Chemistry Reference
In-Depth Information
extracts from bacteria [
79
,
80
] or wheat-germs [
81
,
82
] added to a mixture of
tRNAs, amino acids, nucleotides, enzymes, and cofactors [
83
-
87
]. The increasing
availability of pure isotope-labeled amino acids has allowed a growing number of
groups to use this approach to produce specifically labeled samples for NMR
[
87
-
97
]. In the case of membrane proteins, lipids or detergents can be included in
the reaction mix to maintain them in a soluble state during the synthesis [
91
,
98
,
99
].
Using these methods it is possible for several milligrams of the target be synthesized
from only a few milliliters of reaction mix.
An exciting development in the cell-free approach tomembrane protein production
is mounting evidence suggesting that additives normally intended to maintain
expressed membrane proteins in solution (e.g., lipids, detergents) might not be
necessary. In the absence of added lipids most of the expressed membrane protein
tends to precipitate into an insoluble fraction; however, it has been shown that this
aggregate can be resolubilized in mild detergents [
96
,
100
-
103
]. The validity of this
method has been supported by functional assays on some of these resolubilized
aggregates [
100
,
101
,
103
], and in one case by highly similar NMR spectra for samples
produced from cell-free precipitates vs conventional
E. coli
-based production systems
[
88
]. Meanwhile the purity of these precipitates tends to be high, potentially
eliminating the requirement for subsequent purification steps. Consequently, in
some cases it is possible to directly resolubilize the cell-free expression pellet in the
desired volume and composition of buffer to allow immediate acquisition of NMR
data. Elimination of chromatography, dialysis, fusion protein cleavage, and protein
concentration steps provides significant time savings, and also reduces material costs,
particularly in the consumption of expensive detergents and lipids [
88
].
In addition to the advantages in sample purity offered by cell-free membrane
protein expression, unique possibilities for amino acid specific labeling are also
introduced with this method [
88
,
92
]. In the case of bacterial expression systems,
metabolic processes can break down amino acids added to the expression media,
leading to label dilution and non-specific incorporation. Use of auxotrophic strains
or a limited subset of amino acids can help to get around these issues [
104
,
105
], but
neither approach has the flexibility of specific amino acid labeling that is charac-
teristic of cell-free expression systems. This feature has been particularly useful for
the development of selective labeling approaches to facilitate backbone assignment
of membrane proteins with poor spectral dispersion. Most rely on combinatorial
labeling approaches, with samples having specific combinations of
15
N-labeled
and/or
13
C-labeled amino acids. This allows inter-residue heteronuclear
15
N-
13
C
coupling, such as that used in the HNCO experiment, to rapidly identify adjacent
pairs of amino acids [
106
] (example described in Fig.
1
). There is a range of
combinatorial strategies that have been developed to maximize the information
that can be obtained from a limited number of differently labeled samples [
102
,
107
,
108
]. Since membrane proteins have unique sequence and spectral
characteristics compared to their water-soluble counterparts, a Monte Carlo method
was developed to determine the optimal labeling strategy for a specific protein
sequence [
88
]. Using this approach it was possible to elucidate structures for three
different membrane protein structures in an impressively short 8-month period.
