We wish to thank all of the members of the Dinman laboratory, including Ashton Belew, Karen Jack, Sharmishtha Musalga, Sergey Sulima, Shivani Reddy, and Michael Rhodin for their help and input on this project. This work was supported by grants to AM from the American Heart Association (AHA 0630163N) and to JDD from the National Institutes of Health (5R01 GM058859-12). JAL was supported by an American Reinvestment and Recovery Act of 2009 supplement to the parent grant (3R01GM058859-11S1).

1. Preparation of a cysteine charged Sulfolink resin
<ol>
<li>Prepare Sulfolink resin (Pierce) at room temperature.</li>
<li>Place a total of 10 ml of a 50% slurry of Sulfolink coupling gel as supplied by the manufacturer in two 10 ml plastic vials, with 5 ml distributed into each vial.</li>
<li>Briefly centrifuge vials at 850 x g and carefully remove supernatants.</li>
<li>Wash the gel three times in coupling buffer (50 mM Tris, pH 8.5, 5 mM EDTA) by resuspending the beads in 5 ml, centrifuging briefly at 850 x g and ...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+highly+active+ribosomes+from+Escherichia+coli.">Rapid purification of highly active ribosomes from Escherichia coli.</a> Anal. Biochem. 105, 369-374 (1980).</li><li>Le goffic, F., Moreau, N., Chevelot, L., Langrene, S., Siegrist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+bacterial+ribosomes+using+chloramphenicol+and+erythromycin+columns.">Purification of bacterial ribosomes using chloramphenicol and erythromycin columns.</a> Biochimie. 62, 69-77 (1980).</li><li>Meskauskas, A., Petrov, A. N., Dinman, J. 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G., Xu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chromatography+system+to+isolate+active+ribosomes+from+pathogenic+bacteria.">A novel chromatography system to isolate active ribosomes from pathogenic bacteria.</a> RNA. 14, 188-195 (2008).</li><li>Leshin, J. A., Rakauskaite, R., Dinman, J. D., Meskauskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+purity,+activity+and+structural+integrity+of+yeast+ribosomes+purified+using+a+general+chromatographic+method.">Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method.</a> RNA. Biol. 7, 354-360 (2010).</li><li>Triana, F., Nierhaus, K. 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Ribosome purification protocols basically involve lysing cells, harvesting a cytosolic fraction from a low speed spin, and then pelleting ribosomes by high speed centrifugation 1. While a few novel methods have been used for purifying bacterial ribosomes, the same had not been true for eukaryotes 2-4. Although additional steps have been added along the years, e.g. salt washes, and glycerol cushions (e.g. see 5), biochemical and structural studies of yeast ribosomes have been hampered by ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Sulfolink Coupling resin</td>
			<td>Pierce</td>
			<td>20402</td>
		</tr>
		<tr>
			<td>Mini bead-beater 16</td>
			<td>Biospec</td>
			<td>607</td>
		</tr>
		<tr>
			<td>Optima Max E ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Legend RT</td>
			<td>Sorvall</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.5 mm Glass beads</td>
			<td>Biospec</td>
			<td>11079105</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich</td>
			<td>252859</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>43815</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>54459</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>60128</td>
		</tr>
		<tr>
			<td>Mg(OAc)2</td>
			<td>Sigma-Aldrich</td>
			<td>M5661</td>
		</tr>
		<tr>
			<td>KOH	</td>
			<td>Sigma-Aldrich</td>
			<td>221473</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P7255</td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Fermentas</td>
			<td>R0461</td>
		</tr>
	</tbody>
</table>

chromatographic purification of highly active yeast ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. 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.

Watch this Scientific Journal Video about Chromatographic Purification of Highly Active Yeast Ribosomes at JoVE.com

Chromatographic Purification of Highly Active Yeast Ribosomes

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

chromatographic-purification-of-highly-active-yeast-ribosomes

Research

JoVE Journal

Biology

16.6K visualizações.  University of Maryland. Contaminação de preparações dos ribossomos eucarióticos purificado por métodos tradicionais de co purificação de proteases e nucleases negativamente impactos sobre a jusante análises bioquímicas e estruturais. Um método de purificação cromatográfica rápida e simples é usado para resolver esse problema usando os ribossomos de levedura como um sistema modelo.