A reaction vessel for cell-free synthesis and a cell-free synthesis method

Abstract

The application provides a reaction container for cell-free synthesis and a cell-free synthesis method, effectively solves the problems of poor heat preservation and heat conduction of the cell-free synthesis reaction container, and can ensure the smooth progress of the cell-free synthesis reaction and improve the reaction efficiency.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a container suitable for cell-free protein synthesis (CFPS) and its uses. Background Technology

[0002] Cell-free protein synthesis (CFPS) is a technique that uses exogenous mRNA or DNA as a template to complete the transcription or synthesis of proteins under in vitro conditions using enzymes, amino acid substrates, and energy.

[0003] Compared with traditional in vivo recombinant expression systems, in vitro cell-free synthesis systems have many advantages, such as the ability to directly use PCR products as templates to synthesize multiple proteins in parallel, enabling high-throughput screening of peptide or protein drugs, and offering fast synthesis speed and high efficiency. In addition, in vitro cell-free synthesis systems also have unique advantages in expressing peptides or proteins that are toxic to cells.

[0004] In existing technologies, the containers used in cell-free synthesis reactions are mostly made of conventional materials such as glass, polypropylene (PP), and polystyrene (PS), but the reaction efficiency is not ideal. Currently, there is no research on reaction container materials for the field of cell-free synthesis.

[0005] This invention has conducted research on reaction vessel materials in the field of cell-free synthesis and screened specific reaction vessel materials. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems existing in the prior art and provide a reaction vessel and a cell-free synthesis method for cell-free synthesis. Using the cell-free synthesis vessel of this application, the stable and smooth progress of the cell-free synthesis reaction can be ensured, and it is less susceptible to interference from environmental factors.

[0007] The first aspect of the present invention provides a reaction vessel for cell-free synthesis, wherein the material of the vessel is selected from at least one of polyester polymers, polyvinyl chloride polymers, polyethylene polymers, or metal alloys.

[0008] In a preferred embodiment, the polyester polymer material is selected from at least one of polycarbonate, polyethylene terephthalate, polybutylene terephthalate, polylactic acid polyester, and polymethyl methacrylate.

[0009] In another preferred embodiment, the metal alloy material is selected from alloy steel; preferably at least one of stainless steel or carbon steel; more preferably 304 stainless steel, 316 stainless steel, etc.

[0010] In another preferred embodiment, the container is made of a transparent, translucent, or opaque material, preferably an opaque or translucent material.

[0011] In another preferred embodiment, the thickness of the bottom or sidewall of the reaction vessel is 2mm-10mm; preferably 2mm-8mm; more preferably 3-6mm; even more preferably 3mm, 4mm or 5mm.

[0012] A second aspect of the present invention provides a cell-free synthesis method, wherein the method uses the reaction vessel for cell-free synthesis described in the first aspect.

[0013] In another preferred embodiment, the cell-free synthesis is performed under light or no light conditions. The light exposure refers to the use of a light source with a wavelength of 390nm-760nm, or illumination under natural indoor light conditions. Preferably, it is performed under no light conditions.

[0014] Compared with the prior art, the present invention has the following beneficial effects or advantages:

[0015] (1) The reaction vessel for cell-free synthesis provided by the present invention, such as PC, PE, PVC, stainless steel containers, etc., has been proven by experiments to ensure cell-free synthesis efficiency and stable and smooth reaction.

[0016] (2) The reaction vessel for cell-free synthesis provided by this invention uses materials that are easy to mold and durable: PVC, PE, etc., can be injection molded to achieve complex cavity structures (such as microgrooves); stainless steel is also easy to mold through welding and other methods; and can be prepared into containers of different shapes according to actual needs to provide a reaction site for cell-free synthesis reactions. In addition, these materials are stable and easy to clean, and can be reused for cell-free protein synthesis.

[0017] (3) The reaction vessel for cellless synthesis provided by the present invention has photostability. Although the materials used in this application have a variety of colors, the materials have been verified by experiments to be stable to light. The cellless synthesis reaction inside the container is basically not affected by light, which is more conducive to the smooth progress of the reaction.

[0018] 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. Detailed Implementation

[0019] This invention, based on extensive and in-depth research and through numerous screenings and trials, proposes for the first time a reaction vessel for cell-free synthesis. The reaction vessel is selected from at least one of polyester polymers, polyvinyl chloride polymers, polyethylene polymers, or metal alloys. The material of this reaction vessel is less affected by the environment during synthesis, maintains reaction stability, is easy to mold, durable, and ensures the smooth progress of cell-free synthesis reactions, thereby improving synthesis efficiency.

[0020] 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.

[0021] The present invention provides a reaction vessel for cell-free synthesis, wherein the material of the vessel is selected from at least one of polyester polymers, polyvinyl chloride polymers, polyethylene polymers, or metal alloys.

[0022] The "cell-free synthesis" described in this invention, also known as "cell-free synthetic reaction," "cell-free system," or "IVTT system," refers to the synthesis of proteins or RNA in an in vitro environment without the need for intact living cells, rather than through secretory expression by intact cells. In this in vitro cell-free system, DNA is used as a template to mimic the in vivo transcription process to generate RNA. This technique allows control over the transcribed genes, the transcription process, and the uses of the transcribed RNA; this technique is called in vitro transcription. 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 existing technologies. After in vitro transcription, further in vitro translation can be performed to obtain proteins or peptides, which is also a process of cell-free protein synthesis. Therefore, cell-free synthesis can be divided into two main aspects based on the different products or reaction steps: cell-free protein synthesis and cell-free RNA synthesis. Unlike traditional cell expression systems, cell-free synthesis reactions can synthesize proteins or RNA in vitro, and have advantages such as high efficiency, convenience, flexibility and high purity, making them one of the most promising emerging technologies in the fields of biomedicine and basic research.

[0023] The term "reaction vessel" refers to equipment or containers used for synthesizing proteins, nucleic acids, and other products under in vitro conditions. It can also be called a "reaction chamber," "reactor," or "reaction tank." Essentially, the substrates required for cell-free synthesis are added to the "reaction vessel," allowing the components to mix and undergo a biosynthetic reaction. Therefore, the reaction vessel needs to provide a stable environment for the reaction substrates and other raw materials, as well as suitable temperature and light conditions. This necessitates that the material of the reaction vessel meets these requirements. The polyester polymers, polyvinyl chloride polymers, polyethylene polymers, or metal alloys selected in this application all possess good strength, wear resistance, fatigue resistance, and tensile strength. They also maintain chemical stability in most environments, are not easily corroded by acids, alkalis, or other chemicals, and have better thermal insulation properties compared to glass products. These materials have been experimentally verified and can meet the requirements of cell-free synthesis.

[0024] In one example, the polyester polymer material is selected from at least one of polycarbonate, polyethylene terephthalate, polybutylene terephthalate, polylactic acid polyester, and polymethyl methacrylate. The polyester polymer material refers to a polymer formed by the polycondensation reaction of a diacid and a diol, mainly including basic polyester materials, functionalized and specialty polyesters, etc. Polycarbonate, polyethylene terephthalate, polybutylene terephthalate, and polylactic acid polyester are commonly used types, possessing not only good strength and stability but also ease of processing and molding. They exhibit good thermal and dimensional stability, making the products less prone to deformation during processing and able to withstand high reaction temperatures, meeting the requirements of cell-free synthesis reactions. Polycarbonate (PC) and polymethyl methacrylate (PMMA) are preferred.

[0025] In one example, the reaction vessel is made of polyvinyl chloride (PVC) polymer. Polyvinyl chloride is a polymer composed primarily of vinyl chloride monomers. PVC is a multifunctional thermoplastic with excellent resistance to acid and alkali corrosion, flame retardancy (due to chlorine atoms), and electrical insulation. It is flexible in processing (welding and thermoforming) and is currently the second most widely used plastic globally. Using PVC as the material for preparing cell-free synthesis reaction vessels offers advantages such as readily available raw materials, simple processing methods, and stable physicochemical properties, preventing unwanted reactions with the reaction liquid. In another example, the reaction vessel is made of polyethylene (PE) polymer. Polyethylene is a polymer compound produced by the polymerization of a large amount of ethylene monomers. Polyethylene is typically colorless, transparent, and possesses high toughness and ductility, exhibiting excellent wear resistance, impact resistance, corrosion resistance, heat resistance, and cold resistance.

[0026] In one example, the metal alloy material is selected from alloy steel; preferably at least one of stainless steel or carbon steel; more preferably 304 stainless steel, 316 stainless steel, etc. Compared with the aforementioned plastic raw materials, metal alloy materials have more robust rigidity and better thermal conductivity, and are therefore suitable for cell-free synthesis reactions. Stainless steel is a preferred material, specifically 304 stainless steel, 316 stainless steel, etc.

[0027] In one example, the container is made of a transparent, translucent, or opaque material, preferably an opaque or translucent material. The transparent material refers to a material with a light transmittance of approximately 75% or higher; the translucent material refers to a material with a light transmittance between 30% and 75%; and the opaque material refers to a material with a light transmittance below 30%. The reaction vessel for cell-free synthesis is preferably made of an opaque or translucent material to prevent light interference and ensure the smooth progress of the reaction.

[0028] In one example, the thickness of the bottom or sidewall of the reaction vessel is 2mm-10mm; preferably 2mm-8mm; more preferably 3mm-6mm; even more preferably 3mm, 4mm, or 5mm. The terms "bottom" and "sidewall" are conventional definitions in the art. Specifically, the bottom refers to the lowest or lowest part of the reaction vessel in spatial position; the bottom thickness refers to the vertical cross-sectional dimension of the lowest load-bearing or sealing structure of the vessel. The sidewall refers to the wall structure of the container or object that stands upright or surrounds the main body, typically connecting the bottom and top, forming the outer support or enclosure of the container. Depending on the specific shape of the container, the sidewall can have different specific shapes; the thickness of the sidewall refers to the dimension of the wall structure of the upright or surrounding part of the container in the direction perpendicular to the container surface. All thicknesses refer to average thickness. An appropriate thickness can ensure the rigidity and insulation of the reaction vessel. At the same time, the thickness also determines the cost. Therefore, the applicant has studied the thickness of the reaction vessel and found that a thickness of 2mm-10mm has good characteristics of supporting the reaction and the cost is reasonable. Specifically, 2mm-8mm can be selected; more preferably 3mm-6mm; and even more preferably 3mm, 4mm or 5mm.

[0029] In another preferred embodiment, a cell-free synthesis method is also provided, which uses the reaction vessel for cell-free synthesis described in the first aspect. Due to the use of the aforementioned reaction vessel with excellent properties, the smooth progress of the cell-free synthesis reaction can be ensured, thereby improving production efficiency.

[0030] The cell-free synthesis method includes adding various raw material components for cell-free reactions, i.e., a cell-free synthesis system, to a cell-free synthesis reaction vessel and incubating under appropriate conditions. The cell-free synthesis system includes a cell-free synthesis reaction solution, an RNA or DNA template, etc. The cell-free synthesis reaction solution contains cell extracts, T7 RNA polymerase, sugars, an amino acid mixture, and an inorganic salt buffer, etc.

[0031] In the in vitro cell-free synthesis system of the present invention, the addition of cellular components to promote the reaction is also 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 the present invention, the intentional addition of a small number of intact cells (e.g., whose protein content does not exceed 30 wt% compared to that provided by cell extracts) is also included within the scope of protection of the present invention.

[0032] 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, celllysate, etc. All refer to substances obtained after cell disruption, 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.

[0033] Specifically, the method for preparing the cell extract of the present invention includes the following steps:

[0034] (i) Provide cells;

[0035] (ii) The cells are washed to obtain washed cells;

[0036] (iii) The washed cells are subjected to cell-breaking treatment to obtain crude cell extract;

[0037] (iv) The crude cell extract is subjected to solid-liquid separation to obtain the liquid fraction, which is the cell extract.

[0038] In this invention, the solid-liquid separation method is not particularly limited, but centrifugation is a preferred method.

[0039] In a preferred embodiment, the centrifugation is performed in a liquid state.

[0040] In this invention, the centrifugation conditions are not particularly limited, but a preferred centrifugation condition is 5000-100000g, and more preferably, 8000-30000g.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In a preferred embodiment, the method for extracting cell extracts according to the present invention specifically includes the following steps:

[0046] a) Preparation of cell seed: Pick a single cell colony and inoculate it in YPD medium to obtain the culture, which is the seed culture;

[0047] b) Cell culture: Seed culture was inoculated into YPD medium (containing 20-70mM (preferably 50mM) 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., 14-16h).

[0048] c) After harvesting the cell culture medium, resuspend it in a pre-cooled 4°C 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°C) for 30 min. After that, centrifuge to harvest the solid cell slurry.

[0049] d) After harvesting the cell slime, resuspend it in a pre-cooled 4°C 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.

[0050] e) After harvesting and cleaning, the cell mud is flash-frozen in liquid nitrogen and then stored at ultra-low temperatures (e.g., -196℃ to -10℃);

[0051] 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℃).

[0052] g) Dissolve the cell fragments in a 4°C pre-cooled lysis buffer (e.g., 10-40 mM, pH 8.0 potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES), 50-150 mM potassium acetate, 1-4 mM magnesium acetate) for 20 min to obtain crude cell extract.

[0053] h) Centrifuge the crude cell extract obtained in step f once at low temperature, with a centrifugal force of 8000-30000g and a temperature of 4℃;

[0054] i) After centrifugation, take the cell extract from the middle layer, and then freeze the resulting solution and store it at -80°C.

[0055] In a preferred example, the cell extract is a yeast cell extract, preferably a Kluyveromyces cell extract. Further, the Kluyveromyces is selected from one or a combination of: Kluyveromyces lactis, Kluyveromyces marx, Kluyveromyces dob, Kluyveromyces salina, non-fermented Kluyveromyces, Kluyveromyces wickheim, heat-resistant Kluyveromyces, Kluyveromyces brittle-walled, Kluyveromyces hubeiensis, Kluyveromyces multisporum, Kluyveromyces siamensis, and Kluyveromyces yarbromentosus. Kluyveromyces lactis is preferred.

[0056] Yeast possesses advantages such as simple cultivation, efficient protein folding, and post-translational modification, making it a suitable 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 simultaneously perform protein post-translational modification, demonstrating significant application potential. In a preferred embodiment of this invention, an in vitro protein expression system based on Kluyveromyces lactis is employed.

[0057] In a preferred example, the Kluyveromyces lactis extract includes endogenously expressed T7 RNA polymerase.

[0058] In another preferred embodiment, the cell-free synthesis reaction solution mainly comprises sugars, an amino acid mixture, and an inorganic salt buffer solution, which are mainly the conventional necessary raw material components in cell-free synthesis and related auxiliary reagents to promote and maintain the normal occurrence of the reaction.

[0059] 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.

[0060] The inorganic salt buffer solution contains inorganic salt ions and a buffering agent. Inorganic salt ions, including potassium ions, are commonly used additives in in vitro protein synthesis systems. These ions play an important role in protein translation, promoting ribosome assembly, improving RNA stability, and facilitating polymerase binding.

[0061] 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.

[0062] 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.

[0063] 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 the buffer in a cell-free synthesis system 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. 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.

[0064] 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.

[0065] 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.

[0066] In a preferred embodiment, the cell-free synthesis reaction solution comprises an RNA synthesis substrate. The substrate for synthesizing RNA is preferably a mixture of nucleotides 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, preferably 0.5-3 mM, 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In a preferred embodiment, the cell-free synthesis reaction system comprises: a protein-encoding mRNA or DNA template, NTPs or their substitutes, a cell extract, polyphosphates or their salts, 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 NTPs or their substitutes in the cell-free synthesis system is greater than or equal to 5 mM, preferably greater than or equal to 6 mM, more preferably greater than or equal to 7 mM; the cell extract accounts for 40%-80% of the total system volume, for example 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%, more preferably 50-80%; the concentration of the polyphosphates or their salts in the system is less than or equal to 20 mM but not zero, preferably 5 mM-15 mM, for example 7.5 mM, 10 mM, 12.5 mM, 15 mM, etc.; and the cell-free synthesis reaction solution accounts for 20-40% of the total system volume.

[0071] In a preferred example, the cell-free synthesis reaction solution comprises: glucose, maltodextrin, tripotassium phosphate, 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes-KOH), potassium acetate, 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 offered by our company.

[0072] In another preferred embodiment, the cell-free synthesis is carried out under either illuminated or unilluminated conditions. Illumination refers to the use of a light source with a wavelength of 390nm-760nm or illumination under natural indoor light conditions. As verified by embodiments of the present invention, when using the cell-free synthesis reaction vessel described in this application, the cell-free synthesis reaction can be successfully completed under both illuminated and unilluminated conditions. Here, illumination refers to conventional illumination, such as fluorescent lamps or natural indoor light, and does not include special light sources such as lasers.

[0073] Example 1

[0074] In this embodiment, reaction vessels of the same thickness and size were prepared using different materials and used in cell-free protein synthesis reactions to verify the effects of different materials.

[0075] The reaction vessels are all of the same size, specifically a square tank with a square bottom, an inner diameter of 40mm, an outer diameter of 46mm, a wall thickness of 3mm, and an inner wall height of 30mm. All materials are commercially available products, and their English abbreviations are common names. PA refers to nylon, i.e., polyamide (PA); PE refers to polycarbonate; POM refers to polyoxymethylene (or polyformaldehyde); ABS (Acrylonitrile Butadiene Styrene) refers to acrylonitrile-butadiene-styrene copolymer; PE refers to polyethylene; PVC refers to polyvinyl chloride; and PS refers to polystyrene.

[0076] The raw materials for cell-free protein synthesis reactions include the following components:

[0077] 80% (v / v) Kluyveromyces lactis extract, 15 mM glucose, 320 mM maltodextrin (molar concentration based on glucose monomer), 24 mM tripotassium phosphate, 22 mM 4-hydroxyethylpiperazine ethanesulfonic acid at pH 7.4, 7 mM nucleoside triphosphate mixture (a mixture of adenine, guanine, cytosine, and uracil triphosphates, each with equal final concentration), 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 with a final concentration of 0.7 mM), 5 mM The ingredients include 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, 6% (w / v) trehalose, and 15 ng / μL target protein DNA (EFGP was used in this example). The Kluyveromyces lactis extract contains endogenously expressed T7 RNA polymerase. Alternatively, cell-free synthesis kits sold by Kangma (Shanghai) Biotechnology, such as the ProteinFactory series, can be used directly.

[0078] The specific operating method is as follows: Add equal amounts of the aforementioned cell-free protein synthesis reaction raw materials to cell-free reaction containers prepared from different materials, mix well, and incubate in an environment of 25-30℃ (with conventional lighting in the room and no separately configured light source in the reaction device area) for 3 hours. After the reaction is completed, collect the reaction solution and read the RFU fluorescence value of EGFP protein with an enzyme-linked immunosorbent assay (ELISA) reader to obtain the protein concentration.

[0079] The specific experimental design and results are shown in Table 1. Each group of experiments was set up with 3 parallel experiments. Unless otherwise noted, all experiments were opaque. The black color mentioned in group I5 refers to wrapping the transparent material with black tape to make the container opaque. Since the laboratory did not have white material available for the PVC material group, only the black group was available.

[0080] Table 1. Experimental design and cell-free synthesis results of Example 1

[0081]

[0082] The experimental results in Table 1 show that the experimental groups using PE, PC, and PVC as cell-free synthesis containers all achieved high protein yields. To make a more accurate assessment, the average protein concentration within each container was calculated, and then compared with the average to identify the material groups with the best performance. The calculation results indicate that PE, PC, and PVC significantly improved the efficiency of cell-free protein synthesis compared to the average. Therefore, PE, PC, and PVC are more suitable as containers for cell-free synthesis reactions than other materials such as PA, PS, and POM. Furthermore, for PE, PC, or PVC, the color or transparency of the material had little effect on the reaction results.

[0083] Example 2

[0084] In this embodiment, reaction vessels of the same thickness and size were prepared using different metal materials and used in cell-free protein synthesis reactions to verify the effects of different materials. Meanwhile, PVC material was used as a positive control.

[0085] Except for the different materials used in the reaction vessel, the shape and size of the reaction vessel, the raw materials for cell-free protein synthesis, and the specific operating methods are all the same as in Example 1.

[0086] See Table 2 for the specific experimental design and results.

[0087] Table 2. Experimental design and cell-free synthesis results of Example 2

[0088] Serial Number Experiment number Material color RFU value 1 I1 PVC black 5425 2 J1 iron metallic primary color 1187 3 K2 Stainless steel metallic primary color 5272 4 L1 copper metallic primary color 2184

[0089] As can be seen from the experimental results in Table 2, iron and copper resulted in very low protein synthesis efficiency when used as reaction vessels for cell-free synthesis, while stainless steel containers achieved better synthesis results, approaching the performance of PVC materials. Therefore, stainless steel can also be used as a container for cell-free synthesis.

[0090] Example 3

[0091] In this embodiment, the photostability of the reaction vessel was verified under different lighting conditions. PE and stainless steel materials were selected, and two groups of experiments were conducted for each material. One group was not shaded (the room was illuminated by conventional fluorescent lights, and the reaction device area was not illuminated by a separate light source), and the other group was shaded, that is, the reactor was placed in a completely shaded environment. Each group had three parallel experiments, and all other conditions were the same as in Example 1.

[0092] See Table 3 for the specific experimental design and results.

[0093] Table 3. Experimental design and cell-free synthesis results of Example 3

[0094] Material color Light-blocking RFU value Non-shading RFU value Floating level PE White 1070 1065 ±0.27％ Stainless steel metallic primary color 1591 1697 ±3.22％

[0095] After obtaining the specific RFU values, the RFU values ​​before and after shading were compared with the average value to calculate the fluctuation level of the reaction effect after shading, i.e., (RFU value - average value) / average value. As can be seen from the experimental results in Table 3, the reaction effects of the PE, stainless steel, and other materials selected in this application are very similar before and after shading, with the numerical fluctuation range remaining within 5%. This indicates that the above materials can eliminate light interference, ensuring that the cell-free synthesis reaction inside the container is not affected by light.

[0096] The above are only some embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments.

[0097] 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. A reaction vessel for cell-free synthesis, characterized in that, The material of the container is selected from at least one of polyester polymer materials, polyvinyl chloride polymer materials, polyethylene polymer materials, polyoxymethylene polymer materials, or metal alloy materials.

2. The reaction vessel for cell-free synthesis according to claim 1, characterized in that, The polyester polymer material is selected from at least one of polycarbonate, polyethylene terephthalate, polybutylene terephthalate, polylactic acid polyester, and polymethyl methacrylate.

3. The reaction vessel for cell-free synthesis according to claim 1, characterized in that, The metal alloy material is selected from alloy steel; preferably at least one of stainless steel or carbon steel.

4. The reaction vessel for cell-free synthesis according to any one of claims 1-3, characterized in that, The container is made of a transparent, semi-transparent, or opaque material.

5. The reaction vessel for cell-free synthesis according to any one of claims 1-4, characterized in that, The thickness of the bottom or sidewall of the reaction vessel is 2mm-10mm.

6. A cell-free synthesis method, characterized in that, In the method described, cell-free synthesis is completed using the reaction vessel described in any one of claims 1-5.

7. The cell-free synthesis method according to claim 6, characterized in that, The cell-free synthesis is carried out under light or no light conditions; Preferably, the illumination refers to the use of a light source with a wavelength of 390-760nm, or illumination under indoor natural light conditions.

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