title: Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation.
abstract: We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and (15)N(2)-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2mg of purified, labeled CB2/L of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two (15)N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of (15)N chemical shifts strongly suggests that the (15)N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation.
authors.affiliation: Institute for Biochemistry and Biotechnology, Martin-Luther University, Halle-Wittenberg, Kurt-Mothes-Str., 3, 06120 Halle, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Berger
authors.forename: Jenny T C
authors.initials: JT
authors.lastname: Ho
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kimura
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.forename: Klaus
authors.initials: K
authors.lastname: Gawrisch
authors.forename: Alexei
authors.initials: A
authors.lastname: Yeliseev
mesh_terms: D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008081:Liposomes / Q000378:metabolism; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D043885:Receptor, Cannabinoid, CB2 / Q000096:biosynthesis* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013696:Temperature
chemical_list: D008081:Liposomes; D009587:Nitrogen Isotopes; D043885:Receptor, Cannabinoid, CB2; D011993:Recombinant Fusion Proteins