cell-free expression—making a mark
TRANSCRIPT
Available online at www.sciencedirect.com
Cell-free expression — making a
markFrank Bernhard1 and Yuzuru Tozawa2Cell-free protein production opens new perspectives for
the direct manipulation of expression compartments in
combination with reduced complexity of physiological
requirements. The technology is therefore in particular
suitable for the general synthesis of difficult proteins
including toxins and membrane proteins as well as for the
analysis of their functional folding in artificial environments.
A further key application of cell-free expression is the fast
and economic labeling of proteins for structural and
functional applications. Two extract sources, wheat
embryos and Escherichia coli cells, are currently employed
for the preparative scale cell-free production of proteins.
Recent achievements in structural characterization include
cell-free synthesized membrane proteins and even larger
protein assemblies may become feasible.
Addresses1 Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic
Resonance, Goethe University, Frankfurt am Main, Germany2 Cell-Free Science and Technology Research Center, Ehime University,
Matsuyama, Japan
Corresponding author: Bernhard, Frank ([email protected])
Current Opinion in Structural Biology 2013, 23:374–380
This review comes from a themed issue on New constructs andexpression of proteins
Edited by Imre Berger and Lorenz M Mayr
For a complete overview see the Issue and the Editorial
Available online 27th April 2013
0959-440X/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.sbi.2013.03.012
IntroductionCell-free (CF) expression systems are rapidly evolving
as an alternative option for general protein production
as well as a primary choice for the synthesis of
difficult targets. Frequent examples are toxic proteins
[1,2] or membrane integrated proteins but also appli-
cations for the CF production of vaccines or small
bioactive peptides come into focus [3,4]. Preparative
scale samples of proteins for structural approaches can
be isolated from only few milliliters of reaction
volumes with crude cell extracts from various organ-
isms [5,6�]. The complete control over the amino acid
pool facilitates the efficient and versatile incorpora-
tion of labeled amino acids as a prerequisite for
structural approaches. CF expression is therefore an
established option for the economic labeling of
proteins analyzed by nuclear magnetic resonance
(NMR) spectroscopy [7].
Current Opinion in Structural Biology 2013, 23:374–380
Currently, either extracts from Escherichia coli cells or
from wheat germ embryos are efficient enough for pre-
parative scale protein production and both systems are
implemented as core platforms in structural genomics
projects [8,9]. However, protocols for extract preparations
from alternative sources such as insect and mammalian
cells, protozoa or reconstituted from purified E. coli trans-
lation components are emerging and continuously being
improved in their efficiencies [10–15]. They provide
valuable options for applications with analytical amounts
of proteins such as characterization of function or folding
pathways [16]. In particular insect lysates could have an
increased potential of post-translational protein modifi-
cations after enrichment with microsome fractions or
other supplements.
CF expression protocols must be considered as result of
subsequent optimization levels addressing yield and
sample quality. Complexity of CF protein expression is
largely reduced to the central transcription/translation
process. The basic yield optimization is therefore a rou-
tine approach resulting into high success rates by con-
sidering fundamental issues such as template design and
reaction compound concentrations. The subsequent sys-
tematic modulation of protein quality by taking
advantage of the open accessibility of CF reactions is a
unique option and can require extensive screening of
multitudes of additives or combinations thereof. Supple-
mented compounds can act already co-translationally at
nascent polypeptide chains and promote folding path-
ways or protein stability. This unique option is in particu-
lar valuable for designing artificial hydrophobic
environments upon membrane protein synthesis and
makes CF expression to the most versatile protein pro-
duction system available (Figure 1). We summarize cur-
rent principles for the preparative scale production of
high quality protein samples in CF systems based on E.coli and wheat germ extracts. A further emphasis will be
on emerging structural approaches with membrane
proteins and on recently established tools for efficient
protein labeling.
Cell-free expression in E. coli extracts: theversatile working horseExtract preparation out of a variety of standard E. coli lab
strains has become a routine and reliable procedure that
can be carried out in most biochemical labs. While CF
expression is still usually directed by the T7 RNA poly-
merase, common promoters recognized by the endogen-
ous E. coli RNA polymerase might be considered in future
[17]. Routine Mg2+ ion optimization is highly recom-
mended for best performance [18]. Specific additives
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Cell-free expression Bernhard and Tozawa 375
Figure 1
Strategies for cell-free protein production
(I) Basic reaction configurations
(a) Batch
(a) (b)
(b) CECF
Dialysismembrane
Micelles
Hybrid Micelles
Membrane protein precipitate CF expression of soluble membrane proteins Reconstituted membrane protein samples
Artificial Membranes
Liposomes
Bicelles
Nanodiscs
Mixed micelles
Lipids
RM
RM
20 hr16ºC
Diffusion
FM
FMRM Waste
ValvePumpunit
Dialysismembraneunit
mRNA
FM
(c) Bilayer (d) Filter-and-Feed
(II) Variation of expression conditions; Reaction modes for membrane protein synthesis
Current Opinion in Structural Biology
Configurations of CF reaction and variations of expression mode for producing membrane proteins. (Ia) One-compartment batch configuration for
throughput optimization or preparative expression; (Ib) Two-compartment CECF configuration with reaction mixture (RM) separated from a feeding
mixture (FM) by a dialysis membrane; (Ic) Bilayer configuration providing improved protein synthesis by slow diffusion mixing of the high-density RM
with the low-density FM; (Id) Filter-and-Feed configuration giving optimized protein yield by programmed intermittent exchange of FM and mRNA
across a dialysis membrane unit. II: Reaction modes for membrane protein production as (a) initial precipitate or (b) solubilized with micelles, hybrid
micelles (combination of different detergents or surfactants), mixed micelles (combination of lipids and detergent), or with artificial membranes (by
adding liposomes, nanodiscs or bicelles).
can significantly improve reaction protocols [19,20] and
efficiencies of the basic one compartment batch configur-
ation (Figure 1) could become sufficient for producing
NMR samples [21]. At special conditions, batch reactions
could even be scaled up to industrial scales in bioreactors
[22]. Still generally more efficient for standard lab scale
applications are continuous exchange cell-free (CECF)
configurations providing extra pools of fresh precursors
during the reaction (Figure 1) and several milligrams of
protein can be obtained per milliliter of reaction on a
routine basis [5,18,23].
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For the expression of each new target, protein yield and/or
quality optimization is often mandatory. It has been ident-
ified that poor initial protein synthesis is often correlated to
inefficient initiation of translation and systematic DNA
template optimization can have dramatic effects
[18,21,24,25]. For improved sample quality, the systematic
co-translational screening of protein stabilizers such as deter-
gents or lipids in case of membrane proteins is highly
recommended [18,23]. The disulfide bond formation in
eukaryotic proteins can be modulated by adjusting redox
conditions directly in the CF reactions [20,22,26–28].
Current Opinion in Structural Biology 2013, 23:374–380
376 New constructs and expressions of proteins
Bacterial CF extracts are routinely used for structural
approaches of soluble proteins of both eukaryotic and
prokaryotic origin. A frequent application is structure deter-
mination by NMR which is based on the excellent labeling
options offered by CF expression (see paragraph below). As
an example, more than 1000 solution NMR structures of
eukaryotic proteins or protein domains have been deter-
mined by the RIKEN Genomic Sciences Center [8].
Wheat germ extracts: mimicking eukaryoticenvironmentsEukaryotic CF translation systems derived from wheat
germ (WG) extracts have been fundamentally reinvented
by improved stability of translation activity, utilization of
non-capped mRNAs in combination with translational
enhancer cis elements [6�]. New translation reactions
and implementation of robotic systems facilitate through-
put approaches [29,30�]. The special bilayer configuration
complements the more common CECF configuration in
WGCF reactions (Figure 1). The recently developed
‘Filter-and-Feed’ translation method (Figure 1) enables
robotic synthesis of up to 50 milligrams protein in reaction
volumes of 10 milliliter in 18 hours [30�]. The WG is kept
in a dehydrated state for hibernation and most organelles
such as plastids, mitochondria and vacuoles are not yet fully
developed. Consequently, activity of amino acid metabo-
lizing enzymes is low. The hexaploid genome of cultivated
wheat crops results into increased grain sizes, thus facil-
itating the preparation of purified embryos. Although
wheat grains as well as other plant seeds contain various
enzyme inhibitors that affect translation, most of them are
localized in the endosperm. After the physical detachment
of the embryo part (germ) from endosperm, further exten-
sive washing of the germ followed by extraction results in
inhibitor-free translation efficient extracts [6�].
In general, preparation of WG extracts is more compli-
cated and time consuming if compared with the prep-
aration of E. coli lysates. User of WGCF systems therefore
may depend more on commercial sources. As an eukar-
yotic system, WG extracts may contain components that
are necessary for promoting correct folds of translated
eukaryotic multi-domain proteins [31]. However, eukar-
yotic as well as prokaryotic proteins can generally be
produced in both systems if protocols have been opti-
mized according to the individual system requirements.
WG extracts are considered for protein structural studies
since the early 2000s and have been adopted as one main
tool for structural studies of eukaryotic proteins by pre-
dominantly NMR approaches [9]. More recently, also
crystallization and X-ray analysis of proteins expressed
in the WGCF system becomes popular [32].
Cell-free production of membrane proteins:particular requirements of challenging targetsCF expression of membrane proteins deserves a particu-
lar consideration as it provides a new and versatile
Current Opinion in Structural Biology 2013, 23:374–380
pipeline for the efficient production of this usually very
difficult to synthesize class of proteins [23,33,34]. CF
extracts from E. coli as well as from WG tolerate a wide
range of detergents as additives for the co-translational
solubilization of CF expressed membrane proteins
(Figure 1) [23,33,35]. The portfolio of potential hydro-
phobic additives for membrane protein solubilization is
continuously increased by new surfactants, amphipols or
other compounds [36–40]. The particular compound(s)
suitable to result into optimal quality of a given mem-
brane protein has to be identified by systematic screening
and compound mixtures might be considered as well
[18,35,41,42].
If detergents should be avoided, the co-translational
insertion into provided lipid bilayers is becoming an
interesting option [42,43]. The addition of vesicles or
liposomes of defined compositions into CF reactions
could result into functionally active membrane proteins
that could be analyzed in natural lipid environments of
desired composition [43,44�,45,46]. The underlying
mechanisms for efficient membrane protein transloca-
tion in the artificial in vitro reaction environment are
currently subject of research and might differ from
known in vivo pathways [44�]. In particular, the recent
combination of CF expression with the nanodisc tech-
nology appears to be promising (Figure 1). The co-
translational or post-translational insertion of CF
expressed membrane proteins into nanodiscs with differ-
ent membrane compositions is emerging as an excellent
tool for their functional and structural characterization
[43,47,48�,49�]. In the absence of hydrophobic additives,
the CF synthesized membrane proteins will precipitate
but could retain in many cases at least partially folded
conformations [27,42,50,51]. After non-denaturating
post-translational solubilization in detergent, such CF
produced membrane protein precipitates are frequently
already suitable for functional and structural studies
[27,41,42,50,51,52�].
Structural studies of CF-expressed membrane proteins
have been approached so far in particular by NMR
spectroscopy (Table 1). The NMR structure of the C-
terminal fragment of human presenilin-1, a subunit of the
g-secretase complex, has been solved with re-solubilized
CF produced precipitates [51]. Systematic NMR screen-
ing of re-solubilized CF produced membrane protein
precipitates can result into the routine identification of
samples suitable for structural evaluation [50,52�]. The
NMR structure of a functional membrane protein cotran-
slational solubilized in the presence of detergent/lipid
mixtures was first solved from a bacterial proteorhodopsin
[53�]. Even the structural evaluation of CF expressed
membrane complexes appears to become feasible [54�]. A
542 kDa bacterial ATP synthase complex composed out
of 25 protomers did fully assemble during CF production
in the presence of detergents.
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Cell-free expression Bernhard and Tozawa 377
Table 1
Structural approaches with CF expressed membrane proteins
Protein Origin Characteristics Method Reference
Proteorhodopsin Bacteria Proton pump NMR [53�]
Rhodopsin II Acetabularia sp. Proton pump X-ray [57�]
Presenilin-1 CTF Human Subunit of g-secretase NMR [51]
ATP synthase Caldalkalibacillus sp. 542 kDa complex EM [54�]
hVDAC1 Human Ion channel X-ray [55]
EmrE E. coli Multidrug transporter X-ray [56]
Transmembrane domains of ArcB, QseC, KdpD E. coli Histidine sensor kinases NMR [50]
Six inner membrane proteins Human Unknown NMR [52�]
Reports on the crystallization of CF expressed membrane
proteins are still emerging and comprise lower resolution
crystals of the human voltage-dependent anion channel-1
[55] and crystals of the selenomethionine labeled deriva-
tive of the bacterial small multidrug transporter EmrE
[56]. A recent highlight was the 3.2 A structure of an
eukaryotic rhodopsin by in meso crystallization after CF
expression in the presence of a mixture of lipids and
detergent [57�].
Protein labeling in CF systems: essentialprerequisites for structural approachesEfficient labeling of proteins with non-natural or chemi-
cally modified amino acids is one of the major benefits of
CF expression systems for NMR and X-ray structural
analysis [5,8,9]. Toxic side effects caused by the incorp-
oration of amino acids modified with isotopes, biotin or
with fluorescent or photoreactive groups into proteins as
well as many scrambling problems resulting from meta-
bolic conversions of the supplied label precursors are
generally eliminated or significantly reduced by using
CF expression. The labeling costs in particular for
NMR are lower than by in vivo expression and can be
even more reduced by regeneration of unstable amino
acids [58]. Moreover, there are no restrictions for selective
or combinatorial labeling schemes. Consequently, WG as
well as E. coli extracts have been established as core
platforms for the preparation of labeled protein samples
suitable for NMR structural analysis and also for crystal-
lization [5,8,9]. At least 1200 entries in the Protein Data
Bank (PDB) can currently be attributed to cell-free
expressed proteins. During the last five years, the annual
number of new entries is relatively constant while first
structures of cell-free expressed membrane proteins start
to appear.
A variety of supplements and extract modifications have
been developed in order to increase label stability and to
reduce residual scrambling problems for NMR appli-
cations in E. coli as well as in WG extracts. Extracts from
scrambling enzyme deficient strains or addition of chemi-
cal inhibitors address specific scrambling problems [59–61]. A more general approach is the broadband inhibition
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of cofactor pyridoxal phosphate containing enzymes by
treatment of the CF extract with NaBH4 [62�]. Besides
uniform and amino acid specific labeling, a variety of
combinatorial labeling schemes have become standard
tools for the structural determination of CF expressed
proteins by NMR. Such specific labeling schemes are in
particular required for membrane proteins due to the
generally increased number of ambiguous assignments
[50,51,52�,53�]. The stereo array isotope labeling (SAIL)
approach by using synthesized amino acids with specific
labeling patterns almost exclusively relies on efficient CF
protein production in smaller volumes [63]. The potential
of SAIL applications for spectral simplification and sen-
sitivity enhancement of in particular larger proteins is still
becoming extended [64]. The selective study of func-
tional sites becomes possible by advanced strategies of
site-specific labeling approaches implementing suppres-
sor tRNAs mischarged with unnatural amino acids either
by chemical acylation or by engineered aminoacyl-tRNA
synthetases [65]. The recently improved protocols for the
CF labeling of proteins for NMR applications comp-
lement the already established tools for efficient incorp-
oration of selenomethionine for X-ray analysis or of
modified amino acids for functional studies [66].
ConclusionsCF expression systems based on either E. coli or WG
extracts are established tools for the preparative scale
production of protein samples for structural applications.
The considerable success rate in protein production in
combination with the high versatility in reaction modi-
fication allows to focus on selected targets by systematic
protocol development. The manifold options to design
artificial expression environments of proteins open new
avenues for their synthesis and characterization. CF
expression appears to become one method of choice
for the analysis of difficult proteins such as membrane
proteins, toxins or complexes and it is already a prime
working platform for the general production of labeled
samples for NMR applications. Future developments
including a broader variety of efficient CF extract
sources and improved protocols for the preparation of
crystallization grade membrane proteins come into sight.
Current Opinion in Structural Biology 2013, 23:374–380
378 New constructs and expressions of proteins
As major challenge, there still remains the preparative
scale CF production of post-translationally modified
proteins.
Acknowledgements
We thank Dr Yaeta Endo for his critical comments on this manuscript. Thiswork was supported by MEXT KAKENHI Grant Number 24117516 toY.T.
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