Cell-free expression, purification, and ligand-binding analysis of drosophila melanogaster olfactory receptors DmOR67a, DmOR85b and DmORCO

 

Lotta Tollstoy Tegler, Karolina Corin, Julia Hillger, Brooke Wassie, Yanmei Yu & Shuguang Zhang

Scientific Reports
2015 vol: 5 article number: 7867 doi: 10.1038/srep07867

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.

View Publication

Topics: Monolith – MicroScale Thermophoresis, MST, Proteins, Publications

 

 

Previous Article
Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores
Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

Next Article
MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of mycoplasma hyopneumoniae
MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of mycoplasma hyopneumoniae