Kol, Stefan ; Braun, Christian ; Thiel, Gerhard ; Doyle, Declan A. ; Sundström, Michael ; Gourdon, Pontus ; Nissen, Poul (2013)
Heterologous expression and purification of an active human TRPV3 ion channel.
In: The FEBS journal, 280 (23)
Artikel, Bibliographie
Kurzbeschreibung (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.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2013 |
| Autor(en): | Kol, Stefan ; Braun, Christian ; Thiel, Gerhard ; Doyle, Declan A. ; Sundström, Michael ; Gourdon, Pontus ; Nissen, Poul |
| Art des Eintrags: | Bibliographie |
| Titel: | Heterologous expression and purification of an active human TRPV3 ion channel. |
| Sprache: | Englisch |
| Publikationsjahr: | 2013 |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | The FEBS journal |
| Jahrgang/Volume einer Zeitschrift: | 280 |
| (Heft-)Nummer: | 23 |
| Kurzbeschreibung (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. |
| Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Plant Membrane Biophyscis (am 20.12.23 umbenannt in Biologie der Algen und Protozoen) |
| Hinterlegungsdatum: | 07 Jan 2014 12:49 |
| Letzte Änderung: | 07 Jan 2014 12:49 |
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