A novel eukaryotic cell-free expression system that does not require an artificial energy regeneration system

Abstract

The present disclosure relates to systems, methods, and kits for the in vitro synthesis of biological macromolecules in reactions utilizing cell lysates containing plastids, mitochondria, and / or chloroplasts, in which creatine phosphate and creatine kinase are not added to the reaction to provide artificial energy regeneration. [Selected Figure] Figure 1A

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 457,073, filed February 9, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to the in vitro production of biopolymers. Some embodiments relate to the production of, for example, polypeptides, polynucleotides, and / or polysaccharides in eukaryotic cell-free systems. Certain embodiments utilize organelles (e.g., plastids, mitochondria, or chloroplasts) to provide energy for the continuous production of biopolymers in cell-free systems, eliminating the need for certain undesirable energy storage molecules in the system, such as creatine phosphate. [Background technology]

[0003] The increasing demand for new therapeutic proteins, technological enzymes, protein engineering, and functional genomics requires rapid and efficient protein production and screening platforms. Leader et al.(2008) Nat.Rev.Drug Discov.7(1):21-39;Swartz(2012) Aiche J.58(1):5-13. The emerging technology of cell-free protein synthesis (CFPS) may help meet this demand. Carlson et al.(2012) Biotechnol.Adv.30(5):1185-94. Compared to cell-based expression, CFPS offers advantages such as short process times and direct control and monitoring of reaction conditions. Swartz(2012), supra.PCR products can be used directly for simultaneous expression of multiple proteins without laborious cloning and transformation steps. Wu et al.(2007)Angew.Chem.Int.Ed.Engl.46(18):3356-8;Yabuki et al.(2007)J.Struct.Funct.Genomics 8(4):173-91;Gan & Jewett(2014)Biotechnol.J.9(5):641-51. The CFPS platform allows for the addition of accessory factors that promote protein folding (Ozawa et al (2005) J. Biomol. NMR 32(3):235-41; Endo et al. (2006) Mol. Biotechnol. 33(3):199-209; Matsuda et al (2006) J. Struct. Ltd. Genomics 7(2):93-100) or the incorporation of unnatural amino acids (Albayrak & Swartz (2013) Nucleic Acids Res. 41(11):5949-63; White et al. (2013) Methods 60:70-4). It also facilitates the expression of cytotoxic proteins that cannot be produced in living cells.Xu et al. (2005) Appl.Biochem.Biotechnol.127(1):53-62;Schwarz et al.(2008)Proteomics 8(19):3933-46;Xun et al.(2009)Protein Expr.Purif.68(1):22-7.

[0004] Escherichia coli cell-free lysates are widely used and advantageous due to their low cost, scalability, and high productivity. Zawada et al. (2011) Biotechnol. Bioeng. 108(7):1570-8; Caschera & Noireaux (2014) Biochimie 99:162-8. However, since the lysates originate from bacteria, they are unsuitable for the production of complex proteins with multiple subdomains due to inefficient oxidative folding and due to the lack of chaperone and glycosylation machinery. Eukaryotic cell-free systems are more suitable for the expression of such proteins and support most forms of post-translational modifications. Chang et al. (2005) J. Mol. Biol. 353(2):397-409; Zhang & Kaufman (2006) Handb. Exp. Pharmacol. (172):69-91. The most frequently used systems are based on wheat germ extract (WGE), insect cell extract (ICE), and rabbit reticulocyte lysate (RLL). However, these systems are expensive and the extract preparation is complicated (Carlson et al. (2012), supra). This has created a demand for additional eukaryotic CFPSs, such as those based on Leishmania tarentolae (Mureev et al (2009) Nat. Biotechnol. 27(8):747-52), Chinese hamster ovary (CHO) cells (Brodel et al (2014) Biotechnol. Bioeng. 111(1):25-36), and Saccharomyces cerevisiae (Hodgman & Jewett (2013) Biotechnol. Bioeng. 110(10):2643-54; Gan & Jewett (2014), supra).

[0005] In vitro protein synthesis using cell-free systems has been limited, for example, by the short reaction times and low protein production that are characteristic of such systems, which lead to poor protein yields and excessive costs per unit of protein produced.

[0006] Using continuous flow systems, longer reaction times can be obtained through the use of continuous translation reactions. Spirin et al. (1988) Science 242:1162-1164. Continuous reactions are carried out over tens (or even hundreds) of hours, and methods that rely on continuous flow require a constant supply of the necessary reaction substrates to the chamber. These reactions therefore require a significant investment of time and resources. Furthermore, translation in "continuous" systems is oriented toward producing large amounts of protein, and the systems differ substantially from those used to perform static ("batch") in vitro translation reactions. Static reactions can be performed in small reaction volumes (e.g., microliters) and are not oriented toward producing preparative amounts (e.g., milligrams) of protein. Such batch reactions can be completed in 1-2 hours. For all the aforementioned reasons, while increasing the reaction duration and protein yield compared to the corresponding batch systems, continuous reaction systems require more expensive reagents.

[0007] Disclosure The general strategy disclosed herein utilizes organelles in cell-free lysate-based reactions to provide energy regeneration within the reaction system. This strategy is useful in some examples to achieve improved in vitro synthesis of biopolymers (e.g., polynucleotides, polypeptides, polysaccharides, and complex carbohydrates). In certain embodiments, the presence of mitochondria in eukaryotic cell-free systems provides an improved reaction system for conventional batch and continuous reactions, for example, by significantly reducing or eliminating the need for the addition of energy delivery reagents (e.g., creatine phosphate and / or creatine kinase) and / or amino acid supplementation, while extending the reaction period. In some examples, the disclosed cell-free polymerization reactions are significantly more efficient than conventional reactions currently utilized in the art.

[0008] Described herein is a method for synthesizing a biopolymer, comprising combining a cell lysate containing organelles (e.g., plastids, chloroplasts, and / or mitochondria), a polymer template, and a monomer unit of a polymer in a reaction volume. In some embodiments, the reaction volume does not include, for example, a phosphocreatine / creatine kinase energy regeneration system, which results in no or minimal phosphate being added to the reaction volume. In certain embodiments, the organelle is a mitochondria. In some embodiments, the cell lysate is a eukaryotic cell lysate; for example, a lysate from a plant (e.g., tobacco, corn, and soybean) cell. The cell lysate in some examples is a lysate from a Bright Yellow-2 (BY-2) tobacco cell. In some embodiments, the polymer template is a DNA molecule or an RNA molecule. Reactions utilizing RNA as a polymer template produce polypeptides as biopolymers from monomeric amino acids through a translation reaction. Reactions utilizing DNA as a polymer template may be used to produce additional nucleic acid molecules (e.g., DNA and RNA) as biopolymers from monomeric nucleotides through in vitro replication or transcription reactions, or to produce polypeptides through translation reactions coupled with transcription from a template. In certain embodiments, methods of energy-free synthesis of biopolymers may include, for example, but not limited to, adding organelles, polymer templates and / or monomer units to a reaction volume, and / or isolating a biopolymer from the reaction volume. In some instances, the reaction volume does not require amino acid supplementation to support protein expression, since components of the lysate can generate amino acids using endogenous biosynthetic pathways beginning with intermediates from the TCA cycle. Thus, in some such instances, the amino acids present in the lysate containing the organelles may be sufficient to support sustained synthesis of the polypeptide.

[0009] The disclosed cell lysate system may be supplemented with a minimal amount of exogenous creatine phosphate, a minimal amount of creatine kinase, or both, so long as the reaction volume contains exogenous creatine phosphate and / or creatine kinase in an amount that would be considered inappropriate for an energy regenerating system. For example, the reaction volume may contain 15 mM or less, 10 mM or less, 5 mM or less, 1 mM or less, 500 μM or less, 100 μM or less, 50 μM or less, or 10 μM or less of added creatine phosphate. In another example, the reaction volume may contain 100 μg / mL or less, 50 μg / mL or less, 10 μg / mL or less, 5 μg / mL or less, 1 μg / mL or less, 0.5 μg / mL or less, or 0.1 μg / mL or less of added creatine kinase. These amounts are not suitable for sustaining biopolymer synthesis (such as for sustaining biopolymer synthesis over the long periods of time disclosed herein) and therefore require the inclusion of organelles such as plastids, mitochondria, or chloroplasts in accordance with the methods and systems disclosed herein.

[0010] Some embodiments include a system for synthesizing biopolymers without the use of an artificial energy regeneration system. In these embodiments, the system includes an aqueous cell lysate, an organelle (endogenous or exogenous), and a polymer template. In certain embodiments, the system also includes a monomer unit of a polymer. Conventional cell-free systems for in vitro biopolymer synthesis further include creatine phosphate and creatine kinase, which are used for energy regeneration within the system as the synthesis reaction proceeds. In embodiments herein, the system is substantially devoid of creatine phosphate, and creatine phosphate and creatine kinase are not added to the system. In certain examples, the system does not include either creatine phosphate or creatine kinase. In certain embodiments, the system for synthesis of biopolymers may further include, for example, but not limited to, a pH buffer, magnesium (e.g., Mg(C5H8NO4)2), potassium (e.g., KC5H8NO4), nucleosides (e.g., nucleotide triphosphates, nucleoside diphosphates, and nucleotide monophosphates), enzymes (e.g., RNA polymerase), and chloramphenicol. In certain embodiments, no amino acids other than glutamate are added to those already present in the system (i.e., amino acids present in the lysate and in the organelles). In some cases, the above-described systems may exhibit long-term activity of more than 20 hours (e.g., about 40 hours) and may produce significantly more (e.g., about 60% more) target protein than identical conventional systems that include added creatine phosphate and creatine kinase.

[0011] Some embodiments include a kit for synthesizing biopolymers without the use of an artificial energy regeneration system. In some embodiments, the kit includes components of a system for synthesizing biopolymers without the use of an artificial energy regeneration system, and written instructions for directing the use of the kit. For example, but not limited to, the kit may include one or more of an aqueous cell lysate, an organelle (endogenous or exogenous), a polymer template, and a monomer unit of a polymer, arranged in one or more separate volumes, along with instructions specifying the mixing of the components of the kit and any exogenous components that do not include creatine phosphate and creatine kinase. As a further example, the kit may further include one or more of a pH buffer, magnesium, potassium, a nucleoside, an enzyme (e.g., RNA polymerase), and chloramphenicol. A kit for the synthesis of a biopolymer according to certain embodiments may include an aqueous cell lysate (e.g., including chloroplasts and / or mitochondria), a monomer unit of a polymer (e.g., nucleosides), and written instructions. In certain such embodiments, the written instructions may instruct the user to combine these components with a desired polymeric template (e.g., a DNA molecule encoding a polypeptide) and any other reagents without adding creatine phosphate (along with creatine kinase for energy regeneration) to the combination.

[0012] The embodiments herein incorporate active mitochondria for energy regeneration in ongoing synthetic reactions, and may thereby be utilized to quantitatively examine compounds or proteins that affect mitochondrial function within the context of in vitro synthesis. Additionally, intermediates of the TCA cycle may be utilized during synthetic reactions to produce amino acids, such that amino acid supplementation is not required for long-term polypeptide synthesis. In some embodiments, cell lysates for use in the methods, systems, and kits herein include chloroplasts, which reduces the oxygen dependency of synthetic reactions. For example, cell lysates may be prepared from photosynthetically active cells such that plastids, chloroplasts, and / or mitochondria are retained in the lysate, while undesirable cellular material is removed. In certain such instances, the methods, systems, and kits herein may utilize plastid-derived energy, mitochondria-derived energy regeneration, chloroplast-derived energy regeneration, or a combination of both.

[0013] The above and other features will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0014] [Figure 1A] Figure 1 includes the components and performance of the system for the synthesis of biopolymers without artificial energy regeneration containing tobacco BY-2 cell lysate (BYL). Figure 1(A) shows a comparison of the performance of the present system with a system containing creatine phosphate (CP) and creatine kinase (CK). A coupled transcription-translation reaction was performed for 52 h at 25 °C using a reporter gene (i.e., eYFP) as a template. The yield of the fluorescent reporter protein was determined by measuring the fluorescence intensity using a fluorescence reader with an excitation of 485 / 20 nm and an emission filter of 528 / 20 nm.) [Figure 1B]Figure 1(B) shows the effect of inhibition of the electron transport chain by sodium azide (azide) and trifluoroacetone (TTA), respectively, on reporter yield. Means and standard deviations were calculated from three independent transcription-translation experiments. [Diagram 2] Figure 2 contains a diagram depicting a proposed mechanism for ATP generation in a system for synthesis without the use of artificial energy regeneration. Glutamate is used to produce reducing equivalents, primarily in the form of NADH, through the TCA cycle located inside the mitochondria. NADH fuels oxidative phosphorylation, oxygen serves as the final electron acceptor, and ADP is converted to ATP. [Figure 3A] Figure 3 includes charts showing the effect of different antimicrobial substances on eYFP yield and microbial growth in a system for synthesis without using artificial energy regeneration. Ligated BYL reactions were carried out at 25°C and 700 rpm for 45 hours using pIVEX_GAAAGA_Omega_Strep-eYFP as a template. The yield of eYFP was determined by measuring the fluorescence intensity using a fluorescence reader. The number of colony forming units (CFU) was determined by plating 0.2 μL of the BYL reaction volume on an LB plate and then incubating it at 37°C for 16 hours. The average and standard deviation from three independent experiments were calculated. Figure 3(A) includes the effect of different concentrations of chloramphenicol on microbial growth (CFU / μl). [Figure 3B] FIG. 3(B) contains the effect of different antimicrobials on eYFP yield, normalized to the yield in a standard reaction without antimicrobial (100%). [Figure 3C] FIG. 3(C) includes the effect of different antimicrobial substances on microbial growth (CFU / μl). [Figure 3D] FIG. 3(D) includes the effect of different concentrations of chloramphenicol on the yield of eYFP (μg / mL). [Figure 4]Figure 4 includes a visual representation of the DoE-based optimization of the NTP mixture used in the system for synthesis without artificial energy regeneration. Response surface and contour plots are presented for eYFP synthesis in a coupled BYL system without creatine phosphate and creatine kinase. The effect of different NTPs and magnesium glutamate on the yield is shown when other components of the reaction volume were maintained at optimal concentrations. The plots show significant interactions between magnesium glutamate and ATP (Figure 4(A)), magnesium glutamate and GTP (Figure 4(B)), and magnesium glutamate and CTP / UTP (Figure 4(C)). The reaction was carried out at 25 °C and 700 rpm for 46 h using the plasmid pIVEX_GAAAGA_Omega_Strep-eYFP as the DNA template. The yield of eYFP (provided in relative fluorescence units, RFU) was determined by measuring the fluorescence intensity using a fluorescence reader. [Diagram 5] Figure 5 includes a bar graph showing the effect of increasing lysate concentration on productivity in a system for synthesis without the use of artificial energy regeneration. Reactions with BY-2 cell lysate prepared with mannitol (blue) were compared to reactions with fresh lysate prepared with sorbitol (green). Reactions were performed in a volume of 50 μL in a 96-well plate. Light bars represent 40% (v / v) lysate reactions, and dark bars represent 60% (v / v) lysate reactions. "M" and "S" refer to the use of mannitol and sorbitol, respectively, for protoplast formation, evacuolation, and washing of evacuolated protoplasts during lysate preparation. Ligation reactions were performed at 25 °C and 700 rpm for 48 h using pIVEX_GAAAGA_Omega_Strep-eYFP as the DNA template. The yield of eYFP was determined by measuring the fluorescence intensity using a fluorescence reader. Means and standard deviations were calculated from two independent experiments. The dates indicate the fusion date of the large "BYL mix" and the preparation date of each new lysate, respectively. [Figure 6]Figure 6 includes bar graphs showing the effect of ectoine, hydroxyectoine, and glucosylglycerol on eYFP production in coupled IVTT reactions without artificial energy regeneration. Ectoine, hydroxyectoine, and glucosylglycerol in amounts of 0-8% (v / v) were added to the samples, and the plasmid pIVX_GAAAGA_Omega_Strep-eYFP was used as a template. Reactions were carried out in 50 μL of lysate aliquots of 60% (v / v) at 25 °C and 500 rpm for 44 h at controlled humidity (70%) using 96-well plates. The amount of eYFP produced was determined by use of a fluorescence reader in comparison to eYFP standards. eYFP standards were produced using the IVTT transcription-translation system and purified by Strep-Tactin® Sepharose®. The concentration of purified eYFP was then determined using a colorimetric assay. Data represent the mean and standard deviation of three independent transcription-translation experiments. [Figure 7] Figure 7 contains a bar graph showing the effect of glucosylglycerol on a coupled IVTT reaction without artificial energy regeneration. The amount of eYFP produced from the plasmid pIVX_GAAAGA_Omega_Strep-eYFP in five different lysate batches was compared. Reactions were carried out with 60% or 80% (v / v) lysate and without or with 0.5% (v / v) glucosylglycerol (GG). Reactions were carried out in a volume of 50 μL in 96-well plates at 25 °C and 500 rpm for 48 h at controlled humidity (70%). Data represent the mean and standard deviation of three independent transcription-translation experiments. [Figure 8]Figure 8 includes a graphical representation of eYFP production over time in coupled IVTT reactions without artificial energy regeneration. Reactions contained either 60% or 80% (v / v) lysate without or with 0.5% (v / v) glucosylglycerol (GG). Plasmid pIVX_GAAAGA_Omega_Strep-eYFP was used as template and the amount of eYFP produced (compared to an eYFP standard) was determined by using a fluorescence reader. Reactions were carried out in 96-well plates in a volume of 50 μL at 25° C. and 500 rpm for 64 h under controlled humidity (70%) in a Kuhner™ shaker. Data represent the mean and standard deviation of three independent transcription-translation experiments using different lysate batches. [Figure 9] Figure 9 includes the effect of branched chain amino acids (BCAAs) on the production of eYFP in coupled IVTT reactions without artificial energy regeneration. 0–2 mM BCAAs were added as the last component to coupled IVTT reactions using the plasmid pIVEX_GAAAGA_Omega_Strep-eYFP as template. Reactions were carried out for 66 h at 25 °C and 500 rpm in a volume of 50 µL in a 96-well plate. The amount of eYFP produced was determined by the use of a fluorescence reader, in comparison to an eYFP standard. Data represent the mean and standard deviation of six independent transcription-translation experiments. Lysates were prepared from shake flasks (SF) or continuous fermentations (CF). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] I. Overview of Some Embodiments Crude lysate-based cell-free protein synthesis (CFPS) systems offer several advantages over in vivo systems and are useful in a wide range of applications, especially in protein engineering, biopharmaceutical manufacturing, and research. Conventional crude lysates contain the components necessary for translation, protein folding, and energy metabolism, so that almost any protein encoded by an RNA template can be synthesized therein in the presence of amino acids, nucleotides, and salts, as long as the lysate is supplemented with energy storage reagents. In coupled transcription / translation systems, RNA polymerase can be added to direct the synthesis of proteins from DNA templates. In contrast to intracellular synthesis, CFPS can tolerate shorter process times, reduced protein hydrolysis, and the ability to express toxic proteins and proteins containing specific chemical groups or unnatural amino acids at defined positions. Furthermore, reactions can be directly controlled and monitored.

[0016] Disclosed herein is a cell-free system for biopolymer synthesis (e.g., polypeptide synthesis) that utilizes cell lysates without an artificial energy regeneration system, eliminating the need for additional energy storage reagents. In embodiments, the system utilizes eukaryotic cell lysates, including organelles (e.g., plastids, mitochondria, or chloroplasts), for energy regeneration during synthesis reactions by oxidative phosphorylation. In one particular example, the cell lysate is a tobacco BY-2 lysate, including mitochondria derived from BY-2 cells. In cellular eukaryotic systems during oxidative phosphorylation, electrons are transferred from electron donors to electron acceptors, such as oxygen, via an electron transport chain located within the inner membrane of mitochondria. These redox reactions release energy, which is used to phosphorylate ADP to ATP. Embodiments herein utilize the energy from this process to drive the continued synthesis of biopolymers in the lysate. Thus, embodiments herein use inhibitors of the electron transport chain and tight conditions to stop energy regeneration by oxidative phosphorylation, thereby stopping synthesis reactions by terminating the translational activity of the system. In some embodiments, the system includes plastids, chloroplasts, that can allow reactions to proceed in anaerobic or substantially anaerobic conditions.

[0017] Conventional eukaryotic cell-free systems (e.g., wheat germ extracts, and insect cell extracts) lack mitochondria. Instead, these systems require the addition of creatine phosphate and creatine kinase to achieve the necessary ATP regeneration to support protein expression. The accumulation of a lot of free phosphate (derived from creatine phosphate) added to the reaction mixture for energy regeneration to support protein expression is a significant limiting factor in the performance of these systems. Ezure et al. (2006) Biotechnol. Prog. 22(6): 1570-7; Takai et al. (2010) Curr. Pharm. Biotechnol. 11: 272-8; Brodel et al. (2014) Biotechnol. Bioeng. 111(1): 25-36; Hodgman & Jewett (2013), supra; Schoborg et al. (2014) Biotechnol. J. 9(5): 630-40. Free phosphate introduced into the system binds to magnesium (necessary for transcription and translation), leading to premature collapse of synthetic performance and low product yields.

[0018] To prolong synthesis performance, currently available eukaryotic cell-free systems use "continuous flow" reactions in dialysis mode to provide a long-lasting energy source and dilute inhibitory components such as phosphate in the reaction compartment. The systems described herein, which do not include an artificial energy regeneration system for biopolymer synthesis, reduce or eliminate the need for reaction dialysis, since they produce less inhibitory components and have their own energy regeneration capabilities. However, the systems herein may be utilized in a continuous flow configuration, if desired, at the discretion of the practitioner.

[0019] Compared with conventional eukaryotic cell-free protein expression systems, the biopolymer synthesis systems described herein are generally cheaper and also produce proteins for longer, resulting in increased biopolymer yields.Furthermore, the systems herein provide the possibility to investigate compounds or pathways that affect mitochondrial and / or chloroplast function, for example, as additional promoters of cell-free protein expression.Thus, the fundamentally different systems of the embodiments herein can be optimized to provide even further benefits.

[0020] Some systems for biopolymer synthesis described herein are capable of supporting microbial growth, which may result in substrate depletion in IVTT reactions and / or proteolysis, reducing target protein termination. Thus, in some embodiments, the system includes chloramphenicol to inhibit microbial growth, which may improve protein expression in these embodiments. In certain embodiments, the system includes chloramphenicol in an amount of, for example, 10-500 μg / mL (e.g., 25-250 μg / mL, 50-200 μg / mL, and 100-200 μg / mL).

[0021] Described herein is the discovery that a particular amount of NTPs in a coupled in vitro transcription / translation (IVTT) reaction can provide surprisingly robust expression in some embodiments, which can be about 20% or more (e.g., 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 18-25% more, 19-23% more, about 20% more) than expected from standard NTP amounts. Thus, in some examples, the system can include about 150 mM ATP, about 40 mM GTP, about 20 mM CTP, and about 20 mM UTP. An additional advantage of utilizing these reduced amounts of NTPs is reduced costs, due to the reduced amount of GTP (the most expensive NTP) than traditional plant cell systems.

[0022] Also described herein are surprising discoveries that some modifications of the system herein can provide considerable improvements in product yield and / or quality. For example, the use of sorbitol during protoplast formation and the devacuolation step of lysate preparation can result in increased protein production while further reducing the cost of the system since sorbitol is less expensive than other commonly used osmotic agents such as mannitol. As a further example, increasing the proportion of lysate in the IVTT reaction to 50-90% (e.g., 55-85%) (v / v) causes higher expression of some target proteins. Thus, specific examples herein include the use of cell lysates prepared with sorbitol during protoplast formation and devacuolation in an amount of, for example, about 60% by volume (e.g., 58%, 59%, 60%, 61%, and 62%), or about 80% by volume (e.g., 78%, 89%, 80%, 81%, and 82%). As yet another example, the addition of glucosylglycerol dramatically increases the protein yield of IVTT reactions. For example, compared to standard reactions without glucosylglycerol, 0.25% to 4% amounts of glucosylglycerol resulted in up to 80% more protein. Without being bound to any particular theory, glucosylglycerol enhances reaction yields, presumably by increasing protein and membrane stability. Thus, in some embodiments, the system includes 0.25 to 4% (e.g., 0.25 to 2%, 0.25 to 1%, about 0.5%, and 1.5%) of glucosylglycerol. As a further example, although not required, the addition of branched amino acids (BCAAs) BCAAs can be utilized to increase protein production. Thus, in some embodiments, the system includes BCAAs in an amount of about 0.25 to 4 mM or in an amount of 0.5 to 2 mM (e.g., 0.48 to 2.2 mM, 0.5 to 2.0 mM, 0.5 to 1 mM, and about 1 mM).

[0023] According to the above modifications, in certain embodiments herein, the production of target protein in a coupled IVTT reaction can be extended for up to 64 hours. In one example, a reaction with 80% lysate by volume and 0.5% glucosylglycerol by volume results in approximately 2.5 mg / mL of eYFP.

[0024] II. Abbreviations AAD-12 Aryloxyalkanoate dioxygenase-12 ADP Adenosine diphosphate ATP Adenosine Triphosphate BCAA Branched Chain Amino Acids BY-2 Bright Yellow-2 BYL BY-2 cell lysate CFPS Cell-Free Protein Synthesis CFU colony forming units CHO Chinese hamster ovary CL Cellulase Enzyme CK Creatine kinase CP Creatine Phosphate Cry1F Bacillus thuringiensis Cry1F delta-endotoxin Cry3A B. thuringiensis Cry3A delta-endotoxin CTP Cytidine triphosphate DMSO Dimethyl sulfoxide Designing DOE experiments DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid eYFP Enhanced Yellow Fluorescent Protein FADH Flavin adenine dinucleotide GTP Guanosine triphosphate ICE insect cell extract IMAC Immobilized Metal Affinity Chromatography IVTT In vitro transcription and translation NADH Nicotinamide adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate NEB New England Biolabs NTP Nucleotide triphosphate PCR Polymerase Chain Reaction PEG Polyethylene glycol RLL Rabbit reticulocyte lysate SEC Size Exclusion Chromatography TCA Cycle Tricarboxylic Acid Cycle ("Krebs Cycle") TTA Thenoyltrifluoroacetone UTP Uridine Triline UTR untranslated region WGE wheat germ extract

[0025] III. Terminology Isolated: An "isolated" biological component (e.g., a nucleic acid or protein) is substantially separated, produced or purified away from other biological components (i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins) in the cells of an organism in which the component naturally occurs, while affecting a chemical or functional change in the component (e.g., a nucleic acid can be isolated from a chromosome by breaking the chemical bonds that connect the nucleic acid to the rest of the DNA in the chromosome). "Isolated" nucleic acid molecules and proteins include nucleic acid molecules and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules, proteins, and peptides.

[0026] Nucleic acid molecule: As used herein, the term "nucleic acid molecule" may refer to polymeric forms of nucleotides, including both sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic and mixed polymers thereof. Nucleotides may refer to ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. As used herein, "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide." Unless otherwise stated, nucleic acid molecules are typically at least 10 bases in length. The term includes single-stranded and double-stranded forms of DNA. Nucleic acid molecules may contain either or both natural and modified nucleotides linked together by natural and / or non-natural nucleotide bonds.

[0027] Nucleic acid molecules may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more natural nucleotides with analogs, internucleotide modifications (e.g., uncharged linkages: e.g., methyl phosphonates, phosphotriesters, phosphoramidates, and carbamates; charged linkages: e.g., phosphorothioates and phosphorodithioates; pendent moieties: e.g., peptides; intercalators: e.g., acridine, psoralens, etc.; chelators; alkylators; and modified linkages: e.g., alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes topological conformations, including single-stranded, double-stranded, partially duplexed, triplexed, hairpin, circular, and padlock conformations.

[0028] Exogenous: The term "exogenous" when applied to components (e.g., plastids, mitochondria, or chloroplasts) added to a cell lysate herein means such components have a different origin from the cell lysate. For example, plastids, mitochondria, or chloroplasts added to the lysate that do not originate from the cell lysate are exogenous to the cell lysate. The term exogenous may be applied to organelles such as plastids, mitochondria, or chloroplasts from the same cell type or different cell types (e.g., cells from different tissues or different species) from the cell type used to derive the cell lysate, so long as they are not derived from the cell lysate itself in either case. In addition, the term exogenous may be used herein to mean components of the energy regeneration system (e.g., creatine phosphate and creatine kinase) that are added separately or additionally to organelles such as plastids, mitochondria, or chloroplasts used in the cell lysate of the system disclosed herein.

[0029] IV. Biopolymer synthesis system The present disclosure provides a system for synthesizing biopolymers without the use of an artificial regeneration system. The system herein includes compositions and methods for achieving enhanced in vitro synthesis of biopolymers, in which oxidative phosphorylation is activated, providing increased yield in the absence of energy storage reagents. Improved yield is obtained by combination of reaction IVTT components, which may be assembled and mixed by the practitioner, or may be provided in a kit as premixed components, unmixed components, or a combination of the two. The practitioner may utilize some or all of the components of the kit in combination with the components he or she provides; for example, in some embodiments, the kit includes all components of a system for protein synthesis except for a DNA or RNA template, which is provided by the practitioner to synthesize a selected protein. Once all components of the system are mixed in a reaction volume under appropriate environmental conditions, the reaction is initiated and proceeds generally according to a conventional in vitro cell-free synthesis reaction, with important modifications as described herein. The reaction may be allowed to proceed until one or more of the components (e.g., NTPs and amino acids) are exhausted in the reaction volume, or until it is stopped by adjusting environmental conditions to terminate the energy regeneration process in the system.

[0030] The methods and compositions disclosed herein mimic the cytoplasmic environment of eukaryotic cells, resulting in significant improvements in protein production and protein folding over prior art methods. For example, due to the presence of organelles, oxidative phosphorylation is active in the reaction volume, so the systems herein reduce or completely avoid the need for secondary energy sources associated with synthesis inhibition.

[0031] The systems of the embodiments herein are useful for the production / replication of biopolymers, including, for example, amplification of DNA, transcription of RNA from DNA or RNA templates, translation of RNA into polypeptides, and synthesis of complex carbohydrates from simple sugars. Enhanced synthesis, in some instances, includes one or more of: an increase in the total or relative amount of biopolymer synthesized in the system; an increase in the total or relative amount of biopolymer synthesized per unit time; an increase in the total or relative amount of biologically active biopolymer (e.g., properly folded and / or post-translationally modified protein) synthesized; an increase in the total or relative amount of soluble biopolymer synthesized, and a reduction in the time and / or cost expenditure required to synthesize a given amount of biopolymer.

[0032] Certain embodiments herein achieve translation of mRNA to produce a polypeptide, which translation may be coupled to in vitro synthesis of mRNA from a DNA template. Such cell-free systems contain all the factors required for translation of mRNA, such as ribosomes, amino acids, tRNAs, aminoacyl synthetases, elongation factors, initiation factors, and ribosome recycling factors. In the examples herein, such cell-free systems include cell lysates prepared by the methods described herein from eukaryotic cells, such as plant cells (e.g., tobacco BY-2 cells).

[0033] cell lysate The embodiments herein can be adapted to utilize any eukaryotic cell lysate. Eukaryotic cell lysate retains various post-translational processing activities. Eukaryotic cell lysate also supports the in vitro translation of a wide variety of viral and other prokaryotic RNAs, as well as eukaryotic mRNAs. Template mRNAs with codon usage that deviates from the codon usage of the organism from which the cell lysate is derived can be used efficiently, for example, by supplementing the system with rare tRNAs and / or amino acids in the organism. A specific example herein utilizes tobacco BY-2 cell lysate, which has been shown to provide simple and cost-effective fermentation in suspension culture in both batch and stirred tank fermenters, and is amenable to well-established genetic modification tools.

[0034] Preparation of cell lysates according to embodiments herein may include, inter alia, disruption / removal of cell walls (in the case of plant cells) and cell membranes, removal of degradative vacuoles, and removal of endogenous mRNA.

[0035] Cell walls and membranes may be disrupted in some embodiments by techniques such as, but not limited to, mechanical disruption, liquid homogenization, enzymatic digestion, high frequency sonication, vacuum, freeze / thaw cycles, and manual grinding. In certain embodiments herein, plant cell lysates are prepared by digesting cell walls using one or more cell wall digestive enzymes (e.g., Cellulase Onozuka RS™, Pectolyase Y-23™, Macerozyme R-10) and liquid enzymes (e.g., Rohament CL™, Rohament PL™, and Rohapect UF™; originally intended for the production of fruit juices and extracts). Rohament CL™ contains a cellulase concentrate, Rohament PL™ is a pectinase concentrate, and Rohapect UF™ contains an enzyme complex containing a dedicated pectinase and arabanases. The use of these enzyme combinations reduced the cost of protoplast formation by more than 100-fold compared to conventional methods.

[0036] Also, during the preparation of the lysate, any degraded vacuoles may be removed. Such vacuoles contain undesirable enzymes, such as proteases and ribonucleases, that interfere with the synthesis of polypeptides and mRNA. In some embodiments herein, the degraded vacuoles are removed by centrifugation in a Percoll gradient or any other density gradient. The vacuoles have a low density and can therefore be separated from the protoplasts, resulting in high-density devacuolated protoplasts. In some instances, a stepwise sucrose density gradient may be utilized for devacuolation, where the protoplasts are applied directly to a Percoll-free upper layer. After centrifugation, the devacuolated protoplasts are separated from the concentrated liquid, for example, at the interface between 40%-70% Percoll layers (depending on the gradient used), while the separated low-density vacuoles are in the low-density gradient and float, for example, on the upper layer.

[0037] The devacuolated protoplasts may then be washed to protect labile cellular components from oxidation, and then disrupted by Dounce tissue grinder or nitrogen digestion. After removal of the nuclei and unbroken cells, the lysate may be treated to destroy endogenous mRNA and minimize background translation, while leaving the integrity of 18S and 28S ribosomal RNA largely unaffected. In a particular example herein, nuclease S7 is used.

[0038] template To direct the synthesis of biopolymers in the systems herein, a template must be present in the reaction so that the stored information can be converted into a polymer. The template for cell-free protein synthesis can be either mRNA or DNA encoding any polynucleotide (DNA) or polypeptide (DNA and / or mRNA) of interest. The coupled transcription / translation system continuously produces mRNA from a DNA template with a recognizable promoter. An endogenous RNA polymerase may be used or an exogenous RNA polymerase (e.g., phage RNA polymerase, commonly T7 or SP6) may be added directly to the reaction mixture. Alternatively, the mRNA may be continuously amplified by inserting the message into a template for QB replicase, an RNA-dependent RNA polymerase. In some embodiments, a vector containing a polyA sequence at one end of the multiple cloning region is used as a template for the IVTT reaction. For example, such a vector may contain an SP6, T7, or T3 RNA polymerase promoter at the opposite end of the multiple cloning region, such that cloning into the vector produces a gene flanked by the RNA polymerase promoters of polyA sequences at the 5' and 3' ends. In embodiments in which mRNA is utilized as the template, the purified mRNA may be stabilized by chemical modification prior to being added to the reaction mixture.

[0039] The nucleotide sequence of the DNA or mRNA sequence utilized as a template according to the embodiments herein may be optimized to achieve higher levels of expression. Several mRNA structural features affect translation efficiency, including untranslated regions (UTRs) at the 5' and 3' ends of the coding sequence. The structure of the 5'UTR influences translation initiation, termination, and mRNA stability. One of the rate-limiting steps of translation initiation is the binding of the mRNA to the 43S preinitiation complex. The translation machinery is recruited by the 5' cap or translation enhancer in the leader sequence. In certain embodiments herein, the template mRNA may contain a non-translated region selected from the group including: 5'UTR in PCITE2a (which contains an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV)); sequence of Barley Yellow Dwarf Virus (BYDV) in vector pF3A; 5'UTR of the baculovirus polyhedrin gene; synthetic 3'UTR containing a polyA sequence; and 5'UTR containing an ARC-1 sequence element (which is complementary to an internal 18S rRNA segment and may promote binding to the 40S ribosomal subunit); Tobacco Mosaic Virus (TMV) 5'-UTR (omega sequence) (which may be improved by adding GAAAGA upstream of the first GUA triplet).

[0040] In some embodiments, DNA molecules are used to produce capped mRNA in vitro, for example, in the presence of the cap analog m7G[5']ppp[5']G. Unincorporated nucleotides and the cap analog may be removed by gel filtration, and the purified mRNA, which serves as a template for polypeptide synthesis, can then be introduced into the cell-free systems described herein.

[0041] monomer In a coupled IVTT reaction, ribonucleotide triphosphates (ATP, GTP, CTP, UTP) and amino acids are required in the system as monomer units used to synthesize the desired biopolymer. In some embodiments herein, the system operates with reduced levels of one or more NTPs relative to a comparable system with a conventional energy regenerative system. In these embodiments, the disclosed system advantageously reduces the cost to operate the system. In certain embodiments, the disclosed system operates with a final ATP concentration of 2-10 mM, e.g., 4-8 mM, or 5-7 mM, of ATP. In certain embodiments, the disclosed system operates with a final GTP concentration of 0.8-2.5 mM, e.g., 1-2 mM, or 1.4-1.8 mM, of GTP. In certain embodiments, the disclosed system operates with a final CTP concentration of 0.4-2.4 mM, e.g., 0.5-2 mM, or 0.6-1.0 mM, of CTP. Also, in certain embodiments, the disclosed system operates with a final UTP concentration of 0.4-2.4 mM, e.g., 0.5-2 mM or 0.6-1.0 mM UTP. For example, the system may operate with a final NTP concentration of 6 mM or about 6 mM ATP, 1.6 mM or about 1.6 mM GTP, 0.8 mM or about 0.8 mM CTP, and 0.8 mM or about 0.8 mM UTP. In certain examples, the synthesis reaction is supplemented with a low concentration NTP mix containing about 150 mM ATP, about 40 mM GTP, about 20 mM CTP, and about 20 mM UTP, and the mix is ​​added to the system in an amount sufficient to provide a final concentration of NTPs. Amino acids may also be added, for example, to a final concentration of 20-500 pM. Radiolabeled amino acids (e.g., 35 S-Methionine and 3 H leucine may be used in the ligation reaction, after which the corresponding amino acid may be omitted from the amino acid mixture.

[0042] salt The concentration of salt is controlled in the system according to the embodiment of the present specification. For example, the system may be added with one or more salts, such as, but not limited to, potassium, magnesium, ammonium, and other biologically relevant salts such as manganese (e.g., acetate or sulfate). One or more of these salts may have an amino acid as a counter anion. There is an interdependence between ionic species with respect to the function of the synthesis reaction. When the concentration of a particular ion in the reaction medium is changed, the concentration of another ion may change accordingly. For example, the concentration of the added salt may be simultaneously controlled according to the change of other components such as nucleotides. Furthermore, the concentration level of a component in a continuous flow reactor may change over time.

[0043] magnesium Magnesium is important for protein translation because it enhances ribosome assembly and enhances the stability of assembled ribosomes. Magnesium also appears to play a role in promoting polymerase binding. In embodiments herein, the magnesium concentration of the cell lysate may be adjusted by additional magnesium compounds. In some embodiments, the additional magnesium compound is a salt, such as magnesium chloride, magnesium acetate, magnesium glutamate. To couple transcription and translation, a sufficient amount of magnesium salt may be added to the lysate to raise the final magnesium concentration to a level where RNA is transcribed from DNA and RNA is translated into protein. In some examples, the final magnesium concentration may be adjusted to 1-20 mM. For example, the final magnesium concentration may be 5-15 mM, 7-13 mM, 2.5 mM-5.5 mM, 2.5 mM-3.5 mM, 2.6 mM-3.0 mM, 3.0 mM-5.25 mM, or 4.0 mM-4.75 mM, depending on the lysate used.

[0044] To provide precise control of magnesium concentration in the systems herein, the magnesium level of the lysate may be measured directly through the use of a magnesium assay prior to adding extra magnesium. For example, the Lancer "Magnesium Rapid Star Diagnostic Kit" (Oxford LabWare Division™, Sherwood Medical Co., St. Louis, MO) is one assay that can accurately measure magnesium levels in biological fluids. Once the magnesium ion concentration is known for a given batch of lysate, additional magnesium can be added to bring the magnesium concentration of the lysate within the desired range.

[0045] As mentioned above, the final magnesium concentration of the reaction is influenced by other conditions and considerations. Thus, for example, as the ribonucleotide triphosphate concentration increases, the optimal magnesium concentration increases at the same time, since ribonucleotide triphosphate tends to associate or chelate with magnesium in solution. Thus, as the ribonucleotide triphosphate concentration increases, additional magnesium is generally also added to the reaction. The optimal concentration of magnesium also varies with the type of cell lysate. The amount of magnesium that needs to be added also varies with the concentration of lysate used in the reaction mixture, with increasing the concentration of lysate increasing the contribution of magnesium from the lysate itself.

[0046] potassium Typically, potassium is also added to the system to achieve the desired level of biopolymer synthesis. Potassium (e.g., potassium acetate and potassium glutamate) is typically present at a concentration of 5-250 mM (e.g., 5-100 mM, 5-75 mM, 5-50 mM, and 5-30). In certain instances, the potassium concentration may be 10-20 mM, or more specifically, about 20 mM. As with magnesium, the final potassium concentration may vary slightly due to the presence of endogenous cell lysate components.

[0047] Additional Ingredients Additional components may also be added to the system in certain embodiments as necessary to improve the efficiency or stability of the synthesis reaction. One common addition to the coupled transcription and translation reaction, while not absolutely necessary, is, for example, a polyamine in an amount sufficient to stimulate the efficiency of chain elongation. Polyamines are known to also affect optimal magnesium levels, slightly lowering the effective magnesium concentration for the translation reaction. Thus, polyamines can replace magnesium at some levels, and in certain instances may tolerate a lowering of the optimal magnesium level for coupled transcription and translation.

[0048] Metabolic inhibitors for undesired enzyme activity may be added to the reaction mixture, alternatively, enzymes or factors involved in undesired activity may be directly removed from the extract, or genes encoding undesired enzymes may be inactivated or deleted from the chromosome.

[0049] Purified or synthetic vesicles from host organisms (see Muller & Blobel (1984) Proc. Natl. Acad. Sci. USA 81:7421-5) can also be added to the system. These can be used to enhance protein synthesis and folding. For example, the systems described herein can be used for cell-free reactions to activate membrane proteins; for example, to insert or transfer proteins or transfer other compounds, and these processes can be assisted in certain embodiments by adding vesicles containing the desired membrane protein.

[0050] In addition to the above components, other materials (particularly useful for protein synthesis) can be added to the system described herein. Such materials can include, for example, but are not limited to, other salts, folinic acid, cyclic AMP, protein or nuclease inhibitors, RNasin, protein synthesis inhibitors or regulators, oxidation / reduction potential adjusters, DTT, chloramphenicol, non-denaturing detergents, buffer components (such as those that can be used in solutions to stabilize reaction pH), PEG, Triton X-100, spermine, spermidine, and putrescine.

[0051] Some embodiments include kits that include components of a system for synthesizing biopolymers without the use of an artificial regeneration system. In certain embodiments, the kits may include a cell lysate. Alternatively, the kits may include cells for culturing and amplifying to obtain cells for preparation of a cell lysate. In certain embodiments, the kits may include one or more of salts, NTPs, enzymes (e.g., polymerases and nucleases), enzyme inhibitors (e.g., RNasin), templates, and other additives (e.g., chloramphenicol). In certain examples, the kits may include naked vectors into which a gene of interest can be cloned for use as a template in the system. In kits that include a cell lysate, the lysate may be a standard or of a type whose salt concentration adjustments have already been made during manufacture, or may additionally include one or more of the components, reagents, or buffers required for coupled transcription and translation. In certain examples, the kit may not include a template, but may instead rely on the user to provide a template. The kit may include a set of instructions or a link to a website containing instructions to inform the user how to utilize the components of the kit to perform a synthesis reaction.

[0052] V. Biopolymer Synthesis Methods The above-described systems may be used in a method for in vitro synthesis of one or more biopolymers. In vitro synthesis refers to cell-free synthesis of biological macromolecules in a reaction mixture containing biological extracts and / or defined reagents. The systems herein may be used to perform cell-free synthesis reactions in batch, continuous flow, and semi-continuous flow configurations, as these configurations are known in the art. In some embodiments, batch cultured cells may be used. In some embodiments, cells may be grown continuously in stirred tank fermenters to ensure a reproducible and homogenous supply of cell material.

[0053] There are differences between the use of static IVTT reactions and continuous or flow-through reactions, which may be considered in some applications but not in others. For example, continuous systems are commonly used for large-scale industrial production of proteins, whereas static system reactions are more suitable for small-scale in vitro translation (e.g., in research situations). Continuous translation is much more expensive to perform and requires investment in equipment, as well as large amounts of reagents. In particular, the levels of RNA polymerase used to perform continuous eukaryotic reaction work may be prohibitive for simple research applications (i.e., 20,000-30,000 U / reaction). Furthermore, continuous reactions are designed to be performed in relatively large volumes, whereas static reactions are typically only on the order of 100 μl or less, so no additional equipment is required and only small amounts of reagents are needed.

[0054] The systems herein may utilize large-scale reactors, small-scale reactors, or may be multiplexed to perform multiple simultaneous syntheses. Continuous reactions may use a feeding mechanism to introduce a flow of reagents and isolate the final product as part of the process. In both continuous and static reactions, additional reagents may be introduced to extend the period of active synthesis. The reactors may be run in any mode, such as batch, extended batch, semi-batch, semi-continuous, fed-batch, and continuous, and the mode may be selected according to the application.

[0055] The reaction may be carried out in any volume, again depending on the application and the equipment used. For example, in small-scale reactions, the reaction volume may be 1-15 μl, at least 15 μl, at least 50 μl, at least 100 μl, at least 0.5 mL, or at least 1 mL, but may be less than 10 mL. In principle, the reaction may be carried out at any scale as long as sufficient oxygen (or other electron acceptor) is supplied. For production of the largest amounts of product, industrial bioreactors may be used.

[0056] The methods herein may utilize a means for isolating the synthesized biopolymer, for example a protein isolation means. In some embodiments, it operates in a continuous operation mode, where the product output from the reactor flows through a membrane and enters a protein isolation means. In a semi-continuous operation mode, the outside or external surface of the membrane is contacted with predetermined solutions that are periodically changed in a predetermined sequence. These solutions may include substrates such as amino acids and nucleotides. At this point, the reactor operates in a dialysis or diafiltration batch or fed-batch mode. A feed solution may be fed to the reactor through the same membrane or a separate injection unit. The synthesized protein accumulates in the reactor and is then isolated and purified following the usual methods of protein purification after completion of the system operation.

[0057] If there is a flow of reagents, the direction of liquid flow can be normal and / or tangential to the membrane. Tangential flow is effective for ATP recycling and preventing clogging of the membrane and can be superimposed on the normal flow. Flow normal to the membrane can be generated or influenced by positive pressure pumps or vacuum aspiration pumps. The solution in contact with the external surface of the membrane can be changed periodically or there can be a constant tangential flow to the membrane. Additionally, the reactor can be internally or externally agitated by suitable stirring means.

[0058] The protein isolation means for selectively isolating the desired protein during protein synthesis in the reactor may comprise a unit packed with particles coated with antibody molecules or other molecules having immobilized moieties for adsorbing the synthesized desired protein and a membrane with pores of appropriate size. The protein isolation means preferably comprises two columns for alternating use.

[0059] The amount of protein produced in a translation reaction can be measured in various ways. One method relies on the availability of an assay that measures the activity of the specific protein being translated. Examples of assays for measuring protein activity are the luciferase assay system, or the chloramphenicol acetyltransferase assay system. These assays measure the amount of functionally active protein produced from a translation reaction. Activity assays do not measure inactive full-length proteins due to improper protein folding or the lack of other post-translational modifications required for protein activity. Alternatively, specific proteins can be detected according to their size by capillary electrophoresis.

[0060] Another method for measuring the amount of protein produced in a coupled in vitro transcription and translation reaction is to 35 S-Methionine, 3 H-Leucine, or l4 The reaction is performed using a known amount of a radiolabeled amino acid, such as C-leucine, and then the amount of radiolabeled amino acid incorporated into the newly translated protein is measured. The incorporation assay will measure the amount of radiolabeled amino acid in all proteins produced in the reaction, including truncated protein products. The radiolabeled proteins may be further separated on a protein gel and autoradiography may be performed to confirm that the products are of the appropriate size and that no secondary protein products are being produced. EXAMPLES

[0061] Example 1: Materials and Methods plant material Tobacco cells (Nicotiana tabacum L. cv. Bright Yellow 2, BY-2) were cultivated in 5 L fermenters (Type 100e, Applicon™ Biotechnology, AC Schiedam, The Netherlands) or shake flasks maintaining 20–25% packed cell volume in the dark at 26 °C. Murashige-Skoog liquid medium (Murashige and Skoog basal salt mixture) supplemented with 3% (w / v) sucrose, 1 mg / L thiamine-HCl, 0.2 mg / L 2,4-dichlorophenoxyacetic acid, 100 mg / L myo-inositol, 250 mg / L potassium dihydrogen orthophosphate, and Pluronic® L-61 antifoam (BASF™, Mount Olive, NJ, USA) was used.

[0062] Preparation of BY-2 cell lysates BY-2 cells were harvested during the exponential growth phase of fermentation at a constant packed cell volume of 20-25%. To prepare protoplasts, they were treated with 3% (v / v) Rohament® CL and 0.2% (v / v) Rohapect® UF (pectinase and arabanase) (AB Enzyme™, Darmstadt, Germany) directly in the fermentation medium. Osmolarity was adjusted by adding 360 mM mannitol.

[0063] To devacuolate the resulting protoplasts, they were layered onto a discontinuous Percoll gradient containing (from bottom to top) 70% (v / v, 3 ml), 40% (v / v, 5 ml), 30% (v / v, 3 ml), 15% (v / v, 3 ml) and 0% (3 ml) Percoll (GE™ Healthcare, Munich, Germany) in 0.7 M mannitol, 20 mM MgCl2 and 5 mM PIPES-KOH (pH 7.0) in 50 mL polypropylene tubes (Greiner Bio-One™, Frickenhausen, Germany). After centrifugation at 6800 × g for 1 h at 25 °C in a swinging-bucket rotor (JS-5.3, Beckmann-Coulter™, Krefeld Germany), devacuolated protoplasts were collected from the 40-70% (v / v) Percoll solution interface and suspended in 3-3.5 volumes of TR buffer (30 mM HEPES-KOH (pH 7.4), 60 mM potassium glutamate, 0.5 mM magnesium glutamate, 2 mM DTT) supplemented with 1 tablet of Complete EDTA-free Protease Inhibitor Mixture (Roche Diagnostics™, Mannheim, Germany) per 50 mL.

[0064] Protoplasts were then disrupted on ice using 15 strokes of a Dounce™ homogenizer (Braun™, Melsungen, Germany) and nuclei and unbroken cells were removed by centrifugation at 500×g for 10 min at 4° C. The supernatant was then frozen in 1 mL aliquots at −80° C. Prior to freezing, the supernatant was optionally supplemented with 0.5 mM CaCl2 and treated with 75 U / mL nuclease S7 (Roche Diagnostics) for 15 min at 20° C., followed by centrifugation with CaCl2. 2+ Nucleases can be inactivated by supplementation with 2 mM EGTA as an ion chelating agent.

[0065] Plasmid constructs The vector pIVEX_GAAAGA_Omega_eYFP-His was prepared by inserting annealed oligonucleotide primer 1 (SEQ ID NO: 1) and oligonucleotide primer 2 (SEQ ID NO: 2), containing the T7 promoter and the Tobacco Mosaic Virus 5' omega leader sequence with GAAAGA as the first 6 nucleotides, into pIVEX1.3_eYFP-His (kindly provided by Dr. Stefan Kubick, Fraunhofer Institute for Cell Therapy and Immunology IZI, Potsdam-Golm, Germany) using the NspI and NcoI sites. For the vector pIVEX_GAAAGA_Omega_Strep-eYFP, containing an N-terminal streptavidin affinity tag, the Strep-eYFP sequence was amplified by PCR using pIX3.0_Strep-eYFP (kindly provided by Dr. Stefan Kubick) as template with oligonucleotide primer 3 (SEQ ID NO: 3) and oligonucleotide primer 4 (SEQ ID NO: 4). The PCR product was digested with PciI and Acc65I and inserted into the NcoI and Acc65I sites of pIVEX_GAAAGA_Omega_eYFP-His.

[0066] pIVEX vectors carrying 21333, 22807, AAD12, Cry2A, Cry3A, Trap8VIP3A, VIP3A, Cry6A, 17912, and Cry1F were generated by PCR amplification of the genes using oligonucleotide primers 5-24 (SEQ ID NOs: 5-24) and subsequent integration of the PCR products into pIVEX_GAAAGA_Omega_eYFP-His cut with NcoI and KpnI by Gibson assembly (NEB™, Frankfurt, Germany).

[0067] [Table 1]

[0068] Coupled transcription-translation cell-free protein synthesis The coupled transcription-translation reaction was carried out in 50 μL aliquots in a thermomixer (HLC by Ditabis™, Pforheim, Germany) at 25° C. and 700 rpm for 40-52 h. The reaction with creatine phosphate and creatine kinase contained 40% (v / v) tobacco BY-2 cell lysate (BYL), 20 mM HEPES-KOH pH 7.8, 10 mM magnesium glutamate, 10 mM potassium glutamate, 3 mM ATP, 1.2 mM GTP, 1.2 mM CTP, 1.2 mM UTP, 100 μg / mL chloramphenicol, 50 ng / μL T7 RNA polymerase, 80 ng / μl plasmid, 30 mM creatine phosphate and 100 μg / ml creatine kinase. The reaction solution without creatine phosphate and creatine kinase contained 40% (v / v) BYL, 20 mM HEPES-KOH (pH 7.8), 9 mM magnesium glutamate, 20 mM potassium glutamate, 4 mM ATP, 1.6 mM GTP, 1.6 mM CTP, 1.6 mM UTP, 100 μg / mL chloramphenicol, 30 ng / μL T7 RNA polymerase, and 40 ng / μL plasmid.

[0069] Analysis of products Fluorescence signals from eYFP were quantified using a Synergy™ HT Multi-Mode Microplate Reader (Biotek™, Bad Friedrichshall, Germany) with 485 / 20 nm excitation and 528 / 20 nm emission filters. The amount of eYFP was determined by generating a standard curve based on different concentrations of eYFP in BYL translation reactions without DNA template. eYFP standards were produced using an in-house in vitro translation system based on E. coli (Zawada (2012) Methods Mol. Biol. 805:31-41) and purified by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). The concentration of purified eYFP was determined using a colorimetric assay. Bradford (1976) Anal. Biochem. 72: 248-54.

[0070] Residue-specific labeling of target proteins To selectively fluorescently label the target proteins with amino acids, the FluoroTect™ GreenLys in vitro Translation Labeling System (Promega™, Mannheim, Germany) was used according to the manufacturer's instructions. (登録商標) The system contains a modified charged lysine tRNA labeled with -FL. Using this system, fluorescently labeled lysine residues are incorporated into nascent proteins at multiple sites during translation.

[0071] JC-1 staining The presence of mitochondria in BYL was demonstrated using the lipophilic cationic probe 5,6-dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl]-1,3-diethyl-iodide (JC-1, Thermo Scientific™, Waltham, MA, USA). In live cells, JC-1 exists as a green fluorescent monomer (490 nm excitation / 530 nm emission) at depolarized mitochondrial membrane potential. At normal and hyperpolarized mitochondrial membrane potentials, JC-1 is concentrated in mitochondria and forms J-aggregates, which shift the emission from 530 nm to 590 nm. Cellular mitochondria show increasingly more highly red fluorescence (590 nm) of J-aggregates with increasingly negative mitochondrial membrane potential. Nuydens et al.(1999) J.Neurosci.Methods 92:153-9;Reers et al.(1995)Methods Enzymol.260:406-17;Salvioli et al.(1997)FEBS Lett.411:77-82.

[0072] For BYL staining, JC-1 was dissolved in DMSO at a concentration of 5 mg / mL. JC-1 stock solution was used at a 1:1000 dilution to stain mitochondria within 10 min at a final concentration of 5 μg / mL.

[0073] Cell-free protein synthesis using inhibitors of oxidative phosphorylation Cell-free protein synthesis (50 μL reactions) was performed with and without the addition of inhibitors of oxidative phosphorylation to the BYL system, both with and without creatine phosphate and creatine kinase. The inhibitors included: sodium azide (0.05%, Bogucka & Wojtczak (1966) Biochim. Biophys. Acta 122:381-92) and 2-thenoyltrifluoroacetone (TTA, 0.5 mM; Tappel (1960) Biochem. Pharmacol. 3:289-96). Sodium azide was dissolved in water. TTA was dissolved in methanol. A negative control performed with methanol showed that this solvent had no effect on protein synthesis at the concentrations used in this study.

[0074] Example 2: Protein synthesis without artificial energy regeneration To reduce the release of phosphate, the artificial regeneration system consisting of creatine phosphate (CP) and creatine kinase (CK) was omitted in the cell-free BYL system. Reactions with variable concentrations of reaction components were performed with and without CP and CK; HEPES-KOH, pH 7.8 (0–80 mM), magnesium glutamate (1–12 mM), potassium glutamate (0–40 mM), plasmid (10–100 ng / μL (4.5–43 nM)), NTPs (i.e., ATP / (GTP / CTP / UTP)) (0.5 / 0.2–4 / 1.6 mM), and T7 RNA polymerase (20–80 ng / μL), with and without CP (0–40 mM) and CK. D esign O f EA Design of Experiment (DoE) based approach (fractional designs and response surface modeling in Design Expert v8.0 (State-Ease™ Inc., MN, USA)) was used to devise a method for determining the concentration of chloramphenicol. The concentration of chloramphenicol was taken from the CP / CK system. From these experiments, favorable reactant concentrations were obtained for both systems (with and without CP / CK). Table 2 [Table 2]

[0075] Comparison of the BYL systems with and without CP / CK revealed that the system without CP / CK exhibited long-term activity for approximately 40 h (compared to 20 h in BYL with CP / CK) and yielded up to 60% more target protein (Figure 1A). The presence of mitochondria in BYL was demonstrated by staining of BYL with a selective dye JC-1 (Thermo Fischer Scientific, Waltham, MA, USA) that fluoresces in red, indicating that mitochondria retain their characteristic membrane potential in BYL. Energy regeneration by oxidative phosphorylation was demonstrated by the use of two different inhibitors of the electron transport chain, sodium azide and thenoyltrifluoroacetone (TTA). Both sodium azide and ATP were found to almost completely inhibit the system, as shown by the dramatically reduced biosynthesis of the target protein eYFP (Figure 1B).

[0076] Energy in a CP / CK-free system is thought to be provided by oxidative phosphorylation. Glutamate from added magnesium and potassium glutamate is metabolized in the citric acid cycle in mitochondria, leading to the production of the reducing equivalents NADH and FADH. Electrons enter the electron transport chain via NADH and FADH to generate ATP through oxidative phosphorylation with the consumption of molecular oxygen. Figure 2.

[0077] Expression of ten target proteins in the BYL system without CP / CK was compared to the BYL system with CP / CK. Coupled BYL transcription-translation reactions with and without creatine phosphate (CP) and creatine kinase (CK) were performed for 40 h at 25 °C. In each case, 2 µL of reaction volume was loaded onto a 4-12% (w / v) gradient SDS-PAGE gel, and the amount of synthesized protein was visualized by Coomassie staining. Several target proteins, including AAD12, Cry3A, and Cry1F, showed significantly higher expression levels in the optimized system (without CP / CK), as represented by intense bands in the Coomassie-stained gel.

[0078] The BYL system, which does not have an artificial energy regeneration system, is cheaper and runs longer, leading to increased levels of recombinant proteins. Furthermore, the BYL system offers the possibility to investigate compounds or proteins that affect mitochondrial function.

[0079] Example 3: Modification of coupled transcription-translation reactions without artificial energy regeneration Use of sorbitol for preparation of BY-2 lysate For the preparation of BY-2 lysates, large amounts of osmotic agents are required to adjust the osmolarity during protoplast formation and devacuolation. Mannitol is routinely used for this purpose and accounts for about 10% of the total cost of lysate preparation. Sorbitol (about 10 times cheaper) was tested as osmotic agent. Parallel experiments using mannitol or sorbitol for protoplast formation and devacuolation revealed that sorbitol was surprisingly not equivalent to mannitol but was superior in terms of both lysate yield and lysate quality as determined by the expression of eYFP (Figure 5). Furthermore, the higher solubility of sorbitol was found to facilitate the preparation of buffers. However, mannitol was also found to be superior to sorbitol for the final washing of devacuolated protoplasts, and the utilization of sorbitol in this step led to lower eYFP yields and a more viscous reaction mixture.

[0080] Inhibition of microbial growth BY-2 lysate has the ability to support microbial growth when IVTT reactions are incubated, resulting in depletion of reaction substrates and consequently reduced yield of target protein. Therefore, several antimicrobial substances were tested in the IVTT reaction system. Chloramphenicol, spectinomycin, streptomycin, ampicillin, and sodium azide were investigated for their effects on eYFP production and microbial growth in IVTT.

[0081] Antimicrobial substances were added to the BYL reaction mixture, and after 45 h of incubation, 0.2 μL of the BYL reaction mixture was plated on LB plates to analyze microbial growth. Spectinomycin, streptomycin, ampicillin, and sodium azide had no inhibitory effect on microbial growth or showed a detrimental effect on the yield of eYFP. For example, sodium azide at a concentration of 0.05% (w / v) almost completely inhibited both microbial growth and translation activity, presumably by inhibiting mitochondrial energy regeneration. Only chloramphenicol was able to inhibit microbial growth without any loss of translation activity (Figure 3B; Figure 3C), which makes its usefulness in the IVTT system at least different from other antimicrobial substances. Dose response studies indicated that the highest eYFP yield was achieved at 100 μg / mL chloramphenicol (Figure 3D), while complete inhibition of microbial growth was obtained at 200 μg / mL chloramphenicol (Figure 3A), and SDS-PAGE analysis also demonstrated the protective effect of chloramphenicol on eYFP yield and protein stability. Unlike reactions performed in the presence of chloramphenicol, reactions without chloramphenicol showed significant protein degradation.

[0082] Nucleoside triphosphates The concentration of NTPs in the system was adjusted by a DoE-based approach. High concentrations of ATP and GTP compared to CTP and UTP were predicted to be beneficial for the coupled IVTT system, since ATP and GTP are used in both transcription and translation, whereas CTP and UTP are used only in transcription. Thus, the concentrations of single NTPs were varied in DoE-based experiments instead of using different amounts of a fixed NTP mix. These were screened for optimal concentrations using a cubic IV-optimal design with 96 experiments. The plasmid template for the IVTT reaction was pIVEX_GAAAGA_Omega_Strep-eYFP. The concentration of magnesium glutamate was adjusted by the binding of NTPs to magnesium. Table 3 shows the concentration range of each factor screened. A quadratic model was fitted to the experimental data. A response surface model was used to predict the value of the factor that would result in the most produced eYFP protein (Zhou et al., 2010). All non-significant terms (p>0.05 by ANOVA) were ignored and the model was shown to be significant in the ANOVA table (Table 4).

[0083] Experiments revealed that the most target protein was produced with 6 mM ATP, 0.8 mM CTP, 0.8 mM UTP, and 1.6 mM GTP in the reaction. Figure 4 These results led to the development of a new NTP mix consisting of 150 / 40 / 20 / 20 mM ATP / GTP / CTP / UTP. Using 2 μl of the new NTP mix, the yield of eYFP increased by 20% in the IVTT system. As an added benefit, the new NTP mix is ​​approximately 40% less costly than the standard mix due to the 10-15 times lower cost of ATP compared to other nucleotides.

[0084] [Table 3]

[0085] [Table 4]

[0086] Use of larger lysate volumes To investigate the effect of increasing the lysate portion of the IVTT reaction on target protein expression, several BY-2 lysates prepared with sorbitol during protoplast formation and devacuolization were used, besides BYL prepared with mannitol, to perform 50 μl reactions with 40% (20 μl) or 60% (30 μl) lysates. Reactions were carried out for 48 h at 25 °C and 700 rpm using the plasmid pIVEX_GAAAGA_Omega_Strep-eYFP as template. Reactions with 60% (v / v) lysate prepared with sorbitol achieved approximately 1 mg / mL of eYFP, which corresponds to an 80% higher yield compared to the standard reaction mixture (Figure 5). It is presumed that the increased amount of ribosomes, translation factors, chaperones, and mitochondria increased or prolonged energy production, thereby resulting in a higher yield. This result indicates that the lysates are free of deleterious factors that generate greater inhibition in such large quantities.

[0087] Expression of target proteins in cell-free systems and wheat germ extracts without artificial energy regeneration systems To confirm that the cell-free expression system without artificial energy regeneration is generally effective across substrates, the system was used to express 10 additional target gene products other than eYFP. Strep-tagged eYFP and 10 other target proteins were expressed in 60% BY-2 cell lysate prepared with sorbitol, 40% BY-2 cell lysate prepared with mannitol, and in the CellFree™ Sciences WGE system. Coupled transcription-translation reactions were carried out in a volume of 50 μL at 25° C. and 700 rpm for 40 hours. Coupled transcription and translation reactions using the WGE system were performed according to the manufacturer's instructions. In each case, 1 μL of the reaction mixture and the mixed biphasic reaction were loaded onto a 4-12% (w / v) gradient SDS PAGE gel, respectively, and the target proteins were visualized by Coomassie staining. The BYL system successfully transcribed and translated each of the different genes tested to produce protein in every case. Indeed, the BYL system consistently produced more intense bands compared to those produced using the WGE system. For Strep-eYFP, protein produced in the three systems was quantified using a fluorescence reader and compared to an eYFP standard curve, revealing that 1115 μg / mL and 441 μg / mL were produced under two sets of conditions in the BYL system, while only 105 μg / mL was produced in the WGE system.

[0088] Addition of glucosylglycerol and use of higher lysate amounts To increase the stability of cell-free lysates without artificial energy regeneration, we hypothesized that small molecules described as cell and / or protein cryoprotectants could maintain the translational activity of the lysates. Such molecules occur naturally in extremophiles and protect them against osmotic stress, heat, desiccation, and UV light, stabilizing membranes and proteins by causing an increase in water density at the surface and promoting the native conformation of proteins. However, it was expected that cryoprotectants would have a strong inhibitory effect on the IVTT system.

[0089] Different concentrations of cryoprotectants, ectoine, hydroxyectoine, and glucosylglyceride, were added to the ligated IVTT reactions to determine the amount of eYFP produced from the template plasmid pIVEX_GAAAGA_Omega_Strep-eYFP. 50 μL IVTT reactions (60% (v / v) BYL) were carried out in 96-well plates at 25° C. and 500 rpm for 44 hours in a Kuhner shaker™ with controlled humidity.

[0090] Ectoine and hydroxyectoine had no effect on the eYFP yield at concentrations up to 1% and 2% (v / v), respectively. Figure 6. In fact, higher concentrations of ectoine and hydroxyectoine inhibited the system. In contrast, glucosylglycerol had a strong positive effect on the eYFP yield. Reactions containing 0.5% (v / v) glucosylglycerol yielded approximately 50% more eYFP after 44 h compared to standard reactions without glucosylglyceride. Figure 6. The positive effect of glucosylglycerol was consistent across five different lysate batches (BYL 08.01.2016, BYL 21.01.2016, BYL 11.03.2016, BYL 01.04.2016, BYL 05.04.2016) in IVTT reactions without and with 0.5% (v / v) glucosylglycerol. 50 μL IVTT reactions were performed in triplicate with 60% and 80% (v / v) lysate portions using the plasmid pIVEX_GAAAGA_Omega_Strep-eYFP as template at 25 °C and 500 rpm for 48 h under controlled humidity (70%) in 96-well plates. Reactions using 60% (v / v) lysate and 0.5% (v / v) glucosylglycerol produced approximately 80% more eYFP compared to reactions without glucosylglycerol. Figure 7 Using 80% (v / v) lysate and 0.5% (v / v) glucosylglycerol, the yield of eYFP increased by approximately 110% (nearly 2 mg / mL eYFP) compared to reactions using 80% (v / v) lysate without glucosylglyceride. Figure 7 The time course of the IVTT reaction in a cell-free system without artificial energy regeneration was determined by measuring the amount of protein produced at different time points up to 64 h. Figure 8 After 16 h, reactions using 60% or 80% lysate with or without 0.5% glucosylglycerol produced approximately the same amount of protein. Figure 8 Without glucosylglycerol, translation activity in both 60% and 80% (v / v) lysates did not increase further after 24 h. Figure 8 However, eYFP production with glucosylglycerol showed an approximately linear increase in productivity up to at least 64 h in both 60% and 80% (v / v) lysates. Figure 8 This result confirms that glucosylglycerol has a stabilizing effect on the system that extends translation activity beyond 64 h. On average, reactions using 80% (v / v) lysate and 0.5% (v / v) glucosylglycerol yielded approximately 2.5 mg / mL of eYFP.

[0091] Addition of branched chain amino acids . The ability of branched chain amino acids (BCAAs) to promote protein synthesis in a cell-free expression system was also tested, and it was observed that the addition of BCAAs increased and stabilized the yield of the target protein. To verify the positive effect of BCAAs on the system, different concentrations of BCAAs were added to concatenated IVTT reactions using four lysates prepared from shake flasks (SF) or continuous fermentation (CF). In 50 μL IVTT reactions with 80% (v / v) lysate in 96-well plates at 25 °C and 500 rpm for 66 h using the plasmid template pIVEX_GAAAGA_Omega_Strep-eYFP in a Kuhner™ shaker at controlled humidity, BCAAs had a positive effect on the yield of eYFP at all concentrations tested, regardless of whether the lysate was prepared from shake flasks or continuous fermentation. Figure 9 Reactions with 1 mM BCAAs yielded approximately 70% more eYFP (average yield of 2.5 mg eYFP per mL) compared to reactions without BCAAs.

Claims

1. A system for synthesis of a polypeptide, said system comprising a reaction vessel, An aqueous cell lysate from tobacco plants containing mitochondria and magnesium, exogenous RNA polymerase, an exogenous nucleic acid template encoding a polypeptide; and nucleoside triphosphates (NTPs), the reaction vessel does not contain added creatine phosphate and creatine kinase that are exogenous to the cell lysate and mitochondria, and the RNA polymerase directs synthesis of mRNA to synthesize a polypeptide encoded by a nucleic acid template.

2. The reaction vessel, pH buffers, Mg(C ５ H ８ NO ４ ) ２ 、 KC ５ H ８ NO ４ 、 and chloramphenicol.

3. The container contains an aqueous cell lysate from plant cells containing mitochondria, a DNA template, HEPES-KOH pH 7.8, Mg(C ５ H ８ NO ４ ) ２ , K.C. ５ H ８ NO ４ 13. The system of claim 1, consisting essentially of: nucleoside triphosphates, RNA polymerase, and chloramphenicol.

4. 1. A method for synthesizing at least one polypeptide, the method comprising combining reaction components in a reaction vessel, the reaction components comprising: Aqueous cell lysate from tobacco plants containing mitochondria and magnesium, exogenous RNA polymerase, an exogenous nucleic acid template encoding a polypeptide; and nucleoside triphosphates, The method, wherein the reaction vessel does not contain the cell lysate and creatine phosphate and creatine kinase exogenous to mitochondria, and the RNA polymerase directs synthesis of mRNA to synthesize a polypeptide encoded by a nucleic acid template.

5. The following conditions: the mitochondria are exogenous to the cell lysate; the nucleic acid template is a DNA molecule; and 5. The method of claim 4, wherein the reaction is stopped by inhibiting the electron transport chain or removing oxygen from the reaction in any combination.

6. The method described in claim 4, wherein synthesis of the polypeptide exceeds 20 hours.

7. A devacuolated aqueous cell lysate from tobacco plant cells containing mitochondria and magnesium, said cell lysate being treated to destroy endogenous ribonucleic acid (RNA); nucleoside triphosphates (NTPs), said NTPs and said aqueous cell lysate being disposed in one or more containers separate from a cell lysate derived from said tobacco plant cells; and 5. A kit comprising: a cell lysate from tobacco plant cells containing mitochondria and magnesium, the NTP, an exogenous RNA polymerase, and an exogenous nucleic acid template encoding the polypeptide in a reaction vessel according to claim 4, wherein the kit does not contain creatine phosphate and creatine kinase that are exogenous to the cell lysate and mitochondria.

8. The kit of claim 7, further comprising a nucleic acid template.

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