Cell-free expression—making a mark

Available online at www.sciencedirect.com

Cell-free expression — making a
markFrank Bernhard1 and Yuzuru Tozawa2
Cell-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].


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].


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].


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


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.



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


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.



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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First solution structure of a CF expressed membrane protein in functionalconformation.

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First in meso crystal structure of a CF-expressed eukaryotic membraneprotein.

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inhibition of PLP enzymes for selective 15N-labelling andproduction of perdeuterated proteins in H2O. J Biomol NMR2011, 50:35-42.

Practical method for the general inhibition of most label scramblingenzymes in CF extracts.

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Cell-free expression—making a mark