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F. Katzen Et Al. the Past, Present and Future of Cell-Free Protein Synthesis

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F. Katzen Et Al. the Past, Present and Future of Cell-Free Protein Synthesis Review TRENDS in Biotechnology Vol.23 No.3 March 2005 The past, present and future of cell-free protein synthesis Federico Katzen1, Geoffrey Chang2 and Wieslaw Kudlicki1 1Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA 2Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, CB-105, La Jolla, CA 92037, USA Recent technical advances have revitalized cell-free Configurations and history expression systems to meet the increasing demands In vitro translation systems are based on the early for protein synthesis. Cell-free systems offer several demonstration that cell integrity is not required for advantages over traditional cell-based expression protein synthesis to occur. In its simplest form, translation methods, including the easy modification of reaction can be accomplished using a crude lysate from any given conditions to favor protein folding, decreased sensitivity organism (that provides the translational machinery, to product toxicity and suitability for high-throughput accessory enzymes, tRNA and factors) in combination strategies because of reduced reaction volumes and with exogenously added RNA template, amino acids and process time. Moreover, improvements in translation an energy supply. This classical in vitro translation efficiency have resulted in yields that exceed a milligram scheme is called ‘uncoupled’ as opposed to the ‘coupled’ of protein per milliliter of reaction mix. We review the or ‘combined’ transcription and translation configuration advances on this expanding technology and highlight in which the mRNA is transcribed in situ from a DNA the growing list of associated applications. template added to the reaction (see [2] for details). Usually coupled systems exhibit higher protein yields and are easier and faster to operate than systems that are not Introduction coupled although they require supplementing the reaction Since the pioneering studies conducted by Nirenberg and with additional NTPs and a highly processive RNA Matthaei more than four decades ago [1] cell-free protein polymerase, such as those encoded by T7, T3 or SP6 synthesis has been a valuable tool for understanding how bacteriophages. The use of plasmid or PCR templates mRNAs are translated into functional polypeptides. In rather than purified mRNAs resulted in the emergence of addition, it has been used for antibiotic drug discovery and a variety of new applications. has been a means for small-scale expression of toxic products. With the advent of the proteomics era, the field has experienced a technical renaissance expanding into a Sources of lysates myriad of applications covering both functional and Although any organism could potentially be used as a structural proteomics. Significant improvements made to source for the preparation of a cell-free protein expression the configuration, energetics and robustness of reactions system, the most popular are those based on Escherichia have led to productivities that far surpass the milligram coli, wheat germ and rabbit reticulocytes (for recent levels of product per milliliter of reaction. Also, important reviews see [2,6,7]). The choice of the system should be progress has been made on key reaction components so determined by the origin and biochemical nature of the that it is now possible to obtain a polypeptide with its protein and the specifics of the downstream application. In corresponding post-translational modifications or, in the general, E. coli-based systems provide higher yields and case of a membrane protein, properly imbedded into a lipid more-homogeneous samples suitable for structural bilayer. Finally, the ability to easily manipulate the studies. The protein yields of E. coli-based systems reaction components and conditions makes in vitro protein range from a few micrograms up to several milligrams synthesis particularly amenable to automation and min- per milliliter of reaction, depending on the protein and the iaturization, enabling application to the fields of protein reaction format [8]. Eukaryotic-based systems, although arrays, in vitro evolution and multiplexed real-time less productive, provide a better platform for functional labeling, among others. In this article we review the key studies particularly for post-translationally modified proteins. Reported protein yields for the rabbit reticulo- aspects of this field, emphasizing the most recently cyte lysate are in the microgram or in the fraction of a developed applications for both basic and applied microgram per milliliter of reaction range whereas protein research. A review of the entire field is beyond the scope yields of the wheat germ extract are typically two orders of of this article and further details in this regard can be magnitude higher [2,6,9]. found in previously published literature [2–6]. Crude cell lysates are not the only source of the enzymatic machinery. Despite being one of the most Corresponding author: Kudlicki, W. ([email protected]). complicated basic cellular processes, the whole transla- Available online 26 January 2005 tional mechanism from E. coli was recently reconstituted www.sciencedirect.com 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.01.003 Review TRENDS in Biotechnology Vol.23 No.3 March 2005 151 in vitro starting with O100 individually purified com- easily applicable to high-throughput processes, which ponents [10]. The system exhibits high translational require miniaturization and automation. efficiency with the added advantage of simpler manipu- To this end several laboratories have focused either lation of the reaction conditions and easy purification of on developing high-throughput-friendly systems or on untagged protein products. maximizing the energetics of the batch reaction. Endo and coworkers have devised a highly efficient bilayer diffusion system devoid of membranes that is compa- Productivity and automation tible with high-throughput formats [9,15] (Figure 1d). The principal limitation of the first generation batch- Swartz’s group has consistently sought more efficient formatted reactions (Figure 1a) is their short lifetimes and economical alternatives to the traditional ATP (!1 hr) and consequently their low yield. This is primarily and/or GTP regeneration systems with promising because of the rapid depletion of the high-energy phos- results. For example, they have recently demonstrated phate pool, which occurs even in the absence of protein that is possible to produce close to a milligram of synthesis [11]. In turn, this leads to the accumulation of protein per milliliter of reaction using a batch format free phosphates, which can apparently complex with (see [16] and references therein). The same group has magnesium, and further inhibit protein synthesis. This recently shown that a higher efficiency cell-free system problem was first overcome by Spirin and coworkers with can be obtained simply by mutating genes involved in the introduction of the continuous-flow cell-free (CFCF) the catabolism of certain amino acids [17]. translation system, which relies on the continuous supply of energy and substrates and the continuous removal of the reaction by-products (Figure 1b) [12]. The reaction Folding and post-translational modifications time can then be extended for 20 h and increases the A key goal for cell-free translation systems is to product yield by two orders of magnitude. Despite the synthesize biologically active proteins. Currently, the improvement in yield, the operational complexities make primary issues are protein folding and post-transla- this system extremely impractical. The technology was tional modifications. A clear advantage that these later simplified by the development of a semi-continuous systems have over in vivo protein synthesis is that or continuous-exchange cell-free (CECF) method, in which the environmental conditions can be adjusted easily. a passive rather than active exchange of substrates and Strategies to improve protein folding and post-transla- by-products extended the reaction lifetime [13,14] tional processing include the addition of a variety of (Figure 1c). However, semi continuous systems are not reagents and folding catalysts to the reaction. (a)Batch (b) Continuous flow Feeding Pump Reaction buffer Reaction + feeding buffer Ultrafiltration membrane Pump (c) Continuous exchange (d) Bilayer Dialysis Feeding buffer membrane Interphase Feeding buffer Reaction Reaction TRENDS in Biotechnology Figure 1. Current formats of cell-free protein expression systems. Formats are classified according to how the reaction is fed: (a) batch (b) continuous-flow cell-free (c) continuous exchange cell-free and (d) bilayer. Reaction components include ribosome, translation factors, tRNA, aminoacyl tRNA synthetases, template (RNA or DNA), and RNA polymerase (when necessary). The feeding buffer includes amino acids, energy components, NTPs (when necessary), co-factors and other accessory reagents. Yellow arrows indicate the flow of buffer components and red arrows represent the flow of protein product. www.sciencedirect.com 152 Review TRENDS in Biotechnology Vol.23 No.3 March 2005 Chaperones bonds [23]. Also, the combination of alkylation of the Reports on the exogenous supply of chaperones to cell-free extract with iodoacetamide, a suitable glutathione redox protein synthesis reactions suggest that the effect that buffer and a disulfide bond isomerase added to the in vitro these catalysts have is protein-dependant. For example reaction can have
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