title: Mammalian expression, purification, and crystallization of rhodopsin variants.
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.
authors.affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, CH-5232, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mattle
authors.forename: Ankita
authors.initials: A
authors.lastname: Singhal
authors.forename: Georg
authors.initials: G
authors.lastname: Schmid
authors.forename: Roger
authors.initials: R
authors.lastname: Dawson
authors.forename: Jörg
authors.initials: J
authors.lastname: Standfuss
mesh_terms: D002460:Cell Line; D018360:Crystallography, X-Ray; D006801:Humans; D008854:Microscopy, Electron; D012160:Retina / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure
chemical_list: C013110:metarhodopsins; D012243:Rhodopsin