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

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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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Abbreviations

DTT:

Dithiothreitol

DMSO:

Dimethyl sulfoxide

PAGE:

Polyacrylamide gel electrophoresis

AMC:

7-Amino-4-methylcoumarin

Suc:

N-Succinyl

TEV:

Tobacco etch virus

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Acknowledgments

We appreciate Prof. Colyn Crane-Robinson (University of Portsmouth, UK) for manuscript proof-reading, Prof. Lan Huang (University of California, USA) for generously providing the expression construct of the 19S subunit, and Dr. Masaru Okabe (Osaka University, Japan) for providing the pCX-EGFP plasmid. We also thank Mikhail L. Vorobiev and Grigoriy I. Shtein (Institute of Cytology RAS, Russia) for technical assistance with fluorescence microscopy experiments. This work was partly supported by a FEBS Collaborative Experimental Scholarship for Central and Eastern Europe [to AST], the Molecular and Cellular Biology program of the Presidium of the Russian Academy of Sciences [to NAB], and the Russian Foundation for Basic Research [13-02-00923 to ANT, 13-04-01024 to NAB].

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Authors and Affiliations

  1. Institute of Cytology, Russian Academy of Sciences, Saint-Petersburg, Russia

    Valentina A. Kulichkova, Julia J. Zaykova, Julia B. Ermolaeva, Nikolai A. Barlev, Alexey N. Tomilin & Anna S. Tsimokha

  2. St.Petersburg State Polytechnical University, Saint-Petersburg, Russia

    Tatiana O. Artamonova & Mikhail A. Khodorkovskii

  3. Department of Biochemistry, Leicester University, Leicester, UK

    Nikolai A. Barlev

Authors
  1. Valentina A. Kulichkova
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  2. Tatiana O. Artamonova
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  3. Julia J. Zaykova
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  4. Julia B. Ermolaeva
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  5. Mikhail A. Khodorkovskii
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  8. Anna S. Tsimokha
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Correspondence to Anna S. Tsimokha.

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Kulichkova, V.A., Artamonova, T.O., Zaykova, J.J. et al. Simultaneous EGFP and Tag Labeling of the β7 Subunit for Live Imaging and Affinity Purification of Functional Human Proteasomes. Mol Biotechnol 57, 36–44 (2015). https://doi.org/10.1007/s12033-014-9799-0

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