title: 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 0.2 mM 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.
authors.affiliation: The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand.
authors.forename: Colm
authors.initials: C
authors.lastname: Carraher
authors.forename: Ali Reza
authors.initials: AR
authors.lastname: Nazmi
authors.forename: Richard D
authors.initials: RD
authors.lastname: Newcomb
authors.forename: Andrew
authors.initials: A
authors.lastname: Kralicek
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D029721:Drosophila Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D008081:Liposomes / Q000737:chemistry; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins; D061987:Sf9 Cells
chemical_list: D029721:Drosophila Proteins; D008081:Liposomes; D021122:Protein Subunits; D018035:Receptors, Odorant; D011994:Recombinant Proteins
keywords: Cell-free; Insect odorant receptor; Liposome; Orco; Protein expression; Purification; Sf9 cell; Wheat germ; Zwittergent 3–16