Molecular cloning and bacterial expression of a general odorant-binding protein from the cabbage armyworm Mamestra brassicae.
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.
Maibèche-Coisne
M
M
INRA, Unité de Phytopharmacie et des Médiateurs Chimiques, Versailles, France.
Longhi
S
S
Jacquin-Joly
E
E
Brunel
C
C
Egloff
M P
MP
Gastinel
L
L
Cambillau
C
C
Tegoni
M
M
Nagnan-Le Meillour
P
P
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
D010675:Pheromones; D012333:RNA, Messenger; D018035:Receptors, Odorant; D011994:Recombinant Proteins; C045648:odorant-binding protein