XIAP protein variant or fusion protein comprising same, and use thereof

Abstract

The present invention relates to a XIAP protein variant or a fusion protein comprising same, and use thereof. The XIAP protein variant according to one embodiment has an effect functionally equivalent to that of a wild-type XIAP protein, promotes ubiquitination of receptor-interacting serine / threonine-protein kinase 2 (RIP2) so as to activate NOD signaling, and has increased residual time and levels in cells relative to the wild-type XIAP protein, and thus can be effectively used in the treatment of inflammatory diseases, particularly diseases caused by NOD signaling defects.

Description

XIAP protein variant or fusion protein comprising same and use thereof

[0001] The present invention relates to a XIAP protein variant or a fusion protein comprising the same and a use thereof.

[0002] X-linked inhibitor of apoptosis protein (XIAP) is a multifunctional protein that interacts with various intracellular proteins involved in the regulation of apoptosis and the inflammatory response to bacterial pathogens. In particular, XIAP protein is known as a key regulator of the nucleotide-binding oligomerization domain (NOD) signaling pathway, which is important in innate immunity. NOD signaling, which is activated in response to intracellular bacterial pathogens, involves the NF-κB / MAPK pathway and activation of receptor-interacting serine / threonine-protein kinase 2 (RIP2). In this process, XIAP protein directly interacts with RIP2 and promotes ubiquitination of RIP2 through the RING domain of XIAP, thereby contributing to the activation of NOD signaling.

[0003] Deficiency of the XIAP protein or pathogenic variants (PVs) of the XIAP gene result in functional defects in the signal transduction pathway, which contributes to the pathogenesis of various inflammatory diseases associated with innate immune dysregulation. In particular, XIAP deficiency has been reported to act as a key element in the pathogenesis of autoimmune or autoinflammatory diseases, including inflammatory bowel disease (IBD) (Sci Rep. 2024;14:853. doi:10.1038 / s41598-023-50932-5). In fact, defects in NOD signaling have been observed in XIAP-deficient IBD patients, and functional assays of NOD signaling are also essential for the diagnostic evaluation of pathogenic variants in the XIAP gene.

[0004] However, to date, no effective drug treatment has been reported for XIAP deficiency, and hematopoietic stem cell transplantation (HSCT) is presented as the only practical treatment. However, HSCT is an extreme treatment that can be life-threatening, and other existing treatments, including chemotherapy and biologics, have little effect in controlling hyperinflammation in patients with XIAP deficiency.

[0005] Therefore, protein replacement therapy (PRT) that restores the function of XIAP protein is necessary for the treatment of patients with XIAP deficiency. In the present invention, an improved XIAP protein mutant suitable for protein replacement therapy (ERT) using the direct injection method while maintaining the activity of XIAP was developed.

[0006] One aspect is a variant of XIAP (X-linked inhibitor of apoptosis protein) protein, which comprises at least one of a variant in which methionine is added to the N-terminus of the XIAP protein having an amino acid sequence of SEQ ID NO: 5; a variant in which glutamic acid at position 134 from the N-terminus of the XIAP protein having an amino acid sequence of SEQ ID NO: 5 is substituted with aspartic acid; and a variant in which isoleucine at position 153 from the N-terminus of the XIAP protein having an amino acid sequence of SEQ ID NO: 5 is substituted with leucine.

[0007] Another aspect is to provide a fusion protein comprising the above mutant and a tag protein.

[0008] Another aspect is to provide a polynucleotide encoding the mutant or the fusion protein.

[0009] Another aspect is to provide a recombinant vector comprising a polynucleotide encoding the mutant or the fusion protein.

[0010] Another aspect is to provide a host cell transformed with a recombinant vector comprising a polynucleotide encoding the mutant or the fusion protein.

[0011] Another aspect is to provide a method for producing the mutant or the fusion protein, comprising a step of culturing the host cell.

[0012] Another aspect is to provide a pharmaceutical composition for preventing or treating an inflammatory disease, comprising as an active ingredient a variant of the XIAP protein or a nucleotide encoding the same; or the fusion protein or a polynucleotide encoding the same.

[0013] Another aspect is to provide a health functional food composition for preventing or improving an inflammatory disease, comprising as an active ingredient a variant of the XIAP protein or a nucleotide encoding the same; or the fusion protein or a polynucleotide encoding the same.

[0014] Another aspect is to provide a feed composition for preventing or improving an inflammatory disease, comprising as an active ingredient a variant of the XIAP protein or a nucleotide encoding the same; or the fusion protein or a polynucleotide encoding the same.

[0015] Another aspect provides a method for preventing or treating an inflammatory disease, comprising administering to a subject in need thereof a mutant of the XIAP protein or a nucleotide encoding the same; or a fusion protein or a polynucleotide encoding the same.

[0016] Another aspect provides the use of a variant of the XIAP protein or a nucleotide encoding the same; or a fusion protein or a polynucleotide encoding the same; for the manufacture of a medicament for the prevention or treatment of an inflammatory disease.

[0017] One aspect provides a variant of XIAP (X-linked inhibitor of apoptosis protein) protein, comprising at least one of: a variant in which methionine is added to the N-terminus of the XIAP protein having an amino acid sequence of SEQ ID NO: 5; a variant in which glutamic acid at position 134 from the N-terminus of the XIAP protein having an amino acid sequence of SEQ ID NO: 5 is substituted with aspartic acid; and a variant in which isoleucine at position 153 from the N-terminus of the XIAP protein having an amino acid sequence of SEQ ID NO: 5 is substituted with leucine.

[0018] As used herein, the term “X-linked inhibitor of apoptosis protein (XIAP)” refers to a protein that has the function of inhibiting apoptosis, and performs various biological functions including inhibition of caspase activity and regulation of the NOD signaling pathway. Specifically, the XIAP protein contains a baculovirus IAP repeat (BIR; BIR1, BIR2, BIR3), a ubiquitin-associated domain (UBA), and a RING domain (RING), wherein the RING domain acts as an E3 ubiquitin ligase that ubiquitinates proteins bound to XIAP.

[0019] The above XIAP protein acts as a key mediator in the NOD (Nucleotide-binding oligomerization domain)-RIP2 (Receptor-interacting serine / threonine-protein kinase 2) signaling pathway, and regulates the activation of the NF-κB pathway by inducing the ubiquitination of RIP2 upon recognition of intracellular pathogens. This signaling is particularly essential for maintaining intestinal mucosal homeostasis, and when there is XIAP deficiency or pathogenic mutation, the binding to RIP2 is inhibited, resulting in a loss of NOD signaling function and, consequently, an excessive mucosal inflammatory response. In the intestinal tissue or immune cells of patients with XIAP mutations, defective RIP2 ubiquitination and protein loss due to auto-ubiquitination of XIAP are observed, demonstrating a direct involvement in the pathophysiology of inflammatory bowel disease (IBD).

[0020] The above XIAP (X-linked inhibitor of apoptosis protein) protein may include a XIAP protein analog, and the XIAP protein analog may be a protein that can biologically perform the function of XIAP even if it has some differences in amino acid sequence from the XIAP protein.

[0021] As used herein, the term "variant" means a sequence having a different sequence due to deletion, insertion, non-conservative or conservative substitution or a combination thereof of one or more amino acid residues in the wild-type amino acid sequence.

[0022] In one specific example, the XIAP variant may comprise any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 8.

[0023] Another aspect provides a fusion protein comprising a XIAP variant and a tag protein, wherein the variant comprises at least one of: a mutation in which Methionine is added to the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5; a mutation in which the 134th glutamic acid from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with Aspartic acid; and a mutation in which the 153rd isoleucine from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with Leucine.

[0024] As used herein, the term "tag protein" refers to a short peptide or protein fused to the N-terminus or C-terminus of a target protein to facilitate expression, detection, purification, etc. of the target protein. According to one aspect of the present invention, by fusing a tag protein to the XIAP protein, the solubility of the XIAP protein can be increased, thereby improving purification efficiency, and detection and quantitative analysis using a specific antibody can be facilitated, and the intracellular retention time and residual amount can be increased.

[0025] In one specific example, the tag protein may be any one selected from the group consisting of Histidine (His), Glutathione S-transferase (GST), Maltose-binding protein (MBP), FLAG (DYKDDDDK), Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Yellow Fluorescent Protein (YFP), Cyan Fluorescent Protein (CFP), Thioredoxin (Trx), Protein disulfide isomerase (PDI), N-utilization substance protein A (NusA), and Hemagglutinin (HA), but is not limited thereto.

[0026] In one specific example, the fusion protein may comprise any one amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 11.

[0027]

[0028] Another aspect provides a polynucleotide encoding the variant or the fusion protein.

[0029] For example, the nucleotide may encode a XIAP variant including at least one of a mutation in which Methionine is added to the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5; a mutation in which the 134th glutamic acid from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with Aspartic acid; and a mutation in which the 153rd isoleucine from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with Leucine.

[0030] For example, the nucleotide may encode a fusion protein comprising a XIAP variant including at least one of a mutation in which Methionine is added to the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5; a mutation in which the 134th glutamic acid from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with Aspartic acid; and a mutation in which the 153rd isoleucine from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with Leucine; and a tag protein.

[0031]

[0032] Another aspect provides a recombinant vector comprising a polynucleotide encoding the variant or a fusion protein comprising the same.

[0033] For example, the recombinant vector may include a polynucleotide encoding a XIAP variant including at least one of a mutation in which methionine is added to the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5; a mutation in which the 134th glutamic acid from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with aspartic acid; and a mutation in which the 153rd isoleucine from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with leucine.

[0034] In addition, the recombinant vector may include a polynucleotide encoding a fusion protein including a XIAP variant including at least one of a mutation in which methionine is added to the N-terminus of the XIAP protein consisting of the amino acid sequence of SEQ ID NO: 5; a mutation in which the 134th glutamic acid from the N-terminus of the XIAP protein consisting of the amino acid sequence of SEQ ID NO: 5 is substituted with aspartic acid; and a mutation in which the 153rd isoleucine from the N-terminus of the XIAP protein consisting of the amino acid sequence of SEQ ID NO: 5 is substituted with leucine; and a tag protein.

[0035] As used herein, the term "vector" refers to a DNA molecule for delivering a desired DNA fragment or nucleic acid molecule into a host cell, and is typically a nucleic acid molecule that replicates after inserting the desired DNA or can replicate independently within the host cell. The term "vector" as used herein may be used interchangeably with terms such as "carrier" or "plasmid."

[0036] The above vector can be produced as an expression vector or a cloning vector depending on the purpose, and can be constructed to enable expression and replication in various host cells such as prokaryotic cells or eukaryotic cells.

[0037] In addition, the recombinant vector according to the present invention can be produced by methods well known to those skilled in the art, such as in vitro recombinant DNA technology, chemical DNA synthesis technology, and in vivo recombination technology. The recombinant vector includes a promoter for transcribing a desired DNA sequence to synthesize mRNA, and the promoter is functionally linked to the DNA sequence. In addition, the recombinant vector may further include a ribosome binding site (RBS) for effective translation initiation and a transcription terminator for transcription termination.

[0038]

[0039] Another aspect provides a host cell transformed with the recombinant vector.

[0040] For example, the host cell may be transformed with a recombinant vector including a nucleotide encoding a XIAP variant including any one or more of a mutation in which methionine is added to the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5; a mutation in which the 134th glutamic acid from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with aspartic acid; and a mutation in which the 153rd isoleucine from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with leucine.

[0041] For example, the host cell may be transformed with a recombinant vector including a polynucleotide encoding a fusion protein including a XIAP variant including at least one of a mutation in which methionine is added to the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5; a mutation in which the 134th glutamic acid from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with aspartic acid; and a mutation in which the 153rd isoleucine from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with leucine; and a tag protein.

[0042] In this specification, the term "transformation" may mean a phenomenon in which the genetic properties of an organism are changed by DNA provided from outside, that is, when DNA, a type of nucleic acid extracted from a cell of a certain lineage of an organism, is introduced into a living cell of a different lineage, the DNA enters that cell and the genetic properties are changed.

[0043] The cell, for example, a eukaryotic cell, may be a cell of yeast, fungi, protozoa, plants, higher plants and insects, or amphibians, or a mammalian cell such as CHO, HeLa, HEK293, and COS-1, and may also be a cultured cell (in vitro), a graft cell, and a primary cell culture (in vitro and ex vivo), and an in vivo cell, as commonly used in the art, and also a mammalian cell including a human. In addition, the organism may be a yeast, fungi, protozoa, plants, higher plants and insects, amphibians, or mammals. In addition, the cell may be an animal cell or a plant cell.

[0044] Insertion of a polynucleotide or a recombinant vector containing the same into a host cell can be accomplished using methods widely known in the art. For example, if the host cell is a prokaryotic cell, the calcium chloride (CaCl2) method or electroporation can be used. If the host cell is a eukaryotic cell, the microinjection method, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and gene bombardment can be used, but are not limited thereto.

[0045]

[0046] Another aspect provides a method for producing the mutant or the fusion protein, comprising a step of culturing a host cell according to one aspect of the present invention.

[0047] In one specific example, the manufacturing method may include a step of transforming a host cell with a recombinant vector including a polynucleotide encoding a variant or fusion protein according to one aspect of the present invention; and a step of culturing the transformed host cell to express the variant or fusion protein.

[0048] In the above culturing step, the medium and culture conditions can be appropriately selected and used according to the host cell and the culture conditions such as temperature and culture time can be adjusted to be suitable for mass production of the fusion protein. The fusion protein expressed through the culturing step can be isolated from the cell lysate. The transformant after overexpression is centrifuged to obtain a cell pellet, and the medium components containing the ruptured cell wall or cell membrane can be removed to obtain an eluate containing the cell lysate. Here, the cell pellet can be lysed by a method well known in the art, for example, the use of alkali, a surfactant (such as CHAPS and SDS), an organic solvent, and an enzyme (such as lysozyme), sonication, and the use of a high-pressure grinder (French Press), and the application of periodic high temperature, pressure, freezing and thawing cycles.

[0049] Additionally, the mutant or fusion protein can be purified and separated through a purification process.

[0050]

[0051] In one specific example, the mutant may promote ubiquitination of RIP2 (Receptor-interacting serine / threonine-protein kinase 2) to induce activation of NOD (Nucleotide-binding oligomerization domain) signaling.

[0052] As used herein, the term "RIP2 (Receptor-interacting serine / threonine-protein kinase 2)" refers to a protein that is a serine / threonine kinase that plays a central role in the NOD (Nucleotide-binding oligomerization domain)-derived signal transduction pathway, is activated through interaction with NOD1 or NOD2 receptors in response to recognition of intracellular pathogens, and induces downstream NF-κB and MAPK signaling pathways. The RIP2 is ubiquitinated by XIAP (X-linked inhibitor of apoptosis protein), and this ubiquitination is known to be an essential process for activating NOD signaling. In particular, the ubiquitination of RIP2 plays a key role in regulating the expression of inflammatory cytokines and maintaining mucosal immune homeostasis, and when this process is inhibited or abnormal, the NOD signaling pathway is abnormally regulated, which can act as a direct cause of the development of various inflammatory diseases, including inflammatory bowel disease.

[0053] In one specific example, the fusion protein may have an increased intracellular retention time and residual amount compared to the wild-type XIAP protein.

[0054] The term "wild-type XIAP protein" as used herein refers to a protein having a structure comprising BIR1, BIR2, BIR3 domains, a ubiquitin-associated domain (UBA) and a RING domain (RING), for example, but not limited to, an amino acid sequence of SEQ ID NO: 5.

[0055] The term "increased intracellular retention time and amount" as used herein means that, compared to wild-type XIAP protein treated under the same conditions, it is maintained at a high level for a longer period of time without being degraded or eliminated within the cell, which means improved stability of the protein and increased duration of action in vivo.

[0056]

[0057] Another aspect is to provide a pharmaceutical composition for preventing or treating an inflammatory disease, comprising as an active ingredient a variant of the XIAP protein or a nucleotide encoding the same; or the fusion protein or a polynucleotide encoding the same.

[0058]

[0059] The term "inflammation" in the above-mentioned "inflammatory disease" refers to one of the biological responses to harmful stimuli in living tissue, such as pathogenic microorganisms, damaged cells, and irritants. Inflammation can be caused by various factors, such as microbial infection, wounds, surgery, burns, frostbite, electrical stimulation, and chemicals. The above-mentioned inflammatory disease refers to a disease in which inflammation is the primary lesion. The inflammatory diseases include, for example, osteoarthritis, sepsis, gastritis, inflammatory bowel diseases (IBD), enteritis, nephritis, hepatitis, Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis, ulcerative colitis, irritable bowel syndrome, chronic ileitis, ischemic colitis, ankylosing spondylitis, spondyloarthropathy, juvenile arthritis, arthritis associated with synovitis or vasculitis syndrome, ulcerative proctitis, reactive arthritis, patellofemoral syndrome, chronic inflammatory arthropathy, neuropathic joint disease, inflammatory pain, migraine, headache, back pain, fibromyalgia, myofascial disease, viral infection, bacterial infection, fungal infection, burns, wounds due to surgical or dental operation, hyperprostaglandin syndrome, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, and It may be one or more selected from the group consisting of, but is not limited to, eczema.

[0060] In one specific example, the inflammatory disease may be at least one selected from the group consisting of osteoarthritis, sepsis, gastritis, inflammatory bowel diseases (IBD), enteritis, nephritis, hepatitis, ulcerative colitis, irritable bowel syndrome, chronic ileitis, ischemic colitis, ulcerative proctitis, reactive arthritis, chronic inflammatory arthropathy, neuropathic joint disease, inflammatory pain, and eczema, and more preferably, it may be at least one selected from the group consisting of inflammatory bowel diseases (IBD), ulcerative colitis, chronic ileitis, ulcerative proctitis, and ischemic colitis.

[0061]

[0062] The term "prevention" as used herein collectively refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its secondary symptoms, preventing the acquisition or reacquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder. The above prevention refers to any act of inhibiting or delaying the onset of an inflammatory or inflammation-related disease, disorder, or symptom by administering a composition according to the present invention.

[0063] The term "improvement" herein may mean any action that at least reduces a parameter associated with the condition being treated, for example, the severity of a symptom.

[0064] As used herein, the term "treatment" refers to any action that improves or beneficially alters a disease, disorder, or its associated symptoms. Examples include suppressing, alleviating, or eliminating the development of inflammation and related conditions.

[0065] The term "pharmaceutical composition" as used herein may refer to a molecule or compound that, when administered to a subject, imparts several beneficial effects. Beneficial effects may include enabling diagnostic determination; improving a disease, symptom, disorder, or condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally counteracting a disease, symptom, disorder, or condition.

[0066] The subject may be a mammal, for example, a rat, a human, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.

[0067] The above pharmaceutical composition may be formulated as a preparation selected from the group consisting of tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections and granules.

[0068] The pharmaceutical composition may be for oral or parenteral administration. Formulations for oral administration include tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections, and granules, and these formulations may be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Formulations for parenteral administration may be creams, lotions, ointments, plasters, solutions, aerosols, fluid extracts, elixirs, infusions, sachets, patches, or injections.

[0069] The pharmaceutical composition may contain conventional fillers, bulking agents, binders, disintegrants, anticoagulants, lubricants, wetting agents, pH regulators, nutrients, vitamins, electrolytes, alginic acid and its salts, pectic acid and its salts, protective colloids, glycerin, flavorings, emulsifiers or preservatives.

[0070] The pharmaceutical composition may further be manufactured by including one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers may include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, the composition may be preferably formulated according to each disease or component using an appropriate method in the art.

[0071]

[0072] Another aspect provides a method for preventing or treating an inflammatory disease, comprising administering to a subject in need thereof a mutant of the XIAP protein or a nucleotide encoding the same; or a fusion protein or a polynucleotide encoding the same.

[0073]

[0074] Another aspect provides the use of a variant of the XIAP protein or a nucleotide encoding the same; or a fusion protein or a polynucleotide encoding the same; for the manufacture of a medicament for the prevention or treatment of an inflammatory disease.

[0075]

[0076] Another aspect is to provide a health functional food composition for preventing or improving an inflammatory disease, comprising as an active ingredient a variant of the XIAP protein or a nucleotide encoding the same; or the fusion protein or a polynucleotide encoding the same.

[0077] The above fusion protein is as described above.

[0078] The term "health functional food" as used herein refers to a food manufactured or processed for the purpose of health supplementation using a specific ingredient as a raw material or a specific ingredient contained in a food raw material through extraction, concentration, purification, mixing, etc., and refers to a food designed and processed so that the above-mentioned ingredient can sufficiently exert bioregulatory functions on the body, such as biodefense, regulation of biological rhythm, and prevention and recovery from disease. The above-mentioned health functional food composition can perform functions related to the prevention and improvement of sepsis and related diseases.

[0079] There are no specific restrictions on the types of the above foods. Examples of foods to which the above substances can be added include formulations selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, and health drinks, and include all health foods in the conventional sense.

[0080] The above health beverage composition may, like conventional beverages, contain various sweeteners, flavoring agents, or natural carbohydrates as additional ingredients. The sweetener may be a natural sweetener or a synthetic sweetener. The natural sweetener may be thaumatin or a stevia extract, and the synthetic sweetener may be saccharin or aspartame.

[0081] The health beverage composition of the present invention may, like conventional beverages, contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates mentioned above may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin. The proportion of the natural carbohydrate may generally be about 0.01 to 10 g, preferably about 0.01 to 0.1 g, per 100 ml of the composition of the present invention.

[0082] The above health functional food may include food additives that are food-related and acceptable, and may include an appropriate carrier commonly used in the manufacture of health functional foods.

[0083] In addition to the above, the composition may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The composition may further include fruit pulp for the production of natural fruit juices, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected within the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0084]

[0085] Another aspect is to provide a feed composition for preventing or improving an inflammatory disease, comprising as an active ingredient a variant of the XIAP protein or a nucleotide encoding the same; or the fusion protein or a polynucleotide encoding the same.

[0086] The above fusion protein is as described above.

[0087] When used as a feed composition, the composition may be a highly concentrated solution of 20 to 90% or may be manufactured in the form of powder or granules. The feed composition may further include one or more of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polyphosphate); and natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. The composition may be formulated in the form of a conventional feed and may include conventional feed ingredients together.

[0088] The feed may further include grains such as ground or shredded wheat, oats, barley, corn and rice; plant-based protein feeds such as feeds mainly composed of rapeseed, soybeans and sunflower; animal-based protein feeds such as blood meal, meat meal, bone meal and fish meal; dry ingredients composed of sugars and dairy products such as various types of milk powder and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters and the like.

[0089] The feed composition may be administered to animals alone or in combination with other feed additives in an edible carrier. Furthermore, the feed additives may be readily administered to animals as a top dressing, by mixing them directly into animal feed, or in an oral formulation separate from the feed. When the feed additives are administered separately from animal feed, they may be prepared as immediate-release or sustained-release formulations by combining them with food-grade edible carriers, as is well known in the art. Such edible carriers may be solid or liquid, such as cornstarch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the feed additive may be in the form of a tablet, capsule, powder, troche, or saccharide tablet, or a top dressing in a microdispersible form. When a liquid carrier is used, the feed additive may be in the form of a gelatin soft capsule, or a syrup, suspension, emulsion, or solution.

[0090] Additionally, the feed may contain additives such as preservatives, stabilizers, wetting or emulsifying agents, and solution accelerators. The feed additives may be added to animal feed by injection, spraying, or mixing.

[0091] The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry, and fish.

[0092] As the mammals, it can be used for pigs, cows, sheep, goats, laboratory rodents, and pets (e.g., dogs, cats), as the poultry, it can be used for chickens, turkeys, ducks, geese, pheasants, and quail, and as the fish, it can be used for trout, but is not limited thereto.

[0093] According to one aspect, a XIAP protein mutant or a fusion protein comprising the same maintains a biological function similar to that of the wild-type XIAP protein, and can activate the NOD (nucleotide-binding oligomerization domain) signaling pathway by promoting the ubiquitination of RIP2 (Receptor-interacting serine / threonine-protein kinase 2). In addition, the mutant or fusion protein has an increased intracellular retention time and residual amount compared to the wild-type XIAP protein, and thus can be usefully utilized in the treatment of inflammatory diseases, particularly diseases caused by NOD signaling defects.

[0094] Figure 1 is a schematic diagram showing the amino acid mutation positions of an XIAP (X-linked inhibitor of apoptosis protein) protein variant according to one aspect of the present invention.

[0095] Figure 2 is a photograph showing the results of a co-immunoprecipitation assay to determine whether wild-type XIAP protein and XIAP protein variants according to one aspect bind to RIP2 (Receptor-interacting serine / threonine-protein kinase 2).

[0096] Figure 3 shows the results of evaluating RIP2 ubiquitination activity by wild-type XIAP protein and XIAP protein mutants according to one aspect through ubiquitination assay.

[0097] Figure 4 is a graph showing the results of an IL-8 ELISA assay to evaluate the effect of treatment with XIAP protein variants according to the daily pattern on the expression of IL-8 in cells of patients with hereditary enteritis.

[0098] Figure 5 is a photograph and a graph quantifying the results of Western blot analysis of the amount of protein remaining in cells after XIAP knock-out (KO) cells were treated with XIAP mutants (GST-XIAP-E134D) and XAIP wild-type protein (GST-XIAP) according to one aspect (100 nM, 6 hours).

[0099] Figure 6 is a photograph showing the results of a Western blot analysis of the intracellular residual amount of a fusion protein according to the daily pattern, and a graph quantifying the results.

[0100] Figure 7 is a photograph showing the results of a Western blot analysis of the intracellular residual amount of a fusion protein over time according to the daily pattern, and a graph quantifying the results.

[0101] Figure 8 is a photograph showing the results of comparative analysis of protein availability by fractionating the cells into an insoluble pellet and a soluble supernatant after expressing a protein fused with a His-tag or GST-tag to a XIAP mutant (XIAP-E134D) according to one aspect.

[0102] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0103]

[0104] Example 1. Preparation of XIAP protein variants

[0105] A XIAP protein mutant was prepared by substituting amino acids with similar properties to existing amino acids in the linker portion between the main domains of the wild-type XIAP (X-linked inhibitor of apoptosis protein) protein so that the function can be maintained without changing the domain structure of the protein.

[0106] Specifically, a wild-type (WT) XIAP construct (pEGFP-XIAP-WT) tagged with green fluorescent protein (GFP) at the N terminus was constructed. The full-length cDNA clone of XIAP (KUGI #hMU005178) was purchased from the Korean Gene Bank (KHGB). The PCR-amplified full-length XIAP open reading frame (ORF) was digested with SalI and BamHI restriction enzymes and then inserted (subcloned) into the pEGFP-C2 vector (Takara Bio, Japan) at the XhoI and BamHI restriction sites.

[0107] Specific mutations were introduced into the nucleotides encoding the wild-type XIAP protein consisting of the nucleotide sequence of SEQ ID NO: 1. The introduced mutations are shown in Table 1.

[0108] Sequence before XIAP change Sequence after change + MATGE134DGAGGACI153LATACTG

[0109] Through the above mutations, a recombinant expression vector capable of expressing the mutations E134D, I153L, or +M was constructed.

[0110] HEK 293T cells (CRL-3216, ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Cytiva, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 1% penicillin-streptomycin, and 2.05 mM L-glutamine at 37°C in 5% CO₂. To express GFP-tagged XIAP WT or XIAP mutants in HEK 293T cells, 1.2 × 10 6 After seeding the cells, 8 μL of Lipofectamine 2000 (ThermoFisher, Waltham, MA) and 1 μg each of pEGFP, pEGFP-XIAP-WT, and pEGFP-XIAP mutant plasmids were mixed and transfection was performed for 6 hours. The culture medium was then replaced with normal medium (DMEM containing 10% FBS).

[0111]

[0112] The XIAP protein variants obtained through the above cell line include a variant in which methionine is added to the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5; a variant in which the 134th glutamic acid from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with aspartic acid; and a variant in which the 153rd isoleucine from the N-terminus of the XIAP protein having the amino acid sequence of SEQ ID NO: 5 is substituted with leucine. The amino acid sequences of each variant are shown in Table 2.

[0113]

[0114] Example 2. Preparation of XIAP protein mutants and GST fusion proteins

[0115] A fusion protein was prepared by adding a tag protein to the XIAP protein produced by the method of Example 1 above.

[0116] The XIAP recombinant protein (GST-XIAP-E134D) according to the present invention was expressed using Escherichia coli Rosetta2 (DE3) pLysS strain. The pGEX-4T-1 vector with a GST (glutathione S-transferase) tag fused to the N-terminus was used as the expression vector, and ampicillin and chloramphenicol were applied as antibiotic selection markers. Protein expression was performed in LB medium, and 0.2 mM IPTG was added at 18°C ​​to induce for 16 hours, thereby minimizing the formation of insoluble protein aggregates (inclusion bodies) and increasing the proportion of soluble protein. As a result of Coomassie blue staining and Western blot analysis, the target protein with a molecular weight of approximately 83 kDa was confirmed in the soluble fraction, suggesting that purification under native conditions was possible.

[0117] Purification was performed using a glutathione affinity column (GSTrap FF, GE Healthcare), taking advantage of the affinity purification properties of the GST tag. IPTG-induced cells were harvested, lysed with lysis buffer, and centrifuged to obtain a supernatant. The supernatant was applied to the GST column. The column was equilibrated at a binding flow rate of 0.7 mL / min and a wash flow rate of 1 mL / min, and the protein was efficiently eluted using a gradient elution method. Each eluted fraction was analyzed by 15% SDS-PAGE, and fractions E9 to E14 containing the target protein were selected and used in the subsequent process.

[0118] The selected eluate was dialyzed against PBS buffer, and the protein concentration was then measured using the Bradford assay. The purified GST-XIAP-E134D protein had an average concentration of 1.221 mg / mL, and a total yield of approximately 4.882 mg was obtained in 4 mL volumes. The protein was diluted in PBS buffer and stored at -20°C.

[0119] The GST tag applied to the present invention not only enhances protein solubility but also enables high-efficiency affinity purification, which is advantageous for the production of functional recombinant proteins in an E. coli expression system. This purification process can be applied to the production of recombinant XIAP proteins that retain physiological activity, providing a foundation advantageous for mass production and pharmaceutical applications.

[0120]

[0121] Experimental Example 1. Evaluation of functional equivalence of XIAP mutants to wild-type XIAP

[0122] 1.1 Evaluation of binding ability of XIAP mutants and RIP2 (Co-immunoprecipitation assay)

[0123] Co-immunoprecipitation assay was performed to confirm the binding of XIAP protein mutants to RIP2 (Receptor-interacting serine / threonine-protein kinase 2).

[0124] Specifically, HEK293T cells were transfected with recombinant expression vectors (XIAP (WT), XIAP-C203Y, XIAP-E134D, XIAP-I153L, XIAP+M) using Lipofectamine 2000 for 6 h. The medium was then replaced with regular culture medium. Twenty-four hours after transfection, before cell harvest, the cells were treated with 10 μM MG132 (Enzo, Farmingdale, NY, USA) for 4.5 h. The cells were then lysed in RIPA buffer supplemented with 1X protease inhibitor, 1X phosphatase inhibitor, and 100 μM PMSF. The lysate was centrifuged at 15,000 rpm for 20 min at 4°C. After quantifying equal amounts of protein using BCA analysis, 10% of the total lysate was used for immunoblotting to confirm the expression of each protein. The remaining supernatant was reacted overnight at 4°C with 1 mg of anti-GFP antibody.

[0125] Proteins bound to antibodies were pulled down using 40 μL of Protein A / G Plus agarose at 4°C for 1 hour. Agarose beads were washed three times with RIPA buffer, and 1X SDS sample loading buffer was added followed by heating. Eluted proteins were subjected to immunoblotting using anti-RFP, anti-GFP, and anti-β-actin antibodies, and the results are shown in Figure 2.

[0126] Figure 2 is a photograph showing the results of a co-immunoprecipitation assay to determine whether wild-type XIAP protein and XIAP protein variants according to one aspect bind to RIP2 (Receptor-interacting serine / threonine-protein kinase 2).

[0127] In FIG. 2, WT refers to the wild-type XIAP protein, C203Y refers to an XIAP variant found in a patient with a hereditary enteritis mutation, E134D refers to an XIAP variant in which the 134th glutamic acid from the N-terminus of the XIAP protein consisting of the amino acid sequence of SEQ ID NO: 5 is substituted with aspartic acid, I153L refers to an XIAP variant in which the 153rd isoleucine from the N-terminus of the XIAP protein consisting of the amino acid sequence of SEQ ID NO: 5 is substituted with leucine, and +M refers to an XIAP variant in which methionine is added to the N-terminus of the XIAP protein consisting of the amino acid sequence of SEQ ID NO: 5.

[0128] Specifically, the E134D variant of the present invention has an amino acid sequence of SEQ ID NO: 7, the I153L variant has an amino acid sequence of SEQ ID NO: 8, and the +M variant has an amino acid sequence of SEQ ID NO: 6.

[0129] As shown in Fig. 2, the C203Y mutant, an XIAP mutant found in patients with hereditary enteritis, showed a significantly reduced binding ability to RIP2 compared to the wild type (WT), and the E134D, I153L, and +M mutants were confirmed to bind to RIP2 at a level similar to the wild type.

[0130] This means that the E134D, I153L, and +M mutants have activities equivalent to those of the wild-type protein.

[0131]

[0132] 1.2 Evaluation of RIP2 ubiquitination activity by XIAP mutants (Ubiquitination assay)

[0133] To determine whether XIAP protein mutants promote ubiquitination of RIP2 (Receptor-interacting serine / threonine-protein kinase 2), a ubiquitination assay was performed.

[0134] Specifically, for ubiquitination analysis, HEK293T cells were transfected with recombinant expression vectors (XIAP (WT), XIAP-C203Y, XIAP-E134D, XIAP-I153L, XIAP+M) using Lipofectamine 2000 for 6 h. The medium was then replaced with regular culture medium. Twenty-four hours after transfection, cells were treated with 10 μM MG132 for 2 h before harvest. Cells were then lysed using co-IP lysis buffer supplemented with 20 mM NEM. Cell lysates were centrifuged at 15,000 rpm for 20 min at 4°C. Equal amounts of proteins in the supernatants were quantified by BCA assay, and 10% of the total lysate was used for immunoblot analysis to determine the expression of each protein. The remaining supernatant was reacted overnight at 4°C with 1 mg of anti-GFP or anti-RFP antibody. Antibody-bound proteins were pulled down using 20 μL of Protein A or G magnetic beads at 4°C for 1 h. The beads were then washed three times with co-immunoprecipitation lysis buffer and placed in 1X SDS sample loading buffer and heated. The eluted proteins were subjected to immunoblotting analysis using anti-RFP, anti-GFP, anti-ubiquitin, or anti-β-actin antibodies, and the results are shown in Figure 3.

[0135] Figure 3 shows the results of evaluating RIP2 ubiquitination activity by wild-type XIAP protein and XIAP protein mutants according to one aspect through ubiquitination assay.

[0136] In FIG. 3, WT refers to the wild-type XIAP protein, C203Y refers to an XIAP variant found in a patient with a hereditary enteritis mutation, E134D refers to an XIAP variant in which the 134th glutamic acid from the N-terminus of the XIAP protein consisting of the amino acid sequence of SEQ ID NO: 5 is substituted with aspartic acid, I153L refers to an XIAP variant in which the 153rd isoleucine from the N-terminus of the XIAP protein consisting of the amino acid sequence of SEQ ID NO: 5 is substituted with leucine, and +M refers to an XIAP variant in which methionine is added to the N-terminus of the XIAP protein consisting of the amino acid sequence of SEQ ID NO: 5.

[0137] Specifically, the E134D variant of the present invention has an amino acid sequence of SEQ ID NO: 7, the I153L variant has an amino acid sequence of SEQ ID NO: 8, and the +M variant has an amino acid sequence of SEQ ID NO: 6.

[0138] As shown in Fig. 3, the C203Y mutant did not exhibit ubiquitination activity toward RIP2, but the E134D, I153L, and +M mutants of the present invention were all confirmed to exhibit RIP2 ubiquitination activity at a level similar to that of the wild-type XIAP protein.

[0139] This means that the E134D, I153L, and +M mutants have activities equivalent to those of the wild-type protein.

[0140]

[0141] 1.3 Evaluation of IL-8 expression by XIAP mutants (IL-8 ELISA assay)

[0142] To determine whether the decreased IL-8 expression due to impaired XIAP function in patients with hereditary enteritis could be restored by treatment with XIAP protein mutants (M-XIAP, XIAP-E134D), IL-8 expression evaluation (IL-8 ELISA assay) was performed.

[0143] Specifically, 2×10 patient PBMCs in 96 wells 5Prepare 96-well plates. Treat with XIAP proteins, and then treat with MDP 6 hours later. After 24 hours, collect cells in 96-well plates, centrifuge (3000g, 10 minutes), and prepare the supernatant separately. Mix 2 ml of calibrator diluent RD5P and 8 ml of water, add 10 μl of the supernatant to 490 μl of the 10 ml solution, and prepare a total of 500 μl. In the same way, combine 10 μl of the standard solutions (0, 62.5, 125, 250, 500, 1000 pg / ml) to prepare 500 μl each. Add 100 μl of assay diluent RD1-85 to the 96-well plate coated with L-8-specific antibody. Add 50 μl of the standard and 500 μl sample to the coated 96-well plate (150 μl total). After incubation for 2 hours, wash four times with 200 μl of 1X wash buffer and suction all wells. Add 100 μl of IL-8 conjugate. After incubation for 1 hour, wash four times with 200 μl of 1X wash buffer and suction all wells. Add 200 μl of substrate solution. After incubation for 30 minutes, add 50 μl of stop solution and measure at 450 nm using Envision equipment. Check the standard curve with the optical density (OD) value for the IL-8 standard solution. Using the standard curve, the OD value of the sample was calculated to calculate the amount of IL-8 in each sample, and the results are shown in Figure 4.

[0144] Figure 4 is a graph showing the results of an IL-8 ELISA assay to evaluate the effect of treatment with XIAP protein variants according to the daily pattern on the expression of IL-8 in cells of patients with hereditary enteritis.

[0145] As shown in Figure 4, when normal cells were treated with MDP (muramyl dipeptide), a NOD2 activator, the expression of IL-8 significantly increased, whereas in cells from patients with hereditary enteritis with impaired XIAP function, IL-8 expression was very low when treated with MDP. This is interpreted as being because the NOD2 signaling pathway does not function properly in patients with XIAP deficiency.

[0146] However, when cells from patients with hereditary enteritis were treated with XIAP mutants (M-XIAP, XIAP-E134D) or wild-type XIAP protein (XIAP-WT Commercial), it was confirmed that IL-8 expression was restored to a level similar to that of the normal group.

[0147] These results indicate that the XIAP mutant (M-XIAP, XIAP-E134D) according to one aspect of the present invention has the effect of restoring the immune regulation function and inflammation regulation function by restoring the NOD2 signal transduction pathway damaged due to XIAP deficiency.

[0148]

[0149] Experimental Example 2. Evaluation of Intracellular Stability of XIAP Variants

[0150] To evaluate the intracellular expression levels of XIAP mutants, XIAP knock-out (KO) cells were treated with GST-XIAP-E134D and GST-XIAP (WT) proteins (100 nM, 6 hours), and the amount of protein remaining in the cells was analyzed by Western blot.

[0151] Specifically, a cell culture solution containing 400,000 cells of the colon cancer cell line HCT116 was dispensed into a culture dish and cultured in a 37℃ incubator for 24 hours. GST-XIAP-E134D and GST-XIAP (WT) proteins were diluted to a concentration of 100 nM each and treated to the cells and cultured in a 37℃ incubator for 6 hours. Afterwards, HCT116 cells were harvested, proteins were extracted, and the protein concentration was measured using the BCA protein quantification method, and proteins were separated by molecular weight using SDS-PAGE (Sodium Dodecyl sulfate-polyacylamide gel electrophoresis). The separated proteins inside the gel were transferred to the membrane through the transfer process and blocked with 5% Skim milk / T-TBS. Afterwards, the primary antibody (antibody (1st; XIAP, β-Actin) was bound to the protein, and the secondary antibody with HRP (horseradish peroxidase) attached was reacted. The secondary HRP oxidized the luminol of the ECL (Enhanced chemiluminescence) solution, and the emitted light was exposed to an X-ray film to confirm the amount of protein expression, and the results are shown in Fig. 5.

[0152] Figure 5 is a photograph and a graph quantifying the results of Western blot analysis of the amount of protein remaining in cells after XIAP knock-out (KO) cells were treated with XIAP mutants (GST-XIAP-E134D) and XAIP wild-type protein (GST-XIAP) according to one aspect (100 nM, 6 hours).

[0153] As shown in Fig. 5, it was confirmed that the expression of XIAP protein in the XIAP mutant (GST-XIAP-E134D) treatment group was maintained at a higher level than in the XAIP wild type (GST-XIAP) treatment group.

[0154] These results indicate that the XIAP variant according to one aspect of the present invention is expressed and maintained at a higher level within cells than the XAIP wild type, and has superior intracellular stability.

[0155]

[0156] Experimental Example 3. Evaluation of Intracellular Stability of Fusion Proteins

[0157] 3.1 Evaluation of intracellular residual amounts of fusion proteins

[0158] To evaluate the intracellular stability of fusion proteins containing XIAP mutants and tag proteins according to the daily pattern, HCT116 cells in which the XIAP gene is knocked out (KO) were treated with fusion proteins containing XIAP mutants and tag proteins (GST-M-XIAP, GST-XIAP-E134D) and XIAP wild-type protein (XIAP-WT) (100 nM, 6 hours), and the amount of protein remaining in the cells was analyzed by Western blot.

[0159] Specifically, a cell culture solution containing 400,000 cells of the colon cancer cell line HCT116 was dispensed into a culture dish and cultured in a 37℃ incubator for 24 hours. GST-M-XIAP, GST-XIAP-E134D, and XIAP-WT without a GST tag were diluted to a concentration of 100 nM each and treated to the cells and cultured in a 37℃ incubator for 6 hours. Afterwards, HCT116 cells were harvested, proteins were extracted, and the protein concentration was measured using the BCA protein quantitation method, and proteins were separated by molecular weight using SDS-PAGE (Sodium Dodecyl sulfate-polyacylamide gel electrophoresis). The separated proteins inside the gel were transferred to the membrane through the transfer process and blocked with 5% Skim milk / T-TBS. Afterwards, the primary antibody (antibody (1st; XIAP, β-Actin) was bound to the protein, and the secondary antibody with HRP (horseradish peroxidase) attached was reacted. The secondary HRP oxidized the luminol of the ECL (Enhanced chemiluminescence) solution, and the emitted light was exposed to an X-ray film to confirm the amount of protein expression, and the results are shown in Fig. 6.

[0160] Figure 6 is a photograph showing the results of a Western blot analysis of the intracellular residual amount of a fusion protein according to one aspect of the present invention, and a graph quantifying the results.

[0161] As shown in Fig. 6, it was confirmed that the expression and residual amount of XIAP protein were maintained at a higher level in the fusion protein (GST-M-XIAP, GST-XIAP-E134D) treatment group containing XIAP mutant and tag protein compared to the XIAP wild-type protein treatment group.

[0162] This means that the fusion protein containing the XIAP mutant and the tag protein has increased intracellular stability.

[0163]

[0164] 3.2 Evaluation of intracellular retention time of fusion proteins over time

[0165] To evaluate the intracellular stability of fusion proteins containing XIAP mutants and tag proteins according to the daily pattern, fusion proteins containing XIAP mutants and tag proteins (GST-XIAP-E134D) and XIAP wild-type protein (XIAP-WT) were treated (100 nM) to HCT116 cells in which the XIAP gene was knocked out (KO), and the amount of protein remaining in the cells was analyzed over time (2, 4, and 8 hours) by Western blot.

[0166] Specifically, the cell culture solution containing 400,000 cells of the colon cancer cell line HCT116 was dispensed into a culture dish and cultured in a 37℃ incubator for 24 hours. GST-XIAP-E134D and XIAP-WT proteins without GST tag were diluted to a concentration of 100 nM each and treated to the cells and cultured in a 37℃ incubator for 2, 4, and 8 hours. Afterwards, HCT116 cells were harvested, proteins were extracted, and the protein concentration was measured using the BCA protein quantitation method, and proteins were separated by molecular weight using SDS-PAGE (Sodium Dodecyl sulfate-polyacylamide gel electrophoresis). The separated proteins inside the gel were transferred to the membrane through the transfer process and blocked with 5% Skim milk / T-TBS. Afterwards, the primary antibody (antibody (1st; XIAP, β-Actin) was bound to the protein, and the secondary antibody with HRP (horseradish peroxidase) attached was reacted. The secondary HRP oxidized the luminol of the ECL (Enhanced chemiluminescence) solution, and the emitted light was exposed to an X-ray film to confirm the amount of protein expression, and the results are shown in Fig. 7.

[0167] Figure 7 is a photograph showing the results of a Western blot analysis of the intracellular residual amount of a fusion protein over time according to one aspect of the present invention, and a graph quantifying the results.

[0168] As shown in Fig. 7, the fusion protein (GST-XIAP-E134D) treatment group containing XIAP mutants and tag proteins showed a consistently higher level of residual XIAP protein than the XIAP wild-type protein (XIAP-WT) treatment group throughout all observation times (2, 4, and 8 hours). In particular, after 8 hours of treatment, the amount of residual protein in the fusion protein (GST-XIAP-E134D) treatment group containing XIAP mutants and tag proteins remained significantly higher than that in the XIAP wild-type protein (XIAP-WT) treatment group.

[0169] This means that the fusion protein according to one aspect of the present invention has superior stability within cells than the XIAP wild-type protein, and thus remains at a high level for a longer period of time.

[0170]

[0171] Experimental Example 4. Evaluation of Purification Yield of Fusion Proteins

[0172] In order to evaluate the purification yield of a fusion protein according to one aspect of the present invention depending on the type of tag protein, XIAP mutants (XIAP-E134D) fused with His-tag or GST-tag were expressed, respectively, and then the intracellular solubility was compared and analyzed.

[0173] Specifically, the fusion protein was expressed and purified in the same manner as in Example 2, except that a XIAP mutant (XIAP-E134D) protein fused with a His-tag or GST-tag was used, and its intracellular solubility was evaluated, and the results are shown in Fig. 8.

[0174] Figure 8 is a photograph showing the results of comparative analysis of protein availability by fractionating the cells into an insoluble pellet and a soluble supernatant after expressing a protein fused with a His-tag or GST-tag to a XIAP mutant (XIAP-E134D) according to one aspect.

[0175] As shown in Fig. 8, the XIAP-E134D protein fused with a His-tag was mainly present as an insoluble pellet, and only a trace amount was observed in the supernatant fraction. On the other hand, the GST-XIAP-E134D protein fused with a GST-tag was confirmed to be expressed at a high level mostly in the supernatant fraction.

[0176] These results imply that by fusing a GST-tag, the water solubility of XIAP protein can be significantly increased, thereby significantly improving the purification yield.

Claims

1. A mutant of XIAP (X-linked inhibitor of apoptosis protein), wherein the mutant is A mutation in which methionine is added to the N-terminus of the XIAP protein consisting of the amino acid sequence of sequence number 5; A mutation in which the 134th glutamic acid from the N-terminus of the XIAP protein consisting of the amino acid sequence of sequence number 5 is substituted with aspartic acid; and A XIAP mutant comprising at least one mutation in which the 153rd isoleucine from the N-terminus of the XIAP protein consisting of the amino acid sequence of sequence number 5 is substituted with leucine.

2. In claim 1, The above variant is a XIAP variant comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 8.

3. A fusion protein comprising a variant of claim 1 and a tag protein.

4. In claim 3, the tag protein is a fusion protein selected from the group consisting of Histidine (His), Glutathione S-transferase (GST), Maltose-binding protein (MBP), FLAG (DYKDDDDK), Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Yellow Fluorescent Protein (YFP), Cyan Fluorescent Protein (CFP), Thioredoxin (Trx), Protein disulfide isomerase (PDI), N-utilization substance protein A (NusA), and Hemagglutinin (HA).

5. A fusion protein according to claim 3, wherein the fusion protein comprises any one amino acid sequence selected from the group consisting of sequence numbers 9 to 11.

6. A polynucleotide encoding a variant of claim 1 or a fusion protein of claim 3.

7. A recombinant vector comprising the polynucleotide of claim 6.

8. A host cell transformed with the recombinant vector of claim 7.

9. In claim 1, The above mutant is an XIAP mutant that promotes ubiquitination of RIP2 (Receptor-interacting serine / threonine-protein kinase 2) and induces activation of NOD (Nucleotide-binding oligomerization domain) signaling.

10. In claim 3, The above fusion protein is a fusion protein having an increased intracellular retention time and residual amount compared to the wild-type XIAP protein.

11. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising as an active ingredient a variant of the XIAP protein of claim 1 or a nucleotide encoding the same; or a fusion protein of claim 3 or a polynucleotide encoding the same.

12. In claim 10, A pharmaceutical composition, wherein the inflammatory disease is at least one selected from the group consisting of osteoarthritis, sepsis, gastritis, inflammatory bowel diseases (IBD), enteritis, nephritis, hepatitis, ulcerative colitis, irritable bowel syndrome, chronic ileitis, ischemic colitis, ulcerative proctitis, reactive arthritis, chronic inflammatory arthrosis, neuropathic joint disease, inflammatory pain, and eczema.

13. A method for preventing or treating an inflammatory disease, comprising administering to a subject in need thereof an effective amount of a variant of the XIAP protein of claim 1 or a nucleotide encoding the same; or a fusion protein of claim 3 or a polynucleotide encoding the same.

14. Use of a variant of the XIAP protein of claim 1 or a nucleotide encoding the same; or a fusion protein of claim 3 or a polynucleotide encoding the same; for the manufacture of a medicament for preventing or treating an inflammatory disease.

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