Purification of proteasomes, proteasome subcomplexes, and proteasome-associated proteins from budding yeast.

The proteasome is a highly complex, ATP-dependent protease, consisting of over 30 subunits, and dedicated mainly to the degradation of ubiquitin-protein conjugates. Proteasomes are evolutionarily conserved in the eukaryotic kingdom, and those of yeast are well suited to serve as a general model. We describe techniques for the purification of ...
proteasomes from budding yeast in milligram amounts via conventional and affinity-based strategies. While both approaches yield highly purified material, the affinity method is faster and easier. In addition, the affinity method is more suitable for identifying proteasome-associated proteins. We also describe methods for purifying the major subassemblies of the proteasome, such as the CP, the RP, the lid, and the base. A variety of activity assays and native gel procedures are available to evaluate purified proteasomes functionally. When coupled with the genetic methods available in yeast, these biochemical procedures allow for detailed functional analysis of this unique protein complex.
Mesh Terms:
Chromatography, Affinity, Proteasome Endopeptidase Complex, Saccharomyces cerevisiae
Methods Mol. Biol.
Date: May. 27, 2005
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