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Cell-free expression, purification, and ligand-binding analysis of Drosophila melanogaster olfactory receptors DmOR67a, DmOR85b and DmORCO
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. MIT, MA 02139 USA.
MIT, MA 02139 USA; University of Witwatersrand, South Africa; University of Witwatersrand, South Africa.
MIT, MA 02139 USA.
MIT, MA 02139 USA.
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2015 (English)In: Scientific Reports, ISSN 2045-2322, Vol. 5, no 7867Article in journal (Refereed) Published
Abstract [en]

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 similar to 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.

Place, publisher, year, edition, pages
Nature Publishing Group: Open Access Journals - Option C / Nature Publishing Group , 2015. Vol. 5, no 7867
National Category
Chemical Sciences
URN: urn:nbn:se:liu:diva-114237DOI: 10.1038/srep07867ISI: 000347978600006PubMedID: 25597985OAI: diva2:788721

Funding Agencies|DARPA QuBE [N66001-10-1-4062]; Yang Trust Fund; WennerGren Foundation; Swedish Chemical Society; Claude Leon Foundation

Available from: 2015-02-16 Created: 2015-02-16 Last updated: 2015-02-26

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