A novel aeukaryotic cell expression system that does not require an artificial energy regeneration system.

Abstract

To provide methods for in vitro production of biopolymers in a cell-free system utilizing organelles (e.g., plastids, mitochondria or chloroplasts).SOLUTION: A method for synthesis of a polypeptide comprises introducing, into an aqueous cellular lysate derived from a tobacco plant that comprises organelles, nucleotide triphosphates (NTPs) and an exogenous nucleic acid template that encodes a polypeptide, where the cellular lysate does not contain exogenous added creatine phosphate or creatine kinase, thereby providing a synthesis reaction volume, and synthesizing the polypeptide in the synthesis reaction volume.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Claim of Priority This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 457,073, filed on Feb. 9, 2017 and its disclosure 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 a cell-free system. Certain embodiments utilize organelles (e.g., plastids, mitochondria or chloroplasts) to provide energy for the continuous production of biopolymers in a cell-free system, eliminating the need for certain undesirable energy storage molecules within the system, such as creatine phosphate.

Background Art

[0003] The increasing demand for new therapeutic proteins, industrial 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. New technologies for cell-free protein synthesis (CFPS) can help meet this demand. Carlson et al. (2012) Biotechnol. Adv. 30(5):1185-94. Compared to cell line expression, CFPS offers advantages such as short process times and direct control and monitoring of reaction conditions. Swartz( ​​​​​​ (2012), multiple tans without the laborious cloning and transformation steps mentioned above. PCR products can be used directly for simultaneous expression of the protein. (Wu et al.) (2007)Angew.Chem.Int.Ed.Engl.46(18):335 6-8;Yabuki et al.(2007)J.Struct.Funct.Ge nomics 8(4):173-91;Gan & Jewett(2014)Bio technol.J.9(5):641-51. The CFPS platform allows for... Addition of accessory factors that promote protein folding (Ozawa et al (200) 5)J.Biomol.NMR 32(3):235-41;Endo et al.( 2006)Mol.Biotechnol.33(3):199-209;Matsud a et al(2006)J.Struct.Ltd.Genomics 7(2): 93-100), or incorporation of non-natural amino acids (Albayrak & Swart z(2013)Nucleic Acids Res.41(11):5949-63) White et al. (2013) Methods 60:70-4) makes this possible. It also promotes the expression of cytotoxic proteins that cannot be produced in living cells. Xu et a l.(2005)Appl.Biochem.Biotechnol.127(1):5 3-62;Schwarz et al. (2008) Proteomics 8(19 ):3933-46;Xun et al. (2009) Protein Expr.P urif.68(1):22-7.

[0004] Cell-free lysates of Escherichia coli are widely used, It is advantageous due to its low cost, scalability, and high productivity. (Zawada et al.) (2011)Biotechnol.Bioeng.108(7):1570-8;C aschera & Noireaux(2014)Biochimie 99:162 -8. However, since the lysates originate from bacteria, inefficient oxidative folding occurs. Furthermore, the lack of chaperone and glycosylation mechanisms results in complexes with multiple subdomains. It is unsuitable for protein production. The aeukaryotic cell system is not suitable for the expression of such proteins. It is more suitable for this purpose and supports most forms of post-translational modification. Chang et al.(2005)J.Mol.Biol.353(2):397-409;Zhan g & Kaufman(2006)Handb.Exp.Pharmacol.(17 2):69-91. The most frequently used systems are wheat germ extract (WGE) and insects. Based on cell extracts (ICE) and rabbit reticulocyte lysates (RLL). However, this The system is expensive, and the extract preparation is complex. Carlson et al. 2012), see above. As a result, Leishmania tarentolae (Mur eev et al(2009) Nat.Biotechnol.27(8):747- 52), Chinese hamster ovary (CHO) cells (Brodel et al (20 14) Biotechnol. Bioeng. 111(1):25-36), and Sa ccharomyces cerevisiae(Hodgman&Jewett(20 13)Biotechnol.Bioeng.110(10):2643-54;Gan & Jewett (2014), and further eukaryotic CFPS needs, such as those based on the above). arose.

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

[0006] Using a continuous flow system, 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 into the chamber. Thus, these reactions require a significant investment of time and resources. Furthermore, translation in a "continuous" system is aimed at producing large amounts of protein, and the system is substantially different from those used to perform static ("batch") in vitro translation reactions. Static reactions can be carried out in small reaction volumes (e.g., microliters) and are not aimed at producing preparative amounts (e.g., milligrams) of protein. Such batch reactions can be completed in 1 - 2 hours. For all of the reasons described above, while increasing the reaction period and protein yield compared to the corresponding batch system, continuous reaction systems require more expensive reagents.

[0007] Disclosure The general strategy disclosed herein utilizes organelles in cell-free lysate reactions​​​​​​​​ This provides energy regeneration within the reaction system. This strategy is used in some cases by bio Modification of polymers (e.g., polynucleotides, polypeptides, polysaccharides, and complex carbohydrates) It is useful for achieving improved in vitro synthesis. In certain embodiments, aeukaryotic cells The presence of mitochondria in the system improves upon conventional batch and continuous reaction processes. This results in a reaction system, which is, for example, an energy delivery reagent (e.g., creatine). Addition of phosphates and / or creatine kinase, and / or amino acid supplementation is necessary. This is achieved by significantly reducing or eliminating the essential components while prolonging the reaction period. In some cases, The disclosed cell-free polymerization reaction is significantly more efficient than conventional reactions currently used in the art. It is efficient.

[0008] In the reaction volume, organelles (e.g., plastids, chloroplasts, and / or mitochondria) This includes combining cell lysates, polymer templates, and polymer monomer units. Methods for synthesizing iopolymers are described herein. In some embodiments, the reaction volume is, for example, For example, it does not contain the creatine phosphate / creatine kinase energy regeneration system, and Therefore, either no phosphate is added to the reaction volume, or only the minimum amount of phosphate is added. Yes. In certain embodiments, the organelle is a mitochondria. In some embodiments, Cell lysates are eukaryotic cell lysates; for example, plants (e.g., tobacco, corn, and dwarf corn). It is a lysate derived from cells. In some cases, the cell lysate is Bright Yel. It is a lysate derived from low-2 (BY-2) tobacco cells. In some embodiments, polymers are used. The template is a DNA molecule or an RNA molecule. Reactions that utilize RNA as a polymer template are , producing polypeptides as biopolymers from monomer amino acids through translation reactions Reactions that utilize DNA as a polymer template are used for in vitro replication or transcription reactions. Through this process, further nucleic acid molecules (e.g., D) are produced from monomer nucleotides as biopolymers. NA and RNA may be produced, or through a translation reaction linked to transcription from a template. Polypeptides may be produced by this method. In certain embodiments, the energy of the biopolymer Gee-free synthesis methods include, for example, but are not limited to, organelles, polymer templates and / Alternatively, adding monomer units to the reaction volume and / or adding biopolymers from the reaction volume. - may include isolating the components of the lysate from the TCA cycle. In some cases, the components of the lysate may be isolated from the TCA cycle. Since amino acids can be produced using an endogenous biosynthetic pathway that begins in the intersynthesis, the reaction volume is protein It does not require amino acid supplementation to support the expression of certain proteins. Therefore, some of these proteins... In this example, the amino acids present in the lysate containing organelles are the sustained polypeptide It may be sufficient to support synthesis.

[0009] The disclosed cell lysate system is considered unsuitable for energy regeneration systems in terms of quantity. As long as the reaction volume contains exogenous creatine phosphate and / or creatine kinase, the minimum Supplementation with a small amount of exogenous creatine phosphate, a minimum amount of creatine kinase, or both. This is also acceptable. For example, the reaction volume may be 15 mM or less, 10 mM or less, 5 mM or less, 1 mM or less, Additives of 500 μM or less, 100 μM or less, 50 μM or less, or 10 μM or less It may contain atin phosphate. In another example, the reaction volume is 100 μg / mL or less, 50 μg / mL or less, 10μg / mL or less, 5μg / mL or less, 1μg / mL or less, 0.5μg / It may contain creatine kinase in amounts of 0.1 μg / mL or less. These amounts are used to maintain biopolymer synthesis (over the long-term period disclosed herein). It is not suitable for maintaining biopolymer synthesis (for example), and therefore, this specification According to the methods and systems disclosed herein, plastids, mitochondria, or chloroplasts, etc. It is necessary to include organelles.

[0010] Some embodiments use biopolymers without using artificial energy regeneration systems. The system includes a system for synthesizing aqueous cell lysates. In these embodiments, the system includes an aqueous cell lysate. , including (endogenous or exogenous) organelles, polymer templates. In certain embodiments, The stem also includes the monomer units of the polymer. Conventional for in vitro biopolymer synthesis. The cell-free system further includes creatine phosphate and creatine kinase, which are compounded As the reaction progresses, it is used to regenerate energy within the system. (Implementation as specified herein) In this state, the system is substantially lacking in creatine phosphate, and creatine phosphate and creatine phosphate Creatin kinase is not added to the system. In certain cases, the system is creatine It does not contain either phosphate or creatine kinase. In certain embodiments, biopoly A system for the synthesis of M is, for example, but is not limited to, a pH buffer, magnesium ( For example, Mg(C5H8NO4)2), potassium (for example, KC5H8NO4), nucleate Osides (for example, nucleotide triphosphates, nucleoside diphosphates, and nucleotide monophosphates) Phosphate), enzymes (e.g., RNA polymerase), and chloramphenicol further It may include. In certain embodiments, amino acids other than glutamate are already present in the system. It is not added to those that are present (i.e., amino acids present in lysates and organelles). In some cases, the aforementioned system is used for longer periods exceeding 20 hours (for example, about 40 hours). It may exhibit activity, except for the addition of creatine phosphate and creatine kinase. It extracts significantly more (e.g., about 60% more) target proteins than the same conventional system. It may be manufactured.

[0011] Some embodiments involve generating biopolymers without using artificial energy regeneration systems. Includes a kit for synthesis. In some embodiments, the kit is used for artificial energy regeneration systems. Components and kits for a system to synthesize biopolymers without using a TEM. Includes written instructions for use. For example, but not limited to, one kit. Aqueous cell lysates, (endogenous or exogenous) organelles, arranged in separate volumes above. The components of the kit, along with one or more of the polymer template and polymer monomer units. Instructions specifying a mixture and not including creatine phosphate and creatine kinase. It may contain any exogenous components. As a further example, the kit may contain a pH buffer, magnesium Um, potassium, nucleosides, enzymes (e.g., RNA polymerase), and chloram It may further contain one or more of the phenicols. Biopoly The kit for synthesizing MAR includes aqueous cell lysates (e.g., chloroplasts and / or mitocones). (including doria), monomer units of polymers (e.g., nucleosides), and written documentation. It may include. In these particular embodiments, the written instructions will be used by the user to combine Adding creatine phosphate (along with creatine kinase for energy regeneration) These components are not used as the target polymer template (for example, the DNA component encoding the polypeptide). It may also instruct to combine with the child (and any other reagents).

[0012] Embodiments herein describe the use of activated mitocs for energy regeneration in ongoing synthetic reactions. By incorporating ndria, the shadow of mitochondrial function within the context of in vitro synthesis is cast. It can be used to quantitatively investigate the compounds or proteins that cause the reaction. Furthermore, TCA The intermediates of Ikul are designed so that amino acid supplementation is not required for long-term polypeptide synthesis. It can be used during the synthesis reaction to produce an acid. In some embodiments, the method described herein Cell lysates for use in systems and kits reduce the oxygen dependence of the synthesis reaction. It contains chloroplasts. For example, the cell lysate contains plastids, chloroplasts and / or mitochondria. The lysate may be prepared from photosynthetically active cells so that it is retained in the lysate, on the other hand, Unsuitable cellular material is removed. In such specific examples, the methods and systems described herein The kit also provides energy derived from plastids, energy regeneration derived from mitochondria, and energy from chloroplasts. Energy can be regenerated, or a combination of both.

[0013] The aforementioned and other features are described in detail below in several embodiments, which proceed with reference to the attached drawings. This will become clearer from the explanation. [Brief explanation of the drawing]

[0014] [Figure 1A]Figure 1 shows the components and performance of a system for the synthesis of biopolymers without artificial energy regeneration, including tobacco BY-2 cell lysates (BYL). Figure 1(A) shows a comparison of the performance of this system with a system including creatine phosphate (CP) and creatine kinase (CK). A linked transcription-translation reaction was performed at 25°C for 52 hours 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 excitation at 485 / 20 nm and absorption filtering at 528 / 20 nm. [Figure 1B] Figure 1(B) shows the effects of inhibition of electron transport chain inhibition by sodium azide (azide) and telenoyltrifluoroacetone (TTA), respectively, on reporter yield. The mean and standard deviation were calculated from three independent transcription-translation experiments. [Figure 2] Figure 2 includes a diagram illustrating the proposed mechanism of ATP production in a system for synthesis without the use of artificial energy regeneration. Glutamate is used to produce a reducing equivalent, primarily in the form of NADH, through the TCA cycle located inside the mitochondria. NADH fuels oxidative phosphorylation, oxygen acts as the final electron acceptor, and ADP is converted to ATP. [Figure 3A] Figure 3 includes a chart showing the effect of different antimicrobial agents on eYFP yield and microbial growth in a system for synthesis without the use of artificial energy regeneration. A coupled BYL reaction was performed using pIVEX_GAAAGA_Omega_Strep-eYFP as a template at 25°C and 700 rpm for 45 hours. eYFP yield was determined by measuring fluorescence intensity using a fluorescence reader. The number of colony-forming units (CFUs) was determined by plating 0.2 μL of the BYL reaction volume onto an LB plate and incubating it at 37°C for 16 hours. The mean 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]Figure 3(B) shows the effects of different antimicrobial agents on eYFP yield, normalized against the yield in the standard reaction without antimicrobial agents (100%). [Figure 3C] Figure 3(C) shows the effects of different antimicrobial agents on microbial growth (CFU / μl). [Figure 3D] Figure 3(D) shows the effect of different concentrations of chloramphenicol on eYFP yield (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 the use of artificial energy regeneration. Response surface and contour plots for eYFP synthesis in a coupled BYL system without creatine phosphate and creatine kinase are presented. The effects of different NTPs and magnesium glutamate on yield are shown when other components of the reaction volume are 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 hours 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 fluorescence intensity using a fluorescence reader. [Figure 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 lysates prepared with mannitol (blue) were compared with reactions with novel lysates prepared with sorbitol (green). Reactions were carried out in volumes of 50 μL in 96-well plates. 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 evacuated protoplasts during lysate preparation. The ligation reaction was carried out at 25°C and 700 rpm for 48 hours using pIVEX_GAAAGA_Omega_Strep-eYFP as the DNA template. The yield of eYFP was determined by measuring fluorescence intensity using a fluorescence reader. The mean and standard deviation were calculated from two independent experiments. The dates indicate the fusion date of the large "BYL mix" and the preparation date of each new solution, respectively. [Figure 6] Figure 6 includes a bar graph showing the effects of ectoin, hydroxyectoin, and glucosylglyceridol on eYFP production in a coupled IVTT reaction without artificial energy regeneration. Ectoin, hydroxyectoin, and glucosylglyceridol were added to samples in amounts of 0–8% (v / v), using the plasmid pIVX_GAAAGA_Omega_Strep-eYFP as a template. The reaction was carried out in a 96-well plate at 25°C and 500 rpm for 44 hours at controlled humidity (70%) in a 50 μL lysate portion at 60% (v / v). The amount of eYFP produced was determined by the use of a fluorescence reader compared to an eYFP standard. The eYFP standard was prepared using an IVTT transcription-translation system and purified with Strep-Tactin® Sepharose®. The concentration of the 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 includes a bar graph showing the effect of glucosylglycerol on the coupled IVTT reaction without artificial energy regeneration. The amount of eYFP produced from plasmid pIVX_GAAAGA_Omega_Strep-eYFP was compared in five different lysate batches. The reaction was carried out using 60% or 80% (v / v) lysate, and with or without 0.5% (v / v) glucosylglycerol (GG). The reaction was performed in a volume of 50 μL in a 96-well plate at 25°C and 500 rpm for 48 hours at controlled humidity (70%). The 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 a coupled IVTT reaction without artificial energy regeneration. The reaction involved either 60% or 80% (v / v) lysate with or without 0.5% (v / v) glucosylglycerol (GG). Plasmid pIVX_GAAAGA_Omega_Strep-eYFP was used as a template, and the amount of eYFP produced (compared to eYFP standard) was determined using a fluorescence reader. The reaction was carried out in a 96-well plate at 25°C and 500 rpm for 64 hours in a Kuhner® shaker under controlled humidity (70%). Data represent the mean and standard deviation of three independent transcription-translation experiments using different lysate batches. [Figure 9] Figure 9 shows the effect of branched-chain amino acids (BCAAs) on eYFP production in a coupled IVTT reaction without artificial energy regeneration. 0–2 mM BCAAs were added as the final component to a coupled IVTT reaction using the plasmid pIVEX_GAAAGA_Omega_Strep-eYFP as a template. The reaction was carried out at 25°C and 500 rpm for 66 hours 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 compared to an eYFP standard. Data represent the mean and standard deviation of six independent transcription-translation experiments. Lysates were prepared from shaking flasks (SF) or continuous fermentation (CF). [Modes for carrying out the invention]

[0015] I. Overview of Some Embodiments Crude lysate-based cell-free protein synthesis (CFPS) systems can be used in vivo. It offers several advantages and has a wide range of applications, particularly in protein engineering and biotechnology. It is useful in pharmaceutical manufacturing and research. Conventional crude lysates are used for translation and protein folding. It contains components necessary for folding and energy metabolism, and therefore encodes using RNA templates. Almost any protein that is lysed will, insofar as energy storage reagents are replenished in the lysate, Synthesized therein in the presence of nucleotides, nucleotides, and salts. Linked transcription / translation In the stem, RNA polymerase is used to direct the synthesis of proteins from the DNA template. It can be added. In contrast to intracellular synthesis, CFPS has a shorter process time. Reduced protein hydrolysis, and identification of toxic proteins at defined locations. It may be permissible to express proteins containing the chemical group or non-natural amino acids. Therefore, the reaction can be directly controlled and monitored.

[0016] Eliminates the need for additional energy storage reagents, without artificial energy regeneration systems. Cell-free biopolymer synthesis (e.g., polypeptide synthesis) using cell lysates A system is disclosed herein. In embodiments, the system is obtained by oxidative phosphorylation. For energy regeneration during synthetic reactions, organelles (e.g., plastids, mitochondria) are used. Alternatively, eukaryotic cell lysates containing chloroplasts are used. In certain cases, the cell lysates are B This is a tobacco BY-2 lysate containing mitochondria derived from Y-2 cells. In the eukaryotic system during cell transformation, electrons are transported by the electron transport chain located within the inner membrane of mitochondria. Electrons are transferred from electron donors to electron acceptors such as oxygen. These redox reactions are It releases energy, which is used to phosphorylate ADP into ATP. The embodiment utilizes the energy from this process to enhance the biopolymer in the solution. It promotes subsequent synthesis. Therefore, in the embodiments herein, an inhibitor of the electron transport system and By using airtight conditions, energy regeneration through oxidative phosphorylation is stopped, thereby, The synthesis reaction can be stopped by terminating the translation of the stem. In this application configuration, the system allows the reaction to proceed under anaerobic or substantially anaerobic conditions. It contains plastids and chloroplasts that can be converted into functional cells.

[0017] Conventional aeukaryotic cell systems (e.g., wheat germ extract and insect cell extract) are mito They lack chondria. Instead, these systems achieve the necessary ATP regeneration. To support protein expression, creatine phosphate and creatine kinase Additional components are needed. They react to support protein expression through energy regeneration. Many accumulations of free phosphate (derived from creatine phosphate) added to the mixture are due to these This is a significant limiting factor in the performance of the system. Ezure et al. (2006) )Biotechnol.Prog.22(6):1570-7;Takai et a l.(2010)Curr.Pharm.Biotechnol.11:272-8;B rodel et al.(2014)Biotechnol.Bioeng.111( 1):25-36; Hodgman & Jewett (2013), see above; Schobor g et al.(2014)Biotechnol.J.9(5):630-40. S The free phosphate introduced into the stem binds to magnesium (which is necessary for transcription and translation), and This results in premature breakdown of growth performance and low product yield.

[0018] To extend the lifespan of synthesis performance, currently available aeukaryotic cell systems are in dialysis mode. Using a "continuous flow" reaction, a long-lasting energy source is provided, and phosphorus in the reaction compartment Dilute the inhibitory component, such as the acid. Artificial energy regeneration system for biopolymer synthesis. The systems described herein that do not contain methicillin produce less inhibitory components, and they Because it possesses the body's energy regeneration capacity, it reduces or eliminates the need for reactive dialysis. However, However, the system described herein may, at the discretion of the implementer, be configured in a continuous flow configuration if desired. It may be used.

[0019] Compared to conventional aeukaryotic cell protein expression systems, the bio-expression system described herein Remer synthesis systems are generally inexpensive, and they also produce proteins over a longer period of time. This results in an increase in biopolymer yield. Furthermore, the system described herein, for example Furthermore, as a further promoter of cell-free protein expression, mitochondria and / or leaves This provides the possibility of investigating compounds or pathways that affect plaque function. The fundamentally different systems of the embodiments of the specification provide even greater benefits. It can be optimized.

[0020] Some of the systems for biopolymer synthesis described herein involve the growth of microorganisms. It has the ability to support, and through such proliferation, the IVTT response and / or protein Depletion of the substrate during degradation can occur, which may reduce the termination of the target protein. In some embodiments, the system uses chloramphenicol, which inhibits microbial growth. This includes, and in these embodiments, it may improve protein expression. Specific embodiments So, the system is, for example, 10-500 μg / mL (for example, 25-250 μg / mL) Chloramphenicol in amounts of 50-200 μg / mL and 100-200 μg / mL. Includes .

[0021] Certain amounts of NTPs in linked in vitro transcription / translation (IVTT) reactions are partially This specification describes the finding that an embodiment of this method can provide remarkably robust expression. Therefore, the expression is approximately 20% or more of what was predicted from the standard NTP amount (for example, 18% or less). Top, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% It could be more than % (18-25% more, 19-23% more, or about 20% more). In some cases, the system produces approximately 150 mM ATP, approximately 40 mM GTP, and approximately 20 mM This may include CTP and approximately 20mM of UTP. These reduced amounts of NTP are utilized. A further advantage of doing so is cost reduction, which is better than conventional plant cell systems. This is due to a reduction in the amount of P (the most expensive NTP).

[0022] Furthermore, some modifications to the system described herein may affect the yield and / or quality of the product. This specification describes a remarkable discovery that may provide further improvement. For example, Protoplus The use of sorbitol during the dehydration step of ion formation and lysate preparation is used in protein production. Sorbitol can lead to an increase in other commonly used substances such as mannitol. Since it is cheaper than osmotic agents, it can further reduce the cost of the system. For example, the ratio of the dissolved substance in the IVTT reaction is 50-90% (e.g., 55-85%) (v / Increasing v) leads to higher expression of several target proteins. Therefore, in this specification, specific examples include, for example, about 60% by volume (e.g., 58%, 59%) 60%, 61%, and 62%, or about 80% by volume (e.g., 78%, 89%, 8%). In amounts of 0%, 81%, and 82%, sorbate is present during protoplast formation and dehydrated vesicle formation. This includes the use of cell lysates prepared using Toll. Another example is glucosylg The addition of lyserol dramatically increases the protein yield in the IVTT reaction. For example, Compared to a standard reaction without cosylglycerol, 0.25% to 4% of the amount of glucosylglycerol Cerol yielded up to 80% more protein. Not bound by any particular theory. Although it is not a direct result, glucosylglycerol is presumably responsible for protein and membrane stability. By increasing it, the reaction yield is increased. Therefore, in some embodiments, the system The percentage is 0.25-4% (for example, 0.25-2%, 0.25-1%, approximately 0.5%, and 1%). Contains 0.5% glucosylglycerol. As a further example, although not necessary, branched By adding branched-chain amino acids (BCAAs), protein production can be increased. Therefore, in some embodiments, the system uses an amount of about 0.25 to 4 mM or 0. Amounts of 5-2 mM (for example, 0.48-2.2 mM, 0.5-2.0 mM, 0.5-1 mM) It contains BCAAs (and approximately 1 mM).

[0023] According to the modifications described herein, in certain embodiments, in a coupled IVTT reaction The production of the target protein can be extended for up to 64 hours. In one example, 80% by volume of lysate And the reaction with 0.5 volume% glucosylglycerol yields approximately 2.5 mg / mL eY It brings about FP.

[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 ovaries CL cellulase enzyme CK Creatine Kinase CP Creatine Phosphate Cry1F Bacillus thuringiensis Cry1F Delta-En dotoxin Cry3A B. thuringiensis Cry3A Delta-Endotoxin CTP (Cytidine Triphosphate) DMSO (Dimethyl Sulfoxide) Design of 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 (Tenoyltrifluoroacetone) UTP Uridinetriline UTR Untranslated Area WGE wheat germ extract

[0025] III. Terminology Isolated: An "isolated" biological component (e.g., nucleic acid or protein) is said to be an isolated component. These components naturally exist in the cells of living organisms, as well as other biological components (i.e., other chromosomes). From extrachromosomal DNA and RNA, as well as proteins, chemically in the said components While influencing the subjective or functional changes, they are substantially separated, produced separately, and It is purified (for example, nucleic acids are chemically bonded to the remaining DNA in the chromosome). (By destroying the bonds, it can be isolated from the chromosome.) "Isolated" nucleic acids As molecules and proteins, nucleic acid molecules and proteins purified by standard purification methods. Nucleic acid is one example. This term also refers to nucleic acids prepared by recombinant expression in host cells. and proteins, as well as chemically synthesized nucleic acid molecules, proteins, and peptides. It includes.

[0026] Nucleic acid molecule: As used herein, the term "nucleic acid molecule" refers to a multimeric type It can refer to nucleotides, and the sense strand and antisequence strand of RNA, cDNA, and genomic DNA. It may include both lance chains, as well as synthetic and mixed polymers thereof. Nucleotides are ribonucleotides, deoxyribonucleotides, or either type. It may refer to a modified form of a nucleotide. As used herein, "nucleic acid molecule" This is synonymous with "nucleic acid" and "polynucleotide." Unless otherwise specified, nucleic acid components The offspring is typically at least 10 base pairs long. The aforementioned terms refer to single-stranded and double-stranded DNA. Including form. Nucleic acid molecules are formed by natural and / or unnatural nucleotide bonds. Contains either or both of the linked native nucleotides and modified nucleotides. That's fine.

[0027] Nucleic acid molecules are chemically or biochemically modified, as can be easily understood by those skilled in the art. It may contain non-natural or derivatized nucleotide bases. Examples of embellishments include labeling, methylation, and substitution of one or more native nucleotides with analogs. , nucleotide-to-nucleotide modifications (e.g., non-charged bonds: e.g., methyl phosphonate, triglyceride phosphate) Tel, phosphoramidates, and carbamates; charged bonds: e.g., phosphorothioates and phosphorodithioates; pendent moieties: For example, peptides; intercalators: for example, acridine, psoralens, etc.; chelators Examples include alkylators and modified bonds (e.g., alpha-anemeric nucleic acids). Furthermore, the term "nucleic acid molecule" can refer to single-stranded, double-stranded, partially double, triple, or hairpin. This includes topological conformations, including type, ring, and padlock conformations.

[0028] Exogenous: The term "exogenous" refers to components added to the cell lysates as used herein (e.g., When applied to plastids, mitochondria, or chloroplasts, the cell lysates and This refers to components that have different origins. For example, those that do not originate from the cell lysate. The plastids, mitochondria, or chloroplasts added to the lysate are beneficial to the cell lysate. It is exogenous. The term exogenous does not, in any case, originate from the cell lysate itself. As long as the cell type used to derive the cell lysate is the same cell type or a different cell type. Plastids, mitochondria, and (for example, cells from different tissues or different species) This may also be applied to organelles such as chloroplasts. Furthermore, the term exogenous is used herein. Plastids, mitochondria, or chloroplasts used in cell lysates of the systems disclosed Components of energy regeneration systems that are added separately or in addition to organelles such as (examples) For example, in this specification, to mean creatine phosphate and creatine kinase It can be used.

[0029] IV. Biopolymer Synthesis Systems This disclosure relates to a system for synthesizing biopolymers without using artificial regeneration systems. The system described herein provides a system in which oxidative phosphorylation is activated and energy storage Biopolymer-enhanced in vitro compound that provides increased yield in the absence of storage reagents The present invention includes compositions and methods for achieving yield improvement through the combination of reaction IVTT components. This is obtained by and may be assembled and mixed by the implementer, or pre-mixed. The kit contains combined ingredients, unmixed ingredients, or a combination of both. It may be provided. The implementer may combine the ingredients they provide with the ingredients of the kit. Or all of them may be used; for example, in some embodiments, the kit may use DNA or It includes all components of a system for protein synthesis other than RNA templates, and DNA or The RNA template is provided by the implementer to synthesize the selected protein. When all the components of the reaction are mixed in the reaction volume under appropriate environmental conditions, the reaction begins. Along with the significant changes described in the specification, the conventional in vitro cell-free synthesis reaction was generally modified. The reaction proceeds as follows: The reaction occurs when one or more of the components (e.g., NTPs and amino acids) reach the reaction volume Until it is used up inside, or to terminate the energy regeneration process within the system By adjusting environmental conditions, progress may be permitted until it is stopped.

[0030] The methods and compositions disclosed herein mimic the cytoplasmic environment of eukaryotic cells and are prior art. This method shows significant improvement in protein production and protein folding compared to the previous method. For example, due to the presence of organelles, oxidative phosphorylation is active in the reaction volume. Therefore, the system described herein reduces or eliminates the need for a secondary energy source associated with synthesis inhibition. Avoid it.

[0031] The systems of the embodiments herein are, for example, for DNA amplification, DNA or RNA templates or The transcription of their RNA, the translation of RNA into polypeptides, and the synthesis of complex carbohydrates from monosaccharides. It is useful for the production / replication of biopolymers, including those containing compounds. Enhanced synthesis is useful in some cases. , an increase in the total or relative amount of biopolymers synthesized in the system; per unit time Increase in the total or relative amount of biopolymers synthesized; synthesis of biologically active biopolymers polymers (e.g., properly folded and / or post-translation modified proteins) Increase in metered or relative volume; increase in total or relative volume of soluble biopolymers synthesized. , as well as the time and / or cost required to synthesize a given amount of biopolymer Includes one or more reductions in spending.

[0032] Certain embodiments of this specification achieve translation of mRNA to produce polypeptides, The translation may be linked to the in vitro synthesis of mRNA from a DNA template. The cellular system contains all the factors necessary for mRNA translation, such as ribosomes, amino acids, tRNA, aminoacyl synthase, elongation factors, initiation factors, and ribosome recycling factors It contains offspring. In the examples herein, such a cell-free system is eukaryotic, for example, plant Cell lysis prepared from cells (e.g., tobacco BY-2 cells) by the method described herein Includes decomposition material.

[0033] cell lysate Embodiments of this specification may be adapted to utilize any eukaryotic cell lysate. Cell lysates retain various post-translational processing activities. Eukaryotic cell lysates also possess a wide range of properties. In vitro translation of various viruses and other prokaryotic RNAs, as well as eukaryotic mRNA. It supports codon usage that deviates from the codon usage of organisms from which cell lysates originate. The template mRNA used is, for example, a rare tRNA and / or amino acid in the organism. It can be used efficiently by refilling the stem. A specific example in this specification is tobacco. BY-2 cell lysates are used in both batch culture and agitated tank fermenters. It has been shown that a simple and cost-effective fermentation method can be provided in suspension culture, and that it is sufficiently reliable. It is suitable for established gene modification tools.

[0034] The preparation of cell lysates according to the embodiments herein is particularly important for the cell wall (in the case of plant cells) and This may include disruption / removal of the cell membrane, removal of the degraded vacuole, and removal of endogenous mRNA.

[0035] In some embodiments, the cell wall and membrane are, for example, mechanically destroyed, liquid, etc. Technologies such as homogenization, enzymatic digestion, high-frequency ultrasonic waves, reduced pressure, freeze / thaw cycles, and manual grinding. It can be destroyed by one or more. In the specific embodiments herein, the plant cell lysate is one or more Cell wall digestive enzymes (e.g., Cellulase Onozuka RS (trademark), Pec Tolyase Y-23 (trademark), Macerozyme R-10), and liquid yeast Elements (for example, Rohament CL (trademark), Rohament PL (trademark), and Rohapect UF (trademark); these were originally used in the manufacture of fruit juices and extracts. It is prepared by digesting the cell wall using (as intended). CL (trademark) contains cellulase concentrate, and Rohament PL (trademark) contains pectinate. Rohapect UF (trademark) is a concentrated extract containing pectinase and araba It contains an enzyme complex including an enzyme enzyme. By using this combination of enzymes, the conventional method Compared to conventional methods, the cost of protoplast formation was reduced by more than 100 times.

[0036] Furthermore, any degrading vacuoles can be removed during the preparation of the lysate. These vacuoles are polyp Desired substances include proteases and ribonucleases that inhibit the synthesis of peptides and mRNA. It contains enzymes. In some embodiments of this specification, the degrading vacuole is Percoll. It is removed by centrifugation at a density gradient or any other desired density gradient. The vacuole has a low density. Therefore, it can be separated from the protoplast, and high-density dehydrated protoplast A result can be obtained. In some cases, a stepwise sucrose density gradient is used for dehydration. Often, the protoplast is coated directly onto the Percoll-free upper layer. After separation, the dehydrated protoplasts consist of, for example, 40% to 70% Percoll layer (used) Depending on the gradient, the concentrated liquid is separated at the interface between them, but the separated low-density liquid Vacuoles are located in low-density gradients and, for example, float above the upper layers.

[0037] Next, the dehydrated protoplasts are washed to protect the unstable cellular components from oxidation. The nuclei and may then be destroyed by a Dounce tissue pulverizer or nitrogen decomposition. After removing undamaged cells, the integrity of 18S and 28S ribosomal RNA is the main factor. Without being affected, the lysate is processed to destroy endogenous mRNA, and background translation is performed. This can be minimized. In certain examples herein, nuclease S7 is used.

[0038] template To direct the synthesis of biopolymers in the system described herein, stored information is used. A template must be present in the reaction for it to be converted to MAR. The template is any polynucleotide (DNA) or polypeptide (DNA and It may be either mRNA or DNA that encodes (or / or mRNA). The transcription / translation system uses a recognizable promoter to convert mRNA from a DNA template. Continuous generation. Endogenous RNA polymerase may be used, or exogenous RNA Polymerases (e.g., phage RNA polymerase, commonly T7 or SP6) It may be added directly to the reaction mixture. Alternatively, mRNA can be added to RNA-dependent RNA. By inserting messages into the template of the polymerase QB replicase, a continuous process It may be amplified. In some embodiments, one end of a plurality of cloning regions is a poly-A sequence. A vector containing is used as a template for the IVTT reaction. For example, such a vector This involves SP6, T7, or T3 RNA polymerases at the opposite ends of multiple cloning regions. -se promoter may be included, and as a result, cloning to the vector is performed at the 5' end. and genes adjacent to the RNA polymerase promoter of the polyA sequence at the 3' end It produces. In embodiments where mRNA is used as a template, the purified mRNA is converted It can be stabilized by chemical modification before being added to the reaction mixture.

[0039] DNA or mRNA sequence nucleos used as templates in the embodiments of this specification The cytoplasmic sequence can be optimized to achieve higher levels of expression. The A structural characteristic includes untranslated regions (UTRs) at the 5' and 3' ends of the coding sequence. This affects translation efficiency. The structure of the 5'UTR is important for translation initiation, termination, and mRNA stability. It affects the sex. One of the rate limiting steps of translation initiation is the mR to the 43S pre-initiation complex. This is NA binding. The translation mechanism involves the 5' cap or translational action in the leader sequence. It is recruited by the enhancer. In certain embodiments of this specification, the template mRN A is the 5'UTR in PCITE2a (which originates from encephalomyocarditis virus (EMCV)). Contains an internal ribosome entry site (IRES); in vector pF3A Sequence of wheat yellow wilt virus (BYDV); 5'UT of the baculovirus polyhedrin gene R; Synthetic 3'UTR containing poly-A sequence; and 5'U containing ARC-1 sequence elements. TR (This is complementary to the internal 18S rRNA segment and 40S ribosome subunit) May promote binding to knits; Tobacco Mosaic Virus (TMV) 5'-UT R (Omega sequence) (By adding GAAAGA upstream of the first GUA triplet) It may contain untranslated regions selected from the group that includes (which can be improved by...).

[0040] In some embodiments, for example, the existence of the cap analog m7G[5']ppp[5']G Underlying this, DNA molecules are used to produce capped mRNA in vitro. Non-integrated nucleotides and cap analogs may be removed by gel filtration. Subsequently, purified mRNA, which serves as a template for polypeptide synthesis, is used. It can be introduced into the cell-free system described in the detailed document.

[0041] monomer In linked IVTT reactions, ribonucleotide triphosphates (ATP, GTP, CT) P, UTP, and amino acids are used to synthesize the desired biopolymer. It is required as a unit in the system. In some embodiments of this specification, the system is For an equivalent system with conventional energy regeneration systems, one or more NTP levels The system operates with reduced noise. In these embodiments, the disclosed system operates with reduced noise. To favorably reduce the costs associated with movement. In certain embodiments, the disclosed system, It operates at a final ATP concentration of 2-10 mM, for example, 4-8 mM or 5-7 mM. In certain embodiments, the disclosed system has a range of 0.8 to 2.5 mM, for example, 1 to It operates at a final GTP concentration of 2 mM or 1.4–1.8 mM. In terms of form, the disclosed system is 0.4–2.4 mM, for example 0.5–2 mM or 0 It operates at a final CTP concentration of 0.6 to 1.0 mM. Furthermore, in certain embodiments, it operates within a specific range. The disclosed system is 0.4-2.4 mM, for example 0.5-2 mM or 0.6-1 mM It operates at a final UTP concentration of 0.0 mM UTP. For example, the system operates at 6 mM or approximately 6 mM. mM ATP, 1.6 mM or approximately 1.6 mM GTP, 0.8 mM or approximately 0.8 mM It can operate with CTP and UTP final NTP concentrations of 0.8 mM or approximately 0.8 mM. In certain cases, the synthesis reaction requires approximately 150 mM ATP, approximately 40 mM GTP, and approximately 20 mM It is supplemented with a low-concentration NTP mixture containing mM CTP and approximately 20 mM UTP, N The mix is ​​added to the system in an amount sufficient to provide the final concentration of TP. The amino acids are Furthermore, for example, it can be added up to a final concentration of 20-500 pM. no acids (for example, 35 S-methionine and 3 H-leucine is used in the coupling reaction, and then the corresponding The amino acid in question may be removed from the amino acid mixture.

[0042] salt The salt concentration is controlled by the system according to the embodiments described herein. For example, the system is For example, but not limited to, potassium, magnesium, ammonium, and (for example, vinegar) Add one or more salts, such as manganese (an acid or sulfuric acid) or other biologically related salts. They may be. One or more of these salts may have an amino acid as a counteranion. It may also be possible. There is interdependence between ionic species regarding the function of the synthesis reaction. When the concentration of one ion is changed, the concentration of another ion may change accordingly. For example The concentration of the added salt is controlled simultaneously in accordance with changes in other components such as nucleotides. Furthermore, the concentration levels of components in a continuous flow reactor can change over time.

[0043] magnesium Magnesium enhances ribosome assembly and stabilizes the assembled ribosomes. Magnesium is important for protein translation in order to strengthen polymerase. It appears to play a role in promoting the synthesis. In the embodiments described herein, the magnesium of the cell lysate is used. The magnesium concentration can be adjusted by additional magnesium compounds. In some embodiments... Further magnesium compounds are salts, such as magnesium chloride and magnesium acetate. Um, magnesium glutamate. A sufficient amount is needed to link transcription and translation. By adding a magnesium salt to the lysate, the final magnesium concentration is determined by the transcription of RNA from DNA. Furthermore, the level may be increased to the point where RNA is translated into protein. In some cases, The final magnesium concentration can be adjusted to 1-20 mM. For example, the final magnesium The concentration varies depending on the solvent used, ranging from 5-15 mM, 7-13 mM, and 2.5 mM-5.5 mM. mM, 2.5mM~3.5mM, 2.6mM~3.0mM, 3.0mM~5.25mM, Alternatively, it could be between 4.0 mM and 4.75 mM.

[0044] To provide precise control of magnesium concentration in the system described herein, dissolved The magnesium level is measured using a magnesium assay before adding extra magnesium. It may also be measured directly through use. For example, Lancer "Magnesium Ra pid Star Diagnostic Kit” (Oxford LabWareD ivision(TM), Sherwood Medical Co., St.Louis s, MO) is one assay that can accurately measure the magnesium level in biological fluids. If the magnesium ion concentration is known for a given batch of dissolved material, then additional magnesium By adding citric acid, the magnesium concentration in the dissolved material can be brought within the desired range.

[0045] As mentioned above, the final magnesium concentration in the reaction is influenced by other conditions and considerations. Therefore, for example, when the ribonucleotide triphosphate concentration increases, ribonucle Since rheotide triphosphate tends to associate with or chelate magnesium in solution, The optimal magnesium concentration increases simultaneously. Therefore, the ribonucleotide triphosphate concentration... As the amount increases, additional magnesium is generally added to the reaction. The concentration also varies depending on the type of cell lysate. The amount of magnesium that needs to be added is... Furthermore, it changes depending on the concentration of the dissolved substance used in the reaction mixture, and as the concentration of the dissolved substance increases... The contribution of magnesium from the dissolved substance itself increases.

[0046] potassium Generally, potassium is also used to achieve the desired level of biopolymer synthesis. Potassium (e.g., potassium acetate and potassium glutamate) is added to the mixture. Generally 5-250 mM (for example, 5-100 mM, 5-75 mM, 5-50 mM, and It exists at concentrations of 5-30. In certain cases, even if the potassium concentration is 10-20 mM Often, more specifically, it may be about 20 mM. As with magnesium, the intrinsic factor The presence of sexually charged cell lysate components may slightly alter the final potassium concentration.

[0047] Additional ingredients Additional components may also be added as needed to improve the efficiency or stability of the synthesis reaction. In the application form, it may be added to the system. It is not absolutely necessary, but linked transcription and One common addition to the translation reaction is, for example, a sufficient amount to stimulate the efficiency of chain elongation. It is a polyamine. Polyamines also affect the optimal magnesium level and the translation reaction. It is known to slightly reduce the effective magnesium concentration for that purpose. Therefore, Polyamines can substitute magnesium at some levels, and in certain cases they can be linked. This may allow for a decrease in the optimal magnesium levels for transcription and translation.

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

[0049] Host organism (Muller & Blobel (1984) Proc. Natl. Acad Purified or synthetic vesicles (see Sci.USA81:7421-5) These can also be added to the system. These enhance protein synthesis and folding. It can be used. For example, the system described herein is used to activate membrane proteins. For example, to insert or move proteins, or to move other compounds. Therefore, these processes may be used for cell-free reactions, and these processes may also be used for the desired membrane protein In certain embodiments, this may be supported by adding vesicles containing chlorine.

[0050] In addition to the above components, other materials (particularly used for protein synthesis) are specified herein. It can be added to the system described above. Such materials include, for example, not limited to, However, other salts, folinic acid, cyclic AMP, protein or nuclease inhibitors, RN ASIN, inhibitors or regulators of protein synthesis, modifiers of oxidation / reduction potential, DTT, Loramphenicol, non-denatured surfactant, buffering agent (to stabilize the reaction pH) (For use in liquids, etc.), PEG, Triton X-100, spermine, sperm This may include luminin and putrescine, among others.

[0051] Some embodiments synthesize biopolymers without using artificial regeneration systems. The kit includes components of the system. In certain embodiments, the kit includes cell lysates. It is possible. Alternatively, the kit allows you to culture and grow cells to obtain cells for the preparation of cell lysates. May include cells for widthening. In certain embodiments, the kit may include salts, NTPs, enzymes (e.g.) For example, polymerases and nucleases), enzyme inhibitors (e.g., RNasin), templates It may contain one or more of the following additives (e.g., chloramphenicol): In certain cases, the kit uses the target gene as a template within the system. May contain naked vectors that can be cloned. Kit containing cell lysates. So, the dissolved substance may be standard, or its salt concentration may have already been adjusted during manufacturing. It may be of the type that is present, or additionally, necessary for concatenated transcription and translation. It may be a type that contains one or more of the following: components, reagents, or buffer solutions. In this example, the kit may not include a mold, but instead relies on the user to provide one. It is also possible to use the kit's components to carry out the synthesis reaction. To inform the user, include a set of instructions or a link to a website containing the instructions. It may include "ku".

[0052] V. Methods for synthesizing biopolymers The above system is used in an in vitro synthesis method for one or more biopolymers. In vitro synthesis may also involve the use of biological extracts and / or defined reagents. This refers to the cell-free synthesis of biological macromolecules in a reaction mixture. Using the system described herein, These configurations are known in the art, including batch, continuous flow, and semi-continuous flow. - Cell-free synthesis reactions can be carried out with this configuration. In some embodiments, batch cultured cells are used This is also acceptable. In some embodiments, in order to ensure a reproducible and homogeneous supply of cell material, Cells can be continuously grown in a stirring tank fermentation vessel.

[0053] There is a difference between using a static IVTT reaction and a continuous or flow-through reaction, and this is It may be considered for some applications, but not for others. For example, continuous systems are generally... While primarily used in large-scale industrial production of proteins, static system reactions are used in small-scale in vitro processes. It is more suitable for translation (e.g., research status). Continuous translation is much more efficient. It is expensive and requires investment in equipment as well as a large amount of reagents. In particular, serial eukaryotic regeneration The level of RNA polymerase used to perform the task is for simple research purposes. It may be prohibited (i.e., 20,000-30,000 U / reaction). Continuous reactions are designed to be carried out in relatively large volumes, while static reactions are typically... Since the amount is only on the order of 100 μl or less, no additional equipment is needed, and small amounts of trial Only medication is needed.

[0054] The system described herein can utilize large-scale reactors, small-scale reactors, or multiple reactors. A multiple system may be used to carry out the simultaneous synthesis of multiple compounds. For sequential reactions, a reagent flow is introduced. A feeding mechanism may be used to isolate the final product as part of the process. In both reactive and static reactions, additional reagents may be introduced to extend the duration of the active synthesis. The reactors are batch, extended batch, semi-batch, semi-continuous, fed-batch, and continuous. It may be run in any mode, and the mode may be selected according to the intended purpose. good.

[0055] The reaction can also be carried out in any volume, depending on the application and the equipment used. For example For small-scale reactions, the reaction volume is 1-15 μl, at least 15 μl, and at least 50 μl. l, at least 100 μl, at least 0.5 mL, or at least 1 mL, Less than 10 mL is acceptable. In principle, sufficient oxygen (or other electron acceptors) As long as supplies are available, the reaction can be carried out on any scale. For the production of the maximum amount of product, Industrial bioreactors may be used.

[0056] The methods described herein are means for isolating synthesized biopolymers, for example, proteins A qualitative isolation method may be used. In some embodiments, it operates in a continuous operation mode, and the reactor The product output flows through the membrane and enters the protein isolation device. In semi-continuous operation mode, The outer surface of the membrane comes into contact with a predetermined solution that is periodically changed in a predetermined order. These solutions may contain substrates such as amino acids and nucleotides. At this point, the reactor It operates in dialysis or diafiltration batch or fed-batch batch mode. The feed solution is the same The synthesized protein may be supplied to the reactor through a membrane or a separate injection unit. The protein is accumulated in the reactor and then, after the completion of the system operation, is processed using the standard method of protein purification. Therefore, it is isolated and purified.

[0057] If there is a reagent flow, the direction of the liquid flow should be perpendicular and / or tangential to the membrane. Yes, it is possible. Tangential flow is effective for ATP recycling and preventing membrane clogging, while vertical flow... It can be superimposed on. Flow perpendicular to the membrane is generated by a positive pressure pump or a vacuum suction pump. or may be affected. The solution in contact with the outer surface of the membrane may be changed periodically, and the membrane A steady tangential flow may be used for this purpose. Furthermore, the reactor may be stirred internally by appropriate stirring means. Alternatively, it may be stirred externally.

[0058] During protein synthesis in the reactor, a protein is used to selectively isolate the desired protein. The protein isolation means solidifies the component for adsorbing antibody molecules or the desired synthesized protein. Units filled with particles coated with other stabilized molecules and having appropriately sized pores The membrane may include a protein isolation method that includes two columns for alternating use. preferable.

[0059] The amount of protein produced in a translation reaction can be measured in various ways. The method depends on the availability of assays that measure the activity of specific proteins being translated. An example of an assay for measuring protein activity is the luciferase assay system. This is a chloramphenicol acetyltransferase assay system. These assays measure the amount of functionally active protein produced from translation reactions. Activity assays can detect improper protein folding or other factors necessary for protein activity. We do not measure full-length proteins that are inactive due to the lack of post-translational modifications. Instead, we use a specific method. Proteins can be detected according to their size by capillary electrophoresis.

[0060] The amount of protein produced in linked in vitro transcription and translation reactions is measured. Another method is, 35 S-methionine, 3 H-leucine, or l4 Release of C-leucine, etc. After carrying out the reaction using a known amount of injectionally labeled amino acid, it was incorporated into a novel translated protein. The amount of radiolabeled amino acids is measured. The integrated assay includes cleaved protein products. Hmm, to measure the amount of radiolabeled amino acids in all the proteins produced in the reaction. Yes. The radiolabeled protein may be further separated on the protein gel, and autoradi The oography confirms that the product is of the appropriate size, and that the secondary protein product is It may be confirmed that it is not being manufactured. [Examples]

[0061] Example 1: Materials and Method plant material Tobacco cells (Nicotiana tabacum L. cv. Bright Yel) Low 2, BY-2) were kept in the dark at 26°C while maintaining a packing cell volume of 20-25% for 5 L Fermentation Tank (Type 100e, Applicon (trademark) Biotechnology) Cultured in AC Schiedam (Netherlands) or shaking flasks. 3% (w / v ) sucrose, 1 mg / L thiamine-HCl, 0.2 mg / L 2,4-dichlorophosphate Noxyacetic acid, 100 mg / L myo-inositol, 250 mg / L dihydrogen orthophosphate Potassium, and Pluronic® L-61 defoaming agent (BASF® trademark, M Murashige-Skoog liquid with added olive oil (NJ, USA) A culture medium (a mixture of Murashige and Skoog base salts) was used.

[0062] Preparation of BY-2 cell lysates BY-2 cells are harvested during the exponential growth phase of fermentation at a constant packed cell volume of 20-25%. To prepare the protoplasts, 3% (v / v) of RoH was directly added to the fermentation medium. ament(registered trademark)CL and 0.2%(v / v) Rohapect(registered trademark) UF (Pectinase and Aravanase) (AB Enzyme (trademark), Darmst It was treated with ADT (Germany). By adding 360 mM mannitol... The volumetric osmolality was adjusted.

[0063] To dehydrate the resulting protoplasts, a 50 mL polypropylene tube (Grei ner Bio-One (trademark), Frickenhausen, Germany) 0.7M Mannitol, 20 mM MgCl2, and 5 mM PIPES-KOH (pH 7). 0) Inside (from bottom to top) 70% (v / v, 3 ml), 40% (v / v, 5 ml), Perc of 30%(v / v, 3ml), 15%(v / v, 3ml) and 0%(3ml) Discontinuous containing oll (GE (trademark) Healthcare, Munich, Germany) Protoplasts were layered on a Percoll gradient. Swinging bucket rotor. (JS-5.3, Beckmann-Coulter(TM), Krefeld Ger After centrifuging at 6800 × g for 1 hour at 25°C (many), 40-70% (v / v) P Dehydrated protoplasts were collected from the ercoll solution interface, and one tablet of C was added per 50 mL. omplete EDTA-free Protease Inhibitor Mix ture (Roche Diagnostics (trademark), Mannheim, Germany) Add 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 It was suspended in DTT.

[0064] Next, the protoplasts are homogenized using a Dounce (trademark) homogenizer (Braun (trademark) ), Melsungen, Germany) used 15 strokes to destroy on the ice, nuclear and Non-destructive cells were removed by centrifugation at 500 × g for 10 minutes at 4°C. The supernatant was frozen in 1 mL aliquots at -80°C. Optionally, 0.5 mL of solution was added to the supernatant before freezing. Supplement with M CaCl2 and incubate at 20°C for 15 minutes with 75 U / mL nuclease S7 (Roc It is treated with (he Diagnostics), and then Ca 2+ As an ion chelating agent The nuclease can be inactivated by supplementing with 2 mM EGTA.

[0065] Plasmid construct Vector pIVEX_GAAAGA_Omega_eYFP-His is T7 Promo Tobacco mosaic virus 5 having GAAAGA as the first 6 nucleotides. 'Omega leader sequence-containing annealed oligonucleotide primer 1 (sequence number) Primer 1) and oligonucleotide primer 2 (SEQ ID NO: 2) are used at the NspI site and Nc Using the oI site, pIVEX1.3_eYFP-His(Dr.Stefan Kub ick(Fraunhofer Institute for Cell Therap y and Immunology IZI (Potsdam-Golm, Germany) Prepared by inserting (provided by choice). N-terminal streptavidin affine Vector containing the Nity tag pIVEX_GAAAGA_Omega_Strep-e Regarding YFP, oligonucleotide primer 3 (SEQ ID NO: 3) and oligonucleotide pIX3.0_Strep-eYFP is used as a template along with Chidoprimer 4 (SEQ ID NO: 4). PCR using (provided courtesy of Dr. Stefan Kubick) Strep-eYFP sequences were amplified. The PCR products were digested with PciI and Acc65I. and inserted into the NcoI site and the Acc65I site of pIVEX_GAAAGA_Omega_eYFP-His.

[0066] 21333, 22807, AAD12, Cry2A, Cry3A, Trap8VIP3 A, VIP3A, Cry6A, 17912, and Cry1F-containing pIVEX vectors were prepared by PCR amplification of the genes using oligonucleotide primers 5 to 24 (SEQ ID NOs: 5 to 24) and subsequent incorporation 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] Linked transcription and translation cell-free protein synthesis The coupled transcription-translation reaction was carried out at 25 °C and 700 rpm for 40 to 52 hours in a 50 μL aliquot in a thermomixer (Ditabis™, Pforheim, Germany by HLC). The reaction solution of creatine phosphate and creatine kinase was 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. contained creatine kinase and did not contain creatine phosphate and creatine kinase The reaction solution contained 40% (v / v) BYL, 20 mM HEPES-KOH (pH 7.8) , 9 mM magnesium glutamate, 20 mM potassium glutamate, 4 mM AT P, 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 the product The fluorescence signal from eYFP was measured using 485 / 20 nm excitation and 528 / 20 nm emission filters on a Synergy(™) HT Multi-Mode Microplate Reader (Biotek(™), Bad Friedrichshall, Germany). The amount of eYFP was determined by generating a standard curve based on the different concentrations of eYFP in a BYL translation reaction without DNA template The eYFP standard was produced using an in-house Escherichia coli-based in vitro translation system (Zawada (2012) Methods Mol. Biol. 805:31-41) and purified by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). The concentration of the purified eYFP was determined using a colorimetric assay (Bradford (1976) Anal. Biochem. 72:248-54) oplate Reader(Biotek(商標)、Bad Friedrichsh all、ドイツ)を使用して定量化した。eYFPの量は、DNA鋳型なしのBYL翻訳 反応におけるeYFPの異なる濃度に基づいて標準曲線を生成することによって決定した 。eYFP標準は、大腸菌に基づく社内でのインビトロ翻訳システム(Zawada(2 012)Methods Mol.Biol.805:31-41)を使用して製造し、 固定化金属親和性クロマトグラフィー(IMAC)およびサイズ排除クロマトグラフィー (SEC)によって精製した。精製されたeYFPの濃度は、比色アッセイを使用して決 定した。Bradford(1976)Anal.Biochem.72: 248-5 4.

[0070] Residue-specific labeling of target proteins To fluorescently label the target protein amino acid selectively, FluoroTect(™) )GreenLys in vitro Translation Labeling System (Promega (trademark), Mannheim, Germany) as per manufacturer's instructions It was used according to the instructions. This product is a BODIPY fluorophore. (登録商標) -FL labeled It contains modified charged lysine-tRNA. This system allows you to use fluorescently labeled lysine. Residues are incorporated into the newly synthesized protein at multiple sites during translation.

[0071] JC-1 staining The presence of mitochondria in BYL indicates the presence of the lipophilic cationic probe 5,6-dichloro-2 -[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzidi Dazole-2-ylidene)-1-propenyl]-1,3-diethyl-iodide (JC-1 , Thermo Scientific™, Waltham, MA, USA). This was demonstrated using the following method: In living cells, JC-1 is green firefly-like in depolarized mitochondrial membrane potential. It exists as a photomonomer (excited at 490 nm / emitted at 530 nm). Normal and hyperpolarized In the mitochondrial membrane potential, JC-1 is concentrated within the mitochondria, forming J aggregates. This shifts the radiation from 530nm to 590nm. Cellular mitochondria increase As the negative mitochondrial membrane potential increases, the number of J aggregates increases, and the red fireflies become more highly concentrated. This shows light (590 nm). Nuydens et al. (1999) J. Neuros ci.Methods 92:153-9;Reers et al.(1995)Me thods 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 minutes at a final concentration of 5 μg / mL for use.

[0073] Cell-free protein synthesis using oxidative phosphorylation inhibitors Cell-free protein synthesis (50 μL reaction) was performed both with and without creatine phosphate and creatine kinase and both with and without the addition of inhibitors of oxidative phosphorylation to the BYL system. The inhibitors contained the following: sodium azide (0.05%, Boguck a & Wojtczak (1966) Biochim.Biophys.Acta 1 22:381-92) 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. Negative controls 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, an 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 the 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)), ​​NTP (i.e., ATP / (GTP / CTP / UTP)) (0.5 / 0.2~4 / 1). 6 mM), and T7 RNA polymerase (20-80 ng / μL), CP (0-40 mM) and with and without CK D esign O f E xperiment( Do E) Based approach (Design Expert v8.0 (State-Eas) Fractional design in e(trademark) Inc., MN, USA It was devised using the igns (IGNs) and response surface models. The concentration of chloramphenicol is The system was adopted from the CP / CK system. From these experiments, both systems (CP / CK) Preferred concentrations of the reaction components were obtained for both with and without the substance. (Table 2) [Table 2]

[0075] A comparison of BYL systems with and without CP / CK is shown below. The stem exhibits long-term activity for approximately 40 hours (compared to 20 hours with BYL with CP / CK). This demonstrated that it could yield up to 60% more target protein. Figure 1A. BY The presence of mitochondria in L is detected by the selective dye JC-1 (Ther (Fischer Scientific, Waltham, MA, USA) As demonstrated by BYL staining, mitochondria have their characteristic membranes in BYL. It was shown that the potential is maintained. Energy regeneration by oxidative phosphorylation is performed by the electron transport chain. Sodium azide and tenoyltrifluoroacetone (TTA) are different inhibitors. This was demonstrated by the use of ). Both sodium azide and ATP target proteins As indicated by the dramatically reduced biosynthesis of eYFP, it almost completely inhibits the system. This was found. Figure 1(B).

[0076] In systems without CP / CK, energy is supplied by oxidative phosphorylation. This is thought to be due to the added magnesium glutamate and potassium glutamate. Glutamate is metabolized in the citric acid cycle within mitochondria, and its reduced equivalent is This leads to the production of NADH and FADH. Electrons are then transferred via NADH and FADH. It then enters the electron transport chain and generates ATP through oxidative phosphorylation via the consumption of molecular oxygen. Figure 2.

[0077] Expression of 10 target proteins in a BYL system without CP / CK, with CP / CK Compared with the BYL system. Creatine phosphate (CP) and creatine kinase ( The linked BYL transcription-translation reactions with and without CK were carried out at 25°C for 40 hours. In each case, a 2 μL reaction volume was measured using an SDS-PAGE gradient of 4-12% (w / v). The protein was loaded into a saturation matrix, and the amount of synthesized protein was visualized using Coomassie staining. AAD1 2. Several target proteins, including Cry3A and Cry1F, are stained by Coomassie staining. As expressed by the strong bands in the middle, the optimized system (without CP / CK) It showed a significantly higher expression level.

[0078] BYL systems, which do not have artificial energy regeneration systems, are cheaper and can run for longer periods. This leads to an increase in the level of recombinant proteins. Furthermore, the BYL system is mitoc This offers the possibility of investigating compounds or proteins that affect ndorian function.

[0079] Example 3: Modification of linked transcription-translation reactions without artificial energy regeneration Use of sorbitol for BY-2 solution preparation For the preparation of BY-2 lysate, volumetric osmosis is performed during protoplast formation and dehydrated vesicular formation. A large amount of permeable substance is needed to adjust the concentration. Mannitol is for this purpose. It is used routinely and accounts for about 10% of the total cost of dissolution preparation. Rubitol (approximately 10 times cheaper) was tested. It was used for protoplast formation and dehydrated vesicle formation. In parallel experiments using sorbitol or sorbitol, sorbitol surprisingly... It is not equivalent to mannitol, and the yield of lysates and solubles is determined by eYFP expression. It was found to be superior in both aspects of the quality of the solution (Figure 5). Furthermore, Sol It was found that the higher solubility of bitol facilitated the preparation of the buffer. However, Mannitol is superior to sorbitol for the final washing of dehydrated protoplasts. It was also found that the use of sorbitol in this process results in lower eYFP yields and This resulted in a highly viscous reaction mixture.

[0080] Inhibition of microbial growth BY-2 lysate supports microbial growth when the IVTT reaction solution is incubated. It has the ability to deplete the reaction substrate, which in turn leads to the depletion of the target protein. The amount decreases. Therefore, several antimicrobial substances were tested using the IVTT reaction system. Regarding the effects of chloramphenicol on eYFP production and microbial growth in VTT, Spectinomycin, streptomycin, ampicillin, and sodium azide I investigated.

[0081] The antimicrobial substance was added to the BYL reaction solution, and after 45 hours of incubation, 0.2 μL of BYL was added. The reaction mixture was spread onto LB plates and microbial growth was analyzed. Spectinomycin, S Treptomycin, ampicillin, and sodium azide inhibit microbial growth. It had no effect or showed a harmful effect on eYFP yield. For example, 0.05% ( Sodium azide at w / v concentrations likely inhibits mitochondrial energy regeneration. This nearly completely inhibited both microbial growth and translational activity. Only Nicole can inhibit microbial growth without any loss of translational activity. (Figure 3B; Figure 3C), and as a result, the usefulness in the IVTT system is at least other It is different from an antimicrobial substance. The dose-response study showed that the most effective dose was 100 μg / mL of chloramphenicol. High eYFP yields were achieved (Figure 3D, with 200 μg / mL chloramphenicol). This indicates that complete inhibition of microbial growth was achieved (Figure 3A), and SDS-PAGE analysis also showed Furthermore, the protective effect of chloramphenicol on eYFP yield and protein stability was also observed. This was shown. Unlike the reaction carried out in the presence of chloramphenicol, The reaction without the lubricant showed significant protein degradation.

[0082] Nucleoside triphosphate The concentration of NTPs in the system was adjusted using a DoE-based approach. GTP is used in both transcription and translation, while CTP and UTP are used only in transcription. Therefore, ATP and GTP are linked to higher concentrations compared to CTP and UTP. It was predicted to be beneficial for the IVTT system. Therefore, the concentration of a single NTP is Instead of using different amounts of fixed NTP mixes, we varied them in DoE-based experiments. These are the results of 96 experiments on cubic IV-optimal design. The optimal concentration was screened using the design. The rasmid mold was pIVEX_GAAAGA_Omega_Strep-eYFP. The concentration of magnesium glutamate is regulated by the binding of NTP to magnesium. Table 3 shows the concentration range of each screened factor. Quadratic model The c model was fitted to the experimental data. Using the response surface model, the most This also predicted the values ​​of factors that produce many eYFP proteins (Zhou et al.). (2010). Ignoring all non-significant terms (p>0.05 by ANOVA), the model The ANOVA table showed statistical significance (Table 4).

[0083] Experiments showed that in the reaction, the target protein produced the most ATP at 6 mM and 0.8 mM It has been revealed that it is produced in CTP, 0.8 mM UTP, and 1.6 mM GTP. Figure 4 shows these results for ATP / GTP / C at 150 / 40 / 20 / 20 mM. This led to the development of a new NTP mix consisting of TP / UTP. A new 2μl NTP Using the mix, eYFP yields increased by 20% in the IVTT system. As a benefit, the new NTP mix has 10-1% ATP compared to other nucleotides. Because it is five times less expensive, it costs about 40% less than the standard mix.

[0084] [Table 3]

[0085] [Table 4]

[0086] Use of a larger amount of dissolved material To investigate the effect of increasing the lysate portion of the IVTT reaction on target protein expression In addition to BYL prepared using mannitol, during protoplast formation and dehydration... Using several BY-2 solutions prepared with sorbitol, 40% (20 μl) A 50 μl reaction was performed using either ) or 60% (30 μl) lysate. Plasmid pI Using VEX_GAAAGA_Omega_Strep-eYFP as a template, 25 The reaction was carried out at °C and 700 rpm for 48 hours. 6 prepared with sorbitol Reactions using a 0% (v / v) solution achieved approximately 1 mg / mL of eYFP, which is standard. This corresponds to an 80% higher yield compared to the reaction mixture (Figure 5). Ribosomes, translation factors, sha The increased amount of perones and mitochondria increases or prolongs energy production. It is presumed that this resulted in a higher yield. This result suggests that such a large amount of odor This indicates that the dissolved product does not contain harmful factors that produce greater inhibition.

[0087] Cell-free systems without artificial energy regeneration systems and the standard in wheat germ extract Expression of target proteins Cell-free expression systems that do not involve artificial energy regeneration are generally effective across the entire substrate. To confirm that it is, the system is used to find 10 additional ones other than eYFP. The target gene product was expressed. Strep-tagged eYFP and 10 other target products were expressed. The protein was prepared using sorbitol in a 60% BY-2 cell lysate, and mannitol In a 40% BY-2 cell lysate prepared using, and CellFree® Sc The gene was expressed using the iences WGE system. The linked transcription-translation reaction was performed in 50 μL of liquid. The experiment was conducted in large quantities at 25°C and 700 rpm for 40 hours using a WGE system. The linked transcription and translation reactions were carried out according to the manufacturer's instructions. In addition, 1 μL of the reaction mixture and the mixed two-layer reaction solution are each added at a concentration of 4-12% (w / v). The gradient was loaded onto an SDS-PAGE gel, and the target protein was visualized by Coomassie staining. The BYL system successfully transcribed each of the different genes tested, In fact, the BYL system produced proteins in all cases by performing translation after transcription. Compared to those manufactured using the WGE system, it consistently produces stronger bands. Regarding Strep-eYFP, the proteins produced by the three systems were fluorescently analyzed. Quantitative analysis using a reader and comparison with the eYFP standard curve revealed a concentration of 1115 μg / mL. 441 μg / mL was produced under the conditions of two sets of BYL systems, and the WGE system It was then revealed that only 105 μg / mL was produced.

[0088] Addition of glucosylglycerol and use of higher solubility amounts To increase the stability of cell-free lysates without artificial energy regeneration, Small molecules described as cryoprotective agents for proteins maintain the translational activity of lysates. We hypothesized that this could be achieved. These molecules exist naturally in extremophilic microorganisms. Furthermore, it protects extremophilic microorganisms from osmotic stress, heat, drought, and UV light, and water on the surface. By causing an increase in density and promoting the natural conformation of proteins, the membrane It stabilizes proteins. However, cryoprotective agents have a strong interference with the IVTT system. It was expected to have harmful effects.

[0089] Different concentrations of cryoprotectants, ectoin, hydroxyectoin, and glucosylglycerin Seride is added to the linked IVTT reaction, and the template plasmid pIVEX_GAAAGA_ The amount of eYFP produced from Omega_Strep-eYFP was determined. 50 μL The IVTT reaction (60% (v / v) BYL) was performed in a 96-well plate at 25°C and 5°C. The experiment was conducted at 00 rpm for 44 hours under the controlled humidity of a Kuhner shaker (trademark).

[0090] Ectoin and hydroxyectoin are available in concentrations of up to 1% and 2% (v / v), respectively. The concentration did not affect the yield of eYFP. Figure 6 In fact, higher concentrations of ectoin did not affect the yield of eYFP. Hydroxyectoin inhibited the system. In contrast, glucosylglycerol , had a strong positive effect on eYFP yield. 0.5% (v / v) glucosylglycerol The reaction solution containing the glucosylglyceride took 44 hours longer to react with the standard reaction solution that did not contain glucosylglyceride. This later resulted in approximately 50% more eYFP. Figure 6 The positive effect of glucosylglycerol is observed with 0.5% (v / v) glucosylglycerol. In the IVTT reaction with and with five different lysate batches (BYL 08.01 .2016, BYL 21.01.2016, BYL 11.03.2016, BYL This was consistent from 01.04.2016 (BYL 05.04.2016). 50 The IVTT reaction was carried out in a 96-well plate at 25°C and 500 rpm for 48 hours. Under humidity (70%), use the plasmid pIVEX_GAAAGA_Omega_ as a template. Using Strep-eYFP with 60% and 80% (v / v) dissolved portions, three The experiment was conducted in series. 60% (v / v) solution and 0.5% (v / v) glucosylglyceride Reactions using rolls yield approximately 80% more compared to reactions without glucosylglycerol. A certain amount of eYFP was produced. Figure 7 shows 80% (v / v) lysate and 0.5% (v / v Using glucosylglycerol, the yield of eYFP is greater than that of glucosylglycerides. Compared to reactions using an 80% (v / v) solution, this is approximately 110% (almost 2 mg / mL). eYFP) increased. Figure 7 The time course of the IVTT response in a cell-free system without artificial energy regeneration was measured in 64 This was determined by measuring the amount of protein produced at different time points up to a certain point in time. (Figure) 8. After 16 hours, 60% or 8% with or without 0.5% glucosylglycerol. The reaction using 0% lysate produced approximately the same amount of protein. Figure 8 Glucosyl Without glycerol, the translational activity in both 60% and 80% (v / v) solubils is However, it did not increase further after 24 hours. Figure 8 However, glucosylglycerol eYFP production using this method was observed in both 60% and 80% (v / v) lysates, at least Productivity showed a nearly linear increase up to 64 hours. (Figure 8) This result suggests that glucosylglyceride is the key factor. Cerol has a stabilizing effect on systems that extend translational activity beyond 64 hours. This supports the idea that, on average, 80% (v / v) dissolved and 0.5% (v / v) gluco The reaction using silglycerol yielded an eYFP concentration of approximately 2.5 mg / mL.

[0091] Addition of branched-chain amino acids . The ability of branched-chain amino acids (BCAAs) to promote protein synthesis in cell-free expression systems was also tested, and it was observed that the addition of BCAAs increased and stabilized the yield of target proteins. To verify the positive effect of BCAAs on the system, different concentrations of BCAAs were added to linked IVTT reactions using four lysates prepared from shaking flasks (SF) or continuous fermentation (CF). In 50 μL IVTT reactions using 80% (v / v) lysates in 96-well plates at 25°C and 500 rpm for 66 hours using the plasmid template pIVEX_GAAAGA_Omega_Strep-eYFP in a Kuhner® shaker with controlled humidity, BCAAs had a positive effect on eYFP yield at all concentrations tested, regardless of whether the lysates were prepared from shaking flasks or continuous fermentation. Figure 9 shows that reactions containing 1 mM BCAA yielded approximately 70% more eYFP (average yield of 2.5 mg of eYFP per mL) compared to reactions without BCAA. The invention described in the original claims of this application is listed below. [1] A system for the synthesis of biopolymers, wherein the system is Plastrids, chloroplasts, mitochondria, or aqueous cell lysates containing chloroplasts and mitochondria, Polymer molds, and A system comprising monomer units of the polymer, wherein the system does not contain added creatine phosphate and creatine kinase that are exogenous to the cell lysate, plastids, chloroplasts, mitochondria, or chloroplasts and mitochondria. [2] pH buffering agent Magnesium, preferably Mg(C) 5 H 8 NO 4 ) 2 、 Potassium, preferably KC 5 H 8 NO 4 、 Nucleoside triphosphate, Enzymes, preferably RNA polymerase, and The system according to [1], further comprising at least one of the following: chloramphenicol. [3] Aqueous cell lysate derived from plant cells containing mitochondria, DNA template, HEPES-KOH pH 7.8, Mg(C) 5 H 8 NO 4 ) 2 KC 5 H 8 NO 4 The system described in [1] essentially consists of a nucleoside triphosphate, RNA polymerase, and chloramphenicol. [4] A method for synthesizing at least one biopolymer, wherein the method comprises combining reactants in a reaction volume, and the reactants are Cell lysates containing plastids, chloroplasts and / or mitochondria, Polymer molds, and The monomer units of the polymer, A method wherein the reaction components do not include creatine phosphate and creatine kinase. [5] The following conditions: The biopolymer produced by the above method is a polypeptide. The above method is for the concatenated synthesis of RNA and polypeptides translated from said RNA. The cell lysate is a eukaryotic cell lysate. The cell lysate contains mitochondria, The plastids, chloroplasts, and / or mitochondria are exogenous to the cell lysate. The reaction component does not contain creatine kinase. The polymer template is an RNA molecule. The polymer template is a DNA molecule. The monomer unit of the polymer is a nucleotide. The monomer units of the polymer are amino acids contained in the cell lysate, and The method according to [4], wherein the reaction is terminated by either inhibiting the electron transport system or by removing oxygen from the reaction, or any combination thereof. [6] The method according to [4], wherein the synthesis of the biopolymer occurs over a period of more than 20 hours, preferably about 40 hours. [7] It's a kit, Aqueous cell lysates, plastids, mitochondria and / or chloroplasts, and monomer units of polymers arranged in one or more separate volumes, The kit includes instructions specifying the kit's components and any additional ingredients that do not contain creatine phosphate. [8] The kit according to [7], wherein the cell lysate comprising the plastids, mitochondria and / or chloroplasts is arranged in one volume, and the monomer units of the polymer are arranged in a separate volume. [9] The kit as described in [7], wherein the instructions describe how the user combines the cell lysate, plastids, mitochondria and / or chloroplasts, and monomer units of the polymer with the polymer template in a reaction volume that does not contain creatine phosphate.

[10] The kit described in [7] further includes a polymer mold.

Claims

1. A method for the synthesis of polypeptides, wherein the method is Aqueous cell lysates derived from tobacco plants, including organelles, Exogenous nucleic acid templates encoding polypeptides; and A method comprising the steps of: combining nucleoside triphosphates (NTPs); the cell lysate comprising chloroplasts, plastids, or mitochondria, and not comprising exogenous added creatine phosphate or creatine kinase, thereby providing a synthesis reaction volume; and synthesizing the polypeptide in the synthesis reaction volume.

2. The method according to claim 1, wherein the aqueous cell lysate containing organelles is dehydrated.

3. The aqueous plant cell lysate containing organelles is subjected to the following process: The process of preparing protoplasts from tobacco cells; A step of dehydrating the protoplast and forming a dehydrated protoplast; A step of destroying the membrane of the dehydrated protoplast; and, The step of removing the nucleus from the dehydrated protoplast, The method according to claim 1, produced by...

4. The method according to claim 1, wherein the aqueous plant cell lysate containing organelles is treated with a ribonuclease to destroy endogenous ribonucleic acid.

5. The method according to claim 1, wherein the synthesis reaction volume further comprises chloramphenicol.

6. The method according to claim 1, wherein the synthesis reaction volume further comprises glucosylglycerol.

7. The method according to claim 1, wherein the exogenous nucleic acid template is deoxyribonucleic acid (DNA).

8. The method according to claim 1, wherein the exogenous nucleic acid template is provided by a vector.

9. The method according to claim 1, wherein the organelle is a chloroplast.

10. The method according to claim 1, wherein the organelle is a plastid.

11. The method according to claim 1, wherein the organelle is a mitochondria.

12. The method according to claim 1, further comprising the step of isolating the polypeptide from the synthesis reaction volume.

13. The method according to claim 1, wherein the method includes a step of synthesizing a polypeptide in the synthesis reaction volume for 20 hours or more.

14. The method according to claim 13, wherein the method includes a step of synthesizing a polypeptide in the synthesis reaction volume for 40 hours or more.

15. The method according to claim 14, further comprising the step of isolating the polypeptide from the synthesis reaction volume after 40 hours or more have elapsed since the production of the synthesis reaction volume.

16. The method according to claim 1, comprising the step of synthesizing an encoded polypeptide of 250 μg / mL or more.

17. The method according to claim 1, comprising the step of synthesizing an encoded polypeptide of 600 μg / mL or more.

18. The method according to claim 1, comprising the step of synthesizing an encoded polypeptide of 1000 μg / mL or more.

19. The method according to claim 1, comprising the step of synthesizing an encoded polypeptide of 2000 μg / mL or more.

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