Staff member

Kumar, Mohit

JTD

This study aims to improve our understanding of the interaction between olfactory receptors and odorants to develop highly selective biosensing devices. Natural nanovesicles (NVs) from Saccharomyces cerevisiae, ~100 nm in diameter, carrying either the human OR17-40 or the chimpanzee OR7D4 olfactory receptor (OR) tagged with the c-myc epitope at their N-terminus, are presented as model systems to quantify the interaction between odorant and olfactory receptors. The level of expression of olfactory receptors was determined at individual NVs using a novel competitive ELISA immunoassay comparing the values obtained against those from techniques involving the solubilization of cell membrane proteins and the identification of c-myc-carrying receptors. Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips indicate that cognate odorants bind to their Ors, thereby quantifying the approximate number of odorants that interact with a given olfactory receptor. The selectivity of OR17-40-carrying NVs towards helional and OR7D4-carrying NVs towards androstenone has been proven in cross-check experiments with non-specific odorant molecules (heptanal and pentadecalactone, respectively) and in control receptors.

JTD

Natural vesicles produced from genetically engineered cells with tailored membrane receptor composition are promising building blocks for sensing biodevices. This is particularly true for the case of G-protein coupled receptors (GPCRs) present in many sensing processes in cells, whose functionality crucially depends on their lipid environment. However, the controlled production of natural vesicles containing GPCRs and their reproducible deposition on biosensor surfaces are among the outstanding challenges in the road map to realize practical biomolecular devices based on GPCRs. In this work we present the production and characterization of membrane nanovesicles from Saccharomyces cerevisiae containing heterologously expressed olfactory receptors - a member of the family of GPCRs - and study their deposition onto substrates used as biosensor supports. We show by direct observation with Atomic Force Microscopy that nanovesicles deposit and flatten without rupturing on glass substrates following approximately a diffusive law. We show that surface coverages larger than 20-25% of the substrate can be reproducibly achieved under practical nanovesicle concentrations and reasonable time scales, while keeping to the minimum the presence of background residuals coming from the nanovesicles production process. Surface chemistry modification of gold substrates indicates a higher affinity of natural nanovesicles for acid modified surfaces as compared to amino or alcohol modified surfaces. Present results constitute an important step in the practical realization of biosensor devices based on natural nanovesicles integrating G-protein coupled membrane receptors.

JTD