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Heterologous expression and purification of an active human TRPV3 ion channel

Kol, Stefan; Braun, Christian; Thiel, Gerhard; Doyle, Declan A; Sundström, Michael; Gourdon, Pontus LU and Nissen, Poul (2013) In The FEBS Journal 280(23). p.21-6010
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... (More)

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.

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publication status
published
keywords
Blotting, Western, Cell Membrane, Cells, Cultured, Chromatography, Affinity, Circular Dichroism, Cloning, Molecular, Escherichia coli, Green Fluorescent Proteins, Humans, Patch-Clamp Techniques, Recombinant Fusion Proteins, TRPV Cation Channels, Temperature, Journal Article, Research Support, Non-U.S. Gov't
in
The FEBS Journal
volume
280
issue
23
pages
12 pages
publisher
Federation of European Neuroscience Societies and Blackwell Publishing Ltd
external identifiers
  • scopus:84888305651
ISSN
1742-464X
DOI
10.1111/febs.12520
language
English
LU publication?
no
id
b0aec484-f6a2-40cc-91fa-baf7797d792b
date added to LUP
2017-04-29 15:29:52
date last changed
2018-01-07 12:01:12
@article{b0aec484-f6a2-40cc-91fa-baf7797d792b,
  abstract     = {<p>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.</p>},
  author       = {Kol, Stefan and Braun, Christian and Thiel, Gerhard and Doyle, Declan A and Sundström, Michael and Gourdon, Pontus and Nissen, Poul},
  issn         = {1742-464X},
  keyword      = {Blotting, Western,Cell Membrane,Cells, Cultured,Chromatography, Affinity,Circular Dichroism,Cloning, Molecular,Escherichia coli,Green Fluorescent Proteins,Humans,Patch-Clamp Techniques,Recombinant Fusion Proteins,TRPV Cation Channels,Temperature,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {23},
  pages        = {21--6010},
  publisher    = {Federation of European Neuroscience Societies and Blackwell Publishing Ltd},
  series       = {The FEBS Journal},
  title        = {Heterologous expression and purification of an active human TRPV3 ion channel},
  url          = {http://dx.doi.org/10.1111/febs.12520},
  volume       = {280},
  year         = {2013},
}