Quantitative assessment of olfactory receptors activity in immobilized nanosomes: a novel concept for bioelectronic nose

Abstract

We describe how mammalian olfactory receptors (ORs) could be used as sensing elements of highly specific and sensitive bioelectronic noses. An OR and an appropriate G(alpha) protein were co-expressed in Saccharomyces cerevisiae cells from which membrane nanosomes were prepared, and immobilized on a sensor chip. By Surface Plasmon Resonance, we were able to quantitatively evaluate OR stimulation by an odorant, and G protein activation. We demonstrate that ORs in nanosomes discriminate between odorant ligands and unrelated odorants, as in whole cells. This assay also provides the possibility for quantitative assessment of the coupling efficiency of the OR with different G(alpha) subunits, without the interference of the cellular transduction pathway. Our findings will be useful to develop a new generation of electronic noses for detection and discrimination of volatile compounds, particularly amenable to micro- and nano-sensor formats.

Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses

“…Heterologous expression of olfactory receptors is extremely difficult, with only a few examples found in the literature (8)(9)(10). The heterologous expression system is often performed in Escherichia coli, yeast, or a mammalian cell line.…”

Bioelectronic Noses Based on Olfactory Receptors

“…Small membrane fragments (nanosomes), obtained from cells expressing a given olfactory receptor can be easily immobilized on commercial L1 Biacore sensorchips. We demonstrate that olfactory receptors maintain their activity in such membrane fragments (Vidic et al, 2006). Since the surface plasma resonance signal is proportional to the molecular weight of the analyte, low-molecular weight molecules, as the majority of odorants, cannot be detected directly by the Biacore3000 system.…”

“…Since the surface plasma resonance signal is proportional to the molecular weight of the analyte, low-molecular weight molecules, as the majority of odorants, cannot be detected directly by the Biacore3000 system. To overcome this problem, we took advantage of the presence of G protein in the nanosomes to monitor receptor activation by an odorant, through the departure of the G α subunit from the preparation (Vidic et al, 2006). The same bell-shaped concentration-dependence responses were obtained as in a whole cells, in terms of threshold concentration and concentration at the maximum, which gives evidence that this receptor functional response in the living cell indeed arises from its own behavior upon odorant stimulation, with no artefactual contribution from the cellular transduction pathway.…”

“…As assessed by surface plasmon, resonance responses monitoring olfactory receptors efficiently discriminate between odorant ligands and unrelated odorants. This system can fruitfully serve to evaluate the comparative coupling efficiency of olfactory receptors to various G a protein subunits, without the interference of cellular contribution (Vidic et al, 2006). Furthermore, this new bioelectronic nose can be applied for the fundamental investigation of molecular mechanisms of olfaction.…”

“…Nanosomes are small vesicles (50-100 nm in diameter). They can be obtained by cell disruption, purification of membrane microsomes and then by size homogenization and miniaturization of microsomes by extensive sonication (Vidic et al, 2006;Casuso et al, 2008). Each of this nanometric vesicular structure was shown to contain one or few receptors of the given type (Casuso et al, 2008;Vidic et al, 2007).…”