title: Expression and purification of the C-terminal fragments of TRPV5/6 channels.
abstract: The transient receptor potential vanniloid 5 and 6 (TRPV5 and TRPV6) Ca(2+)-ion channels are crucial for the regulation of minute-to-minute whole body calcium homeostasis. They act as the gatekeepers of active Ca(2+) reabsorption in kidney and intestine, respectively. In spite of the great progress in the TRP channels characterization, very little is known at the atomic level about their structure and interactions with other proteins. To the major extent it is caused by difficulties in obtaining suitable samples. Here, we report expression and purification of 36 intracellular C-terminal fragments of TRPV5 and TRPV6 channels, for which no structural information is reported thus far. We demonstrate that these proteins contain intrinsically disordered regions and identify fragments suitable for biophysical characterization. By combining bioinformatic predictions and experimental results, we propose several criteria that may aid in designing a scheme for large-scale production of difficult proteins.
authors.affiliation: Department of Protein Biophysics, IMM, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands. n.kovalevskaya@science.ru.nl
authors.forename: Nadezda V
authors.initials: NV
authors.lastname: Kovalevskaya
authors.forename: Nathalie
authors.initials: N
authors.lastname: Schilderink
authors.forename: Geerten W
authors.initials: GW
authors.lastname: Vuister
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011817:Rabbits; D016415:Sequence Alignment; D050916:TRPV Cation Channels / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; C120776:TRPV6 channel