title: Simultaneous EGFP and tag labeling of the β7 subunit for live imaging and affinity purification of functional human proteasomes.
abstract: The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation, playing important functions in various biological processes. The C-terminus of the β7 (PSMB4) proteasome subunit was tagged with EGFP and with a composite element for affinity purification and TEV cleavage elution (HTBH). When the construct was retrovirally delivered into HeLa cells, virtually all of the β7-EGFP-HTBH fusion protein was found to be incorporated into fully functional proteasomes. This ensured that subcellular localization of the EGFP signal in living HeLa cells could be attributed to β7-EGFP-HTBH within the proteasome complex rather than to free protein. The β7-EGFP-HTBH fusion can, therefore, serve as a valuable tool for in vivo imaging of proteasomes as well as for high-affinity purification of these complexes and associated molecules for subsequent analyses.
authors.affiliation: Institute of Cytology, Russian Academy of Sciences, Saint-Petersburg, Russia.
authors.forename: Valentina A
authors.initials: VA
authors.lastname: Kulichkova
authors.forename: Tatiana O
authors.initials: TO
authors.lastname: Artamonova
authors.forename: Julia J
authors.initials: JJ
authors.lastname: Zaykova
authors.forename: Julia B
authors.initials: JB
authors.lastname: Ermolaeva
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Khodorkovskii
authors.forename: Nikolai A
authors.initials: NA
authors.lastname: Barlev
authors.forename: Alexey N
authors.initials: AN
authors.lastname: Tomilin
authors.forename: Anna S
authors.initials: AS
authors.lastname: Tsimokha
mesh_terms: D002470:Cell Survival; D002846:Chromatography, Affinity / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D057054:Molecular Imaging; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification* / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D059748:Proteolysis; D011993:Recombinant Fusion Proteins
chemical_list: D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C108898:PSMB4 protein, human; D046988:Proteasome Endopeptidase Complex