Simultaneous EGFP and Tag Labeling of the β7 Subunit for Live Imaging and Affinity Purification of Functional Human Proteasomes

Overview

Journal Mol Biotechnol

Publisher Springer

Specialties Biotechnology
Molecular Biology

Date 2014 Aug 29

PMID 25164490

Citations 8

Authors

Valentina A Kulichkova

Tatiana O Artamonova

Julia J Zaykova

Julia B Ermolaeva

Mikhail A Khodorkovskii

Nikolai A Barlev

Alexey N Tomilin

Anna S Tsimokha

Affiliations

Soon will be listed here.

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.

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