Tài liệu Báo cáo khoa học: Cell-free translation systems for protein engineering docx

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 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. 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 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 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]. protein synthesis on ribosome Aminoacylation amino acid tRNA AT P 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 termination, as well as components for aminoacylation 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. 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 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. Y. Shimizu et al. Cell-free translation for protein engineering FEBS Journal 273 (2006) 4133–4140 ª 2006 The Authors Journal compilation ª 2006 FEBS 4135 How 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 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 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. 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 techniques and bypasses the need for living cells. At present, there are a number of ways to link geno- type and phenotype 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]. Y. Shimizu et al. Cell-free translation for protein engineering FEBS Journal 273 (2006) 4133–4140 ª 2006 The Authors Journal compilation ª 2006 FEBS 4137 molecular 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. 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EMBO J 22, 24–35. 60 Samatov TR, Chetverina HV & Chetverin AB (2005) Expressible molecular colonies. Nucleic Acids Res 33, e145. Cell-free translation for protein engineering Y. Shimizu et al. 4140 FEBS Journal 273 (2006) 4133–4140 ª 2006 The Authors Journal compilation ª 2006 FEBS . 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. MINIREVIEW Cell-free translation systems for protein engineering Yoshihiro Shimizu 1 , Yutetsu Kuruma 2 , Bei-Wen