
A
D001665:Binding Sites; D002458:Cell Fractionation / Q000379:methods; D002845:Chromatography / Q000379:methods; D004340:Drug Contamination; D057075:Enzyme Assays; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D012335:RNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015003:Yeasts / Q000737:chemistry* / Q000166:cytology
D012335:RNA, Ribosomal; D029701:Saccharomyces cerevisiae Proteins; D012260:Ribonucleases; D010447:Peptide Hydrolases
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