Open AccessMolecular cloning and bacterial expression of a general odorant‐binding protein from the cabbage armyworm Mamestra brassicae
Author(s) -
MaibècheCoisne Martine,
Longhi Sonia,
JacquinJoly Emmanuelle,
Brunel Carole,
Egloff MariePierre,
Gastinel Louis,
Cambillau Christian,
Tegoni Mariella,
NagnanLe Meillour Patricia
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2580768.x
Subject(s) - complementary dna , biology , recombinant dna , microbiology and biotechnology , affinity chromatography , biochemistry , molecular cloning , inclusion bodies , escherichia coli , size exclusion chromatography , gene , enzyme
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.