MINIREVIEW
Cell-free translation systems for protein engineering
Yoshihiro Shimizu
1
, Yutetsu Kuruma
2
, Bei-Wen Ying
1
, So Umekage
3
and Takuya Ueda
1
1 Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa-shi,
Chiba, Japan
2 ‘Enrico Fermi’ Center, Compendio del Viminale, Rome, Italy
3 Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho,
Toyohashi, Aichi, Japan
Introduction
Although noncoding RNAs play significant roles in
cellular function [1,2], especially in higher organisms, it
is proteins that dominate most cellular processes. Pro-
teins are the most abundant cellular components and
are responsible for structural, metabolic and regulatory
functions both inside and outside of cells. Thus, inves-
tigation of proteins and elucidation of the molecular
mechanisms underlying their activities are crucial to
our understanding of life.
Generally, owing to their low cost and high produc-
tivity, proteins are prepared using in vivo gene expres-
sion systems. However, the problems associated with
using living cells for recombinant protein expression

Cell-free translation systems have developed significantly over the last two
decades and improvements in yield have resulted in their use for protein
production in the laboratory. These systems have protein engineering appli-
cations, such as the production of proteins containing unnatural amino
acids and development of proteins exhibiting novel functions. Recently, it
has been suggested that cell-free translation systems might be used as the
fundamental basis for cell-like systems. We review recent progress in the
field of cell-free translation systems and describe their use as tools for pro-
tein production and engineering.
Abbreviations
EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; PDI, protein disulfide isomerase; PURE, protein synthesis using
recombinant elements; scFv, single-chain variable fragment of antibody; Sec, secretory; SR, signal recognition particle receptor; SRP, signal
recognition particle.
FEBS Journal 273 (2006) 4133–4140 ª 2006 The Authors Journal compilation ª 2006 FEBS 4133
several hours. As PCR products can be used, synthes-
ized protein may be obtained rapidly from a small
amount of cDNA. In addition, control can be achieved
easily via modified reaction conditions, such as the
addition of accessory elements or removal of inhibitory
substances. Thus, cell-free translation has the potential
to meet many of the needs of preparatory protein
science, and further improvements will accelerate
exploitation of this technology.
In this article, we focus on the techniques relating
to cell-free translation systems for enhancing the syn-
thesis of biologically active proteins, the creation of
cell-like compartments and the synthesis of artificial
proteins.
Overview
Cell-free translation systems are based on the cellular

buted the productivity of the system.
An alternative to cell-extract based systems is repre-
sented by protein synthesis using recombinant elements
(PURE) system [10], which comprises individually
purified components of the E. coli translation appar-
atus. This system is currently not well established, yet
as a fully reconstituted system, it may provide a
greater degree of control than the conventional S30-
directed translation processes. Hence, we believe that
further analyses and developments of the system will
improve the system as a strong tool for producing pro-
teins.
Production of biologically active
proteins
In order for the cell-free translation system to produce
biologically active proteins, additional proteins such as
molecular chaperones may be required to ensure cor-
rect folding [11,12]. In E. coli, these chaperones include
the DnaK system (with its cochaperones DnaJ and
GrpE), trigger factor, and the chaperonin GroEL sys-
tem (with its cochaperonin GroES). Even in S30 sys-
tems in which intrinsic chaperones are present in
abundance, molecular chaperones are supplied to reac-
tions in order to increase synthesis of active-state
proteins [13,14]; this practice has been employed suc-
cessfully in the production of luciferase [15] and active
single-chain variable fragment of antibody (scFv) [13].
Similarly, integration of the chaperonin GroEL system
has also been found to assist folding in rabbit reticulo-
cyte lysates [16].

creatine phosphate (CP) ⁄ creatine kinase (CK). Cell extracts provide
translation factors and enzymes for aminoacylation, whereas in
reconstituted cell-free translation systems [10] the purified compo-
nents are added individually.
Cell-free translation for protein engineering Y. Shimizu et al.
4134 FEBS Journal 273 (2006) 4133–4140 ª 2006 The Authors Journal compilation ª 2006 FEBS
Taking advantage of the absence of such molecular
chaperones in the reconstituted cell-free translation
system [10], it has been used to evaluate the chaperone
dependency on the folding of newly synthesized pro-
teins. The enzymatic activity of MetK could be detec-
ted only in the presence of GroEL ⁄ ES [17], whereas
for anti-BSA scFv, the proportion of soluble and ⁄ or
functional protein increased with the addition of the
DnaK system and trigger factor, but not GroEL ⁄ ES
[18]. Thus, further exhaustive analyses of such depend-
encies will provide not only the reconstituted cell-free
translation system itself but the S30 systems with the
specific supplementation strategies for efficient synthe-
sis of biologically active proteins.
Correct disulfide bond formation in proteins such as
antibodies can be facilitated by the addition of the
redox-dependent chaperone protein disulfide isomerase
(PDI) [19], disulfide oxidoreductase and ⁄ or modification
of the redox conditions. The greatest solubility and
activity of newly synthesized single-chain antibodies
were observed in both E. coli (B W. Ying, H. Taguchi
and T. Ueda, unpublished data, and [13]), and wheat
germ [20] systems when PDI was used under oxidative
conditions. Similarly, the large fragment (Fab) of the

tein, the main player of the multiple cellular functions.
Yu et al. [26] performed the first liposome-encapsu-
lated cell-free protein synthesis using E. coli cell
extracts to synthesize a green fluorescence protein
(GFP-mut1) within egg phosphatidyl choline ⁄ choles-
terol liposomes. As they are easily detected, other
GFPs such as red-shifted GFP or enhanced GFP
(EGFP) have been produced effectively to illustrate the
utility of minimal cell development. For example,
Ishikawa et al. have demonstrated a unique cascading
expression system using a double expression plasmid
carrying genes encoding GFP and T7 RNA polym-
erase, under control of the T7 and SP6 promoters,
respectively [27]. The plasmid, cell-free expression sys-
tem, and SP6 RNA polymerase were trapped inside
liposomes, and production of GFP was then observed,
demonstrating that the two-level cascade actually took
place within the lipid vesicles. Sequential protein
expression (first T7 RNA polymerase, then GFP) was
proven using flow cytometry analysis. In a recent
report that did not involve liposomes, Luisi et al. [28]
divided the cell-free components into several premix-
tures (i.e., plasmids carrying the gene encoding EGFP,
amino acids and E. coli extract), then trapped them in
individual water-in-oil emulsions. Following the pre-
paration of each compartment, all three emulsions
were mixed and EGFP synthesis was observed as com-
partments fused and exchanged their contents, bringing
the reaction components together.
Although there have been many reports in recent

brane integration and translocation were reproduced
as sequential reactions coupled with translation. The
results indicate that the minimum additional cytosolic
factors for membrane integration and translocation are
the signal recognition particle (SRP) ⁄ SRP receptor
(SR) [31] and SecA [32], respectively.
In considering membrane components, the secretory
(Sec) translocon is known to play an important role as
a protein-conducting channel for membrane integra-
tion and translocation [33]. The majority of membrane
proteins integrated through the Sec translocon, which
in E. coli is formed primarily by the essential proteins
SecY and SecE. The Sec translocon binds with high
affinity to the large ribosomal subunit, containing the
elongating nascent polypeptides, which are then integ-
rated cotranslationally. In addition, a Sec-independent
pathway using YidC [34] has been implicated in the
integration of some small molecular mass proteins,
such as the Foc subunit of FoF1-ATP synthase [35].
According to these reports, if either the Sec translocon
and ⁄ or YidC are incorporated into the lipid bilayer of
liposomes (proteoliposomes) in addition to SRP ⁄ SR,
the corresponding synthetic cell has the ability to
generate functional membrane proteins (Fig. 2). Thus,
current studies on protein expression within vesicles
may extend to the biosynthesis of lipid soluble proteins,
several of which play important roles in minimal cells.
Synthesis of artificial proteins
Over the last few decades, several applied technologies,
such as incorporation of unnatural amino acids, have

rated into the lipid bilayer through the force
of peptide elongation. In contrast, some
small membrane proteins are targeted to
YidC, possibly via an SRP ⁄ SR pathway, and
are integrated through YidC alone. Direct
targeting of nascent polypeptides to the Sec
translocon or YidC may occur in the artificial
compartments.
Cell-free translation for protein engineering Y. Shimizu et al.
4136 FEBS Journal 273 (2006) 4133–4140 ª 2006 The Authors Journal compilation ª 2006 FEBS
additional codon–anticodon interactions and expand-
ing the genetic code [42,43]. Thus, reconstituted cell-
free systems have enabled a rewriting of the genetic
code and the incorporation of unnatural amino acids
into proteins [44,45].
Recently, a protein evolution system based on cell-
free translation has been developed (Fig. 3). This
technology is an expanded version of the SELEX (sys-
tematic evolution of ligands by exponential enrich-
ment) system [46], in which functional RNA molecules
can be selected from large libraries through successive
cycles of selection, RNA reverse transcription and
DNA amplification. Because proteins cannot be ampli-
fied by themselves, genotype and phenotype are physi-
cally linked in the system, enabling enrichment of
specific genotypes through successive selection of the
synthesized proteins. Although similar methodology,
such as phage display [47], is widely used for the same
purpose, amplification of the initial library through the
cell-free system enables the use of simple manipulation

on the adjustment of the concentration of DNA and
the size of the emulsions to express a single molecule
of DNA in each compartment. Because these novel
technologies are performed using a DNAÆprotein com-
plex, they have the potential to overcome the unrelia-
bility of RNAÆprotein complex selection, which
is subject to the instability of RNA. Finally, compart-
mentalization has also been achieved using the
Fig. 3. A system for protein evolution based
on cell-free translation. An initial DNA library
is used as the template for cell-free transla-
tion. Following genotype–phenotype
(RNAÆprotein or DNAÆprotein) complex for-
mation, the complexes are selected accord-
ing to protein function. Subsequently, the
RNA of the selected complex is reverse
transcribed (this stage can be omitted for
DNAÆprotein complexes), amplified by PCR
and used as the template for cell-free trans-
lation. Successive rounds of selection result
in enrichment of the desired genotype–phe-
notype complex. Typical complex formations
include: ribosome display, which utilizes a
protein–tRNA–ribosome–mRNA complex
[48]; mRNA display [49] or in vitro virus [50],
which utilize a protein–puromycin–mRNA
complex; CIS display, which utilizes a pro-
tein–RepA–DNA complex [57]; and streptavi-
din–biotin linkage in emulsions (STABLE)
display, which utilizes a protein–streptavi-

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