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Heterologous expression and purification of an active human TRPV 3 ion channel
Author(s) -
Kol Stefan,
Braun Christian,
Thiel Gerhard,
Doyle Declan A.,
Sundström Michael,
Gourdon Pontus,
Nissen Poul
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12520
Subject(s) - trpv , transient receptor potential channel , chemistry , heterologous expression , recombinant dna , escherichia coli , biophysics , ion channel , biochemistry , chromatography , microbiology and biotechnology , biology , receptor , trpv1 , gene
The transient receptor potential vanilloid 3 ( TRPV 3) 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 TRPV 3 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 TRPV 3 channel heterologously will aid future functional and structural studies. Structured digital abstractTRPV3 and TRPV3 bind by molecular sieving ( 1 , 2 )TRPV3 and TRPV3 bind by blue native page ( 1 , 2 , 3 )