Abstract
For a successful characterization of channelrhodopsins with biophysical methods like FTIR, Raman, EPR and NMR spectroscopy and X-ray crystallography, large amounts of purified protein are requested. For proteins of eukaryotic origin, which are poorly expressing in bacterial systems or not at all, the yeast Pichia pastoris represents a promising alternative for overexpression. Here we describe the methods for cloning, overexpression and mutagenesis as well as the purification procedures for channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) and the scaffold protein MSP1D1 for reconstitution of the membrane proteins into nanodiscs. Finally, protocols are provided to study CaChR1 by FTIR difference spectroscopy and by time-resolved UV/Vis spectroscopy.
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Acknowledgements
We thank Nils Krause and Vera Muders for developing protocols and Kirsten Hoffmann and Dorothea Heinrich for technical assistance. Financial support was granted from the Deutsche Forschungsgemeinschaft (SFB1078 TP B4).
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Walter, M., Schlesinger, R. (2021). Nanodisc Reconstitution of Channelrhodopsins Heterologously Expressed in Pichia pastoris for Biophysical Investigations. In: Dempski, R. (eds) Channelrhodopsin. Methods in Molecular Biology, vol 2191. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0830-2_3
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DOI: https://doi.org/10.1007/978-1-0716-0830-2_3
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