title: Cell-free expression, purification, and ligand-binding analysis of Drosophila melanogaster olfactory receptors DmOR67a, DmOR85b and DmORCO.
abstract: Insects transmit numerous devastating diseases, including malaria, dengue fever, and sleeping sickness. Olfactory cues guide insects to their hosts, and are thus responsible for disease transmission. Understanding the molecular basis of insect olfaction could facilitate the development of interventions. The first step is to heterologously overexpress and purify insect olfactory receptors (ORs). This is challenging, as ORs are membrane proteins. Here, we show that insect ORs and their co-receptor can be expressed in an E. coli cell-free system. After immunoaffinity chromatography, the ORs are ~95% pure, and up to 1 mg/10 ml reaction is obtained. Circular dichroism together with microscale thermophoresis indicate that each receptor is properly folded, and can bind its respective ligand. This is the first time insect ORs have been expressed in an E. coli system. The methods described here could facilitate future structure-function studies, which may aid in developments to alleviate the suffering of millions caused by insect-transmitted diseases.
authors.affiliation: 1] Center for Bits and Atoms, Massachusetts Institute of Technology, 77 Massachusetts, Cambridge, MA 02139-4307, USA [2] Division of Chemistry, Department of Physics, Chemistry and Biology, Linkoping University, SE-581 83 Linkoping, Sweden.
authors.forename: Lotta Tollstoy
authors.initials: LT
authors.lastname: Tegler
authors.affiliation: 1] Center for Bits and Atoms, Massachusetts Institute of Technology, 77 Massachusetts, Cambridge, MA 02139-4307, USA [2] Biomedical Engineering Research Group, School of Electrical and Information Engineering University of the Witwatersrand, Johannesburg, South Africa [3] Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: Karolina
authors.initials: K
authors.lastname: Corin
authors.affiliation: Center for Bits and Atoms, Massachusetts Institute of Technology, 77 Massachusetts, Cambridge, MA 02139-4307, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Hillger
authors.affiliation: Center for Bits and Atoms, Massachusetts Institute of Technology, 77 Massachusetts, Cambridge, MA 02139-4307, USA.
authors.forename: Brooke
authors.initials: B
authors.lastname: Wassie
authors.affiliation: 1] Center for Bits and Atoms, Massachusetts Institute of Technology, 77 Massachusetts, Cambridge, MA 02139-4307, USA [2] College of Electronics and Information Engineering, Sichuan University, Chengdu, China 610065.
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Center for Bits and Atoms, Massachusetts Institute of Technology, 77 Massachusetts, Cambridge, MA 02139-4307, USA.
authors.forename: Shuguang
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002474:Cell-Free System; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004331:Drosophila melanogaster / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D008024:Ligands; D018034:Olfactory Receptor Neurons / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D018035:Receptors, Odorant / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D029721:Drosophila Proteins; D008024:Ligands; C530080:Or67d protein, Drosophila; C492465:Orco protein, Drosophila; D011956:Receptors, Cell Surface; D018035:Receptors, Odorant