title: Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans.
abstract: High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack post-translational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work, we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with (15)N,(13)C by providing them with isotopically labeled bacteria. (2)H labeling also was achieved by growing C. elegans in the presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the "test" GPCR to demonstrate the viability of this approach. Although the worms' cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization.
authors.affiliation: Polgenix, Cleveland, OH 44106, USA; Department of Pharmacology, Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: David
authors.initials: D
authors.lastname: Salom
authors.affiliation: Department of Pharmacology, Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Pengxiu
authors.initials: P
authors.lastname: Cao
authors.affiliation: Department of Pharmacology, Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Yiyuan
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: Center for Proteomics and Bioinformatics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Masaru
authors.initials: M
authors.lastname: Miyagi
authors.affiliation: Department of Pharmacology, Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address: john.feng@case.edu.
authors.forename: Zhaoyang
authors.initials: Z
authors.lastname: Feng
authors.affiliation: Department of Pharmacology, Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address: kxp65@case.edu.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012243:Rhodopsin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D012243:Rhodopsin
keywords: Caenorhabditis elegans; G protein-coupled receptors (GPCRs); Heterologous expression; Isotopic labeling; Nuclear magnetic resonance (NMR); Rhodopsin