Biomedical Engineering Reference
In-Depth Information
acid-specific labelling. However, due to the complexity of the yeast expression
system, and particularly the induction conditions, labelled expression is more
costly than in E. coli.
Three yeast expression hosts are currently available. Pichia pastoris is able to
achieve high cell densities, with expression controlled by methanol via the
alcohol oxidase promoter (AOXI). A recent study demonstrated expression of
20 functional GPCRs in Pichia pastoris; a variety of different receptors from
different organisms were studied and eight were optimised sufficiently to
enable structural studies.
46
Other studies have demonstrated expression of the
human CB1 cannabinoid receptor,
47
5HT
5A
serotonin receptor,
48,49
b
2
-
adrenergic receptor,
48
m-opioid receptor,
50
endothelin-A and endothelin-B
receptors,
51,52
and CB2 cannabinoid receptor.
53
Saccharomyces cerevisiae is typically cultured to lower expression levels than
P. pastoris, and is galactose-inducible, under the control of the GAL promoter.
Recently, the A
2A
receptor was expressed in 1 mg L
21
quantities.
54
Such
amounts represent the highest yet reported for A
2A
, indicating the potential of
this system.
55
Schizosaccharomyces pombe, with vectors under the control of
thiamine, can be used to express functional GPCRs,
56
but has proved less
popular than the two aforementioned systems.
Whilst the position of yeast between bacterial and mammalian expression
systems seems to offer many advantages, a reduced range of post-translational
modifications and heterogeneous addition of such modifications may result in
difficulties for structural studies. Purification is also hampered by the thick cell
walls and the promoters used may necessitate isotope-labelled inducing agents,
increasing the cost of labelling. Differences in membrane lipid composition
from mammalian cells may also cause difficulties in expression of functional
protein.
To date, Pichia pastoris seems to be the most popular expression host and a
number of membrane proteins have been purified and crystallised including
potassium channels
57,58
and aquaporins.
59,60
Economical approaches for
15
N/
13
C-labelling have been demonstrated
61
whilst partial deuteration is also
possible.
62
Amino-acid-type selective labelling has also been demonstrated on
a prototrophic strain, with successful incorporation of [a-
15
N]Cys, -Leu, -Lys
and -Met.
63
Recently uniformly
13
C/
15
N-labelled fungal microbial-type seven-
transmembrane helical proteorhodopsin from Leptosphaeria maculans has
been expressed in P. pastoris permitting the recording of high-resolution magic
angle spinning solid-state NMR spectra.
64
12.2.3 Baculovirus/Insect Expression
Insect cells offer a system much closer to that of mammalian cells, and provide
a more established system for functional expression of GPCRs.
65-67
Insect cells
provide a range of post-translational modifications and contain a number of
GPCR-interacting proteins (G
sa
,G
oa
,G
ia
in Sf9 cells) making this a system
with native ability to produce functional receptors.
21
Difficulties may remain
