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

  • Kulichkova V
  • Artamonova T
  • Zaykova J
 et al. 
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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.

Author-supplied keywords

  • Affinity purification
  • EGFP
  • Intracellular localization
  • Proteasome
  • Retroviral infection

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Authors

  • Valentina A. Kulichkova

  • Tatiana O. Artamonova

  • Julia J. Zaykova

  • Julia B. Ermolaeva

  • Mikhail A. Khodorkovskii

  • Nikolai A. Barlev

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