title: Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.
abstract: The vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules, where it is activated by the extracellular arginine vasopressin. In men, inactivating and activating mutations cause nephrogenic diabetes insipidus and the nephrogenic syndrome of inappropriate antidiuresis respectively. Like most GPCRs, V2R's third intracellular loop (V2R-i3) is involved in the binding and activation of its major effector, the GαS protein. We overexpressed the V2R₂₂₄₋₂₇₄ fragment corresponding to V2R-i3 as a fusion protein with thioredoxin A at the N-terminus and a hexahistidine tag between the two proteins. Recombinant V2R-i3 was designed to harbor N- and C-terminal cysteines, in order to introduce a disulfide bond between N- and C-terminal extremities and hence reproduce the hairpin fold presumably present in the full-length receptor. The fusion protein was produced as inclusion bodies in Escherichia coli and purified by nickel affinity chromatography under denaturing conditions. After a refolding step, thioredoxin and hexahistidine tags were specifically cleaved with the tobacco etch virus protease. The hydrolysis yield, initially very low, increased up to 80% thanks to optimization of buffers and refolding methods. The cleaved fragment, V2₂₂₄₋₂₇₄, devoid of any tag, was then eluted with low imidazole concentrations in a second nickel affinity chromatography in denaturing conditions. The final yield was sufficient to prepare a ¹⁵N-¹³C labeled NMR sample suitable for triple resonance experiments. We assigned all NMR resonances and confirmed the correct peptide sequence. As expected, the peptide forms a hairpin stabilized by a disulfide bond between its N- and C-terminal parts, thus mimicking its native structure in the full-length receptor. This study may provide a strategy for producing and studying the structure/function relationship of GPCR fragments.
authors.affiliation: INSERM U1054, Centre de Biochimie Structurale, 29 Rue de Navacelles, 34090 Montpellier Cedex, France.
authors.forename: Gaëtan
authors.initials: G
authors.lastname: Bellot
authors.forename: Robert
authors.initials: R
authors.lastname: Pascal
authors.forename: Christiane
authors.initials: C
authors.lastname: Mendre
authors.forename: Serge
authors.initials: S
authors.lastname: Urbach
authors.forename: Bernard
authors.initials: B
authors.lastname: Mouillac
authors.forename: Hélène
authors.initials: H
authors.lastname: Déméné
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies; D042541:Intracellular Space; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D017483:Receptors, Vasopressin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D017483:Receptors, Vasopressin; D011993:Recombinant Fusion Proteins