Expression and characterization of a lepidopteran general odorant binding protein

Area of Science:

• Insect olfaction research
• Molecular biology of sensory proteins
• Biochemistry of odorant binding

Background:

• Olfaction relies on odorant binding proteins (OBPs) to transport hydrophobic odorants.
• General odorant binding proteins (GOBPs) are a subfamily of OBPs with unknown ligand specificities.
• GOBP1 and GOBP2 share high identity within their subfamily and with pheromone binding proteins (PBPs).

Purpose of the Study:

• To investigate the odorant specificities of GOBP1 and GOBP2 from Manduca sexta.
• To characterize recombinant GOBPs (rGOBPs) for binding studies.
• To explore the potential function of GOBP2 in insect olfaction.

Main Methods:

• Expressed recombinant GOBP1 and GOBP2 (rGOBP1, rGOBP2) in E. coli.
• Purified soluble and refolded rGOBPs using isoelectric focusing, gel filtration, and ion-exchange FPLC.
• Utilized cross-reactivity assays, photoaffinity labeling, and binding competition assays with plant odors.
• Confirmed protein structure using circular dichroism (CD).

Main Results:

• rGOBP2, but not rGOBP1, showed cross-reactivity with an anti-GOBP2 antiserum and could be photoaffinity labeled.
• rGOBP2 demonstrated significant binding competition with plant odors like (Z)-3-hexen-1-ol, geraniol, geranyl acetate, and limonene.
• Binding specificity of rGOBP2 was sensitive to pH and salt concentrations.
• CD spectroscopy indicated rGOBP2 is predominantly alpha-helical, similar to PBPs.

Conclusions:

• Recombinant GOBP2 (rGOBP2) from Manduca sexta can be successfully expressed, refolded, and purified.
• rGOBP2 exhibits binding affinity for a range of plant-derived odors, suggesting a broad tuning.
• GOBP2 likely plays a role in detecting "green" and floral scents in insects.
• Further characterization of rGOBP1 is needed, but rGOBP2 is a viable target for olfactory studies.