Biomedical Engineering Reference
In-Depth Information
cell-free based methods. These in vitro approaches provide much greater
control over the membrane protein production process and have been shown
to achieve yields appropriate for biophysical and structural studies.
Furthermore, they avoid problems with toxicity of over-expression, proteolytic
degradation and appropriate targeting to membrane compartments. Cell-free
expression typically uses E. coli, wheat germ or eukaryotic (e.g., rabbit
reticulocyte) cell lysate. Cell-free expression methods have been successfully
used for expression of a number of membrane proteins.
86-88
Three approaches
are available for membrane proteins: proteins may be expressed in the absence
of detergent resulting in the formation of aggregates, which can be solubilised
by addition of detergents without the need for refolding and avoiding the need
for further purification from the reaction components, which remain soluble;
proteins may be expressed directly into detergents or other surfactants e.g.,
fluorinated surfactants or amphipols although the detergents used must be
mild to avoid affecting the transcription and translation machinery; or proteins
may be expressed into lipid-based structures e.g., liposomes, cell-membrane
fractions, lipomicelles, bicelles or nanodiscs.
89
Cell-free approaches are particularly well suited to NMR studies since they
allow extremely high flexibility in labelling approaches enabling, in addition to
uniform
13
C- and
15
N-labelling, labelling of individual amino acids.
90
Furthermore, by the use of engineered tRNA molecules, specific amino acid
types can be labelled.
91
This approach should enable the simplification of
complex spectra of large membrane proteins, providing a key avenue to NMR
structure determination.
92
The small reaction volumes make efficient use of
expensive labelling reagents and approaches to allow deuteration have also
been proposed.
90,93-95
Difficulties remain in optimising cell-free systems and many solubilising
detergents are known to inhibit the cell-free synthesis method. Issues of
scalability also need to be solved and the systems remain expensive. However,
considerable advances are being made, which offer the promise of a more
systematic approach. For example, recent studies have identified key
detergents which inhibit the cell-free methods including n-octyl-b-
D
-glucopyr-
anoside, n-dodecyl-b-
D
-maltoside (DDM), and CHAPS (3-[(3-cholamidopro-
pyl)dimethylammonio]-1-propanesulfonate, whilst the polyoxyethylene-alkyl-
ether detergents (brij-35, brij-58, brij-78, and brij-98) and digitonin were found
to be suitable for cell-free approaches.
96,97
(For structures of detergents and
other surfactants discussed in the text, see Figure 12.1.) Furthermore, high
protein yields via this system are not always compatible with production of
functional protein, and may be particularly dependent on the solubilising
detergent; however, with appropriate optimisation, functional membrane
proteins can be produced.
93
Overall, this system appears to offer considerable
promise for structural studies of a range of membrane proteins, as
demonstrated by a recent study which used cell-free expression, combined
with sequence-optimised combinatorial
15
N,
13
C-labelling, to achieve rapid
backbone assignment and subsequent backbone structure determination of the
