Production of Olfactory Receptors and Nanosomes Using Yeast System for Bioelectronic Nose - Bioelectronic Nose: Integration of Biotechnology and Nanotechnology - page 126

from 38 to 90 %, with a hypervariability of amino acids lining the odorant binding
pocket (TMIII to VII and extracellular loop 2) [ 25 - 30 ], related to the diversity of
the odorants that ORs can bind. Final PCR products were sequenced so as to iden-
tify the ORs expressed in the responsive neurons. Although this multi-step approach
is risky, this protocol allowed us to identify several ORs for a given target odorant.
Finding several receptors responding to one odorant is not surprising because the
number of ORs specific for a given odorant is still unknown [ 31 ] and can vary great-
ly depending on the odorant. However, since single cell approach often gives false
positive results, it is necessary to check the functional response of the identified
ORs by expressing them in heterologous expression systems, such as S. cerevisiae.

8.3 Yeast Plasmid Constructs and Yeast Strains

The yeast S. cerevisiae growth characteristics and genetics have been well known
for years, making it a suitable host for OR expression.

8.3.1 Plasmids Constructs

The pJH2 plasmid used for the first time for a successful expression of a GPCR
in yeast [ 9 ] also allowed expression of 2 different ORs in S. cerevisiae , under the
control of the galactose-inducible GAL1 promoter combined to the GAL4 synthesis
under the control of the GAL10 inducible promoter [ 16 , 17 ]. Indeed, overexpres-
sion of the GAL4 protein has been shown to increase the expression of the OR
cDNA under the control of the GAL1 promoter [ 32 ]. Insertion of the OR cDNA in
pJH2 was obtained by homologous recombination. However, this plasmid was not
useable to create cassette plasmids.
Following these pioneers' works, new expression vectors (pESC vectors) were
developed by Stratagene to express heterologous proteins in yeast cells. From these
vectors, three different types of shuttle plasmids were developed.
Because the N-terminal region of GPCRs often plays an important role in ex-
pression or membrane trafficking of the protein and the C-terminal region interacts
with the G protein, Radhika et al. constructed an expression cassette vector which
contains an insert encoding the N-terminal part (amino acids 1 to 61) and the C-ter-
minal part (amino acids 295 to 327) of the rat ORI7, separated by multiple cloning
sites in which the ligand-binding pocket of any OR can be inserted in frame [ 18 ].
Fukutani et al. also used two different N terminal parts of the I7 cDNA sequences
to construct chimeric ORs, and cloned them into a pESC vector [ 19 ]. These chime-
ric ORs (32 or 58 amino acids from the ORI7) were translated with the c-myc tag of
the vector at their N-terminus.
Alternately, pESC vectors were modified as described in Wade et al. to allow the
expression of ORs tagged at their N terminus with either the c-myc or the human