Biomedical Engineering Reference
In-Depth Information
reproducible reconstitution cannot be guaranteed [
27
]. Therefore, the reconstitu-
tion of recombinant GPCRs is normally carried out with artificial lipid membranes
[
27
]. Various artificial membrane compositions should be tested for reconstitution
with membrane-mimetic environments [
14
]. The purification and reconstitution of
recombinant membrane protein is often a matter of trial and error, using detergents.
Since there is no well-established strategy for the systematic and efficient pro-
duction of GPCRs from inclusion bodies, there are not many examples reported. For
the successful expression of GPCRs in
E. coli
at high-level, optimization of several
factors is needed, including the promoter and origin in the bacterial expression vec-
tor, fusion partner, strain of
E. coli
, culture condition and induction condition [
62
].
These conditions should be different for each GPCR, and the optimization process
can be time consuming and labor intensive [
63
]. The systematic evaluation of ex-
pression methodologies for obtaining a large amount of inclusion bodies of GPCRs
in
E. coli
has been carried out on 100 mammalian GPCRs, by exploring various dif-
ferent expression vectors and
E. coli
strains [
42
]. The Gateway bacterial expression
vectors and the C43
E. coli
strain allowed a large amount of insoluble GPCRs. It is
also demonstrated by other reports that the Gateway vector and the C43 strain offer
high-level expression of GPCRs as inclusion bodies [
28
,
31
,
62
-
64
]. The produc-
tion process was scaled up using large-scale fermentation, and several 100 mg of
GPCRs per liter were produced [
42
,
65
].
9.2.2
Production of Olfactory Receptors from Bacterial Cells
With advances in the production of GPCRs, the production of olfactory receptors
from bacterial cells has become easier, and allowed efficient recognition elements
for bioelectronic noses, with human-like performances.
The first example for successful production of an olfactory receptor from bacte-
rial cell is the production of olfactory receptor (OR) 5 [
29
]. The OR5 was produced
from
E. coli
as a gluathione-S-transferase (GST) fusion protein, in the form of
inclusion body. A mutant form of OR5, which contained some positive charge in
the loop connecting transmembrane domain 1 and 2, was constructed, to block the
insertion into cell membranes. It is known that the loops with some positive charge
make it difficult to translocate to the periplasmic side in bacterial cells [
66
,
67
]. The
toxicity along with the translocation in membranes was reduced, and the mutant
form of OR5 was successfully expressed as the inclusion body. The overexpressed
OR5 was solubilized with strong anionic detergent. It failed to use a GST affin-
ity column for the purification of the receptor, which was probably because the
GST domain was denatured and misfolded, under the condition of strong anionic
detergent. However, a Ni-NTA agarose column allowed successful purification of
hexahistidine tagged olfactory receptor. After the purification, the detergent condi-
tion was replaced with digitonin, which is a well-known detergent for the stabiliza-
tion of GPCR [
68
], and reconstituted in lipid vesicles composed of artificial lipids.
This study suggested that the difficulties in expression of OR might be because of
the toxicity, during the insertion of receptors into the bacterial membranes. Fur-
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