Biomedical Engineering Reference
In-Depth Information
Membrane proteins are typically expressed to the cell membrane where they
can be extracted in folded form, for example pSRII.
19
A variety of tags are
available to target proteins to the membrane such as the signal peptide of
periplasmic maltose binding protein (MBP),
31,32
and the membrane-integrat-
ing protein, mistic.
33
C-Terminal thioredoxin fusions have also been reported
to have a stabilising effect,
31
as well as other tags such as glutathione S-
transferase (GST)
34
and ketosteroid isomerase.
35
Alternatively, membrane proteins may be targeted to inclusion bodies where
they are protected from proteases and can subsequently be extracted and
refolded. Since proteins remain unfolded, the lack of eukaryotic post-
translational modifications in E. coli does not affect the expression level. In
a recent study which investigated expression of y100 GPCRs in E. coli,
reasonable yields were obtained for 23 receptors, with considerably higher
yields obtained from expression in a fermenter rather than flasks.
36
Whilst
optimisation over a range of conditions is advised, Gateway vectors, especially
pDEST17oi, E. coli strain C43, 37 uC and fermenter expression proved a
particularly promising choice.
36
Subsequent refolding of two expressed targets,
the mouse cannabinoid receptor 1 (muCB1R) and the human parathyroid
hormone receptor 1 (huPTH1R), was achieved using SDS to inhibit
aggregation and solubilise the inclusion bodies, producing large quantities of
functional receptor.
37
Whilst to date very few other GPCRs have been
produced by refolding
34,35,38
this approach has been successfully used for other
membrane proteins
13
suggesting that this may prove a suitable approach.
E. coli expression is considerably more difficult for GPCRs as key post-
translational modifications, such as glycosylation, are absent. Whilst this can
enable production of homogenous populations of protein, many GPCRs such
as rhodopsin and the somatostatin, b
2
-adrenergic, human thyrotropin, and
bombesin BB2 receptors are known to need modifications for ligand binding
and/or G protein-coupling.
39-43
However, in some cases modifications have
been found to be unnecessary for function e.g., palmitoylation in the M2
receptor.
32
The lipid composition of E. coli membranes is also significantly
different from mammalian cell membranes, particularly due to the lack of
cholesterol; whilst in some cases this seems to have no influence
32
in other cases
it appears to have a significant effect on expression and functionality.
44,45
12.2.2
Yeast
A host that appears to be extremely promising for the expression of GPCRs in
particular, is yeast cells. Like E. coli, they can be cultured to high cell density
and are easily scalable. As a eukaryotic cell, they possess a variety of
intracellular membrane compartments into which membrane proteins can be
targeted. Yeast also provides some post-translational modifications which can
aid expression of functional protein to high levels. For the purpose of NMR
studies, minimal-medium approaches have been developed for yeast expression
enabling uniform
15
N,
13
Cand
2
H-labelling, as well as the potential for amino-
