Production of Olfactory Receptors and Nanosomes Using Yeast System for Bioelectronic Nose - Bioelectronic Nose: Integration of Biotechnology and Nanotechnology - page 129

Fig. 8.2 Effect of galactose induction and temperature on the functional expression of the ORI7
in yeast. Strains were grown in minimal media with 2 % glucose (uninduced) or in minimal media
with 2 % galactose (induced) at 15 or 30 °C. A Western blot analysis of the ORI7 expression levels
was performed on yeast membrane fraction (20 µg of protein per lane). The band near 52 kDa is
attributed to the receptor dimer. (Adapted from Fig. 4 in [ 17 ], with permission)
al., induction was thus performed at 15-19 °C for 60 h, before testing the functional
response or ORs by growth in selective (histidine-deprived) medium, or at 20 °C
for 48 h prior to testing the functional response or ORs by hygromycin-resistance
growth (see below; [ 16 ]). Galactose induction at temperatures higher than 20 °C
prevented the detection of a functional expression of the OR in terms of differen-
tial growth of the yeast strain in response to odorant stimulation. Temperature thus
seems to play a major role in optimizing OR expression under galactose induction
in these yeast strains.
Minic et al. monitored the effects of temperature on OR expression upon ga-
lactose induction by western blotting (Fig. 8.2 ) [ 17 ]. They observed that the ORI7
expression level was increased in membrane fractions from yeasts induced by ga-
lactose, at 15 °C rather than 30 °C. This effect can be ascribed in part to the low
temperature positively affecting the yield of properly folded proteins along the traf-
ficking pathways, and the insertion of the transmembrane domains in the plasma
membrane [ 39 ]. An exceptionally high recombinant production of a membrane pro-
tein, human aquaporin-1 (8.5 % of total membrane protein content), was obtained in
S. cerevisiae at 15 °C, a temperature which was mentioned as crucial for the correct
protein folding [ 40 ]. It was also reported that a temperature downshift to 10-18 °C
leads to an induction of specific cold shock proteins, some of them able to serve as
molecular chaperones [ 41 ]. Interestingly, among these yeast cold shock proteins,
YOP1 is the homologous of mouse REEP1, one of the transmembrane proteins
(RTP1, RTP2 and REEP1) known to increase the functional expression of several
ORs in mammalian cells (HEK293) [ 42 ]. Chaperone proteins, as members of the
Hsp70 family, are also endogenously expressed in mouse mature olfactory neurons
and play an important role in ORs expression [ 43 ]. Moreover, at 15 °C the yeast
membrane composition changes to increase sterols concentration [ 44 ], allowing a
better cryo-protection for cells. It was also reported that sterols play a decisive role