Chromatographic Purification of Highly Active Yeast Ribosomes

Summary

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.

Abstract

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.

Protocol

1. Preparation of a cysteine charged Sulfolink resin

1. Prepare Sulfolink resin (Pierce) at room temperature.
2. 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.
3. Briefly centrifuge vials at 850 x g and carefully remove supernatants.
4. 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 removing the supernatant by pipette.
5. Add five ml of a 50 mM solution of L-cysteine in coupling buffer to each tube and mix the slurry for 1 h....

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sulfolink Coupling resin	Pierce, Thermo Scientific	20402
Mini bead-beater 16	Biospec Products	607
Optima Max E ultracentrifuge	Beckman Coulter Inc.
Legend RT	Sorvall, Thermo Scientific
0.5 mm Glass beads	Biospec Products	11079105
Tris	Sigma-Aldrich	252859
EDTA	Sigma-Aldrich	E9884
DTT	Sigma-Aldrich	43815
HEPES	Sigma-Aldrich	54459
NH4Cl	Sigma-Aldrich	A9434
KCl	Sigma-Aldrich	60128
Mg(OAc)2	Sigma-Aldrich	M5661
KOH	Sigma-Aldrich	221473
Glycerol	Sigma-Aldrich	G5516
Puromycin	Sigma-Aldrich	P7255
GTP	Fermentas	R0461