IL3 fusion protein with improved expression levels and solubility, and use thereof

Abstract

The present invention relates to an IL3 fusion protein with enhanced solubility and expression levels in various E. coli strains using a Protein Disulfide Isomerase (PDI) or Maltose-Binding Protein (MBP) tag. The fusion protein and IL3 isolated from the fusion protein not only maintain biological activity but also induce a greater TF-1 cell proliferation or differentiation effect compared to commercial IL3. The TF-1 cell proliferation effect of the fusion protein and IL3 isolated from the fusion protein can be utilized for the treatment of hematopoietic disorders.

Description

Technology Field

[0001] The present invention relates to an IL3 fusion protein with improved expression levels and solubility in various Escherichia coli strains and its uses. Background Technology

[0002] Human interleukin 3 (IL3) is a multifunctional cytokine involved in regulating cell development, growth, and activation in the hematopoietic and immune systems. Due to the diversity of its functions, IL3 is also referred to by various alternative names, ranging from multi-colony stimulating factor and persisting cell stimulating factor to hematopoietic cell growth factor and stem cell activating factor.

[0003] IL3 belongs to the β common chain cytokines, which include human granulocyte-macrophage colony-stimulating factor (hGM-CSF) and human interleukin-5 (hIL5). These cytokines coordinate various inflammatory responses to rapidly eliminate pathogens, while playing an important role in the pathogenesis of chronic inflammation. Functionally, IL3 promotes the proliferation and differentiation of various hematopoietic cells derived from hematopoietic stem cells, such as macrophages, dendritic cells, mast cells, basophils, and eosinophils. From a clinical perspective, IL3 enhances the expansion of hematopoietic progenitors after bone marrow transplantation, improves the recovery of stem cells, CD34+ cells, platelets, eosinophils, and T cells, acts as an adjuvant for aplastic anemia and chemotherapy protocols, and provides potential treatments for leukemia and allergic diseases. Furthermore, as IL3 is known as an essential factor for the in vitro maturation of various hematopoietic cells from stem cell progenitors, the efficient production of IL3, which plays a pivotal role in hematopoiesis and potential therapeutic applications, is important for both biomedical research and clinical purposes.

[0004] However, the efficient production of recombinant IL3 is a difficult task. IL3 is a secreted protein composed of 133 amino acids, characterized by intramolecule disulfide bonds and two putative N-linked glycosylations. Conventional E. coli ( Escherichia coli or E. coli There have been attempts to produce IL3 in ), but when IL3 is expressed in E. coli, it shows low expression levels due to aggregation into inclusion bodies, and complex solubilization and refolding methods are required to solve this. Therefore, there is a need for an efficient production system for recombinant IL3 that maintains biological activity.

[0005] The inventors prepared a recombinant IL3 fusion protein with improved solubility and expression levels in various E. coli strains using a Protein Disulfide Isomerase (PDI) or Maltose-Binding Protein (MBP) tag. Furthermore, using the fusion protein, they paved the way for the mass production of recombinant IL3 that maintains biological activity in E. coli. The problem to be solved

[0006] One aspect provides a fusion protein comprising protein disulfide isomerase (PDI) or maltose-binding protein (MBP); and human interleukin-3 (IL3).

[0007] Another aspect is to provide a recombinant IL3 protein from which the protein disulfide isomerase (PDI) or maltose-binding protein (MBP) portion is removed from the fusion protein.

[0008] Another aspect is to provide a nucleotide encoding the fusion protein and a recombinant vector containing it.

[0009] Another aspect is to provide host cells transformed with the aforementioned recombinant vector.

[0010] Another aspect provides a method for producing a recombinant IL3 protein with improved expression levels and solubility, comprising the step of culturing the recombinant microorganism in a medium to express the fusion protein.

[0011] Another aspect is to provide a composition for promoting cell proliferation or differentiation comprising the recombinant IL3 protein.

[0012] Another aspect provides a composition for improving hematopoietic function comprising a fusion protein or recombinant IL3 protein according to the present invention.

[0013] Another aspect provides a composition for improving hematopoietic function comprising a fusion protein or recombinant IL3 protein according to the present invention.

[0014] Another aspect provides a pharmaceutical composition for the prevention or treatment of hematopoietic disorders comprising a fusion protein or recombinant IL3 protein according to the present invention.

[0015] Another aspect provides a health functional food composition for the prevention or improvement of hematopoietic disorders comprising a fusion protein or recombinant IL3 protein according to the present invention. means of solving the problem

[0016] To achieve the above objective, one aspect provides a fusion protein comprising protein disulfide isomerase (PDI) or maltose-binding protein (MBP); and human interleukin-3 (IL3).

[0017] In this specification, the term "fusion protein" refers to an artificial recombinant protein that is expressed by linking one or more genes of other proteins to a gene encoding a specific protein.

[0018] In one embodiment, the fusion protein may have the amino acid sequence of any fusion protein in which PDI or MBP is combined with IL3, but preferably, PDI or MBP may be fused to the N-terminus of IL3. For example, the fusion protein may be a structure in which PDI or MBP is fused to the N-terminus of IL3 (PDI-IL3 or MBP-IL3).

[0019] In one embodiment, the fusion protein may include DNA encoding a protease cleavage (recognition) site. The protease cleavage site is included to facilitate the separation of IL3 protein from the expressed PDI or MBP-IL3 fusion protein, and the protease may be one or more selected from the group consisting of factor Xa protease, thrombin, enterokinase, and tobacco etch virus (TEV) protease, and preferably may be TEV (Tobacco Etch Virus) protease.

[0020] In one embodiment, the fusion protein may have a tobacco etch virus (TEV) protease cleavage site inserted therein. Since the TEV protease recognizes the ENLYFQ↓G (Glu - Asn - Leu - Tyr - Phe -Gln ↓ Gly) sequence and cleaves the labeled region, the IL3 protein can be easily recovered from the expressed PDI or MBP-IL3 fusion protein by inserting DNA encoding the sequence into the vector. Specifically, the TEV protease cleavage site may be introduced at the N-terminus of the IL3 sequence. For example, the fusion protein may have a TEV protease cleavage site (ENLYFQ / G) inserted between the PDI or MBP tag and the IL3 coding sequence so that the PDI or MBP tag can be removed in a subsequent purification step, but is not limited thereto. More specifically, the PDI may be a PDIb'a' domain, and the fusion protein may have the amino acid sequence of SEQ ID NO. 1.

[0021] In one embodiment, the fusion protein may include a hexahistidine residue at the N-terminus of PDI or MBP to facilitate purification.

[0022] The term "protein disulfide isomerase (PDI)" in this specification refers to an enzyme that has the function of causing disulfide bonds to occur at the correct location when proteins synthesized in cells are folded and packed into a three-dimensional structure. In the present invention, PDI is inserted as a protein tag at one end of an IL3 protein to perform the role of increasing the expression level and solubility of IL3 within E. coli.

[0023] In one embodiment, the protein disulfide isomerase (PDI) may be a PDI b'a' domain (protein disulfide isomerase b'a' domain), but is not limited thereto.

[0024] The term "maltose-binding protein (MBP)" in this specification is Escherichia coli As a protein found in bacteria such as E. coli and primarily present in the cell membrane of malt, in the present invention, MBP is inserted as a protein tag at one end of the IL3 protein to perform the role of increasing the expression level and stability of IL3 within E. coli.

[0025] The term "Human interleukin-3 (IL3)" in this specification refers to a protein encoded by the human IL3 gene located on chromosome 5q31.1, said IL3 being produced as a monomer by activated T cells, monocytes / macrophages and stromal cells and regulating the concentration of various types of blood cells. Additionally, it induces the proliferation and differentiation of both early pluripotent stem cells and reproductive progenitor cells, and also influences platelet regeneration or early antibody isoform conversion.

[0026] The PDI protein, MBP protein, or IL3 protein constituting the above fusion protein includes not only those having their natural amino acid sequences but also amino acid sequence variants thereof. The term "amino acid sequence variant of the PDI protein, MBP protein, or IL3 protein" refers to a protein having a sequence different from the natural amino acid sequence of the PDI protein, MBP protein, or IL3 protein by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues. The above PDI protein, MBP protein, IL3 protein, and their variants may be extracted from nature, synthesized, or produced by genetic recombination methods based on DNA sequences.

[0028] Another aspect is to provide a recombinant IL3 protein from which the protein disulfide isomerase (PDI) or maltose-binding protein (MBP) portion is removed from the fusion protein.

[0029] The recombinant IL3 protein above may be one in which the protein disulfide isomerase (PDI) or maltose-binding protein (MBP) portion is removed from the fusion protein above, and may include a polypeptide having the amino acid sequence represented by SEQ ID NO. 2 and a functional equivalent of said polypeptide.

[0030] The term "functional equivalent" in this specification refers to a polypeptide that exhibits substantially the same physiological activity as the PDI protein, MBP protein, recombinant IL3 protein, or fusion protein thereof, having at least 80%, more preferably 90% or more, sequence homology with the amino acid sequence as a result of the addition, substitution, or deletion of amino acids. Methods for exchanging amino acids of a protein without significantly altering the physiological activity of the protein are known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The proteins may, in some cases, be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc.

[0031] The definitions of the above "protein disulfide isomerase (PDI)", "maltose-binding protein (MBP) portion", and "recombinant IL3 protein" are as previously stated.

[0033] Another aspect provides a nucleotide encoding the fusion protein.

[0034] The above polynucleotide may be a single-stranded or double-stranded having a base sequence, may be DNA or RNA, and the above polynucleotide may be isolated from nature or prepared by chemical synthesis.

[0035] In the present invention, the gene capable of encoding the PDI protein, MBP protein, recombinant IL3 protein and / or fusion protein thereof can be artificially synthesized using a nucleic acid synthesizer or the like, or can be prepared by performing PCR using genomic DNA of the gene capable of encoding the PDI protein, MBP protein, recombinant IL3 protein and / or fusion protein thereof as a template, and using oligonucleotides having sequences complementary to both ends of the gene capable of encoding the desired PDI protein, MBP protein, recombinant IL3 protein and / or fusion protein thereof as primers.

[0036] Meanwhile, due to the optimization of codons, genes capable of encoding the PDI protein, MBP protein, recombinant IL3 protein and / or fusion proteins thereof according to the present invention may exist in various nucleotide sequences, and all nucleotide sequence variants of said proteins are included within the scope of the present invention. Specifically, genes capable of encoding the PDI protein, MBP protein, recombinant IL3 protein and / or fusion proteins thereof may each include nucleotide sequences having sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more. The "% of sequence homology" for said polynucleotides is determined by comparing two optimally arranged sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal arrangement of the two sequences.

[0038] Another aspect provides a recombinant vector comprising a nucleotide encoding the fusion protein. In the present invention, the recombinant vector may be any recombinant vector in which a polynucleotide encoding the PDI or MBP-IL3 fusion protein is inserted into a vector, but preferably may be a recombinant vector inserted into a vector comprising a promoter that can be expressed in prokaryotic cells.

[0039] The term "vector" in this specification refers to a self-replicating DNA molecule used to carry a clonal gene (or other fragment of clonal DNA). The vector may be, but is not limited to, a plasmid, a phage particle, or simply a potential genomic insert. When the vector is transformed into a suitable host, it may replicate and function independently of the host genome, or parts of it may be incorporated into the genome itself. Specifically, it refers to a plasmid, virus, or other medium into which a polynucleotide sequence encoding the fusion protein may be inserted or introduced. For example, the above plasmids include plasmids derived from E. coli (pBR322, pBR325, pUC118 and pUC119, pET-22b(+)), plasmids derived from Bacillus subtilis (pUB110 and pTP5), and plasmids derived from yeast (YEp13, YEp24 and YCp50), and the above viruses may be animal viruses such as retroviruses, adenoviruses, or vaccinia viruses, or insect viruses such as baculoviruses. Specifically, a vector suitable for introducing a polynucleotide that carcinogenizes the fusion protein of the present invention into a host cell may be used, and preferably, an expression vector designed to facilitate the induction of protein expression and the isolation of the expressed protein may be used.

[0040] The term "expression vector" in this specification refers to a recombinant DNA molecule comprising a nucleic acid sequence essential for expressing a target sequence and an coding sequence linked so as to be operable in a specific host organism. Specifically, a polynucleotide sequence encoding a fusion protein according to the present invention may be operablely linked to an expression regulatory sequence, and said operablely linked gene sequence and expression regulatory sequence may be included in a single expression vector that also includes a selection marker and a replication origin.

[0041] The expression vector may preferably include one or more selectable markers. The marker is a nucleic acid sequence having characteristics that can typically be selected by chemical means, and includes any gene capable of distinguishing transformed cells from non-transformed cells. Examples include, but are not limited to, antibiotic resistance genes such as ampicillin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, and can be appropriately selected by a person skilled in the art.

[0042] The term “operably linked gene sequence” in this specification may be a gene and an expression control sequence linked in such a manner that gene expression is enabled when a suitable molecule is bound to the expression control sequence. The “expression control sequence” means a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a specific host cell. Such a control sequence includes a promoter for carrying out transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0043] The term "Gateway Vector system" in this specification refers to a DNA cloning technology used to effectively deliver DNA fragments. Specifically, it consists of an entry vector and a destination vector, wherein the entry vector is a vector having attL1 and attL2 at both ends of the destination gene, and the destination vector is a vector containing attR1 and attR2. The entry vector and the destination vector undergo an LR reaction with attR1 and attR2 of the destination vector by means of a recombinase, and in this process, the destination gene contained in the entry vector is transferred to the destination vector, and attR1 and attR2 are replaced with the sequences attB1 and attB2.

[0044] In one embodiment, the expression vector of the present invention can be produced using the BP and LR reactions of the Gateway cloning system. For example, it can be produced by cloning a codon-optimized IL3 gene into a pDONR207 vector to generate an entry clone through a BP recombination system, and then recombining the IL3 entry clone with a target vector (pDest-H-MBP, pDest-H-PDIb'a') using an LR recombination system, but is not limited thereto.

[0046] Another aspect provides a host cell transformed with the above-mentioned recombinant vector.

[0047] A recombinant vector comprising a polynucleotide encoding a fusion protein of the present invention can be introduced into a host cell using methods known in the art. For example, methods for introducing the recombinant vector according to the present invention into a host cell may include, but are not limited to, calcium chloride (CaCl2) and heat shock methods, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and precipitation with PEG (polyethylenglycol).

[0048] In one embodiment, the host cell may be a prokaryotic cell or a eukaryotic cell depending on the type of recombinant vector. Preferably, the host cell may be a bacterium, specifically E. coli, and more specifically, BL21, ClearColi, LOBSTR, Origami 2, Rosetta-gami B and SHuffle T7 It may be any one selected from the group consisting of, but is not limited thereto.

[0050] Another aspect provides a method for producing a recombinant IL3 protein with improved expression level and solubility, comprising the step of culturing the host cell in a medium to express the fusion protein of claim 1.

[0051] The above method comprises culturing the transformed host cells under appropriate media and conditions so that a polynucleotide encoding the fusion protein of the present invention is expressed within the transformed cells. A method for culturing the transformed cells to express a recombinant protein is known in the art; for example, protein expression can be induced by inoculating the transformed cells into a suitable medium for growth and performing a seed culture, and then inoculating the cells into a main culture medium and culturing them under suitable conditions.

[0052] The term "culture" in this specification refers to growing microorganisms under appropriately artificially controlled environmental conditions. The recombinant microorganisms can be grown in ordinary media, and, for example, can be cultured in a nutrient broth medium. The medium contains nutrients required by the microorganisms to be cultured, i.e., the microorganisms serving as the culture medium, in order to cultivate specific microorganisms, and may contain substances for special purposes that are additionally added and mixed. The medium is also referred to as a culture medium or culture solution, and is a concept that encompasses natural media, synthetic media, and selective media. The recombinant microorganisms can be cultured according to ordinary culture methods.

[0053] Specifically, the medium used for the above culture must satisfy the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc., within a conventional medium containing suitable carbon sources, nitrogen sources, amino acids, vitamins, etc. Carbon sources that can be used include a mixed sugar of glucose and xylose as the primary carbon source, and additionally include sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products may be used. These nitrogen sources may be used alone or in combination. The medium may contain monopotassium phosphate, dipotassium phosphate, and corresponding sodium-containing salts as phosphorus. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts may be used as phosphorus. Additionally, inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used. Finally, essential growth substances such as amino acids and vitamins may be used in addition to the above materials.

[0054] In addition, suitable precursors may be used in the culture medium. The aforementioned raw materials may be added to the culture in a batch, fed-batch, or continuous manner in a manner suitable for the culture process, but are not particularly limited thereto. The pH of the culture may be controlled by using basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, or acid compounds such as phosphoric acid or sulfuric acid in a suitable manner.

[0055] The term "solubility" in this specification refers to a characteristic indicating how well a protein dissolves in a solvent (generally water), and is influenced by the characteristics, structure, and surrounding environment of the protein. In recombinant microorganisms transformed using a recombinant vector for producing fusion proteins containing the PDI or MBP protein, the recombinant IL3 protein folds normally and is overexpressed in water-soluble form, so the recombinant IL3 protein can be mass-produced using this.

[0056] In one embodiment, the step of expressing the fusion protein may be to culture at 16 to 40°C for 3 to 20 hours, preferably 18 to 38 It can be cultured in, but is not limited to.

[0057] In one embodiment, the method for producing a recombinant IL3 protein with improved expression levels and solubility may further comprise the steps of: treating a fusion protein with a protease to remove a protein disulfide isomerase (PDI) or maltose-binding protein (MBP) portion; and isolating and purifying the recombinant IL3 protein from which the protein disulfide isomerase (PDI) or maltose-binding protein (MBP) has been removed.

[0058] The step of isolating and purifying the above IL3 protein can be performed using conventional protein isolation methods. For example, after lysing the cells, the lysate can be purified by applying techniques such as salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.), dialysis, gel filtration, chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion), electrophoresis, fractional solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction, either alone or in combination, but is not limited thereto.

[0059] In one embodiment, the step of isolating and purifying the IL3 recombinant protein may involve using IMAC (Immobilized Metal Affinity Chromatography) and / or (ion-exchange chromatography, IEC).

[0060] The term "IMAC (Immobilized Metal Affinity Chromatography)" in this specification refers to a chromatography technique that purifies proteins by utilizing the specific binding between a specific metal ion and a protein. A target protein having a histidine tag can bind to a metal ion in the chromatography matrix to selectively purify the protein.

[0061] The term "ion-exchange chromatography (IEC)" in this specification refers to a method of separating chemical species using a charge difference, wherein cation-exchange chromatography having an anionic functional group as the stationary phase can be used when the target molecule has a positive charge, and anion-exchange chromatography having a cationic functional group as the stationary phase can be used when the target molecule has a negative charge.

[0063] Another aspect provides a composition for promoting cell proliferation or differentiation comprising a fusion protein or recombinant IL3 protein according to the present invention.

[0064] The term "promotion of cell proliferation" in this specification refers to a phenomenon regulated by cell growth factors, growth hormones, cytokines, etc., which may lead to an increase in the number of cells. Such cell proliferation is involved in various biological processes, such as tissue growth, development, maintenance, and repair of damaged tissues.

[0065] Another aspect provides a composition for improving hematopoietic function comprising a fusion protein or recombinant IL3 protein according to the present invention.

[0066] Another aspect provides a composition for improving hematopoietic function comprising a fusion protein or recombinant IL3 protein according to the present invention.

[0067] Another aspect provides a pharmaceutical composition for the prevention or treatment of hematopoietic disorders comprising a fusion protein or recombinant IL3 protein according to the present invention.

[0068] The term "hematopoietic stem cell" in this specification refers to a cell that performs the hematopoietic function through self-proliferation and differentiation, also referred to as a hematopoietic stem cell. Hematopoietic stem cells mature into final cells by passing through the stage of progenitor cells, which are intermediate stages of various blood cells, and differentiate into various blood cells such as red blood cells, platelets, neutrophils, eosinophils, basophils, monocytes, T cells, B cells, natural killer cells, or dendritic cells; thus, the development of red blood cells or lymphocytes can be promoted by the differentiation of the hematopoietic stem cells. Accordingly, a composition according to one aspect can be used as a treatment for anemia by promoting the proliferation or differentiation of hematopoietic stem cells. Furthermore, since it can induce cell proliferation or differentiation in tissues such as adipose tissue, pancreas, muscle, blood vessels, and skin that express erythropoietin receptors, it can be used for the prevention or treatment of diseases requiring cell proliferation effects, such as obesity and diabetes, muscle regeneration, blood vessel regeneration, and skin regeneration.

[0069] The term "hematopoietic disorder" in this specification refers to a condition in which blood cell production is not carried out normally due to a defect in hematopoietic function, and hematopoietic disorder can cause various diseases. Specifically, the disease may be aplastic anemia, malignant lymphoma or leukemia, chronic liver disease, renal failure, severe infection, myelopathic thrombocytopenia, idiopathic thrombocytopenic purpura (ITP), thrombocytopenia, lymphopenia, neutropenia, mononucleosis, granulocytopenia, myelodysplastic syndrome, or myeloproliferative disease.

[0070] The above composition can promote the development of red blood cells or lymphocytes by promoting the proliferation or differentiation of hematopoietic stem cells, and thus can be used for the prevention or treatment of anemic diseases. The anemic disease may be, for example, acute or chronic anemia, anemia due to kidney disease, anemia due to renal failure, anemia due to blood disease, radiation therapy-induced anemia, chemotherapy-induced anemia, anemia due to surgery, anemia due to infection, nutritional deficiency anemia, abnormal red blood cell production, early anemia of premature birth, or a combination thereof.

[0071] The composition according to one embodiment may include one or more peptides selected from the group consisting of SEQ ID NOs 1 to 28 in an amount of 0.001% to 80% by weight based on the total weight of the composition. Additionally, the dosage of the composition may be 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. However, the dosage may vary depending on factors such as the formulation method, administration method, patient's age, body weight, gender, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The number of administrations may be one or two or more within the range of clinically acceptable side effects, and the administration site may be one or two or more. For animals other than humans, the dosage per kg may be the same as that for humans, or an amount calculated by converting the above dosage based on, for example, the ratio of organ (e.g., heart) volumes between the target animal and humans (e.g., average value). Possible routes of administration may include oral, sublingual, parenteral (e.g., subcutaneous, intramuscular, intra-arterial, intraperitoneal, intradural, or intravenous), rectal, local (including transdermal), inhalation, and injection, or insertion of an implantable device or substance. Examples of animals targeted for treatment according to one embodiment include humans and other mammals intended for treatment, specifically including humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs, etc.

[0072] A pharmaceutical composition according to one embodiment may include pharmaceutically acceptable carriers and / or additives. For example, it may include sterile water, physiological saline, a common buffer (phosphate, citric acid, other organic acids, etc.), a stabilizer, a salt, an antioxidant (ascorbic acid, etc.), a surfactant, a suspending agent, an isotonic agent, or a preservative. For topical administration, it may also include combinations with organic materials such as biopolymers, inorganic materials such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. When a pharmaceutical composition according to one embodiment is prepared in a formulation suitable for injection, the peptide may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution.

[0073] A pharmaceutical composition according to one embodiment may appropriately include, if necessary depending on the method of administration or dosage form, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic agent, a buffer, a reducing agent, an antioxidant, etc. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, 19th ed., 1995]. A pharmaceutical composition according to one embodiment may be prepared in a unit dose form or contained in a multi-dose container by formulating using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art to which the invention pertains. In this case, the dosage form may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granule, tablet, or capsule.

[0074] Another aspect provides a health functional food composition for the prevention or improvement of hematopoietic disorders comprising a fusion protein or recombinant IL3 protein according to the present invention.

[0075] The details regarding the above "hematopoietic disorder," "hematopoietic stem cells," etc. are as previously described.

[0076] The above health food composition may be used in combination with other foods or food ingredients in addition to the peptide, and may be used appropriately according to conventional methods. The amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a health functional food, the composition of this specification may be added in an amount of 15 parts by weight or less relative to the raw material. There are no special restrictions on the type of health food.

[0077] For details regarding the genetic engineering technology used in the present invention, reference can be made to the literature of Sambrook et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) and the literature of Frederick et al. (Frederick M. Ausubel et al., Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)). Effects of the invention

[0078] The present invention relates to a recombinant IL3 fusion protein with enhanced solubility and expression levels in various E. coli strains using a Protein Disulfide Isomerase (PDI) or Maltose-Binding Protein (MBP) tag, and to a novel IL3 fusion protein and its use in promoting cell proliferation and differentiation. The IL3 fusion protein and the TF-1 cell proliferation effect of IL3 isolated from the fusion protein can be utilized in the treatment of hematopoietic disorders. Brief explanation of the drawing

[0079] Figure 1 is a schematic diagram showing the structure of a fusion protein (MBP-IL3 or PDIb'a'-IL3) and the plasmid construction process for the production of the fusion protein: Figure 1a is a schematic diagram showing the structures of MBP-LI3 and PDIb'a'-LI3, and Figure 1b is a schematic diagram showing the generation of the entry vector pENTR-IL3 through the BP reaction of the gateway system and the generation of the expression vector pDEST-H-tags-LI3 through the LR reaction of the gateway system. Figure 2 shows various E. coli strains ( BL21, ClearColi, LOBSTR, Origami 2, Rosetta-gami B , SHuffle T7 This is the result of confirming the expression levels of fusion proteins (MBP-IL3 (59.2 kDa), PDIb'a'-IL3 (50 kDa)) overexpressed in ): Figure 2a shows the results of quantifying expression and solubility levels by analyzing the total bacterial lysate, supernatant, and pellet fractions using SDS-PAGE, and Figure 2b is a graph showing the results of quantitative analysis of fusion protein expression levels and solubility ((1st y-axis) blue bar graph: expression percentage, orange bar graph: solubility percentage, (2nd y-axis) black line: expression level X solubility). Fig. 3 is at 30°C ClearColi This is the result of confirming the expression level of the fusion protein in a 1L flask culture: Figure 3a is a graph showing the expression profile of the PDIb'a'-IL3 fusion protein (PDIb'a'-IL3), and Figure 3b is an SDS-PAGE image showing the expression levels of the fusion protein at various time intervals after IPTG induction. The black arrows in the graph indicate the optimal IPTG induction moment corresponding to the peak rate of cell growth. Figure 4 shows the results of purification and analysis of recombinant IL3 extracted from PDIb'a'-IL3 produced in E. coli: FIG. 4a is a schematic diagram detailing the sequential purification steps used for the separation of recombinant IL3, and FIG. 4b shows a two-step chromatography process including IMAC and anion-exchange chromatography. ClearColi Figure 4c shows the results of SDS-PAGE analysis of the purified recombinant IL3 protein under both reducing and non-reducing conditions (M; protein molecular weight marker, Lane 1; total cellular protein before IPTG induction (negative control), Lane 2; total protein disrupted by ultrasound after IPTG induction, Lane 3; soluble protein fraction from total cellular protein, Lane 4: PDIb'a'-IL3 fusion protein purified by IMAC, Lane 5: recombinant IL3 protein after cleaving with TEV, Lane 6: final recombinant IL3 protein purified by IEC), Figure 4c shows the results of SDS-PAGE analysis of the purified recombinant IL3 protein under both reducing and non-reducing conditions (M; protein molecular weight marker, R; reducing condition (treated with DTT), NR; non-reducing condition (not treated with DTT)), and Figure 4d shows a silver-stained SDS-PAGE gel image of recombinant IL3 under reducing conditions (M; protein molecular weight marker). Figure 5 is a graph showing the mass spectrometry results of purified recombinant IL3. Figure 6 is a graph showing the results of a comparison of the dose-response effects of IL3, PDIb'a'-IL3, and PDIb'a' on TF-1 cell proliferation: Figure 6a is a graph showing the biological effects (cell proliferation promoting effect) of the purified recombinant IL3 of the present invention and the commercial IL3 in TF-1 cells, and Figure 6b is a graph showing the biological effects of PDIb'a'-IL3 and PDIb'a' in TF-1 cells. Specific details for implementing the invention

[0080] The following examples will be explained in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0081] Example 1. Plasmid Design for IL3 Fusion Protein Production

[0082] E. coli To evaluate the effects of various protein tags on the expression levels and solubility of IL3 in an expression system, plasmids were designed to generate constructs (PDIb'a'-IL3, MBP-IL3) in which PDIb'a' and MBP are fused to the N-terminus of IL3. Specifically, E. coli To enhance the solubility of IL3 in the cytoplasm, two soluble enhancers, MBP and PDIb'a', were tagged to the IL3 sequence spanning amino acids 20–152, excluding the signal peptide. A TEV protease plasticizer site (ENLYFQ / G) was inserted between each tag and the IL3 coding sequence so that the tags could be removed in a subsequent purification step. Additionally, hexahistidine residues were included at the N-terminus of PDIb'a' and MBP to facilitate purification.

[0083] This codon-optimized sequence was initially cloned into the pDONR207 plasmid using the BP reaction of the Gateway cloning system, resulting in the generation of a pEntry construct containing the IL3 gene. Subsequently, the IL3 gene was transferred to an expression vector containing a tag protein (MBP or PDIb'a') using the LR reaction to generate MBP-IL3 and PDIb'a'-IL3. The amino acid sequence of the fusion protein (PDIb'a'-IL3), the amino acid sequence of the recombinant IL3 protein, and the above codon-optimized sequence are shown in Table 1.

[0084] order order number Amino acid sequence of the fusion protein (PDIb'a'-IL3) MKHHHHHHHHEGGGGLIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPE SEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSF PTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVGTGSYITSLYKKAGFENLYFQGMAPMTQTTPLKTSWVNCSNMIDEI ITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF 1 Recombinant IL3 protein amino acid sequence MAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF 2 IL3 codon optimization sequence ATGGCTCCCATGACGCAGACTACTCCCTTGAAAACTAGTTGGGTTAACTGCTCCAATATGATAGACGAGATAATAACACACCTTAAACAACCGCCTCTGCCACTGTTGGACTTTAATAACTTAAACGGTGAAGACCAAGACATCCTGATGGAAAATAATCTTCGGCGGCCGAACCTGGAAGCGTTCAATCGTGCGGTAAAA TCGTTGCAGAACGCTTCAGCAATTGAAAGTATTTTGAAGAACTTGTTACCATGTCTGCCGCTTGCAACAGCGGCACCCACGCGCCACCCGATACACATTAAAGACGGAGACTGGAATGAGTTTCGCCGTAAATTGACGTTTTACCTTAAAACTCTTGAGAACGCTCAAGCGCAACAGACGACTTTGAGTCTGGCGATCTTC 3

[0085] Figure 1 is a schematic diagram showing the structure of a fusion protein (MBP-IL3 or PDIb'a'-IL3) and the plasmid construction process for the production of the fusion protein:

[0086] Figure 1a is a schematic diagram showing the structures of MBP-LI3 and PDIb'a'-LI3, and Figure 1b is a schematic diagram showing the generation of the entry vector pENTR-IL3 through the BP reaction of the gateway system and the generation of the expression vector pDEST-H-tags-LI3 through the LR reaction of the gateway system.

[0087] The integrity and accuracy of the final expression vector were verified through DNA sequencing.

[0089] Example 2. Analysis of fusion protein expression levels and solubility in various Escherichia coli strains

[0090] To find the optimal E. coli strain and conditions for recombinant IL3 production, the expression vector prepared by the method of Example 1 was used with a heat shock method BL21, ClearColi, LOBSTR, Origami 2, Rosetta-gami B and SHuffle T7 Each E. coli strain was transformed, and the expression level and solubility of the fusion protein were measured.

[0091] Specifically, transformed E. coli were grown on LB agar plates supplemented with 100 μg / mL ampicillin to identify high-expression colonies. Selected colonies were incubated overnight at 37°C with shaking at 200 rpm in LB medium containing the same concentration of ampicillin (100 μg / mL). Subsequently, the overnight culture was diluted (1:100) with 10 mL of fresh LB medium containing 100 μg / mL ampicillin. The diluted culture was OD 600 The cultures were cultured under the same conditions as described above until the measured value reached between 0.5 and 0.6. The culture medium was evenly distributed into three separate tubes, and protein expression was induced using 1 mM IPTG. After protein expression induction, each culture medium was cultured under three distinct culture conditions (4 hours at 37°C, 6 hours at 30°C, and overnight at 18°C ​​with shaking at 200 rpm). After culture, bacterial pellets were harvested by centrifugation at 13,500 µg for 5 minutes at 4°C, and the harvested pellets were lysed using an ultrasonic cell disruptor at 35% amplitude for 10 repetitions of a 5-second ON / 5-second OFF cycle.

[0092] To separate the soluble and insoluble protein fractions, the lysate was centrifuged at 13,500 µg for 5 minutes at 4°C. To quantify the expression and solubility of the fusion protein, 10% trisine SDS-PAGE was performed and quantified using GelAnalyzer software (http: / / www.gelanalyzer.com). Using the following formula BL21, ClearColi, LOBSTR, Origami 2, Rosetta-gami B and SHuffle T7 The expression level and solubility of the fusion protein were evaluated, and the results are shown in Table 1, Table 2 and Figure 2.

[0093] [Formula 1]

[0094] Expression level = Amount of fusion protein / Total cellular protein after IPTG induction

[0095] Solubility Level = Amount of Soluble Fusion Protein / Total Amount of Fusion Protein

[0096] Table 2 is E. coli This shows the results of measuring the expression level of the fusion protein after culturing each strain at 37°C for 4 hours, at 30°C for 6 hours, and at 18°C ​​overnight with shaking at 200 rpm.

[0097] E. coli strains MBP-IL3 PDIb'a'-IL3 37°C 30°C 18°C 37°C 30°C 18°C BL21 32 ± 2 21 ± 3 19 ± 2 29 ± 4 30 ± 2 34 ± 8 ClearColi 43 ± 3 33 ± 3 23 ± 2 33 ± 10 43 ± 7 34 ± 5 LOBSTR 26 ± 6 24 ± 4 21 ± 4 13 ± 2 12 ± 2 21 ± 3 Origami 2 38 ± 11 40 ± 12 36 ± 9 16 ± 2 23 ± 2 37 ± 4 Rosetta-gami B 24 ± 5 24 ± 3 21 ± 4 14 ± 3 22 ± 4 27 ± 8 SHuffle T7 28 ± 5 37 ± 6 35 ± 7 10 ± 2 25 ± 4 28 ± 8

[0098] As shown in Table 2, the PDIb'a'-IL3 and MBP-IL3 fusion proteins showed high expression levels at 37°C and 30°C.

[0100] Table 3 is E. coli This shows the results of measuring the solubility of the fusion protein after culturing each strain at 37°C for 4 hours, 30°C for 6 hours, and 18°C ​​overnight with shaking at 200 rpm.

[0101] E. coli strains MBP-IL3 PDIb'a'-IL3 37°C 30°C 18°C 37°C 30°C 18°C BL21 46 ± 5 82 ± 11 90 ± 5 53 ± 7 88 ± 5 98.2 ± 0.3 ClearColi 62 ± 15 70 ± 6 85 ± 9 78 ± 11 97 ± 3 98.1 ± 0.5 LOBSTR 46 ± 2 65 ± 2 70 ± 8 45 ± 11 67 ± 8 82 ± 6 Origami 2 61 ± 11 70 ± 8 84 ± 9 71 ± 11 91 ± 7 97 ± 2 Rosetta-gami B 51 ± 15 80 ± 8 63 ± 9 45 ± 13 93 ± 3 87 ± 7 SHuffle T7 64 ± 5 95 ± 2 94 ± 3 83 ± 7 97 ± 1 98.6 ± 0.8

[0102] As shown in Table 3, the PDIb'a'-IL3 and MBP-IL3 fusion proteins showed high solubility at 30°C and 18°C.

[0104] Figure 2 shows various E. coli strains ( BL21, ClearColi, LOBSTR, Origami 2, Rosetta-gami B , SHuffle T7 This is the result of confirming the expression levels of fusion proteins (MBP-IL3 (59.2 kDa), PDIb'a'-IL3 (50 kDa)) overexpressed in ):

[0105] Figure 2a shows the results of quantifying expression and solubility levels by analyzing the total bacterial lysate, supernatant, and pellet fractions using SDS-PAGE, and Figure 2b is a graph showing the results of quantitative analysis of fusion protein expression levels and solubility ((1st y-axis) blue bar graph: expression percentage, orange bar graph: solubility percentage, (2nd y-axis) black line: expression level X solubility).

[0106] As shown in Fig. 2, Diverse expression and solubility profiles of the fusion proteins MBP-IL3 and PDIb'a'-IL3 were observed depending on the specific E. coli strain. However, across all strains, both proteins consistently showed enhanced solubility and expression levels at low temperatures of 18°C ​​and 30°C. In particular, the MBP-IL3 fusion protein reached maximum expression and solubility levels when expressed in BL21 at 30°C.

[0108] Example 3. Analysis of fusion protein expression levels and solubility in a large-volume flask

[0109] Considering the expression and solubility indicators of the fusion protein and the purification advantages provided by various E. coli strains, among the fusion proteins expressed by the method of Example 2, the one expressing PDIb'a'-IL3 at 30℃ ClearColi Selected and cultured in a 1L flask.

[0110] The results of monitoring cell growth and expression profiles in a 1L flask culture are shown in Figure 3.

[0111] Fig. 3 is at 30°C ClearColi This is the result of confirming the expression level of the fusion protein in a 1L flask culture:

[0112] Figure 3a is a graph showing the expression profile of the PDIb'a'-IL3 fusion protein (PDIb'a'-IL3), and Figure 3b is an SDS-PAGE image showing the expression levels of the fusion protein at various time intervals after IPTG induction. The black arrows in the graph indicate the optimal IPTG induction moment corresponding to the peak rate of cell growth.

[0113] As shown in Figure 3, E. coli cells maintained a consistent growth trajectory for up to about 11 hours, after which a plateau occurred, and after IPTG induction, the expression of the fusion protein increased significantly, reaching a peak 2 hours after induction.

[0114] Meanwhile, at 30°C ClearColi Specific figures for the expression profile, such as the expression level and solubility of the fusion protein (PDIb'a'-IL3), according to 1L flask culture are shown in Table 4 below.

[0115] Endpoint OD 600 1.31 0.06 Wet cell weight (g) 1.79 0.24 Expression level (%) 22 1 Solubility level (%) 46 2

[0116] As shown in Figure 3 and Table 4, it was confirmed that the expression level and solubility of IL3 fusion protein were high even when cultured in large quantities.

[0117] According to existing studies on IL3 expression in E. coli, there are few reported cases regarding IL3 protein expression levels or solubility, and even when reported, the mainstream view is that IL3 proteins are observed as inclusion bodies (Boyle 1999, PMID 10512796, Dagar 2017, PMID 27864159). This means that most expressed IL3 proteins are insoluble. Therefore, the present invention implies that it is possible to efficiently produce large quantities of highly soluble IL3 proteins compared to previously known IL3 protein expression systems.

[0119] Example 4. Isolation and purification of IL3 fusion protein expressed in E. coli

[0120] 4.1 Collection of Transformed E. coli

[0121] An aliquot of the transformed bacterial culture from Example 2 was taken and inoculated into 10 mL of LB medium supplemented with 100 μg / mL ampicillin, and cultured overnight at 37°C with shaking at 200 rpm. The overnight culture was diluted 100-fold with 1 L of fresh LB medium containing 100 μg / mL ampicillin, and the OD 600 Shaking incubation was performed at 200 rpm at 37°C until the measured value was between 0.5 and 0.7. OD 600 When the measured value reached 0.5 to 0.7, 1 mM IPTG was added and the culture was incubated for an additional 6 hours at 30°C. Afterwards, the culture medium was centrifuged at 3,500 µg at 4°C for 45 minutes to collect the bacteria, the supernatant was discarded, and the resulting bacterial pellet was stored at -20°C for subsequent use.

[0122] 4.2 Isolation and Purification of PDIb'a'-IL3

[0123] The PDIb'a'-IL3 fusion protein was isolated from the transformed E. coli of Example 4.1 and purified using an IMAC HisTrap FF column.

[0124] Specifically, a pellet derived from a 0.5 L E. coli culture was resuspended in 50 mL of Tris buffer (50 mM Tris-HCl, 5% glycerol, pH 8) containing a 1 x protease inhibitor cocktail. The E. coli cells were lysed using an ultrasonic cell disruptor in an ice bath to prevent overheating. After sonication, the lysate was centrifuged at 11,000 H g at 4 °C for 25 minutes to pellet the cellular debris. Subsequently, the purified supernatant was filtered through a 0.45 μm filter, and the salt concentration of the supernatant was adjusted with 500 mM NaCl and 20 mM imidazole. The lysate was then loaded into a pre-equilibrated IMAC HiTrap FF 5 mL column using an equilibration buffer containing 50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, and 5% glycerol (pH 8.0). After sample loading, the column was washed with equilibration buffer at a minimum of 5 CV to remove loosely bound proteins. To increase protein purity, additional washing was performed using 10 CV of 7% elution buffer (50 mM Tris-HCl, 500 mM NaCl, 1 M imidazole, and 5% glycerol, pH 8.0). Subsequently, the fusion protein was eluted using 400 mM imidazole, equivalent to 40% of the elution buffer. The eluted fusion protein fraction was diluted 1:10,000 and dialyzed in Tris buffer (50 mM Tris-HCl, pH 8.0) to remove residual NaCl and imidazole.

[0125] 4.3 Purification of recombinant IL3 with the PDIb'a' tag cleaved

[0126] Following the IMAC prepared by the method of Example 4.2, the dialyzed fusion protein was treated with TEV protease at a ratio of 20:1 (fusion protein:TEV protease weight) to cleave the PDIb'a' tag. This cleavage reaction was carried out at 18°C ​​for 16-20 hours.

[0127] After cleaving PDIb'a'-IL3, the samples were buffered with MES (pH 6.5) and loaded onto a 5 mL HiTrap Q HP column pre-equilibrated with 50 mM MES buffer at pH 6.5. After loading, IL3 was found mainly in the flow-through, and was subsequently desalted and buffered with PBS (pH 7.4). To obtain the required concentration, the purified IL3 was centrifuged at 3,000 µg and 4°C using a 3K Amicon Ultra filter. The fractions obtained from each purification step were evaluated using 10% Tricine SDS-PAGE, and protein concentration was measured using BCA assay according to the manufacturer's instructions; the results are shown in Figure 4.

[0128] Figure 4 shows the results of purification and analysis of recombinant IL3 extracted from PDIb'a'-IL3 produced in E. coli:

[0129] FIG. 4a is a schematic diagram detailing the sequential purification steps used for the separation of recombinant IL3, and FIG. 4b shows a two-step chromatography process including Immobilized Metal Affinity Chromatography (IMAC) and anion-exchange chromatography. ClearColiFigure 4c shows the results of SDS-PAGE analysis of the purified recombinant IL3 protein under both reducing and non-reducing conditions (M; protein molecular weight marker, Lane 1; total cellular protein before IPTG induction (negative control), Lane 2; total protein disrupted by ultrasound after IPTG induction, Lane 3; soluble protein fraction from total cellular protein, Lane 4: PDIb'a'-IL3 fusion protein purified by IMAC, Lane 5: recombinant IL3 protein after cleaving with TEV, Lane 6: final recombinant IL3 protein purified by IEC), Figure 4c shows the results of SDS-PAGE analysis of the purified recombinant IL3 protein under both reducing and non-reducing conditions (M; protein molecular weight marker, R; reducing condition (treated with DTT), NR; non-reducing condition (not treated with DTT)), and Figure 4d shows a silver-stained SDS-PAGE gel image of recombinant IL3 under reducing conditions (M; protein molecular weight marker).

[0130] As shown in Figure 4, the isolated IL3 protein was observed at the expected monomer size under both reducing and non-reducing SDS-PAGE conditions, and its purity was measured using silver staining and showed a purity of 93 ± 4%.

[0131] More specifically, the results regarding the purity and yield of PDIb'a'-IL3 or isolated IL3 at each purification step in 0.5 L cell culture are shown in Table 5.

[0132] Purification step Total protein (mg) Purity (%) Target protein (mg) Yield (%) Supernatant of whole E. coli lysate 81.4 14.6 42.4 8.9 33.8 4 100 IMAC 17.8 4.1 30.5 9.1 5.3 1.3 15.9 0.05 IEC 0.38 0.02 95.8 3.6 0.36 0.02 6.6 1.4

[0133] As shown in Table 5, the purity of the PDIb'a'-IL3 fusion protein at the IMAC stage was 30.5 ± 9.1%, and after the ICE stage, it was 95.8 It was confirmed that it increased to 3.6%. In addition, approximately 0.36 ± 0.02 mg of IL3 was obtained from a 0.5 L E. coli culture expressing PDIb'a'-IL3, which represents a yield of 6.6 ± 1.4%.

[0135] Example 5. Mass analysis of purified IL3 protein

[0136] LC-MS / MS analysis was performed to verify the IL3 protein purified by the method of Example 3. After treating the purified IL3 protein with trypsin, the protein identification results were confirmed through LC-MS / MS.

[0137] Specifically, purified IL3 proteins were identified using Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS / MS). Protein bands were cleaved from an SDS-PAGE gel and washed, followed by treatment with Trypsin to generate peptides. For the LC step, samples were fed into a PepMap RSLC 75 x 50 column using solutions A (0.1% formic acid in water) and B (0.1% formic acid in acetaminophen). Peptides were separated for 200 minutes at flow rates of 200 to 280 nL / min, and a gradient elution of solution B ranging from 5% to 90% was applied for 180 minutes. In the Mass Spectrometry step, the first segment (MS1) was operated at a resolution of 70,000 with a packing time of 50 milliseconds for the top 10 precursors. The second segment (MS2) was set to a resolution of 17,500, with a packing time of 150 milliseconds and automatic gain control applied at a peak of 1e5. Finally, for protein identification, all MS / MS samples were analyzed using Proteome Discoverer. Sequence databases were searched using Sequest HT, and criteria included a fragment mass tolerance of 0.02 Da, a precursor mass tolerance of 10 PPM, a fixed correction for carbamidomethylation of cysteine, and a variable correction for oxidation of methionine. The results of the LC-MS / MS analysis are shown in Figure 5.

[0138] Figure 5 is a graph showing the mass spectrometry results of purified recombinant IL3.

[0139] As shown in Figure 5, LC-MS / MS analysis identified masses in various segments and showed comprehensive 100% coverage. This means that the IL3 protein purified by the method of Example 3 is a protein identical to IL3.

[0141] Example 6. Evaluation of the biological activity of purified recombinant IL3 and PDIb'a'-IL3

[0142] The biological activity (cell proliferation promotion) of recombinant IL3 extracted from the PDIb'a'-IL3 fusion protein by the method of Example 4 was evaluated through TF-1 cell proliferation assay.

[0143] Specifically, TF-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS (tumor neoplasm culture medium), 3.5 ng / mL GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor), and 1% penicillin-streptomycin. Prior to evaluating biological activity, the cells were washed twice with RPMI 1640 to remove any remaining cytokines. Subsequently, 1 x 10⁶ cells were placed in a 96-well plate per well. 4 TF-1 cells were seeded at a cell density of 10, and GM-CSF and FBS were removed for 24 hours. For the activity test, TF-1 cells were exposed to IL3 concentrations in the range of 0-2 μM and cultured for 48 hours in a chamber at 37°C and appropriate humidity containing 5% CO2.

[0144] TF-1 cells treated with PBS (negative control) and a medium at the maximum IL3 concentration without cells (blank control) were set as controls. Each concentration was analyzed three times. After incubation, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in the dark at 37°C for 4 hours. Subsequently, absorbance was measured at 450 nm using a microplate reader. The obtained data were analyzed using Equation 2 (Hill Equation) below and Microsoft Excel.

[0145] [Equation 2]

[0146] Re = Bl + (max - Bl) / (1 + (EC 50 / conc) Hs )

[0147] Here, Re represents the cellular response, Bl represents the baseline response at low protein concentration, Max represents the maximum observed response, conc represents the protein concentration, and Hs represents the Hill stimulation factor.

[0148] The results of measuring the biological activity of the cells are shown in Figure 6.

[0149] Figure 6 is a graph showing the results of a comparison of the dose-response effects of IL3, PDIb'a'-IL3, and PDIb'a' on TF-1 cell proliferation:

[0150] Figure 6a is a graph showing the biological effects (cell proliferation promoting effect) of the purified recombinant IL3 of the present invention and the commercial IL3 in TF-1 cells, and Figure 6b is a graph showing the biological effects of PDIb'a'-IL3 and PDIb'a' in TF-1 cells.

[0151] As shown in Fig. 6a, it was confirmed that the purified recombinant IL3 of the present invention and the conventional IL3 exhibit a two-step proliferation-promoting effect at specific concentrations and stimulate TF-1 cell proliferation. It was confirmed that in the low concentration range, the purified recombinant IL3 of the present invention (160%) stimulates TF-1 cell proliferation more than the commercial IL3 (132%).

[0152] Meanwhile, as shown in Figure 6b, the biological effects of PDIb'a'-IL3 and PDIb'a' were also analyzed in TF-1 cells. It was confirmed that PDIb'a'-IL3 also induced a two-step proliferation response in TF-1 cells. However, PDIb'a' showed a proliferation-promoting effect in TF-1 cells only at high concentrations. Table 6 shows the specific values ​​of the proliferation parameters of TF-1 cells.

[0153] Purified IL3 Commercial IL3 PDIb'a'-IL3 PDIb'a' EC 50 (1st stimulation) 0.07 ± 0.01 pM 0.18 ± 0.06 pM 195 ± 82 pM 184 ± 25 nM Hill coefficient (1st stimulation) 1.6 ± 0.2　 1.17 ± 0.18　 1.09 ± 0.1　 1.5 ± 0.2　 Maximum response (1st stimulation) 160 ± 18%　 132 ± 5%　 165 ± 4%　 350 ± 79%　 EC 50 (2nd stimulation) 151 ± 25 nM 262 ± 16 nM 396 ± 47 nM None Hill coefficient (2nd stimulation) 1.6 ± 0.1　 1.75 ± 0.35　 1.9 ± 0.1　 None Maximum response (2nd stimulation) 345 ± 14%　 419 ± 31%　 345 ± 19%　 None

[0154] As shown in Table 6, the purified recombinant IL3 of the present invention has an EC of 0.07 ± 0.01 pM and 151 ± 25 nM. 50 TF-1 cells exhibited a two-stage proliferation pattern characterized by... Commercial IL3 had an EC of 0.18 ± 0.06 pM and 262 ± 16 nM. 50 Eul, the PDIb'a'-IL3 fusion protein had ECs of 195 ± 82 pM and 396 ± 47 nM. 50 It exhibited a two-stage proliferation pattern of TF-1 cells characterized by [specific characteristics]. This implies that the purified recombinant IL3 of the present invention not only maintains biological activity but also causes a greater TF-1 cell proliferation effect compared to commercial IL3. Furthermore, it implies that the PDIb'a'-IL3 fusion protein also has a TF-1 cell proliferation effect. By utilizing the TF-1 cell proliferation effect of this purified recombinant IL3, it can be used for the treatment of hematopoietic disorders.

[0156] Example 7. Statistical Analysis

[0157] Data were expressed as mean ± standard error of the mean. All statistical calculations were performed using Microsoft Excel. Paired t-tests were used for Pirwise comparisons. One-way Analysis of Variance (ANOVA) was applied to comparisons between groups, followed by Bonferroni post-hoc tests to adjust for multiple comparisons. For all tests, the statistical significance level was set at p < 0.05.

Claims

Claim 1 A fusion protein comprising protein disulfide isomerase (PDI) or maltose-binding protein (MBP); and human interleukin-3 (IL3). Claim 2 A fusion protein according to claim 1, wherein the protein disulfide isomerase (PDI) is a PDI b'a' domain (protein disulfide isomerase b'a' domain). Claim 3 The fusion protein of claim 1, wherein the fusion protein has a protease cleavage site inserted therein. Claim 4 A fusion protein according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO.

1. Claim 5 A recombinant IL3 protein in which the protein disulfide isomerase (PDI) or maltose-binding protein (MBP) portion is removed from the fusion protein of claim 1. Claim 6 A recombinant IL3 protein according to claim 5, wherein the recombinant IL3 protein has the amino acid sequence of SEQ ID NO.

2. Claim 7 A nucleotide encoding the fusion protein of claim 1. Claim 8 A recombinant vector comprising the nucleotide of claim 7. Claim 9 Isolated host cells transformed with the recombinant vector of claim 8. Claim 10 In claim 9, the host cell is E. coli ( E.coli A host cell that is ) Claim 11 In claim 10, the E. coli is BL21, ClearColi, LOBSTR, Origami 2, Rosetta-gami B and SHuffle T7 A host cell that is any one selected from a group consisting of Claim 12 A method for producing a recombinant IL3 protein, comprising the step of culturing the transformed host cell of claim 9 in a medium to express the fusion protein of claim 1. Claim 13 A method for producing a recombinant IL3 protein according to claim 12, wherein the step of expressing the fusion protein is to culture at 16 to 40°C for 3 to 20 hours. Claim 14 A method for producing a recombinant IL3 protein according to claim 13, further comprising: a step of treating the fusion protein with a protease to remove a protein disulfide isomerase (PDI) or maltose-binding protein (MBP) portion; and a step of isolating and purifying the IL3 recombinant protein from which the protein disulfide isomerase (PDI) or maltose-binding protein (MBP) has been removed. Claim 15 A method for producing a recombinant IL3 protein according to claim 13, wherein the step of isolating and purifying the IL3 recombinant protein utilizes Immobilized Metal Affinity Chromatography (IMAC) and Ion Exchange Chromatography (IEC).

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