Proteins are pro-duced only when template DNA or mRNA is added to the reaction mixture, followed by incubation for Keywords cell-free protein synthesis; chaperone; disulfide bond formati
Trang 1Cell-free translation systems for protein engineering
Yoshihiro Shimizu1, Yutetsu Kuruma2, Bei-Wen Ying1, So Umekage3and Takuya Ueda1
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 include protein degradation and aggregation, or loss of template DNA Furthermore, it requires several labori-ous experimental steps including DNA cloning in the vector, DNA transformation in cells, and overexpres-sion of the desired protein in cells Thus, there are limitations associated with using in vivo technology for protein production
Cell-free translation represents an alternative to
in vivo expression, and rapid progress is being made in this field, which is gaining attention for its simplicity and high degree of controllability Proteins are pro-duced only when template DNA or mRNA is added
to the reaction mixture, followed by incubation for
Keywords
cell-free protein synthesis; chaperone;
disulfide bond formation; in vitro selection;
liposome; minimal cell; ribosome display;
translation; unnatural amino acid
Correspondence
T Ueda, Department of Medical Genome
Sciences, Graduate School of Frontier
Sciences, University of Tokyo, FSB401,
5-1-5, Kashiwanoha, Kashiwa-shi, Chiba
prefecture 277-8562, Japan
Fax: +81 4 7136 3642
Tel: +81 4 7136 3641
E-mail: ueda@k.u-tokyo.ac.jp
(Received 8 May 2006, revised 20 June
2006, accepted 26 June 2006)
doi:10.1111/j.1742-4658.2006.05431.x
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.
Trang 2several 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
ribosomal protein synthesis system Generally, the
sys-tem is composed of a cell extract (referred to as the
S30 fraction) from Escherichia coli, wheat germ, or
rabbit reticulocytes These extracts are supernatants
from a 30 000 g centrifugation and contain
compo-nents such as ribosomes, translation factors,
amino-acyl-tRNA synthetases, and tRNAs, which are
required for production of protein Efficient protein
production may require supplementation of the S30
extract with additional RNA polymerase, as well as
several enzymes for energy regeneration and their
sub-strates (Fig 1)
The productivity of S30-directed, cell-free translation
systems has improved greatly over the last two
dec-ades In 1988, the continuous-flow cell-free system [3]
represented the first demonstration that cell-free
trans-lation could be utilized as a tool for producing protein
This system relied upon a continuous supply of energy
source and amino acids, resulting in a significant
increase in productivity Although this method was not
used widely due to its complexity and variable
repro-ducibility of yield, the concept resulted in the
subse-quent development of the continuous-exchange
cell-free [4] and the bilayer cell-free systems [5] Using
these processes, milligram quantities of product were
achieved from a 1 mL reaction Furthermore, the
developments of the reaction condition such as,
opti-mization of the E coli system [6,7], improved
prepar-ation of wheat germ cell extract [8] and development
of the energy regeneration system [9], have also
contri-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 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]
protein synthesis on ribosome
Aminoacylation amino acid tRNA ATP
aminoacyl-tRNA
Transcription temlate DNA ATP/GTP/CTP/UTP RNA polymerase
mRNA Energy regeneration system
Enzymes Substrates (PEP/PK system CP/CK system etc.)
ATP/GTP
Translation factor Initiation factor Elongation factor Termination factor
Fig 1 The cell-free protein synthesis system Efficient protein syn-thesis requires transcription of mRNA, aminoacyl tRNA, energy pro-vision, and translation factors Transcription of mRNA requires template DNA, ribonucleotides and enzymes such as T7 and SP6 RNA polymerases Translation requires factors for initiation, elonga-tion and terminaelonga-tion, as well as components for aminoacylaelonga-tion of tRNA, such as amino acids, tRNA and ATP The energy regener-ation system requires enzymes and their substrates such as phosphoenolpyruvate (PEP) ⁄ phophoenolpyruvate kinase (PK) and 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.
Trang 3Taking 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
catalytic antibody 6D9, which comprises several
disul-fide bonds, was expressed successfully under oxidative
conditions [21] In the reconstituted cell-free system,
biologically active alkaline phosphatase has also been
found to be synthesized under oxidative conditions [22]
Therefore, these studies indicate that expression of
correctly folded and functional proteins can be
achieved in cell-free systems by the addition of folding
helpers, and that the flexibility of these systems
repre-sents a powerful means of generating mature protein
Synthesis of membrane proteins for
minimal cells
The goal of the new and rapidly developing field of
synthetic biology is the development of a minimal cell,
also called an artificial cell [23] Minimal cells are
designed to comprise the least number of molecular
components and genes [24], while still being considered
alive The classical approach involves entrapment of
components (genes, enzymes, ribosomes, etc.) in a
syn-thetic compartment, in order to separate them from the
external environment These compartments are usually
produced by lipid vesicles or liposomes, because they
closely resemble the cellular envelope Based on the
concept that translation is one of the central cellular
processes required for life, cell-free transcription⁄
trans-lation systems have been widely used in the
develop-ment of simple cellular models [25] Indeed, when
functional protein synthesis occurs inside liposomes, it provides a platform for simulating a complex cellular activity because the product of the system is the pro-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 years of cell-free expression in liposomes, no one has succeeded in synthesizing functional membrane pro-teins in these systems However, Noireaux and Libc-haber have succeeded in synthesizing a-hemolysin (from Staphylococcus aureus) within liposomes, using
an E coli extract cell-free system [29] a-Hemolysin is
a water soluble monomeric protein that is able to self-assemble in a lipid bilayer as a homoheptamer, gener-ating a selectively permeable pore They used the a-hemolysin pore as a gate for nutrient transportation into the liposomes, and by supplementing energy and substrates from outside the liposome, were able to extend protein synthesis up to four days Furthermore, using the ability of a-hemolysin to self-assemble, an a-hemolysin-EGFP fusion protein was successfully formed on the membrane surface [25] However, although these results appear to represent impressive achievements in minimal cell development, it must be remembered that a-hemolysin is a water soluble (not lipid soluble) protein
Trang 4How can we generate integral membrane proteins
within liposomes, and is there any way to integrate
proteins into the lipid bilayer in the proper
conforma-tion? Recent progress in answering these questions
arose from an experiment in which we combined
PURE system and the membrane integration⁄
translo-cation system, in vesicles prepared from inverted
E coli cell membranes [30] Using this system,
mem-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 translocaintegra-tion [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 taken advantage of advances in cell-free translation systems The use of tRNA, mischarged with an un-natural amino acid through a chemical acylation method originally developed by Hecht et al [36], was first applied to cell-free translation systems by Schultz and coworkers [37] They mischarged suppressor tRNA that recognizes amber codons (UAG) with an unnatural amino acid, thereby altering a nonsense codon to a sense codon corresponding to the specific unnatural amino acid Alternatively, mischarged tRNA can be prepared through the use of engineered aminoa-cyl-tRNA synthetases [38,39] and ribozymes [40] that can catalyze aminoacylation of tRNA with specific unnatural amino acids In addition to amber codons, other target codons have been utilized for the same purpose Artificial tRNAs that recognize four-base co-dons have created novel codon–anticodon interactions [41] Furthermore, two unnatural nucleobases that form a novel Watson–Crick-like base pair have been introduced into tRNA and mRNA, generating
Fig 2 Model for integration of membrane proteins into minimal cells Nascent poly-peptides that are being synthesized on ribosomes become associated with signal recognition particle (SRP) The ribosome– polypeptide–SRP complex is targeted to the Sec translocon, which is embedded in the membrane through interaction with the SRP receptor (SR) Following release of SRP and
SR, polypeptides are cotranslationally integ-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.
Trang 5additional 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
techniques and bypasses the need for living cells
At present, there are a number of ways to link
geno-type and phenogeno-type within the cell-free translation
sys-tem (Fig 3) The first technique to be demonstrated
was ribosome display [48]; this technique utilizes the
ribosome complex that has peptidyl-tRNA and mRNA
bound noncovalently to the ribosome, to form a link
consisting of protein⁄ tRNA ⁄ ribosome ⁄ mRNA In the
in vitro virus or mRNA display, a covalently linked
mRNA⁄ puromycin ⁄ protein complex that is formed by the ribosome via a peptide bond is substituted for the stalled ribosome complex in ribosome display [49,50] These methodologies select functional peptides or pro-teins from large libraries and have been used to isolate antibodies or scaffolding proteins that bind specific proteins with high affinity [51,52], streptavidin-binding peptide [53] and ATP-binding protein [54] In addition, these methods have also been used for proteomic ana-lyses of protein–protein interactions [55,56]
Recently, CIS display achieved noncovalent linkage between DNA and the synthesized protein in a cell-free, coupled transcription⁄ translation system [57] CIS display uses fusions between DNA encoding random peptides and the DNA replication initiator protein (RepA), which binds exclusively to the DNA from which it has been expressed, resulting in a selectable library of proteinÆDNA complexes The formation of proteinÆDNA complexes can also be achieved by using cell-free translation system compartmentalized in water-in-oil emulsions [58,59] This technology is based
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-din–biotin–DNA complex [58].
Trang 6molecular colony technique, in which reactions are
separated by two-dimensional geometry in an
acryla-mide gel [60]
Conclusion
Proteins are attractive polymers that exhibit an
enor-mous variety of structures and functions However,
this variation can sometimes cause problems for
pro-duction and handling, for as long as propro-duction is
con-strained by in vivo expression, improvements are
limited by the difficulty in introducing expression
sub-systems into host cells In contrast, a large variety of
systems can be integrated into cell-free translation,
simply by adding the supplements required for the
pro-tein product Furthermore, prompt and reliable
evalua-tion of both supplement and product can be achieved
in vitro Thus, we believe that further progress in the
development of subsystems, as well as improvement of
the cell-free translation system itself, will make these
techniques more widely available and will contribute
greatly to the field of protein science
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