title: Molecular cloning and bacterial expression of a general odorant-binding protein from the cabbage armyworm Mamestra brassicae.
abstract: A cDNA clone encoding a general odorant-binding protein (GOBP2) was isolated from antennal RNA of Mamestra brassicae by reverse transcription-PCR (RT-PCR) and RACE-PCR. The cDNA encoding the GOBP2 was further used for bacterial expression. Most of the recombinant GOBP2 (>90%) was found to be insoluble. Purification under denaturing conditions consisted of solubilisation of inclusion bodies, affinity chromatography, refolding and gel filtration. The refolded rGOBP2 was cross-reactive with a serum raised against the GOBP2 of the Lepidoptera Antheraea polyphemus. The purified refolded rGOBP2 was further characterised by native PAGE, IEF, N-terminal sequencing, and two-dimensional NMR. A functional characterisation of the rGOBP2 was carried out by testing its ability to bind pheromone compounds. The yields of production and purification fulfil the requirements of structural studies.
authors.affiliation: INRA, Unité de Phytopharmacie et des Médiateurs Chimiques, Versailles, France.
authors.forename: M
authors.initials: M
authors.lastname: Maibèche-Coisne
authors.forename: S
authors.initials: S
authors.lastname: Longhi
authors.forename: E
authors.initials: E
authors.lastname: Jacquin-Joly
authors.forename: C
authors.initials: C
authors.lastname: Brunel
authors.forename: M P
authors.initials: MP
authors.lastname: Egloff
authors.forename: L
authors.initials: L
authors.lastname: Gastinel
authors.forename: C
authors.initials: C
authors.lastname: Cambillau
authors.forename: M
authors.initials: M
authors.lastname: Tegoni
authors.forename: P
authors.initials: P
authors.lastname: Nagnan-Le Meillour
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D007313:Insecta / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010675:Pheromones / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding; D012333:RNA, Messenger / Q000235:genetics; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D010675:Pheromones; D012333:RNA, Messenger; D018035:Receptors, Odorant; D011994:Recombinant Proteins; C045648:odorant-binding protein