Method for producing protein

Abstract

The present disclosure provides a method for producing a target protein, said method comprising culturing, under conditions that activate protein kinase C (PKC), cells that contain a nucleic acid that encodes the target protein and is operably linked to a promoter that contains a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.

Description

Protein manufacturing method

[0001] This patent application claims priority to Japanese Patent Application No. 2021-144198, the entire contents of which are incorporated herein by reference. The present disclosure relates to a method for producing a protein.

[0002] Biological products (biopharmaceuticals) such as antibodies have made it possible to treat many chronic and acute diseases that were previously difficult to treat, and are becoming essential pharmaceuticals in modern medicine. However, the price of biopharmaceuticals is extremely high, and not many people are able to enjoy their benefits. The main reason for this is that cultured cells are usually used to produce biopharmaceuticals, and the production volume per culture volume is limited.

[0003] Specifically, biopharmaceuticals are produced by linking the cDNA of a target protein (antibody, bioactive peptide, etc.) downstream of a strong promoter such as the CMV promoter and expressing it in cultured mammalian cells such as HEK or CHO. Since the production volume of a biopharmaceutical is determined by the protein production capacity of the cells, the culture volume, and the culture time, it is necessary to improve the protein production capacity of the cells in order to increase production volume while suppressing production costs.

[0004] An object of the present disclosure is to increase the yield of a target protein in protein production using cultured cells.

[0005] The present inventors have demonstrated that activating PKC can increase the transcriptional activity of promoters such as the CMV promoter, and have discovered a new method for activating PKC, thereby increasing the production of a target protein in cultured cells.

[0006] Thus, in one aspect, the present disclosure provides a method for producing a protein of interest, comprising culturing cells comprising a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1 under conditions that activate PKC, wherein the culturing under conditions that activate PKC is any of the following: (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor, (2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor, (3) expressing a nucleic acid encoding a peptide comprising a Pro region or a RING region in the cells and culturing in the presence of a PKC activator, and (4) The cells are cultured in the presence of a compound selected from the group consisting of:

[0007] In one aspect, the disclosure provides a method for enhancing transcription of a gene of interest, comprising culturing cells comprising a nucleic acid of the gene operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1 under conditions that activate PKC, wherein the culturing under conditions that activate PKC is any of the following: (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor; (2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor; (3) expressing a nucleic acid encoding a peptide comprising a Pro region or a RING region in the cells and culturing in the presence of a PKC activator; and (4) culturing the cells in the presence of Compound X and a compound selected from Compounds #1 to #7, or an ester, salt, or solvate thereof.

[0008] In one aspect, the present application provides a kit for producing a protein of interest, comprising: (1) a PKC activator and a calmodulin inhibitor; (2) a nucleic acid encoding an activated form of PKC and a calmodulin inhibitor; (3) a nucleic acid encoding a PKC activator and a peptide comprising a Pro region or a RING region; or (4) compound X and a compound selected from compounds #1 to #7, or an ester, salt, or solvate thereof; wherein the production of the protein of interest comprises culturing cells comprising the nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.

[0009] In one aspect, the present application provides an expression construct comprising: a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1; a nucleic acid encoding a protein; an internal ribosome entry site (IRES) or a 2A self-cleaving peptide (2A peptide) sequence; and a nucleic acid encoding an activated form of PKC, all of which are operably linked.

[0010] In one aspect, the present application provides an expression construct comprising: a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1, operably linked thereto; a nucleic acid encoding a protein; and a nucleic acid encoding a peptide comprising a Pro region or a RING region.

[0011] In one aspect, the present application provides a composition for activating PKC, comprising Compound X and a compound selected from Compounds #1 to #7, or an ester, salt, or solvate thereof.

[0012] The present disclosure allows for increased production of proteins of interest.

[0013]

[0033] Figure 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X or any of Compounds #1 to #7, and in the presence or absence of a PKC inhibitor.

[0034] Figure 1 shows changes in luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence or absence of Compound X.

[0035] Figure 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X or any of PKC activators #1 to #4, and in the presence or absence of a PKC inhibitor. Figure 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X or any of PKC activators #5 to #6, and in the presence or absence of a PKC inhibitor. Figure 2 shows luciferase activity in HEK293A cells transfected with pRL-CAG or pRL-EF1, in the presence of Compound X or any of Compounds #1 to #5, and in the presence or absence of a PKC inhibitor. Figure 3 shows luciferase activity in HEK293A cells transfected with pRL-CAG, in the presence of Compound X, Compounds #6 to #7, and any of PKC activators #1 to #6, and in the presence or absence of a PKC inhibitor.

[0033] Figure 1 shows luciferase activity in HEK293A cells transfected with pRL-EF1 in the presence of any of Compound X, Compounds #6 to #7, and PKC activators #1 to #6, and in the presence or absence of a PKC inhibitor.

[0034] Figure 1 shows the phosphorylation state of PKC substrate proteins in HEK293A cells in the presence of any of Compound X, Compounds #1 to #7, PKC activators #1 to #6, Compound SC, and Compound SB, and in the presence or absence of a PKC inhibitor.

[0035] Figure 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X and / or Compound SC, and in the presence or absence of a PKC inhibitor.

[0033] Figure 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X, Compound SC, and / or SB.

[0034] Figure 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X and any of Compounds #1 to #7, and in the presence or absence of Compound SB or Compound SB + SC.

[0035] Figure 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X and any of PKC activators #1 to #6, and in the presence or absence of Compound SB or Compound SB + SC.

[0039] Figure 1 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla luciferase) in the presence of Compound X or Compound X + Compound SC, and in the presence of various histone deacetylase inhibitors. HEK293A cells were transfected with an expression construct in which a mouse IgG antibody heavy chain cDNA was linked under the control of a CMV promoter, and an expression construct in which a mouse IgG antibody light chain cDNA was linked under the control of a CMV promoter, and the cells were cultured in a medium supplemented with Compound X, Compounds #1 to #5, or PKC activators #1 to #6, or in the presence or absence of Compound SB + SC, and the concentration of mouse IgG antibody in the medium was measured. HEK293A cells that constitutively express human proinsulin under the control of the CMV promoter were cultured in a medium supplemented with Compound X, Compounds #1 to #5, PKC activator #1 to #3, #5, or #6, and in the presence or absence of Compound SB+SC, and the concentration of human proinsulin in the medium was measured. HEK293A cells were transfected with an expression construct in which human leptin cDNA was linked under the control of the CMV promoter, and the cells were cultured in a medium supplemented with Compound X, Compounds #1 to #5, or PKC activator #1 to #6, and in the presence or absence of Compound SB+SC, and the concentration of human leptin in the medium was measured.

[0073] As in Figures 14 to 16, cells transiently or constitutively expressing mouse IgG antibody heavy and light chains, human proinsulin, or human leptin were cultured in medium supplemented with Compound X, Compound #6, or Compound #7, and in the presence or absence of Compound SB + SC, and the concentrations of each protein in the medium were measured.

[0074] Figure 14 shows the results of HEK293A cells constitutively expressing mouse IgG antibody heavy and light chains, human proinsulin, or human leptin under the control of a CMV promoter, cultured in the presence or absence of Compound X or Compound X + SB, and the concentrations of each protein in the medium were measured over 3 or 4 days.

[0075] Figure 14 shows a schematic diagram of an expression construct linking a CMV promoter, a protein cDNA (XXX), an IRES, and an activated PKC cDNA.

[0033] Figure 1 shows the results of transfecting HEK293A cells with an expression construct linking a CMV promoter, Rluc cDNA, IRES, and activated PKC cDNA, followed by measurement of luciferase activity. Figure 1 shows the results of transfecting HEK293A cells with an expression construct linking a CMV promoter, Rluc cDNA, IRES or P2A, and activated PKC cDNA, followed by measurement of luciferase activity. Figure 1 shows the results of transfecting HEK293A cells with an expression construct linking a CMV promoter, mouse IgG antibody heavy chain cDNA, IRES, and activated PKC cDNA, and an expression construct linking a CMV promoter, mouse IgG antibody light chain cDNA, IRES, and activated PKC cDNA, followed by measurement of the IgG antibody concentration in the medium over a period of 4 days. HEK293A cells were transfected with an expression construct linking a CMV promoter, human leptin cDNA, IRES, and activated PKC cDNA, and the leptin concentration in the culture medium was measured over a period of 4 days. Luciferase activity in HEK293A cells transfected with pRL-CMV in the presence of a naphthalenesulfonamide derivative and in the presence or absence of Compound X is shown. A schematic diagram of an expression construct linking a CMV promoter, Rluc cDNA, IRES, and PML (wild-type or deletion-type) cDNA is shown.

[0039] Figure 1 shows luciferase activity in the presence or absence of Compound X in HEK293A cells transfected with an expression construct comprising a CMV promoter, Rluc cDNA, IRES, and PML (wild-type or deleted) cDNA. Figure 2 shows luciferase activity in the presence or absence of Compound X, SB, Compound SC, or a combination thereof in HEK293A cells transfected with an expression construct comprising a CMV promoter, Rluc cDNA, IRES, and PMLΔ9 or PML-Ring cDNA. BLAST analysis was performed on the amino acid sequence of the RING region of PML, and a phylogenetic tree between genes with homologous amino acid sequences is shown. A schematic diagram of an expression construct ligated with a CMV promoter, Rluc cDNA, IRES, and RING region cDNA is shown.

[0039] Figure 1 shows luciferase activity in HEK293A cells transfected with an expression construct linking a CMV promoter, Rluc cDNA, and cDNAs for the IRES and RING regions, in the presence or absence of Compound X.

[0040] Figure 1 shows a schematic diagram of an expression construct linking a CMV promoter, Rluc cDNA, IRES, and PML (with a stop codon introduced) and an expression construct linking a CMV promoter, Rluc cDNA, and PML (wild-type or deletion-type) cDNA.

[0049] Figure 1 shows luciferase activity in the presence or absence of Compound X in HEK293A cells transfected with an expression construct linking a CMV promoter, Rluc cDNA, IRES, and PML (with a stop codon introduced) cDNA, and an expression construct linking a CMV promoter, Rluc cDNA, and PML (wild-type or deleted) cDNA.

[0014] Unless otherwise specified, terms used in this disclosure have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Below, definitions of some terms used in this disclosure are provided, but these definitions take precedence over common understandings in this disclosure.

[0015] The method for producing a protein of interest disclosed herein utilizes genetic engineering techniques, in which a nucleic acid encoding the protein of interest is introduced into cultured cells, and the protein is expressed and recovered. Such genetic engineering techniques are well known in the art and can be carried out in accordance with methods described in the literature (e.g., Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983) and DNA Cloning, D.M. Glover, IRL PRESS (1985)).

[0016] In the present disclosure, the promoter may be any promoter that contains a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1. The promoter may further contain a binding site for other transcription factors, for example, CREB. In some embodiments, the promoter contains binding sites for SP1, CEBP, AP1, NF-κB, and YY1. In some embodiments, the promoter contains binding sites for SP1, CEBP, AP1, NF-κB, YY1, and CREB. Examples of promoters that can be used include the CMV promoter, the CAG promoter, and the EF1 promoter, particularly the CMV promoter. These transcription factors, binding sites, and promoters are well known in the art and can be used in accordance with methods described in the literature.

[0017] In the present disclosure, the term "operably linked" means that regulatory sequence elements such as a promoter, an IRES, and a 2A peptide sequence are linked to a nucleic acid encoding a protein in a manner that allows expression of the protein, and the 3' end of each DNA may be directly linked to the 5' end of the downstream DNA, or any DNA sequence may be present between them.

[0018] The protein of interest is not limited as long as it does not cause unacceptable toxicity to the cultured cells used. For example, the protein of interest may be an active ingredient of a biopharmaceutical, such as immunoglobulin, leptin, insulin, or a fragment thereof. A nucleic acid encoding the protein of interest can be produced, for example, by conventional DNA synthesis or amplification by RT-PCR, based on the amino acid sequence information of the protein and the sequence information of the nucleic acid encoding it.

[0019] An expression construct can be prepared by incorporating a promoter and a nucleic acid encoding a protein of interest into an expression vector. The expression vector used here can be appropriately selected depending on the host used, the purpose, etc., and includes plasmids, phage vectors, viral vectors, etc. Examples include plasmid vectors such as pKCR, pCDM8, pGL2, pcDNA3.1, pRc / RSV, and pRc / CMV, and viral vectors such as retroviral vectors, adenoviral vectors, and adeno-associated viral vectors. The vector may appropriately contain factors such as a selectable marker gene and a terminator.

[0020] By transforming a host cell with the expression construct, a cell containing a promoter and a nucleic acid encoding a protein of interest can be produced. The host cell is typically an animal cell, such as HEK293A cell, HEK293T cell, CHO cell, COS cell, Vero cell, HeLa cell, L929 cell, BALB / c3T3 cell, or C127 cell.

[0021] The expression vector can be introduced into the host cell by any conventional method suitable for the host cell. Specific examples include the calcium phosphate method, the DEAE-dextran method, electroporation, and lipofection. The transformed cells thus obtained may transiently express the target protein, or a cell line that stably expresses the target protein may be established.

[0022] By culturing transformed cells under conditions that activate PKC, a target protein can be efficiently produced. Culture conditions, such as the medium, culture time, and culture temperature, suitable for each cell type are well known to those skilled in the art and can be selected appropriately. For example, the cells are cultured under conditions that activate PKC for 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 48 hours or more, and 60 hours or less, 48 ​​hours or less, 36 hours or less, 30 hours or less, or 24 hours or less, and then the target protein is recovered. The obtained protein can be further isolated and purified by common biochemical purification methods. Examples of purification methods include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, and gel filtration chromatography.

[0023] In one embodiment, the conditions that activate PKC are (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor.

[0024] PKC is a type of protein kinase that phosphorylates the hydroxyl groups of serine and threonine residues in substrate proteins, and more than 10 isozymes are known. Isozymes are classified into three subfamilies: conventional (α, βI, βII, γ), novel (δ, ε, θ, η), and atypical (ζ, Mζ, ι / λ) depending on their structure, activation mechanism, and physiological activity. In the present disclosure, the PKC is preferably a conventional PKC isozyme or a novel PKC isozyme, particularly preferably PKCα or PKCδ.

[0025] In the present disclosure, the term "PKC activator" refers to a substance that enhances the kinase activity of PKC. Two or more types of PKC activators may be used in combination.

[0026] In one embodiment, the PKC activator is a compound of formula (I): {In the formula, R １ is H, halogen, -OH, C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Alkoxy, C６－１４ Aryl or —OC(O)R ３ where C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ The alkoxy or aryl may be substituted with 1 to 3 halogen atoms, which may be the same or different; R ２ is C ６－１２ Alkyl, C ６－１２ Alkenyl, C ６－１２ Alkynyl or C ６－１２ Alkoxy, where C ６－１２ Alkyl, C ６－１２ Alkenyl, C ６－１２ Alkynyl or C ６－１２ The alkoxy may be substituted with 1 to 3 halogen atoms, which may be the same or different; R ３ is C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, amino or C ６－１４ aryl, where C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl or C ６－１４ The aryl may be substituted with 1 to 3 halogen atoms, which may be the same or different,}, Formula (II): {In the formula, R ４ is H, halogen, -OH, C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ Alkynyl, C １－１８ Alkoxy or —OC(O)R ６ where C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ Alkynyl or C １－１８ The alkoxy may be substituted with 1 to 3 halogen atoms, which may be the same or different; R ５ is C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ alkynyl or amino, wherein C １－６ Alkyl, C２－６ alkenyl or C ２－６ The alkynyl may be substituted with 1 to 3 halogen atoms, which may be the same or different, and R ６ is C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ alkynyl or amino, where C １－１８ Alkyl, C ２－１８ alkenyl or C ２－１８ The alkynyl may be substituted with 1 to 3 halogen atoms, which may be the same or different, or a group represented by formula (III): {In the formula, R ７ is H, C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ Alkynyl or —C(O)R ９ where C １－１８ Alkyl, C ２－１８ alkenyl or C ２－１８ The alkynyl may be substituted with 1 to 3 halogen atoms, which may be the same or different, and R ８ is H, C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ Alkynyl or —C(O)R ９ where C １－１８ Alkyl, C ２－１８ alkenyl or C ２－１８ The alkynyl may be substituted with 1 to 3 halogen atoms, which may be the same or different, and R ９ is C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ alkynyl or amino, where C １－１８ Alkyl, C ２－１８ alkenyl or C ２－１８ and the alkynyl may be substituted with 1 to 3 halogen atoms, which may be the same or different.} or an ester, salt or solvate thereof may be used.

[0027] In this disclosure, the term "halogen" refers to an atom selected from fluorine, chlorine, bromine, and iodine. In this disclosure, the term "alkyl" refers to a saturated, straight or branched chain hydrocarbon group. In this disclosure, the term "alkenyl" refers to a straight or branched chain hydrocarbon containing one or more double bonds. In this disclosure, the term "alkynyl" refers to a straight or branched chain hydrocarbon containing one or more triple bonds. In this disclosure, the term "alkoxy" refers to -O-alkyl, where alkyl is as defined in this disclosure.

[0028] In this disclosure, the term "aryl" refers to a monovalent aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Aryl typically includes phenyl and naphthyl. In this disclosure, the term "amino" refers to -NH ２ means a group represented by the formula:

[0029] In the present disclosure, "ester" refers to an ester that can be hydrolyzed in vivo or in vitro, including those that readily decompose to release the parent compound or its salt. Suitable ester groups include, for example, those derived from aliphatic carboxylic acids, particularly alkanoic acids, alkenoic acids, cycloalkanoic acids, and alkanedioic acids (wherein each alkyl or alkenyl group has, for example, 6 or fewer carbon atoms). Examples of specific esters include formates, acetates, propionates, butyrates, acrylates, and ethylsuccinates.

[0030] In the present disclosure, a "salt" may be a salt of a compound with an inorganic or organic acid. Preferred salts are salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, or sulfuric acid, or salts with organic carboxylic or sulfonic acids, such as acetic acid, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, or methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, or naphthalenedisulfonic acid.

[0031] Salts may also be salts with conventional bases, for example alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. calcium or magnesium salts), or ammonium salts, especially sodium salts, derived from ammonia or organic amines (e.g. diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabietylamine, methylpiperidine, L-arginine, creatine, choline, L-lysine, ethylenediamine, benzathine, ethanolamine, meglumine or tromethamine).

[0032] In the present disclosure, "solvate" means a compound that forms a complex in the solid or liquid state by coordination with solvent molecules. Preferred solvates are hydrates.

[0033] In one embodiment, in formula (I), R １ is H or —OC(O)R ３ and R ２ is C ６－１２ Alkyl or C ６－１２ alkenyl, and R ３ is C １－６ Alkyl or C ６－１４ It is aryl.

[0034] In one embodiment, in formula (I), R １ is H or —OC(O)R ３ and R ２ is nonyl or 1,3-nonadienyl, R ３ is methyl or phenyl.

[0035] In certain embodiments, the compound of formula (I) is Compound X, Compound #1, or Compound #2, described below.

[0036] In one embodiment, in formula (II), R ４ is H or —OC(O)R ６ and R ５ is C １－６ Alkyl or C ２－６ alkenyl, and R ６ is C １－１８It is alkyl.

[0037] In one embodiment, in formula (II), R ４ is H or —OC(O)R ６ and R ５ is methyl, propyl, sec-butyl or butenyl, R ６ is propyl, nonyl or tridecyl.

[0038] In certain embodiments, the compound of formula (II) is Compound #3, Compound #4, Compound #5, TPA, phorbol 12,13-dibutyrate, or prostratin, as described below, particularly Compound #3, Compound #4, or Compound #5.

[0039] In one embodiment, in formula (III), R ７ is H or —C(O)R ９ and R ８ is H or —C(O)R ９ and R ９ is C １－１８ Alkyl or C ２－１８ It is alkenyl.

[0040] In one embodiment, in formula (III), R ７ is H or —C(O)R ９ and R ８ is H or —C(O)R ９ and R ９ is pentadecyl or butenyl.

[0041] In certain embodiments, the compound of formula (III) is Compound #6, Compound #7, or ingenol 3-angelate, particularly Compound #6 or Compound #7, described below.

[0042] The compounds of formulae (I) to (III) or esters, salts or solvates thereof may be added to the culture medium at a concentration of, for example, 0.1 to 10 μg / ml, 0.1 to 1 μg / ml, 0.1 to 1000 ng / ml, 2 to 500 ng / ml or 5 to 200 ng / ml.

[0043] In some embodiments, the PKC activator is a compound of formula (I) or an ester, salt, or solvate thereof, which may be added to the culture medium at a concentration of, for example, 0.1-10 μg / ml, 0.1-1 μg / ml, 0.1-1000 ng / ml, 20-500 ng / ml, or 50-200 ng / ml.

[0044] In certain embodiments, the PKC activator is a compound selected from Compound X and Compounds #1-#7 below, or an ester, salt, or solvate thereof.

[0045] In some embodiments, the PKC activator is Compound X or an ester, salt, or solvate thereof.

[0046] Known PKC activators may be used, including 12-O-tetradecanoylphorbol 13-acetate (TPA, also known as phorbol 12-myristate 13-acetate (PMA)), prostratin, bryostatin 1, bryostatin 2, FR236924, (−)-indolactam V, PEP005, phorbol 12,13-dibutyrate, 1-oleoyl-2-acetyl-sn-glycerol, and 1-O-hexadecyl-2-O-arachidonyl. Examples of PKC activators include, but are not limited to, 1,2-dioctanoyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, PIP2, resiniferatoxin, phorbol 12,13-dihexanoate, mezerein, ingenol 3-angelate, RHC-80267, DCP-LA, lipoxin A4, (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam, and the like. In some embodiments, the PKC activator is TPA, prostratin, (-)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate, or (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam. Known PKC activators may be used as appropriate according to methods known in the art, for example, methods recommended by the manufacturer.

[0047] In certain embodiments, the PKC activator is a compound selected from Compound X, Compounds #1-#7, TPA, prostratin, (-)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate, and (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam, or an ester, salt, or solvate thereof.

[0048] Calmodulin is an acidic protein that functions as a calcium sensor and regulates intracellular calcium levels. Calmodulin inhibitors include compounds represented by formula (IV): wherein n is an integer from 1 to 8, and R is C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Alkoxy, C ６－１４ aryl, amino, hydroxy, COOH or COOR′, where R′ is C １－６ is alkyl} or an ester, salt or solvate thereof may be used.

[0049] In one embodiment, in formula (IV), n is an integer from 4 to 6; and R is C １－６ Alkyl, C ６－１４ It is aryl or amino.

[0050] In certain embodiments, in formula (IV), n is an integer of 4 or 6; and R is methyl, phenyl, or amino.

[0051] Compounds of formula (IV) include, for example, SC-9, SC-10, and W-7. The compounds of formula (IV) may be used at a concentration of, for example, 0.5 to 100 μg / ml, 1 to 50 μg / ml, or 2 to 10 μg / ml.

[0052] Known calmodulin inhibitors, such as W-7, calmidazolium, bisindolylmaleimide I, trifluoperazine, ruthenium red, ophiobolin A, CaM kinase II (290-309), E6 berbamine, mastoparan, compound 48 / 80, phenoxybenzamine, W-7 isomer, polystesmastoparan, A-7, fluphenazine-N-2-chloroethane, W-13, W-13 isomer, CGS 9343B, W-5 isomer, W-12, N-(5-aminopentyl)-5-chloro-2-naphthalenesulfonamide, and W-5, may be used as appropriate according to methods known in the art, for example, methods recommended by the manufacturer. Two or more calmodulin inhibitors may also be used in combination.

[0053] In one embodiment, the condition for activating PKC is (2) expressing activated PKC in the cells and culturing the cells in the presence of a calmodulin inhibitor.

[0054] "Activated PKC" refers to a PKC mutant that constitutively exhibits kinase activity. Activated PKC may be a PKC lacking the N-terminal regulatory region (Molecular and Cellular Biology 19(2):1313-24, 1999). Activated PKC may also have a mutation that enhances kinase activity (PNAS 115(24):E5497-E5505. 2018). Examples of activated PKC include PKCδ-CA (SEQ ID NO: 1) and PKCαCA-M489V (SEQ ID NO: 2).

[0055] The kinase activity of a PKC mutant can be measured by various methods known in the art, such as a method in which a PKC mutant is overexpressed in cultured cells and the phosphorylation level of the substrate is detected by Western blotting using an antibody specific to the phosphorylated substrate (e.g., THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 279, No. 27, pp. 27986-27993, 2004), an ELISA method (e.g., Cell Death and Differentiation (2015) 22, 2078-2086), or an in vitro method. ３２A method for assessing the incorporation of a phosphate group into a substrate using P-γ-ATP (e.g., THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 262, No. 20, pp. 9569-9573, 1987) can be used. Various kits for measuring kinase activity, such as a PKC kinase activity kit (Enzo Life Science, #ADI-EKS-420A), may also be used.

[0056] In one embodiment, cells expressing a PKC mutant are cultured in the presence and absence of a PKC inhibitor, and the amount of phosphorylated protein is measured by an immunological technique using an antibody that specifically recognizes the phosphorylated protein. If the amount of phosphorylated protein is reduced by the PKC inhibitor, the PKC mutant can be determined to be an activated PKC. Examples of immunological techniques include flow cytometry analysis, radioisotope immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemical staining.

[0057] For example, a protein of interest and activated PKC can be expressed in a cell using an expression construct comprising an operably linked promoter, a nucleic acid encoding the protein of interest, an internal ribosome entry site (IRES) or 2A self-cleaving peptide (2A peptide) sequence, and a nucleic acid encoding activated PKC. In one embodiment, the promoter, the nucleic acid encoding the protein of interest, the IRES or 2A peptide sequence, and the nucleic acid encoding activated PKC are operably linked in this order.

[0058] By using an IRES to express a protein of interest and activated PKC from a single expression construct, the protein of interest can be produced efficiently. An IRES is an RNA region that can recruit eukaryotic ribosomes to mRNA, allowing cap-independent initiation of translation as part of the protein synthesis process. Many IRESs have been identified in viral and eukaryotic genomes, and synthetic IRESs have also been developed.

[0059] For example, IRES can be derived from various viruses, including enteroviruses (e.g., human papillomavirus 1, human coxsackievirus B); rhinoviruses (e.g., human rhinovirus); hepatoviruses (hepatitis A virus); cardioviruses (encephalomyocarditis virus ECMV and Theiler's encephalomyelitis virus); aphthoviruses (foot-and-mouth disease virus, equine rhinitis A virus, equine rhinitis B virus); pestiviruses (e.g., bovine viral diarrhea virus and hog cholera virus; hepaciviruses (e.g., hepatitis C virus) and GB virus B). Alternatively, the IRES can be derived from viruses of the Retroviridae family, such as members of the lentivirus family (e.g., simian immunodeficiency virus and human immunodeficiency virus 1); BLV-HTLV retroviruses (e.g., human T-lymphotropic virus type 1); and the mammalian C-type retrovirus family (e.g., Moloney murine leukemia virus, Friend murine leukemia virus, Harvey murine sarcoma virus, avian reticuloendotheliosis virus, murine leukemia virus (envRNA), Rous sarcoma virus). IRESs derived from eukaryotic mRNAs include, for example, the IRESs of BiP, Drosophila antennapedia (exons d and e), c-myc, and the X-linked inhibitor of apoptosis (XIAP) gene. Additionally, various synthetic IRES have been developed, see, e.g., De Gregorio et al. (1999) EMBO J. 75:4865-74; Owens et al. (2001) PNAS 4:1471-6; and Venkatesan et al. (2001) Molecular and Cellular Biology 21:2826-37. For additional IRES known in the art, see, e.g., rangueil.inserm.fr / IRESdatabase. In one embodiment, an IRES from the encephalomyocarditis virus (ECMV) is used.

[0060] The 2A peptide sequence induces ribosome skipping during protein translation. When a 2A peptide sequence is present in the amino acid sequence of a protein, the protein is translated as two polypeptides cleaved at the C-terminus of the 2A peptide sequence. Examples of 2A peptides include those described in Kim, JH, et al., PLoS One. 6(4), e18556 (2011), and known examples include P2A peptide (SEQ ID NO: 3: (GSG)ATNFSLLKQAGDVEENPGP), T2A peptide (SEQ ID NO: 4: (GSG)EGRGSLLTCGDVEENPGP), E2A peptide (SEQ ID NO: 5: (GSG)QCTNYALLKLAGDVESNPGP), and F2A peptide (SEQ ID NO: 6: (GSG)VKQTLNFDLLKLAGDVESNPGP) (in each sequence, the N-terminal GSG may or may not be present).

[0061] In the condition (2), the calmodulin inhibitor can be used in the same manner as in the above (1).

[0062] In one embodiment, the condition for activating PKC is (3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in the cell and culturing the cell in the presence of a PKC activator.

[0063] The protein of interest can be efficiently produced by using an expression construct containing a nucleic acid encoding the protein of interest and a nucleic acid encoding a peptide comprising a Pro or RING region. For example, an expression construct containing a promoter, a nucleic acid encoding the protein, and a nucleic acid encoding the peptide comprising a Pro or RING region, all operably linked, can be used. In one embodiment, the promoter, the nucleic acid encoding the protein, and the nucleic acid encoding the peptide comprising a Pro or RING region are operably linked in this order.

[0064] A nucleic acid encoding a peptide comprising a Pro region or a RING region may or may not be translated into a peptide. If it is to be translated, an IRES or 2A peptide sequence may be inserted between the nucleic acid encoding the protein of interest and the nucleic acid encoding the peptide comprising a Pro region or a RING region. The IRES and 2A peptide sequence may be used in the same manner as in (2) above. For example, an expression construct may be used that includes a promoter, a nucleic acid encoding a protein, an IRES or 2A peptide sequence, and a nucleic acid encoding a peptide comprising a Pro region or a RING region, all operably linked together. In one embodiment, the promoter, the nucleic acid encoding the protein, the IRES or 2A peptide sequence, and the nucleic acid encoding the peptide comprising a Pro region or a RING region are operably linked in this order.

[0065] In the present disclosure, the Pro region or RING region may be derived from any species, such as mouse, rat, hamster, rabbit, cat, dog, cow, pig, sheep, monkey, human, etc., particularly human. Preferably, the Pro region or RING region is derived from the same species as the cell used.

[0066] Pro regions refer to regions containing proline-rich amino acid sequences and mediate specific interactions with functional domains such as WW domains and SH3 domains. Pro regions include repetitive short proline-rich amino acid sequences, tandemly repeated proline-rich amino acid sequences, non-repetitive proline-rich amino acid sequences, and hydroxyproline-rich amino acid sequences. Pro regions can be found in a variety of proteins, including, but not limited to, nuclear proteins, transcription factors, integral membrane proteins such as transporters, channels, and receptors, globular proteins, hormones, neuropeptides, mucins, immunoglobulins, and extracellular matrix proteins.

[0067] The Pro region may be derived from any protein. Examples of proteins containing a Pro region include PML, ARHGEF1, aggrecan-1, RALGDS, DGKK, SPATA21, rabphilin-3A, TEAD3, SPPL2B, and FLJ43093.

[0068] RING domains, also called RING finger domains, bind to a pair of zinc atoms and mediate protein-protein interactions. RING domains generally have the following consensus sequence: C-X ２ -C-X ９－３９ -C-X １－３ -H-X ２－３ -C-X ２ -C-X ４－４８－ C-X ２ -C {wherein C is a cysteine ​​residue, H is a histidine residue, and X is any amino acid residue.} These cysteine ​​and histidine residues are necessary for forming a structure mediated by binding with the zinc atom and are highly conserved.

[0069] The RING region may be derived from any protein. Examples of proteins containing a RING region include TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22, KIAA0182, TRIM65, RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML, and TRIM56. In some embodiments, the RING region is a RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1, or HTLF.

[0070] In one embodiment, the Pro region or RING region is derived from the promyelocytic leukemia protein (PML). PML is required for the assembly of intranuclear structures called PML bodies. PML bodies have diverse functions and are suggested to be involved in a wide range of intracellular processes. Multiple isoforms of human PML are known, and all isoforms share the same amino acid sequence at the N-terminus.

[0071] The amino acid sequence and nucleotide sequence of human wild-type (WT) PML isoform 5 (Gene ID: 5371, NCBI Reference Sequence: NP_150247.2) are shown in SEQ ID NOs: 7 and 8. The amino acid sequence consists of 560 amino acids, and has the regions shown in Figure 25. Positions 1 to 45 of the amino acid sequence of SEQ ID NO: 7 (SEQ ID NO: 9) are a Pro region, and positions 46 to 105 (SEQ ID NO: 10) are a RING region. Positions 1 to 135 of the nucleotide sequence of SEQ ID NO: 8 (SEQ ID NO: 11) encode a Pro region, and positions 136 to 315 (SEQ ID NO: 12) encode a RING region.

[0072] A Pro region can be a region of a protein that matches the region from positions 1 to 45 of SEQ ID NO:7 when the amino acid sequence of the protein and the amino acid sequence of SEQ ID NO:7 are aligned optimally (maximum amino acid identity). In one embodiment, the Pro region comprises or consists of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:9. In one embodiment, the Pro region comprises the amino acid sequence of SEQ ID NO:9. In one embodiment, the Pro region consists of the amino acid sequence of SEQ ID NO:9. In one embodiment, the Pro region is encoded by a nucleotide sequence having at least 90% identity with or comprising the nucleotide sequence of SEQ ID NO:11. In one embodiment, the Pro region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:11. In one embodiment, the Pro region is encoded by the nucleotide sequence of SEQ ID NO:11.

[0073] A RING region can be a region of a protein that corresponds to a region from positions 46 to 105 of SEQ ID NO:7 when the amino acid sequence of the protein and the amino acid sequence of SEQ ID NO:7 are optimally aligned. In one embodiment, the RING region comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:10. In one embodiment, the RING region comprises the amino acid sequence of SEQ ID NO:10. In one embodiment, the RING region consists of the amino acid sequence of SEQ ID NO:10. In one embodiment, the RING region is encoded by a nucleotide sequence having at least 90% identity to or comprising the nucleotide sequence of SEQ ID NO:12. In one embodiment, the RING region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:12. In one embodiment, the RING region is encoded by the nucleotide sequence of SEQ ID NO:12.

[0074] In the present disclosure, the identity of nucleotide sequence or amino acid sequence refers to the degree of sequence similarity between nucleic acids or proteins, and is determined by comparing two sequences that are optimally aligned (maximizing the number of nucleotide or amino acid matches) across the region of the sequences being compared.The numerical value (%) of sequence identity is calculated by determining the identical nucleotides or amino acids present in both sequences to determine the number of matching sites, then dividing this number of matching sites by the total number of nucleotides or amino acids in the region of the sequences being compared, and multiplying the resulting number by 100.Algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly available to those skilled in the art (for example, BLAST algorithm, FASTA algorithm, etc.).Sequence identity can be determined using sequence analysis software such as BLAST, FASTA, etc.

[0075] In the condition (3), the PKC activator can be used in the same manner as in the above (1).

[0076] In one embodiment, the condition for activating PKC is (4) culturing the cells in the presence of a compound selected from Compound X and Compounds #1 to #7, or an ester, salt, or solvate thereof. These compounds are included in the PKC activators described in (1) above and can be used as described above.

[0077] Compound X and compounds #1 to #7 may be obtained by chemical synthesis or extracted from plants containing them. For example, compound X can be extracted from Lowdaphne Stringbush, compounds #1 and #2 from Lilac Daphne, compounds #3 to #5 from Croton, and compounds #6 to #7 from Cape Euphorbia. Plant processed products containing these compounds, such as plant extracts, may also be used.

[0078] In one embodiment, there is provided a composition for activating PKC, comprising Compound X and a compound selected from Compounds #1 to #7, or an ester, salt, or solvate thereof. The composition may contain, for example, a suitable carrier, excipient, additive, etc., and may also contain other active ingredients.

[0079] When activating PKC under the conditions (1) to (4), a histone deacetylase inhibitor may be further added to the medium. Histone deacetylase is an enzyme that deacetylates histones, which are major components of chromatin structure, and plays an important role in gene transcription regulation. Known histone deacetylase inhibitors include, for example, trichostatin A, M344, butyrate, phenylbutyrate, apicidin, valproic acid, BML-210, depudecin, romidepsin (FK-228), HC toxin, oxamflatin, scriptaid, splitomycin, suberoyl bis-hydroxamic acid, vorinostat, dacinostat (LAQ-824), panobinostat (LBH-589), belinstat (PXD- 101), phenyl acetate, IF2357, FK-228, entinostat (MS-275), mocetinostat (MGCD0103), or tacedinaline (CI994), preferably sodium butyrate, valproic acid, trichostatin A, vorinostat, apicidin, entinostat, or tacedinaline, particularly preferably sodium butyrate, may be used as appropriate according to methods well known in the art, for example, methods recommended by the manufacturer. For example, sodium butyrate may be used at a concentration of 0.1 to 10 mM, 0.5 to 5 mM, or 1 to 3 mM. Two or more histone deacetylase inhibitors may be used in combination.

[0080] When PKC is activated under the conditions (3) or (4), a calmodulin inhibitor may be further added to the medium. The calmodulin inhibitor may be used in the same manner as in the above (1).

[0081] The present disclosure also provides a method for enhancing transcription of a gene of interest, comprising culturing cells containing a nucleic acid of the gene operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1 under conditions that activate PKC. The culturing under conditions that activate PKC is any of the methods (1) to (4) described in connection with the method for producing a protein.

[0082] Kits that can be used in the methods of the present disclosure are also provided. Each component included in the kit can be provided separately or, if possible, mixed together, dissolved in water or an appropriate buffer, or lyophilized, and contained in an appropriate container. Suitable containers include bottles, vials, test tubes, tubes, plates, etc. The containers can be made of a variety of materials, such as glass, plastic, or metal. The containers can also have labels. The kits can further include other components that are desirable from a commercial and user standpoint, such as documentation (e.g., written or storage media) containing instructions for use.

[0083] Furthermore, the present inventors have found that the transcriptional activity of the CMV promoter can be enhanced by expressing a nucleic acid encoding a peptide comprising a Pro or RING region. Accordingly, in one aspect, the present application provides a method for producing a protein of interest, comprising expressing in cells and culturing an expression construct comprising: a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1; a nucleic acid encoding the protein of interest; and a nucleic acid encoding a peptide comprising a Pro or RING region, all of which are operably linked. This method can be carried out in accordance with (3) above, except that it does not use a PKC activator.

[0084] For example, the following embodiments are provided: [1] A method for producing a protein of interest, comprising culturing cells containing a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1 under conditions that activate PKC, wherein the culturing under conditions that activate PKC is any of the following: (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor, (2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor, (3) expressing a nucleic acid encoding a peptide comprising a Pro region or a RING region in the cells and culturing in the presence of a PKC activator, and (4) culturing the cells in the presence of compound X and a compound selected from compounds #1 to #7, or an ester, salt, or solvate thereof. [2] The method according to item 1, comprising a step of recovering the protein of interest.

[0085] [3] A method for enhancing transcription of a gene of interest, comprising culturing cells containing a nucleic acid of the gene operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1 under conditions that activate PKC, wherein the culturing under conditions that activate PKC is any of the following: (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor; (2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor; (3) expressing a nucleic acid encoding a peptide comprising a Pro region or a RING region in the cells and culturing in the presence of a PKC activator; and (4) culturing the cells in the presence of compound X and a compound selected from compounds #1 to #7, or an ester, salt, or solvate thereof.

[0086] [4] The method of any of items 1 to 3, wherein the promoter comprises binding sites for SP1, CEBP, AP1, NF-κB, and YY1. [5] The method of any of items 1 to 4, wherein the promoter further comprises a binding site for CREB. [6] The method of any of items 1 to 5, wherein the promoter is a CMV promoter, a CAG promoter, or an EF1 promoter. [7] The method of any of items 1 to 6, wherein the promoter is a CMV promoter.

[0087] [8] The method of any of items 1 to 7, wherein the culturing under conditions that activate PKC is (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor. [9] The method of any of items 1 to 7, wherein the culturing under conditions that activate PKC is (2) causing the cells to express activated PKC and culturing in the presence of a calmodulin inhibitor.

[10] The method of item 9, wherein the cells comprise an expression construct comprising, operably linked together, a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding activated PKC.

[11] The method of item 10, wherein a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding activated PKC are operably linked in this order.

[12] The method of any of items 9 to 11, wherein the activated PKC is PKCδ-CA or PKCαCA-M489V.

[0088]

[13] The method according to any one of items 1 to 7, wherein the culturing under conditions that activate PKC is (3) expressing a nucleic acid encoding a peptide comprising a Pro region or a RING region in the cells, and culturing in the presence of a PKC activator.

[14] The method according to item 13, wherein a nucleic acid encoding a peptide comprising a RING region is expressed in the cells.

[15] The RING region is C-X ２ -C-X ９－３９ -C-X １－３ -H-X ２－３ -C-X ２ -C-X ４－４８－ C-X２ 15. The method according to item 14, wherein the amino acid sequence is: -C {wherein C is a cysteine ​​residue, H is a histidine residue, and X is any amino acid residue}.

[16] The method of claim 14, wherein the RING region is the RING region of TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22, KIAA0182, TRIM65, RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML, or TRIM56.

[17] The method of any of items 14 to 16, wherein the RING region is the RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1 or HTLF.

[18] The method of any of items 14 to 17, wherein the RING region is the RING region of PML.

[19] The method of any of items 14 to 18, wherein the RING region consists of an amino acid sequence having at least 90% or more identity with the amino acid sequence of SEQ ID NO: 10.

[20] The method of any of items 14 to 19, wherein the RING region is encoded by a nucleotide sequence having at least 90% or more identity with the nucleotide sequence of SEQ ID NO: 12.

[0089]

[21] The method of item 13, wherein a nucleic acid encoding a peptide comprising a Pro region is expressed in a cell.

[22] The method of item 21, wherein the Pro region is the Pro region of PML, ARHGEF1, aggrecan-1, RALGDS, DGKK, SPATA21, rabfilin-3A, TEAD3, SPPL2B, or FLJ43093.

[23] The method of item 21 or 22, wherein the Pro region is the Pro region of PML.

[24] The method of any of items 21 to 23, wherein the Pro region consists of an amino acid sequence having at least 90% or more identity with the amino acid sequence of SEQ ID NO: 9.

[25] The method of any of items 21 to 24, wherein the nucleic acid encoding the peptide comprising a Pro region consists of a nucleotide sequence having at least 90% or more identity with the nucleotide sequence of SEQ ID NO: 11.

[26] The method according to any one of items 13 to 25, wherein a nucleic acid encoding a peptide comprising a Pro region and a RING region is expressed in a cell.

[0090]

[27] The method of any of items 13 to 26, wherein the cells comprise an expression construct comprising, operably linked, a promoter, a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide comprising a Pro region or a RING region.

[28] The method of item 27, wherein a promoter, a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide comprising a Pro region or a RING region are operably linked in this order.

[29] The method of item 27 or 28, wherein the expression construct further comprises a nucleic acid encoding an internal ribosome entry site or a 2A peptide sequence.

[30] The method of any of items 27 to 29, wherein a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding a peptide comprising a Pro region or a RING region are operably linked in this order.

[31] The method of any of items 13 to 30, wherein the cells are cultured in the presence of a calmodulin inhibitor.

[0091]

[32] The method according to any one of items 8 and 13 to 31, wherein the PKC activator is a compound of formula (I), formula (II), or formula (III), or an ester, salt, or solvate thereof.

[33] The method according to item 32, wherein the PKC activator is a compound of formula (I), or an ester, salt, or solvate thereof.

[34] R １ is H or —OC(O)R ３ and R ２ But C ６－１２ Alkyl or C ６－１２ alkenyl, and R ３ But C １－６ Alkyl or C ６－１４

[35] R １ is H or —OC(O)R ３ and R ２ is nonyl or 1,3-nonadienyl, and R ３

[36] The method according to item 32, wherein the PKC activator is a compound of formula (II) or an ester, salt or solvate thereof. ４ is H or —OC(O)R ６ and R ５ But C １－６ Alkyl or C ２－６ alkenyl, and R ６ But C １－１８

[38] R ４ is H or —OC(O)R ６ and R ５ is methyl, propyl, sec-butyl or butenyl, and R ６

[39] The method according to item 32, wherein the PKC activator is a compound of formula (III) or an ester, salt or solvate thereof. ７ is H or —C(O)R ９ and R ８ is H or —C(O)R ９ and R ９ But C １－１８Alkyl or C ２－１８

[41] R ７ is H or —C(O)R ９ and R ８ is H or —C(O)R ９ and R ９ is pentadecyl or butenyl.

[42] The method of any of items 8 and 13 to 31, wherein the PKC activator is a compound selected from compound X, compounds #1 to #7, TPA, prostratin, (-)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate, and (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam, or an ester, salt, or solvate thereof.

[43] The method of item 42, wherein the PKC activator is a compound selected from compound X and compounds #1 to #7, or an ester, salt, or solvate thereof.

[44] The method of item 42 or 43, wherein the PKC activator is compound X, or an ester, salt, or solvate thereof.

[0092]

[45] The method of any one of items 1 to 7, wherein the culturing under conditions that activate PKC is (4) culturing the cells in the presence of a compound selected from compound X and compounds #1 to #7, or an ester, salt, or solvate thereof.

[46] The method of item 45, wherein the cells are cultured in the presence of compound X, or an ester, salt, or solvate thereof.

[47] The method of item 45 or 46, wherein the cells are cultured in the presence of a calmodulin inhibitor.

[48] The method of any one of items 8 to 12, 31, and 47, wherein the calmodulin inhibitor is a compound of formula (IV) or an ester, salt, or solvate thereof.

[49] The method of item 48, wherein the calmodulin inhibitor is SC-9, SC-10, or W-7.

[50] The method of item 48 or 49, wherein the calmodulin inhibitor is SC-10.

[51] The method of any one of items 1 to 50, wherein the cells are cultured in the presence of a histone deacetylase inhibitor.

[52] The method of item 51, wherein the histone deacetylase inhibitor is sodium butyrate, valproic acid, trichostatin A, vorinostat, apicidin, entinostat, or tacedinaline.

[53] The method of item 51 or 52, wherein the histone deacetylase inhibitor is sodium butyrate.

[0093]

[54] A kit for producing a protein of interest, comprising: (1) a PKC activator and a calmodulin inhibitor, (2) a nucleic acid encoding an activated PKC and a calmodulin inhibitor, (3) a PKC activator and a nucleic acid encoding a peptide comprising a Pro region or a RING region, or (4) Compound X and a compound selected from Compounds #1 to #7, or an ester, salt, or solvate thereof, wherein the production of the protein of interest comprises culturing cells comprising a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.

[55] An expression construct comprising: a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1, all operably linked thereto, a nucleic acid encoding the protein, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding an activated PKC.

[56] The expression construct according to item 55, wherein a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding activated PKC are operably linked in this order.

[57] An expression construct comprising a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1, a nucleic acid encoding a protein, and a nucleic acid encoding a peptide comprising a Pro region or a RING region, all of which are operably linked.

[58] The expression construct according to item 57, wherein a promoter, a nucleic acid encoding a protein, and a nucleic acid encoding a peptide comprising a Pro region or a RING region are operably linked in this order.

[59] The expression construct according to item 57 or 58, comprising a nucleic acid encoding an internal ribosome entry site or a 2A peptide sequence.

[60] The expression construct according to any one of items 57 to 59, wherein a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding a peptide comprising a Pro region or a RING region are operably linked in this order.

[61] A composition for activating PKC, comprising Compound X and a compound selected from Compounds #1 to #7, or an ester, salt, or solvate thereof.

[0094] All documents cited in this specification are incorporated herein by reference. The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples. The above descriptions are all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible within the scope of the technical idea thereof.

[0095] Constructs: pRL-CMV (Promega, #E2261) was used to measure luciferase activity driven by the CMV promoter. The CAG promoter was derived from the pCAGGS vector, and the EF1 promoter was derived from the pEFBOS vector. These constructs were created by replacing the CMV region of pRL-CMV. Mouse IgG antibody (heavy chain, light chain), human proinsulin, and human leptin expression constructs were created by inserting these cDNAs downstream of the CMV promoter in the pcDNA vector.

[0096] HEK293A cells (human embryonic kidney cells) were cultured in DMEM medium containing 10% FCS and 1% penicillin / streptomycin at 37°C and 5% CO ２ The cells were cultured under conditions of 0°C and 90% humidity. HEK293A cell lines, which constitutively express mouse IgG antibody, human proinsulin, and leptin, were transfected with constructs in which these cDNAs were inserted downstream of the CMV promoter, and then single clones were formed by long-term culture. Expression of the target protein from each clone was then confirmed using ELISA, and cell lines were established. These cell lines were also cultured under the same conditions.

[0097] Transfection: HEK293A cells were transfected into 24-well plates at 1.0 × 10 ５Unless otherwise specified, transfection was performed by adding 50 ng of expression construct per well, 1 μl of Plus Reagent, and 1 μl of Lipofectamine LTX to each well of a 24-well plate containing cultured cells, and culturing at 37°C for 24 hours.

[0098] Luciferase Assay: 24 hours after transfection, the medium was removed from the wells, and medium containing various concentrations of compounds was added. The cells were then cultured for another 24 hours. The medium was then removed, washed with 500 μl of PBS, and lysed with 50 μl of 1x Glo Lysis buffer (Promega #E2661). 5 μl of this solution was used for luciferase activity measurement, and 5 μl was used for protein concentration measurement. Luciferase activity was measured by detecting the luminescence of coelenterazine h (FUJIFILM #035-22991) to quantify Rluc expression. Protein concentration was quantified using the BCA method, and each luciferase activity was calculated as the Rluc / BCA value. Luminescence detection and BCA measurement were performed using a Multilabel Reader 2030 ARVO™ X (Perkin Elmer).

[0099] Western blot: HEK293A cells were plated in a 6-well plate at 5.0 × 10 ５Cells were seeded at 1000 cells / well and cultured for 24 hours. After treatment with each compound, the medium was replaced. After 3 hours, the cells were washed with 2 ml of PBS and then lysed in 200 μl of RIPA buffer. The lysed cells were disrupted by sonication and centrifuged (20,000 g, 15 minutes, 4°C). The supernatant was collected and the protein concentration was quantified. Equal volumes of the lysates were combined, 1x SDS sample buffer was added, and the mixture was heated at 95°C for 3 minutes. The resulting mixture was used for SDS-PAGE (5 μg of protein per lane). SDS-PAGE gels were SuperSep™ Ace 5-20%, 17-well (Wako) gels, and electrophoresis was performed at 500 V, 40 mA, and 35 minutes per gel. The gels were then transferred to PVDF membranes using a blocking device (ATTO) at 500 V, 100 mA, and 60 minutes per gel. After transfer, the membrane was permeated with blocking solution (3% BSA / TBS-T) for 30 minutes at room temperature. The primary antibody reaction was performed overnight at 4°C using a 3000-fold dilution of the primary antibody (Phospho-PKC Substrate Motif [(R / K)XpSX(R / K)] MultiMab™ Rabbit mAb mix, Cell Signaling Technology #6967) in blocking solution. The membrane was then washed three times with TBS-T. The secondary antibody reaction was performed for 3 hours at room temperature using a 5000-fold dilution of the HRP-conjugated secondary antibody (Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey, Cytiva #NA934-1ML) in TBS-T. The membrane was then washed three times with TBST and detected using Chemi-Lumi One (Nacalai #07880-70) and ImageQuant LAS4010.

[0100] ELISA method: Each secreted protein-expressing cell was plated on a 24-well plate at 1.0 × 10 ４Cells were seeded at 1 cell / well and cultured for 24 hours. Each compound was then treated with a 1 ml / well medium change. 120 μl of medium was collected every 24 hours and stored refrigerated. The concentration of target proteins in the medium was then measured using the appropriate ELISA kits (mouse IgG, Betyl Lab #E99-131; human proinsulin: Mercodia #10-1118-01; human leptin: Proteintech #KE00095). Measurements were performed using a Multilabel Reader 2030 ARVO™ X (Perkin Elmer).

[0101] Compounds The compounds in the table below were used.

[0102] Test 1: Identification of Compounds that Enhance CMV Promoter Transcriptional Activity. HEK293T cells were transfected with a plasmid containing a luciferase gene inserted downstream of the CMV promoter at 20 ng per well of a 24-well plate. After 24 hours, the medium was replaced with one containing 30 μg / ml of plant extract. After 24 hours, CMV promoter activity was measured using a luciferase assay. Approximately 1,000 plant extracts were used in this study. From a proprietary plant (herbal medicine) extract library, five plant extracts that dramatically enhance CMV promoter transcriptional activity and the compounds responsible for this activity (eight compounds listed below) were identified.

[0103] Test 2: Compound activity is inhibited by protein kinase C (PKC) inhibitors. HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in medium containing Compound X or Compounds #1 to #7 at the concentrations shown in Figure 1, in the presence or absence of a PKC inhibitor, and luciferase activity was measured. The results are shown in Figure 1. Transcriptional activity of the CMV promoter was enhanced in the presence of the compounds, and this enhancement was inhibited by a PKC inhibitor.

[0104] HEK293A cells constitutively expressing pRL-CMV were cultured in medium containing Compound X (100 ng / ml) for 24 hours, and luciferase activity was measured over time. The results are shown in Figure 2. Enhancement of CMV promoter transcription activity by Compound X was observed starting 1 hour after Compound X addition.

[0105] Test 3: CMV promoter activity is also enhanced by known PKC activators. HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in medium containing Compound X or PKC activators #1 to #4 at the concentrations shown in Figure 3, in the presence or absence of a PKC inhibitor, and luciferase activity was measured. The results are shown in Figure 3. CMV promoter transcription activity was also enhanced by known PKC activators.

[0106] Compound X, compounds #1 to #7, and PKC activators #1 to #4 belong to the terpene group, collectively known as diterpenes. They are classified into the following three types based on their skeletons:

[0107] Test 4: CMV promoter activity is also enhanced by PKC activators other than diterpenes. HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in medium containing Compound X or PKC activators #5 to #6 at the concentrations shown in Figure 4, in the presence or absence of a PKC inhibitor (Ro-318425), and luciferase activity was measured. The results are shown in Figure 4. CMV promoter transcription activity was also enhanced by PKC activators other than diterpenes.

[0108] Test 5: Compounds also enhance the activity of the CAG promoter and EF1 promoter. HEK293A cells were transfected with pCAG-Rluc or pEF1-Rluc and cultured in medium containing Compound X, Compounds #1 to #7, or PKC activators #1 to #6 at the concentrations shown in Figures 5 to 7. The transcriptional activity of the CAG promoter derived from the pCAGGS vector and the EF1 promoter derived from the pEFBOS vector was measured. The results are shown in Figures 5 to 7. The transcriptional activity of the CAG promoter and EF1 promoter was enhanced in the presence of the compounds or PKC activators, but this enhancement was inhibited by a PKC inhibitor. These promoters contain binding sites for transcription factors such as SP1, CEBP, AP1, NF-κB, and YY1. This suggests that these compounds and PKC activators activate these promoters via a common transcription factor.

[0109] Test 6: Compounds activate PKC. HEK293A cells were cultured for 3 hours in medium containing Compound X, Compounds #1 to #7, PKC activators #1 to #6, Compound SC, or sodium butyrate (SB) at the same concentrations as in Tests 2 to 5, in the presence or absence of a PKC inhibitor (3 μM). Cells were lysed, and Western blot analysis was performed using an antibody that specifically recognizes proteins phosphorylated by PKC. The results are shown in Figure 8. Compound X, Compounds #1 to #7, and PKC activators #1 to #6 activated PKC. Compound SC and sodium butyrate did not activate PKC.

[0110] Test 7: Identification of new CMV promoter activators HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in medium containing Compound X and / or Compound SC at the concentrations shown in Figure 9 in the presence or absence of a PKC inhibitor, and luciferase activity was measured. The results are shown in Figure 9. The transcriptional activity of the CMV promoter was also enhanced by Compound SC, but this enhancement was not inhibited by the PKC inhibitor. The combined use of Compound X and Compound SC further enhanced the transcriptional activity.

[0111] Experiment 8: CMV promoter transcriptional activity is further enhanced by the combined use of a PKC activator, Compound SC, and a histone deacetylase inhibitor. HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in medium containing Compound X, Compound SC, and / or sodium butyrate (SB) at the concentrations shown in Figure 10, and luciferase activity was measured. The results are shown in Figure 10. CMV promoter transcriptional activity was highest when Compound X, Compound SC, and sodium butyrate were used in combination. Similar results were obtained with Compounds #1 to #7 and PKC activators #1 to #6 (Figures 11 and 12). Similar results were obtained when the histone deacetylase inhibitors valproic acid, trichostatin A (TSA), vorinostat (SAHA), apicidin, entinostat (MS-275), or tacedinaline (CI994) were used instead of sodium butyrate (Figure 13).

[0112] Experiment 9: Increased Protein Production with PKC Activators. Expression constructs were prepared by inserting cDNA for mouse IgG antibody heavy chain, mouse IgG antibody light chain, human proinsulin, or human leptin downstream of the CMV promoter in a pcDNA vector. The mouse IgG antibody and human leptin constructs were transfected into HEK293A cells, respectively. HEK293A cells were transfected with a human proinsulin construct to generate HEK293A cells constitutively expressing human proinsulin. Cells were cultured for 24 hours in medium containing Compound X, Compounds #1 to #7, and PKC activators #1 to #6 at the concentrations shown in Figures 14 to 17, in the presence or absence of Compound SC or Compound SC plus sodium butyrate (SB), and the concentrations of each protein in the medium were measured by ELISA. The results are shown in Figures 14 to 17. Furthermore, for Compound X, HEK293A cells constitutively expressing mouse IgG antibody heavy and light chains, human proinsulin, or human leptin were used, and protein concentrations were measured every 24 hours for 4 days in the presence or absence of sodium butyrate (SB). The results are shown in Figure 18. The production of IgG antibodies, proinsulin, and leptin was enhanced by these compounds. The production of IgG antibodies and proinsulin was further enhanced when these compounds were combined with Compound SC and sodium butyrate. The production of leptin was most enhanced when these compounds were combined with Compound SC, and although it decreased in the presence of SB, it was still higher than when cultured in a medium without any of the substances.

[0113] Experiment 10: Activation of the CMV promoter by genetically engineered PKC activation. PKC was activated by genetic engineering to examine the transcriptional activity of the CMV promoter. Expression constructs were used in which the protein cDNA (XXX), an IRES, and an activated PKC cDNA were ligated downstream of the CMV promoter. The activated PKCs used were the 334-695 amino acid region of human PKCδ (PKCδ-CA) and the constitutively activated form of human PKCα (PKCαCA-M489V), in which the methionine at position 489 in the 326-672 amino acid region was changed to valine. Furthermore, genes encoding Rluc, mouse IgG antibody (heavy chain, light chain), and human leptin were inserted into the XXX region. A schematic diagram of these constructs is shown in Figure 19.

[0114] Expression constructs were prepared by inserting Rluc cDNA, an IRES, and either PKCδ-CA or PKCαCA-M489V cDNA downstream of the CMV promoter in a pcDNA vector. Control constructs lacking PKC were also prepared. These constructs were transfected into HEK293A cells at concentrations ranging from 6.25 to 200 ng / well, cultured for 24 hours, and luciferase activity was measured. The results are shown in Figure 20. High luciferase activity was observed in the PKC group. This result indicates that the transcriptional activity of the CMV promoter is enhanced by positive feedback. Similar results were obtained with an expression construct that used the P2A peptide sequence, a 2A self-cleaving peptide sequence, instead of the IRES (Figure 21).

[0115] Expression constructs were prepared by inserting mouse IgG antibody heavy or light chain cDNA, an IRES, and PKCδ-CA or PKCαCA-M489V cDNA downstream of the CMV promoter in a pcDNA vector. Control constructs lacking PKC were also prepared. The IgG antibody heavy and light chain constructs were combined and transfected into HEK293A cells at a concentration of 100 ng / well. The cells were cultured for 24 hours, and the IgG antibody concentration in the medium was measured by ELISA every 24 hours over 4 days. The results are shown in Figure 22. IgG antibody production was enhanced in the PKC group. These results demonstrate that genetically engineered PKC activation can activate the CMV promoter and enhance protein production.

[0116] An expression construct was prepared by inserting human leptin cDNA, an IRES, and PKCαCA-M489V cDNA downstream of the CMV promoter in a pcDNA vector. A control construct lacking PKC was also prepared. These constructs were transfected into HEK293A cells at a concentration of 100 ng / well and cultured for 24 hours. The leptin concentration in the medium was measured over 4 days by ELISA. The results are shown in Figure 23. Leptin production was enhanced in the PKC group. This result demonstrates that genetically engineered PKC activation can activate the CMV promoter and enhance protein production.

[0117] Test 11: Search for target proteins of compound SC Naphthalenesulfonamide derivatives having the following structure, including compound SC, were examined for their synergistic effect with compound X.

[0118] HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in the presence or absence of compound X (100 ng / ml) in a medium containing one of the naphthalenesulfonamide derivatives at the concentrations shown in Figure 24, and luciferase activity was measured. The results are shown in Figure 24. The transcriptional activity of the CMV promoter did not change when the naphthalenesulfonamide derivative was used alone, but was enhanced when the naphthalenesulfonamide derivative was used in combination with compound X. The naphthalenesulfonamide derivative W-7 is used as a calmodulin inhibitor, and compounds SC and SC-9 also act as calmodulin inhibitors, presumably exhibiting a synergistic effect with compound X.

[0119] Experiment 12: Transcriptional activity of the CMV promoter is enhanced by PKC activators and genetically engineered PML expression. An expression construct was prepared by inserting Rluc cDNA, an IRES, and cDNA for wild-type PML or deletion mutant PML (PMLΔ1-10 or PML-Ring) downstream of the CMV promoter in a pcDNA vector (Figure 25). HEK293A cells were transfected with wild-type PML and PMLΔ1-10 constructs and cultured for 24 hours in the presence or absence of Compound X, and luciferase activity was measured. The results are shown in Figure 26. Luciferase activity in the presence of Compound X was higher in cells transfected with wild-type PML, PMLΔ1, and PMLΔ6-10 compared to control (-) cells. These results suggest that PML further enhances the transcriptional activity of the CMV promoter, which is enhanced by Compound X, and that the Pro region of PML, including positions 1 to 46, is responsible for this activity. Furthermore, in cells transfected with wild-type PML, PMLΔ1, and PMLΔ6-10, luciferase activity was higher than that in control (−) cells, even in the absence of Compound X.

[0120] Furthermore, constructs containing PMLΔ9 or PML-Ring were transfected into HEK293A cells and cultured for 24 hours in the presence or absence of Compound X, SB, Compound SC, or a combination thereof, and luciferase activity was measured. The results are shown in Figure 27. Luciferase activity was higher in cells transfected with PMLΔ9 or PML-Ring than in control (Mock) cells under all conditions. This result suggests that the RING region containing positions 47 to 106 of PML also further enhances the transcriptional activity of the CMV promoter, which is enhanced by Compound X. Furthermore, luciferase activity was higher in cells transfected with PMLΔ9 or PML-Ring than in control (-) cells, even in the absence of Compound X.

[0121] Experiment 13: Genetic expression of a PKC activator and a peptide containing a RING domain enhances the transcriptional activity of the CMV promoter. The RING domain has a common sequence among various proteins. BLAST analysis of the amino acid sequence of the RING domain of PML was performed, and the proteins shown in Figure 28 were identified as proteins with similar sequences. High conservation was observed in several cysteine ​​and histidine residues in the RING domains of these proteins. Several proteins with different degrees of similarity to the RING domain of PML were selected and used in the following analysis.

[0122] Expression constructs were prepared by inserting Rluc cDNA, an IRES, and the cDNA of the RING domain of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1, or HTLF downstream of the CMV promoter in a pcDNA vector (Figure 29). These expression constructs were transfected into HEK293A cells, which were cultured for 24 hours in the presence or absence of Compound X, and luciferase activity was measured. The results are shown in Figure 30. In cells transfected with the RING domain, luciferase activity was higher in the presence of Compound X than in control (-) cells. This result suggests that the RING domains of various proteins further enhance the transcriptional activity of the CMV promoter, which is enhanced by Compound X. Furthermore, in cells transfected with the RING domain, luciferase activity was higher than in control (-) cells, even in the absence of Compound X.

[0123] Experiment 14: Enhancement of CMV promoter transcriptional activity by PML depends on mRNA expression. Expression constructs were prepared by inserting Rluc cDNA, an IRES, and cDNAs for wild-type PML or deletion mutant PML (PMLΔ9, PMLΔ9 with a stop codon introduced at position 2, or PMLΔ9 with a stop codon introduced at position 7) downstream of the CMV promoter in a pcDNA vector (Figure 31, left). Expression constructs were also prepared by inserting Rluc cDNA and cDNAs for wild-type PML or deletion mutant PML (PMLΔ9, PMLΔ10, or PML-RING) downstream of the CMV promoter in a pcDNA vector (Figure 31, right). When these constructs were introduced into cells, PML mRNA was transcribed but not translated into protein. These constructs were transfected into HEK293A cells, cultured for 24 hours in the presence or absence of Compound X, and luciferase activity was measured. The results are shown in Figure 32. For all constructs, higher luciferase activity was observed in the presence and absence of Compound X than in the control. This result suggests that the enhancement of the transcriptional activity of the CMV promoter by PML depends on the expression of mRNA encoding the Pro or RING region of PML, rather than on the PML protein.

[0124] The present disclosure can be used in the production of biopharmaceuticals, as it allows for increased production of proteins of interest.

Claims

1. A method for producing a protein of interest, comprising culturing cells containing a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1 under conditions that activate protein kinase C (PKC); The method, wherein the culturing under conditions that activate PKC is any of the following: (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor; (2) expressing activated PKC in the cells and culturing them in the presence of a calmodulin inhibitor; (3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in the cells and culturing the cells in the presence of a PKC activator; and, （４） 【Chemical 1】 The cells are cultured in the presence of a compound selected from the group consisting of:

2. The method of claim 1, wherein the culturing under conditions that activate PKC is (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor.

3. The method according to claim 1, wherein the culturing under conditions that activate PKC comprises (2) expressing activated PKC in the cells and culturing the cells in the presence of a calmodulin inhibitor.

4. The method of claim 3, wherein the cell contains an expression construct comprising, operably linked together, a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding an activated form of PKC.

5. The method of claim 1, wherein the culturing under conditions that activate PKC is (3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in a cell and culturing the cell in the presence of a PKC activator.

6. The method of claim 5, wherein a nucleic acid encoding a peptide comprising a RING region is expressed in a cell.

7. 7. The method of claim 6, wherein the RING region is the RING region of TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22, KIAA0182, TRIM65, RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML, or TRIM56.

8. 7. The method of claim 6, wherein the RING region is a RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1 or HTLF.

9. The method according to claim 5, wherein a nucleic acid encoding a peptide comprising a Pro region is expressed in a cell.

10. The method of claim 5, wherein the cell contains an expression construct comprising, operably linked together, a promoter, a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide comprising a Pro region or a RING region.

11. The PKC activator has the formula (I): 【Chemistry 2】 {During the ceremony, R １ is H, halogen, -OH, C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Alkoxy, C ６－１４ Aryl or —OC(O)R ３ where C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ The alkoxy or aryl may be substituted with 1 to 3 halogen atoms, which may be the same or different; R ２ is C ６－１２ Alkyl, C ６－１２ Alkenyl, C ６－１２ Alkynyl or C ６－１２ Alkoxy, where C ６－１２ Alkyl, C ６－１２ Alkenyl, C ６－１２ Alkynyl or C ６－１２ The alkoxy may be substituted with 1 to 3 halogen atoms, which may be the same or different; R ３ is C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, amino or C ６－１４ aryl, where C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl or C ６－１４ the aryl may be substituted with 1 to 3 halogen atoms, which may be the same or different; Formula (II): 【Chemistry 3】 {During the ceremony, R ４ is H, halogen, -OH, C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ Alkynyl, C １－１８ Alkoxy or —OC(O)R ６ where C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ Alkynyl or C １－１８ The alkoxy may be substituted with 1 to 3 halogen atoms, which may be the same or different; R ５ is C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ alkynyl or amino, wherein C １－６ Alkyl, C ２－６ alkenyl or C ２－６ The alkynyl may be optionally substituted with 1 to 3 halogens, which may be the same or different; R ６ is C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ alkynyl or amino, where C １－１８ Alkyl, C ２－１８ alkenyl or C ２－１８ alkynyl may be substituted with 1 to 3 halogen atoms, which may be the same or different; or Formula (III): 【Chemistry 4】 {During the ceremony, R ７ is H, C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ Alkynyl or —C(O)R ９ where C １－１８ Alkyl, C ２－１８ alkenyl or C ２－１８ The alkynyl may be optionally substituted with 1 to 3 halogens, which may be the same or different; R ８ is H, C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ Alkynyl or —C(O)R ９ where C １－１８ Alkyl, C ２－１８ alkenyl or C ２－１８ The alkynyl may be optionally substituted with 1 to 3 halogens, which may be the same or different; R ９ is C １－１８ Alkyl, C ２－１８ Alkenyl, C ２－１８ alkynyl or amino, where C １－１８ Alkyl, C ２－１８ alkenyl or C ２－１８ The alkynyl may be substituted with 1 to 3 halogen atoms, which may be the same or different. or an ester, salt or solvate thereof.

12. The method of any one of claims 2 and 5 to 10, wherein the PKC activator is a compound selected from compound X, compounds #1 to #7, TPA, prostratin, (-)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate, and (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam, or an ester, salt, or solvate thereof.

13. The method according to any one of claims 2 and 5 to 10, wherein the PKC activator is a compound selected from Compound X and Compounds #1 to #7, or an ester, salt, or solvate thereof.

14. Calmodulin inhibitors Formula (IV) 【Chemistry 5】 {During the ceremony, n is an integer from 1 to 8, R is C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Alkoxy, C ６－１４ aryl, amino, hydroxy, COOH or COOR′, where R′ is C １－６ alkyl} or an ester, salt or solvate thereof.

15. The method according to claim 1, wherein the culturing under conditions that activate PKC is culturing the cells in the presence of (4) a compound selected from Compound X and Compounds #1 to #7, or an ester, salt, or solvate thereof.

16. The method according to any one of claims 1 to 10, wherein the cells are cultured in the presence of a histone deacetylase inhibitor.

17. 17. The method of claim 16, wherein the histone deacetylase inhibitor is sodium butyrate.

18. A method for enhancing transcription of a gene of interest, comprising culturing a cell containing nucleic acid of the gene operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1 under conditions that activate PKC; The method, wherein the culturing under conditions that activate PKC is any of the following: (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor; (2) expressing activated PKC in the cells and culturing them in the presence of a calmodulin inhibitor; (3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in the cells, and culturing the cells in the presence of a PKC activator; and （４） 【Chemistry 6】 The cells are cultured in the presence of a compound selected from the group consisting of:

19. A kit for producing a protein of interest, comprising: (1) PKC activators and calmodulin inhibitors, (2) a nucleic acid encoding an activated PKC and a calmodulin inhibitor; (3) a nucleic acid encoding a PKC activator and a peptide containing a Pro region or a RING region, or （４） 【Chemistry 7】 or an ester, salt or solvate thereof, Including, A kit, wherein the production of a protein of interest comprises culturing cells containing a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.

20. An expression construct comprising a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1, operably linked thereto, a nucleic acid encoding a protein, an internal ribosome entry site or a 2A peptide sequence, and a nucleic acid encoding an activated form of PKC.

21. An expression construct comprising: a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1, operably linked thereto; a nucleic acid encoding a protein; and a nucleic acid encoding a peptide comprising a Pro region or a RING region.

22. [Chemical 8] A composition for activating PKC, comprising a compound selected from the group consisting of:

23. A method for producing a protein of interest, comprising expressing in cells and culturing an expression construct comprising: a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1, all operably linked to the promoter; a nucleic acid encoding the protein of interest; and a nucleic acid encoding a peptide comprising a Pro region or a RING region.

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