Abstract
After 25 years of intensive research, the understanding of how photoreceptors in the eye perceive light and convert it into nerve signals has largely advanced. Central to this is the structural and mechanistic exploration of the G protein-coupled receptor rhodopsin acting as a dim-light sensing pigment in the retina. Investigation of rhodopsin by X-ray crystallographic, electron microscopic, and biochemical means depends on the ability to produce and isolate pure rhodopsin protein. Robust and well-defined protocols permit the production and crystallization of rhodopsin variants to investigate the inactive ground, the fully activated metarhodopsin II state, or disease-causing rhodopsin mutations. This chapter details how we express and purify biologically active variants of rhodopsin from HEK293S GnTI− cells in a quality and quantity suitable for biochemical assays, crystallization, and structure determination.
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Acknowledgments
We thank Marcello Foggetta, Martin Siegrist, and Agnese Baronina for technical assistances and valuable discussions. We are grateful for the financial support from the Roche Postdoctoral Research Fellowship RPF298 (to D.M.) and from the Swiss National Science Foundation (SNSF) grant 31003A_141235 (to J.S.).
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Mattle, D., Singhal, A., Schmid, G., Dawson, R., Standfuss, J. (2015). Mammalian Expression, Purification, and Crystallization of Rhodopsin Variants. In: Jastrzebska, B. (eds) Rhodopsin. Methods in Molecular Biology, vol 1271. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2330-4_3
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DOI: https://doi.org/10.1007/978-1-4939-2330-4_3
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