Novel peptide derived from NDST3 or NDST4 with deacetylase or sulfonotransferase activity

Abstract

The present invention relates to a peptide having deacetylase or sulfate-transferase activity, a method for preparing the same, and uses thereof, by identifying a domain having deacetylase or sulfate-transferase activity in NDST3 and NDST4, producing a water-soluble peptide containing the amino acid sequence of said domain, and confirming the deacetylase or sulfate-transferase activity of said peptide. The peptide of the present invention is expected to be used as a platform for developing protein drugs or inhibitors thereof utilizing NDST3 and NDST4 proteins.

Description

Technology Field

[0001] The present invention relates to a peptide having deacetylase or sulfate-transferase activity, a method for preparing the same, and uses thereof, which is completed by identifying a domain having deacetylase or sulfate-transferase activity in NDST3 and NDST4, producing a water-soluble peptide containing the amino acid sequence of said domain, and confirming the deacetylase or sulfate-transferase activity of said peptide. Background Technology

[0003] NDST3 and NDST4 are members of the NDST family and are enzymes that play a crucial role primarily in the biosynthesis of heparan sulfate proteoglycans (HSPGs). NDST3 and NDST4 regulate the chemical modification and structural diversity of HSPGs, performing essential functions for intercellular signaling, cell growth, and tissue development; through this, they influence neurodevelopment, neuroregeneration, and pathological mechanisms in certain diseases.

[0004] NDST3 is highly expressed in specific tissues or cell types during the early embryonic stage, with a particularly prominent role in the nervous system. The known functions of NDST3 are as follows: 1) NDST3 regulates the modification of HSPGs, which play a crucial role in the growth and differentiation of nerve cells. The structure of heparan sulfate regulated by NDST3 plays a vital role in regeneration following nerve injury as well as in neural development due to its binding affinity with specific growth factors and extracellular matrix proteins. 2) NDST3-modified HSPGs bind to various growth factors such as FGF and VEGF to regulate cell signaling, thereby enhancing signals related to cell proliferation, differentiation, and survival. 3) By regulating the composition of HSPGs associated with immune cell activity, it modulates inflammatory responses, participates in the migration, adhesion, and activation of immune cells, and can serve as an important target in inflammatory and autoimmune diseases.

[0005] NDST3 plays a particularly important role in the nervous system. It is essential for neuronal growth, axon guidance, and synapse formation, plays a crucial role in signal transmission between neurons, and facilitates the proper growth and communication of neurons, thereby performing important functions in neurodevelopment and growth regulation. Furthermore, NDST3 participates in the regeneration process following neuronal injury to promote the regeneration of damaged neurons and is involved in protecting neurons from damage caused by external factors or stress. Modifications or mutations in NDST3 also affect the process of amyloid plaque accumulation, which is a cause of Alzheimer's disease. Therefore, the NDST3 protein can provide therapeutic and neuroprotective effects by promoting neuronal regeneration in cases of spinal cord injury or traumatic brain injury. It can be utilized as a protein therapeutic to overcome various neurological injuries or neurotoxic environments, and can serve as a new drug development platform targeting neurological diseases such as Alzheimer's disease and Parkinson's disease.

[0006] As such, NDST3 and NDST4 proteins play roles such as promoting neuroregeneration and providing neuroprotective effects. However, despite the research results reported on NDST3 and NDST4 to date, many aspects have not been experimentally verified, and the mechanisms by which they directly regulate nerve cells to promote regeneration or provide protection remain unknown. Consequently, NDST3 and NDST4 are attracting attention as new targets for the treatment of neurological diseases. While the development of new drugs based on these enzymes holds significance as targets for innovative therapies for various neurological conditions, including neurological disorders, neuroinjury, and developmental disorders, research on the development of such treatments is minimal. To develop drugs utilizing the mechanisms of NDST3 and NDST4, which remain largely unidentified to date, research into the signaling pathways that induce activation and inhibition, as well as the search for protein activators and inhibitors of NDST3 and NDST4, are tasks that must be undertaken for the development of treatments for neurodevelopment and neurological diseases.

[0007] As a result of diligent research to meet such requirements, the inventor obtained water-soluble proteins by cloning the functional sites of NDST3 and NDST4 proteins, specifically N-deacetylase and N-sulfotransferase or a specific portion containing the respective N-deacetylase and N-sulfotransferase domains, and completed the present invention by confirming that these water-soluble NDST3 and NDST4 proteins would be useful as a platform for developing protein drugs or inhibitors. Prior art literature

[0009] Current Opinion in Cell Biology, 18(5), 562-568.Nature, 446(7139), 1030-1037Nature Neuroscience, 15(3), 423-433 The problem to be solved

[0010] The technical problem that the present invention aims to solve is to identify a deacetylase or sulfotransferase active domain in NDST3 and NDST4, and to provide a peptide containing said domain derived from said NDST3 or NDST4 as a deacetylase or sulfotransferase.

[0011] In addition, the present invention provides a method for mass-producing the peptide in a water-soluble form using E. coli, and aims to provide the peptide for use in treating neurological diseases.

[0012] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below. means of solving the problem

[0014] In this specification, amino acid sequences are listed in order from the N-terminus to the C-terminus.

[0015] The human NDST3 protein consists of the amino acid sequence of SEQ ID NO. 1, and the human NDST4 protein consists of the amino acid sequence of SEQ ID NO. 5.

[0016] The inventors confirmed that the domain exhibiting deacetylase activity in the NDST3 protein is at position 72-569 aa, and the domain exhibiting sulfotransferase activity is at position 569-873 aa.

[0017] In addition, the inventors confirmed that the domain exhibiting deacetylase activity in the NDST4 protein is at position 72-569 aa, and the domain exhibiting sulfate transferase activity is at position 569-873 aa.

[0018] Accordingly, the present invention provides a peptide having deacetylase activity.

[0019] The peptide having deacetylase activity may include or be composed of the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 7. If the peptide having deacetylase activity includes the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 7, the peptide may include a tag sequence for purification at the N-terminus of SEQ ID NO. 3 or SEQ ID NO. 7, and the total amino acid length does not exceed 800 a.a.

[0020] In addition, the present invention provides a peptide having sulfate transferase activity.

[0021] The peptide having sulfate-transferase activity may include or be composed of the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 8. If the peptide having sulfate-transferase activity includes the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 8, the peptide may include a tag sequence for purification at the N-terminus of SEQ ID NO. 4 or SEQ ID NO. 8, and the total amino acid length does not exceed 800 a.a.

[0022] The amino terminus of the peptide of the present invention may be bonded to a protecting group such as an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG), and the carboxyl terminus of the peptide may be modified to a hydroxyl group (-OH), an amino group (-NH2), an azide (-NHNH2), etc. Additionally, fatty acids, oligosaccharide chains, all nanoparticles (gold particles, liposomes, heparin, hydrogels, etc.), amino acids, carrier proteins, etc., may be bonded to the terminus of the peptide of the present invention or to the R-remote group (R-group) of an amino acid. The modification of the amino acid described above serves to improve the potency and stability of the peptide of the present invention. In this specification, the term "stability" refers not only to in vivo stability but also to storage stability (including storage stability at room temperature, refrigerated, and frozen).

[0023] Meanwhile, each peptide constituting the peptide of the present invention may have one or more amino acid sequences substituted, modified, or deleted within the range of maintaining its function.

[0024] In addition, the present invention provides a polynucleotide encoding a peptide having deacetylase activity or a peptide having sulfate transferase activity.

[0025] In addition, the present invention provides the peptide expression cassette comprising the polynucleotide.

[0026] In addition, the present invention provides a peptide having deacetylase activity or a peptide having sulfate transferase activity comprising the above expression cassette.

[0027] In addition, the present invention provides a transformant transformed by the above vector.

[0028] In one embodiment of the present invention, the transformant may be Escherichia coli (E. coli).

[0029] The term "polynucleotide" as used herein refers to a polymer of deoxyribonucleotides or ribonucleotides existing in a single-stranded or double-stranded form. It encompasses RNA genome sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless specifically noted otherwise.

[0030] In the present invention, the base sequence encoding the peptide includes not only the base sequence encoding the amino acid described by each sequence number, but also, without limitation, any base sequence encoding a protein that exhibits an efficacy substantially identical or corresponding to each protein, which has homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with said sequence. Furthermore, it is obvious that the scope of the present invention also includes cases where some sequences have deleted, modified, substituted, or added amino acid sequences, provided that the sequence has homology with said sequence and has biological activity identical or corresponding to the conjugate protein of said sequence number described.

[0031] The term "homology" in this specification refers to the degree of similarity between a base sequence encoding a protein or an amino acid sequence constituting a protein, and when homology is sufficiently high, the expression product of the gene and the protein may have the same or similar activity. Additionally, homology may be expressed as a percentage according to the degree of correspondence with a given amino acid sequence or base sequence. In this specification, a homologous sequence having the same or similar activity as a given amino acid sequence or nucleotide sequence is indicated as "% homology." For example, this can be verified by using standard software, specifically BLAST 2.0, to calculate parameters such as score, identity, and similarity, or by comparing sequences through hybridization experiments performed under defined stringent conditions, and the defined appropriate hybridization conditions may be determined by methods well known to those skilled in the art within the scope of the relevant technology.

[0032] In addition, due to the degeneracy of codons, the polynucleotide encoding the above peptide may undergo various modifications to its coding region within a range that does not alter the amino acid sequence of the protein expressed from the coding region, taking into account the codons preferred by the organism intended to express the protein. Therefore, the polynucleotide may be included without limitation as long as it is a polynucleotide sequence encoding each of the proteins.

[0033] In addition, the polynucleotide comprises not only a nucleotide sequence encoding the amino acid sequence of the peptide, but also a sequence complementary to that sequence. The complementary sequence comprises not only a perfectly complementary sequence, but also a substantially complementary sequence, which means a sequence that can be hybridized, for example, with the nucleotide sequence encoding the amino acid sequence of the peptide under stringent conditions known in the art.

[0034] Hybridization requires that two polynucleotides have complementary sequences, even though a mismatch between bases may be possible depending on the degree of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, regarding DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the present application may also include substantially similar polynucleotide sequences as well as isolated polynucleotide fragments that are complementary to the entire sequence.

[0035] In this specification, the term "expression vector" refers to a recombinant vector capable of being introduced into a suitable host cell to express a target protein, and is a genetic construct comprising essential regulatory elements operably linked to enable the expression of a gene insert. The term "operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding the target protein are functionally linked to perform a general function. Operatory linkage with the recombinant vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be easily performed using enzymes or the like generally known in the art.

[0036] Suitable expression vectors of the present invention may include signal sequences for membrane targeting or secretion in addition to expression regulatory elements such as promoters, start codons, stop codons, polyadenylation signals, and enhancers. The start codon and stop codon are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein, must exhibit action in the individual when the gene construct is administered, and must be in frame with the coding sequence. General promoters can be constitutive or inducible, and in the case of prokaryotic cells, lac, tac, T3 and T7 promoters; in the case of eukaryotic cells, monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV), e.g., HIV long terminal repeat (LTR) promoter, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Rhoese's sarcoma virus (RSV) promoters, as well as β-actin promoters, human hemoglobin, human muscle creatine, human metallothionein-derived promoters, etc., but are not limited thereto.

[0037] Additionally, the expression vector may include a selectivity marker for selecting host cells containing the vector. The selectivity marker is intended to select cells transformed with the vector, and markers conferring selectable phenotypes, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or the expression of surface proteins, may be used. Since only cells expressing the selectivity marker survive in an environment treated with a selectivity agent, the transformed cells can be selected. Furthermore, if the vector is a replicable expression vector, it may include a replication origin, which is a specific nucleic acid sequence at which replication is initiated.

[0038] Various types of vectors, such as plasmids, viruses, and cosmids, can be used as recombinant expression vectors for inserting foreign genes. The type of recombinant vector is not particularly limited as long as it functions to express the desired gene and produce the desired protein in various host cells, including prokaryotic and eukaryotic cells; however, specifically, vectors capable of producing large quantities of foreign proteins in a form similar to their natural state while possessing a promoter exhibiting potent activity and strong expression capacity may be utilized.

[0039] To express the protein of the present invention, various combinations of hosts and vectors may be used. Expression vectors suitable for eukaryotic hosts may include, but are not limited to, pCMV, pFLAG, pMYC, pHA, etc., expression regulatory sequences derived from SV40, papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors suitable for bacterial hosts may include, but are not limited to, bacterial plasmids obtained from Escherichia coli including, pET21a, pET, pRSET, pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9, or derivatives thereof, plasmids having a broader host range such as RP4. gt10, It may include phage DNA that can be exemplified by phage lambda derivatives such as gt11 or NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. Yeast cells 2 Plasmids or their derivatives may be used, and pVL941 may be used in insect cells.

[0040] The above cells, for example, eukaryotic cells, may be cells of yeast, fungi, protozoa, plants, higher plants and insects, or amphibians, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, and may be, for example, cultured cells (in vitro), graft cells and primary cell cultures (in vitro and ex vivo), and in vivo cells commonly used in the industry, and may also be mammalian cells including humans. Additionally, the above organism may be yeast, fungi, protozoa, plants, higher plants and insects, amphibians, or mammals.

[0041] However, in the present invention, the host cell may be a prokaryote to meet the purpose of mass production of the peptide described above, and may be E. coli considering cost and time.

[0042] In addition, the present invention provides a method for mass production of a peptide having deacetylase activity or a peptide having sulfate transferase activity.

[0043] The above method may include the following steps (1) to (5):

[0044] (1) A step of producing an expression vector comprising a gene encoding a peptide having deacetylase activity or a peptide having sulfate transferase activity;

[0045] (2) A step of transforming E. coli with the above-mentioned expression vector;

[0046] (3) A step of culturing the transformed E. coli;

[0047] (4) A step of obtaining a cell precipitate by crushing and centrifuging the transformed E. coli; and

[0048] (5) A step of purifying the peptide having deacetylase activity or the peptide having sulfate transferase activity from the cell precipitate.

[0049] The above method may include the following steps (a) to (d).

[0050] (a) a step of preparing E. coli transformed with an expression vector comprising a gene encoding a peptide having deacetylase activity or a peptide having sulfate transferase activity;

[0051] (b) a step of culturing the transformed E. coli above;

[0052] (c) a step of crushing and centrifuging the above E. coli to obtain a cell precipitate; and

[0053] (d) A step of purifying a peptide having deacetylase activity or a peptide having sulfate transferase activity from the cell precipitate.

[0054] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases comprising, as an active ingredient, a peptide having deacetylase activity or a peptide having sulfate transferase activity.

[0055] In the present invention, the term "neurological disease" is used to include neurological diseases and degenerative brain diseases.

[0056] The term "treatment" in this specification refers to any act of improving or beneficially altering the symptoms of a neurological disease by administering a composition of the present invention.

[0057] The term "prevention" in this specification refers to any act in which the possibility of the onset of a neurological disease or disease is suppressed or delayed by the administration of the composition of the present invention.

[0058] The above pharmaceutical composition may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the injected compound. Here, "pharmaceutically acceptable" means that the subject of application (prescription) does not possess toxicity beyond an tolerable level without inhibiting the activity of the active ingredient.

[0059] Any carrier that is commonly used and pharmaceutically acceptable in the art may be used as the carrier that can be used in the present invention. Non-limiting examples of said carriers include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used alone or in a mixture of two or more. The said pharmaceutical composition may be prepared as an oral formulation or a parenteral formulation according to the route of administration by conventional methods known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient.

[0060] The above pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions, each according to conventional methods. When formulating the above pharmaceutical composition, it may be prepared by adding diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, or surfactants.

[0061] When the above pharmaceutical composition is prepared as an oral formulation, it may be prepared in the form of powder, granules, tablets, pills, coated tablets, capsules, liquids, gels, syrups, suspensions, wafers, etc., in accordance with methods known in the art together with a suitable carrier. Examples of pharmaceutically acceptable suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinylpyrrolidone; water; methylhydroxybenzoate; propylhydroxybenzoate; magnesium stearate; mineral oil; malt; gelatin; talc; polyols; vegetable oils, etc. In the case of formulation, the formulation may include diluents and / or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants as needed.

[0062] When the above pharmaceutical composition is prepared as a parenteral formulation, it may be formulated in the form of an injectable, transdermal, nasal inhalant, and suppository according to methods known in the art with a suitable carrier. When formulated as an injectable, suitable carriers may include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof; preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose, etc. When formulated as a transdermal formulation, it may be formulated in the form of an ointment, cream, lotion, gel, topical solution, paste, liniment, aerosol, etc. In the case of nasal inhalers, they can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide, and when formulated as suppositories, the bases may include Witepsol, Tween 61, polyethylene glycols, cocoa starch, laurin starch, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, and sorbitan fatty acid esters.

[0063] The above pharmaceutical composition may be administered in a pharmaceutically effective amount, wherein the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dose level may be determined based on factors including the severity of the disease, drug activity, patient's age, weight, health, gender, patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and elimination rate, the duration of treatment, drugs combined or used concurrently with the composition of the present invention used, and other factors well known in the medical field. The above pharmaceutical composition may be administered alone or in combination with a component known to exhibit therapeutic effects for known cancer diseases. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration.

[0064] The dosage of the above pharmaceutical composition may be determined by a person skilled in the art by taking into consideration the purpose of use, the degree of toxicity of the disease, the patient's age, weight, gender, medical history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered to an adult at a dose of about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg. The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or divided into several doses. The above dosage or frequency of administration does not limit the scope of the present invention in any way.

[0065] In addition, the present invention provides a method for preventing or treating a neurological disease comprising the step of administering the peptide of the present invention to an individual.

[0066] In the present invention, the term "individual" includes, without limitation, mammals including rats, livestock, and humans, birds, reptiles, farmed fish, etc., that have developed or are at risk of developing a neurological disease.

[0067] The above pharmaceutical composition may be administered as a single or multiple doses in pharmaceutically effective amounts. In this case, the composition may be administered in the form of a liquid, powder, aerosol, injection, intravenous fluid (Ringer), capsule, pill, tablet, suppository, or patch. The route of administration of the above pharmaceutical composition for the prevention or treatment of cancer may be any general route as long as it can reach the target tissue.

[0068] The above pharmaceutical composition is not particularly limited thereto, but may be administered via routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, nasal administration, pulmonary administration, or rectal administration, depending on the purpose.

[0069] Specific information regarding NDST3 (T0) or NDST4 (T4) and variants thereof (T1~T3 and T5~T7) described in this specification is as shown in Table 1 below.

[0070] In this specification, peptides derived from NDST3 and NDST4 that function as enzymes are referred to as fragments of NDST3 or NDST4 or variants thereof.

[0071] T0 = Human N-Deacetylase And N-Sulfotransferase 3 (1-873 a.a) MSFIMKLHRHFQRTVILLATFCMVSIIISAYYLYSGYKQENELSETASEVDCGDLQHLPYQLMEVKAMKLFDASRTDPTVLVFVESQYSSLGQDIIMILESSRFQYHIEIAPGKGDLPVLIDKMKGKYILIIYENILKYINMDSWNRSLLDKYCVEYGVGVIGFHKTSEKSVQSFQLKGFPFSIYGNLAVKDCCINPHSPLIRVTKSSKLEKGSLPGTDWTVFQINHSAYQPVIFAKVKTPENLSPSISKGAFYATIIHDLGLHDGIQRVLFGNNLNFWLHKLIFIDAISFLSGKRLTLSLDRYILVDIDDIFVGKEGTRMNTNDVKALLDTQNLLRAQITNFTFNLGFSGKFYHTGTEEEDEGDDCLLGSVDEFWWFPHMWSHMQPHLFHNESSLVEQMILNKKFALEHGIPTDMGYAVAPHHSGVYPVHVQLYEAWKKVWNIKITSTEEYPHLKPARYRRGFIHKNIMVLPRQTCGLFTHTIFYKEYPGGPKELDKSIQGGELFFTVVLNPISIFMTHLSNYGNDRLGLYTFVNLANFVKSWTNLRLQTLPPVQLAHKYFELFPDQKDPLWQNPCDDKRHRDIWSKEKTCDRLPKFLVIGPQKTGTTALYLFLVMHPSILSNSPSPKTFEEVQFFNRNNYHRGIDWYMDFFPVPSNVTTDFLFEKSANYFHSEEAPKRAASLVPKAKIITILIDPSDRAYSWYQHQRSHEDPAALKFSFYEVISAGPRAPSELRALQKRCLVPGWYASHIERWLVYFPPFQLLIIDGQQLRTDPATVMDEVQKFLGVLPHYNYSEALTFDSHKGFWCQLLEEGKTKCLGKSKGRKYPPMDSDSRTFLSSYYRDHNVELSKLLHKLGQPLPSWLRQELQKVR 서열번호 1 T1 = △TM_Human N-Deacetylase And N-Sulfotransferase 3 (72 - 873 a.a) DASRTDPTVLVFVESQYSSLGQDIIMILESSRFQYHIEIAPGKGDLPVLIDKMKGKYILIIYENILKYINMDSWNRSLLDKYCVEYGVGVIGFHKTSEKSVQSFQLKGFPFSIYGNLAVKDCCINPHSPLIRVTKSSKLEKGSLPGTDWTVFQINHSAYQPVIFAKVKTPENLSPSISKGAFYATIIHDLGLHDGIQRVLFGNNLNFWLHKLIFIDAISFLSGKRLTLSLDRYILVDIDDIFVGKEGTRMNTNDVKALLDTQNLLRAQITNFTFNLGFSGKFYHTGTEEEDEGDDCLLGSVDEFWWFPHMWSHMQPHLFHNESSLVEQMILNKKFALEHGIPTDMGYAVAPHHSGVYPVHVQLYEAWKKVWNIKITSTEEYPHLKPARYRRGFIHKNIMVLPRQTCGLFTHTIFYKEYPGGPKELDKSIQGGELFFTVVLNPISIFMTHLSNYGNDRLGLYTFVNLANFVKSWTNLRLQTLPPVQLAHKYFELFPDQKDPLWQNPCDDKRHRDIWSKEKTCDRLPKFLVIGPQKTGTTALYLFLVMHPSILSNSPSPKTFEEVQFFNRNNYHRGIDWYMDFFPVPSNVTTDFLFEKSANYFHSEEAPKRAASLVPKAKIITILIDPSDRAYSWYQHQRSHEDPAALKFSFYEVISAGPRAPSELRALQKRCLVPGWYASHIERWLVYFPPFQLLIIDGQQLRTDPATVMDEVQKFLGVLPHYNYSEALTFDSHKGFWCQLLEEGKTKCLGKSKGRKYPPMDSDSRTFLSSYYRDHNVELSKLLHKLGQPLPSWLRQELQKVR 서열번호 2 T2 = Human NDST3_N-Deacetylase domain (72 - 569 a.a) DASRTDPTVLVFVESQYSSLGQDIIMILESSRFQYHIEIAPGKGDLPVLIDKMKGKYILIIYENILKYINMDSWNRSLLDKYCVEYGVGVIGFHKTSEKSVQSFQLKGFPFSIYGNLAVKDCCINPHSPLIRVTKSSKLEKGSLPGTDWTVFQINHSAYQPVIFAKVKTPENLSPSISKGAFYATIIHDLGLHDGIQRVLFGNNLNFWLHKLIFIDAISFLSGKRLTLSLDRYILVDIDDIFVGKEGTRMNTNDVKALLDTQNLLRAQITNFTFNLGFSGKFYHTGTEEEDEGDDCLLGSVDEFWWFPHMWSHMQPHLFHNESSLVEQMILNKKFALEHGIPTDMGYAVAPHHSGVYPVHVQLYEAWKKVWNIKITSTEEYPHLKPARYRRGFIHKNIMVLPRQTCGLFTHTIFYKEYPGGPKELDKSIQGGELFFTVVLNPISIFMTHLSNYGNDRLGLYTFVNLANFVKSWTNLRLQTLPPVQLAHKYFELFPDQK 서열번호 3 T3 = Human NDST3_ N-Sulfotransferase domain (569 - 873 a.a) KDPLWQNPCDDKRHRDIWSKEKTCDRLPKFLVIGPQKTGTTALYLFLVMHPSILSNSPSPKTFEEVQFFNRNNYHRGIDWYMDFFPVPSNVTTDFLFEKSANYFHSEEAPKRAASLVPKAKIITILIDPSDRAYSWYQHQRSHEDPAALKFSFYEVISAGPRAPSELRALQKRCLVPGWYASHIERWLVYFPPFQLLIIDGQQLRTDPATVMDEVQKFLGVLPHYNYSEALTFDSHKGFWCQLLEEGKTKCLGKSKGRKYPPMDSDSRTFLSSYYRDHNVELSKLLHKLGQPLPSWLRQELQKVR 서열번호 4 T4 = Human N-Deacetylase And N-Sulfotransferase 4 (1-873 a.a) MNLIVKLRRSFRTLIVLLATFCLVSIVISAYFLYSGYKQEMTLIETTAEAECTDIKILPYRSMELKTVKPIDTSKTDPTVLLFVESQYSQLGQDIIAILESSRFQYHMVIAPGKGDIPPLTDNGKGKYTLVIYENILKYVSMDSWNRELLEKYCVEYSVSIIGFHKANENSLPSTQLKGFPLNLFNNLALKDCFVNPQSPLLHITKAPKVEKGPLPGEDWTIFQYNHSTYQPVLLTELQTEKSLSSLSSKTLFATVIQDLGLHDGIQRVLFGNNLNFWLHKLIFIDAISFLSGKRLTLSLDRYILVDIDDIFVGKEGTRMNVKDVKALLETQNLLRTQVANFTFNLGFSGKFYHTGTEEEDEGDDLLLRSVDEFWWFPHMWSHMQPHLFHNESSLVEQMILNKEFALEHGIPINMGYAVAPHHSGVYPVHIQLYAAWKKVWGIQVTSTEEYPHLKPARYRKGFIHNSIMVLPRQTCGLFTHTIFYKEYPGGPQELDKSIRGGELFLTILLNPISIFMTHLSNYGNDRLGLYTFVNLVNFVQSWTNLKLQTLPPVQLAHQYFELFPEQKDPLWQNPCDDKRHKDIWSREKTCDHLPKFLVIGPQKTGTTALYLFLLMHPSIISNLPSPKTFEEVQFFNGNNYHKGIDWYMDFFPTPSNTTSDFLFEKSANYFHSEEAPRRAASLVPKAKIITILIDPSDRAYSWYQHQRSHEDPAALRFNFYEVISTGHWAPSDLKTLQRRCLVPGWYAVHIERWLTYFATSQLLIIDGQQLRSDPATVMDEVQKFLGVTPRYNYSEALTFDPQKGFWCQLLEGGKTKCLGKSKGRKYPPMDPESRTFLSNYYRDHNVELSKLLHRLGQPLPSWLRQELQKVR 서열번호 5 T5 = △TM_Human N-Deacetylase And N-Sulfotransferase 4 (72 - 873 a.a) DTSKTDPTVLLFVESQYSQLGQDIIAILESSRFQYHMVIAPGKGDIPPLTDNGKGKYTLVIYENILKYVSMDSWNRELLEKYCVEYSVSIIGFHKANENSLPSTQLKGFPLNLFNNLALKDCFVNPQSPLLHITKAPKVEKGPLPGEDWTIFQYNHSTYQPVLLTELQTEKSLSSLSSKTLFATVIQDLGLHDGIQRVLFGNNLNFWLHKLIFIDAISFLSGKRLTLSLDRYILVDIDDIFVGKEGTRMNVKDVKALLETQNLLRTQVANFTFNLGFSGKFYHTGTEEEDEGDDLLLRSVDEFWWFPHMWSHMQPHLFHNESSLVEQMILNKEFALEHGIPINMGYAVAPHHSGVYPVHIQLYAAWKKVWGIQVTSTEEYPHLKPARYRKGFIHNSIMVLPRQTCGLFTHTIFYKEYPGGPQELDKSIRGGELFLTILLNPISIFMTHLSNYGNDRLGLYTFVNLVNFVQSWTNLKLQTLPPVQLAHQYFELFPEQKDPLWQNPCDDKRHKDIWSREKTCDHLPKFLVIGPQKTGTTALYLFLLMHPSIISNLPSPKTFEEVQFFNGNNYHKGIDWYMDFFPTPSNTTSDFLFEKSANYFHSEEAPRRAASLVPKAKIITILIDPSDRAYSWYQHQRSHEDPAALRFNFYEVISTGHWAPSDLKTLQRRCLVPGWYAVHIERWLTYFATSQLLIIDGQQLRSDPATVMDEVQKFLGVTPRYNYSEALTFDPQKGFWCQLLEGGKTKCLGKSKGRKYPPMDPESRTFLSNYYRDHNVELSKLLHRLGQPLPSWLRQELQKVR 서열번호 6 T6 = Human NDST4_N-Deacetylase domain (72 - 569 a.a) DTSKTDPTVLLFVESQYSQLGQDIIAILESSRFQYHMVIAPGKGDIPPLTDNGKGKYTLVIYENILKYVSMDSWNRELLEKYCVEYSVSIIGFHKANENSLPSTQLKGFPLNLFNNLALKDCFV NPQSPLLHITKAPKVEKGPLPGEDWTIFQYNHSTYQPVLLTELQTEKSLSSLSSKTLFATVIQDLGLHDGIQRVLFGNNLNFWLHKLIFIDAISFLSGKRLTLSLDRYILVDIDDIFVGKEGTR MNVKDVKALLETQNLLRTQVANFTFNLGFSGKFYHTGTEEEDEGDDLLLRSVDEFWWFPHMWSHMQPHLFHNESSLVEQMILNKEFALEHGIPINMGYAVAPHHSGVYPVHIQLYAAWKKVWGI QVTSTEEYPHLKPARYRKGFIHNSIMVLPRQTCGLFTHTIFYKEYPGGPQELDKSIRGGELFLTILLNPISIFMTHLSNYGNDRLGLYTFVNLVNFVQSWTNLKLQTLPPVQLAHQYFELFPEQK Sequence No. 7 T7 = Human NDST4_ N-Sulfotransferase domain (569 - 873 aa) KDPLWQNPCDDKRHKDIWSREKTCDHLPKFLVIGPQKTGTTALYLFLLMHPSIISNLPSPKTFEEVQFFNGNNYHKGIDWYMDFFPTPSNTTSDFLFEKSANYFHSEEAPRRAASLVPKAKIITILIDPSDRAYSWYQHQRSHEDPAALRFN FYEVISTGHWAPSDLKTLQRRCLVPGWYAVHIERWLTYFATSQLLIIDGQQLRSDPATVMDEVQKFLGVTPRYNYSEALTFDPQKGFWCQLLEGGKTKCLGKSKGRKYPPMDPESRTFLSNYYRDHNVELSKLLHRLGQPLPSWLRQELQKVR Sequence No. 8 Effects of the invention

[0073] The present invention identifies N-deacetylase or N-sulfotransferase active domains in NDST3 and NDST4 proteins and confirms that said peptides can be mass-produced in a soluble form in E. coli using a vector containing a peptide gene derived from NDST3 or NDST4 containing said domains. Therefore, the peptides of the present invention are expected to be used as a platform for developing protein drugs or inhibitors thereof utilizing NDST3 and NDST4 proteins. Brief explanation of the drawing

[0075] Figure 1a is a schematic diagram showing the functional domain of the human NDST3 protein. Figure 1b is a schematic diagram showing the functional domain of the human NDST4 protein. Figure 2a is a schematic diagram of a human-derived NDST3-derived protein expression DNA vector. Figure 2b is a schematic diagram of a human-derived NDST4-derived protein expression DNA vector. Figure 3 shows the results of confirming the production of the water-soluble protein designed in the present invention. E. coli transformed with an expression vector encoding each human DNST3 domain were cultured and lysed, and then Western blot was performed to confirm the degree of protein expression and yield. 'M' is a protein marker (kDa), 'I-' is protein expression induction, 'I+' is for the cell lysate after protein expression induction, 'S' is for the supernatant obtained by centrifugation after cell lysis, and 'P' indicates the state of the expressed insoluble protein. Figure 4 shows the results of confirming the production of the water-soluble protein designed in the present invention. E. coli transformed with an expression vector encoding each domain of human DNST4 was cultured and lysed, and then Western blot was performed to confirm the degree of protein expression and yield. 'M' is a protein marker (kDa), 'I-' is protein expression induction, 'I+' is for the cell lysate after protein expression induction, 'S' is for the supernatant obtained by centrifugation after cell lysis, and 'P' indicates the state of the expressed insoluble protein. FIGS. 5a to 5c show the results of isolating and purifying the water-soluble proteins designed in the present invention by performing affinity chromatography and size exclusion chromatography using a nickel (Ni-NTA) column after culturing and lysing E. coli transformed with an expression vector encoding each domain of human DNST3. 'C' refers to the cell lysate after protein expression induction, 'S' refers to the supernatant obtained by centrifugation after cell lysis, 'FT' refers to the column pass-through, '4%' refers to the column pass-through after a 20 mM imidazole wash, and '10%' refers to the column pass-through after a 50 mM imidazole wash. 'Before cut' and 'After cut' refer to the state before and after cutting eight histidine tags, respectively, using PreScission protease. 'E' or elution refers to the fractions according to the imidazole concentration gradient or each fraction separated and purified by protein exclusion chromatography. Figures 6a and 6b show the results of isolating and purifying the water-soluble proteins designed in the present invention by performing affinity chromatography and size exclusion chromatography using a nickel (Ni-NTA) column after culturing and lysing E. coli transformed with an expression vector encoding each domain of human DNST4. 'C' refers to the cell lysate after protein expression induction, 'S' refers to the supernatant obtained by centrifugation after cell lysis, 'FT' refers to the column pass-through, '4%' refers to the column pass-through after a 20 mM imidazole wash, and '10%' refers to the column pass-through after a 50 mM imidazole wash. 'Before cut' and 'After cut' refer to the state before and after cutting eight histidine tags, respectively, using PreScission protease. 'E' or elution refers to the fractions according to the imidazole concentration gradient or each fraction separated and purified by protein exclusion chromatography. Figure 7 shows the results of evaluating the enzyme activity of human NDST3 and NDST4-derived proteins in vitro. Specific details for implementing the invention

[0076] The present invention is capable of various modifications and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0077] [Example]

[0078] Example 1. Amplification and expression of human NDST3 and NDST4 variant genes

[0079] 1-1. Amplification of human NDST3 and NDST4 variant genes

[0080] As described in SEQ ID NO. 2, the amino acid sequence from the N-terminus of aspartic acid (D) at position 72 to arginine (R) at position 873; as described in SEQ ID NO. 3, the amino acid sequence from the N-terminus of aspartic acid (D) at position 72 to lysine (K) at position 569; as described in SEQ ID NO. 4, the amino acid sequence from the N-terminus of lysine (K) at position 569 to arginine (R) at position 873; and as described in SEQ ID NO. 6, the amino acid sequence from the N-terminus of aspartic acid (D) at position 72 to arginine (R) at position 873; as described in SEQ ID NO. 7, the amino acid sequence from the N-terminus of aspartic acid (D) at position 72 to lysine (K) at position 569. As described in SEQ ID NO. 8, the coding sequence of a human NDST4 variant protein having an amino acid sequence from the 569th lysine (K) terminal (N-terminus) to the 873rd amino acid arginine (R) was used, and to facilitate the purification of the produced protein, a histidine fusion (His-tag) protein, glutathione S-transferase (GST), or thioredoxin (trx) may be conjugated to the N-terminus. Each sequence is derived from the human NDST3 or NDST4 protein, and each domain of the said protein is indicated in Figures 1a and 1b. For the synthesis and amplification of the gene, a PCR reaction was performed using the following primers synthesized using a nucleic acid synthesizer. A PCR reaction was performed using a gene amplifier using the primers synthesized for the amplification of each gene fragment (Table 2).

[0081] Template DNA direction Primer sequence (5' --> 3') Sequence number HUMAN NDST3 full (1-873 aa) Forward direction 5`- caccggatccatgagttttatcatgaagcttc-3` 9 reverse direction 5`- gctaggcatctcgagttactatcttactttctgcagctc-3` 10 HUMAN NDST3 △TM (72-873 aa) Forward direction 5`- caccggatccatggatgcctcaaggacagac-3` 11 reverse direction 5`- gctaggcatctcgagttactatcttactttctgcagctc-3` 12 HUMAN NDST3 N-Deacetylase (1-569 aa) Forward direction 5`- caccggatccatgagttttatcatgaagcttc-3` 13 reverse direction 5` gctaggcatctcgagttactatttctgatcaggaaacagc -3` 14 HUMAN NDST3 △TM N-Deacetylase (72-569 aa) Forward direction 5`- caccggatccatggatgcctcaaggacagac -3` 15 reverse direction 5`- gctaggcatctcgagttaCTAtttctgatcaggaaacagc -3` 16 HUMAN NDST3 N-Sulfotransferase (569-873 aa) Forward direction 5`- caccggatccatggaccctctctggcagaatcc -3` 17 reverse direction 5`- gctaggcatctcgagttactatcttactttctgcagctc -3` 18 HUMAN NDST4 Full (1-873 aa) Forward direction 5`- caccggatccatgaatcttattgtgaa -3` 19 reverse direction 5`- gctaggcatctcgagttatctcactttctgcagttcc -3` 20 HUMAN NDST4 △TM (72-873 aa) Forward direction 5`- caccggatccatggacacatccaaaaacgg-3` 21 reverse direction 5`- gctaggcatctcgagttatctcactttctgcagttcc-3` 22 HUMAN NDST4 N-Deacetylase (1-569 aa) Forward direction 5`- caccggatccatgaatcttattgtgaa -3` 23 reverse direction 5`- gctaggcatctcgagttagtctttctgctcagggaag-3` 24 HUMAN NDST4 △TM N-Deacetylase (72-569 aa) Forward direction 5`- caccggatccatggatgcctcaaggacagac -3` 25 reverse direction 5`- gctaggcatctcgagttagtctttctgctcagggaag-3` 26 HUMAN NDST4 N-Sulfotransferase (569-873 aa) Forward direction 5`- caccggatccatggaccctctatggcagaa 27 reverse direction 5`- gctaggcatctcgagttatctcactttctgcagttcc-3` 28

[0083] The above forward primers contain nucleotide sequences corresponding to the BamH1 restriction enzyme recognition site (Sequence Nos. 9, 11, 13, 14, 16, 18), and the reverse primers contain nucleotide sequences corresponding to the XhoI restriction enzyme recognition site (Sequence Nos. 10, 12, 15, 17). PCR was performed as follows using Human NDST3 and Human NDST4 as templates. A reaction solution was prepared by adding 79 µl of distilled water to a mixture of 1 µl of Human NDST3 and Human NDST4 full-length cDNA, 8 µl of 2.5 mM dNTPs, 1 µl each of 100 pmol forward primers (sequence numbers: 9, 11, 13, 14, 16, 18) and reverse primers (sequence numbers: 10, 12, 15, 17), 1 µl of PfuTaq DNA polymerase (5 U / µl, Stratagene, USA), and 10 µl of PCR buffer (Stratagene). Then, the reaction solution was repeated 30 times at a temperature of 82 ℃ for 2 minutes, 94 ℃ for 1 minute, 94 ℃ for 30 seconds, 56 ℃ for 1 minute 30 seconds, and 72 ℃ for 4 minutes 30 seconds. The above reaction solution was separated by electrophoresis on a 0.8% agarose gel to elute the genes. To 16 µl of the obtained eluate, 2 µl of 10-fold restriction enzyme reaction buffer and 1 µl each of restriction enzymes BamH1 (NEB (New England Biolabs, USA)) and Xho I (NEB, USA) were added, and the reaction was carried out at 37°C for 16 hours. The resulting reaction solution was separated by electrophoresis on a 0.8% agarose gel to extract the DNA fragments coding for the desired human NDST3 and NDST4 protein variants, which were then dissolved in 50 µl of distilled water and respectively HUMAN_NDST3 △ TM (hNDST3N △ TM) B / X, HUMAN NDST3_N-Deacetylasea (hNDST3DA) B / X, HUMAN_NDST3 △ TM N-Deacetylase (hNDST3 △TMDA) B / X, HUMAN NDST3 N-Sulfotransferase (hNDST3ST) B / X, HUMAN_NDST4 △ TM (hNDST4N △ TM) B / X, HUMAN NDST4_N-Deacetylasea (hNDST4DA) B / X, HUMAN_NDST4 △ TM N-Deacetylase (hNDST4 △ It was named TMDA) B / X and HUMAN NDST4 N-Sulfotransferase (hNDST4ST) B / X.

[0085] 1-2. Preparation of human NDST3 and NDST4 variant expression vectors

[0086] Plasmid pET-32a (Novagene Inc., USA) expresses thioredoxin and six histidines at the N-terminus, pGEX4T-1 (Novagene Inc., USA) expresses glutathione S-transferase at the N-terminus, and pET-28a (Novagene Inc., USA) expresses six histidine residues at the N-terminus; after treating these with restriction enzymes BamHI and XhoI, DNA fragments of approximately 920 bp were isolated by electrophoresis and named pET-32aB / X, pGEX4T-1B / X, and pET-28aB / X. 0.5 µg of hNDST3N △ TM B / X, hNDST3DA B / X, hNDST3 △ TMDA B / X, hNDST3ST B / X, hNDST4N △ TM B / X, hNDST4DA B / X, hNDST4 △After adding TMDA B / X and hNDST4ST B / X to a reaction tube along with 0.1 µg of pET-32aB / X, pGEX4T-1B / X and pET-28a B / X, 2 µl of 10x ligation reaction solution (20 mM Hepe-HCl, pH 7.8, 100 mM MgCl2, 100 mM DTT and 10 mM ATP) and 10 U of T4 DNA ligase were added, distilled water was added to make the total volume 20 µl, and the reaction was carried out at 16 °C for 12 hours. This reaction solution was added to competent E. coli BL21(DE3) (Novagene Inc., USA) or Rosetta(DE3) (Novagene Inc., USA) cells to transform them, and the E. coli transformants were plated on 1) kanamycin medium containing 50 µg / ml LB (1% bactotryptone, 0.5% yeast extract, and 1% sodium chloride) and 2) ampicillin medium containing 100 µg / ml LB (1% bactotryptone, 0.5% yeast extract, and 1% sodium chloride). Plasmids were extracted from these, and recombinant plasmids were obtained by restriction enzyme and DNA sequencing analysis.

[0087] Plasmids were extracted from this, and by restriction enzyme and sequence analysis, the hNDST3N prepared in Example 1-1 was analyzed into plasmids pET-32aB / X, pGEX4T-1B / X, and pET-28aB / X. △ TM B / X, hNDST3DA B / X, hNDST3 △ TMDA B / X, hNDST3ST B / X, hNDST4N △ TM B / X, hNDST4DA B / X, hNDST4 △ hNDST3N with TMDA B / X and hNDST4ST B / X fragments connected △ TM, hNDST3DA, hNDST3 △ TMDA, hNDST3ST, hNDST4N △ TM, hNDST4DA, hNDST4 △It was confirmed that TMDA and hNDST4ST were obtained. The process of cloning the coding genes of the above HUMAN NDST3 and NDST4 variant proteins into E. coli expression vectors pET32a, pGEX4T-1, and pET28a, respectively, is illustrated in Figures 2a and 2b.

[0089] 1-3. Expression of NDST3 and NDST4 Variants in Escherichia coli

[0090] The expression vector pET-32a-hNDST3N prepared in Examples 1-2 above △ TM, pGEX4T-1-hNDST3N △ TM, pET-28a-hNDST3N △ TM, pET-32a-hNDST3DA, pGEX4T-1-hNDST3DA, pET-28a-hNDST3DA, pET-32a-hNDST3 △ TMDA, pGEX4T-1-hNDST3 △ TMDA, pET-28a-hNDST3 △ TMDA, pET-32a-hNDST3ST, pGEX4T-1-hNDST3ST, pET-28a-hNDST3ST, pET-32a-hNDST4N △ TM, pGEX4T-1-hNDST4N △ TM, pET-28a-hNDST4N △ TM, pET-32a-hNDST4DA, pGEX4T-1-hNDST4DA, pET-28a-hNDST4DA, pET-32a-hNDST4 △ TMDA, pGEX4T-1-hNDST4 △ TMDA, pET-28a-hNDST4 △Escherichia coli BL21 (De3) (Novagen Inc., USA) or Rosetta (DE3) (Novagen Inc., USA) were transformed by conventional methods with TMDA, pET-32a-hNDST4ST, pGEX4T-1-hNDST4ST, and pET-28a-hNDST4ST, respectively. The transformed Escherichia coli strains were shaken and cultured for 12 hours in LB medium containing 100 µg / ml kanamycin and 100 µg / ml ampicillin. Then, 1 ml was taken and added to 100 ml of LB medium (containing 100 µg / ml kanamycin and 100 µg / ml ampicillin), and cultured at 37°C until the absorbance of the culture medium at 600 nm was approximately 0.6. Then, the culture temperature was lowered to 18°C, and IPTG (isopropyl-β-D-galactopyranoside) was added to a final concentration of 0.6 mM. 1 mL of the culture medium was taken before the addition of IPTG and 16 hours after addition, respectively, and the cell precipitates were collected after centrifugation at 10,000 g for 2 minutes. The collected precipitates were subjected to 15% SDS-polyacrylamide gel electrophoresis, and the proteins were analyzed by staining them with Coomassie Brilliant Blue (Bio-Rad 161-0400). The obtained protein hNDST3N △ TM, hNDST3DA, hNDST3 △ TMDA, hNDST3ST, hNDST4N △ TM, hNDST4DA, hNDST4 △ Figures 3 and 4 show the results of SDS-polyacrylamide gel electrophoresis of cell precipitates obtained by culturing E. coli transformed with E. coli expression vectors pET32a, pGEX4T-1, or pET28a containing coding genes for TMDA and hNDST4ST.

[0092] Example 2. Expression of the recombinant hNDST3N△TM (72-873aa) gene in E. coli

[0093] The expression vector pET32a-hNDST3N obtained in the above step△ TM (72-873aa) was transformed into the expression host Escherichia coli BL21 (RIL) using conventional methods. pET32a- hNDST3N △ After incubating an E. coli strain transformed with TM (72-873aa) in 5.5 ml of LB liquid medium containing 100 µg / ml ampicillin at 37°C for 18 hours, 50 µl was taken and subcultured in 5.5 ml of LB liquid medium (containing 100 µg / ml ampicillin), and then incubated at 37°C until the absorbance of the culture medium at 600 nm reached approximately 0.2–0.3. Then, the incubation temperature was lowered to 18°C ​​and incubated until the absorbance of the culture medium reached approximately 0.5–6, after which IPTG (isopropyl-β-D-galactopyranoside) was added to a final concentration of 0.5 mM.

[0094] Cells cultured with IPTG for 18 hours were centrifuged, and the respective cell precipitates were collected. Each collected precipitate was suspended in 0.5 mL of cell lysis buffer (300 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, 5 mM beta-mercaptoethanol), and the cells were lysed using a mini sonic dismembrator (Sonic Dismembrator, Fisher, USA) in an ice bath. The resulting solution was centrifuged at 13,000 rpm for 10 minutes to separate the supernatant and precipitate, followed by 15% SDS-polyacrylamide gel electrophoresis. The protein pET32a-hNDST3N was then analyzed using Coomassie Brilliant Blue (Bio-Rad 161-0400). △ TM (72-873aa) was stained and analyzed. The results for each are shown in Figure 3. (M: Protein marker, I-: Before IPTG induction, I+: Cell fragmentation sample after IPTG induction, S: Supernatant after centrifugation of IPTG-induced sample, P: Precipitate after centrifugation of IPTG-induced sample)

[0096] Example 3. Purification of recombinant protein hNDST3N△TM (72-873aa) in E. coli

[0097] 3-1. Culture and Disruption of E. coli Cells

[0098] In the same manner as in Example 2, E. coli cells expressing the recombinant protein were cultured in 8 L volumes, and the E. coli cell precipitate was collected by centrifuging at 3,600 rpm for 20 minutes. Then, 25 mL of a buffer solution composed of 400 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, and 10 mM beta-mercaptoethanol was added per L of cells to suspend the cells, and the cells were lysed using a sonic dismembrator (Fisher, USA) in an ice bath. The obtained solution was centrifuged at 18,000 rpm for 1 hour to obtain the supernatant.

[0100] 3-2. Purification by Column Chromatography

[0101] pET32a-hNDST3N obtained in Example 3-1 above △ The TM (72-873aa) supernatant was fed onto a nickel (Ni-NTA) column (GE Healthcare Life Sciences, USA) pre-equilibrated with the above buffer solution. Then, using the same buffer as above as the eluent, proteins were eluted from the column using a 500 mM imidazole concentration gradient. Protein-containing fractions were identified by 10% SDS-polyacrylamide gel electrophoresis and collected. pET32a- hNDST3N △ The results for TM (72-873aa) are shown in Fig. 5a. (M: protein marker, I-: before IPTG induction, I+: cell fragmentation sample after IPTG induction, S: supernatant after centrifugation of IPTG-induced sample, P: precipitate after centrifugation of IPTG-induced sample)

[0103] 3-3. Purification of Proteins by Ion Exchange Chromatography

[0104] pET32a- hNDST3N obtained in step 3-2 of the above Example 3 △ The volume of TM (72-873aa) protein was concentrated to about 5 ml and injected into a gel permeation column (Superdex 200, 10 / 300 GL, GE Healthcare Life Sciences, USA) that had been pre-equilibrated with the same buffer solution as above. The protein was then eluted with a buffer solution composed of 200 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, and 10 mM betamercaptoethanol to separate the proteins by molecular weight.

[0105] pET32a- hNDST3N by performing SDS-acrylamide gel electrophoresis △ The fraction corresponding to the TM (72-873aa) protein is collected. Each of the above results is shown in Fig. 5a.

[0107] Example 4. Expression of the recombinant protein hNDST3△TMDA gene in E. coli

[0108] The expression vector pET32a-hNDST3 obtained in the above step △ TMDA (72-569aa) was transformed into the expression host Escherichia coli BL21 (RIL) by conventional methods. pET32a- hNDST3 △ After incubating an E. coli strain transformed with TMDA (72-569aa) in 5.5 ml of LB liquid medium containing 100 µg / ml ampicillin at 37°C for 18 hours, 50 µl was taken and subcultured in 5.5 ml of LB liquid medium (containing 100 µg / ml ampicillin), and then incubated at 37°C until the absorbance of the culture medium at 600 nm reached approximately 0.2–0.3. Then, the incubation temperature was lowered to 18°C ​​and incubated until the absorbance of the culture medium reached approximately 0.5–6, after which IPTG (isopropyl-β-D-galactopyranoside) was added to a final concentration of 0.5 mM.

[0109] Cells cultured with IPTG for 18 hours were centrifuged to collect the respective cell precipitates. Each collected precipitate was suspended in 0.5 mL of cell lysis buffer (300 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, 5 mM beta-mercaptoethanol) and lysed using a mini sonic dismembrator (Sonic Dismembrator, Fisher, USA) in an ice bath. The resulting solution was centrifuged at 13,000 rpm for 10 minutes to separate the supernatant and precipitate. After 15% SDS-polyacrylamide gel electrophoresis, the protein pET32a-hNDST3 (72-569aa) was stained with Coomassie Brilliant Blue (Bio-Rad 161-0400) for analysis. The results for each are shown in Figure 3. (M: Protein marker, I-: Before IPTG induction, I+: Cell fragmentation sample after IPTG induction, S: Supernatant after centrifugation of IPTG-induced sample, P: Precipitate after centrifugation of IPTG-induced sample)

[0111] Example 5. Purification of recombinant protein hNDST3△TMDA in E. coli

[0112] 5-1. Culture and Disruption of E. coli Cells

[0113] In the same manner as in Example 2, E. coli cells expressing the recombinant protein were cultured in a volume of 8 L, and the E. coli cell precipitate was collected by centrifuging at 3,600 rpm for 20 minutes. Then, 15 mL of a buffer solution composed of 400 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, and 10 mM beta-mercaptoethanol was added per L of cells to suspend the cells, and the cells were lysed using a sonic dismembrator (Fisher, USA) in an ice bath. The obtained solution was centrifuged at 18,000 rpm for 1 hour to obtain the supernatant.

[0115] 5-2. Purification by Column Chromatography

[0116] pET32a-hNDST3 obtained in Step 1 above △ The TMDA (72-569aa) supernatant was fed onto a nickel (Ni-NTA) column (GE Healthcare Life Sciences, USA) pre-equilibrated with the above buffer solution. Then, using the same buffer as above as the eluent, proteins were eluted from the column using a 500 mM imidazole concentration gradient. Protein-containing fractions were identified by 10% SDS-polyacrylamide gel electrophoresis and collected. pET32a-hNDST3 △ The results for TMDA (72-569aa) are shown in Fig. 5b. (M: protein marker, I-: before IPTG induction, I+: cell fragmentation sample after IPTG induction, S: supernatant after centrifugation of IPTG-induced sample, P: precipitate after centrifugation of IPTG-induced sample)

[0118] 5-3. Purification of Proteins by Ion Exchange Chromatography

[0119] pET32a-hNDST3 obtained in Example 5-2 above △ TMDA (72-569aa) protein was diluted by more than three times with a buffer solution composed of 20 mM Hepes, pH 7.8, and 10 mM beta-mercaptoethanol, and flowed through a Q HP column (GE Healthcare Life Sciences, USA) equilibrated with the above buffer solution, and then the protein was eluted from the column using the same buffer solution as the eluent and a 1 M sodium chloride concentration gradient.

[0120] 15% SDS-polyacrylamide gel electrophoresis was performed, and pET32a-hNDST3 △ The results of TMDA (72-569aa) are shown in Fig. 5b.

[0122] Example 6. Expression of the hNDST3ST(569-873aa) recombinant gene in E. coli

[0123] The expression vector pET32a-hNDST3ST (569-873aa) obtained in the above step was transformed into the expression host Escherichia coli BL21 (RIL) by a conventional method. The Escherichia coli strain transformed with pET32a-hNDST3ST (569-873aa) was cultured at 37°C for 18 hours in 5.5 ml LB liquid medium containing 100 μg / ml ampicillin, then 50 μl was taken and subcultured in 5.5 ml LB liquid medium (containing 100 μg / ml ampicillin), and then cultured at 37°C until the absorbance of the culture medium at 600 nm reached approximately 0.2–0.3. Then, the culture temperature was lowered to 18 ℃, and cultured until the absorbance of the culture medium was about 0.5 to 6, and then IPTG (isopropyl-ß-D-galactopyranoside) was added to reach a final concentration of 0.5 mM.

[0124] Cells cultured with IPTG for 18 hours were centrifuged to collect the respective cell precipitates. Each collected precipitate was suspended in 0.5 mL of cell lysis buffer (300 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, 5 mM beta-mercaptoethanol) and lysed using a mini sonic dismembrator (Sonic Dismembrator, Fisher, USA) in an ice bath. The resulting solution was centrifuged at 13,000 rpm for 10 minutes to separate the supernatant and precipitate. After 15% SDS-polyacrylamide gel electrophoresis, the protein pET32a-hNDST3ST (569-873aa) was stained with Coomassie Brilliant Blue (Bio-Rad 161-0400) for analysis. The results are shown in Figure 5c. (M: Protein marker, I-: Before IPTG induction, I+: Cell fragmentation sample after IPTG induction, S: Supernatant after centrifugation of IPTG-induced sample, P: Precipitate after centrifugation of IPTG-induced sample)

[0126] Example 7. Purification of recombinant protein hNDST3ST (569-873aa) in E. coli

[0127] 7-1. Culture and Disruption of E. coli Cells

[0128] In the same manner as in Example 2, E. coli cells expressing the recombinant protein were cultured in 4 L volumes, and the E. coli cell precipitate was collected by centrifuging at 3,600 rpm for 20 minutes. Then, 25 mL of a buffer solution composed of 400 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, and 10 mM beta-mercaptoethanol was added per L of cells to suspend the cells, and the cells were lysed using a sonic dismembrator (Fisher, USA) in an ice bath. The obtained solution was centrifuged at 18,000 rpm for 1 hour to obtain the supernatant.

[0130] 7-2. Purification by Column Chromatography

[0131] The supernatant of pET32a-hNDST3ST (569-873aa) obtained in 7-1 above was fed into a nickel (Ni-NTA) column (GE Healthcare Life Sciences, USA) pre-equilibrated with the aforementioned buffer solution. Then, using the same buffer as above as the eluent, proteins were eluted from the column using a 500 mM imidazole concentration gradient. Protein-containing fractions were identified by SDS-polyacrylamide gel electrophoresis and collected. The results for pET32a-hNDST3ST (569-873aa) are shown in Fig. 5c. (M: Protein marker, I-: Before IPTG induction, I+: Cell fragmentation sample after IPTG induction, S: Supernatant after centrifugation of IPTG-induced sample, P: Precipitate after centrifugation of IPTG-induced sample)

[0133] 7-3. Separation and Purification of Proteins by Column Chromatography

[0134] The volume of the pET32a- hNDST3ST (569-873aa) protein obtained in 7-2 above was concentrated to about 5 ml and injected into a gel permeation column (Superdex 200, 26 / 60, GE Healthcare Life Sciences, USA) that had been pre-equilibrated with the same buffer solution as above, and the proteins were separated by molecular weight by eluting with a buffer solution composed of 400 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, and 10 mM betamercaptoethanol.

[0135] SDS-acrylamide gel electrophoresis was performed to collect the fraction corresponding to pET32a-hNDST3ST (569-873aa) proteins. The results are shown in Figure 3.

[0136] To cleave the N-terminal His tag and Trx tag of the collected proteins, thrombin protease was treated (1 unit of thrombin protease per 1 mg of target protein) and reacted at 4°C for 18 hours, after which SDS-polyacrylamide gel electrophoresis was performed, and the results for pET32a-hNDST3ST (569-873aa) are shown in Figure 3.

[0138] 7-4. Protein Denaturation with Urea and Protein Purification by Column Chromatography

[0139] The pET32a- hNDST3ST (569-873aa) protein obtained in step 3 above is dissolved in a buffer solution composed of 200mM NaCl, 20mM Hepes, pH 7.4, and 8M Urea, and rotated at room temperature for 18 hours to sufficiently expose it to urea.

[0140] The pET32a-hNDST3ST (569-873aa) proteins, cleaved with His Tek and Trx Tek, were fed at room temperature into a nickel (Ni-NTA) column (GE Healthcare Life Sciences, USA) pre-equilibrated with the buffer solution, and the pass-through was collected in a constant volume. Finally, the proteins were eluted from the column using a 500 mM imidazole concentration gradient. 15% SDS-polyacrylamide gel electrophoresis was performed, and the results for pET32a-hNDST3ST (569-873aa) are shown in Fig. 5c.

[0142] Example 8. Expression of the hNDST4△TMDA (72-569aa) recombinant gene in Escherichia coli

[0143] The expression vector pET32a-hNDST4 obtained in the above step △ TMDA (72-569aa) was transformed into the expression host Escherichia coli BL21 (RIL) by conventional methods. pET32a- hNDST4 △ After incubating an E. coli strain transformed with TMDA (72-569aa) in 5.5 mL of LB liquid medium containing 100 μg / mL ampicillin at 37 °C for 16 hours, 50 μl was taken and subcultured in 5.5 mL of LB liquid medium (containing 100 μg / mL ampicillin), and then incubated at 37 °C until the absorbance of the culture medium at 600 nm was approximately 0.2–0.3. Then, the incubation temperature was lowered to 18 °C and incubated until the absorbance of the culture medium was approximately 0.6, at which point IPTG (isopropyl-β-D-galactopyranoside) was added to a final concentration of 0.5 mM.

[0144] Cells cultured with IPTG for 18 hours were centrifuged to collect the respective cell precipitates. Each collected precipitate was suspended in 0.5 mL of cell lysis buffer (200 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, 5 mM beta-mercaptoethanol), and the cells were lysed using a mini sonic dismembrator (Sonic Dismembrator, Fisher, USA) in an ice bath. The resulting solution was centrifuged at 13,000 rpm for 10 minutes to separate the supernatant and precipitate, followed by 15% SDS-polyacrylamide gel electrophoresis. The protein pET32a-hNDST4 was then analyzed using Coomassie Brilliant Blue (Bio-Rad 161-0400). △ TMDA (72-569aa) was stained and analyzed. The results for pET32a-hNDST4TMDA (72-569aa) are shown in Figure 4. (M: Protein marker, I-: Before IPTG induction, I+: Cell fragmentation sample after IPTG induction, S: Supernatant after centrifugation of IPTG-induced sample, P: Precipitate after centrifugation of IPTG-induced sample)

[0146] Example 9. Purification of recombinant protein hNDST4△TMDA (72-569aa) in E. coli

[0147] 9-1. Culture and Disruption of E. coli Cells

[0148] In the same manner as in Example 2, E. coli cells expressing the recombinant protein were cultured in a volume of 8 L, and the E. coli cell precipitate was collected by centrifuging at 3,600 rpm for 20 minutes. Then, 15 mL of a buffer solution composed of 400 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, and 10 mM beta-mercaptoethanol was added per L of cells to suspend the cells, and the cells were lysed using a sonic dismembrator (Fisher, USA) in an ice bath. The obtained solution was centrifuged at 18,000 rpm for 1 hour to obtain the supernatant.

[0150] 9-2. Purification by Column Chromatography

[0151] pET32a-hNDST4 obtained in Step 1 above △ The TMDA (72-569aa) supernatant was fed onto a nickel (Ni-NTA) column (GE Healthcare Life Sciences, USA) pre-equilibrated with the above buffer solution under 100 mM NaCl conditions. Then, using the same buffer as above as the eluent, proteins were eluted from the column using a 500 mM imidazole concentration gradient. Protein-containing fractions were identified by SDS-polyacrylamide gel electrophoresis and collected. pET32a- hNDST4 △ The results for TMDA (72-569aa) are shown in Fig. 6a. (M: protein marker, I-: before IPTG induction, I+: cell fragmentation sample after IPTG induction, S: supernatant after centrifugation of IPTG-induced sample, P: precipitate after centrifugation of IPTG-induced sample)

[0153] Example 10. Expression of the recombinant hNDST4ST(569-873aa) gene in Escherichia coli

[0154] The expression vector pET32a-hNDST4ST (569-873aa) obtained in the above step was transformed into the expression host Escherichia coli BL21 (RIL) by a conventional method. The Escherichia coli strain transformed with pET32a-hNDST4ST (569-873aa) was cultured at 37 °C for 16 hours in 5.5 mL of LB liquid medium containing 100 μg / mL ampicillin, then 50 μl was taken and subcultured in 5.5 mL of LB liquid medium (containing 100 μg / mL ampicillin), and then cultured at 37 °C until the absorbance of the culture medium at 600 nm was about 0.2–0.3. Then, the culture temperature was lowered to 18 ℃, and cultured until the absorbance of the culture medium was about 0.6, and then IPTG (isopropyl-ß-D-galactopyranoside) was added to reach a final concentration of 0.5 mM.

[0155] Cells cultured with IPTG for 18 hours were centrifuged to collect the respective cell precipitates. Each collected precipitate was suspended in 0.5 mL of cell lysis buffer (200 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, 5 mM beta-mercaptoethanol) and lysed using a mini sonic dismembrator (Sonic Dismembrator, Fisher, USA) in an ice bath. The resulting solution was centrifuged at 13,000 rpm for 10 minutes to separate the supernatant and precipitate, followed by 15% SDS-polyacrylamide gel electrophoresis. The protein GST-ZnF_hUSP3 was then stained with Coomassie Brilliant Blue (Bio-Rad 161-0400) for analysis. The results for pET32a-hNDST4 are shown in Figure 4. (M: Protein marker, I-: Before IPTG induction, I+: Cell fragmentation sample after IPTG induction, S: Supernatant after centrifugation of IPTG-induced sample, P: Precipitate after centrifugation of IPTG-induced sample)

[0157] Example 11. Purification of recombinant protein hNDST4ST (569-873aa) in E. coli

[0158] 11-1. Culture and Disruption of E. coli Cells

[0159] In the same manner as in Example 2, E. coli cells expressing the recombinant protein were cultured in a volume of 8 L. The E. coli cell precipitate was collected by centrifuging at 3,600 rpm for 20 minutes. Then, 27 mL of a buffer solution composed of 400 mM sodium chloride, 20 mM Hepes-hydrochloric acid buffer, pH 7.5, 10 mM beta-mercaptoethanol, and 5% glycerol was added per L of cells to suspend the cells. The cells were then lysed using a sonic dismembrator (Fisher, USA) in an ice bath. The resulting solution was centrifuged at 18,000 rpm for 1 hour to obtain the supernatant.

[0161] 11-2. Purification by Column Chromatography

[0162] The supernatant of pET32a-hNDST4ST (569-873aa) obtained in 11-1 above was fed into a nickel (Ni-NTA) column (GE Healthcare Life Sciences, USA) pre-equilibrated with the aforementioned buffer solution. Then, using the same buffer as above as the eluent, proteins were eluted from the column using a 500 mM imidazole concentration gradient. The protein-containing fractions were identified by SDS-polyacrylamide gel electrophoresis and collected. The results for pET32a-hNDST4ST (569-873aa) are shown in Fig. 6b. (M: Protein marker, I-: Before IPTG induction, I+: Cell-disintegrated sample after IPTG induction, S: Supernatant after centrifugation of IPTG-induced sample, P: Precipitate after centrifugation of IPTG-induced sample)

[0164] 11-3. Separation and Purification of Proteins by Column Chromatography

[0165] The volume of the pET32a-hNDST4ST (569-873aa) protein obtained in Example 11-2 above was concentrated to about 5 ml and injected into a gel permeation column (Superdex 200, 26 / 60, GE Healthcare Life Sciences, USA) that had been pre-equilibrated with the same buffer solution as above. The protein was then eluted with a buffer solution composed of 400 mM sodium chloride, 20 mM Hepes-hydrochloride buffer, pH 7.5, 10 mM beta-mercaptoethanol, 5% glycerol, and 2 mM TCEP (Tris[2-carboxyethyl]phosphine hydrochloride) to separate the proteins by molecular weight.

[0166] Perform 15% SDS-acrylamide gel electrophoresis to collect the fraction corresponding to pET32a-hNDST4ST (569-873aa) protein.

[0167] To cleave the N-terminal His tag and Trx tag of the collected proteins, thrombin protease was treated (1 unit of thrombin protease per 1 mg of target protein) and reacted at 4°C for 3 days, after which SDS-polyacrylamide gel electrophoresis was performed, and the results for pET32a-hNDST4ST (569-873aa) are shown in Figure 6b.

[0169] 11-4. Protein Denaturation with Urea and Purification by Column Chromatography

[0170] Since the pET32a-hNDST4ST (569-873aa) protein obtained in Example 11-2 above was in a precipitated state, it was washed at different concentrations using 1M, 2M, and 4M urea to dissolve it back into a liquid state. The precipitate was then dissolved in 8M urea and passed through a nickel (Ni-NTA) column (GE Healthcare Life Sciences, USA) pre-equilibrated with a buffer solution composed of 200mM sodium chloride, 20mM HEPES-hydrochloric acid buffer, pH 7.5, and 8M urea. 3ml of the resulting solution was collected and subjected to SDS-acrylamide gel electrophoresis to confirm that the pET32a-hNDST4 protein was purified. The results for pET32a-hNDST4ST (569-873aa) are shown in Figure 6b.

[0172] Example 12. Evaluation of enzyme activity of human NDST3 and NDST4 variants in vitro

[0173] To measure the enzymatic activity of NDST3 (hNDST3TMDA, hNDST3ST) and NDST4 (hNDST4TMDA, hNDST4ST) cleaved on polysaccharide substrates, 250 nM NDST3 variants (hNDST3TMDA, hNDST3ST) and NDST4 variants (hNDST4) were prepared by varying the concentrations of K5 or HS polysaccharides (low-sulfur fraction; Iduron, GAG HS-I). △ TMDA, hNDST4ST), 350 nM Sult1a1-K65E-R68G, 20 μM PAPS, and 4 mM 4MU-sulfate were reacted in 50 mM Tris-HCl pH 7.5, 15 mM MgCl2, and 1 mM DTT. A control group excluding polysaccharides was excluded from the reaction prior to further analysis. To measure pseudo-primary reaction activity for PAPS, PAPS diluted 2-fold at 50 μM was reacted with 0.5 mg / mL HS, NDST3 variant (hNDST3 △ TMDA, hNDST3ST) and NDST4 variant (hNDST4 △Reactions were carried out with TMDA (hNDST4ST), 350 nM Sult1a1-K65E-R68G, and 4 mM 4MU-sulfate in 50 mM Tris-HCl pH 7.5, 15 mM MgCl2, and 1 mM DTT. A control group excluding PAPS was excluded from the reaction prior to further analysis. All reactions were performed in black half-area 96-well plates (Greiner) with a total reaction volume of 20 μL. All reactions were initiated by the addition of polysaccharides, and fluorescence changes were recorded using a Clariostar plate reader (SpectraMax® M3, Molecular Devices) with the 4MU preset. The change in 4MU during the reaction was calculated using an 8-point 2-fold dilution standard curve of 4MU (starting at 10 μM) in 50 mM Tris-HCl pH 7, 15 mM MgCl2. The results are shown in Figure 7.

[0175] Although the embodiments have been described above with reference to the limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, suitable results may be achieved even if the described techniques are performed in a different order than described, and / or if the components of the described system, structure, device, circuit, etc. are combined or assembled in a form different from described, or replaced or substituted by other components or equivalents.

[0176] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims set forth below.

Claims

Claim 1 A peptide having deacetylase activity, wherein the peptide comprises the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO.

7. Claim 2 A method for producing the peptide of claim 1, comprising: a step of producing an expression vector containing a gene encoding the peptide of claim 1; a step of transforming Escherichia coli with said expression vector; a step of culturing said transformed Escherichia coli; a step of crushing and centrifuging said transformed Escherichia coli to obtain a cell precipitate; and a step of purifying the peptide of claim 1 from said cell precipitate. Claim 3 A pharmaceutical composition for treating neurological diseases comprising the peptide of claim 1 as an active ingredient. Claim 4 A peptide having sulfotransferase activity, wherein the peptide comprises the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO.

8. Claim 5 A method for producing the peptide of claim 4, comprising: a step of producing an expression vector containing a gene encoding the peptide of claim 4; a step of transforming E. coli with said expression vector; a step of culturing said transformed E. coli; a step of crushing and centrifuging said transformed E. coli to obtain a cell precipitate; and a step of purifying the peptide of claim 1 from said cell precipitate. Claim 6 A pharmaceutical composition for treating neurological diseases comprising the peptide of claim 4 as an active ingredient.

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