Expression and purification of the C-terminal fragments of TRPV5/6 channels

• Published in: Protein expression and purification 2011-11, Vol.80 (1), p.28-33
• Main Authors: Kovalevskaya, Nadezda V., Schilderink, Nathalie, Vuister, Geerten W.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; C-terminus; Circular Dichroism; Cloning, Molecular; Escherichia coli - genetics; Gene Expression; Humans; Intrinsically disordered; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Protein Conformation; Rabbits; Regulation; Sequence Alignment; TRP channels; TRPV Cation Channels - chemistry; TRPV Cation Channels - genetics; TRPV Cation Channels - isolation & purification; TRPV5; TRPV6
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2011.05.021

► We report on large scale purification of difficult proteins. ► C-termini of TRPV5/6 channels are difficult proteins for structural studies. ► C-termini of TRVP5/6 channels feature intrinsically disordered fragments. 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.