KR20020010445A - Cell-transducing HIV-1 Tat-superoxide dismutase fusion protein and use of the fusion protein - Google Patents

Abstract

The present invention is a fusion incorporating HIV-1 Tat protein to transport Cu, Zn-superoxide dismutase (SOD), which is known to play an important role in biological defense mechanisms into cells and to have activity in cells. Protein Tat-SOD, a polynucleotide encoding the fusion protein Tat-SOD, a vector for producing the fusion protein, a method for introducing the fusion protein into a cell, and a use thereof, wherein the Tat-SOD fusion protein is used in a pharmaceutical composition and Useful for applications such as cosmetics.

Description

Cell-transducing HIV-1 Tat-superoxide dismutase fusion protein and use of the fusion protein}

The present invention relates to a cell penetrating Cu, Zn-Superoxide dismutase (hereinafter referred to as "Cu, Zn-Superoxide dismutase", "superoxide dismutase", " SOD "," Cu, Zn-SOD "are used interchangeably, all have the same meaning), recombinant polynucleotide encoding this fusion protein, expression vector of this fusion protein, and thio-superoxide dismutase fusion The present invention relates to a method for introducing a protein into a cell and its use, and more particularly, a fusion protein in which six histidine residues and nine HIV-1 Tat proteins are bound to the N-terminal side of superoxide dismutase using a recombinant vector. The present invention relates to a superoxide dismutase which is permeated into cells with high efficiency by preparing and purifying them.

As used herein, the term "target protein" refers to a therapeutic molecule, a preventive molecule, a diagnostic molecule, and the like, which are covalently bound to the HIV-1 Tat transport domain to be introduced into a cell and exhibit activity, and are not limited thereto. Peptides, polypeptides, glycoproteins associated with sugars, peptidoglycans, and the like are used collectively.

In addition, the specification is used interchangeably with the expressions "transport", "penetration", "transport", "delivery", "transmission", "pass" for "introducing" a protein, peptide, organic compound into a cell.

Reactive oxygen species are inevitably produced as by-products of various intracellular metabolism in all living organisms that use oxygen for energy. These reactive oxygen species damage biopolymers such as proteins, nucleic acids and fats in cells. It is deeply involved in the progress of various diseases in the human body. In particular, it is involved in carcinogenesis, stroke, arthritis, arteriosclerosis, radiation damage and inflammatory reactions, and plays an important role in promoting aging even in normal aging [Floyd, RA (1990) FASEB J. 4,2587-2597.Anderson , WF (1998) Human gene therapy. Nature 392, 25-30. Halliwell B. and Gutteridge J.M.C. (1999) Free radicals in biology and medicine, Oxford University Press, Oxford.

A brief description of the diseases associated with Cu, Zn-superoxide dismutase is shown in Table 1 below.

category Concrete example Inflammatory / immune damage Glomerulonephritis, vasculitis, autoimmune diseases, etc. Ischemia Stroke, myocardial infarction, arrhythmia, angina, etc. Drug and Toxic Induction Reactions skip Iron Overload: Tissue and Plasma Idiopathic hemochromatosis, etc. Radiation-related damage Nuclear exposure, radiotherapy, etc. Aging Progesteritis, disease-related aging Red blood cells Sickle cell disease, malaria, etc. Bronchus As a result of cigarette smoking, emphysema Heart and cardiovascular system Cardiomyopathy, etc. kidney Autoimmune nephrotic syndrome Camouflage Betelnut-related oral cancer Brain / nerve system / nerve muscle abnormalities Hyperbaric oxygen, Alzheimer's disease, Parkinson's disease, etc. Eye Cataracts, etc. skin Diseases due to UV irradiation

* Source: Free radicals in biology and medicine, Oxford University Press, Oxford pp 618 ~ 619.

Known active oxygen species include ₁ O ₂ , OH, O ₂ , H ₂ O ₂ , which are produced by various enzyme reactions in vivo, and play an important role in biosynthesis, immune function, and metabolism of various bioactive substances. However, if it is overproduced by external radiation, ultraviolet rays, pollution, and various stresses, it may cause damage to the living body, and thus the living body may use enzymes such as SOD, catalase, and peroxidase as a protective function. When aging begins, the balance of the skin is broken and skin protection against various free radicals is reduced.

Therefore, there is a need to protect cells from these active oxygen species, SOD, lactoferrin, antioxidants, etc. have been used or developed as an active oxygen remover as a cosmetic raw material. However, SOD is poorly soluble in lipids and poor in stability due to its nature as an enzyme protein, despite its ability to remove active oxygen, and its molecular weight is more than 30,000 Daltons (Dalton). It didn't work.

Cu, Zn-superoxide dismutase is an important defense enzyme in cells that prevents cell damage from free radical detoxification and oxygen damage [Fridovich, I. (1995) Annu. Rev. Biochem. 64, 97-112.]. Since all in vivo polymers are always exposed to the harmful effects of oxygen radicals, there is a growing interest in using Cu, Zn-superoxide dismutase for therapeutic purposes in various diseases.

Recently, many methods have been tried to clinically apply Cu, Zn-superoxide dismutase. In vivo transport methods of Cu, Zn-superoxide dismutase developed to date can be largely classified into three types. First, a method of conjugating polyethylene glycol, ficoll, lecithin, albumin, etc. to Cu, Zn-superoxide dismutase [Del Zoppo, GJ, Wagner, S. and Tagaya, M. (1997) Drugs 54, 9-38. Muzykantov, V.R., Atochina, E.N., Ischiropoulos, H., Danilov, S.M. and Fisher, A.B. (1996) Proc. Natl. Acad. Sci. USA 93, 5213-5218.], Second, a method for encapsulating Cu, Zn-superoxide dismutase using liposomes [Perdereau, B., Campana, F., Vilcoq, JR, de la Rochefordiere, A., Barbaroux, C., Fourquet, A. and Magdelenat, H. (1994) Bull. Cancer 81, 659-669.] Third, gene therapy that induces overexpression of enzymes in cells by transducing the Cu, Zn-superoxide dismutase gene has been attempted [Okumura, K., Nishiguchi]. , K., Tanigawara, Y., Mori, S., Iwakawa, S. and Komada, F. (1997) Pharm. Res. 14, 1223-1227. Lehmann, T.G., Wheeler, M.D., Schoonhoven, R., Bunzendahl, H., Samulski, R.J. and Thurman, R. G. (2000) Transplantation 69, 1051-1057. Liu, R., Oberley, T. D. and Oberley, L. W. (1997) Hum. Gene Ther. 8, 585-595.].

Most notable among them is gene therapy, and many studies have been conducted to apply Cu, Zn-superoxide dismutase gene therapy to diseases. However, gene therapy is not easy to transport genes into cells, has low expression rate in target cells, short duration of protein expression in expressed cells, and artificially controls the amount of protein expressed in target cells. Has many problems, such as very difficult [Verma, IM and Somia, N. (1997) Nature 389, 239-242.

Another way to move drugs or proteins for treatment into cells is to think of direct delivery of the target protein across the cell membrane. However, therapeutic drugs or proteins are very difficult to cross cell membranes because of their size or various biochemical properties. In general, substances having a molecular weight of 600 or more are known to be almost impossible to pass through the cell membrane.

Recently, the Tat (transactivator of transcription) protein, a type of Human Immunodeficiency Virus type-1 protein, efficiently moves through the cell membrane and easily migrates into the cytoplasm. This function appears due to the nature of the protein transduction domain, which is the intermediate region of the Tat protein, and its exact mechanism is still unknown [Frankel, A.D. and Pabo, C. O. (1988) Cell 55, 1189-1193. Green, M. and Loewenstein, P. M. (1988) Cell 55, 1179-1188. Ma, M. and Nath, A. (1997) J. Virol. 71, 2495-2499. Vives, E., Brodin, P. and Lebleu, B. (1997) J. Biol. Chem. 272, 16010-16017.]. However, it is understood that no specific receptors or carriers are involved in the transmembrane of Tat protein and that the protein transduction site of Tat protein occurs by directly interacting with the lipid bilayer of the membrane [Vives, E., Brodin, P. and Lebleu , B. (1997) J. Biol. Chem. 272, 16010-16017. Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G. and Prochiantz, A. (1996) J. Biol. Chem. 271, 18188-18193.].

In recent studies, heterologous proteins such as ovalbumin, β-galactosidase, and horseradish peroxidase were fused with HIV-1 Tat protein in vivo tissue and culture. It is shown to be directly transported into the cells of the cells. Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, LL, Pepinsky, B. and Barsoum, J. (1994) Proc. Natl. Acad. Sci. USA 91, 664-668. Schwartze, S.R., Ho, A., Vocero-Akbani, A. and Dowdy, S. F. (1999) Science. 285, 1569-1572. Watson, K. and Edward, R.J. (1999) Biochem. Pharmacol. 58, 1521-1528. These results indicate that the Tat protein has the ability to transport not only itself but also other giant proteins of its kind into the cell.

However, not all proteins are carried by the Tat protein. In addition, it is not yet clear whether all proteins carried into cells by Tat show biological activity.

The present invention seeks to solve the problems of the prior art in introducing or expressing superoxide dismutase into cells and to introduce them into cells with high efficiency and to have activity in cells.

In addition, the present invention is to provide a cosmetic having excellent removal ability of active oxygen species by penetrating the skin epidermis, dermis layer, subcutaneous fat layer using superoxide dismutase fused with Tat protein.

In order to achieve the above object, the present inventors confirmed that the fusion protein was effectively permeated into cells by fusing the protein transduction site of Tat protein and human Cu, Zn-superoxide dismutase, in an experiment using HeLa cells. In addition, a method of producing and purifying a large amount of Tat-superoxide dismutase fusion protein has been developed.

1A discloses a method for preparing a Tat-SOD expression vector (pTat-SOD) using a pET15b vector.

Figure 1B is a schematic diagram of the expressed fusion protein Tat-SOD and SOD used as a control protein.

Figure 2 shows the results of 15% SDS-PAGE and Western blot analysis of the protein fraction recovered in each step of purification after the expression of Tat-SOD in E. coli.

A shows E. coli cell lysate and B shows SDS-PAGE results for purified fusion proteins.

Lane 1: A lysate of cells containing only the pET vector

Lane 2: lysate from cells containing the pTat-SOD vector

Lane 3: lysate of the cell containing the pSOD vector

Lane 1: B purified Tat-SOD

Lane 2: refined SOD

C shows the results of Western blot analysis using human Cu, Zn-SOD monoclonal antibody against Tat-SOD and SOD purified as B.

Lane 1: Tat-SOD

Lane 2: SOD

D shows the results of Western blot analysis using human Cu, Zn-SOD monoclonal antibody to determine the HeLa cell permeability of Tat-SOD fusion protein. Administration of Tat-SOD and SOD of 1 μ M in a cell culture medium, and analyzed the amount of fusion protein in 1 hour into a transmission cell by Western blot.

Lane 1: Urea Denatured Tat-SOD

Lane 2: Denatured SOD by Urea

Lane 3: undenatured Tat-SOD

Lane 4: undenatured SOD

Figure 3A shows the results of Western blot analysis using human Cu, Zn-SOD monoclonal antibody to determine the HeLa cell permeability of denatured Tat-SOD over time. 1 results when the administration of Tat-SOD and SOD in μ M in the medium for 5-1 minutes.

Figure 3B shows the results of Western blot analysis using human Cu, Zn-SOD monoclonal antibody to determine the HeLa cell permeability of denatured Tat-SOD according to the concentration. Western blot results of administration of the Tat-SOD and SOD of 0.25-2 μ M to the culture medium for 1 hour.

Figure 4 is a graph showing the change in SOD enzyme activity in cells when denatured Tat-SOD administered to HeLa cells.

A when 1 μ M Tat-SOD and SOD was administered to the medium for 5 minutes-1 hour.

B, when administered to Tat-SOD and SOD of 0.25-2 μ M to the culture medium for 1 hour.

5 shows a polynucleotide sequence encoding a Tat-SOD fusion protein.

Figure 6 is a photograph of the rat skin cell permeation experiment results. A and B are epidermal and dermal areas of the control and experimental groups, respectively, and C and D represent subcutaneous fat tissue areas of the control and experimental groups, respectively. The arrow in the picture indicates the color development site.

7 is a graph showing the change in activity over time by measuring the activity of intracellular superoxide dismutase after rat skin cell permeation experiment for Tat-SOD and SOD.

The present invention is a fusion incorporating HIV-1 Tat protein to transport Cu, Zn-superoxide dismutase (SOD), which is known to play an important role in biological defense mechanisms into cells and to have activity in cells. Protein Tat-SOD, a vector for producing this fusion protein Tat-SOD, a method for producing and purifying a fusion protein using the vector. The present invention also provides a pharmaceutical composition and cosmetic composition using the fusion protein Tat-SOD.

To this end, a Tat-SOD expression vector was developed that can overexpress and easily purify the Tat-SOD fusion protein. This expression vector (pET-Tat-SOD) is a cDNA capable of expressing 9 amino acids (Tat 49-57) at the human Cu, Zn-SOD, Tat transduction sites, and 6 histidine residues at the amino acid terminus. It includes.

Using this expression vector, the Tat-SOD fusion protein was overexpressed in E. coli and purified easily and conveniently by metal-chelating affinity chromatography in denaturing and natural state.

Tat-SOD fusion protein of the present invention can be prepared by a general chemical binding method in addition to gene recombination method.

The denatured Tat-SOD fusion protein was confirmed by Western blot to be delivered to cells in time and concentration dependent manner in cultured HeLa cells. On the other hand, Tat-SOD purified in nature and SOD used as a control protein were not transported into cells.

The activity of SOD enzyme in cells treated with Tat-SOD fusion protein increased in proportion to the amount of protein carried into the cells. These results indicate that Tat can move SOD into cells and artificially increase the activity of SOD in cells.

The present invention provides a cell-penetrating Tat-superoxide dismutate in which an HIV-1 Tat transduction site 49-57 residue (Tat 49-57) is covalently bonded to an amino terminus of Cu, Zn-superoxide dismutase or a derivative thereof. Fusion protein.

In addition, the present invention is the Tat- is encoded by binding the DNA sequence of 49-57 residues (Tat 49-57) HIV-1 Tat transduction site to the 5 'side of Cu, Zn- superoxide dismutase or its derivative cDNA. It relates to a recombinant polynucleotide encoding a superoxide dismutase fusion protein.

The present invention also relates to a vector expressing a cell penetrating Tat-superoxide dismutase fusion protein constructed as shown in the restriction map of FIG. 1A including the recombinant polynucleotide to express the fusion protein.

In addition, the present invention comprises the steps of expressing the vector in a microorganism;

Purifying the expressed Tat-superoxide dismutase fusion protein;

It relates to a method of introducing a Tat- superoxide dismutase fusion protein consisting of; adding the purified Tat-superoxide dismutase fusion protein into the cell.

In addition, in the purification step, the Tat-superoxide dismutase fusion protein, characterized in that the purified Tat- superoxide dismutase fusion protein in a denatured state using 6M urea into the cell. It is about a method of introduction.

The present invention also relates to a pharmaceutical composition comprising the Tat-SOD fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

Further, the present invention is glomerulonephritis, vasculitis, autoimmune disease, stroke, myocardial infarction, arrhythmia, angina, idiopathic hemochromatosis, disease by radiation therapy, premature ejaculation, disease-related aging, sickle cell disease, malaria, emphysema, Kim Geun Tat-SOD fusion protein used as a therapeutic agent for SOD dependent diseases such as syndrome, autoimmune nephrotic syndrome, Betelnet-related oral cancer, hyperbaric oxygen, Alzheimer's disease, Parkinson's disease, cataract, etc. as an active ingredient and a pharmaceutically acceptable carrier It relates to a pharmaceutical composition comprising a.

In addition, the present invention relates to a cosmetic composition comprising the Tat-SOD fusion protein as an active ingredient and having a function of removing active oxygen species.

In addition, the present invention is a cosmetic composition is a functional lotion, gel, water-soluble liquid, oil-in-water (O / W) type and water-in-oil (W / O) type Tat-SOD fusion protein as an active ingredient and removes the active oxygen species It relates to a cosmetic composition comprising a.

Furthermore, the present invention relates to a method for preventing or treating SOD dependent diseases using the fusion protein or recombinant polynucleotides for producing the fusion protein.

In addition, the present invention relates to a method for preventing or treating SOD dependent diseases using a microorganism transformed with a vector expressing the cell penetrating Tat-superoxide dismutase fusion protein.

In addition, the present invention comprises the steps of expressing the vector in a microorganism; Purifying the expressed Tat-superoxide dismutase fusion protein or purifying by denaturation with 6M urea; Adding a purified Tat-superoxide dismutase fusion protein to a cell; and a method of preventing or treating SOD dependent disease by introducing a Tat-superoxide dismutase fusion protein into a cell. It is about.

Furthermore, the present invention provides a method for preventing or treating a SOD dependent disease using a method of introducing the fusion protein into cells using the fusion protein, the recombinant polynucleotide, the recombinant vector, and the recombinant vector. Dependent diseases include glomerulonephritis, vasculitis, autoimmune diseases, stroke, myocardial infarction, arrhythmia, angina pectoris, idiopathic hemochromatosis, disease caused by radiation therapy, premature ejaculation, disease-related aging, sickle cell disease, malaria, emphysema, kim myopathy, The present invention relates to a method for preventing or treating SOD-dependent diseases characterized by autoimmune nephrotic syndrome, Betelnet-related oral cancer, hyperbaric oxygen, Alzheimer's disease, Parkinson's disease and cataracts.

Pharmaceutical compositions containing the Tat-SOD fusion protein as an active ingredient can be formulated orally or by injection by conventional methods in combination with a carrier that is conventionally acceptable in the pharmaceutical art. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, may contain diluents (e.g. lactose, textose, sucrose, mannitol, sorbitol, cellulose and / or glycine), suspending agents (e.g. silica, Talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols, and the tablets also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpi It is preferred to contain a disintegrant (eg starch, agar, alginic acid or its sodium salt) or a boiling mixture and / or absorbents, colorants, antiseptics and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries (eg, preservatives, stabilizers, wetting or emulsifier solution promoters, salts or buffers for controlling osmotic pressure). In addition, they may contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be administered orally as desired, or parenterally, i.e., intravenously, subcutaneously, intraperitoneally, or topically, and the dosage may be from 0.001 to 100 mg / kg. It can be administered in 1 to several times. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

In addition, the cosmetic composition of the present invention can be formulated and used as a cream, ointment, gel, lotion, lotion, and the like, which formulation is used and can be determined very easily by those skilled in the art to which the present invention belongs. .

In addition, lotions, gels, essences, gels, creams, lotions, etc., each of which is characterized in that the active ingredient of the Tat-superoxide dismutase fusion protein of the present invention and has the function of removing active oxygen species, are prepared in general It can be easily prepared in any form according to the method, and can be conveniently added to basic cosmetics and the like and used as an anti-aging and improving agent for skin.

As an example, in the preparation of creams, the Tat-superoxide dismutase fusion protein of the present invention is contained in a creamy oil-in-water (O / W) or water-in-oil (W / O) cream base. While pigments, antioxidants and preservatives may be used, synthetic or natural materials such as proteins, minerals and vitamins may be used in combination for the purpose of improving the properties.

Hereinafter, the configuration of the present invention will be described in detail by examples. However, the scope of the present invention is not limited to the following examples.

material

Restriction enzymes and T4 DNA ligase were purchased from Promega (USA) and Pfu polymerase was purchased from stratagene (USA). Tat oligonucleotides were synthesized by Gibco BRL custom primer (USA). IPTG was purchased from Duchefa (Netherland). pET-15b and BL21 (DE3) plasmids were purchased from Novagen (USA) and Ni-nitrilo-triactic acid sepharose superflow was purchased from Qiagen (Germany).

Human Cu, Zn-Superoxide Dismutase (hereafter interchanged with meaning "Cu, Zn-SOD", "SOD") cDNA is referred to as Polymerase Chain Reaction (hereinafter referred to as "PCR"). ) Was isolated from human placental cDNA library [Kang, JH, Choi, BJ and Kim, S. M. (1997) J. Biochem. Mol. Biol. 30, 60-65]. All other reagents were used as express products.

In addition, in the embodiment of the present invention, a fusion protein was prepared using human Cu, Zn-superoxide dismutase, but the scope of the present invention is a fusion protein using human-derived Cu, Zn-superoxide dismutase. Not only does it affect, but it turns out that the effect of this invention can be acquired also when using yeast origin, bacteria derived enzyme, etc.

SOD enzyme activity measurement

In the embodiment of the present invention, the activity of SOD was measured by spectrophotometer to determine the degree of inhibition of ferricytochrome c by xanthine / xanthine oxidase reaction according to McCord and Fridovich's method (1969). It was measured by observing with [McCord, JM and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055.

Standard assays were performed in 50 mM potassium phosphate buffer (pH 7.8) containing 3 ml of 0.1 mM EDTA at 25 ° C. The reaction mixture is to measure the degree of reduction of 10 μ M Perry cytochrome c (ferricytochrome c), 50 μ M xanthine and sufficient amounts of xanthine oxidase and containing Perry cytochrome c at 550nm. The amount of superoxide dismutase that reduces cytochrome c by 50% under these conditions was defined as 1 unit.

Western blot analysis

Western blot analysis was performed in the Examples of the present invention as follows.

Proteins in the cell mill were separated by 15% SDS polyacrylamide gel electrophoresis, and then the proteins in the gel were electrophoresed to a nitrocellulose membrane (Amersham, UK). The protein-shifted nitrocellulose membrane was treated with 5% bovine serum albumin solution and then treated with monoclonal antibody (hSOD mAb-1.4) against human Cu, Zn-SOD for 1 hour. After washing, the membrane was reacted with a goat anti-mouse IgG (Sigma, 1: 5,000 dilution) for 1 hour with alkaline phosphatase. Finally, an alkaline phosphatase conjugate substrate kit (Bio-Rad, USA) was treated to identify protein bands that respond to human Cu, Zn-SOD monoclonal antibodies.

Example 1 Preparation and Transformation of Tat-SOD Expression Vector

In order to overexpress Tat-SOD fusion protein, pET-Tat-SOD expression vector containing Cu, Zn-SOD, transduction site of HIV-1 Tat (Tat49-57) and cDNA for 6 histidines in sequence (Fig. 1A). In order to overexpress SOD, a control protein for the fusion protein, only the transduction site of Tat (Tat49-57) was included and the other sites were prepared with the same pET-SOD expression vector.

First, in order to produce a Tat-SOD fusion protein, a pET-Tat expression vector containing a basic domain of HIV-1 Tat (ie, amino acids 49-57) was prepared.

Two types of oligonucleotides corresponding to the Tat base domain (top strand, 5'-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3 '; bottom strand, 5'-TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC-3') were cut with NdeI-XhoI restriction enzyme Ligation was inserted into pET15b. Then, two kinds of oligonucleotides were synthesized based on the sequences of cDNAs of human Cu, Zn-SOD. Forward primer (Forward primer) is, sequence of, and has a Xho I restriction site in the reverse primer (reverse primer) was 5'- GGATCC TTATTGGGCGATCCCAATTAC-3 '5'- CTCGAG GCGACGAAGGCCGTGTGCGTG-3 has a BamH I restriction site into.

Polymerase chain reaction (PCR) was performed in a thermal cycler (Perkin-Elmer, model 9600). The reaction mixture was placed in a 50 μl silicon tube (siliconized reaction tube) and heated at 94 ° C. for 5 minutes. PCR reactions consisted of 30 extensions for 40 seconds at 94 ° C, denaturation for 1 minute at 54 ° C, annealing for 3 minutes at 70 ° C, and final extension for 5 minutes at 20 ° C for 10 minutes at 72 ° C. final extension). After PCR, the reactants were separated by agarose gel electrophoresis and ligated to TA cloning vectors (Invitrogen, Sandiego, USA). The vector was then transformed into transformant cells and the plasmids were isolated from the transformed bacteria by alkaline lysis method [Sambrook, J., Fritsch, F.E. and Maniatis, T (1989) Molecular cloning, Cold spring harbor laboratory press, Cold spring harbor. Human SOD TA vector containing cDNAXho IandBamH IAnd then inserted into pET-15b and pET-15b-Tat expression vectors (FIG. 1A). Expression of the vector is under the control of the T7 promoter and lacO-operator.

E. coli BL21 (DE3) transformed with pET-SOD and pET-Tat-SOD was selected, followed by inoculation of colonies in 100ml LB medium and addition of IPTG (0.5 mM) into the medium to recombine SOD and Tat. Overexpression of -SOD was induced. A schematic diagram of the expressed fusion protein Tat-SOD and the SOD used as the control protein is shown in FIG. 1B.

E. coli cells, which induced overexpression of the fusion protein with IPTG, were disrupted by sonication at 4 ° C, and then centrifuged to separate proteins from the supernatant by 15% SDS-polyacrylamide gel electrophoresis. 2A shows protein bands stained with Coomassie brilliant blue. The arrowed portions of lanes 2 (Tat-Cu, Zn-SOD) and lanes 3 (Cu, Zn-SOD) of the gels represent overexpressed fusion proteins and are very high compared to the control lane 1 (pET-15b vector). It is well shown that the concentration of the fusion protein is overexpressed.

Overexpressed SOD and Tat-SOD were confirmed by SDS-polyacrylamide gel electrophoresis (FIG. 2A) and Western blot analysis.

Example 2 Purification of Recombinant Tat-SOD Fusion Proteins

Transformed E. coli BL21 was put in LB medium containing ampicillin and incubated at 37 ° C. at 200 rpm. When the bacterial concentration in the culture medium showed OD 600 = 0.5 to 1.0, IPTG was added to the medium to bring the final concentration to 0.5 mM, followed by further incubation for 3 hours. The cultured cells were collected by centrifugation and sonicated with 5 ml binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). In this study, fusion proteins were purified in both denatured and native conditions to compare their permeability with each other. To obtain a partially denatured fusion protein, the cells were sonicated in binding buffer containing 6M urea and immediately purified.

The supernatant was immediately loaded by centrifugation into a 2.5 ml Ni ²⁺ -nitrilotriacetic acid sepharose column and 10-fold volume of binding buffer and 6-volume washing buffer (60 mM imidazole). , 0.5M NaCl, 20mM Tris-HCl, pH 7.9), and then the fusion protein was eluted with elution buffer (1M imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9). Subsequently, the fractions containing the fusion protein were collected, and Sephadex G-15 column chromatography was performed to remove the salts contained in the fractions. Since the fusion protein contains six histidines at the N-terminus, the fusion protein was purified almost purely (purity> 90%) in a single step immobilized metal-chelate affinity chromatography (FIG. 2B). ).

Table 2 shows the amount and SOD activity of the fusion protein purified from 100 ml of E. coli culture. As shown in the table, the fusion protein purified in the denatured state was more than two times higher than that in the natural state. This may be due to the prevention of aggregation of the fusion protein due to overexpression in the denatured state. As expected, however, degeneration of the fusion protein lost most of the SOD activity. Sat activity of the purified Tat-SOD fusion protein in its natural state was not reduced by 50% compared to the expression of only SOD.

Protein concentrations of the fractions were determined by Bradford method using bovine serum albumin as standard [Bradford, M.A. (1976) Anal. Biochem. 72, 248-254.

Purified fusion protein was once again confirmed by Western blot analysis. As shown in FIG. 2C, Tat-SOD and SOD bands responding to SOD monoclonal antibodies were observed at the same positions as the protein bands of FIG. 2B, respectively.

Protein (mg) SOD specific activity (U / mg protein) SOD (D) 5.2 8.7 Tat-SOD (D) 2.3 5.3 SOD (N) 2.5 1540 Tat-SOD (N) 0.9 882

* () In () is denatured state, N is natural state.

Example 3 HeLa Cell Culture and Intracellular Permeation of Recombinant Tat-SOD

HeLa cells provide 95% air and 5% CO ₂ at 37 ° C with 20 mM HEPES / NaOH (pH 7.4), 5 mM NaHCO ₃ , 10% fetal bovine serum ("FBS") and antibiotics (100 μg / cultured in Dulbecco's Modified Eagle's Medium containing ml streptomycin, 100 U / ml penicillin).

To observe intracellular penetration of Tat-SOD, HeLa cells were incubated for 4-6 hours in 6-well plates, replaced with 1 ml fresh culture medium without FBS, and treated with different concentrations of recombinant Tat-SOD in the culture. It was. After 1 hour the cells were treated with trypsin-EDTA (Gibco BRL) and washed thoroughly with phosphate buffered saline (PBS). After crushing the cells, the amount and activity of the SOD permeated into the cells was measured by SOD activity analysis and Western blot analysis.

First, the degree of permeation of purified Tat-SOD into HeLa cells in the denatured and natural state was measured. 1 μ to Tat-SOD of the M time was treated in a cell culture medium for 1 hour Tat-SOD in the modified state, but can determine by Western blot that the good transmission into cells SOD used in the natural Tat-SOD or control protein is any membrane It did not penetrate (Fig. 2D).

Then, it was observed how the HeLa cell permeation of the denatured Tat-SOD fusion protein changes with time and concentration. The time and concentration dependence of HeLa cell permeation of denatured Tat-SOD is shown in Figures 3A and B. Results of 1 when the administration of Tat-SOD and SOD in μ M in the medium for 5-1 minutes (A), 0.25-2 when administered Tat-SOD and SOD in μ M to the culture medium for 1 hour (B) to be. The amount of protein permeated into cells was analyzed by Western blot.

As shown in FIG. 3, the denatured Tat-SOD migrated into HeLa cells in a time and concentration dependent manner. However, the control protein SOD could not observe the fusion protein permeated into cells even after 1 hour treatment. When the degree of intracellular permeation of Tat-SOD was observed by time, the fusion protein permeated after 5 minutes of administration was confirmed, and the intracellular permeation increased in proportion to the treatment time (FIG. 3A). In addition, it was confirmed that the amount of Tat-SOD permeated into the cell increased in proportion to the concentration of the fusion protein administered (FIG. 3B).

Fusion proteins penetrated into cells must maintain their inherent activity before they can be applied to protein therapy. Therefore, the degree of biological activity of the fusion protein penetrated into the cell is a very important problem. Tat-SOD expressed in this experiment was used as a denatured fusion protein without activity, but it was observed that SOD activity was restored in the cytoplasm after HeLa cell permeation.

4 is a change in SOD enzyme activity in cells when denatured Tat-SOD is administered to HeLa cells. Results of 1 when the administration of Tat-SOD and SOD in μ M in the medium for 5-1 minutes (A), 0.25-2 when administered Tat-SOD and SOD in μ M to the culture medium for 1 hour (B) to be. Experimental results are expressed as the mean ± standard error of five experiments. Asterisk and cross are statistically significant at p <0.05 and p <0.01, respectively. Statistical analysis was performed by students t- test. As shown in FIG. 4, SOD activity in HeLa cells treated with denatured Tat-SOD was increased in time and concentration. SOD activity in cells without administration of the fusion protein was 2.46 ± 0.39 U / mg protein SOD activity when was administered to 2 μ M Tat-SOD was significantly increased by 8.30 ± 1.49 U / mg protein (p <0.01) .

These results suggest that the structure of the denatured Tat-SOD is restored to its original state in HeLa cells.

The denatured Tat-SOD fusion protein was confirmed by Western blot to be delivered to cells in time and concentration dependent manner in cultured HeLa cells. On the other hand, Tat-SOD purified in nature and SOD used as a control protein were not transported into cells.

The activity of SOD enzyme in cells treated with Tat-SOD fusion protein increased in proportion to the amount of protein carried into the cells. These results indicate that HIV-1 Tat protein can artificially increase the activity of SOD in cells by moving SOD into cells. Thus, the delivery of these Tat-SOD fusion proteins into cells offers the potential for a variety of applications in the research and treatment of over 100 diseases known to be related to SOD.

<Example 4> Animal Skin Cell Penetration of Tat-SOD

As an experimental animal Male SD rats of about 200 g body weight were used. Experimental animals were used with 10 control groups (SOD application) and 15 experimental groups (Tat-SOD application).

Each animal was fasted for 24 hours, followed by induction of general anesthesia with 3% isoflurane in a gas mixture of nitrogen and oxygen at 7: 3, followed by anesthesia with 2.5% isoflurane in the same gas. 0.3 mg of SOD and Tat-SOD were applied to the abdominal skin, respectively.

The normal group and each experimental group were intraperitoneally injected with ketamine (30 mg / kg) after 2 hours of application, followed by general anesthesia, then slaughtered to cut the skin of the applied site, and then prepared a 10 μm frozen tissue section in a conventional manner. It was.

The prepared tissue sections were fixed with 4% paraformaldehyde for 10 minutes and washed with 0.1M phosphate buffered saline (PBS, pH 7.4) three times for 10 minutes to remove the fixed solution in the tissue. Then, in order to prevent nonspecific immunoreactivity, the reaction was diluted with 0.1 M PBS (pH 7.4) in a solution of 0.3% Triton X-100 and 10% normal goat serum at room temperature for 1 hour. Thereafter, rabbit anti-histidine IgG (primary antibody) was diluted 1: 500 and reacted at room temperature for 24 hours. After washing three times with 0.1M PBS for 10 minutes to remove the primary antibody in the tissue section and 0.1M of the secondary antibody biotinylated goat anti-rabbit IgG (Vector Laboratories, USA) Diluted 1: 200 with PBS and reacted at room temperature for 1 hour. After completion of the reaction, the tissues were washed three times with 0.1 M PBS for 10 minutes, and then diluted with peroxidase-conjugated streptavidin (Vector Laboratories, USA) at 1: 300 (in 0.1 M PBS). Reaction was carried out for 1 hour.

The tissue sections of the immune response were developed with substrate solution (40 mg diaminobenzidine / 0.045% H2O2 in 100 ml PBS) for 3 minutes. In order to avoid excessive color development, the degree of color development was continuously checked under an optical microscope. The colored tissue sections were attached to the slides, washed several times with distilled water and 0.1 M PB, and dehydrated in a conventional manner to prepare permanent specimens.

As a result, almost no histidine immune response was observed in the SOD group (FIG. 6A and FIG. 6), but in the Tat-SOD group, a strong histidine immune response was observed throughout the epidermal layer, the dermal layer and the entire subcutaneous tissue layer (FIG. 6, B and D). The histidine immune response observed in the Tat-SOD-applied group was mainly observed in the nucleus, and the cells in which the immune response was observed were evenly observed in epidermal cells, fibroblasts and hair follicles and the surrounding cells.

Example 5 Determination of Tat-SOD Enzyme Activity Through Animal Skin Cells Over Time

As an experimental animal Male SD rats of about 200 g body weight were used. Experimental animals were used for the control group (SOD application) and the experimental group (Tat-SOD application), respectively.

Each animal was fasted for 24 hours, followed by induction of general anesthesia with 3% isoflurane in a gas mixture of nitrogen and oxygen at 7: 3, followed by anesthesia with 2.5% isoflurane in the same gas. 0.3 mg of SOD and Tat-SOD were applied to the abdominal skin, respectively.

The normal group and each experimental group were intraperitoneally injected with ketamine (ketamine) (30 mg / kg) 2 hours after application, and then slaughtered to cut the skin of the applied site.

After that, the cut skin tissue was thoroughly washed with physiological saline, and 1.5 ml of PBS was added to cut the tissue with scissors. After homogeneous grinding of the tissue with a polygraph, the supernatant was separated by centrifugation (17,000 rpm, 10 min), and the activity of SOD was measured by the method described above.

The results are shown in the graph of FIG. As time passed, the activity of Tat-SOD in HeLa cells increased significantly, indicating that it was infiltrated into cells in a time-dependent manner.

The present invention is the first experimental report to permeate human Cu, Zn-superoxide dismutase (Cu, Zn-SOD) directly into cells at the protein level.

In particular, Tat-SOD administered intracellularly suggests that Tat-SOD can be efficiently used for protein treatment by restoring SOD activity.

Free radicals damage cellular biopolymers. Reportedly, it is closely related to the progress of more than 100 diseases. Therefore, the present invention can effectively utilize Tat-SOD in the treatment of protein by treating SOD, which plays a major role in removing such reactive oxygen species, into cells.

In addition, as a result of using the Tat-SOD of the present invention to remove the active oxygen species causing skin aging, wrinkles, etc., it was confirmed that the skin penetrates smoothly to the skin epidermis, dermis and subcutaneous fat layer. Tat-SOD fusion protein can be utilized as a cosmetic.

Based on this technology, SOD, a type of antioxidant enzyme, can be delivered directly into cells to remove free radicals that are harmful to our human body. Therefore, this technology can be widely used in cosmetics and health food industries as well as various diseases.

Claims (14)

Cell penetrating Tat-superoxide dismutase fusion protein covalently bonded to the HIV-1 Tat transduction site 49-57 residue (Tat 49-57) at the amino terminus of Cu, Zn-superoxide dismutase or a derivative thereof . The Tat-superoxide of claim 1 is bound to the 5 'side of the Cu, Zn-superoxide dismutase or derivative thereof cDNA by binding the DNA sequence of 49-57 residue (Tat 49-57) coding for HIV-1 Tat transduction site. SEQ ID NO: 6 or an equivalent recombinant polynucleotide encoding a dismutase fusion protein. A vector expressing a cell penetrating Tat-superoxide dismutase fusion protein comprising the recombinant polynucleotide of claim 2 to express the fusion protein of claim 1. The vector expressing the cell penetrating Tat-superoxide dismutase fusion protein according to claim 3, wherein the expression vector is configured as the restriction map of FIG. 1A. Expressing the vector of claim 3 or 4 in a microorganism; Purifying the expressed Tat-superoxide dismutase fusion protein; Adding the purified Tat-superoxide dismutase fusion protein to the cell; the method comprising introducing a Tat-superoxide dismutase fusion protein into the cell. The method of claim 5, wherein the purification step introduces the Tat-superoxide dismutase fusion protein into the cell by denatured using 6M urea. How to let. A cosmetic composition comprising the Tat-superoxide dismutase fusion protein of claim 1 as an active ingredient and a function of removing active oxygen species. According to claim 7, wherein the cosmetic composition is an active ingredient Tat-superoxide dismutase fusion protein of the lotion, gel, water-soluble liquid, oil-in-water (O / W) type and water-in-oil (W / O) type as an active ingredient Cosmetic composition characterized by having a function of removing oxygen species. A pharmaceutical composition comprising the Tat-superoxide dismutase fusion protein of claim 1 as an active ingredient and a pharmaceutically acceptable carrier. 10. The method according to claim 9, which comprises: glomerulonephritis, vasculitis, autoimmune diseases, stroke, myocardial infarction, arrhythmia, angina, idiopathic hemochromatosis, disease by radiation therapy, premature ejaculation, disease related aging, sickle cell disease, malaria, emphysema, Tat-superoxide dismutase fusion protein, which is used as a therapeutic agent for SOD-dependent diseases such as muscle myalgia, autoimmune nephrotic syndrome, Betelnet-related oral cancer, hyperbaric oxygen, Alzheimer's disease, Parkinson's disease, cataract, etc. A pharmaceutical composition, characterized in that it comprises a pharmaceutically acceptable carrier. A method for preventing or treating a SOD dependent disease using the fusion protein of claim 1 or the recombinant polynucleotide of claim 2. A method for preventing or treating SOD dependent disease using a microorganism transformed with the vector of claim 3 or 4. A method of preventing or treating SOD dependent disease using the method of claim 5 or 6. The disease according to any one of claims 11 to 13, wherein the SOD dependent disease is glomerulonephritis, vasculitis, autoimmune disease, stroke, myocardial infarction, arrhythmia, angina pectoris, idiopathic hemochromatosis, disease by radiation therapy, prematurity Fusion protein of claim 1, wherein the disease-related aging, sickle cell disease, malaria, emphysema, myositis, autoimmune nephrotic syndrome, Betelnet-related oral cancer, hyperbaric oxygen, Alzheimer's disease, Parkinson's disease, cataract A method of preventing or treating SOD dependent disease using the recombinant polynucleotide of claim 2.