Recombinant expression, detergent solubilisation and purification of insect odorant receptor subunits

Abstract

Insect odorant receptors (ORs) are seven transmembrane domain proteins that comprise a novel family of ligand-gated non-selective cation channels. The functional channel is made up of an odour activated ligand-binding OR and the OR co-receptor, Orco. However, the structure, stoichiometry and mechanism of activation of the receptor complex are not well understood. Here we demonstrate that baculovirus-mediated Sf9 cell expression and wheat germ cell-free expression, but not Escherichia coli cell-based or cell-free expression, can be used successfully to over-express a selection of insect ORs. From a panel of 19 detergents, 1%w/v Zwittergent 3–16 was able to solubilise five Drosophila melanogaster ORs produced from both eukaryotic expression systems. A large-scale purification protocol was then developed for DmOrco and the ligand-binding receptor, DmOr22a. The proteins were nickel-affinity purified using a deca-histidine tag in a buffer containing 0.2mM Zwittergent 3–16, followed by size exclusion chromatography. These purified ORs appear to form similarly sized protein-detergent complexes when isolated from both expression systems. Circular dichroism analysis of both purified proteins suggests they are folded correctly. We also provide evidence that when DmOrco is expressed in Sf9 cells it undergoes post translational modification, probably glycosylation. Finally we show that the recombinant ORs can be incorporated into pre-formed liposomes. The ability to recombinantly express and purify insect ORs to homogeneity on a preparative scale, as well as insert them into liposomes, is a major step forward in enabling future structural and functional studies, as well as their use in OR based biosensors.