Production of Olfactory Receptors and Nanosomes Using Yeast System for Bioelectronic Nose - Bioelectronic Nose: Integration of Biotechnology and Nanotechnology - page 133

indicates the presence of 90 receptors per cell with a Ka value of 0.1 × 10 4 M −1 [ 18 ].
This is in agreement with the 10-100 µM threshold levels seen for this ligand and
the view that ORs are generally low-affinity receptors [ 51 , 52 ].

8.5.3 Fluorescence

Radhika et al. monitored the GFP response of WIF-1α-ORI7 cells to characterize
the functionality of this prototypic yeast strain [ 18 ]. This strain was engineered with
the mammalian olfactory signalling pathway so that the functional response of OR
to the specific odorant stimulation induces fluorescence emission after synthesis
of the reporter protein GFP. Indeed, the cells were exposed to a range of octanal
concentrations, known to be one of the preferential ligands of the ORI7 receptor,
and GFP expression was measured in a microplate reader at intervals of 1 h,
revealing a ligand dose-dependent increase in GFP expression. The maximal levels
of fluorescence of the cells were reached by 3 h. Moreover, the GFP response of the
cells is well correlated with the known different affinity of ORI7 for closely related
aliphatic aldehydes (heptanal or hexanal), indicating that GFP is a useful reporter to
study the OR response in WIF-1α-OR strains. This strategy was then used by these
authors to identify an OR capable of detecting 2,4-dinitrotoluene (DNT). The WIF-
1α-OR transformants, obtained by the cloning of a library of OR cDNAs and trans-
formation of the yeast cells, were exposed to 50 µM DNT and scored for emitted
fluorescence. Several DNT-positive clones were identified, which were enriched
by serial dilution, and their response to DNT 25 µM monitored by fluorescence
microscopy. The recovery of the plasmids from positive cells allowed to sequence
the insert in the expression vector. This strategy involving the GFP reporter allowed
the authors to identify a new rat olfactory receptor, exhibiting an extensive sequence
homology with two different mouse olfactory receptors (Olfr2 and mOR226-1) that
can detect DNT. This system thus appears as reliable, easy to handle, and useful to
identify OR ligands.

8.5.4 Luminescence

To address the functional integrity of the olfactory receptors expressed in yeast
cells, two labs have developed a luciferase reporter bioassay by modifying the yeast
pheromonal signal transduction pathway (see yeast strain constructs). The firefly
luciferase gene is used as luminescence reporter gene due to its high sensitivity.
Fukutani et al. tested the sensing abilities of 3 chimeric ORs for DNT [ 19 ]. They
investigated the role of the N-terminal region of ORs on their functional expres-
sion (see plasmid constructs). After induction of the ORs expression in galactose
medium, the cells were incubated in media containing the ligand. At 1 mM DNT,
the 2 chimeric ORs obtained by replacing the N-terminal region of OR226 by the
N-terminal region of ORI7 exhibited a measurable functional response. The chi-
meric OR for which the replacement was up to the first intracellular loop of ORI7