title: Large scale expression and purification of the rat 5-HT2c receptor.
abstract: 5-HT2c G-protein coupled receptors located in the central nervous system bind the endogenous neurotransmitters serotonin and couple to G protein to mediate excitatory neurotransmission, which inhibits dopamine and norepinephrine release in the brain. Thus, 5-HT2c receptors play important roles in cognitive function and are potent drug targets. Structural information is needed to elucidate the molecular mechanism of ligand-binding and receptor-activation of the 5-HT2c receptor. Lacking of an efficient expression system that produces sufficient amounts of active and homogenous receptors hinders progress in the functional and structural characterization of the 5-HT2c receptor. We present here a protocol which can be used easily to obtain milligram amount of purified rat 5-HT2c receptors. We established this protocol by protein engineering and optimization of expression and purification based on radioligand-binding assay. The purified and well-characterized rat 5-HT2c receptors are active, stable, homogenous, and ready for 2-dimensional and 3-dimensional crystallization experiments.
authors.affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: He
authors.affiliation: Heptares Therapeutics, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AX, UK.
authors.forename: Nathan
authors.initials: N
authors.lastname: Robertson
authors.affiliation: Heptares Therapeutics, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AX, UK.
authors.forename: Ali
authors.initials: A
authors.lastname: Jazayeri
authors.affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland.
authors.forename: Antonietta Geroni
authors.initials: AG
authors.lastname: Gasperina
authors.affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland.
authors.forename: Gebhard
authors.initials: G
authors.lastname: Schertler
authors.affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland. Electronic address: xiao.li@psi.ch.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D003460:Crystallization; D003902:Detergents / Q000378:metabolism; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D055550:Protein Stability; D011869:Radioligand Assay; D051381:Rats; D044405:Receptor, Serotonin, 5-HT2C / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013696:Temperature
chemical_list: D003902:Detergents; D044405:Receptor, Serotonin, 5-HT2C; D011994:Recombinant Proteins
keywords: Expression; G protein-coupled receptor; Purification; Serotonin receptor; Structural and functional studies