Expression and characterization of a lepidopteran general odorant binding protein.
Olfaction in months involves the transport of volatile, hydrophobic odorant molecules through the aqueous interior of the antennal sensory hairs by soluble odorant binding proteins. Two subfamilies of the 17 kDa general odorant binding proteins, GOBP1 and GOBP2, are 47-57% identical to each other and 21-57% to the pheromone binding proteins (PBPs); identity within a GOBP subfamily exceeds 78% in all lepidopteran species examined. However, the ligands for GOBPs are unknown. In order to investigate odorant specificities of GOBPs, recombinant proteins were expressed in Escherichia coli using PCR-prepared expression cassettes based on the cDNA sequences of GOBP1 and GOBP2 from Manduca sexta. Both soluble and insoluble recombinant GOBPs (rGOBPs) were obtained, and the inclusion body GOBPs were solubilized, refolded and purified. The soluble and refolded rGOBPs were purified by preparative isoelectric focusing (IEF), gel filtration, and finally by ion-exchange fast protein liquid chromatography (FPLC). Only rGOBP2, but not rGOBP1, was crossreactive with an anti-GOBP2 (Antheraea polyphemus) antiserum. rGOBP2, but not rGOBP1, could be photoaffinity labelled by the diazoacetate pheromone analog [3H]-6E, 11Z, 16:Dza. For rGOBP2, plant odors such as (Z)-3-hexen-1-ol (3Z-6:OH), geraniol, geranyl acetate, and limonene showed significant competition for binding; binding specificity was sensitive to pH and to salt concentrations. Circular dichroism (CD) confirms that, as with the pheromone binding proteins, GOBP2 is predominantly alpha-helical. Although the characterization of rGOBP1 has resisted analysis, rGOBP2 is readily prepared and studied. We suggest that GOBP2 may be broadly tuned to a class of "green" and floral odors.
Feng
L
L
Department of Chemistry, University at Stony Brook, NY 11794-3400, USA.
Prestwich
G D
GD
D000818:Animals; D015870:Gene Expression; D018402:Manduca / Q000737:chemistry* / Q000235:genetics; D018035:Receptors, Odorant / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; C045648:odorant-binding protein