Biomedical Engineering Reference
In-Depth Information
in terms of lipid composition, 68 although baculovirus membranes are a
considerably more favourable environment than their bacterial analogues. As
with other expression systems, widely varying conditions and yields are seen
for different GPCRs: in a recent study which looked at 16 different GPCRs in
three different cell lines, no one set of optimal conditions could be identified
and even closely related receptors required different conditions. 69 Whilst
baculovirus has been successfully used for protein expression for crystal-
lography, 65,66,70 NMR studies are limited due to the difficulties in selective
labelling. Amino-acid type-specific 15 N-labelling has been demonstrated, 71,72
however, since it is not possible to grow insect cells on minimal media, uniform
isotope labelling requires the use of costly labelled media. 73 Nevertheless,
successes have been reported for uniform 13 C/ 15 N labelling of Abl kinase. 74,75
Deuteration, which is toxic for higher eukaryotic cells, remains an obstacle and
further development will be needed for this method to be of widespread use to
NMR.
12.2.4 Mammalian
For expression of mammalian membrane proteins, and especially GPCRs,
mammalian systems such as Chinese hamster ovary (CHO) cells, or HEK293
cells present an attractive solution since GPCRs are natively expressed in
functional form. 76,77 Nevertheless, considerable development is required in this
area as problems remain with the stability of cell lines, toxicity of over-
expression, scalability and isotope labelling. As with baculovirus systems,
isotope labelling is complicated as it is not possible to grow mammalian cell
cultures in minimal media. Consequently approaches typically rely on uniform
labelling using 15 N- or 15 N/ 13 C-labelled amino acids, which can be purified
from labelled bacterial or algal cultures, supplemented with glutamine and
cysteine 78 and this has led to a number of successful NMR studies. 79
Commercial media have also been used for isotope labelling, enabling a study
of the GPCR rhodopsin. 80 The cost of these approaches as well as toxicity of
perdeuteration means that many studies rely on amino-acid type-selective
labelling. 81-83
Despite these difficulties, a number of studies show promise. A 2A receptor
expression has been demonstrated at levels equivalent to 2 mg L 21 , 84 whilst in
another study, the b 2 -adrenergic receptor was expressed at 1.7 mg L 21 . 85 More
recently CHO cells have been used in a receptor-stabilisation strategy 76 whilst
HEK293 cells have been used to express constitutively active rhodopsin. 77
Nevertheless, considerable development will be needed to enable this system to
be of widespread benefit to NMR.
12.2.5 Cell-Free Expression
An alternative approach to in vivo expression, which seems to offer
considerable general promise for membrane protein expression, is the use of
Search WWH ::




Custom Search