Purification and analysis of recombinant 11S activators of the 20S proteasome: Trypanosoma brucei PA26 and human PA28 alpha, PA28 beta, and PA28 gamma.
Proteasomes perform the bulk of nonlysosomal degradation of aberrant, damaged, misfolded, and naturally short-lived regulatory proteins in eukaryotic cells. They are approximately 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.
Masters
Eugene I
EI
Department of Biology, University of Utah, Salt Lake City, UT 84103, USA.
Pratt
Gregory
G
Förster
Andreas
A
Hill
Christopher P
CP
D000818:Animals; D019843:Archaeal Proteins / Q000302:isolation & purification; D001324:Autoantigens / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003460:Crystallization; D004789:Enzyme Activation / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods; D006801:Humans; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides; D046988:Proteasome Endopeptidase Complex / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D015800:Protozoan Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D013194:Staining and Labeling / Q000379:methods; D013822:Thermoplasma; D014346:Trypanosoma brucei brucei
D019843:Archaeal Proteins; D001324:Autoantigens; D005456:Fluorescent Dyes; C099558:Ki antigen; D009124:Muscle Proteins; C402204:PA26 protein, Trypanosoma brucei; C085047:PSME1 protein, human; D010455:Peptides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C486207:PSME2 protein, human; D046988:Proteasome Endopeptidase Complex