Method for producing protein, method for producing cultured meat, additive used in method for producing cultured meat, and kit used in method for producing protein

Abstract

To provide a method for producing a protein that self-proliferates an energy-regenerating enzyme.SOLUTION: The problem can be solved by a method for producing a protein, the method comprising a protein translation step for translating a protein using a translation template mRNA in the absence of a cell and in the presence of an element for translating the translation template mRNA into the protein, wherein the translation template mRNA includes a translation template mRNA for translating an energy-regenerating enzyme.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The disclosure in this application relates to a method for producing a protein, a method for producing cultured meat, an additive for use in the method for producing cultured meat, and a kit for use in the method for producing a protein. [Background technology]

[0002] A cell-free protein synthesis system is a system in which a medium containing intracellular elements involved in protein synthesis is prepared and protein synthesis is performed cell-free. Various cell-free protein synthesis systems are known. Such synthesis systems include a system in which a template DNA, which is a transcription template, is applied to a medium to synthesize a protein, which is the final product, and a system in which a translation template mRNA is applied to a medium to synthesize a protein.

[0003] Known cell-free protein synthesis systems use extracts prepared from E. coli, insect cells, wheat germ, animal cells, etc., and kits are commercially available from several companies. Proteins are synthesized using energy sources such as ATP and GTP, with mRNA as a translation template. However, when the energy source is consumed by protein synthesis, ATP is converted to AMP or ADP, and GTP is converted to GDP, resulting in a shortage of energy sources for protein synthesis. For this reason, it is known to add energy regenerating enzymes to cell-free protein synthesis systems that regenerate AMP or ADP to ATP and GDP to GTP. For example, Patent Document 1 discloses creatine kinase, myokinase, nucleoside diphosphate kinase (NDK), etc., as energy regenerating enzymes.

[0004] If the amount of energy regenerating enzyme added to the cell-free protein synthesis system is too small, the amount of energy source regenerated will also be small, resulting in a small amount of protein synthesis. Therefore, it is necessary to add a certain amount of energy regenerating enzyme to the cell-free protein synthesis system, and Patent Document 2 describes, for example, the use of creatine kinase at a concentration of 0.1 mg / mL to 0.5 mg / mL. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 4061043 [Patent Document 2] JP 2013-158342 A Summary of the Invention [Problem to be solved by the invention]

[0006] Among the energy regenerating enzymes, for example, creatine kinase is derived from rabbit muscle, and myokinase is derived from yeast. However, since energy regenerating enzymes are derived from living organisms, they require a process of isolating the enzymes, which makes them relatively expensive. In addition, in order to prevent the energy regenerating enzymes from being inactivated, it is necessary to pay attention to the addition of stabilizers and storage temperature control.

[0007] The disclosure of the present application has been made to solve the above-mentioned conventional problems. As a result of intensive research, the present inventors have newly discovered that an energy regeneration system can be constructed without adding an isolated energy regeneration enzyme itself to a cell-free protein synthesis system by (1) using a translation template mRNA for translating an energy regeneration enzyme and (2) self-propagating the energy regeneration enzyme in a cell-free protein synthesis system.

[0008] In other words, the object of the disclosure of this application is to provide a method for producing a protein that self-replicates an energy regenerating enzyme, a method for producing cultured meat, an additive for use in the method for producing cultured meat, and a kit for use in the method for producing a protein. [Means for solving the problem]

[0009] The disclosure of the present application relates to the following: a protein production method, a cultured meat production method, an additive used in the cultured meat production method, and a kit used in the protein production method.

[0010] (1) A method for producing a protein, comprising a protein translation step of translating a protein using a translation template mRNA in the absence of cells and in the presence of elements for translating the translation template mRNA into a protein, The translation template mRNA is Contains the translation template mRNA for translating the energy-regenerating enzyme Methods for producing proteins. (2) The translation template mRNA further includes a translation template mRNA for translating a target protein. A method for producing the protein described in (1) above. (3) The protein translation step a first protein translation step of translating an energy regenerating enzyme using a translation template mRNA for translating the energy regenerating enzyme; a second protein translation step of translating a target protein using a translation product of the first protein translation step and a translation template mRNA for translating the target protein; Includes A method for producing the protein described in (2) above. (4) The protein translation step The translation is carried out in the presence of a translation template mRNA for translating the energy regenerating enzyme and a translation template mRNA for translating the target protein. A method for producing the protein described in (2) above. (5) The energy regenerating enzyme is at least one selected from the group consisting of creatine kinase, nucleoside diphosphate kinase, arginine kinase, and adenylate kinase. A method for producing the protein according to any one of (1) to (4) above. (6) The enzyme further comprises a translation template mRNA for translating at least one enzyme selected from the group consisting of inorganic diphosphatase, inorganic pyrophosphatase, and disulfide isomerase. A method for producing the protein according to any one of (1) to (4) above. (7) A translation template mRNA for translating the energy regenerating enzyme, and a translation template mRNA for translating the target protein, using a transcription template DNA including a region encoding the translation template mRNA and a promoter region located at the 5' end of the region encoding the translation template mRNA, Translated using the transcription template DNA in the absence of cells and in the presence of elements for transcribing the transcription template DNA into mRNA. A method for producing the protein described in (2) above. (8) The energy regenerating enzyme and the target protein are from the same organism. A method for producing the protein described in (2) above. (9) The target protein is a growth factor. A method for producing the protein according to any one of (2) to (4), (7) and (8) above. (10) A method for producing cultured meat, comprising: The method includes a step of culturing cells in a culture medium containing, as an additive, the translation product produced by the method for producing a protein according to (9) above. How cultured meat is produced. (11) An additive for use in a cultured meat production method, comprising: The protein includes a translation product produced by the method for producing the protein described in (9) above. additives. (12) A kit for use in a method for producing a protein, the kit comprising: a translation template mRNA for translating an energy regenerating enzyme; an element for translating a translation template mRNA for translating said energy regenerating enzyme into a protein in the absence of a cell; Includes kit. (13) Further comprising a translation template mRNA for translating a target protein. The kit according to (12) above. (14) The energy regenerating enzyme and the target protein are from the same organism. The kit according to (13) above. Effect of the Invention

[0011] The protein production method, cultured meat production method, additives used in the cultured meat production method, and kits used in the protein production method disclosed in the present application can construct an energy regeneration system using an energy regeneration enzyme translated from a translation template by a cell-free protein synthesis system, and therefore there is no need to add an isolated energy regeneration enzyme to the cell-free protein synthesis system. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a graph showing the time course of the GFP synthesized in Reference Example 1. [Diagram 2] FIG. 2 is a schematic diagram showing the transcription template DNA of CK. [Diagram 3] FIG. 3 is a diagram showing an outline of the procedure for preparing CK_DNA by PCR. [Figure 4] FIG. 4 is a photograph substituting a drawing, which is a Western blot showing the results of self-amplification of an energy-regenerating enzyme (CK) using a cell-free protein synthesis system in Example 1. [Diagram 5] FIG. 5 is a photograph substituting a drawing, which is a Western blot showing the results of self-amplification of energy-regenerating enzymes (CK) from various animals using a cell-free protein synthesis system in Example 2. [Figure 6] FIG. 6 is a graph showing the results of synthesizing GFP using the translation product obtained in Example 1 in Example 3. [Figure 7] FIG. 7 shows an outline of transcription template DNA for chicken, tuna, and eel FGF2. [Figure 8]FIG. 8 is a schematic diagram showing the transcription template DNA of bovine FGF2. [Figure 9] FIG. 9 shows an outline of the procedure for preparing FGF2_DNA by PCR. [Figure 10] FIG. 10 is a Western blot showing the results of synthesizing a target protein (FGF2) using the energy regenerating enzyme derived from the same organism in Example 4. [Figure 11] FIG. 11 is a graph showing that in Example 5, in the presence of a translation template mRNA for translating an energy regenerating enzyme and a translation template mRNA for translating a target protein, an energy regeneration system is self-assembled and the target protein can be synthesized. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The protein production method, cultured meat production method, additives used in the cultured meat production method, and kits used in the protein production method disclosed in this application are described in detail below. Note that the following explanations are provided for ease of understanding, and the scope of the technical matters disclosed in this application is not limited to the following explanations. Needless to say, in addition to the following examples, appropriate modifications can be made without departing from the spirit of the disclosure of this application.

[0014] (First embodiment of the method for producing a protein) The method for producing a protein according to the first embodiment includes a protein translation step of translating a protein using a translation template mRNA in the absence of cells and in the presence of elements for translating the translation template mRNA into a protein, the translation template mRNA including a translation template mRNA for translating an energy-regenerating enzyme, which is a protein.

[0015] The cell-free protein synthesis system is a method of synthesizing proteins in a test tube by adding a translation template mRNA or a transcription template DNA to a solution containing amino acids, energy molecules such as ATP and GTP, an energy regeneration system, salts such as magnesium ions, and the like, to a cell-free extract containing translation components such as ribosomes, tRNA, aminoacylated tRNA synthetase, translation initiation factors, translation elongation factors, and translation termination factors. The protein production method disclosed in the present application adds a translation template mRNA for translating the energy regeneration enzyme instead of adding an isolated energy regeneration enzyme to the cell-free protein synthesis system. The method is characterized in that the energy regeneration system is self-constructed by using the energy regeneration enzyme translated from the translation template mRNA by the cell-free protein synthesis system. Therefore, when the term "elements for translating the translation template mRNA into a protein in the absence of cells" is used in this specification, it means a solution (translation solution) containing a cell-free extract, amino acids, energy molecules, and salts. In addition, the "element for translating a translation template mRNA into a protein in the absence of cells" can also be said to be an element for which "an isolated energy regenerating enzyme is not added at the start of protein synthesis". Incidentally, energy regenerating enzymes are generally isolated from animals. Therefore, "an isolated energy regenerating enzyme is not added at the start of protein synthesis" can be rephrased as "an animal-derived energy regenerating enzyme is not added at the start of protein synthesis". As described above, the protein production method disclosed in the present application is characterized in that an energy regenerating system is self-constructed by using an energy regenerating enzyme translated from a translation template mRNA by a cell-free protein synthesis system, but from a technical point of view, the addition of an isolated animal-derived energy regenerating enzyme does not inhibit the self-construction of the energy regenerating system. Therefore, it does not prevent the addition of a trace amount of an isolated energy regenerating enzyme in order to smoothly proceed with the initial cycle of self-constructing the energy regenerating system. When an isolated energy regenerating enzyme is added, it is preferable to add the same enzyme as the energy regenerating enzyme that translates from the translation template mRNA so that the added energy regenerating enzyme is not treated as an impurity.

[0016] As described above, the cell-free protein synthesis system disclosed in the present application is characterized in that no energy regenerating enzyme isolated at the start of synthesis is added. Meanwhile, creatine kinase, an example of an energy regenerating enzyme, is mainly present in animal muscles. Therefore, when a cell-free extract derived from animal cells is used, there is a risk that creatine kinase will be contained in the cell-free extract. Therefore, the cell-free extract disclosed in the present application is preferably a non-animal cell-free extract. The non-animal cell-free extract is not limited, but may be, for example, one extracted by a known method from wheat germ or the like.

[0017] The amino acids used may be various naturally occurring amino acids, but may also be non-naturally occurring amino acids.

[0018] The energy molecule may be a known molecule used in protein synthesis. Examples of the energy molecule include nucleotide triphosphate, creatine phosphate, and formyl folate. Here, the nucleotide triphosphate may include ATP, GTP, CTP, and UTP. When producing a protein from a translation template mRNA, ATP and GTP may be used as the energy molecule. When producing a protein from a transcription template DNA, ATP, GTP, CTP, and UTP may be used.

[0019] Energy regenerating enzymes include enzymes known in the field of protein synthesis, including, but not limited to, creatine kinase, nucleoside diphosphate kinase, arginine kinase, pyruvate kinase, and adenylate kinase.

[0020] Creatine kinase catalyzes the reaction of creatine phosphate and AMP to regenerate ADP into ATP. Nucleoside diphosphate kinase uses polyphosphate as a substrate and regenerates AMP and ADP into ATP, and GDP into GTP. Arginine kinase phosphorylates arginine (Arg) to generate arginine phosphate (PArg), and regenerates ATP by transferring the phosphate of PArg generated to ADP. Pyruvate kinase transfers a phosphate group from phosphoenolpyruvate to ADP to regenerate ATP. Adenylate kinase is an enzyme that generates one molecule of AMP and one molecule of ATP from two molecules of ADP.

[0021] There are no particular limitations on the translation template mRNA for translating the energy regenerating enzyme as long as it can translate the energy regenerating enzyme. The translation template mRNA includes a coding region that encodes the amino acid sequence of the energy regenerating enzyme, and non-translated regions may be appropriately linked to the 5' and 3' sides of the coding region. Examples of 5' and 3' non-translated regions for improving protein translation efficiency are described in International Publication Nos. 2022 / 185664 and 2021 / 070616 filed by the present applicant. The translation template mRNA disclosed in this application may use the non-translated regions described in International Publication Nos. 2022 / 185664 and 2021 / 070616 as necessary. The matters described in International Publication Nos. 2022 / 185664 and 2021 / 070616 are incorporated herein by reference.

[0022] The salts to be used may be any known salts used in cell-free protein synthesis systems, including, but not limited to, magnesium acetate, potassium acetate, calcium chloride, and the like.

[0023] The method for producing a protein according to the first embodiment has the following advantages. (1) An energy regeneration system can be constructed using the energy regeneration enzyme translated by a cell-free protein synthesis system, which eliminates the need for isolated energy regeneration enzymes and reduces costs. (2) Proteins produced by a cell-free protein synthesis system can be used as medicines or foods. However, when a protein derived from a biological species that has never been ingested is ingested as a medicine or food, there is a risk of allergic reactions or the like. For example, the options of biological species from which commercially available energy regenerating enzymes are derived are limited, such as creatine kinase derived from rabbits. On the other hand, in the present application, the energy regenerating enzyme is obtained by translating a translation template mRNA, and therefore, an energy regenerating enzyme derived from a desired biological species can be translated. This reduces the risk of allergies and the like. (3) When the produced protein is to be used in food or pharmaceutical products, it may not be possible to use raw materials from a specific species for religious reasons or to avoid contamination with zoonotic viruses. However, the protein production method of the present application can obtain an energy regenerating enzyme derived from a desired species by translation, thereby solving problems caused by religious reasons or contamination with zoonotic viruses. (4) Due to the influence of prion diseases, there is a tendency to avoid using animal-derived raw materials to produce food and medicines. In the protein production method of the present application, even if the type of energy regenerating enzyme is animal, it is possible to produce proteins using only non-animal raw materials. Therefore, there is no risk of diseases derived from animal raw materials.

[0024] (Second embodiment of the method for producing a protein) Next, a second embodiment of the protein production method will be described. The protein production method according to the second embodiment differs from the protein production method according to the first embodiment in that it contains a translation template mRNA for translating the target protein, but other points are the same as the protein production method according to the first embodiment. In order to avoid redundant description, the second embodiment of the protein production method will be described mainly with respect to the points different from the first embodiment. Therefore, it goes without saying that the matters already described in the first embodiment can be applied to the second embodiment even if they are not explicitly described in the second embodiment.

[0025] The target protein is not particularly limited as long as it can be synthesized by a cell-free protein synthesis system. Examples of the target protein include growth factors, secretory proteins such as insulin and amylase, and plasma proteins such as transferrin, myoglobin, and albumin. Examples of animal species of the target protein include fish such as salmon and eel, mammals such as humans, cows, and rabbits, and echinoderms such as sea urchins.

[0026] The protein translation step in the protein production method according to the second embodiment is not particularly limited as long as the target protein can be produced using an energy regenerating enzyme translated from a translation template mRNA for translating the energy regenerating enzyme.

[0027] An example of the protein translation process is A first protein translation step is carried out by translating an energy regenerating enzyme using a translation template mRNA for translating the energy regenerating enzyme; Next, a second protein translation step is carried out to translate the target protein using the translation product of the first protein translation step and a translation template mRNA for translating the target protein. An example is given below (hereinafter, sometimes referred to as the "first example").

[0028] Other examples of the protein translation process include: The translation is carried out in the presence of a translation template mRNA for translating the energy regenerating enzyme and a translation template mRNA for translating the target protein. An example can be given (hereinafter, sometimes referred to as the "second example").

[0029] In the first example, since the energy regenerating enzyme is translated first from the translation template mRNA in the first protein translation step, the amount of the energy regenerating enzyme to be translated can be adjusted by adjusting the reaction time. In addition, the amount of the translation product of the first protein translation step added when the second protein translation step is performed can also be adjusted. Therefore, in the first example, the amount of the energy regenerating enzyme contained in the cell-free protein synthesis system when translating the target protein can be adjusted. By adjusting the amount of the energy regenerating enzyme, the translation efficiency of the target protein can be optimized. On the other hand, in the second example, the translation of the energy regenerating enzyme and the target protein is performed simultaneously, so the translation efficiency of the target protein is lower than in the first example, but the convenience of protein production is improved because there is no need to separate the protein translation steps.

[0030] The translation template mRNA for translating the energy regenerating enzyme and the target protein may be synthesized by a nucleic acid synthesizer or transcribed from a transcription template DNA. The transcription template DNA may contain a region encoding the translation template mRNA and a promoter sequence located at the 5' end of the region encoding the translation template mRNA. The translation template mRNA can be synthesized by translating the transcription template DNA in the absence of cells and in the presence of elements for transcribing the transcription template DNA into mRNA. The promoter sequence and elements for transcribing the transcription template DNA into mRNA may be those known in the art. Examples of promoter sequences include, but are not limited to, T7 promoter sequences, SP6 promoter sequences, T3 promoter sequences, and the like.

[0031] The method for producing a protein according to the second embodiment has the following advantages in addition to the advantages (1) to (4) achieved by the method for producing a protein according to the first embodiment. (5) In the effects (2) to (4) achieved by the method for producing a protein according to the first embodiment, the effect is achieved by replacing "energy regenerating enzyme" with "target protein".

[0032] [Optional additional items that can be adopted in the embodiment of the protein production method] Next, optional additional items that can be adopted in the embodiment of the method for producing a protein will be described. (Addition of translation template mRNA for translating other enzymes) In the protein translation step, in addition to the energy regenerating enzyme, an enzyme for decomposing by-products (e.g., inorganic pyrophosphate) generated during transcription / translation and / or a translation template mRNA for translating an enzyme for folding (activating) the target protein may be added. Examples of enzymes for decomposing by-products include, but are not limited to, inorganic diphosphatase, inorganic pyrophosphatase, etc. Examples of enzymes for folding the target protein include, but are not limited to, disulfide isomerase, etc.

[0033] In the case of the first example of the protein translation step, the enzyme for decomposing by-products and / or the translation template mRNA for translating the enzyme for folding the target protein may be added during the first protein translation step, during the second protein translation step, or during the first and second protein translation steps. In the case of the second example of the protein translation step, the enzyme for decomposing by-products and / or the translation template mRNA for translating the enzyme for folding the target protein may be added together with the translation template mRNA for translating the energy regenerating enzyme and the translation template mRNA for translating the target protein. In the case of preparing the translation template mRNA for translating other enzymes by transcribing it from the transcription template DNA, the transcription template DNA may be designed to include a region encoding the translation template mRNA and a promoter region located at the 5' end of the region encoding the translation template mRNA. In the protein translation step, the coexistence of the enzyme for decomposing by-products and / or the enzyme for folding the target protein improves the translation efficiency of the target protein. It should be noted that the enzymes for decomposing by-products and / or the enzymes for folding the target protein are merely preferred examples, and other enzymes may be used as long as they improve the translation efficiency of the target protein.

[0034] When a translation template mRNA for translating another enzyme is added, the following effects are achieved in addition to the effects achieved by the first and second embodiments of the protein production method. (6) By-products that inhibit efficient synthesis in cell-free protein synthesis systems can be removed, improving protein production efficiency. In addition, the produced proteins can be activated.

[0035] (About the energy regenerating enzyme and the target protein) The combination of the energy regenerating enzyme and the target protein may be derived from the same biological species. In this case, the nucleic acid sequences of the energy regenerating enzyme and the target protein of the same biological species may be obtained from a publicly known database, and a translation template mRNA may be designed. In addition, when the translation template mRNA is produced by transcribing it from a transcription template DNA, a transcription template DNA may be designed that includes a region encoding the translation template mRNA and a promoter region located at the 5' end of the region encoding the translation template mRNA.

[0036] When the energy regenerating enzyme and the target protein are derived from the same biological species, the following effect is achieved in addition to the effects achieved by the first and second embodiments of the protein production method. (7) When the produced protein is to be used in food, mixing proteins derived from different species may be problematic. By combining the energy regenerating enzyme and the target protein from the same species, the risk of problematic mixing can be reduced.

[0037] (About the target protein) The target protein may be a growth factor or cytokine. In recent years, cultured meat has been attracting attention from the perspectives of responding to population growth and increased food production, environmental issues due to the increase in pasture land for meat animals, safety of administering antibiotics to animals, and ethics of taking the lives of animals. Cultured meat is produced by culturing stem cells extracted from animals in a culture medium containing substances necessary for cell growth, such as amino acids and carbohydrates, and growth factors are required for the culture. Cytokines are also required for cell differentiation. Examples of growth factors or cytokines include, but are not limited to, the following growth factors: ·EGF (Epidermal growth factor): Epidermal growth factor, ·IGF(Insulin-like growth factor): Insulin-like growth factor, ·TGF(Transforming growth factor): Transforming growth factor, bFGF or FGF2 (basic fibroblast growth factor): Basic fibroblast growth factor, NGF (Nerve growth factor): ·BDNF(Brain-derived neurotrophic factor): Brain-derived neurotrophic factor, ·VEGF (Vesicular endothelial growth factor): Vascular endothelial growth factor, ·G-CSF (Granulocyte-colony stimulating factor): · GM-CSF (Granulocyte-macrophage-colony stimulating factor): Granulocyte-macrophage colony stimulating factor, ·PDGF (Platelet-derived growth factor): Platelet-derived growth factor, EPO (Erythropoietin): Erythropoietin, TPO (Thrombopoietin): Thrombopoietin, ·HGF (Hepatocyte growth factor): Hepatocyte growth factor. ·LIF(leukemia inhibotory factor): Leukemia inhibitory factor

[0038] When the target protein is a growth factor, the following effect is achieved in addition to the effects achieved by the first and second embodiments of the protein production method. (8) Growth factors are very expensive, but by using the protein production method disclosed in the present application, growth factors can be produced at low cost.

[0039] The protein production method disclosed in the present application has been described above by giving some specific embodiments and optional additional items that can be adopted, but various modifications may be made without being limited to these embodiments as long as they are within the scope of the technical ideas disclosed in the present application. In addition, the optional additional items that can be adopted as examples may be added alone, or any two or more additional items may be combined. When any two or more additional items are used in combination, the respective effects are synergistically exhibited. In addition, if necessary, an energy regenerating enzyme and / or a sequence for adding a protein tag to the N-terminus and / or C-terminus of the target protein may be linked to the translation template mRNA or transcription template DNA.

[0040] (Embodiments of the cultured meat production method and embodiments of the additive) In the method for producing a protein, when a growth factor is produced as the target protein, the translation product can be used in the method for producing cultured meat. The method for producing cultured meat may be carried out by a step of culturing cells using a culture medium containing the translation product as an additive. The culture medium may be a known culture medium. As described above, the cells may be cells of the animal to be cultured. There is no particular limitation on the animal to be cultured as long as it is consumed as meat. Examples of the animal to be cultured include, but are not limited to, mammals such as cows, pigs, horses, and sheep; birds such as chickens, ducks, and quails; fish such as salmon, eels, and tuna; and crustaceans such as shrimp and crabs.

[0041] Furthermore, in the case where a growth factor is produced as the target protein in the protein production method, the translation product can be used as an additive in the cultured meat production method.

[0042] Embodiments of the cultured meat production method and embodiments of the additive have the following advantages. (9) The cost of cultured meat can be reduced because growth factors are inexpensive to obtain. (10) When using energy-regenerating enzymes and growth factors from the same species as the animal being cultured, it is possible to produce cultured meat that does not contain proteins derived from other types of animals.

[0043] (Embodiments of kits used in the method for producing proteins) Next, an embodiment of a kit used in a protein production method will be described. As described above, in conventional cell-free protein synthesis systems, an energy regenerating enzyme isolated from an animal or the like is used. On the other hand, the present inventors have newly discovered that, in a cell-free protein synthesis system, an energy regenerating enzyme obtained from a translation template mRNA for translating the energy regenerating enzyme is used to construct an energy regenerating system. Therefore, a kit used in a protein production method including a translation template mRNA for translating the energy regenerating enzyme and an element for translating the translation template mRNA into a protein in the absence of cells is a new invention. In addition, the kit may further include a translation template mRNA for translating the target protein, and the energy regenerating enzyme and the target protein may be from the same organism.

[0044] The "translation template mRNA for translating the energy regenerating enzyme", "elements for translating the translation template mRNA into a protein in the absence of cells", and "translation template mRNA for translating a target protein" have already been explained in the embodiment of the protein production method. Therefore, detailed explanation will be omitted to avoid duplication.

[0045] The "translation template mRNA for translating the energy regenerating enzyme", "elements for translating the translation template mRNA into a protein in the absence of cells" and the "translation template mRNA for translating the target protein" may be provided separately and mixed at the time of use. Alternatively, the "translation template mRNA for translating the energy regenerating enzyme" and the "elements for translating the translation template mRNA into a protein in the absence of cells" may be provided mixed together, and the "translation template mRNA for translating the target protein" may be provided separately. Further alternatively, the "translation template mRNA for translating the energy regenerating enzyme", "elements for translating the translation template mRNA into a protein in the absence of cells" and the "translation template mRNA for translating the target protein" may all be provided mixed together.

[0046] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the scope of the invention disclosed in the present application. EXAMPLES

[0047] <Reference example 1> [The importance of creatine kinase in protein production using cell-free protein synthesis systems] First, we confirmed the difference in protein synthesis between the case where creatine kinase purified from rabbits was used and the case where it was not used during cell-free protein synthesis of the fluorescent protein GFP. The experimental procedure is described below.

[0048] (1) Raw materials WGE: Wheat germ extract, prepared according to the procedure described in JP 2006-288320 A. CK: Creatine kinase (product number 10127566001) from Roche Diagnostics Deutschland GmbH AAM: Amino acid substrate. The amino acid substrate included with the PSS5100 manufactured by NUProtein was used. mRNA: Designed to contain a coding region encoding the amino acid sequence of GFP from water jellyfish. The mRNA was obtained by transcribing a transcription template DNA in which a sequence encoding GFP (Acc. No. P42212, purchased from Eurofins Genomics) was inserted into the CK_Organisum portion of Table 3 in <Example 1> described below, in the same manner as in Example 1. (2) Translation reaction A translation reaction solution with the composition shown in Table 1 below was prepared in a 1.5 mL tube. The units of the compositions shown in Table 1 below are μL. The stock concentration of the added CK was 20 mg / mL, and the final concentration was 200 ng / μL. For X in Table 1, CK+ is 77.8 and CK- is 80.

[0049] [Table 1]

[0050] A reaction solution having the composition shown in Table 1 was added to a 96-well flat-bottom titer plate, and the plate was reacted in a plate reader at room temperature (about 23° C.).

[0051] [Fluorescence measurement of synthesized GFP] 200 μL of the 220 μL solution containing GFP synthesized in Reference Example 1 was used as a sample, and 475 nm excitation light was irradiated for 0.5 seconds, and the fluorescence intensity from GFP was measured every hour for 15 hours using a plate reader (absorption filter 500-550 nm). The plate reader used was a GloMax (registered trademark) plate reader (Promega). The measurement results are shown in FIG. 1.

[0052] As is clear from Figure 1, the amount of GFP synthesis increased over time by adding CK, an energy regenerating enzyme, to the cell-free protein synthesis system (CK+). On the other hand, when CK was not added (CK-), almost no GFP was synthesized. From these results, it was confirmed that the target protein cannot be synthesized efficiently if the cell-free protein synthesis system does not have an energy regenerating system.

[0053] [Confirmation of self-propagation of energy-regenerating enzymes using a cell-free protein synthesis system] <Example 1> (1) Creatine kinase sequence Among the energy regenerating enzymes derived from various organisms, the sequence information (ACC. No.), organism species, and source of acquisition of creatine kinase (CK) are shown in Table 2. In Example 1, No. 4 CK_T (Thunnus, tuna) was used. Nos. 1 to 3 and 5 were used in Examples 2 to 4 described below. [Table 2]

[0054] (2) Transcription template DNA FIG. 2 shows an outline of the CK transcription template DNA (hereinafter, sometimes referred to as "CK_DNA"). Table 3 shows the sequences of each region of CK_DNA. Note that the CK sequences of Organism Nos. 1 to 5 above are inserted into CK_Organism in the following Table 3. [Table 3]

[0055] The procedure for preparing CK_DNA by PCR will be described with reference to FIG. 3. CK_DNA was prepared by two-step PCR using primers designed as shown in FIG. 3. Table 4 shows the PCR primers, Tables 5 and 6 show the PCR reaction solution composition, and Table 7 shows the PCR program. The method for synthesizing the transcription template DNA was performed in accordance with the PSS5100 instruction manual (May-2021 / Ver.3.00) of NUProtein Co., Ltd. The reagents and machines used are as follows: PCR enzyme: Toyobo KOD-Plus-Neo Primer, artificial genes: Eurofins Genomics, Inc. Custom synthesis services Thermal Cycler: Eppendorf Mastecycler X50s High-speed micro refrigerated centrifuge: Tommy Kogyo Co., Ltd. MX-307

[0056] [Table 4]

[0057] [Table 5]

[0058] [Table 6]

[0059] [Table 7]

[0060] (3) Transcription reaction Next, the prepared transcription template DNA was used to prepare a translation template mRNA. The transcription reaction was carried out in a 1.5 mL tube using the following reaction solution of PSS5100 manufactured by NUProtein, Inc., and 2.5 μL of the previously prepared second PCR reaction solution (containing the transcription template DNA), and was incubated at 37°C for 3 hours.

[0061] [Table 8]

[0062] 10 μL of 4M ammonium acetate was added to 25 μL of the transcription reaction solution and mixed well, and 100 μL of 100% ethanol was added and mixed. After flushing with a tabletop centrifuge, the mixture was left to stand at -20°C for 10 minutes. Then, it was centrifuged (12,000g, 15 minutes, 4°C). After removing the supernatant, it was flushed using a tabletop centrifuge. The supernatant was removed again, and the mixture was left to stand until the precipitate dried. Then, 80 μL of ultrapure water was added to 25 μL of the transcription reaction solution to dissolve the precipitate. This was used as the translation template mRNA solution.

[0063] (4) Translation reaction Next, a translation reaction solution with the following composition was prepared in a 1.5 mL tube, and was reacted in an incubator set at 23°C for 15 hours. The composition shown in Table 9 was obtained by adding CK in addition to the translation template mRNA of CK_T. The composition shown in Table 10 was obtained by adding only the translation template mRNA of CK_T, and no CK was added to the translation reaction solution. For comparison, the translation reaction was carried out by administering the translation template mRNA of GFP prepared in <Reference Example 1> instead of the translation template mRNA of CK_T in Table 10. The units of values ​​in Tables 9 and 10 are μL, and X in Table 9 is 77.8 and X in Table 10 is 80. The stock concentration of the added CK is 20 mg / mL, and the final concentration is 200 ng / μL.

[0064] [Table 9] [Table 10]

[0065] After the reaction, the 1.5 mL tube was centrifuged (15,000 g, 15 minutes, 4° C.), and the supernatant was used as the protein solution after the translation was completed. The obtained protein was subjected to Western blotting.

[0066] <Reagents used> Gel: 4-15% Tris-Glycine gel (Bio-Rad Laboratories) Membrane: 0.2 μm PVDF membrane (Bio-Rad Laboratories) ·Primary antibody: HA antibody (manufactured by Proteintech) ·Secondary antibody: goat anti mouse HRP antibody (manufactured by Southern Biotech) Western blot luminescence reagent: SuperSignal West Pico (Thermo)

[0067] The results are shown in FIG. 4. In FIG. 4, L2 is the result of the translation product obtained with the composition shown in Table 9, L3 is the result of the translation product obtained with the composition shown in Table 10, and L1 is the result of the translation product obtained with the composition using the translation template mRNA of GFP. As is clear from FIG. 4, in L3, in which no isolated CK was added to the translation reaction solution, a predetermined amount of CK_T was translated from the translation template mRNA, although it was less than in L2. From the above results, it is considered that when the translation reaction is performed with the composition shown in Table 10, CK_T is translated from the translation template mRNA by the elements contained in the translation reaction solution, and the translated CK_T establishes an energy regeneration system, causing CK_T to self-multiply.

[0068] <Example 2> An experiment was carried out in the same manner as in Example 1, except that CK_B (Bovine), CK_C (Chicken), and CK_U (Unagi) were used instead of CK_T (Thunnus) in Example 1, and primers with the following sequences were used as 1st_CK_NF and 1st_CK_NR_01. The CK_R (Rabbit) primer used in Example 4 described later is also listed. In Table 11, the species of organism is listed after CK to specify which species of organism the primer is for. [Table 11]

[0069] The results are shown in Figure 5. For comparison, CK_T (Thunnus, tuna) was also loaded into the lane and Western blotting was performed. As is clear from Figure 5, it was confirmed that for cows, a mammal; chickens, a bird; and tuna and eels, a fish, CK of each species was translated from the translation template mRNA by the elements contained in the translation reaction solution, and the translated CK constructed an energy regeneration system, which allowed CK to self-multiply. From the above results, it can be said that the self-construction of an energy regeneration system is not limited to any particular species.

[0070] [Synthesis of proteins other than energy regenerating enzymes] Next, we synthesized proteins using a system that self-replicates energy regenerating enzymes. <Example 3> GFP was synthesized as a target protein using CK_T (L3 in FIG. 4), which is the translation product obtained in the above <Example 1>. Table 12 shows the composition of the translation reaction solution. The translation product CK_T was added in different amounts as shown in No. 1 to 4, and experiments were performed. The target protein was GFP, and the translation template mRNA described in Reference Example 1 was used. The negative control (NC) was without CK, CK_T, and GFP mRNA, and the positive control (PC) was CK instead of the translation product CK_T. The translation reaction was performed for about 15 hours with the composition in Table 11, and the experiment was performed in the same manner as in the above <Reference Example 1> to measure the fluorescence intensity of the synthesized GFP. The unit of the values ​​in Table 11 is μL. The stock concentration of the added CK was 20 mg / mL, and the final concentration was 200 ng / μL. [Table 12]

[0071] The results are shown in Figure 6. Almost no GFP production was observed in No. 1, in which no translation product of CK_T was added, but GFP production was confirmed in Nos. 2 to 4. From the above results, it was confirmed that the protein production method disclosed in the present application can produce a target protein using CK_T, which is a translation product translated from a translation template mRNA.

[0072] <Example 4> Using the translation products of bovine, chicken, tuna, and eel CK obtained in Example 2 above, synthesis was carried out for the target protein FGF2 corresponding to each of the biological species listed in Table 13 below. The specific procedure is described below.

[0073] [Table 13]

[0074] (1) FGF2 transcription template DNA (chicken, tuna, eel) Figure 7 shows an outline of the transcription template DNA (hereinafter, sometimes referred to as "FGF2_DNA") of chicken, tuna, and eel FGF2. Table 14 shows the sequences of each region of FGF2_DNA. Note that the FGF2 sequences of Chicken, Thunnus, and Unagi in Table 13 above are inserted into FGF2_Organism in the following Table 13. [Table 14]

[0075] (2) FGF2 transcription template DNA (bovine) Figure 8 shows an outline of the transcription template DNA of bovine FGF2. Table 14 shows the sequences of each region of the transcription template DNA of bovine FGF2. Note that the FGF2 sequence of Bovine in Table 13 above is inserted into FGF2_Bovine in Table 15 below. [Table 15]

[0076] The procedure for preparing FGF2_DNA by PCR will be described with reference to FIG. 9. Note that in the case of bovine, the structure of the transcription template DNA shown in FIG. 9 is different, but the procedure is the same. Primers were designed as shown in FIG. 9, and FGF2_DNA was prepared by two-step PCR. Table 16 shows the 1st PCR primers for various species, Table 17 shows the 2nd PCR primers for chicken, tuna, and eel, and Table 18 shows the 2nd PCR primers for bovine. Note that "2nd_NCF_U01" and "2nd_FLAG_CR1_E15" in Table 18 correspond to "2nd_CF1" and "2nd_FLAG_CR1_01" in FIG. 9, respectively. "2nd_NCR_U01" is common regardless of the species.

[0077] The composition of the PCR reaction solution is the same as that in Tables 5 and 6 of Example 1, and the PCR program is the same as that in Table 7 of Example 1. The method for synthesizing the transcription template DNA, the reagents, and the machine are also the same as those in Example 1.

[0078] [Table 16]

[0079] [Table 17] [Table 18]

[0080] (3) Transcription reaction Transcription reactions were carried out in the same manner as in Example 1 using the transcription template DNAs (chicken, tuna, eel, and cow) of FGF2 described in (1) and (2) above.

[0081] (4) Synthesis of FGF2 FGF2 was synthesized using various CKs, which are the translation products obtained in <Example 2> above, and the translation template mRNA obtained in (3) above. Table 19 shows the composition of the translation reaction solution. The experiment was performed using the composition in Table 19 and the same procedure as in <Example 1> above. Note that the following primary antibodies were used instead of the primary antibodies in Example 1. Primary antibody: Anti-DYKDDDDK tag (monoclonal antibody Code No. 018-22381; Fujifilm Wako Pure Chemical Industries, Ltd.)

[0082] [Table 19]

[0083] The results are shown in Figure 10. As is clear from the results in Figure 10, it was confirmed that the target protein can be synthesized using the energy regenerating enzyme derived from the same organism species.

[0084] [Co-expression of energy regenerating enzyme and target protein] <Example 5> Next, a translation reaction was carried out in the presence of both a translation template mRNA for translating the energy regenerating enzyme and a translation template mRNA for translating the target protein. The experimental procedure is described below. (1) Energy regenerating enzymes A translation template mRNA was prepared in the same manner as in Example 1 using CK_R shown in No. 1 of Table 2 in Example 1. (2) Target protein The translation template mRNA of GFP described in Reference Example 1 was used. (3) Co-expression of energy regenerating enzyme and target protein The composition of the translation reaction solution is shown in Table 20. The negative control (NC) contained no CK, CK_Rabbit or GFP mRNA, and the positive control (PC) used CK instead of the translation template mRNA of CK_Rabbit. The translation reaction was carried out for about 15 hours with the composition in Table 20, and the experiment was carried out in the same manner as in the above <Reference Example 1>, and the fluorescence intensity of the synthesized GFP was measured.

[0085] [Table 20]

[0086] The results are shown in Figure 11. As is clear from Figure 11, it was confirmed that the energy regeneration system was self-assembled and the target protein could be synthesized even in the presence of both the translation template mRNA for translating the energy regeneration enzyme and the translation template mRNA for translating the target protein. [Industrial Applicability]

[0087] The protein production method, cultured meat production method, additives used in the cultured meat production method, and kits used in the protein production method disclosed in this application make it possible to construct an energy regeneration system for cell-free protein synthesis using an energy regeneration enzyme translated from a translation template mRNA. Therefore, this is useful in industries that require cell-free protein synthesis, such as the food industry, pharmaceutical industry, and research institutes.

Claims

1. A method for producing a protein, comprising a protein translation step of translating a protein using a translation template mRNA in the absence of a cell and in the presence of elements for translating the translation template mRNA into a protein, comprising: The translation template mRNA is Contains a translation template mRNA for translating an energy regenerating enzyme Methods for producing proteins.

2. The translation template mRNA further comprises a translation template mRNA for translating a target protein. A method for producing the protein described in claim 1.

3. The protein translation step comprises: a first protein translation step of translating an energy regenerating enzyme using a translation template mRNA for translating the energy regenerating enzyme; a second protein translation step of translating a target protein using a translation product of the first protein translation step and a translation template mRNA for translating the target protein; Includes A method for producing the protein described in claim 2.

4. The protein translation step comprises: The translation is carried out in the presence of a translation template mRNA for translating the energy regenerating enzyme and a translation template mRNA for translating the target protein. A method for producing the protein described in claim 2.

5. The energy regenerating enzyme is at least one selected from the group consisting of creatine kinase, nucleoside diphosphate kinase, arginine kinase, and adenylate kinase. A method for producing the protein according to any one of claims 1 to 4.

6. The enzyme further comprises a translation template mRNA for translating at least one enzyme selected from the group consisting of inorganic diphosphatase, inorganic pyrophosphatase, and disulfide isomerase. A method for producing the protein according to any one of claims 1 to 4.

7. a translation template mRNA for translating the energy regenerating enzyme, and a translation template mRNA for translating the target protein, using a transcription template DNA including a region encoding the translation template mRNA and a promoter region located at the 5' end of the region encoding the translation template mRNA, Translated using the transcription template DNA in the absence of cells and in the presence of elements for transcribing the transcription template DNA into mRNA. A method for producing the protein described in claim 2.

8. The energy regenerating enzyme and the target protein are from the same organism. A method for producing the protein described in claim 2.

9. The target protein is a growth factor. A method for producing the protein according to any one of claims 2 to 4, 7 and 8.

10. 1. A method for producing cultured meat, comprising: The method includes a step of culturing cells in a culture medium containing, as an additive, the translation product produced by the method for producing a protein according to claim 9. How cultured meat is produced.

11. An additive for use in a cultured meat production method, comprising: The protein includes a translation product produced by the method for producing the protein according to claim 9. additives.

12. A kit for use in a method for producing a protein, the kit comprising: a translation template mRNA for translating an energy regenerating enzyme; an element for translating the translation template mRNA for translating said energy regenerating enzyme into a protein in the absence of a cell; Includes kit.

13. Further comprising a translation template mRNA for translating the target protein. The kit of claim 12.

14. The energy regenerating enzyme and the target protein are from the same organism. The kit of claim 13.

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