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.

journal_name

Mol Biotechnol

journal_title

Molecular biotechnology

authors

Kulichkova VA,Artamonova TO,Zaykova JJ,Ermolaeva JB,Khodorkovskii MA,Barlev NA,Tomilin AN,Tsimokha AS

doi

10.1007/s12033-014-9799-0

subject

Has Abstract

pub_date

2015-01-01 00:00:00

pages

36-44

issue

1

eissn

1073-6085

issn

1559-0305

journal_volume

57

pub_type

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