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 isolate d from antennal RNA of Mamestra brassicae by reverse transcription-PCR (RT- PCR) and RACE-PCR. The cDNA encoding the GOBP2 was further used for bacteri al expression. Most of the recombinant GOBP2 (>90%) was found to be insolub le. Purification under denaturing conditions consisted of solubilisation of inclusion bodies, affinity chromatography, refolding and gel filtration. T he: refolded rGOBP2 was cross-reactive with a serum raised against the GOBP 2 of the Lepidoptera Antheraea polyphemus. The purified refolded rGOBP2 was further characterised by native PAGE, IEE N-terminal sequencing, and two-d imensional NMR. A functional characterisation of the rGOBP2 was carried out by testing its ability to bind pheromone compounds. The yields of producti on and purification fulfil the requirements of structural studies.