Protein production method, cultured meat production method, additive for use in cultured meat production method, and kit for use in protein production method

Abstract

The present invention addresses the problem of providing a method for producing a protein in which an energy-regenerating enzyme is self-proliferated. A method for producing a protein includes a protein translation step for translating a protein using a translation template mRNA containing a translation template mRNA for translating an energy-regenerating enzyme, in the absence of a cell and in the presence of an element for translating the translation template mRNA into a protein.

Description

Technical Field

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

[0002] A synthesis system for synthesizing protein in a cell-free state is a system in which a medium containing intracellular elements related to protein synthesis is prepared and protein synthesis is performed in a cell-free state. Various such cell-free protein synthesis systems are known. As such synthesis systems, there are systems in which a template DNA as a transcription template is applied to a medium to synthesize a protein as a final product, and systems in which mRNA as a translation template is applied to a medium to synthesize a protein.

[0003] In the cell-free protein synthesis system, there is a known system using an extract prepared from Escherichia coli, insect cells, wheat germ and animal cells, and the material in the kit is commercially available from several companies. Protein is synthesized using energy sources such as ATP and GTP using mRNA as a translation template. However, when the energy source is consumed by protein synthesis, ATP changes to AMP or ADP, and GTP changes to GDP, so the energy source for protein synthesis is insufficient. Therefore, it is known to add an energy regeneration enzyme that regenerates AMP or ADP into ATP and GDP into GTP in the cell-free protein synthesis system. As an energy regeneration enzyme, for example, creatine kinase, myokinase or nucleoside diphosphate kinase (NDK) is disclosed in Patent Document 1.

[0004] If the amount of energy regeneration enzyme added to the cell-free protein synthesis system is too small, the regenerated energy source will also decrease, and as a result, the amount of protein synthesis will also decrease. Therefore, in the cell-free protein synthesis system, a predetermined amount of energy regeneration enzyme needs to be added, and Patent Document 2 records that creatine kinase is used at a concentration of 0.1 mg / mL to 0.5 mg / mL, for example.

[0005] Prior Art Literature

[0006] Patent Literature

[0007] Patent document 1: Japanese Patent No. 4061043;

[0008] Patent document 2: Japanese Patent Application Publication No. 2013-158342. Summary of the invention

[0009] Problem that the invention aims to solve

[0010] Among energy regeneration enzymes, for example, creatine kinase is known to be derived from rabbit muscle, and myokinase is known to be derived from yeast. However, since energy regeneration enzymes are derived from organisms, a process of isolating the enzyme is required, which results in a high cost. In addition, in order to prevent the inactivation of the energy regeneration enzyme, attention needs to be paid to the addition of stabilizers, storage temperature management, and the like.

[0011] The disclosure in the present application is completed to solve the above-mentioned conventional problems. The present inventors have conducted in-depth research and have newly discovered that: (1) using a translation template mRNA for translating an energy regeneration enzyme, (2) by utilizing a cell-free protein synthesis system to self-propagate the energy regeneration enzyme, an energy regeneration system can be constructed without adding the separated energy regeneration enzyme itself to the cell-free protein synthesis system.

[0012] That is, the purpose of the present application is to provide a method for producing a protein that self-proliferates an energy-regenerating enzyme, a method for producing cultured meat, an additive used in a method for producing cultured meat, and a kit for use in a method for producing a protein.

[0013] Means of solving the problem

[0014] The disclosure of the present application relates to a method for producing a protein, a method for producing cultured meat, an additive used in the method for producing cultured meat, and a kit used in the method for producing a protein as shown below.

[0015] (1) A method for producing a protein, comprising:

[0016] The protein translation step is to translate the protein using the translation template mRNA in the absence of cells and in the presence of elements for translating the translation template mRNA into protein.

[0017] The translation template mRNA comprises a translation template mRNA for translating an energy regeneration enzyme.

[0018] (2) The method for producing a protein according to (1) above, wherein the translation template mRNA further comprises a translation template mRNA for translating a target protein.

[0019] (3) The method for producing a protein according to (2) above, wherein:

[0020] The protein translation process comprises:

[0021] A first protein translation step of translating the energy regenerating enzyme using a translation template mRNA for translating the energy regenerating enzyme; and

[0022] The second protein translation step is to translate the target protein using the translation product of the first protein translation step and the translation template mRNA for translating the target protein.

[0023] (4) The method for producing a protein according to (2) above, wherein the protein translation step is performed in the presence of a translation template mRNA for translating the energy regenerating enzyme and a translation template mRNA for translating the target protein.

[0024] (5) The method for producing a protein according to any one of (1) to (4) above, wherein the energy regeneration enzyme is at least one selected from the group consisting of creatine kinase, nucleoside diphosphate kinase, arginine kinase and adenylate kinase.

[0025] (6) The method for producing a protein according to any one of (1) to (4) above, further comprising a translation template mRNA for translating at least one selected from the group consisting of inorganic diphosphatase, inorganic pyrophosphatase and disulfide isomerase.

[0026] (7) A method for producing a protein according to (2) above, wherein the translation template mRNA for translating the energy regeneration enzyme and the translation template mRNA for translating the target protein are obtained by translating the transcription template DNA in the absence of cells and in the presence of an element for transcribing the transcription template DNA into mRNA, wherein the transcription template DNA comprises a region encoding the translation template mRNA and a promoter region provided at the 5' end of the region encoding the translation template mRNA.

[0027] (8) The method for producing a protein according to (2) above, wherein the energy-regenerating enzyme and the target protein are from the same biological species.

[0028] (9) The method for producing a protein according to any one of (2) to (4), (7) and (8) above, wherein the target protein is a growth factor.

[0029] (10) A method for producing cultured meat, comprising the step of culturing cells using a culture medium, wherein the culture medium contains a translation product produced by the method for producing a protein according to (9) above as an additive.

[0030] (11) An additive for use in a method for producing cultured meat, the additive comprising a translation product produced by the method for producing a protein according to (9) above.

[0031] (12) A kit for use in a method for producing a protein, the kit comprising:

[0032] A translation template mRNA for translating an energy regenerating enzyme; and

[0033] Elements for translating the translation template mRNA for translating the energy regenerating enzyme into protein in the absence of cells.

[0034] (13) The kit according to (12) above, further comprising a translation template mRNA for translating the target protein.

[0035] (14) The kit according to (13) above, wherein the energy regenerating enzyme and the target protein are of the same biological species.

[0036] Effects of the Invention

[0037] The protein production method, cultured meat production method, additive for use in the cultured meat production method, and kit for use 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. Therefore, there is no need to add a separate energy regeneration enzyme to the cell-free protein synthesis system. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a diagram showing the temporal changes of GFP synthesized in <Reference Example 1>.

[0039] Figure 2 This is a diagram schematically showing the transcription template DNA of CK.

[0040] Figure 3 This is a diagram schematically showing the procedure for producing CK_DNA based on PCR.

[0041] Figure 4 This is a photograph replacing a drawing, and is a Western blot showing the result of self-growth of the energy regenerating enzyme (CK) using the cell-free protein synthesis system in Example 1.

[0042] Figure 5 This is a photograph replacing a drawing, and is a Western blot showing the results of self-proliferation of energy regeneration enzyme (CK) of various animals using the cell-free protein synthesis system in Example 2.

[0043] Figure 6 This is a diagram showing the results of synthesizing GFP using the translation product obtained in Example 1 in Example 3.

[0044] Figure 7 This is a diagram schematically showing the transcription template DNA of FGF2 of chicken, tuna, and eel.

[0045] Figure 8This is a diagram schematically showing the transcription template DNA of bovine FGF2.

[0046] Fig. 9 This is a diagram schematically showing the procedure for producing FGF2_DNA by PCR.

[0047] Fig.10 This is a Western blot showing the results of synthesizing the target protein (FGF2) in Example 4 using the energy regeneration enzyme derived from the same biological species.

[0048] Fig.11 This is a diagram showing that in Example 5, in a state where a translation template mRNA for translating an energy-regenerating enzyme and a translation template mRNA for translating a target protein coexist, an energy-regenerating system is self-constructed and the target protein can be synthesized. DETAILED DESCRIPTION

[0049] Hereinafter, the method for producing protein, the method for producing cultured meat, the additive used in the method for producing cultured meat, and the kit for producing protein disclosed in the present application are described in detail. In addition, the following description is for easy understanding, and the scope of the technical matters disclosed in the present application is not limited to the following description. In addition to the following examples, it is of course possible to make appropriate changes within the scope of the subject matter disclosed in the present application without damaging it.

[0050] (First Embodiment of the Method for Producing Protein)

[0051] The method for producing a protein according to the first embodiment comprises a protein translation step, in which a translation template mRNA is used to translate a protein in the absence of cells and in the presence of elements for translating a translation template mRNA into a protein. Furthermore, the translation template mRNA includes a translation template mRNA for translating an energy regeneration enzyme as a protein.

[0052] The cell-free protein synthesis system is a method of synthesizing protein in a test tube by adding translation template mRNA or transcription template DNA to a cell-free extract containing translation components such as ribosomes, tRNA, aminoacyl tRNA synthetase, translation initiation factor, translation elongation factor, and translation termination factor in a solution containing energy molecules such as amino acids, ATP, GTP, energy regeneration system, and salts such as magnesium ions. In the production method of protein disclosed in the present application, instead of adding an isolated (separately manufactured as an additive) energy regeneration enzyme in the cell-free protein synthesis system, a translation template mRNA for translating the energy regeneration enzyme is added. Moreover, it is characterized in that the energy regeneration system is self-constructed by using the energy regeneration enzyme translated from the translation template mRNA using the cell-free protein synthesis system. Therefore, in the case of being recorded as "an element for translating the translation template mRNA into protein in the absence of cells" in this specification, it refers to a solution (translation liquid) containing cell-free extracts, amino acids, energy molecules, and salts. In addition, "an element for translating the translation template mRNA into protein in the absence of cells" can also be referred to as an element of "an element in which the isolated energy regeneration enzyme is not added at the beginning of protein synthesis". However, energy regeneration enzymes are usually isolated from animals. Therefore, "no isolated energy regeneration enzyme is added at the beginning of protein synthesis" can also be said to be "no isolated energy regeneration enzyme derived from animals is added at the beginning of protein synthesis". In addition, as described above, the protein production method disclosed in the present application is characterized in that the energy regeneration system is self-constructed by using the energy regeneration enzyme translated from the translation template mRNA using a cell-free protein synthesis system, but from a technical point of view, the addition of the isolated energy regeneration enzyme derived from animals does not hinder the self-construction of the energy regeneration system. Therefore, in order to smoothly carry out the circulation of the initial stage of the self-constructed energy regeneration system, it is not hindered to add a very small amount of isolated energy regeneration enzyme. In addition, in the case of adding the isolated energy regeneration enzyme, in order not to treat the added energy regeneration enzyme as an impurity, it is preferred to add the same enzyme as the energy regeneration enzyme translated from the translation template mRNA.

[0053] As described above, the cell-free protein synthesis system disclosed in the present application is characterized in that no separated energy regeneration enzyme is added at the beginning of synthesis. However, creatine kinase, which is an example of an energy regeneration enzyme, is mainly present in the muscles of animals. Therefore, when a cell-free extract derived from animal cells is used, the cell-free extract may contain creatine kinase. Therefore, the cell-free extract disclosed in the present application is preferably a non-animal cell-free extract. There is no limitation on the non-animal cell-free extract, and for example, a cell-free extract extracted from wheat germ or the like by a known method can be cited.

[0054] As the amino acid, various natural amino acids may be used, or non-natural amino acids may be used.

[0055] Energy molecules can be any known molecules used for protein synthesis. Examples of energy molecules include nucleotide triphosphates, creatinine phosphate, and formylfolate. Here, nucleotide triphosphates include ATP, GTP, CTP, and UTP. When protein is produced from translation template mRNA, ATP and GTP can be used as energy molecules. When protein is produced from transcription template DNA, ATP, GTP, CTP, and UTP can be used.

[0056] Examples of the energy regeneration enzyme include enzymes known in the field of protein synthesis, and examples thereof include, but are not limited to, creatine kinase, nucleoside diphosphate kinase, arginine kinase, pyruvate kinase, and adenylate kinase.

[0057] Creatine kinase catalyzes the reaction of creatine phosphate and AMP, regenerating ADP into ATP. Nucleoside diphosphate kinase uses polyphosphate as a substrate to regenerate AMP and ADP into ATP, and GDP into GTP. Arginine kinase phosphorylates arginine (Arg) to generate arginine phosphate (PArg), and transfers the phosphate of the generated PArg to ADP, thereby regenerating ATP. Pyruvate kinase transfers the phosphate group from phosphoenolproestilate to ADP, regenerating ATP. Adenylate kinase is an enzyme that generates one molecule of AMP and one molecule of ATP from two molecules of ADP.

[0058] The translation template mRNA for translating the energy regeneration enzyme is not particularly limited as long as it can translate the above-mentioned energy regeneration enzyme. The translation template mRNA includes a coding region encoding the amino acid sequence of the energy regeneration enzyme, and the non-translated region is appropriately connected to the 5' and 3' sides of the coding region. As an example of a 5' non-translated region and a 3' non-translated region for improving the translation efficiency of a protein, it is recorded in International Publication No. 2022 / 185664 and International Publication No. 2021 / 070616 applied by the applicant. The translation template mRNA disclosed in the present application can use the non-translated region recorded in International Publication No. 2022 / 185664 and International Publication No. 2021 / 070616 as needed. Matters recorded in International Publication No. 2022 / 185664 and International Publication No. 2021 / 070616 are included in this specification by reference.

[0059] As the salt, any known salt used in a cell-free protein synthesis system may be used, and there are no limitations thereon, and examples thereof include magnesium acetate, potassium acetate, and calcium chloride.

[0060] The method for producing a protein according to the first embodiment exerts the following effects.

[0061] (1) At the start of protein synthesis, the energy regeneration enzyme is not added, and only the energy regeneration enzyme translated from the translation template mRNA for translating the energy regeneration enzyme is used to construct the energy regeneration system. Therefore, since the separated energy regeneration enzyme is not required, the cost can be reduced.

[0062] (2) Proteins produced by the cell-free protein synthesis system can be used as medicines and foods. However, when proteins derived from biological species that have not been ingested are ingested as medicines and foods, allergic reactions may occur. For example, creatine kinase is derived from rabbits, etc., and the selection of biological species from which commercially available energy regeneration enzymes are derived is limited. On the other hand, in the present application, since the energy regeneration enzyme is obtained by translating from the translation template mRNA, the desired energy regeneration enzyme derived from the biological species can be translated. Therefore, the possibility of allergies, etc. can be reduced.

[0063] (3) When the produced protein is used in food or medicine, it is sometimes impossible to use raw materials from specific biological species in order to avoid contamination by religious reasons or zoonotic viruses. However, in the protein production method according to the present application, since the desired energy-regenerating enzyme derived from the biological species is obtained through translation, the problems caused by religious reasons or zoonotic virus contamination can be solved.

[0064] (4) Due to the influence of prion diseases, etc., the use of raw materials derived from animals to manufacture food and medicines tends to be avoided. In the protein production method of the present application, even if the type of energy regeneration enzyme is animal, protein can be produced only from non-animal raw materials. Therefore, there is no risk of diseases derived from animal raw materials.

[0065] (Second Embodiment of the Method for Producing Protein)

[0066] Next, the second embodiment of the method for producing protein is described. The method for producing protein in the second embodiment is different from the method for producing protein in the first embodiment in that it comprises a translation template mRNA for translating the target protein, and is the same as the method for producing protein in the first embodiment in other aspects. In order to avoid repeated description, in the second embodiment of the method for producing protein, the point different from the first embodiment is described as the center. Therefore, in the second embodiment, even if it is not clearly described, the matters described in the first embodiment can certainly be applied to the second embodiment.

[0067] The target protein is not particularly limited as long as it can be synthesized by a cell-free protein synthesis system. For example, growth factors; secretory proteins such as insulin and amylase; plasma proteins such as transferrin, myoglobin, and albumin; and the like. In addition, examples of animal species of the target protein include fish such as salmon and eels; mammals such as humans, cattle, and rabbits; and echinoderms such as crabs.

[0068] The protein translation step of the method for producing a protein according to the second embodiment is not particularly limited as long as the target protein can be produced using the energy regenerating enzyme translated from the translation template mRNA for translating the energy regenerating enzyme.

[0069] As an example of a protein translation process, the following example can be cited: first, a first protein translation process is performed to translate the energy regenerating enzyme using a translation template mRNA for translating the energy regenerating enzyme, and then a second protein translation process is performed to translate the target protein using the translation product of the first protein translation process and a translation template mRNA for translating the target protein (hereinafter, sometimes recorded as the "first example").

[0070] Another example of the protein translation step is an example in which the translation is performed in the presence of a translation template mRNA for translating the energy regenerating enzyme and a translation template mRNA for translating the target protein (hereinafter, sometimes referred to as "second example").

[0071] In the case of the first example, since the energy regeneration enzyme is first translated from the translation template mRNA in the first protein translation step, the amount of the energy regeneration 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 implemented can also be adjusted. Therefore, in the case of the first example, the amount of the energy regeneration enzyme contained in the cell-free protein synthesis system when the target protein is translated can be adjusted. By adjusting the amount of the energy regeneration enzyme, the effect of optimizing the translation efficiency of the target protein is achieved. On the other hand, in the case of the second example, since the translation of the energy regeneration enzyme and the target protein is implemented simultaneously, the translation efficiency of the target protein is lowered compared to the first example, but since there is no need to separate the protein translation step, the convenience of protein production is improved.

[0072] The translation template mRNA for translating the energy regeneration enzyme and the target protein can use the mRNA synthesized by the nucleic acid synthesis device, or can use the mRNA transcribed by the transcription template DNA. The transcription template DNA can be synthesized by translating the transcription template DNA in the absence of cells and in the presence of the elements for transcribed transcription template DNA into mRNA. The promoter sequence, the elements for transcribed transcription template DNA into mRNA, can use the elements known in the art. Although it is illustrative and not limiting, the promoter sequence can include T7 promoter sequence, SP6 promoter sequence, T3 promoter sequence, etc.

[0073] The method for producing a protein according to the second embodiment produces the following effects in addition to the effects described in (1) to (4) produced by the method for producing a protein according to the first embodiment.

[0074] (5) Among the effects described in (2) to (4) exerted by the method for producing a protein according to the first embodiment, an effect of replacing the "energy regenerating enzyme" with the "target protein" is exerted.

[0075] [Optional Additional Matters That Can Be Adopted in Embodiments of the Method for Producing Protein]

[0076] Next, optional additional matters that can be adopted in the embodiment of the method for producing a protein are described.

[0077] (Addition of translation template mRNA for translation by other enzymes)

[0078] In the protein translation step, in addition to the energy regeneration enzyme, an enzyme for decomposing byproducts (e.g., inorganic pyrophosphate) generated by transcription / translation and / or a translation template mRNA for translating an enzyme used in the folding of the target protein (activation of the target protein) may be added. The enzyme for decomposing byproducts is not limited, and examples thereof include inorganic diphosphatase, inorganic pyrophosphatase, etc. In addition, the enzyme used in the folding of the target protein is not limited, and examples thereof include disulfide isomerase, etc.

[0079] In the case where the protein translation step is the first example, the enzyme for decomposing the byproduct and / or the translation template mRNA for translating the enzyme used in the folding of the target protein can be added during the first protein translation step, can be added during the second protein translation step, and can be added during the first protein translation step and the second protein translation step. In the case where the protein translation step is the second example, together with the translation template mRNA for translating the energy regeneration enzyme and the translation template mRNA for translating the target protein, the enzyme for decomposing the byproduct and / or the translation template mRNA for translating the enzyme used in the folding of the target protein can be added. In the case where the translation template mRNA for translating other enzymes is made by transcribing from the transcription template DNA, it is sufficient to design the transcription template DNA containing the region encoding the translation template mRNA and the promoter region at the 5' end of the region encoding the translation template mRNA. In the protein translation step, the translation efficiency of the target protein is improved by the coexistence of the enzyme for decomposing the byproduct and / or the enzyme for folding the target protein. In addition, the enzyme for decomposing the byproduct and / or the enzyme for folding the target protein is only a preferred example, as long as it is an enzyme that improves the translation efficiency of the target protein, it can also be other enzymes.

[0080] 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 method for producing a protein.

[0081] (6) Byproducts that inhibit the efficient synthesis of cell-free protein synthesis systems can be removed, thereby improving protein production efficiency. In addition, the produced proteins can be activated.

[0082] (Energy regeneration enzyme and target protein species)

[0083] The combination of energy regeneration enzyme and target protein can be derived from the same biological species. In this case, the nucleotide sequence of energy regeneration enzyme and target protein of the same biological species is obtained from a known database (DB), and the translation template mRNA is designed. In addition, in the case of making by transcribing the translation template mRNA from the transcription template DNA, it is only necessary to design the transcription template DNA containing the region encoding the translation template mRNA and the promoter region at the 5' end of the region encoding the translation template mRNA.

[0084] When the energy-regenerating enzyme and the target protein are derived from the same biological species, the following effects are achieved in addition to the effects achieved by the first and second embodiments of the method for producing a protein.

[0085] (7) When the produced protein is used in food, it is sometimes necessary to avoid mixing proteins derived from different species. By combining the energy-regenerating enzyme and the target protein as those derived from the same species, the possibility of avoiding this is reduced.

[0086] (About target protein)

[0087] The target protein may be a growth factor or a cytokine. In recent years, cultured meat has received attention from the perspectives of responding to population growth / increased food production, environmental issues associated with the increase in pastureland as feed for carnivorous animals, the safety of giving antibiotics to animals, and the ethics of taking animal lives. Cultured meat uses a culture medium to which substances necessary for cell proliferation, such as amino acids and carbohydrates, are added to culture stem cells taken from animals, but growth factors are required for the culture. In addition, cytokines are required for cell differentiation. There is no limitation to growth factors or cytokines, and examples thereof include the following growth factors.

[0088] EGF (Epidermal growth factor): epidermal growth factor;

[0089] IGF (Insulin-like growth factor): insulin-like growth factor;

[0090] ·TGF (Transforming growth factor): transforming growth factor;

[0091] bFGF or FGF2 (basic fibroblast growth factor): basic fibroblast growth factor;

[0092] NGF (Nerve growth factor): nerve growth factor;

[0093] BDNF (Brain-derived neurotrophic factor): Brain-derived neurotrophic factor;

[0094] VEGF (Vesicular endothelial growth factor): vascular endothelial growth factor;

[0095] G-CSF (Granulocyte-colony stimulating factor): granulocyte-stimulating factor;

[0096] GM-CSF (Granulocyte-macrophage-colony stimulating factor): Granulocyte-macrophage colony stimulating factor;

[0097] PDGF (Platelet-derived growth factor): platelet-derived growth factor;

[0098] EPO (Erythropoietin): erythropoietin;

[0099] TPO (Thrombopoietin): thrombopoietin;

[0100] HGF (Hepatocyte growth factor): Hepatocyte growth factor;

[0101] ·LIF (leukemia inhibotory factor): leukemia inhibitory factor.

[0102] When the target protein is a growth factor, in addition to the effects exerted by the first and second embodiments of the method for producing a protein, the following effects are exerted.

[0103] (8) Growth factors are very expensive. However, by using the method for producing the protein disclosed in the present application, growth factors can be produced at low cost.

[0104] In the above, several specific embodiments and any additional matters that can be adopted are listed and the production method of the protein disclosed in the present application is described, but as long as it is within the scope of the technical idea disclosed in the present application, it is not limited to these embodiments, and various changes can be made. In addition, any additional matters that can be adopted as examples can be added alone, or any two or more additional matters can be combined. When any two or more additional matters are used in combination, their respective effects are exerted synergistically. In addition, as needed, the energy regeneration enzyme and / or the sequence for adding a protein tag to the N-terminal side and / or C-terminal side of the target protein can also be connected to the translation template mRNA or the transcription template DNA.

[0105] (Embodiments of the method for producing cultured meat and embodiments of the additive)

[0106] In the method for producing 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 can be implemented by a step of performing cell culture using a culture medium containing the translation product as an additive. The culture medium can use a known culture medium. The cells can use cells of an animal that is the object of culture as described above. There is no particular limitation on the animal that is the object of culture as long as it is an animal that is ingested as edible meat. Although there is no limitation, examples include mammals such as cattle, pigs, horses, and sheep; birds such as chickens, ducks, and quails; fish such as salmon, rabbits, and tuna; and crustaceans such as shrimps and crabs.

[0107] In addition, in the case of producing a growth factor as a target protein in a protein production method, the translation product can be used as an additive in a production method for cultured meat.

[0108] The embodiments of the method for producing cultured meat and the embodiments of the additives have the following effects.

[0109] (9) Since growth factors can be obtained cheaply, the cost of cultured meat can be reduced.

[0110] (10) When using energy-regenerating enzymes and growth factors from the same species as the animal to be cultured, cultured meat can be produced that does not contain other types of animal-derived proteins.

[0111] (Embodiments of Kit for Use in Methods of Producing Proteins)

[0112] Next, the embodiment of the kit for the production method of protein is described. As mentioned above, in the existing cell-free protein synthesis system, the energy regeneration enzyme uses an enzyme isolated from animals, etc. On the other hand, in the cell-free protein synthesis system, it is newly discovered by the inventor to use the energy regeneration enzyme obtained from the translation template mRNA for translating the energy regeneration enzyme to construct the energy regeneration system. Therefore, the kit for the production method of protein comprising the translation template mRNA for translating the energy regeneration enzyme and the elements for translating the translation template mRNA into protein in the absence of cells is a new invention. In addition, the kit for the production method of protein involved in the embodiment only uses the energy regeneration enzyme translated from the translation template mRNA for translating the energy regeneration enzyme to construct the energy regeneration system, so it can also be called a kit without adding the energy regeneration enzyme. In addition, the kit can further include the translation template mRNA for translating the target protein, and the energy regeneration enzyme and the target protein can be the same biological species.

[0113] "Translation template mRNA for translating energy regeneration enzyme", "element for translating translation template mRNA into protein in the absence of cells", "translation template mRNA for translating target protein" have been described in the embodiment of the protein production method. Therefore, since it is repeated, the detailed description is omitted.

[0114] "Translation template mRNA for translating energy regeneration enzyme", "elements for translating translation template mRNA into protein in the absence of cells" and "translation template mRNA for translating target protein" can be provided separately and mixed when used. Alternatively, "translation template mRNA for translating energy regeneration enzyme" and "elements for translating translation template mRNA into protein in the absence of cells" can be provided in a mixed manner, and "translation template mRNA for translating target protein" can be provided separately. Further alternatively, "translation template mRNA for translating energy regeneration enzyme", "elements for translating translation template mRNA into protein in the absence of cells" and "translation template mRNA for translating target protein" can all be provided in a mixed manner.

[0115] The following examples are given to specifically illustrate the embodiments disclosed in the present application, but these examples are only used to illustrate the embodiments and are not intended to limit or restrict the scope of the invention disclosed in the present application.

[0116] Example

[0117] <Reference Example 1>

[0118] [The importance of creatine kinase in protein production using cell-free protein synthesis systems]

[0119] First, in the case of cell-free protein synthesis of GFP, which is a fluorescent protein, the difference in protein synthesis between the case where creatine kinase purified from rabbit was used and the case where it was not used was confirmed. The experimental procedure is described below.

[0120] (1) Raw materials

[0121] WGE: wheat germ extract. It was prepared according to the procedure described in Japanese Patent Application Laid-Open No. 2006-288320.

[0122] CK: Creatine kinase manufactured by Roche Diagnostics Deutschland GmbH (Product No.: 10127566001).

[0123] · AAM: amino acid substrate. The amino acid substrate attached to PSS5100 manufactured by NUProtein was used.

[0124] mRNA: designed to contain a coding region encoding the amino acid sequence of GFP of jellyfish. In addition, mRNA was obtained by transcribing a transcription template DNA having a sequence encoding GFP (sequence information P42212, purchased from Eurofins Genomics) inserted into the portion of CK_organism in Table 3 of <Example 1> described later in the same order as Example 1.

[0125] (2) Translation reaction

[0126] A translation reaction solution of the composition described in the following Table 1 was prepared in a 1.5 mL tube. In addition, the unit of the composition solution described in the following Table 1 is μL. The stock solution concentration of the added CK was 20 mg / mL, and the final concentration was 200 ng / μL. Regarding X in Table 1, CK+ is 77.8 and CK- is 80.

[0127] [Table 1]

[0128]

[0129] The reaction solution having the composition described in Table 1 was added to a 96-well flat-bottom titer plate and reacted in a plate reader at room temperature (about 23° C.).

[0130] [Fluorescence measurement of synthetic GFP]

[0131] 200 μL of the 220 μL solution containing GFP synthesized in Reference Example 1 was used as a sample, and irradiated with excitation light of wavelength 475 nm for 0.5 seconds. The fluorescence intensity from GFP was measured every hour for 15 hours using an ELISA reader (absorption filter 500-550 nm). The ELISA reader used was a GloMax (registered trademark) ELISA reader (Promega). The measurement results are shown in Figure 1 .

[0132] Depend on Figure 1 It can be seen that by adding CK (CK+) as an energy regeneration enzyme to the cell-free protein synthesis system, the amount of GFP synthesis increases over time. On the other hand, when CK (CK-) is not added, GFP is almost not synthesized. From the above results, it is confirmed that when the cell-free protein synthesis system does not have an energy regeneration system, the target protein cannot be efficiently synthesized.

[0133] [Confirmation of self-growth of energy-regenerating enzyme using a cell-free protein synthesis system]

[0134] <Example 1>

[0135] (1) Creatine kinase sequence

[0136] Among energy regeneration enzymes derived from various organisms, the sequence information (ACC. No.), biological species, and acquisition sources of creatine kinase (CK) are shown in Table 2. In Example 1, CK_T (Thunnus, tuna) with No. 4 was used. Nos. 1 to 3 and 5 were used in Examples 2 to 4 described below.

[0137] [Table 2]

[0138] serial number mark Protein name Sequence information organism company 1 CK_R Creatine kinase NM_001082239.1 rabbit GenScript 2 CK_B Creatine kinase NP_777198.2 ox Eurofins Genomics 3 CK_C Creatine kinase NP_990838.1 chicken Eurofins Genomics 4 CK_T Creatine kinase XP_042286032.1 tuna Eurofins Genomics 5 CK_U Creatine kinase XP_035288992.1 eel Eurofins Genomics

[0139] (2) Transcription template DNA

[0140] Figure 2 The outline of the transcription template DNA of CK (hereinafter, sometimes described as "CK_DNA") is shown in Table 3. In addition, the sequence of each region of CK_DNA is shown in Table 3. In addition, the CK sequence of the organisms No. 1 to 5 above is inserted into the CK_organism in the following Table 3.

[0141] [Table 3]

[0142]

[0143] Reference Figure 3 , the preparation sequence of CK_DNA based on PCR is described. Figure 3 Primers were designed as shown, and CK_DNA was prepared by two-stage PCR. Table 4 shows PCR primers, Tables 5 and 6 show the composition of the reaction solution for PCR, and Table 7 shows the PCR program. In addition, the synthesis method of the transcription template DNA was implemented in accordance with the instruction manual of PSS5100 of New Protein Co., Ltd. (May 2021 / Version 3.00). In addition, the reagents and machines used are as follows.

[0144] PCR enzyme: Toyobo Co., Ltd., KOD-Plus-Neo

[0145] Primers, artificial genes: Eurofins Genomics Co., Ltd., contract synthesis service

[0146] Thermal Cycler: Eppendorf, Mastercycler X50s

[0147] Micro high-speed cooling centrifuge: Tomys Industries, Ltd., MX-307

[0148] [Table 4]

[0149]

[0150] [Table 5]

[0151] Composition of the first PCR reaction solution

[0152] Reagents Volume 10xPCR Buffer 5μL 2mM dNTPs 5μL <![CDATA[25mM MgSO4]]> 3μL 10μM 1st_CK_NF 1μL 10μM 1st_CK_NR_01 1μL 1ng / μL artificial gene 1μL PCR DNA polymerase 1μL Ultrapure water 33μL total 50μL

[0153] [Table 6]

[0154] Second PCR reaction mixture composition

[0155] Reagents Volume 10xPCR Buffer 5μL 2mM dNTPs 5μL <![CDATA[25mM MgSO4]]> 3μL 10μM 2nd_NF2 1μL 100nM 2nd_HA_NF1 1μL 10μM 2nd_NCR_U01 1μL First PCR product 1μL PCR DNA polymerase 1μL Ultrapure water 32μL total 50μL

[0156] [Table 7]

[0157] First / Second PCR Procedure

[0158]

[0159] (3) Transcriptional response

[0160] Next, the prepared transcription template DNA was used to prepare the translation template mRNA. The transcription reaction was prepared using the following reaction solution of PSS5100 manufactured by Neoprotein Co., Ltd., and 2.5 μL of the previously prepared second PCR reaction solution (containing the transcription template DNA) was used in a 1.5 mL tube and incubated at 37°C for 3 hours.

[0161] [Table 8]

[0162] Reagents Volume 10x Transcription Buffer 2.5μL 25 mM NTP mix 2.5μL T7 RNA polymerase 1μL 100mM DTT 1.25μL Second PCR product 2.5μL Ultrapure water 15.25μL total 25μL

[0163] Relative to 25 μL of transcription reaction solution, add 10 μL of 4M ammonium acetate and mix thoroughly, then add 100 μL of 100% ethanol and mix, after flash evaporation using a desktop centrifuge, let stand at -20°C for 10 minutes. Then, centrifuge (12,000g, 15 minutes, 4°C). After removing the supernatant, flash evaporation is performed using a desktop centrifuge. Remove the supernatant again and let stand until the precipitate is dry. Then, add 80 μL of ultrapure water to 25 μL of transcription reaction solution to dissolve the precipitate. Use it as a translation template mRNA solution.

[0164] (4) Translation reaction

[0165] Next, a translation reaction solution of the following composition was prepared in a 1.5 mL tube and placed in an incubator set to 23° C. for reaction for 15 hours. The composition recorded in Table 9 is the addition of CK to the translation template mRNA of CK_T. The composition recorded in Table 10 is only the translation template mRNA of CK_T, and CK is not added to the translation reaction solution. In addition, for comparison, instead of the translation template mRNA of CK_T in Table 10, the translation template mRNA of GFP prepared in <Reference Example 1> was given to implement the translation reaction. In addition, the units of the numerical values ​​in Tables 9 and 10 are μL, X in Table 9 is 77.8, and X in Table 10 is 80. The stock solution concentration of the added CK is 20 mg / mL, and the final concentration is 200 ng / μL.

[0166] [Table 9]

[0167]

[0168] [Table 10]

[0169]

[0170] After the reaction, the 1.5 mL tube was centrifuged (15,000 g, 15 minutes, 4° C.), and the supernatant was used as a protein solution after translation. The obtained protein was subjected to Western blotting.

[0171] <Reagents used>

[0172] Gel: 4-15% Tris-Glycine gel (Biolad Laboratories)

[0173] Membrane: 0.2 μm PVDF membrane (manufactured by Biolad Laboratories)

[0174] Primary antibody: HA antibody (Proteintech)

[0175] Secondary antibody: goat anti-mouse HRP antibody (manufactured by Southern Biotech)

[0176] Western blotting luminescent reagent: SuperSignal West Pico (manufactured by Thermo)

[0177] Figure 4 Indicates the result. Figure 4 L2 is the result of the translation product obtained by the composition described in Table 9, L3 is the result of the translation product obtained by the composition described in Table 10, and L1 is the result of the translation product obtained by the composition of the translation template mRNA using GFP. Figure 4 It can be seen that in L3, to which the separated CK was not added to the translation reaction solution, a predetermined amount of CK_T was translated from a smaller amount of translation template mRNA than in L2. Based on the above results, it is believed that if the translation reaction is carried out with the composition described 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 constructs an energy regeneration system, thereby self-propagating CK_T.

[0178] <Example 2>

[0179] The experiment was conducted in the same order 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 the primers of the following sequences were used as 1st_CK_NF and 1st_CK_NR_01. In addition, the primers of CK_R (Rabbit) used in Example 4 described later are also described. In Table 11, the species of the primers are identified by describing the species after CK.

[0180] [Table 11]

[0181]

[0182]

[0183] Figure 5 In addition, for comparison, CK_T (Thunnus, tuna) was also added to the lane and protein blotting was performed. Figure 5 It can be seen that for cows as mammals, chickens as birds, and tuna and eels as fish, CK of each species is translated from the translation template mRNA by the elements contained in the translation reaction solution, and the translated CK constructs an energy regeneration system, thereby confirming that CK self-proliferates. Based on the above results, it can be said that the self-construction of the energy regeneration system is not limited to biological species.

[0184] [Synthesis of proteins other than energy regenerating enzymes]

[0185] Next, protein synthesis is carried out using a system that allows energy-regenerating enzymes to self-multiply.

[0186] <Example 3>

[0187] Using the translation product CK_T obtained in the above <Example 1> ( Figure 4L3), synthesis of GFP as the target protein was carried out. Table 12 shows the composition of the translation reaction solution. CK_T as the translation product is shown in numbers 1 to 4, and the added amount is changed for the experiment. The target protein is GFP, and the translation template mRNA described in Reference Example 1 is used. The negative control (NC) uses CK, CK_T and mRNA without GFP, and the positive control (PC) uses CK instead of the translation product CK_T. The translation reaction is carried out for about 15 hours with the composition of Table 11, and the experiment is carried out in the same order as the above-mentioned <Reference Example 1> to measure the fluorescence intensity of the synthesized GFP. In addition, the unit of the numerical value of Table 11 is μL. The stock solution concentration of the added CK is 20mg / mL, and the final concentration is 200ng / μL.

[0188] [Table 12]

[0189]

[0190]

[0191] The results are shown in Figure 6 In the translation product No. 1 to which CK_T was not added, almost no GFP was produced, but GFP production was confirmed in Nos. 2 to 4. The above results confirmed that the method for producing a protein disclosed in the present application can produce a target protein using the translation product CK_T translated from the translation template mRNA.

[0192] <Example 4>

[0193] Using the CK of cow, chicken, tuna, and eel as the translation product obtained in <Example 2>, the target protein FGF2 corresponding to each biological species listed in Table 13 below was synthesized. The specific steps are described below.

[0194] [Table 13]

[0195] mark Protein name Sequence information organism company FGF2_B FGF2 P03969 ox Eurofins Genomics FGF2_C FGF2 NP_990764.1 chicken Eurofins Genomics FGF2_T FGF2 XP_042274368.1 tuna Eurofins Genomics FGF2_U FGF2 XP_035281190.1 eel Eurofins Genomics

[0196] (1) Transcription template DNA of FGF2 (chicken, tuna, eel)

[0197] Figure 7 The outline of the transcription template DNA (hereinafter sometimes described as "FGF2_DNA") of FGF2 of chicken, tuna and eel is shown. In addition, the sequence of each region of FGF2_DNA is shown in Table 14. In addition, the FGF2 sequence of chicken, tuna and eel in the above Table 13 was inserted into the FGF2_organism in the following Table 13.

[0198] [Table 14]

[0199]

[0200]

[0201] (2) Transcription template DNA of FGF2 (cow)

[0202] Figure 8 The outline of the transcription template DNA of bovine FGF2 is shown. In addition, the sequence of each region of the transcription template DNA of bovine FGF2 is shown in Table 14. In addition, the bovine FGF2 sequence of the above Table 13 is inserted into the FGF2_cow in the following Table 15.

[0203] [Table 15]

[0204]

[0205] Reference Fig. 9 , the preparation procedure of FGF2_DNA based on PCR is described. Fig. 9 The structures of the transcription template DNA shown are different, but the sequences are the same. Fig. 9 As shown in Table 16, primers were designed and FGF2_DNA was prepared by two-stage PCR. Table 16 shows the first PCR primers for various species, Table 17 shows the second PCR primers for chicken, tuna, and eel, and Table 18 shows the second PCR primers for cattle. In addition, "2nd_NCF_U01" and "2nd_FLAG_CR1_E15" in Table 18 correspond to Fig. 9 "2nd_CF1" and "2nd_FLAG_CR1_01" are used for the same biological species. "2nd_NCR_U01" is used for all biological species.

[0206] The composition of the PCR reaction solution is the same as that in Table 5 and Table 6 of Example 1, and the PCR program is also the same as that in Table 7 of Example 1. In addition, the synthesis method, reagents and machinery of the transcription template DNA are also the same as those in Example 1.

[0207] [Table 16]

[0208]

[0209]

[0210] [Table 17]

[0211] <Chicken, tuna, eel>

[0212]

[0213] [Table 18]

[0214] <Cow>

[0215]

[0216] (3) Transcriptional response

[0217] A transcription reaction was carried out in the same manner as in Example 1 using the FGF2 transcription template DNA (chicken, tuna, eel, and cattle) described in (1) and (2) above.

[0218] (4) Synthesis of FGF2

[0219] Using various CKs obtained as translation products in the above <Example 2> and the translation template mRNA obtained in the above (3), FGF2 was synthesized. Table 19 shows the composition of the translation reaction solution. The experiment was carried out in the same steps as in the above <Example 1> with the composition of Table 19. In addition, regarding the primary antibody, the following primary antibody was used instead of the primary antibody of Example 1.

[0220] Primary antibody: anti-DYKDDDDK tag (monoclonal antibody, product number 018-22381; Fujifilm Wako Pure Chemical Industries, Ltd.)

[0221] [Table 19]

[0222]

[0223] The results are shown in Fig.10 .Depend on Fig.10 The results showed that the target protein could be synthesized using the energy-regenerating enzyme derived from the same biological species.

[0224] [Co-expression of energy regeneration enzyme and target protein]

[0225] <Example 5>

[0226] Next, a translation reaction is performed in the presence of 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.

[0227] (1) Energy regeneration enzyme

[0228] Using CK_R listed in No. 1 in Table 2 of Example 1, the translation template mRNA was prepared according to the same procedure as in Example 1.

[0229] (2) Target protein

[0230] The GFP translation template mRNA described in Reference Example 1 was used.

[0231] (3) Co-expression of energy regeneration enzyme and target protein

[0232] Table 20 shows the composition of the translation reaction solution. Negative controls (NC) used CK, CK_rabbit and mRNA without GFP, and positive controls (PC) used CK instead of CK_rabbit translation template mRNA. The translation reaction was carried out for about 15 hours with the composition of Table 20, and the experiment was carried out in the same order as in the above-mentioned <Reference Example 1> to measure the fluorescence intensity of the synthesized GFP.

[0233] [Table 20]

[0234] PC No. 1 No. 2 No. 3 NC Remark WGE(-CK) 20 20 20 20 20 AAM 40 40 40 40 40 CK 2.2 - - - - 20mg / mL CK_rabbit - 20 10 10 - mRNA GFP 40 20 30 40 - mRNA MilliQ X X X X X total 220 220 220 220 220

[0235] Fig.11 Indicates the result. Fig.11 It was found that even in the state where the translation template mRNA for translating the energy-regenerating enzyme and the translation template mRNA for translating the target protein coexist, the energy-regenerating system is self-constructed and the target protein can be synthesized.

[0236] Industrial Applicability

[0237] The protein production method, cultured meat production method, additive for use in the cultured meat production method, and kit for use in the protein production method disclosed in the present application can be used to construct an energy regeneration system for cell-free protein synthesis using an energy regeneration enzyme translated from a translation template mRNA. Therefore, it is useful in industries requiring cell-free protein synthesis, such as the food industry, the pharmaceutical industry, and research institutions.

Claims

1. A method for producing a protein, comprising: The protein translation step is to translate the protein using the translation template mRNA in the absence of cells and in the presence of elements for translating the translation template mRNA into protein. The translation template mRNA comprises a translation template mRNA for translating an energy regeneration enzyme, At the start of protein synthesis, the energy regeneration enzyme is not added, and only the energy regeneration enzyme translated from the translation template mRNA for translating the energy regeneration enzyme is used to construct the energy regeneration system.

2. The method for producing a protein according to claim 1, wherein: The translation template mRNA also includes a translation template mRNA for translating a target protein.

3. The method for producing a protein according to claim 2, wherein: The protein translation process comprises: A first protein translation step of translating the energy regenerating enzyme using a translation template mRNA for translating the energy regenerating enzyme; and The second protein translation step is to translate the target protein using the translation product of the first protein translation step and the translation template mRNA for translating the target protein.

4. The method for producing a protein according to claim 2, wherein: The protein translation step is performed in the presence of a translation template mRNA for translating the energy regenerating enzyme and a translation template mRNA for translating the target protein.

5. The method for producing a protein according to any one of claims 1 to 4, wherein The energy regeneration enzyme is at least one selected from the group consisting of creatine kinase, nucleoside diphosphate kinase, arginine kinase and adenylate kinase. 6 . The method for producing a protein according to claim 1 , further comprising a translation template mRNA for translating at least one selected from the group consisting of inorganic diphosphatase, inorganic pyrophosphatase and disulfide isomerase.

7. The method for producing a protein according to claim 2, wherein: The translation template mRNA for translating the energy regeneration enzyme and the translation template mRNA for translating the target protein are obtained 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, wherein the transcription template DNA includes a region encoding the translation template mRNA and a promoter region set at the 5' end of the region encoding the translation template mRNA.

8. The method for producing a protein according to claim 2, wherein: The energy regeneration enzyme and the target protein are from the same biological species.

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

10. A method for producing cultured meat, comprising: The process of culturing cells using culture medium. The culture medium contains the translation product produced by the method for producing a protein according to claim 9 as an additive.

11. A kit for use in a method for producing a protein, the kit comprising: Translation template mRNA for translating energy regenerating enzymes; as well as elements for translating the translation template mRNA for translating the energy regenerating enzyme into protein in the absence of cells, and, The energy regeneration system is constructed using only the energy regeneration enzyme translated from the translation template mRNA for translating the energy regeneration enzyme, and therefore the energy regeneration enzyme is not added.

12. The kit according to claim 11, further comprising a translation template mRNA for translating a target protein.

13. The kit according to claim 12, wherein The energy regeneration enzyme and the target protein are of the same biological species.

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