Purification, crystallization, and X-ray analysis of the yeast 20S proteasome

Intracellular protein degradation is one of the most precisely regulated processes in living cells. The main component of the degradation machinery is the 20S proteasome present in eukaryotes as well as in prokaryotes. We have developed successful purification protocols for the 20S proteasome in its native state using an affinity tag strategy. This chapter describes in detail the purification protocols, proteolytic activity assays, crystallization, and structure determination for the yeast 20S proteasome. The crystal structure of the eukaryotic proteasome opens new possibilities for identifying, characterizing, and elucidating the mode of action for natural and synthetic inhibitors, which affect its function. Some of these compounds may find therapeutic applications in contemporary medicine.