KR101567329B1 - Pharmaceutical composition for autosomal dominant polycystic kidney disease containing FK506 binding protein fusion protein - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of autosomal dominant polycystic kidney, and more particularly, to a pharmaceutical composition for preventing or treating monocanthus, comprising a fusion protein of FK506 binding protein which is permeable to polycystic kidney cells, ≪ / RTI >

Description

[0001] The present invention relates to a pharmaceutical composition for preventing and treating polycystic kidney disease containing FK506 binding protein fusion protein,

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing and treating polycystic kidney disease, and more particularly, to a pharmaceutical composition for prevention and treatment of polycysticosis comprising, as an active ingredient, a fusion protein of FK506 binding protein capable of tissue and intracellular permeability .

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease with an incidence rate of at least 1 per 500 to 1,000 people. Autosomal dominant danangsin is characterized by the formation of a large number of liquid cysts in both kidneys and a 4 to 8 fold increase in kidney compared to healthy individuals. Until now, there has been no effective clinical treatment for polycystic kidney disease, and most of the polycystic kidneys progress to end stage renal disease (Gabow, 1993; Grantham, 1996). Cyst formation in human polycystic kidney is accompanied by increased cell proliferation and magnetic cell death of cystic epidermal cells.

Although many anticancer therapies have been developed so far, disappointing clinical results have been reported due to negative feedback inhibition.

WT 9-7 cells are basically polycontogenic cells with activated mTOR signal. Therefore, WT 9-7 cells should die when using therapeutic agents. Rapamycin is the most widely known treatment for polycystic kidney disease. Rapamycin is known to be a therapeutic agent for polycystic kidney disease by replacing S6, which is a signal of mTOR, activated in WT 9-7 cells with p-S6, but in contrast, it exhibits side effects of activating the inflammatory factor Erk signal.

FK506 binding protein (FK506BP) is one of the immunophilin families, such as cyclophilin A, and is a rich cytosolic receptor protein with peptidyl-prolyl-isomerase activity receptor protein. FK506BP binds to the immunosuppressive drug FK506 and rapamycin in the cell membrane to form a complex that regulates the expression of cytokines involved in signaling for T cell activation. FK506BP is also reported to be involved in Ca ²⁺ signaling.

There is a growing interest in using in vivo polymers for therapeutic purposes. At present, gene therapy is the focus of attention. Gene therapy, however, is not easy to carry out, and it is difficult to artificially regulate the amount of protein expressed in target cells because of low gene expression, low expression in target cells, .

In order to transfer a therapeutic drug or protein into a cell, a method of directly delivering the target protein through the cell membrane may be considered. However, proteins are very difficult to pass through cell membranes because of their size and their biochemical nature. In general, substances with a molecular weight of 600 Daltons or more are known to be almost impossible to pass through cell membranes.

In recent years, it has been found that one of the methods of protein transport is to use a PEP-1 peptide to carry a naturally occurring heterologous protein into a cell. The PEP-1 peptide consists of 21 amino acids (KETWWETWWTEW SQP KKKRKV) and has three domains (hydrophobic domain, spacer, hydrophilic domain). Until now, studies using PEP-1 peptides have revealed that when the PEP-1 peptide and the external protein are simultaneously administered to the cells, the proteins can be transported into the cells in a natural state. In addition, the PEP peptide has several advantages as a protein therapeutic agent compared with Tat protein, that is, it penetrates the protein very efficiently into the cell, shows stability in physiological buffer solution, lack of sensitivity to serum, and the like. However, it has been confirmed that PEP peptides are effectively transported into cells only if they are administered at a constant ratio with external proteins, such as green fluorescent protein (GFP), β-galactosidase, and β-Gal. . However, it is not yet clear whether all proteins including therapeutic proteins can be delivered into cells by PEP-1.

The present invention aims to provide a more effective protein therapeutic agent by selecting proteins that inhibit mTOR, which is a target of polycyclic therapy, and confirming mechanism and protection effect against polycystic kidney using protein permeation technique.

The aim of the present invention is to find a therapeutic agent for polycyclic antineoplastic protein that does not activate the Erk signal while changing the signal S6 below the mTOR signal to p-S6. FK506BP is known to inhibit the following signals by directly inhibiting mTOR by complexing with rapamycin.

In order to achieve the above object, the present inventors have studied to develop a protein drug that is an immunosuppressive drug, which enhances penetration ability. The effect of penetration of protein preparation, the activity of mTOR and the activity of Erk The expression of FK506BP fusion protein was confirmed to be reduced, and the effect of the FK506BP fusion protein on the polycationic effect was confirmed, thereby completing the present invention.

The present inventors have found that the PEP-1 peptide that transports natural proteins into cells and the N- and / or C-peptide of one of the HIV-1 Tat peptides, as an external protein, FK506BP (hereinafter referred to as "FK506 binding protein" And the fusion protein was overexpressed in E. coli and purified easily and conveniently by metal chelating affinity chromatography. In addition, it has been confirmed through experiments that the purified fusion protein effectively penetrates into the polycystic kidney cell line and reduces the mTOR signal and the Erk signal. The present invention has raised the possibility that the FK506BP fusion protein can be used as a protein therapeutic agent for autosomal dominant polycystine.

According to one aspect of the present invention, there is provided a fusion protein (hereinafter referred to as " fusion protein ") of an FK506 binding protein in which a protein transport domain such as PEP-1 or Tat is covalently bonded to one or more of N-terminal and C- And "FK506BP fusion protein"). ≪ / RTI >

In the present invention, the permeability of the fusion protein of FK506 binding protein into the polycystic kidney cell line was confirmed by time and dose, and the stability of the FK506BP fusion protein after cell penetration was confirmed.

As a result, the FK506BP fusion protein according to the present invention effectively penetrated into polycystic kidney cells in a time-dependent and concentration-dependent manner, and was stable for at least 60 hours after infiltration. In addition, treatment with FK506BP fusion protein in polycystic kidney cell line decreased mTOR signal and Erk signal.

The present invention relates to a pharmaceutical composition for the prevention and treatment of autosomal dominant polycystine comprising a fusion protein in which a protein transport domain is covalently bonded to one or more of N-terminal and C-terminal of FK506 binding protein.

The present invention also relates to the use of

a) a hydrophilic domain consisting of 15 to 30 amino acids and containing at least 5 tryptophan, a hydrophilic domain containing 4 or more lysines, and a spacer for separating the two domains spacer), < / RTI >

b) a protein transport domain consisting of 6 to 12 amino acid residues and containing 3/4 or more arginine or lysine residues,

c) an oligo lysine protein transport domain consisting of 6-12 lysines,

d) an oligoarginine protein transport domain consisting of 6 to 12 arginines and

e) an oligo (lysine, arginine) protein transport domain consisting of 6 to 12 lysine or arginine, and derivatives of a) to e).

In addition, the present invention is characterized in that the fusion protein is one selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO:

In addition, the present invention is characterized in that the composition further comprises rapamycin in addition to the FK506BP fusion protein.

The pharmaceutical composition containing the PEP-1-FK506BP fusion protein as an active ingredient can be formulated into an injection form or a coating agent by a conventional method in combination with a carrier that is conventionally acceptable in the pharmaceutical field. The injectable compositions are preferably isotonic aqueous solutions or suspensions, and the compositions mentioned are sterile and / or contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agent accelerators, salts for controlling osmotic pressure, or buffers. They may also contain other therapeutically valuable substances.

The pharmaceutical preparation thus prepared may be administered parenterally, that is, subcutaneously, muscularly, or topically, depending on the purpose, and the dose is preferably in the range of 0.001 μg to 10 mg / kg per day, It can be divided into several doses. The dosage level for a particular patient may vary depending on the patient's body weight, age, sex, health condition, time of administration, method of administration, severity of the disease, and the like.

In addition, the coating agent containing the fusion protein of the present invention as an active ingredient can be easily produced in any form according to a conventional production method. For example, when preparing a cream type coating agent, the PEP-1-FK506BP fusion protein of the present invention is contained in a cream base of a general water type (O / W) or water in water (W / O) Antioxidants, preservatives and the like may be used as needed, while synthetic or natural materials such as proteins, minerals, and vitamins may be used in combination for the purpose of improving physical properties.

The intracellular delivery of the FK506BP protein molecule according to the present invention comprises 15-30 amino acids in FK506BP, and includes a hydrophobic domain containing 5 or more tryptophan, a hydrophilic domain containing 4 or more lysines terminus of the target protein, FK506BP, and / or the N-terminal of the target protein FK506BP, in which the cell-permeable transport domain including Pep-1 or the HIV Tat cell permeable domain in which the transport domain consisting of a spacer Terminus and a covalently linked form of the fusion protein with the C-terminus. Examples of the transport domain of the present invention include PEP-1 peptide consisting of 21 amino acids and having the amino acid sequence as shown in SEQ ID NO: 1 and HIV Tat 49-57 peptide having amino acid sequence as shown in SEQ ID NO: 2. However, the protein transport domain of the present invention is not limited to the PEP-1 peptide of SEQ ID NO: 1, the HIV Tat 49 to 57 residue peptide of SEQ ID NO: 2, and may be substituted or deleted with some substitution or deletion of the amino acid sequence of PEP- It is easy for a person skilled in the art to prepare a peptide having a function similar to that of PEP-1 peptide or HIV Tat peptide, so that it is composed of 15 to 30 amino acids and contains 5 or more tryptophan A protein transport domain composed of a hydrophilic domain, a hydrophilic domain containing four or more lysines, and a spacer separating the two domains, and a protein transport domain comprising the same or similar amino acid partial substitution A protein transport domain that carries a protein transport function, or 6 to 12 amino acid residues A transport domain comprising at least 3/4 of an arginine or lysine residue, an oligo lysine transport domain consisting of 6-12 lysines, an oligoarginine transport domain consisting of 6-12 arginines, or 6-12 lysine or arginine It will be appreciated that the oligo- (lysine, arginine) transport domains also fall within the scope of the invention.

In particular, the present invention relates to a pharmaceutical composition for the prevention or treatment of autosomal dominant polycystic acid comprising FK506BP fusion protein covalently linked to the protein transport domain as described above.

The definitions of the main terms used in the description of the present invention and the like are as follows.

"FK506BP fusion protein" means a covalent complex formed by genetic fusion or chemical bonding between a protein transport domain and FK506BP and a protein transport domain and a cargo molecule (i.e., FK506BP in the present invention). As used herein, the term "PEP-1-FK506BP" refers to FK506BP fusion protein in which the PEP-1 protein transport domain is bound to the N-terminus of FK506BP .

In addition, the term "genetic fusion" means a link consisting of a linear, covalent bond formed through genetic expression of a DNA sequence encoding a protein.

The term "target cell" refers to a cell to which a cargo molecule is delivered by a transport domain. That is, the target cell is a cell, that is, a living organism or a cell or organ It is meant to include microorganisms that are found. In addition, the target cell means an extracellular cell, that is, a cultured animal cell, a human cell or a microorganism. Specifically, the target cell in the present specification refers to a polycyclic kidney cell or the like.

As used herein, the term "protein transport domain" refers to a peptide or protein that is covalently bonded to a cargo molecule (target protein) peptide or protein and can introduce the peptide or protein into cells without requiring additional receptor, carrier, energy, For example, PEP-1 peptide (SEQ ID NO: 1).

As used herein, the term "target protein" refers to a molecule that is covalently bound to a PEP-1 protein transduction domain and introduced into cells to exhibit activity. It is synonymous with "cargo molecule".

Also, in the present specification, the term "introduction", "transport", "penetration", "transport", "transfer", "permeation", "pass" Respectively.

In the present invention, the protein transport domain is a protein transport domain composed of 15 to 30 amino acids and composed of a non-hydrophilic domain containing 5 or more tryptophan, a hydrophilic domain containing many lysines, and a spacer separating the two domains, A transport domain comprising 6 to 12 amino acid residues and comprising ¾ or more arginine or a lysine residue, an oligolysine transport domain consisting of 6 to 12 lysines, an oligoroginalin transport domain consisting of 6 to 12 arginines, Or an oligo (lysine, arginine) transport domain consisting of 12 lysine or arginine. In addition, the target protein (cargo molecule) is FK506BP. The protein transport domain and the target protein may be replaced by other amino acid (s) of similar polarity in which one or more amino acids functionally equivalently function in the sequence according to the silent change. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, the hydrophobic amino acid class includes alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positive basic amino acids include arginine, lysine and histidine. Acidic amino acids with negative charge include aspartic acid and glutamic acid. In addition, fragments or derivatives thereof having the same or similar biological activity within a certain range of homology, such as 85-100%, between the fusion protein of the present invention and the amino acid sequence are included in the scope of the present invention.

In addition, the present invention is characterized in that the cell-inducing FK506BP fusion protein has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO:

Also, the present invention is characterized in that the protein transducing domain ("PTD") is covalently bonded to one or both of the carboxy terminal and the amino terminal of the FK506BP protein.

The present invention also relates to a pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney characterized by containing the cell-transducing FK506BP fusion protein as an active ingredient.

The present invention also relates to a composition comprising the cell-inducing FK506BP fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The FK506BP fusion protein according to the present invention infiltrated into polycystic kidney cells in a time-dependent and dose-dependent manner.

In addition, the FK506BP fusion protein according to the present invention penetrated into cells and stably maintained for at least 60 hours.

In addition, the FK506BP fusion protein according to the present invention decreased mTOR signal and Erk signal in polycystic kidney cells.

Therefore, the FK506BP fusion protein according to the present invention is expected to be useful as a pharmaceutical composition for the prevention or treatment of autosomal dominant polycystine.

FIG. 1 is a photograph of WT 9-7 cells treated with 5 μM FK506BP fusion protein at different times for 0-120 minutes followed by Western blotting. As a control, FK506BP in which the protein transport domain was not bound was used. "C" refers to a group not treated with FK506BP fusion protein or FK506BP protein.
FIG. 2 is a photograph of WT 9-7 cells subjected to treatment with 0.5-5 .mu.M for 2 hours at different concentrations to treat FK506BP fusion protein and Western blotting. FIG. As a control, FK506BP in which the protein transport domain was not bound was used. "C" refers to a group not treated with FK506BP fusion protein or FK506BP protein.
FIG. 3 is a photograph of WT 9-7 cells treated with 5 μM of FK506BP fusion protein for two hours, followed by Western blotting over time. The fusion proteins remained stable until 60 hours.
FIG. 4 is a photograph of WT 9-7 cells treated with 5 μM FK506BP fusion protein for two hours and then photographed using a confocal microscope. "Control" is a no-treatment group.
FIG. 5 shows the results of MTT analysis of cell viability by adding FK506BP fusion protein, rapamycin and the like to WT 9-7 cells.
FIG. 6 shows the effect of inhibiting mTOR signal by adding FK506BP fusion protein, rapamycin, etc. to WT 9-7 cells.
FIG. 7 shows the effect of suppressing Erk signal by adding FK506BP fusion protein, rapamycin, etc. to WT 9-7 cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are intended to further illustrate the present invention and that the scope of the present invention is not limited by the scope of these embodiments. Particularly, as a result of each experimental example, data on the PEP-1-FK506BP fusion protein among the various fusion proteins prepared in the specific examples have been described, but other fusion proteins have also been used as the samples for PEP-1-FK506BP fusion protein The results are similar to those of the one result.

<Material>

Restriction enzymes and T4 DNA ligase were purchased from Promega (USA), and Pfu polymerase was purchased from stratagene (USA). Tat oligonucleotides were synthesized with 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-triacetic acid Sepharose Superflow was purchased from Qiagen (Germany). Human FK506 binding protein (FK506BP) cDNA was isolated from human liver cDNA library by PCR method. All of the other reagents were made by high-grade products.

&Lt; Example 1: Preparation of expression vector of PEP-1-FK506BP fusion protein and transformation >

In order to analyze the ability of PEP-1 peptide to transfer the protein into cells, a fusion protein expression vector was constructed which can express the protein FK506BP Respectively.

First, PEP-1-FK506BP A pET-PEP expression vector containing a PEP-1 peptide (KETWWE TWWTEW SQP KKKRKV) (SEQ ID NO: 1) was prepared to produce a fusion protein. Two kinds of oligonucleotides corresponding to PEP-1 peptide nucleotide (top chain, 5'-TATGAAAGAAACCTGGTGGGAAACCTGGTGGACCGAATGGTCTCAGCCGAAAAAAAAACGTAAAGTGC-3 '(SEQ ID NO: 15); lower chain, 5'-TCGAGCACTTTACGTTTTTTTTTCGGCTGACACCATTCGGTCCACCAGGTTTCCCACCAGGTTTCTTTCC-3' (SEQ ID NO: 16)) the NdeⅠ - XhoⅠ limit And inserted into pET-15b cut with an enzyme. Two kinds of oligonucleotides were synthesized based on the sequence of human FK506BP cDNA. Forward primer has a 5'-CTCGAGATGGGAGTGC AGGTGGAAACCATC-3 '(SEQ ID NO: 17), and to have a restriction site XhoⅠ reverse primer 5'-GGATCCTCATTCCAGTTTTAGAAGCTCCAC-3' BamHⅠ restriction site (SEQ ID NO: 18).

Polymerase chain reaction (PCR) was performed in a warm circulation reactor (Perkin-Elmer, model 9600). The reaction mixture was placed in a 50 占 퐇 silicone tube and heated at 94 占 폚 for 5 minutes. PCR reaction was performed. After the PCR, the reaction product was separated by agarose gel electrophoresis, and the reaction product was ligated to a TA cloning vector (Invitrogen, Sandiego, USA), transformed into competent cells that were easy to clone Plasmids were isolated from the transformed bacteria by an alkaline lysis method. Cutting the TA vector containing the human cDNA in FK506BP XhoⅠ BamHⅠ and then was inserted into the PEP expression vector. E. coli BL21 (DE3) transformed with PEP-1-FK506BP was selected and colonies were inoculated into 100 ml LB medium and IPTG (0.5 mM) was added to the medium to induce overexpression of the recombinant PEP-1-FK506BP fusion protein Lt; / RTI &gt; Overexpressed PEP-1-FK506BP fusion protein was confirmed by SDS-PAGE and Western blot analysis.

The above method was modified to prepare FK506BP-PEP-1 fusion protein and PEP-1-FK506BP-PEP-1 fusion protein.

&Lt; Example 2: Expression and purification of PEP-1-FK506BP fusion protein &

E. coli BL21 (DE3) cells containing the human FK506BP cDNA in the form of PEP-1-FK506BP prepared as in Example 1 were placed in LB medium containing ampicillin and cultured at 37 ° C with stirring 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 a final concentration of 0.5 and 1 mM, followed by further incubation at 30 ° C for 12 hours. The cultured cells were collected by centrifugation, and then 5 ml of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) was added and sonication was carried out using an ultrasonic grinder. The supernatant was immediately centrifuged Ni ²⁺ - acrylonitrile triazol setik acid Sepharose Super flow ^{(Ni 2+ -nitrilotriacetic acid sepharose super flow} ) load the column and binding buffer of 10 times by volume with 6-fold volume wash buffer (60mM of Imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and then eluted the fusion protein with elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Then, the fractions containing the fusion protein were collected and subjected to PD-10 column chromatography to remove the salt contained in the fraction.

The purified protein concentration was measured by the Bradford method using bovine serum albumin as a standard.

The FK506BP-PEP-1 fusion protein and the PEP-1-FK506BP-PEP-1 fusion protein were overexpressed and purified in the same manner as described above.

&Lt; Example 3: Preparation of Tat-FK506BP fusion protein expression vector and transformation >

To overexpress the Tat-FK506BP fusion protein, a pET-Tat-FK506BP expression vector was prepared in which FK506BP, HIV-1 Tat transduction site (Tat49-57) and cDNA for 6 histidine were consecutively contained. First, a pET-Tat expression vector containing the basic domain of HIV-1 Tat (i.e., amino acids 49-57) was prepared. (Top strand, 5'-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3 '(SEQ ID NO: 19); bottom strand, 5'-TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC-3' (SEQ ID NO: ) Was ligated to pET15b cut with NdeI-XhoI restriction enzyme and inserted. Two kinds of oligonucleotides were synthesized based on the sequence of human FK506BP cDNA. The forward primer has an Xho I restriction site in SEQ ID NO: 17 in Example 1 and the reverse primer has a BamH I restriction site in SEQ ID NO: 18 in Example 1 above.

Polymerase chain reaction (PCR) was performed in a thermal cycler (Perkin-Elmer, model 9600) and the reaction mixture was placed in a 50 μl siliconized reaction tube and heated at 94 ° C for 5 minutes. The PCR reaction was carried out at 94 ° C for 30 seconds, 30 minutes extension, 54 ° C for 1 minute, 70 ° C for 3 minutes, annealing at 72 ° C for 10 minutes, final extension). After the PCR, the reaction product was separated by agarose gel electrophoresis and ligated to TA cloning vector (Invitrogen, Sandiego, USA). This vector was then transformed into competent cells and the plasmid was isolated from the transformed bacteria by an alkaline lysis method (Sambrook, J., Fritsch, F.E. and Maniatis, T (1989) Molecular cloning, Cold spring harbor laboratory press, Cold spring harbor]. The TA vector containing human FK506BP cDNA was digested with Xho I and BamH I and inserted into the pET-15b and pET-15b-Tat expression vectors. Expression of the vector is under the control of the T7 promoter and lacO-operator.

E. coli BL21 (DE3) transformed with pET-Tat-FK506BP was selected and colony was inoculated into 100 ml of LB medium and IPTG (0.5 mM) was added to the medium to induce overexpression. Escherichia coli cells inducing overexpression of fusion proteins with IPTG were disrupted by sonication at 4 ° C and then centrifuged to separate proteins of the supernatant by 15% SDS-polyacrylamide gel electrophoresis. Overexpressed SOD and Tat-SOD were confirmed by SDS-polyacrylamide gel electrophoresis and Western blot analysis.

The above method was modified to prepare FK506BP-Tat fusion protein and Tat-FK506BP-Tat fusion protein.

&Lt; Example 4: Overexpression and purification of Tat-FK506BP fusion protein >

The transformed E. coli BL21 was added to LB medium containing ampicillin and cultured at 37 DEG C with stirring at 200 rpm. When the bacterial concentration in the culture medium exhibited OD 600 = 0.5 to 1.0, IPTG was added to the culture medium to a final concentration of 0.5 mM, followed by further incubation for 3 hours. The cultured cells were harvested by centrifugation and sonicated with 5 ml binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9).

The supernatant was immediately centrifuged and loaded onto a 2.5 ml Ni 2+ -nitrile nitrilotriacetic acid sepharose column and washed with a 10-fold volume of binding buffer and 6-fold volume of wash buffer (60 mM imidazole , 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and eluted the fusion protein with elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Fractions containing the fusion protein were then collected and subjected to Sephadex G-15 column chromatography to remove the salt contained in the fraction. Because the fusion protein contains six histidines at the N-terminus, the fusion protein was purified to near pure (purity> 90%) in a single step of immobilized metal-chelate affinity chromatography. The protein concentration of the fraction was determined by the Bradford method using bovine serum albumin as a standard (Bradford, M.A. (1976) Anal. Biochem. 72, 248-254].

The FK506BP-Tat fusion protein and the Tat-FK506BP-Tat fusion protein were overexpressed and purified in the same manner as described above.

&Lt; Example 5: Preparation of sample >

The various FK506BP fusion proteins purified in Examples 2 and 4 were dissolved in physiological saline at 5 占 / / 5 占 퐇 and used as a sample in the following experiment.

Experimental Example 1: Intracellular penetration efficiency of FK506BP fusion protein according to time and dose

1-1. Cell line and drug treatment

Autosomal dominant polycystic tubule cells WT9-7 were cultured in DMEM / F12 (Welgene, Korea) medium containing 10% (v / v) fetal calf serum and penicillin-streptomycin. The cells were cultured at 37 ° C in a humidified condition of 5% CO ₂ and 95% air. These cells were treated with 2 ~ 4 μM 5-aza-dC (5-aza-2'-deoxycytidine, Sigma-Aldrich, St Louis, MO, USA) or 100 μM zebularine (Calbiochem, San Diego, ) For 14 days. The medium was changed every day or two and added a new 5-aza-dC or zebularin.

1-2. Cell penetration efficiency by treatment time

After 5 × 10 ⁵ cells were seeded in a 60 mm culture dish, 5 μM FK506BP fusion protein was treated with WT9-7 cells for 10, 30, 60, and 120 minutes, respectively. Then, the cells were washed twice with phosphate-buffered saline, For 1 minute, the cells were removed from the culture dish and centrifuged (1300 rpm, 5 minutes) to obtain cells. The cells thus obtained were dissolved in a Ripa buffer solution, which was a cell lysis solution, and then the intracellular penetration efficiency was confirmed by Western blotting using an antihistidin antibody.

As a result, as shown in Fig. 1, it was confirmed that the FK506BP fusion protein infiltrated into WT9-7 cells in a time-dependent manner. In contrast, the FK506BP protein without the transport domain bound did not penetrate into the cells.

1-3. Cell Penetration Efficiency by Treatment Concentration

After 5 × 10 ⁵ cells were seeded in a 60 mm culture dish, FK506BP fusion protein was treated with WT9-7 cells at 0.5, 1, 3 and 5 μM for two hours, followed by 2 washes with phosphate buffered saline The cells were treated with trypsin for 1 minute, and the cells were removed from the culture dish and centrifuged (1300 rpm, 5 minutes) to obtain cells. The cells thus obtained were dissolved in a Ripa buffer solution, which was a cell lysis solution, and then the intracellular penetration efficiency was confirmed by Western blotting using an antihistidin antibody.

As a result, as shown in Fig. 2, it was confirmed that the FK506BP fusion protein infiltrated into WT9-7 cells in a concentration (dose) -dependent manner. In contrast, the FK506BP protein without the transport domain bound did not penetrate into the cells.

Experimental Example 2: Stability of FK506BP fusion protein infiltrated into cells

1 x 10 ⁴ WT9-7 cells in cover glasses were treated with 5 [mu] M FK506BP fusion protein for 2 hours. After washing twice with phosphate-buffered saline, the cells were fixed with paraformaldehyde. The cells were treated with a solution containing 3% bovine serum albumin and 0.1% Tween 20 in phosphate buffered saline to make the cells permeable and then treated with the primary antibody, antihistamine, for 3 hours. After treatment with Alexa 488, a secondary fluorescent antibody, DAPI (4 ', 6-diamidino-2-phenylindole) was treated with blue fluorescence to specifically stain the nuclei of the cells. This was observed with a confocal microscope. The results are shown in Fig. As a result, it was confirmed that the FK506BP fusion protein was successfully infiltrated into WT9-7 cells. On the other hand, FK506BP protein did not penetrate into cells.

The infected FK506BP fusion protein was washed twice with phosphate buffered saline for 1, 3, 6, 12, 24, 36, 48, 60 hours after each time, and treated with trypsin for 1 minute to remove the cells from the culture dish And then centrifuged (1300 rpm, 5 minutes) to obtain cells. The cells thus obtained were dissolved in a Ripa buffer solution, which was a cell lysis solution, and the stability was confirmed by Western blotting using an antihistidin antibody. As a result, as shown in Fig. 3, the fusion protein was stably maintained for 60 hours or more.

Experimental Example 3: MTT analysis of FK506BP fusion protein in WT 9-7 cells

WT 9-7 cells with mTOR signal activated were treated with rapamycin, which is known to be a therapeutic agent for polycystic kidney disease. 1 x 10 ⁴ cells were cultured in a 96 well culture dish. After 12 hours, the medium without bovine serum was treated with 5uM FK506BP fusion protein, 100nM rapamycin, respectively for 3 hours. Then, the medium was changed to a medium without bovine serum and cultured for 12 hours. The mitochondria of living cells were stained with MTT media staining for 2 hours at 570 nm. Cells were lysed with isopropanol and the absorbance at 570 nm was measured. As a result, as shown in FIG. 5, the cell death was confirmed even when the FK506BP fusion protein was treated, and it was confirmed that when the FK506BP fusion protein and the rapamycin were simultaneously treated, apoptosis was remarkably active (1: control, 2 : FK506BP, 3: FK506BP + rapamycin, 4: PEP-1-FK506BP fusion protein, 5: rapamycin, 6: PEP-1-FK506BP fusion protein + rapamycin). In the lane 3, it seems that FK506BP and rapamycin are not able to function as an initial complex, and lane 6 shows that when PEP-1-FK506BP fusion protein and rapamycin are treated together, And the effect was excellent.

Experimental Example 4: Study of ADPKD signal of PEP-FK506BP fusion protein in WT 9-7 cells

WT 9-7 cells are cells with activated mTOR signal and expression. The FK506BP fusion protein or rapamycin did not alter the amount of mTOR expression and confirmed its function on the mTOR signal from the immediately following S6 signal. 5 × 10 ⁵ cells were placed in a 60 mm culture dish and treated with 5 μM FK506BP fusion protein and 100 nM rapamycin, respectively, for 2 hours. After washing twice with phosphate-buffered saline, the cells were treated with trypsin for 1 min. The cells were removed from the culture dish and centrifuged (1300 rpm, 5 min) to obtain cells. The cells thus obtained were dissolved in a cell lysis solution, Ripa buffer, and their signals were confirmed by Western blotting using anti-mTOR, anti-S6, anti-p6 antibody. it can be confirmed that there is no function for the mTOR signal and no change is observed for the S6 signal (Fig. 6). However, in the case of p-S6, the difference in the amount of expression is obvious. As shown in the figure, the PEP-1-FK506BP fusion protein and rapamycin have almost the same effect. When the PEP-1-FK506BP fusion protein and rapamycin are treated together, the function is very close (1: 2: FK506BP, 3: FK506BP + rapamycin, 4: PEP-1-FK506BP fusion protein, 5: rapamycin, 6: PEP-1-FK506BP fusion protein + rapamycin).

Experimental Example 5: Study of p-Erk signal of PEP-FK506BP fusion protein in WT 9-7 cells

In the case of rapamycin, the function of inhibiting S6, which is the signal below mTOR, is very good, but it shows side effects as a therapeutic agent by increasing the inflammatory signal Erk signal. 5 × 10 ⁵ cells were placed in a 60 mm culture dish and treated with 5 μM FK506BP fusion protein and 100 nM rapamycin, respectively, for 2 hours. After washing twice with phosphate-buffered saline, the cells were treated with trypsin for 1 min. The cells were removed from the culture dish and centrifuged (1300 rpm, 5 min) to obtain cells. The cells thus obtained were dissolved in a cell lysis solution, Ripa buffer, and their signals were confirmed by Western blotting using the anti-Erk and anti-p-Erk antibodies. It was confirmed that the PEP-1-FK506BP fusion protein inhibits the p-Erk signal and also suppresses the p-Erk signal in the WT 9-7 cell. In the case of rapamycin, it was confirmed that p-Erk was activated. However, when the PEP-1-FK506BP fusion protein + rapamycin was treated together, it was confirmed that p-Erk was reduced (FIG. 7) (1: control, 2: FK506BP, 3: FK506BP + rapamycin, 1-FK506BP fusion protein, 5: rapamycin, 6: PEP-1-FK506BP fusion protein + rapamycin).

From the above experimental results, it can be seen from the above experimental results that FK506BP fusion protein is smoothly penetrated into cells and exhibits a stability of at least 60 hours when the FK506BP fusion protein is treated on polycystic kidney, and inhibits S6 signal and Erk signal, Is expected to be useful as a pharmaceutical composition for preventing or treating polycystic kidney disease.

<110> Industry-Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for arthritis containing fk506 binding          protein fusion protein <130> sychoi-fk506bp-arthritis <160> 20 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> protein transducing domain called PEP-1 <400> 1 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val              20 <210> 2 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 2 Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 <210> 3 <211> 404 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide encoding PEP-1-FK506BP fusion protein <400> 3 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatgggagtg caggtggaaa ccatctcccc aggagacggg cgcaccttcc 120 ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat ggaaagaaat 180 ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag caggaggtga 240 tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc aaactgacta 300 tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca ccacatgcca 360 ctctcgtctt cgatgtggag cttctaaaac tggaatgagg atcc 404 <210> 4 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-FK506BP fusion protein <400> 4 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Gly Val Gln Val Glu Thr Ile Ser              20 25 30 Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val          35 40 45 His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg      50 55 60 Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile  65 70 75 80 Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala                  85 90 95 Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro             100 105 110 Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu         115 120 125 Lys Leu Glu     130 <210> 5 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding FK506BP-PEP-1 fusion protein <400> 5 atgggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 60 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaatt tgattcctcc 120 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 180 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 240 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 300 gatgtggagc ttctaaaact ggaatgagga tcctaaaaga aacctggtgg gaaacctggt 360 ggaccgaatg gtctcagccg aaaaaaaaac gtaaagtgta g 401 <210> 6 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> FK506BP-PEP-1 fusion protein <400> 6 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe   1 5 10 15 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu              20 25 30 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys          35 40 45 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val      50 55 60 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp  65 70 75 80 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala                  85 90 95 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Lys Glu Thr Trp             100 105 110 Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys         115 120 125 Val Gly Ser     130 <210> 7 <211> 472 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide encoding PEP-1-FK506BP-PEP-1 fusion protein <400> 7 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatgggagtg caggtggaaa ccatctcccc aggagacggg cgcaccttcc 120 ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat ggaaagaaat 180 ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag caggaggtga 240 tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc aaactgacta 300 tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca ccacatgcca 360 ctctcgtctt cgatgtggag cttctaaaac tggaatgagg atcctaaaag aaacctggtg 420 ggaaacctgg tggaccgaat ggtctcagcc gaaaaaaaaa cgtaaagtgt ag 472 <210> 8 <211> 154 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-FK506BP-PEP-1 fusion protein <400> 8 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Gly Val Gln Val Glu Thr Ile Ser              20 25 30 Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val          35 40 45 His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg      50 55 60 Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile  65 70 75 80 Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala                  85 90 95 Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro             100 105 110 Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu         115 120 125 Lys Leu Glu Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu     130 135 140 Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 145 150 <210> 9 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding Tat-FK506BP fusion protein <400> 9 aggaagaagc ggagacagcg acgaagactc gagatgggag tgcaggtgga aaccatctcc 60 ccaggagacg ggcgcacctt ccccaagcgc ggccagacct gcgtggtgca ctacaccggg 120 atgcttgaag atggaaagaa atttgattcc tcccgggaca gaaacaagcc ctttaagttt 180 atgctaggca agcaggaggt gatccgaggc tgggaagaag gggttgccca gatgagtgtg 240 ggtcagagag ccaaactgac tatatctcca gattatgcct atggtgccac tgggcaccca 300 ggcatcatcc caccacatgc cactctcgtc ttcgatgtgg agcttctaaa actggaatga 360 ggatcc 366 <210> 10 <211> 119 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Tat-FK506BP fusion protein <400> 10 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Gly Val Gln Val   1 5 10 15 Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln              20 25 30 Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe          35 40 45 Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys      50 55 60 Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val  65 70 75 80 Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala                  85 90 95 Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp             100 105 110 Val Glu Leu Leu Lys Leu Glu         115 <210> 11 <211> 361 <212> DNA <213> Artificial Sequence <220> <223> nucleotide encoding FK506BP-Tat fusion protein <400> 11 atgggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 60 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaatt tgattcctcc 120 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 180 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 240 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 300 gatgtggagc ttctaaaact ggaaggatcc taggaagaag cggagacagc gacgaagata 360 g 361 <210> 12 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> FK506BP-Tat fusion protein <400> 12 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe   1 5 10 15 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu              20 25 30 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys          35 40 45 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val      50 55 60 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp  65 70 75 80 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala                  85 90 95 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Arg Lys Lys Arg             100 105 110 Arg Gln Arg Arg Arg         115 <210> 13 <211> 395 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide encoding Tat-FK506BP-Tat fusion protein <400> 13 taggaagaag cggagacagc gacgaagact cgagatggga gtgcaggtgg aaaccatctc 60 cccaggagac gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg 120 gatgcttgaa gatggaaaga aatttgattc ctcccgggac agaaacaagc cctttaagtt 180 tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt 240 gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc 300 aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaagg 360 atcctaggaa gaagcggaga cagcgacgaa gatag 395 <210> 14 <211> 128 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Tat-FK506BP-Tat fusion protein <400> 14 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Gly Val Gln Val   1 5 10 15 Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln              20 25 30 Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe          35 40 45 Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys      50 55 60 Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val  65 70 75 80 Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala                  85 90 95 Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp             100 105 110 Val Glu Leu Leu Lys Leu Glu Arg Lys Lys Arg Arg Gln Arg Arg Arg         115 120 125 <210> 15 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1 <400> 15 tatgaaagaa acctggtggg aaacctggtg gaccgaatgg tctcagccga aaaaaaaacg 60 taaagtgc 68 <210> 16 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Anticoding sequence for PEP-1 <400> 16 tcgagcactt tacgtttttt tttcggctga caccattcgg tccaccaggt ttcccaccag 60 gtttctttcc 70 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ctcgagatgg gagtgcaggt ggaaaccatc 30 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ggatcctcat tccagtttta gaagctccac 30 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> top strand for Tat peptide <400> 19 taggaagaag cggagacagc gacgaagac 29 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> bottom strand for Tat peptide <400> 20 tcgagtcttc gtcgctgtct ccgcttcttc c 31

Claims (4)

A fusion protein comprising a protein transport domain covalently bound to one or more of the N-terminal and C-terminal ends of an FK506 binding protein, wherein the fusion protein comprises an FK506 binding protein fusion protein consisting of the amino acid sequence of SEQ ID NO: 4, A pharmaceutical composition for therapeutic use.
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