[ 25 ] Purification and Analysis of Recombinant 11 S Activators of the 20 S Proteasome : Trypanosoma brucei PA 26 and Human PA 28 , PA 28 , and PA 28

Proteasomes perform the bulk of nonlysosomal degradation of aberrant, damaged, misfolded, and naturally short‐lived regulatory proteins in eukaryotic cells. They are 700‐kDa assemblies whose hollow architecture sequesters the proteolytic sites inside a central chamber, thereby ensuring that the activity of isolated proteasomes is repressed. In vivo, proteasomes are activated by protein complexes, including the 11S activators (PA28 and PA26), which bind to one or both ends of the barrel‐shaped structure. This chapter describes protocols for the purification of recombinant 11S regulators, characterization of their ability to stimulate proteasome activity, and crystallization of proteasome complexes.