Principles to recover copper-conducting CTR proteins for the purpose of structural and functional studies

Niloofar Nayeri, Ping Li, Kamil Górecki, Karin Lindkvist-Petersson, Pontus Gourdon*

*Corresponding author for this work

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Abstract

Transition metals such as copper and zinc are essential elements required for the survival of most organisms, from bacteria to humans. Yet, elevated levels of these elements are highly toxic. The Copper TRansporter protein family (CTRs) represents the only identified copper uptake proteins in eukaryotes and hence serves as key components for the maintenance of appropriate levels of the metal. Moreover, CTRs have been proposed to serve as an entry point into cells of certain cancer drugs and to constitute attractive drug-targets for novel antifungals. Nevertheless, the structure, function, and regulation of the CTRs remain elusive, limiting valuable information also for applied sciences. To this end, here we report procedures to isolate a range of CTR members using Saccharomyces cerevisiae as a production host, focusing on three homologs, human CTR1, human CTR2, and Candida albicans CTR. Using forms C-terminally-linked to a protease cleavage sequence, Green Fluorescent Protein (GFP), and a His-tag, assessment of the localization, quantification and purification was facilitated. Cellular accumulation of the proteins was investigated via live-cell imaging. Detergents compatible with acceptable solubilization yields were identified and fluorescence-detection size-exclusion-chromatography (F-SEC) revealed preferred membrane extraction conditions for the targets. For purification purposes, the solubilized CTR members were subjected to affinity chromatography and SEC, reaching near homogeneity. The quality and quantity of the CTRs studied will permit downstream efforts to uncover imperative biophysical aspects of these proteins, paving the way for subsequent drug-discovery studies.

Original languageEnglish
Article number106213
JournalProtein Expression and Purification
Volume203
Number of pages8
ISSN1046-5928
DOIs
Publication statusPublished - 2023

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Keywords

  • Biophysical studies
  • Copper homeostasis
  • CTRs
  • Protein production
  • Protein purification

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