title: Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits.
abstract: The proteasome is a multi-subunit proteolytic complex that plays a central role in protein degradation in all eukaryotic cells. It regulates many vital cellular processes therefore its dysfunction can lead to various pathologies including cancer and neurodegeneration. Isolation of enzymatically active proteasomes is a key step to the successful study of the proteasome regulation and functions. Here we describe a simple and efficient protocol for immunoaffinity purification of the functional 20S proteasomes from human HEK 293T cells after transient overexpression of specific proteasome subunits tagged with 3xFLAG. To construct 3xFLAG-fusion proteins, DNA sequences encoding the 20S proteasome subunits PSMB5, PSMA5, and PSMA3 were cloned into mammalian expression vector pIRES-hrGFP-1a. The corresponding recombinant proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG were transiently overexpressed in human HEK 293T cells and were shown to be partially incorporated into the intact proteasome complexes. 20S proteasomes were immunoprecipitated from HEK 293T cell extracts under mild conditions using antibodies against FLAG peptide. Isolation of highly purified 20S proteasomes were confirmed by SDS-PAGE and Western blotting using antibodies against different proteasome subunits. Affinity purified 20S proteasomes were shown to possess chymotrypsin- and trypsin-like peptidase activities confirming their functionality. This simple single-step affinity method of the 20S proteasome purification can be instrumental to subsequent functional studies of proteasomes in human cells.
authors.affiliation: Institute of Cytology, Russian Academy of Science, Tikhoretsky ave. 4, 194064 Saint Petersburg, Russia; Institute of Nanobiotechnologies, Saint Petersburg State Polytechnical University, Polytechnicheskaya 29, 195251 Saint Petersburg, Russia.
authors.forename: Veronika A
authors.initials: VA
authors.lastname: Livinskaya
authors.affiliation: Institute of Cytology, Russian Academy of Science, Tikhoretsky ave. 4, 194064 Saint Petersburg, Russia; Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom.
authors.forename: Nickolai A
authors.initials: NA
authors.lastname: Barlev
authors.affiliation: Institute of Cytology, Russian Academy of Science, Tikhoretsky ave. 4, 194064 Saint Petersburg, Russia; Institute of Nanobiotechnologies, Saint Petersburg State Polytechnical University, Polytechnicheskaya 29, 195251 Saint Petersburg, Russia. Electronic address: nikiforovan@hotmail.com.
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Nikiforov
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D009842:Oligopeptides / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D009842:Oligopeptides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D046988:Proteasome Endopeptidase Complex
keywords: 20S proteasome; 3xFLAG peptide; Immunoaffinity purification; PSMA3; PSMA5; PSMB5