W0345

Purification of Drosophila Expressed Odorant Binding Proteins. Elena J. Rossi, Laurie A. Graham, Tara Signorelli, Peter L. Davies, David R. Rose, Medical Biophysics, Univ. of Toronto, 610 University Ave, Toronto, ON M5G 2M9 CANADA.

Insect odorant-binding proteins (OBPs) are small, soluble proteins found in the lymph fluid surrounding olfactory-receptor neurons. They are thought to solubilize hydrophobic odorant molecules and shuttle them to olfactory receptors on dendrites of the antennal sensory neurons. Other OBP functions may include odorant clearance and protection of odorants from degradation. Additional OBPs are found in tissues unrelated to olfaction and may function as carriers of small hydrophobic non-odorant compounds.

To date, two OBP structures, from the beetle and the moth, have been solved. Two Drosophila melanogaster odorant-binding proteins, LUSH and Obp83d, have been over-expressed in D. melanogaster tissue cells in order to purify the proteins in large quantities for x-ray crystallographic studies. Vector constructs were made with the OBP cDNA preceded by a metallothionein promoter, a BIP secretory signal sequence, a HIS tag and a TEV protease recognition site. The constructs were transfected into Drosophila cells and positively expressing single-cell colonies were selected for large-scale growth. Efforts in purification of the secreted OBPs from cell culture media will be presented.