A novel energy regeneration system, cell-free protein synthesis system comprising the same, kit and method

Abstract

The application provides a novel energy regeneration system, a cell-free synthesis system, a kit and a method. By using a nucleoside monophosphate or a salt thereof as a main component of the energy regeneration system, a conventional expensive NTP energy system component is replaced, and the problem of high cost of cell-free synthesis is effectively solved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to the field of synthetic biotechnology, and more specifically, to a novel energy regeneration system, a cell-free protein synthesis system comprising the same, a reagent kit, and a method thereof. Background Technology

[0002] In cell-free in vitro systems, RNA is generated by mimicking the in vivo transcription process using DNA as a template. This technique allows control over the transcribed genes, the transcription process, and the use of the transcribed RNA. The main components of in vitro transcription include template DNA, enzymes and enzyme-catalyzed reactants, and buffer solutions. Temperature and time conditions need to be controlled during the reaction, and NTPs are an essential component in current technologies.

[0003] Proteins are among the most important molecules in living organisms, possessing an extremely wide range of functions and roles. Traditionally, researchers have used cell expression systems to prepare the proteins they need and conduct in-depth studies on them. However, cell expression systems face some bottlenecks and challenges due to limitations such as cell growth, maintenance, and contamination, especially in high-throughput production and customized protein manufacturing.

[0004] To overcome these limitations and provide more options for efficient protein preparation and research, cell-free protein expression technology has emerged. It has been used in life sciences as a basic research tool for over fifty years, and recent technological advancements have facilitated high-yield cell-free protein expression. Unlike traditional cell expression systems, cell-free synthesis reactions can complete protein synthesis under in vitro conditions and offer advantages such as high efficiency, convenience, flexibility, and high purity, making it one of the most promising emerging technologies in the fields of biomedicine and basic research.

[0005] Cell-free protein expression, also known as cell-free protein synthesis (CFPS), is an in vitro recombinant protein expression technique. It involves synthesizing proteins in vitro using cell lysates containing essential components for protein synthesis (ribosomes, transfer RNA, initiation / elongation / termination factors, guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, uridine triphosphate, PO4+, Mg2+, and K+). Cell-free protein expression is suitable for preparing various types of proteins, including difficult-to-express, toxic, and complex proteins. It has gained widespread attention and application in drug research, biomanufacturing, and life sciences, encompassing research, development, and commercial applications. Currently, cell-free protein expression is primarily used in drug development, such as antibody preparation and biopharmaceutical production. Although cell-free protein expression systems can perform large-scale protein expression, the required prokaryotic and eukaryotic cell extracts, some raw materials and reagents, and synthetic equipment are relatively expensive, creating a certain economic burden for their application.

[0006] In particular, the energy regeneration system is one of the main factors limiting cell-free expression systems in vitro.

[0007] Traditional cell-free protein synthesis relies on phosphoenolpyruvate (PEP) and pyruvate kinase (PK), or creatine phosphate (CP) and creatine kinase (CK), or other similar compounds, for energy supplementation. As the cell-free protein synthesis reaction proceeds, the energy is depleted, and the reaction can no longer continue. Traditional in vitro transcription requires ATP as an energy source, and while ATP or NTPs can be added to supplement energy, their high cost significantly increases the cost, which is detrimental to industrial production. Therefore, there is an urgent need for an energy alternative that can provide a continuous energy supply while reducing costs. Summary of the Invention

[0008] The purpose of this invention is to overcome the problems existing in the prior art and provide a cell-free synthetic reaction with a novel energy regeneration system, a kit containing the system, and a method for synthesizing RNA or proteins in vitro using the system. The cell-free synthetic reaction of this application can significantly reduce the cost of cell-free in vitro synthesis.

[0009] The first aspect of the present invention provides an energy regeneration system, the energy regeneration system comprising: (a) Cell extract; (b) Nucleoside monophosphate or its salt.

[0010] In a preferred embodiment, the monophosphate nucleoside or its salt is dissolved in an alkaline solution in the energy regeneration system; the preferred alkaline solution is sodium hydroxide or potassium hydroxide.

[0011] In a preferred embodiment, in the energy regeneration system, the monophosphate nucleoside is selected from any one or more combinations of adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, and uridine monophosphate.

[0012] In a preferred embodiment, the monophosphate nucleoside in the energy regeneration system includes adenosine monophosphate and / or guanosine monophosphate.

[0013] In a preferred embodiment, the salt in the energy regeneration system refers to the disodium salt of a monophosphate nucleoside.

[0014] In a preferred embodiment, in the energy regeneration system, the concentration of the monophosphate nucleoside or its salt in the cell-free synthesis system is greater than or equal to 5 mM or 8 mM.

[0015] In a preferred embodiment, the cell extract in the energy regeneration system is a yeast cell extract, preferably one or more of Saccharomyces cerevisiae, Kluyveromyces var. kanamycin, and Pichia pastoris.

[0016] A second aspect of the present invention provides a cell-free synthesis system, characterized in that the system includes the energy regeneration system described in the first aspect.

[0017] In a preferred embodiment, the cell-free synthesis system further includes any one or more combinations of the following components: a cell-free synthesis reaction solution, and an RNA or DNA template; The cell-free synthesis reaction solution contains any one or more combinations of T7 RNA polymerase, sugars, amino acid mixtures, and inorganic salt buffer.

[0018] A third aspect of the present invention provides a cell-free synthesis kit, the kit comprising: The components of the cell-free synthetic system described in the second aspect.

[0019] A fourth aspect of the present invention provides an in vitro synthesis method, the method comprising the following steps: (i) Provide an RNA or DNA template; (ii) The template is added to a cell-free synthesis system comprising the energy regeneration system described in the first aspect and the cell-free synthesis reaction solution described in the second aspect, and an incubation reaction is carried out under suitable conditions to perform in vitro synthesis, preferably, the in vitro synthesis being in vitro transcription and / or in vitro translation.

[0020] Compared with the prior art, the present invention has the following beneficial effects or advantages: (1) This invention addresses the long-standing problem in cell-free synthesis that high energy regeneration costs and difficulty in sustaining synthetic reactions. Through long-term research, it has been found that a low-cost energy regeneration system can replace the most expensive component in commonly used eukaryotic cell-free protein synthesis—the NTP system (guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, uridine triphosphate). For example, at the same concentration of 7 mM, in a 1500-liter reaction system, the cost of using a conventional energy system (NTP) is approximately RMB 16,000, while the cost of using mononucleotide monophosphates (such as AMP and its sodium salt, GMP and its sodium salt, alone or in combination) is approximately RMB 2,000. Therefore, this replacement can significantly reduce the overall cost of cell-free synthesis while ensuring a continuous energy supply, extending the reaction time for proteins or RNA, and reducing the cost of cell-free synthesis to less than one-eighth of the original cost without significantly altering expression levels. This helps the industrialization of cell-free synthesis and increases the reaction volume of cell-free synthesis.

[0021] (2) This invention uses monophosphate nucleosides such as AMP and GMP as energy regeneration systems, which does not affect the cell-free synthesis process and conditions, and the expression level of the target product can be basically close to that of commonly used energy systems (NTPs). The applicant also found that monophosphate nucleoside salts, such as AMP-2Na, can also achieve similar effects, and the cost can be further reduced. In addition, the combination of AMP and GMP can further enhance the energy regeneration capacity, achieve a better replacement effect, and the overall cost remains low.

[0022] (3) The present invention also provides corresponding reagent kits and synthesis methods. The reagent kits are easy to use and the methods are simple and easy to implement, which provides convenience for research and development and production in the field of cell-free synthesis.

[0023] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0024] Figure 1 The study demonstrated the regeneration effect of different concentrations of AMP on the energy system. Detailed Implementation

[0025] This invention, based on extensive and in-depth research, and through numerous screenings and trials, proposes for the first time a cell-free synthesis system that uses low-cost monophosphate nucleosides to replace conventional nucleoside triphosphate energy regeneration systems. By combining AMP or its sodium salt with GMP, the yield of protein or RNA can be essentially achieved as that of conventional energy regeneration systems, but at a significantly lower cost.

[0026] The present invention will be further illustrated below with reference to specific embodiments and examples. For the specific methods or materials used in the examples, those skilled in the art can make conventional substitutions based on the technical concept of the present invention and existing technologies, and are not limited to the specific descriptions in the embodiments of the present invention.

[0027] The present invention discloses an energy regeneration system for cell-free synthetic reactions, the energy regeneration system comprising: (a) Cell extract; (b) Nucleoside monophosphate or its salt.

[0028] In this invention, "cell-free synthesis reaction" refers to the reaction that synthesizes proteins (including peptides), RNA, etc. in an in vitro cell-free environment synthesis system.

[0029] "Cell-free protein synthesis," also known as "in vitro protein synthesis" or "in vitro cell-free protein synthesis," refers to the synthesis of proteins in an in vitro cell-free environment, including at least the translation process. This includes, but is not limited to, IVT (in vitro translation), IVTT (in vitro transcription-translation), and IVDTT (in vitro replication-transcription-translation). The IVTT reaction, corresponding to the IVTT system, is the process of transcribing and translating DNA into protein in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as a D2P system, D-to-P system, or DNA-to-Protein system; and the corresponding in vitro protein synthesis methods are also called D2P methods, D-to-P methods, or DNA-to-Protein methods. In this invention, "cell-free," "cell-free synthetic reaction," "cell-free system," or "IVTT system" refers to the synthesis of proteins or RNA in vitro, which is not achieved through secretion and expression by intact cells. It should be noted that in the in vitro cell-free synthetic system of this invention, the addition of cellular components to promote the reaction is permitted, but the added cells are not primarily intended for the secretion and expression of exogenous target proteins or RNA. Furthermore, in the D2P system constructed under the guidance of this invention, the intentional addition of a small number of intact cells (e.g., whose protein content does not exceed 30 wt% compared to the protein content provided by cell extracts) is also included within the scope of protection of this invention.

[0030] The terms "cell extract," "cell extract solution," and "cell lysate" used in this invention are interchangeable and can be described in English as cell extract, cell lysate, etc. All refer to substances obtained after cell lysis, and the cell extract does not contain intact cells. Typical cell extracts contain ribosomes for protein translation, transfer RNA, aminoacyl-tRNA synthetase, initiation factors, elongation factors, and termination release factors required for protein synthesis, and are important raw materials for cell-free synthesis reactions. The cell extract described is an aqueous extract of yeast cells.

[0031] Specifically, the method for preparing the cell extract of the present invention includes the following steps: (i) Provide cells; (ii) The cells are washed to obtain washed cells; (iii) The washed cells are subjected to cell-breaking treatment to obtain crude cell extract; (iv) The crude cell extract is subjected to solid-liquid separation to obtain the liquid fraction, which is the cell extract.

[0032] In this invention, the solid-liquid separation method is not particularly limited, but centrifugation is a preferred method.

[0033] In a preferred embodiment, the centrifugation is performed in a liquid state.

[0034] In this invention, the centrifugation conditions are not particularly limited, but a preferred centrifugation condition is 5000-100000g, and more preferably, 8000-30000g.

[0035] In this invention, the centrifugation time is not particularly limited, but a preferred centrifugation time is 0.5 min to 2 h, and more preferably, 20 min to 50 min.

[0036] In this invention, the temperature of the centrifugation is not particularly limited. Preferably, the centrifugation is carried out at 1-10°C, and more preferably, at 2-6°C.

[0037] In this invention, the washing treatment method is not particularly limited. A preferred washing treatment method is to use a washing solution at a pH of 7-8 (preferably 7.4). The washing solution is not particularly limited, and a typical washing solution is selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

[0038] In this invention, the method of cell disruption is not particularly limited, but a preferred method of cell disruption includes high-pressure disruption and freeze-thaw (e.g., liquid nitrogen cryogenic) disruption.

[0039] In a preferred embodiment, the method for extracting cell extracts according to the present invention specifically includes the following steps: a) Preparation of cell seeds: Pick a single cell colony and inoculate it in YPD medium to obtain the culture, which is the seed culture; b) Cell culture: Seed culture was inoculated into YPD medium (containing 20-70 mM (preferably 50 mM) PO33+) for large-scale culture. Cells were harvested by centrifugation at room temperature in the middle and late logarithmic phase of cell growth (e.g., 14-16 h).

[0040] c) After harvesting the cell culture medium, resuspend it in a pre-cooled 4 ℃ wash buffer (such as 10-40 mM, pH 8.0 potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES), 50-150 mM potassium acetate, 1-4 mM magnesium acetate), and then perform cold shock (0-8 ℃) for 30 min. After that, centrifuge to harvest the solid cell slurry.

[0041] d) After harvesting the cell slime, resuspend it in a pre-cooled 4 ℃ wash buffer (such as 10-40 mM, pH 8.0 potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES), 50-150 mM potassium acetate, 1-4 mM magnesium acetate), and centrifuge. Repeat this step 2-4 times. e) After harvesting and cleaning, the cell slime is flash-frozen in liquid nitrogen and then stored at ultra-low temperatures (e.g., -196 ℃ to -10 ℃); f) The cells are broken up using liquid nitrogen mechanical disruption (e.g., using a liquid nitrogen cryogenic stirring and disruption machine (LN Cryo-Blender) or a liquid nitrogen cryogenic pulverizer (LN Cryo-Grinder)). The resulting cell powder is then packaged and stored at ultra-low temperatures (e.g., -196℃ to -20℃). g) Dissolve the cell fragments in a 4°C pre-cooled lysis buffer (e.g., 10-40 mM potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES), 50-150 mM potassium acetate, or 1-4 mM magnesium acetate) for 20 min to obtain a crude cell extract. h) Centrifuge the crude cell extract obtained in step f once at low temperature, with a centrifugal force of 8000-30000 g and a temperature of 4℃; i) After centrifugation, take the cell extract from the middle layer, and then freeze the resulting solution and store it at -80 °C.

[0042] In this invention, the terms "energy system," "energy system," and "energy supply system" have equivalent meanings and can be used interchangeably. Similarly, "energy regeneration system" and "energy regeneration system" have equivalent meanings and can be used interchangeably. The "energy regeneration system" refers to a composition capable of continuously releasing energy from ATP. In the energy regeneration system of this application, monophosphates are used, such as adenosine monophosphate (AMP) and guanosine monophosphate (GMP). It is known that AMP can be converted to ADP under the catalysis of polyphosphokinases, and ADP can further interconvert with ATP, thereby enabling the continuous regeneration of the energy system. The applicant has discovered that using GMP or a combination of different monophosphates can also provide energy. Although the required kinases or triphosphates are not added to the energy regeneration system of this application, this application has found that utilizing natural kinases contained in cell extracts can ensure the reaction proceeds.

[0043] In one example, in the energy regeneration system, the monophosphate nucleoside or its salt is dissolved in an alkaline solution; preferably, the alkaline solution is sodium hydroxide or potassium hydroxide. The applicant has found that dissolving the monophosphate nucleoside or its salt in an alkaline solvent, compared to direct dissolution in water, better preserves the bioactivity of the monophosphate nucleoside or its salt, thus stimulating its energy substitution potential.

[0044] In one example, in the energy regeneration system, the monophosphate nucleoside is selected from any one or a combination of adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, and uridine monophosphate. Preferably, any one of adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, and uridine monophosphate can be used alone; more preferably, a combination of adenosine monophosphate and guanosine monophosphate is used.

[0045] In a preferred embodiment, the monophosphate nucleosides in the energy regeneration system include adenosine monophosphate (AMP) and / or guanosine monophosphate (GMP). The applicant has found that AMP or GMP, whether used alone or in combination, effectively provide reaction energy to cell-free reaction systems, with the combination yielding even better results.

[0046] In a preferred embodiment, the salt in the energy regeneration system refers to the disodium salt of a nucleoside monophosphate. The disodium salt includes disodium adenosine monophosphate, disodium guanosine monophosphate, disodium cytidine monophosphate, or disodium guanosine monophosphate. Generally, disodium salts of nucleoside monophosphates are more stable and easier to store and transport. However, experiments have shown that disodium salts can also serve as substitutes, therefore, disodium salts can also be used.

[0047] In a preferred embodiment, in the energy regeneration system, the concentration of the monophosphate nucleoside or its salt in the cell-free synthesis system is greater than or equal to 5 mM or 8 mM. Unless otherwise specified, the concentrations used in this application refer to the concentration in the entire cell-free synthesis system. Preferably, it is greater than or equal to 6 mM, more preferably greater than or equal to 7 mM, even more preferably greater than or equal to 8 mM, and even more preferably greater than or equal to 9 mM. Since the regeneration of the energy system is a dynamic equilibrium reaction, adding a larger amount of monophosphate nucleoside or its salt will not significantly enhance the promoting effect on the protein synthesis reaction. Therefore, considering both cost and output, a concentration of AMP and its disalt of 5 mM or higher is considered suitable for production.

[0048] In a preferred embodiment, the cell extract in the energy regeneration system is a yeast cell extract, preferably one or more of Saccharomyces cerevisiae, Kluyveromyces var. kanamycin, and Pichia pastoris.

[0049] Furthermore, the Kluyveromyces is selected from one or a combination of the following: Kluyveromyces lactis, Kluyveromyces marx, Kluyveromyces dob, Kluyveromyces var. sacchari, non-fermenting Kluyveromyces, Kluyveromyces wickheim, heat-resistant Kluyveromyces, Kluyveromyces brittle-walled, Kluyveromyces hubeiensis, Kluyveromyces multisporum, Kluyveromyces siamensis, and Kluyveromyces yarbromentosus. Kluyveromyces lactis is preferred.

[0050] Yeast possesses advantages such as simple cultivation, efficient protein folding, and post-translational modification, making it a valuable raw material for preparing in vitro translation systems. This means that yeast extracts can be used for in vitro protein translation expression. Commonly used yeasts for in vitro protein expression include Pichia pastoris and Saccharomyces cerevisiae. Kluyveromyces is an ascospore-forming yeast, with Kluyveromyces marxianus and Kluyveromyces lactis being widely used industrially. Compared to other yeasts, Kluyveromyces lactis has many advantages, such as superior secretion capacity, better large-scale fermentation characteristics, food safety standards, and the ability to perform post-translational modification, giving it enormous application potential and making it a commonly used model strain.

[0051] A second aspect of the present invention provides a cell-free synthesis system, characterized in that the system includes the energy regeneration system described in the first aspect. Due to the use of the aforementioned energy regeneration system, the reaction time of the cell-free synthesis system is extended, thereby improving the utilization of the reaction substrate and increasing the yield of the target substance.

[0052] In a preferred embodiment, the cell-free synthesis system further includes any one or more combinations of the following components: a cell-free synthesis reaction solution, and an RNA or DNA template; The cell-free synthesis reaction solution contains any one or more combinations of T7 RNA polymerase, sugars, amino acid mixtures, and inorganic salt buffer.

[0053] In a preferred example, the Kluyveromyces lactis extract includes endogenously expressed T7 RNA polymerase.

[0054] The cell-free synthesis reaction solution mainly contains sugars, amino acid mixtures, and inorganic salt buffer solutions, which are the conventional necessary raw material components in cell-free synthesis as well as related auxiliary reagents to promote and maintain the normal occurrence of the reaction.

[0055] Amino acids are the basic building blocks of proteins, and amino acid mixtures are the fundamental raw materials for protein synthesis in cell-free systems. The amino acid mixture includes at least the amino acid mixture required for the synthesis of exogenous proteins. Preferably, the amino acid mixture is a mixture of natural and / or non-natural amino acids. Representative amino acids include (but are not limited to) 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. The concentration of each amino acid is typically 0.01-0.5 mM, preferably 0.02-0.2 mM, such as 0.05, 0.06, 0.07, or 0.08 mM.

[0056] The inorganic salt buffer solution contains inorganic salt ions and a buffering agent. Inorganic salt ions are commonly used additives in in vitro protein synthesis systems, including magnesium ions and potassium ions. These ions play an important role in protein translation, promoting ribosome assembly, improving RNA stability, and facilitating polymerase binding.

[0057] The main functions of buffers include maintaining a stable pH environment, reducing protein degradation and aggregation, and optimizing ionic strength and charge environment, thereby improving the efficiency and purity of protein synthesis.

[0058] In a preferred example, the magnesium ion source is preferably selected from: magnesium aspartate, magnesium acetate, magnesium glutamate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, or combinations thereof; in the protein synthesis system, the concentration of the magnesium ions is 1-10 mM, more preferably 1-5 mM, and even more preferably 2-4 mM.

[0059] In a preferred embodiment, the potassium ion source is preferably selected from: potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or combinations thereof. In the protein synthesis system, the concentration of the potassium ions is 30-210 mM, more preferably 30-150 mM, and even more preferably 30-60 mM.

[0060] In a preferred embodiment, the buffer is selected from the group consisting of 4-hydroxyethylpiperazine ethanesulfonic acid, tris(hydroxymethyl)aminomethane, or combinations thereof. The main functions of buffers in cell-free synthesis systems include maintaining a stable pH environment, reducing protein degradation and aggregation, and optimizing ionic strength and charge environment, thereby improving protein synthesis efficiency and purity. 4-hydroxyethylpiperazine ethanesulfonic acid and tris(hydroxymethyl)aminomethane are commonly used buffers. The concentration of the buffer is not strictly limited, typically 5-50 mM, preferably 10-50 mM, more preferably 15-30 mM, and even more preferably 20-25 mM.

[0061] Sugars can provide more energy and prolong reaction time. The T7 RNA polymerase can be added exogenously or by modifying yeast genes to ensure that the obtained cell extract contains T7 RNA polymerase.

[0062] DNA or RNA templates are often provided in the form of DNA or RNA solutions. These templates serve as gene templates for the target substances such as proteins or RNA produced during transcription and translation, determining the structure and type of the produced protein or RNA. They are essential starting substrates for cell-free synthesis. Sugars and phosphate compounds present in the system provide some energy, which is released slowly, further ensuring the smooth progress of the cell-free synthesis reaction.

[0063] The cell-free synthesis reaction solution includes an RNA synthesis substrate. The substrate for synthesizing RNA is preferably a nucleotide mixture selected from: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof. The nucleoside triphosphate mixture is adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and uracil nucleoside triphosphate. In another preferred embodiment, in the protein synthesis system, the concentration of the substrate for synthesizing RNA is 0.1-5 mM, more preferably 0.5-3 mM, and even more preferably 1-1.5 mM. The concentration of each mononucleotide is not particularly limited, but typically the concentration of each mononucleotide is 0.5-5 mM, preferably 1.0-2.0 mM.

[0064] Furthermore, the cell-free synthesis reaction solution also includes a crowding agent; the crowding agent is preferably polyethylene glycol, polyvinyl alcohol, polypropylene, dextran, sucrose polymer, polyethylene (vinylpyrrolidone), etc., or a combination of the above components; polyethylene glycol is preferred.

[0065] The concentration of the polyethylene glycol or its analogue is not particularly limited, but typically the concentration (w / v) of the polyethylene glycol or its analogue is 0.1-8%, preferably 0.5-4%, and more preferably 1-2%, based on the total weight of the protein synthesis system. Representative PEGs are selected from the group consisting of PEG3000, PEG3350, PEG6000, PEG8000, or combinations thereof. Furthermore, the polyethylene glycol includes polyethylene glycol with a molecular weight (Da) of 200-10000, such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc., and preferably, polyethylene glycol with a molecular weight of 3000-10000.

[0066] In a preferred embodiment, the cell-free synthesis reaction system comprises: a protein-encoding mRNA or DNA template, a monophosphate nucleoside or its salt, a cell extract, and a cell-free synthesis reaction solution. The concentration of the protein-encoding mRNA or DNA template is 10-20 ng / µL; the concentration of the monophosphate nucleoside or its salt in the cell-free synthesis system is greater than or equal to 5 mM; the cell extract accounts for 40%-80% of the total system volume, and the cell-free synthesis reaction solution accounts for 20-40% of the total system volume.

[0067] In a preferred example, the cell-free synthesis reaction solution comprises: glucose, maltodextrin, tripotassium phosphate, 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes-KOH), potassium acetate, adenine triphosphate (ATP), guanine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), an amino acid mixture, dithiothreitol (DTT), magnesium L-aspartate, polyethylene glycol, and trehalose. The cell-free reaction solution can be prepared in-house or directly from a cell-free synthesis kit, such as the Fast series, High Yield, and ProteinFactory series products sold by our company.

[0068] In subsequent specific embodiments, the cell-free expression system (IVTT system) used comprises the following components in the cell-free synthesis reaction solution: 15 mM glucose, 320 mM maltodextrin (measured by molar concentration of glucose monomer), 24 mM tripotassium phosphate, 22 mM 4-hydroxyethylpiperazine ethanesulfonic acid at pH 7.4, 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine, each amino acid having a final concentration of 0.7 mM), 5 mM L-aspartate magnesium, 80 mM potassium acetate, 0.44 mM dithiothreitol, 2% (w / v) polyethylene glycol 8000, 9.78 mM pH 8.0 Tris·HCl buffer, and 6% (w / v) trehalose.

[0069] A third aspect of the present invention provides a cell-free synthesis kit, the kit comprising: The components of the cell-free synthetic system described in the second aspect.

[0070] Using the aforementioned cell-free synthesis kit, in vitro cell-free synthesis reactions can be performed to efficiently synthesize target exogenous proteins, etc. The containers include reagent containers and reaction containers, used to hold different components of the aforementioned expression system, respectively. The reaction container is used to mix the reaction materials and initiate protein or RNA synthesis reactions. The material of the containers is not particularly limited; it only needs to be suitable for holding solvents and reactions, and not react with the reaction materials or products. Common materials include glass, polyethylene, and polypropylene.

[0071] Optionally, the kit may further include a nucleic acid template encoding a foreign protein or RNA; optionally, the kit may also include protein or RNA isolation and / or analytical materials. For kits containing known target proteins or RNA species, the corresponding encoding nucleic acids can be directly included as part of the kit for production, sale, and use. To further enhance the kit's functionality and facilitate subsequent separation and analysis of protein products, materials for protein or RNA isolation and analysis may also be included, such as magnetic beads and chromogenic reagents for separating and purifying target proteins. Those skilled in the art can select and add these materials according to the characteristics of the target protein.

[0072] A fourth aspect of the present invention provides an in vitro synthesis method, the method comprising the following steps: (i) Provide an RNA or DNA template; (ii) The template is added to a cell-free synthesis system comprising the energy regeneration system described in the first aspect and the cell-free synthesis reaction solution described in the second aspect, and an incubation reaction is carried out under suitable conditions to synthesize in vitro, preferably, the in vitro synthesis being in vitro transcription and / or in vitro translation.

[0073] Furthermore, in step (ii) of the synthesis method, the mixed reaction components are incubated in an environment of 20-35 °C. These temperature conditions are designed to simulate the temperature of biological cells, promoting the normal progress of the reaction. It is preferable to conduct the reaction at room temperature, as this accommodates most synthesis reactions, saves production costs, and allows for fine-tuning based on the characteristics of the cell extract or DNA template used.

[0074] In a preferred example, the method further includes "(iii) Optionally, isolating or detecting the exogenous protein or RNA from the cell-free synthesis system."

[0075] The separation or detection methods mentioned above commonly include the detection of synthetic protein concentration, protein separation and purification, etc., which can all be carried out using methods commonly used in the field, such as spectrophotometry, enzyme-linked immunosorbent assay (ELISA), magnetic bead separation, etc.

[0076] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention. It should be noted that since the experiments in each batch were not conducted simultaneously, and the concentrations of cell extracts from different batches varied, there were differences in the specific values ​​of the final protein synthesis reaction results. However, this is within the normal error range and does not affect the judgment of the conclusion. Experimental methods in the following embodiments that do not specify conditions are generally performed under conventional conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) and "Cell-Free Protein Synthesis Laboratory Manual" "Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008", or according to the conditions recommended by the manufacturer. Unless otherwise specified, the experimental materials or strains used in the following embodiments are all commercially available.

[0077] Example 1 In this embodiment, AMP is used as a component of the energy regeneration system to verify its effectiveness.

[0078] Experimental Methods: In this embodiment, 10 experimental groups (hereinafter referred to as AMP groups) and 2 control groups were set up, with 3 parallel experiments in each group. In each experiment of the experimental groups, 150 μL of Kluyveromyces lactis (a commonly used engineered strain, strain model abbreviation L1) cell extract, 60 μL of cell-free synthesis reaction solution, and 10 μL of DNA (encoding DNA of green fluorescent protein) template solution were taken and added to different volumes of AMP stock solution (dissolved in 0.1M KOH) to make the final concentration of AMP from 1 mM to 10 mM. The above solutions were mixed and diluted with ultrapure water to make the total reaction volume of a single sample 300 μL. After thorough shaking and mixing, the solution was placed in a 24-well plate and reacted in a shaker at 30°C for 3 hours. After 3 hours, 10 μL of the reaction solution was taken and added to a black 384-well plate. The 384-well plate was placed in a Tecan microplate reader to detect the eGFP protein concentration and obtain the RFU absorbance value. The mean and standard deviation of each group were calculated and statistically analyzed.

[0079] In the control group, one group served as a blank control (NC group), that is, no energy regeneration system components were added, and the other group was added with conventional energy regeneration system components (PC group), namely NTP (guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, uridine triphosphate), with a total NTP concentration of 7 mL (each component was mixed in equal proportions). Other operations and parameters were the same as those in the experimental group.

[0080] Experimental results: Figure 1 The RFU values ​​of the proteins obtained from each reaction group are shown. Table 1 further shows the percentage of protein produced by different concentrations of AMP relative to the protein produced by the conventional energy system group (PC group). Note: The costs in the following tables refer to the cost of AMP, GMP or their salts used in the experimental groups, and the cost of NTP used for the control.

[0081] Table 1 Experimental Design and Results of Example 1

[0082] The experimental results showed that the NC histone yield was very low, indicating that it is difficult to achieve continuous protein synthesis without the addition of an energy regeneration system. However, the addition of AMP significantly increased the protein product concentration, demonstrating AMP's positive effect on energy regeneration. Comparing the target protein concentrations, considering both production cost and yield, at an AMP concentration of 4 mM, the protein yield in the system reached 50% of that of the conventional energy regeneration system group. At 5 mM and above, the protein yield reached over 60% of the conventional energy regeneration system group, significantly reducing costs while maintaining high protein content, demonstrating a good replacement effect. However, beyond a certain AMP concentration, the system's energy regeneration capacity does not significantly improve further. Therefore, a concentration within the 5 mM-10 mM range can be selected based on cost and actual reaction requirements. In terms of cost, in a 1 L system, the cost of 7 mM NTP is approximately 11 yuan, while the cost of 7 mM AMP is only about 1.5 yuan. This shows that using AMP as a substitute can greatly reduce synthesis costs.

[0083] (The NC group and PC group in this article have the same settings as in Example 1, and will not be repeated hereafter.) Example 2 In this embodiment, monophosphate nucleosides were added in different forms to screen for the optimal addition method.

[0084] Experimental Methods: In this example, a total of 6 experimental groups and 1 control group were set up, with 3 parallel experiments in each group. In the experimental groups, AMP was dissolved in 0.1M NaOH solution in groups 1 and 2, and the final concentration of AMP in the cell-free protein synthesis system was different. AMP was dissolved in 0.1M KOH solution in groups 3 and 4. AMP powder was added directly to groups 5 and 6. The control group was the same as in Example 1. Other operating methods, raw material components, and parameters were the same as in Example 1. The specific concentration settings and experimental results of each experimental group are shown in Table 2 below.

[0085] Table 2 Experimental Design and Results of Example 2

[0086] The experimental results show that, compared to adding AMP powder directly to the cell-free protein synthesis reaction system, dissolving AMP in an alkaline solvent before adding it can replace more than 60% of the NTP energy system, resulting in a better energy substitution effect.

[0087] Example 3 In this embodiment, AMP+GMP is used as the components of the energy regeneration system to verify the effect of using the two together.

[0088] Experimental Methods: In this embodiment, 10 experimental groups and 1 control group were set up, with 3 parallel experiments in each group. The experimental groups were further divided into two categories: one group with only GMP added (hereinafter referred to as the GMP group), and the other group with the same amount of AMP added to the groups with different concentrations of GMP (hereinafter referred to as the A+G group). GMP was dissolved in purified water (in subsequent embodiments, unless otherwise stated, GMP was dissolved using the above method), and other methods and conditions were the same as in Example 1. The specific concentration settings and experimental results of each experimental group are shown in Table 3 below.

[0089] Table 3 Experimental Design and Results of Example 3

[0090] Experimental results: Experimental results show that GMP alone can promote energy regeneration within the reaction system (above 2 mM can achieve a substitution effect of over 50%). The protein yield produced by the combination of AMP and GMP is significantly higher than that of GMP alone, and the protein yield of the combined combination is close to that of the PC group, indicating a better energy regeneration effect. Regarding dosage, 6 mM AMP and 1 mM or higher GMP both produce good results. In terms of cost, in a 1L system, the cost of 7 mM NTP is approximately 11 yuan, the cost of a combination of 6 mM AMP and 1 mM GMP is only about 1.4 yuan, and the cost of 5 mM GMP is less than 1 yuan. Therefore, using GMP alone or in combination with AMP can significantly reduce synthesis costs while achieving a certain substitution effect.

[0091] Example 4 In this embodiment, AMP+GMP is used as the component of the energy regeneration system, and the effect is compared with that of using AMP alone.

[0092] Experimental Methods: In this embodiment, 14 experimental groups and 1 control group were set up, with 3 parallel experiments in each group. The first 7 experimental groups were supplemented with only AMP (hereinafter referred to as AMP group), with concentrations ranging from 4mM to 10mM. The latter 7 experimental groups were supplemented with both AMP and GMP (hereinafter referred to as A+G group), with AMP concentrations ranging from 4mM to 10mM and GMP concentration fixed at 2mM. The control group was the PC group. Other methods and conditions were the same as in Example 1.

[0093] Experimental results: The protein concentrations in each group of experiments were statistically analyzed, and the specific results are shown in Table 4 below.

[0094] Table 4 Experimental Design and Results of Example 4

[0095] Compared to using AMP alone, adding a small amount of GMP to AMP significantly improves energy regeneration and overall protein yield. This demonstrates that GMP further enhances the energy replacement effect of AMP. With the same total dosage, the effect of AMP + GMP is significantly higher than AMP alone, and the combined effect (AMP 10mM + GMP 2mM) is close to that of the PC group. In terms of cost, in a 1L system, the cost of 7mM NTP is approximately 11 yuan, while the combination of 10mM AMP and 2mM GMP, although using a slightly higher dosage, only costs about 2.4 yuan. This shows that using the combination of AMP and GMP achieves better replacement effects while significantly reducing synthesis costs. Example 5 In this embodiment, AMP-2Na is used as a component of the energy regeneration system to verify its effectiveness.

[0096] Experimental Methods: In this embodiment, a total of 14 experimental groups and 2 control groups were set up, with 3 parallel experiments in each group. The first 7 experimental groups only added AMP-2Na (referred to as A-Na group), with concentrations ranging from 4mM to 10mM. The other group added AMP-2Na and GMP (referred to as A-Na+G group), where the AMP-2Na concentration ranged from 4mM to 10mM, and the GMP concentration was fixed at 2mM. The control groups were NC and PC groups. AMP-2Na was dissolved in purified water, and other methods and conditions were the same as in Example 1.

[0097] Experimental results: The protein concentrations in each group of experiments were statistically analyzed, and the specific results are shown in Table 5 below.

[0098] Table 5 Experimental Design and Results of Example 5

[0099] The experimental results above show that AMP-2Na alone can promote energy regeneration within the reaction system, achieving approximately 50% of the protein yield of the PC group. In terms of cost, the price of AMP-2Na is similar to that of AMP. Furthermore, the energy regeneration effect is significantly improved when AMP-2Na is combined with GMP; the combined effect is even better. A combination of 4mM AMP-2Na and 2mM GMP can achieve over 70% of the PC group yield. In terms of cost, 7mM NTP costs approximately 11 yuan, while the combination of 4mM AMP-2Na and 2mM GMP costs only about 1.1 yuan. Therefore, under the same yield conditions, using a combination of AMP and GMP can significantly reduce synthesis costs. Example 6 In this embodiment, AMP+GMP-2Na is used as the component of the energy regeneration system to verify its effectiveness.

[0100] Experimental Methods: In this embodiment, four experimental groups and two control groups were set up, with three parallel experiments in each group. The first two experimental groups only added AMP (referred to as the AMP group), with concentrations of 8 mM and 9 mM, respectively. The other group added AMP and GMP-2Na (referred to as the A+G-Na group), with AMP concentrations of 8 mM and 9 mM, and the GMP-2Na concentration fixed at 2 mM. The control groups were NC and PC. GMP-2Na was dissolved in purified water, and other methods and conditions were the same as in Example 1.

[0101] Experimental results: The protein concentrations in each group of experiments were statistically analyzed, and the specific results are shown in Table 6 below.

[0102] Table 6 Experimental Design and Results of Example 6

[0103] The experimental results above show that, compared to using AMP alone, combining AMP with GMP-2Na can further promote energy regeneration within the reaction system and increase protein synthesis. The combination of 8mM AMP and 2mM GMP-2Na can achieve over 80% of the protein yield of the 7mM PC group. In terms of cost, the cost of 7mM NTP in a 1L system is approximately 11 yuan, while the cost of the combination of 8mM AMP and 2mM GMP-2Na is only about 2 yuan. Therefore, using the combination of AMP and GMP-2Na can significantly reduce synthesis costs. Thus, the use of monophosphate nucleosides and monophosphate nucleoside salts can also be considered as an alternative to the conventional energy regeneration system.

[0104] Example 7 In this embodiment, different combinations of monophosphate nucleosides were used as components of the energy regeneration system to verify its effectiveness.

[0105] Experimental Methods: Two experimental groups and one control group were set up in this example, with three parallel experiments in each group. The experimental groups were each supplemented with the same concentration of a mixture of AMP and NMP (AMP+GMP+CMP+UMP). The AMP concentration in the experimental group was 2.83 mM, and the total concentration of the NMP mixture in the experimental group was 5.65 mM (GMP, CMP, and UMP concentrations were 0.94 mM, and AMP concentration was 2.83 mM). The control group was the PC group with a concentration of 7 mM. Except for AMP, which was dissolved in KOH, all other components were dissolved in purified water. Other methods and conditions were the same as in Example 1.

[0106] Experimental results: The protein concentrations in each group of experiments were statistically analyzed, and the specific results are shown in Table 7 below.

[0107] Table 7 Experimental Design and Results of Example 7

[0108] The experimental results show that the NMP mixture can also be used as an energy substitute to replace the conventional NTP energy system, and can achieve protein synthesis effects close to those of the NTP energy system. In terms of cost, the cost of 7mM NTP in a 1L system is approximately 11 yuan, while the cost of the 5.65mM NMP group is only about 4 yuan.

[0109] All the above embodiments used laboratory-modified Kluyveromyces lactis strains, numbered L1, and the modification did not involve energy system conversion.

[0110] Example 8: Effects of different engineered bacterial strain cell extract systems In this embodiment, cell extracts from engineered strains (referring to strains modified from wild-type strains) (such as different types of Kluyveromyces lactis, Kluyveromyces marxi, etc.) were used to replace the Kluyveromyces lactis cell extract in the previous embodiment to verify whether other types of strains could achieve the same or similar effects. Each strain was divided into experimental and control groups. In the experimental group, AMP and GMP (5mM AMP + 3mM GMP) were used as components of the energy regeneration system. The control groups were PC and NC groups, respectively. The PC group was supplemented with 6.5mL NTP as an energy supply component, while the NC group was not supplemented with any additional nucleotide energy components or energy substitutes. Except for the difference in the energy system components, all other components in the experimental and control groups were the same as in Example 1. The specific experimental design and results are shown in Table 8. Three parallel experiments were set up in each group.

[0111] Table 8 Experimental Design and Results of Example 8

[0112] The experimental results show that in other different yeast engineered strain systems, the addition of AMP and GMP can replace NTP as an energy substitute to complete cell-free protein synthesis reactions.

[0113] Example 9: Effects of different wild-type strain cell extract systems In this embodiment, cell extracts from different wild yeast strains (such as Kluyveromyces lactis and Kluyveromyces marxi) were used to replace the cell extracts of the engineered strains in the previous embodiments to verify whether the same or similar effects could be obtained in the wild yeast cell extract system. Each wild strain was divided into an experimental group and a control group. In the experimental group, AMP and GMP (5mM AMP + 3mM GMP) were used as components of the energy regeneration system. The control groups were PC group and NC group, respectively. The PC group was supplemented with 6.5mL NTP as the energy supply component. In both the experimental and control groups, except for the difference in the energy system components, an appropriate amount of T7 RNA polymerase was added to the reaction system. Other components were the same as in Example 1. The specific experimental design and results are shown in Table 9. Three parallel experiments were set up in each group.

[0114] Table 9 Experimental Design and Results of Example 9

[0115] The experimental results show that adding AMP and GMP as energy substitutes can successfully complete cell-free protein synthesis reactions in different wild yeast strain cell extract systems, and the substitution effect is very good, even better than the NTP energy system.

[0116] The above are only some embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments.

[0117] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing description of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An energy regeneration system, characterized in that, The energy regeneration system includes: (a) Cell extract; (b) Nucleoside monophosphate or its salt.

2. The energy regeneration system according to claim 1, characterized in that, The monophosphate nucleoside or its salt is dissolved in an alkaline solution; preferably, the alkaline solution is sodium hydroxide or potassium hydroxide.

3. The energy regeneration system according to claim 1 or 2, characterized in that, The monophosphate nucleoside is selected from any one or a combination of adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, and uridine monophosphate.

4. The energy regeneration system according to claim 3, characterized in that, The monophosphate nucleosides in the energy regeneration system include adenosine monophosphate and / or guanosine monophosphate.

5. The energy regeneration system according to any one of claims 1-4, characterized in that, The salt mentioned refers to the disodium salt of a monophosphate nucleoside.

6. The energy regeneration system according to claim 4, characterized in that, The concentration of the monophosphate nucleoside or its salt used in the cell-free synthesis system is greater than or equal to 5 mM or 8 mM.

7. The energy regeneration system according to claim 1, characterized in that, The cell extract is a yeast cell extract, preferably one or more of Saccharomyces cerevisiae, Kluyveromyces var. kanamycin, and Pichia pastoris.

8. A cell-free synthesis system, characterized in that, The system includes the energy regeneration system according to any one of claims 1-7.

9. The cell-free synthesis system according to claim 8, characterized in that, It also includes any one or more combinations of the following components: cell-free synthetic reaction solution, RNA or DNA template; The cell-free synthesis reaction solution contains any one or more combinations of T7 RNA polymerase, sugars, amino acid mixtures, and inorganic salt buffer.

10. A cell-free synthesis kit, characterized in that, The kit includes: The components of the cell-free synthesis system according to claim 8 or 9.

11. An in vitro synthesis method, characterized in that, The method includes the following steps: (i) Provide an RNA or DNA template; (ii) The template is added to a cell-free synthesis system comprising the energy regeneration system of any one of claims 1-7 and the cell-free synthesis reaction solution of claim 9, and an incubation reaction is carried out under suitable conditions to perform in vitro synthesis, preferably, the in vitro synthesis being in vitro transcription and / or in vitro translation.

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