Biomedical Engineering Reference
In-Depth Information
acetylcholine, by integration with swCNT-FET. The mAChR-based neurotransmit-
ter sensor showed high selectivity and sensitivity to acetylcholine [
84
]. In addition,
the class B GPCR, human parathyroid hormone receptor (hPTHR), was also suc-
cessfully expressed as an inclusion body in
E. coli,
and purified [
80
]. The produced
hPTHR was applied to the development of a hormone sensor, by integration with
the conducting polymer nanoparticle field effect transistor, and offered high selec-
tivity and sensitivity for peptide hormone detection, with human-like performance.
In these works, the Gateway vector system also allowed high-level expression of
GPCR in
E. coli
.
9.3
Production of Olfactory Receptors Using
Animal Cells
9.3.1
Production of Olfactory Receptors Using
Mammalian Cells
The mammalian cells present a suitable environment, including a lipid membrane
composition closed to native tissues, for the production of fully functional ORs [
15
,
27
,
39
,
85
,
86
]. In addition, these cell systems offer native machineries, including
G-protein and ion channels, for OR-mediated signal transduction [
87
-
89
]. Thus, the
mammalian cell system can be the most appropriate for functional study of ORs,
and also for the development of a cell-based bioelectronic nose, using whole cell
expressing ORs in cell membranes [
88
,
90
-
93
]. About 20 ORs have been identified
for their specific odorants, using the screening assay with mammalian cell systems
[
88
,
89
,
95
-
101
]. Although there are many advantages, it has been difficult to uti-
lize the mammalian cell system for the production of ORs for the development of
a cell-based bioelectronic nose and for functional study, because of difficulties in
the expression of ORs [
15
,
27
,
88
]. The mammalian cell system cannot produce the
recombinant proteins in large amount, compared with the bacterial cell system [
15
,
27
,
88
]. Furthermore, ORs are easily degraded in the proteosome, when expressed
in mammalian cells [
36
,
37
,
101
].
Since ORs do not possess the import sequence, fusion tags and accessory pro-
teins are required for the enhancement of expression of ORs in cell membranes [
9
,
88
,
102
]. HEK-293 cells offer a high-level expression of rhodopsin, with efficient
translocation to the plasma membrane [
103
]. By taking this advantage, the first 20
amino acids of bovine rhodopsin, a rho-tag, was found, that facilitates translocation
of synthesized ORs to the plasma membrane [
88
]. This approach has been widely
used as a model system for the study of ligand specificity and function of ORs
[
88
,
100
,
104
,
105
]. In addition, there are also some examples of the use of import
sequences from other GPCRs, which allowed efficient membrane integration for
ORs [
99
,
106
]. The receptor transporting proteins (RTPs) were also screened as
accessory proteins for targeting ORs to the cell surface [
39
,
107
]. It was discovered
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