Heterologous expression and purification of an active human TRPV3 ion channel

Authors

  • Stefan Kol,

    Corresponding author
    1. Protein Function and Interactions, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Denmark
    Current affiliation:
    1. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark
    • Correspondence

      S. Kol, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allè 6, DK-2970 Hørsholm, Denmark

      Fax: +45 4525 8001

      Tel.: +45 2465 4189

      E-mail: stko@biosustain.dtu.dk

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  • Christian Braun,

    1. Plant Membrane Biophysics, Technische Universität Darmstadt, Germany
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  • Gerhard Thiel,

    1. Plant Membrane Biophysics, Technische Universität Darmstadt, Germany
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  • Declan A. Doyle,

    1. Centre for Biological Sciences, University of Southampton, UK
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  • Michael Sundström,

    1. Protein Function and Interactions, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Denmark
    Current affiliation:
    1. Karolinska Development, Solna, Sweden
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  • Pontus Gourdon,

    1. Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Aarhus University, Denmark
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  • Poul Nissen

    1. Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Aarhus University, Denmark
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Abstract

The transient receptor potential vanilloid 3 (TRPV3) cation channel is widely expressed in human tissues and has been shown to be activated by mild temperatures or chemical ligands. In spite of great progress in the TRP-channel characterization, very little is known about their structure and interactions with other proteins at the atomic level. This is mainly caused by difficulties in obtaining functionally active samples of high homogeneity. Here, we report on the high-level Escherichia coli expression of the human TRPV3 channel, for which no structural information has been reported to date. We selected a suitable detergent and buffer system using analytical size-exclusion chromatography and a thermal stability assay. We demonstrate that the recombinant purified protein contains high α-helical content and migrates as dimers and tetramers on native PAGE. Furthermore, the purified channel also retains its current inducing activity, as shown by electrophysiology experiments. The ability to produce the TRPV3 channel heterologously will aid future functional and structural studies.

Structured digital abstract

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