Cell-free expression, purification, and ligand-binding analysis of Drosophila melanogaster olfactory receptors DmOR67a, DmOR85b and DmORCO
Name
Tegler-2015-Cell-free expression.pdf
Size
827.82 KB
Format
Adobe PDF
Checksum (MD5)
a5a7dbc9ccc79d1b702a0f8cd5663d7c
Author(s)
Tegler, Lotta Tollstoy
Corin, Karolina A.
Hillger, Julia
Wassie, Brooke
Yu, Yanmei
Zhang, Shuguang
Date Issued
January 2015
Journal
Scientific Reports
Publisher
Nature Publishing Group
Citation
Tegler, Lotta Tollstoy, Karolina Corin, Julia Hillger, Brooke Wassie, Yanmei Yu, and Shuguang Zhang. “Cell-Free Expression, Purification, and Ligand-Binding Analysis of Drosophila Melanogaster Olfactory Receptors DmOR67a, DmOR85b and DmORCO.” Sci. Rep. 5 (January 19, 2015): 7867.
Version
Final published version
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.
MIT Department
Massachusetts Institute of Technology. Center for Bits and Atoms
Massachusetts Institute of Technology. Media Laboratory
Terms of Use
Creative Commons Attribution
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1038/srep07867
