KR20150101098A - Pharmaceutical composition for autosomal dominant polycystic kidney disease containing PRAS40 fusion protein - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating an autosomal dominant polycystic kidney disease and, more specifically, to a pharmaceutical composition for preventing or treating a polycystic kidney disease comprising, as an active ingredient, a PRAS40 fusion protein capable of penetrating into polycystic kidney disease cells.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing and treating polycystic kidney disease containing PRAS40 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 polycystic kidney disease containing PRAS40 fusion protein capable of tissue and intracellular permeability as an active ingredient.

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.

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.

It is an object of the present invention to provide an effective prophylactic and therapeutic agent for polycyclic acid.

The present invention aims to provide a more effective protein therapeutic by identifying Erk-inhibiting proteins that are targets of polycyclic therapy and identifying mechanism and protective effect against polycystic kidney using protein permeation technique.

In order to achieve the above object, the present inventors have studied to develop a protein preparation which is an immunosuppressive drug, which enhances the penetration ability, and that the penetration effect of a protein preparation and the PRAS40 fusion protein in a polycystic kidney cell line, The expression of PRAS40 fusion protein was confirmed, and thus the present invention was completed.

In the present invention, the protein transport domain carrying the protein into cells and tissues was fused to the external protein PRAS40, and the fusion protein was overexpressed in E. coli and easily and conveniently purified by metal chelating affinity chromatography.

In one specific embodiment of the present invention, a PRAS40 fusion protein capable of overexpressing and easily purifying the PRAS40 fusion protein Expression vector was developed. This expression vector has the human PRAS40 cDNA (SEQ ID NO: 8), the protein transport domain and six histidines connected in series. Using this expression vector, the PRAS40 fusion protein was over-expressed in E. coli and purified using a Ni ²⁺ -affinity chromatography column. Overexpression of PRAS40 fusion protein was significantly higher, resulting in a higher amount of purified protein. In addition, it was confirmed through experiments that the purified fusion protein effectively penetrates into the polycystic kidney cell line and reduces Erk signal and Akt signal. The present invention has raised the possibility that the PRAS40 fusion protein can be used as a protein therapeutic agent for autosomal dominant polycystine.

In one embodiment of the present invention, the inventors first developed a Tat-PRAS40 expression vector capable of overexpressing and easily purifying Tat-PRAS40 fusion protein. This expression vector contains human PRAS40, nine amino acids (Tat 49-57) at the Tat transduction site, and cDNA capable of expressing six histidine residues at the amino terminal end.

In the present invention, permeability of the PRAS40 fusion protein into the polycystic kidney cell line was confirmed by time and dose, and the stability of the PRAS40 fusion protein was confirmed after cell infiltration.

As a result, the PRAS40 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 36 hours after infiltration. In addition, treatment of PRAS40 fusion protein in polycystic kidney cell line decreased Erk and Akt signals.

The pharmaceutical composition containing the PRAS40 fusion protein and the transport domain fusion protein as an active ingredient can be formulated together with a carrier that is conventionally acceptable in the pharmaceutical field and can be formulated by oral method or injection form by a conventional method. Oral compositions include, for example, tablets and gelatin capsules, which may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, , Magnesium stearate, stearic acid and its magnesium or calcium salt and / or polyethylene glycol) and the tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone ), And may optionally contain a disintegrant (e.g., starch, agar, alginic acid or a sodium salt thereof) or a boiling mixture and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The injectable composition is preferably an isotonic aqueous solution or suspension, and the composition mentioned is sterilized and / or contains adjuvants such as preservatives, stabilizers, wetting or emulsifying solution accelerators, salts for controlling osmotic pressure and / or buffering agents. They may also contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be administered orally, parenterally, that is, intravenously, subcutaneously, intraperitoneally or topically, depending on the purpose, and in the case of application to asthma, As known to those of ordinary skill in the art, it can be formulated into a formulation by inhalation or spray. The dose may be administered in a single daily dose of 0.0001 to 100 mg / kg dividedly in 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, excretion rate, severity of disease, and the like.

Further, the present invention provides a pharmaceutical composition for the prevention or treatment of autosomal dominant polycystine, comprising the PRAS40 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides a health functional food composition comprising the PRAS40 fusion protein as an active ingredient and preventing or improving autosomal dominant polycystic kidney.

The present invention also provides a method for efficiently delivering the PRAS40 protein into cells. Intracellular delivery of the PRAS40 protein molecule according to the present invention is carried out by constructing a fusion protein in which a protein transport domain including HIV Tat is covalently linked. Examples of the transport domain of the present invention include amino acids such as SEQ ID NOS: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, HIV Tat < / RTI > peptide. However, the protein transport domain of the present invention is not limited to the Tat peptides of SEQ ID NOS: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33, It is easy for a person skilled in the art to make a peptide having a function similar to that of a Tat peptide due to the addition or absence thereof. Therefore, it is preferable that the peptide is composed of 7 to 15 amino acids and contains 4 or more lysine or arginine And a fusion protein using a protein-transporting domain that performs the same or similar protein-transporting function with a partial substitution of an amino acid from the protein-transporting domain, is also within the scope of the present invention.

Specifically, the present invention relates to a pharmaceutical composition for treating and preventing polycystic acid including a PRAS40 fusion protein, a health functional food, and the like.

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

"PRAS40 fusion protein" means a covalent complex formed by genetic fusion or chemical bonding of a protein transport domain and PRAS40, and a transport domain and a target protein (i. E., PRAS40 in the present invention). In the present specification, "Tat-PRAS40 "," PRAS40 fusion protein "

"Target protein" is a molecule which is not originally able to enter the target cell, or which is not a transport domain or a fragment thereof that can not enter the target cell at an inherently useful speed, as a molecule itself before being fused with the transport domain, Means the target protein portion. The target protein includes a polypeptide, a protein, and a peptide. In the present invention, it means PRAS40.

"Fusion protein" means a complex comprising a transport domain and one or more target protein fragments, formed by genetic fusion or chemical bonding of the transport domain and the target protein.

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 target protein is delivered by a transport domain, and the target cell refers to a cell in the body or in vitro. That is, the target cell is meant to include a body cell, that is, a living animal, or a cell or living organism of a human organ or tissue, or a microorganism found in a human being. In addition, the target cell means an extracellular cell, that is, a cultured animal cell, a human cell or a microorganism.

The term "protein transport domain" in the present invention refers to a protein transport domain that is covalently bonded to a polymer organic compound such as an oligonucleotide, peptide, protein, oligosaccharide or polysaccharide to introduce the organic compound into cells without requiring additional receptor, It can be said.

Also, in the present specification, the terms "transport", "penetration", "transport", "delivery", "permeation" and "passage" are used interchangeably with respect to "introducing" proteins, peptides and organic compounds into a cell.

The present invention relates to a PRAS40 fusion protein comprising 9 to 15 amino acid residues and having a transport domain comprising 3/4 or more of arginine or lysine residues covalently bonded to at least one terminal of PRAS40 (Proline-rich Akt substrate 40) The present invention provides a pharmaceutical composition for the prevention and treatment of autosomal dominant polycystic kidney disease.

In addition, the present invention is characterized in that said transport domain is at least one of HIV Tat 49-57 residue, Pep-1 peptide, oligo lysine, oligoarginine or oligo (lysine, arginine).

In addition, the present invention is characterized in that the amino acid sequence of the PRAS40 fusion protein is the same as SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, In accordance with the selection of the restriction site sequence in the production of the PRAS40 fusion protein, fusion proteins of various sequences can be obtained, which is obvious to a person having ordinary skill in the art. It is to be understood that the above amino acid sequence is only exemplary and that the amino acid sequence of PRAS40 fusion protein is not limited to the sequence listed above.

In addition, the present invention provides a pharmaceutical composition for preventing and treating autosomal dominant polycystine comprising the PRAS40 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides a health functional food composition comprising the PRAS40 fusion protein as an active ingredient and having an effect of preventing and improving autosomal dominant polycystic kidney.

The present invention relates to a pharmaceutical composition comprising a cell-transducing PRAS40 fusion protein consisting of 9 to 15 amino acids and having a protein transport domain comprising at least 4 lysine or arginine covalently bonded to at least one end of the PRAS40 protein ≪ / RTI > Also, depending on the silent change, one or more amino acids within the sequence may be replaced with other amino acid (s) of similar polarity functionally equivalent. 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. Also included within the scope of the invention are fragments or derivatives thereof having homologous homology, for example within the range of 85-100%, between the fusion protein of the present invention and the amino acid sequence, having the same similar biological activity.

The PRAS40 fusion protein according to the present invention infiltrated into polycystic kidney cells in a time-dependent and dose-dependent manner.

In addition, the PRAS40 fusion protein according to the present invention penetrated into cells and stably maintained for 36 hours or more.

In addition, the PRAS40 fusion protein according to the present invention decreased Erk signal and Akt signal in polycystic kidney cells.

Therefore, the PRAS40 fusion protein according to the present invention is expected to be useful as a pharmaceutical composition for the prevention or treatment of autosomal dominant polycystic kidney.

FIG. 1 shows the results of purification of PRAS40 protein and Tat-PRAS40 fusion protein followed by SDS-PAGE. Further, Western blot analysis showed that the PRAS protein and the Tat-PRAS40 fusion protein were purified.
FIG. 2 is a photograph of western blot analysis after treatment of WT 9-7 cells with 3 μM of Tat-PRAS40 fusion protein at different times for 0 to 60 minutes. As a control, PRAS40 protein without a protein transduction domain was used.
FIG. 3 is a photograph of western blot analysis after treatment of WT 9-7 cells with 0.25 ~ 2 μM of Tat-PRAS40 fusion protein at different concentrations for 1 hour. As a control, PRAS40 protein without a protein transduction domain was used.
FIG. 4 is a photograph of western blot analysis of WT 9-7 cells treated with 3 μM of Tat-PRAS40 fusion protein for 1 hour. The fusion protein remained stable until 36 hours.
FIG. 5 is a photograph of WT 9-7 cells treated with 3 μM Tat-PRAS40 fusion protein for 1 hour and then photographed using a confocal microscope. "Control" is a no-treatment group.
FIG. 6 shows the inhibitory effect of Tat-PRAS40 fusion protein on p38, ERK and JNK signaling in WT 9-7 cells.
Fig. 7 shows the inhibitory effect of Tat-PRAS40 fusion protein on the Akt signaling mechanism in WT 9-7 cells.
Figure 8 shows the inhibitory effect of Tat-PRAS40 fusion protein on the p65 and IκBα signaling mechanism in WT 9-7 cells.
FIG. 9 shows Western blot analysis of WT 9-7 cells to examine the inhibitory effect of Tat-PRAS40 fusion protein on COX2, iNOS, IL-6, IL-1β, and TNF-α signaling mechanism.
FIG. 10 shows RT-PCR of WT 9-7 cells to inhibit COX2, iNOS, IL-6, IL-1β, and TNF-α signaling mechanism of Tat-PRAS40 fusion protein.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments. Particularly, as a result of each experimental example, data on the Tat-PRAS40 fusion protein among the various fusion proteins prepared in the specific examples were described, but other fusion proteins were also similar to the results obtained using the Tat-PRAS40 fusion protein as a sample And the results are shown.

material

Restriction endonuclease and T4 DNA ligase were purchased from Promega (Madison, Wis., USA). Plasmid pET-15b and Escherichia coli strain BL21 (DE3) were purchased from Novagen (Hilden, Germany). Oligonucleotides were synthesized in Bioneer (Dajeon, Korea). Ni ^{2 +} -nitrile trichlorosilicate Sepharose Superflow column was obtained from Qiagen (Valencia, CA, USA). Fetal serum and antibiotics were purchased from Gibco BRL (Grand Island, NY, USA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted and the best available analytical grade products were used.

PRAS40 Fusion protein  Expression and purification

PRAS40 and Tat-PRAS40 fusion protein expression vectors were constructed. The sense primer sequence is 5'-CTCGAGCGCAGCCCC-3 '(SEQ ID NO: 6) and comprises the Xho I restriction site and the antisense primer sequence is 5'-GGATCCTCAATATTTCCGCTTCAG-3' (SEQ ID NO: 7) and contains the Bam HI restriction site. Polymerase chain reaction (PCR) was performed using PRAS40 primers and cDNA template, and PCR products were inserted into the TA cloning vector. The TA vector containing the PRAS40 cDNA was ligated to two pET-15b vectors with PRAS40 cDNA digested with Xho I and Bam HI and digested . One vector contains the Tat sequence and six consecutive histidine sequences as Tat-PRAS40 expression vectors. The other vector contains only six consecutive histidine sequences as the PRAS40 expression vector.

E. coli BL21 (DE3) cells were transformed with each vector construct to produce PRAS40 and Tat-PRAS40 fusion proteins. The transformed bacteria were cultured in 100 ml of LB medium at 37 ° C. until the optical density of 600 nm became 0.5, and then 0.5 mM IPTG (isopropyl-β-D-thiogalactoside) was added thereto, followed by induction at 37 ° C. for 4 hours . Cells were harvested and lysed and lysed by centrifugation at 4 ° C in binding buffer (10 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The supernatant was added to a sepharose sepharose super flow column with 2.0 ml of Ni ^{2 +} -nitrile. The column was washed with 10 times of binding buffer and 7 times of wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). PRAS40 and Tat-PRAS40 fusion proteins were eluted with elution buffer (280 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The salts contained in the purified protein were removed with a PD-10 desalting column (Amersham, Braunschweig, Germany). Protein concentration was determined by Bradford method using bovine serum albumin as a standard.

Cell culture and PRAS40 Fusion protein  Introduction

WT 9-7 cells were cultured in EMEM (Eagle's Minimum) containing 20 mM HEPES / NaOH (pH 7.4), 5 mM NaHCO3, 10% fetal calf serum (FBS) and antibiotics (100 ug / ml streptomycin, 100 U / ml penicillin) Essential Medium) medium at 37 ° C, 95% air and 5% CO ₂ .

To confirm that the PRAS40 and PRAS40 fusion proteins were introduced into WT 9-7 cells in a dose-dependent manner, cells were grown in a 60-mm diameter dish and the PRAS40 fusion proteins at various concentrations (0.25 to 2 μM) And cultured together.

Cells were treated with PRAS40 fusion protein (3 μM) for various times (10-60 min) to see if each protein was time-dependent for cell introduction.

Cells were harvested to produce cell extracts and Western blot analysis.

Western blot  analysis

Proteins from each cell lysate were analyzed by 12% SDS-PAGE. The analyzed proteins were electroporated into nitrocellulose membranes blocked with PBS containing 5% skim milk. The membranes were probed with anti-histidine polyclonal antibodies or various antibodies (1: 15,000; Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and then incubated with horseradish peroxidase-conjugated goat anti- rabbit immunoglobulin ; Sigma-Aldrich). The conjugated antibody complexes were visualized as enhanced chemiluminescent as directed by the manufacturer (Immobilon Western Chemiluminescent AP Substrate; Millipore, Billerica, MA, USA).

Confocal  Fluorescence microscope

WT 9-7 cells were placed on glass cover slips and incubated with PRAS40 and Tat-PRAS40 fusion protein (3 μM) for one hour at 37 ° C. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 3 minutes. Cells were fixed with PBS containing 3% bovine serum albumin, 0.1% Triton X-100 and washed with PBS-BT. The anti-histidine primary antibody was diluted 1: 2,000 and incubated with each sample for one and a half hours at room temperature. Alexa fluor 488 conjugated secondary antibody (Invitrogen, Carlsbad, Calif., USA) was diluted 1: 15,000 and incubated at room temperature for 1 hour under dark conditions. Nuclei were stained with 4'6-diamidino-2-phenylindole (Roche Applied Science, Basel, Switzerland) diluted 1: 3,000 with 1 μg / ml for 3 minutes. Fluorescence was analyzed with an Olympus FV-300 confocal fluorescence microscope (Olympus, Tokyo, Japan).

iNOS  And COX -2 expression level measurement

WT 7-9 cells were cultured in 6-well plates for 12 hours to achieve 70% confluence. After incubation, the cells were pretreated with Tat-PRAS40 (0.25 1 [mu] M) for 2 hours and the culture medium was harvested. Expression of RNA levels as well as iNOS and COX-2 protein expression was determined by Western blot analysis and RT-PCR.

RT - PCR  analysis

Total RNA was isolated from Raw 264.7 cells using the Easy blue kit according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif., USA). RNA (2 μg) was then reverse transcribed using reverse transcriptase and oligo- (dT) primers. PCR amplification for cDNA was performed using the following sense and antisense primers. COX-2 sense, 5'-CAAAGGCCTCCATTGACCAGA-3 '; COX-2 antisense, 5'-TGGACCAGGTTTTTCCACCG-3 '; IL-1? Sense, 5'-TGCAGAGTTCCCCAACTGGTACTC-3 '; IL-1? Antisense, 5'-GTGCTGCCTAATGTCCCTTGAATC-3 '; IL-6 sense, 5'-CAAGAAAGACAAAGCCAGAGTCCTT-3 '; IL-6 antisense, 5'-TGGATGGTCTTGGTCCCTTAGCC-3 '; TNF-α sense, 5'-AAGTTCCCAAATGGCCTCCC-3 '; TNF-a antisense, 5'-TGGCACCACTAGTTGGTTGTCTTT-3 '; And GADPH sense, 5'-TGAAGGTCGGTGAACGGATTTGG-3 '; GAPDH antisense, 5'-CATGTAGGCCATGAGGTCCAC CAC-3 '. The PCR primer kit (Intron Biotechnology, Seoul, Korea) was used for PCR and the ECH primer was terminated by heating to 72 ° C for 5 minutes. The PCR products were separated on 1% agarose gel, stained with ethidium bromide and observed with UV light.

Experimental Example  One: PRAS40 Fusion protein  Expression and purification

PRAS40 and PRAS40 fusion protein expression vectors were prepared as described above. As in Figure 1, both PRAS40 and PRAS40 fusion proteins were over-expressed and confirmed by SDS-PAGE and Western blot analysis. The molecular weights of the purified PRAS40 and PRAS40 fusion proteins were about 40 and 43 kDa, respectively.

Experimental Example  2: Time and dose dependent PRAS40  Of the fusion protein Intracellular  Penetration efficiency

1) Cell line and drug treatment

Autosomal dominant polycystic tubule cells WT 9-7 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.

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 WT 9-7 cells for 10, 20, 30, and 60 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. 2, it was confirmed that the PRAS40 fusion protein infiltrated into WT 9-7 cells in a time-dependent manner. In contrast, the PRAS40 protein that did not bind the transport domain did not penetrate into the cells.

3) Cell penetration efficiency according to treatment concentration

After 5 × 10 ⁵ cells were seeded in a 60 mm culture dish, the PRAS40 fusion protein was treated with WT 9-7 cells at 0.25, 0.5, 1 and 2 μM for two hours, followed by two washes with phosphate buffered saline The cells were treated with fructosyne 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. 3, it was confirmed that the PRAS40 fusion protein infiltrated into WT 9-7 cells in a concentration-dependent manner. In contrast, the PRAS40 protein that did not bind the transport domain did not penetrate into the cells.

Experimental Example  3: penetrating into cells PRAS40  Stability of fusion protein

1 x 10 ⁴ WT 9-7 cells in a cover glass were treated with 3 μM PRAS40 fusion protein for 1 hour. 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 PRAS40 fusion protein was successfully infiltrated into WT 9-7 cells. On the other hand, PRAS40 protein did not penetrate into cells.

In addition, PRAS40 fusion proteins infiltrated were washed twice with phosphate-buffered saline for 1, 6, 12, 18, 24, 30, and 36 hours after each time, treated with trypsin for 1 minute, Cells were obtained by centrifugation (1300 rpm, 5 minutes). 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. 4, the fusion protein was stably maintained for 36 hours or more.

Experimental Example  4: WT  In 9-7 cells PRAS40  Mechanism of fusion protein

One) p38 , ERK , JNK  Signal mechanism

Western blot analysis was performed on WT 9-7 cells, and the p38, ERK, and JNK signaling mechanisms were examined using PRAS40 protein and PRAS40 fusion protein. As a result, PRAS40 fusion protein was found to have a p38, ERK and JNK signaling mechanism , Respectively.

2) Akt  Signal mechanism

Western blot analysis was performed on WT 9-7 cells. As a result of confirming the Akt signaling mechanism using PRAS40 protein and PRAS40 fusion protein, it was confirmed that PRAS40 fusion protein inhibited the Akt signaling mechanism in a concentration-dependent manner.

3) p65  And IκBα signaling mechanism

Western blot analysis was performed on WT 9-7 cells, and the p65 and IκBα signaling mechanisms were examined using PRAS40 protein and PRAS40 fusion protein. As a result, PRAS40 fusion protein inhibited p65 and IκBα signaling in a concentration-dependent manner I could confirm.

4) COX2 , iNOS , IL -6, IL -1β and TNF -α signal mechanism

Western blot analysis was performed on WT 9-7 cells, and the mechanism of COX2, iNOS, IL-6, IL-1β and TNF-α signaling was examined using PRAS40 protein and PRAS40 fusion protein. As a result, PRAS40 fusion protein Inhibition of COX2, iNOS, IL-6, IL-1β and TNF-α signaling in a dose-dependent manner.

5 ) COX2 , iNOS , IL -6, IL -1β and TNF -α signal mechanism

As shown in FIG. 10, the expression of PRAS40 fusion protein was confirmed by RT-PCR in WT 9-7 cells and the signaling mechanism of COX2, iNOS, IL-6, IL-1β and TNF-α using PRAS40 protein and PRAS40 fusion protein Inhibition of COX2, iNOS, IL-6, IL-1β and TNF-α signaling in a dose-dependent manner.

From the above experimental results, it was found that when PRAS40 fusion protein is applied to polycystic kidney cells, it penetrates smoothly into cells and exhibits a stability of 36 hours and reduces Erk signal and Akt signal. Therefore, And is expected to be useful as a pharmaceutical composition.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for autosomal dominant polycystic          kidney disease containing PRAS40 fusion protein <130> hallym-PRAS40-Polycystickidney <160> 33 <170> Kopatentin 1.71 <210> 1 <211> 29 <212> DNA <213> Human immunodeficiency virus type 1 <400> 1 taggaagaag cggagacagc gacgaagac 29 <210> 2 <211> 31 <212> DNA <213> Human immunodeficiency virus type 1 <400> 2 tcgagtcttc gtcgctgtct ccgcttcttc c 31 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ctcgagatgg cagaaccgca gcccccgtcc 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ctcgagatga aggtagaggt gctgcctgcc 30 <210> 5 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 5 Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ctcgagcgca gcccc 15 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggatcctcaa tatttccgct tcag 24 <210> 8 <211> 771 <212> DNA <213> Homo sapiens <400> 8 atggcgtcgg ggcgccccga ggagctgtgg gaggccgtgg tgggggccgc tgagcgcttc 60 cgggcccgga ctggcacgga gctggtgctg ctgaccgcgg ccccgccgcc accaccccgc 120 ccgggcccct gtgcctatgc tgcccatggt cgaggagccc tggcggaggc agcgcgccgt 180 tgcctccacg acatcgcact ggcccacagg gctgccactg ctgctcggcc tcctgcgccc 240 ccaccagcac cacagccacc cagtcccaca cccagcccac cccggcctac cctggccaga 300 gaggacaacg aggaggacga ggatgagccc acagagacag agacctccgg ggagcagctg 360 ggcattagtg ataatggagg gctctttgtg atggatgagg acgccaccct ccaggacctt 420 ccccccttct gtgagtcaga ccccgagagt acagatgatg gcagcctgag cgaggagacc 480 cccgccggcc cccccacctg ctcagtgccc ccagcctcag ccctacccac acagcagtac 540 gccaagtccc tgcctgtgtc tgtgcccgtc tggggcttca aggagaagag gacagaggcg 600 cggtcatcag atgaggagaa tgggccgccc tcttcgcccg acctggaccg catcgcggcg 660 agcatgcgcg cgctggtgct gcgagaggcc gaggacaccc aggtcttcgg ggacctgcca 720 cggccgcggc ttaacaccag cgacttccag aagctgaagc ggaaatattg a 771 <210> 9 <211> 256 <212> PRT <213> Homo sapiens <400> 9 Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala   1 5 10 15 Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr              20 25 30 Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala          35 40 45 His Gly Arg Gly Ala Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp      50 55 60 Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro  65 70 75 80 Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro                  85 90 95 Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu             100 105 110 Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu         115 120 125 Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys     130 135 140 Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr 145 150 155 160 Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro                 165 170 175 Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly             180 185 190 Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly         195 200 205 Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala     210 215 220 Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro 225 230 235 240 Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr                 245 250 255 <210> 10 <211> 858 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding Tat-PRAS40 fusion protein <400> 10 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggcgtcg gggcgccccg aggagctgtg ggaggccgtg 120 gtgggggccg ctgagcgctt ccgggcccgg actggcacgg agctggtgct gctgaccgcg 180 gccccgccgc caccaccccg cccgggcccc tgtgcctatg ctgcccatgg tcgaggagcc 240 ctggcggagg cagcgcgccg ttgcctccac gacatcgcac tggcccacag ggctgccact 300 gctgctcggc ctcctgcgcc cccaccagca ccacagccac ccagtcccac acccagccca 360 ccccggccta ccctggccag agaggacaac gaggaggacg aggatgagcc cacagagaca 420 gagacctccg gggagcagct gggcattagt gataatggag ggctctttgt gatggatgag 480 gacgccaccc tccaggacct tccccccttc tgtgagtcag accccgagag tacagatgat 540 ggcagcctga gcgaggagac ccccgccggc ccccccacct gctcagtgcc cccagcctca 600 gccctaccca cacagcagta cgccaagtcc ctgcctgtgt ctgtgcccgt ctggggcttc 660 aaggagaaga ggacagaggc gcggtcatca gatgaggaga atgggccgcc ctcttcgccc 720 gacctggacc gcatcgcggc gagcatgcgc gcgctggtgc tgcgagaggc cgaggacacc 780 caggtcttcg gggacctgcc acggccgcgg cttaacacca gcgacttcca gaagctgaag 840 cggaaatatt gaggatcc 858 <210> 11 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Tat-PRAS40 fusion protein <400> 11 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Ser Gly Arg   1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro Pro          35 40 45 Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala      50 55 60 Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His  65 70 75 80 Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro Pro Ala Pro Gln                  85 90 95 Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu             100 105 110 Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly         115 120 125 Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu     130 135 140 Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu 145 150 155 160 Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro                 165 170 175 Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala             180 185 190 Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg         195 200 205 Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Ser Pro     210 215 220 Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu 225 230 235 240 Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn                 245 250 255 Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr             260 265 <210> 12 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding PRAS40-Tat fusion protein <400> 12 catcatcatc atcatcacag cagcggcctg gtgccggcgg cagccactcg agatggcgtc 60 ggggcgcccc gaggagctgt gggaggccgt ggtgggggcc gctgagcgct tccgggcccg 120 gactggcacg gagctggtgc tgctgaccgc ggccccgccg ccaccacccc gcccgggccc 180 ctgtgcctat gctgcccatg gtcgaggagc cctggcggag gcagcgcgcc gttgcctcca 240 cgacatcgca ctggcccaca gggctgccac tgctgctcgg cctcctgcgc ccccaccagc 300 accacccca cccagtccca cgaggaggac gaggatgagc ccacagagac agagacctcc ggggagcagc tgggcattag 420 tgataatgga gggctctttg tgatggatga ggacgccacc ctccaggacc ttcccccctt 480 ctgtgagtca gaccccgaga gtacagatga tggcagcctg agcgaggaga cccccgccgg 540 cccccccacc tgctcagtgc ccccagcctc agccctaccc acacagcagt acgccaagtc 600 cctgcctgtg tctgtgcccg tctggggctt caaggagaag aggacagagg cgcggtcatc 660 agatgaggag aatgggccgc cctcttcgcc cgacctggac cgcatcgcgg cgagcatgcg 720 cgcgctggtg ctgcgagagg ccgaggacac ccaggtcttc ggggacctgc cacggccgcg 780 gcttaacacc agcgacttcc agaagctgaa gcggaaatat ggatcctagg aagaagcgga 840 gacagcgacg aagatag 857 <210> 13 <211> 265 <212> PRT <213> Artificial Sequence <220> <223> PRAS40-Tat fusion protein <400> 13 Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala   1 5 10 15 Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr              20 25 30 Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala          35 40 45 His Gly Arg Gly Ala Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp      50 55 60 Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro  65 70 75 80 Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro                  85 90 95 Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu             100 105 110 Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu         115 120 125 Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys     130 135 140 Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr 145 150 155 160 Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro                 165 170 175 Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly             180 185 190 Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly         195 200 205 Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala     210 215 220 Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro 225 230 235 240 Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr                 245 250 255 Arg Lys Lys Arg Arg Gln Arg Arg Arg             260 265 <210> 14 <211> 886 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding Tat-PRAS40-Tat fusion protein <400> 14 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggcgtcg gggcgccccg aggagctgtg ggaggccgtg 120 gtgggggccg ctgagcgctt ccgggcccgg actggcacgg agctggtgct gctgaccgcg 180 gccccgccgc caccaccccg cccgggcccc tgtgcctatg ctgcccatgg tcgaggagcc 240 ctggcggagg cagcgcgccg ttgcctccac gacatcgcac tggcccacag ggctgccact 300 gctgctcggc ctcctgcgcc cccaccagca ccacagccac ccagtcccac acccagccca 360 ccccggccta ccctggccag agaggacaac gaggaggacg aggatgagcc cacagagaca 420 gagacctccg gggagcagct gggcattagt gataatggag ggctctttgt gatggatgag 480 gacgccaccc tccaggacct tccccccttc tgtgagtcag accccgagag tacagatgat 540 ggcagcctga gcgaggagac ccccgccggc ccccccacct gctcagtgcc cccagcctca 600 gccctaccca cacagcagta cgccaagtcc ctgcctgtgt ctgtgcccgt ctggggcttc 660 aaggagaaga ggacagaggc gcggtcatca gatgaggaga atgggccgcc ctcttcgccc 720 gacctggacc gcatcgcggc gagcatgcgc gcgctggtgc tgcgagaggc cgaggacacc 780 caggtcttcg gggacctgcc acggccgcgg cttaacacca gcgacttcca gaagctgaag 840 cggaaatatg gatcctagga agaagcggag acagcgacga agatag 886 <210> 15 <211> 278 <212> PRT <213> Artificial Sequence <220> <223> Tat-PRAS40-Tat fusion protein <400> 15 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Ser Gly Arg   1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro Pro          35 40 45 Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala      50 55 60 Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His  65 70 75 80 Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro Pro Ala Pro Gln                  85 90 95 Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu             100 105 110 Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly         115 120 125 Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu     130 135 140 Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu 145 150 155 160 Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro                 165 170 175 Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala             180 185 190 Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg         195 200 205 Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Ser Pro     210 215 220 Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu 225 230 235 240 Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn                 245 250 255 Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr Gly Ser Arg Lys Lys             260 265 270 Arg Arg Gln Arg Arg Arg         275 <210> 16 <211> 895 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding Pep-PRAS40 fusion protein <400> 16 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccataa aagaaacctg 60 gtgggaaacc tggtggaccg aatggtctca gccgaaaaaa aaacgtaaag tgctcgagat 120 ggcgtcgggg cgccccgagg agctgtggga ggccgtggtg ggggccgctg agcgcttccg 180 ggcccggact ggcacggagc tggtgctgct gaccgcggcc ccgccgccac caccccgccc 240 gggcccctgt gcctatgctg cccatggtcg aggagccctg gcggaggcag cgcgccgttg 300 cctccacgac atcgcactgg cccacagggc tgccactgct gctcggcctc ctgcgccccc 360 accagcacca cagccaccca gtcccacacc cagcccaccc cggcctaccc tggccagaga 420 ggacaacgag gaggacgagg atgagcccac agagacagag acctccgggg agcagctggg 480 cattagtgat aatggagggc tctttgtgat ggatgaggac gccaccctcc aggaccttcc 540 ccccttctgt gagtcagacc ccgagagtac agatgatggc agcctgagcg aggagacccc 600 cgccggcccc cccacctgct cagtgccccc agcctcagcc ctacccacac agcagtacgc 660 caagtccctg cctgtgtctg tgcccgtctg gggcttcaag gagaagagga cagaggcgcg 720 gtcatcagat gaggagaatg ggccgccctc ttcgcccgac ctggaccgca tcgcggcgag 780 catgcgcgcg ctggtgctgc gagaggccga ggacacccag gtcttcgggg acctgccacg 840 gccgcggctt aacaccagcg acttccagaa gctgaagcgg aaatattgag gatcc 895 <210> 17 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> Pep-PRAS40 fusion protein <400> 17 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 Ala Ser Gly Arg Pro Glu Glu Leu              20 25 30 Trp Glu Ala Val Val Gly Ala Ala Glu Arg Phe Arg Ala Arg Thr Gly          35 40 45 Thr Glu Leu Val Leu Leu Thr Ala Pro Pro Pro Pro Arg Pro      50 55 60 Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala Leu Ala Glu Ala  65 70 75 80 Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His Arg Ala Ala Thr                  85 90 95 Ala Ala Arg Pro Pro Ala Pro Pro Ala Pro Gln Pro Pro Ser Pro             100 105 110 Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu Asp Asn Glu Glu         115 120 125 Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly Glu Gln Leu Gly     130 135 140 Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu Asp Ala Thr Leu 145 150 155 160 Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu Ser Thr Asp Asp                 165 170 175 Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro Thr Cys Ser Val             180 185 190 Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala Lys Ser Leu Pro         195 200 205 Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg Thr Glu Ala Arg     210 215 220 Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Pro Asp Leu Asp Arg 225 230 235 240 Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu Ala Glu Asp Thr                 245 250 255 Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn Thr Ser Asp Phe             260 265 270 Gln Lys Leu Lys Arg Lys Tyr         275 <210> 18 <211> 894 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding PRAS40-Pep fusion protein <400> 18 catcatcatc atcatcacag cagcggcctg gtgccggcgg cagccactcg agatggcgtc 60 ggggcgcccc gaggagctgt gggaggccgt ggtgggggcc gctgagcgct tccgggcccg 120 gactggcacg gagctggtgc tgctgaccgc ggccccgccg ccaccacccc gcccgggccc 180 ctgtgcctat gctgcccatg gtcgaggagc cctggcggag gcagcgcgcc gttgcctcca 240 cgacatcgca ctggcccaca gggctgccac tgctgctcgg cctcctgcgc ccccaccagc 300 accacccca cccagtccca cgaggaggac gaggatgagc ccacagagac agagacctcc ggggagcagc tgggcattag 420 tgataatgga gggctctttg tgatggatga ggacgccacc ctccaggacc ttcccccctt 480 ctgtgagtca gaccccgaga gtacagatga tggcagcctg agcgaggaga cccccgccgg 540 cccccccacc tgctcagtgc ccccagcctc agccctaccc acacagcagt acgccaagtc 600 cctgcctgtg tctgtgcccg tctggggctt caaggagaag aggacagagg cgcggtcatc 660 agatgaggag aatgggccgc cctcttcgcc cgacctggac cgcatcgcgg cgagcatgcg 720 cgcgctggtg ctgcgagagg ccgaggacac ccaggtcttc ggggacctgc cacggccgcg 780 gcttaacacc agcgacttcc agaagctgaa gcggaaatat ggatcctaaa agaaacctgg 840 tgggaaacct ggtggaccga atggtctcag ccgaaaaaaa aacgtaaagt gtag 894 <210> 19 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> PRAS40-Pep fusion protein <400> 19 Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala   1 5 10 15 Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr              20 25 30 Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala          35 40 45 His Gly Arg Gly Ala Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp      50 55 60 Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro  65 70 75 80 Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro                  85 90 95 Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu             100 105 110 Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu         115 120 125 Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys     130 135 140 Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr 145 150 155 160 Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro                 165 170 175 Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly             180 185 190 Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly         195 200 205 Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala     210 215 220 Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro 225 230 235 240 Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr                 245 250 255 Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln             260 265 270 Pro Lys Lys Lys Arg Lys Val         275 <210> 20 <211> 960 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding Pep1-PRAS40-Pep1 fusion protein <400> 20 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccataa aagaaacctg 60 gtgggaaacc tggtggaccg aatggtctca gccgaaaaaa aaacgtaaag tgctcgagat 120 ggcgtcgggg cgccccgagg agctgtggga ggccgtggtg ggggccgctg agcgcttccg 180 ggcccggact ggcacggagc tggtgctgct gaccgcggcc ccgccgccac caccccgccc 240 gggcccctgt gcctatgctg cccatggtcg aggagccctg gcggaggcag cgcgccgttg 300 cctccacgac atcgcactgg cccacagggc tgccactgct gctcggcctc ctgcgccccc 360 accagcacca cagccaccca gtcccacacc cagcccaccc cggcctaccc tggccagaga 420 ggacaacgag gaggacgagg atgagcccac agagacagag acctccgggg agcagctggg 480 cattagtgat aatggagggc tctttgtgat ggatgaggac gccaccctcc aggaccttcc 540 ccccttctgt gagtcagacc ccgagagtac agatgatggc agcctgagcg aggagacccc 600 cgccggcccc cccacctgct cagtgccccc agcctcagcc ctacccacac agcagtacgc 660 caagtccctg cctgtgtctg tgcccgtctg gggcttcaag gagaagagga cagaggcgcg 720 gtcatcagat gaggagaatg ggccgccctc ttcgcccgac ctggaccgca tcgcggcgag 780 catgcgcgcg ctggtgctgc gagaggccga ggacacccag gtcttcgggg acctgccacg 840 gccgcggctt aacaccagcg acttccagaa gctgaagcgg aaatatggat cctaaaagaa 900 acctggtggg aaacctggtg gaccgaatgg tctcagccga aaaaaaaacg taaagtgtag 960                                                                          960 <210> 21 <211> 302 <212> PRT <213> Artificial Sequence <220> <223> Pep1-PRAS40-Pep1 fusion protein <400> 21 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 Ala Ser Gly Arg Pro Glu Glu Leu              20 25 30 Trp Glu Ala Val Val Gly Ala Ala Glu Arg Phe Arg Ala Arg Thr Gly          35 40 45 Thr Glu Leu Val Leu Leu Thr Ala Pro Pro Pro Pro Arg Pro      50 55 60 Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala Leu Ala Glu Ala  65 70 75 80 Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His Arg Ala Ala Thr                  85 90 95 Ala Ala Arg Pro Pro Ala Pro Pro Ala Pro Gln Pro Pro Ser Pro             100 105 110 Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu Asp Asn Glu Glu         115 120 125 Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly Glu Gln Leu Gly     130 135 140 Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu Asp Ala Thr Leu 145 150 155 160 Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu Ser Thr Asp Asp                 165 170 175 Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro Thr Cys Ser Val             180 185 190 Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala Lys Ser Leu Pro         195 200 205 Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg Thr Glu Ala Arg     210 215 220 Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Pro Asp Leu Asp Arg 225 230 235 240 Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu Ala Glu Asp Thr                 245 250 255 Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn Thr Ser Asp Phe             260 265 270 Gln Lys Leu Lys Arg Lys Tyr Gly Ser Lys Glu Thr Trp Trp Glu Thr         275 280 285 Trp Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val     290 295 300 <210> 22 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding 9lys-PRAS40 fusion protein <400> 22 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaactcgag atggcgtcgg ggcgccccga ggagctgtgg gaggccgtgg 120 tgggggccgc tgagcgcttc cgggcccgga ctggcacgga gctggtgctg ctgaccgcgg 180 ccccgccgcc accaccccgc ccgggcccct gtgcctatgc tgcccatggt cgaggagccc 240 tggcggaggc agcgcgccgt tgcctccacg acatcgcact ggcccacagg gctgccactg 300 ctgctcggcc tcctgcgccc ccaccagcac cacagccacc cagtcccaca cccagcccac 360 cccggcctac cctggccaga gaggacaacg aggaggacga ggatgagccc acagagacag 420 agacctccgg ggagcagctg ggcattagtg ataatggagg gctctttgtg atggatgagg 480 acgccaccct ccaggacctt ccccccttct gtgagtcaga ccccgagagt acagatgatg 540 gcagcctgag cgaggagacc cccgccggcc cccccacctg ctcagtgccc ccagcctcag 600 ccctacccac acagcagtac gccaagtccc tgcctgtgtc tgtgcccgtc tggggcttca 660 aggagaagag gacagaggcg cggtcatcag atgaggagaa tgggccgccc tcttcgcccg 720 acctggaccg catcgcggcg agcatgcgcg cgctggtgct gcgagaggcc gaggacaccc 780 aggtcttcgg ggacctgcca cggccgcggc ttaacaccag cgacttccag aagctgaagc 840 ggaaatattg aggatcc 857 <210> 23 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> 9Lys-PRAS40 fusion protein <400> 23 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Leu Glu Met Ala Ser Gly Arg   1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro Pro          35 40 45 Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala      50 55 60 Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His  65 70 75 80 Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro Pro Ala Pro Gln                  85 90 95 Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu             100 105 110 Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly         115 120 125 Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu     130 135 140 Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu 145 150 155 160 Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro                 165 170 175 Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala             180 185 190 Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg         195 200 205 Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Ser Pro     210 215 220 Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu 225 230 235 240 Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn                 245 250 255 Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr             260 265 <210> 24 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding PRAS40-9Lys fusion protein <400> 24 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccactc gagatggcgt 60 cggggcgccc cgaggagctg tgggaggccg tggtgggggc cgctgagcgc ttccgggccc 120 ggactggcac ggagctggtg ctgctgaccg cggccccgcc gccaccaccc cgcccgggcc 180 cctgtgccta tgctgcccat ggtcgaggag ccctggcgga ggcagcgcgc cgttgcctcc 240 acgacatcgc actggcccac agggctgcca ctgctgctcg gcctcctgcg cccccaccag 300 caccacagcc acccagtccc acacccagcc caccccggcc taccctggcc agagaggaca 360 acgaggagga cgaggatgag cccacagaga cagagacctc cggggagcag ctgggcatta 420 gtgataatgg agggctcttt gtgatggatg aggacgccac cctccaggac cttcccccct 480 tctgtgagtc agaccccgag agtacagatg atggcagcct gagcgaggag acccccgccg 540 gcccccccac ctgctcagtg cccccagcct cagccctacc cacacagcag tacgccaagt 600 ccctgcctgt gtctgtgccc gtctggggct tcaaggagaa gaggacagag gcgcggtcat 660 cagatgagga gaatgggccg ccctcttcgc ccgacctgga ccgcatcgcg gcgagcatgc 720 gcgcgctggt gctgcgagag gccgaggaca cccaggtctt cggggacctg ccacggccgc 780 ggcttaacac cagcgacttc cagaagctga agcggaaata tggatccaaa aaaaaaaaaa 840 aaaaaaaaaa aaaatag 857 <210> 25 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> PRAS40-9Lys fusion protein <400> 25 Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala   1 5 10 15 Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr              20 25 30 Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala          35 40 45 His Gly Arg Gly Ala Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp      50 55 60 Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro  65 70 75 80 Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro                  85 90 95 Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu             100 105 110 Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu         115 120 125 Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys     130 135 140 Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr 145 150 155 160 Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro                 165 170 175 Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly             180 185 190 Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly         195 200 205 Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala     210 215 220 Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro 225 230 235 240 Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr                 245 250 255 Gly Ser Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys             260 265 <210> 26 <211> 884 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding 9Lys-PRAS40-9Lys fusion protein <400> 26 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaactcgag atggcgtcgg ggcgccccga ggagctgtgg gaggccgtgg 120 tgggggccgc tgagcgcttc cgggcccgga ctggcacgga gctggtgctg ctgaccgcgg 180 ccccgccgcc accaccccgc ccgggcccct gtgcctatgc tgcccatggt cgaggagccc 240 tggcggaggc agcgcgccgt tgcctccacg acatcgcact ggcccacagg gctgccactg 300 ctgctcggcc tcctgcgccc ccaccagcac cacagccacc cagtcccaca cccagcccac 360 cccggcctac cctggccaga gaggacaacg aggaggacga ggatgagccc acagagacag 420 agacctccgg ggagcagctg ggcattagtg ataatggagg gctctttgtg atggatgagg 480 acgccaccct ccaggacctt ccccccttct gtgagtcaga ccccgagagt acagatgatg 540 gcagcctgag cgaggagacc cccgccggcc cccccacctg ctcagtgccc ccagcctcag 600 ccctacccac acagcagtac gccaagtccc tgcctgtgtc tgtgcccgtc tggggcttca 660 aggagaagag gacagaggcg cggtcatcag atgaggagaa tgggccgccc tcttcgcccg 720 acctggaccg catcgcggcg agcatgcgcg cgctggtgct gcgagaggcc gaggacaccc 780 aggtcttcgg ggacctgcca cggccgcggc ttaacaccag cgacttccag aagctgaagc 840 ggaaatatgg atccaaaaaa aaaaaaaaaa aaaaaaaaaa atag 884 <210> 27 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> 9Lys-PRAS40-9Lys fusion protein <400> 27 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ser Ser Gly Leu Val Pro Arg   1 5 10 15 Gly Ser His Leu Glu Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu              20 25 30 Ala Val Val Gly Ala Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu          35 40 45 Leu Val Leu Leu Thr Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro      50 55 60 Cys Ala Tyr Ala Ala His Gly Arg Gly Ala Ala Gul Ala Ala Arg  65 70 75 80 Arg Cys Leu His Asp Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala                  85 90 95 Arg Pro Pro Ala Pro Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro             100 105 110 Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu         115 120 125 Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser     130 135 140 Asp Asn Gly Gly Leu Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp 145 150 155 160 Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser                 165 170 175 Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro             180 185 190 Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser         195 200 205 Val Pro Val Trp Gly Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser     210 215 220 Asp Glu Glu Asn Gly Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala 225 230 235 240 Ala Ser Met Arg Ala Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val                 245 250 255 Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys             260 265 270 Leu Lys Arg Lys Tyr Gly Ser Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys         275 280 285 <210> 28 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding 9Arg-PRAS40 fusion protein <400> 28 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaga agaagaagaa 60 gaagaagaag aagactcgag atggcgtcgg ggcgccccga ggagctgtgg gaggccgtgg 120 tgggggccgc tgagcgcttc cgggcccgga ctggcacgga gctggtgctg ctgaccgcgg 180 ccccgccgcc accaccccgc ccgggcccct gtgcctatgc tgcccatggt cgaggagccc 240 tggcggaggc agcgcgccgt tgcctccacg acatcgcact ggcccacagg gctgccactg 300 ctgctcggcc tcctgcgccc ccaccagcac cacagccacc cagtcccaca cccagcccac 360 cccggcctac cctggccaga gaggacaacg aggaggacga ggatgagccc acagagacag 420 agacctccgg ggagcagctg ggcattagtg ataatggagg gctctttgtg atggatgagg 480 acgccaccct ccaggacctt ccccccttct gtgagtcaga ccccgagagt acagatgatg 540 gcagcctgag cgaggagacc cccgccggcc cccccacctg ctcagtgccc ccagcctcag 600 ccctacccac acagcagtac gccaagtccc tgcctgtgtc tgtgcccgtc tggggcttca 660 aggagaagag gacagaggcg cggtcatcag atgaggagaa tgggccgccc tcttcgcccg 720 acctggaccg catcgcggcg agcatgcgcg cgctggtgct gcgagaggcc gaggacaccc 780 aggtcttcgg ggacctgcca cggccgcggc ttaacaccag cgacttccag aagctgaagc 840 ggaaatattg aggatcc 857 <210> 29 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> 9Arg-PRAS40 fusion protein <400> 29 Arg Arg Arg Arg Arg Arg Arg Arg Leu Glu Met Ala Ser Gly Arg   1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro Pro          35 40 45 Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala      50 55 60 Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His  65 70 75 80 Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro Pro Ala Pro Gln                  85 90 95 Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu             100 105 110 Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly         115 120 125 Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu     130 135 140 Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu 145 150 155 160 Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro                 165 170 175 Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala             180 185 190 Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg         195 200 205 Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Ser Pro     210 215 220 Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu 225 230 235 240 Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn                 245 250 255 Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr             260 265 <210> 30 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding PRAS40-9 Arg fusion protein <400> 30 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccactc gagatggcgt 60 cggggcgccc cgaggagctg tgggaggccg tggtgggggc cgctgagcgc ttccgggccc 120 ggactggcac ggagctggtg ctgctgaccg cggccccgcc gccaccaccc cgcccgggcc 180 cctgtgccta tgctgcccat ggtcgaggag ccctggcgga ggcagcgcgc cgttgcctcc 240 acgacatcgc actggcccac agggctgcca ctgctgctcg gcctcctgcg cccccaccag 300 caccacagcc acccagtccc acacccagcc caccccggcc taccctggcc agagaggaca 360 acgaggagga cgaggatgag cccacagaga cagagacctc cggggagcag ctgggcatta 420 gtgataatgg agggctcttt gtgatggatg aggacgccac cctccaggac cttcccccct 480 tctgtgagtc agaccccgag agtacagatg atggcagcct gagcgaggag acccccgccg 540 gcccccccac ctgctcagtg cccccagcct cagccctacc cacacagcag tacgccaagt 600 ccctgcctgt gtctgtgccc gtctggggct tcaaggagaa gaggacagag gcgcggtcat 660 cagatgagga gaatgggccg ccctcttcgc ccgacctgga ccgcatcgcg gcgagcatgc 720 gcgcgctggt gctgcgagag gccgaggaca cccaggtctt cggggacctg ccacggccgc 780 ggcttaacac cagcgacttc cagaagctga agcggaaata tggatccaga agaagaagaa 840 gaagaagaag aagatag 857 <210> 31 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> PRAS40-9 Arg fusion protein <400> 31 Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala   1 5 10 15 Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr              20 25 30 Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala          35 40 45 His Gly Arg Gly Ala Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp      50 55 60 Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro  65 70 75 80 Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro                  85 90 95 Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu             100 105 110 Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu         115 120 125 Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys     130 135 140 Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr 145 150 155 160 Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro                 165 170 175 Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly             180 185 190 Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly         195 200 205 Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala     210 215 220 Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro 225 230 235 240 Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr                 245 250 255 Gly Ser Arg Arg Arg Arg Arg Arg Arg Arg Arg             260 265 <210> 32 <211> 884 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding 9Arg-PRAS40-9Arg fusion protein <400> 32 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaga agaagaagaa 60 gaagaagaag aagactcgag atggcgtcgg ggcgccccga ggagctgtgg gaggccgtgg 120 tgggggccgc tgagcgcttc cgggcccgga ctggcacgga gctggtgctg ctgaccgcgg 180 ccccgccgcc accaccccgc ccgggcccct gtgcctatgc tgcccatggt cgaggagccc 240 tggcggaggc agcgcgccgt tgcctccacg acatcgcact ggcccacagg gctgccactg 300 ctgctcggcc tcctgcgccc ccaccagcac cacagccacc cagtcccaca cccagcccac 360 cccggcctac cctggccaga gaggacaacg aggaggacga ggatgagccc acagagacag 420 agacctccgg ggagcagctg ggcattagtg ataatggagg gctctttgtg atggatgagg 480 acgccaccct ccaggacctt ccccccttct gtgagtcaga ccccgagagt acagatgatg 540 gcagcctgag cgaggagacc cccgccggcc cccccacctg ctcagtgccc ccagcctcag 600 ccctacccac acagcagtac gccaagtccc tgcctgtgtc tgtgcccgtc tggggcttca 660 aggagaagag gacagaggcg cggtcatcag atgaggagaa tgggccgccc tcttcgcccg 720 acctggaccg catcgcggcg agcatgcgcg cgctggtgct gcgagaggcc gaggacaccc 780 aggtcttcgg ggacctgcca cggccgcggc ttaacaccag cgacttccag aagctgaagc 840 ggaaatatgg atccagaaga agaagaagaa gaagaagaag atag 884 <210> 33 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> 9Arg-PRAS40-9Arg fusion protein <400> 33 Arg Arg Arg Arg Arg Arg Arg Arg Ser Ser Gly Leu Val Pro Arg   1 5 10 15 Gly Ser His Leu Glu Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu              20 25 30 Ala Val Val Gly Ala Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu          35 40 45 Leu Val Leu Leu Thr Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro      50 55 60 Cys Ala Tyr Ala Ala His Gly Arg Gly Ala Ala Gul Ala Ala Arg  65 70 75 80 Arg Cys Leu His Asp Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala                  85 90 95 Arg Pro Pro Ala Pro Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro             100 105 110 Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu         115 120 125 Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser     130 135 140 Asp Asn Gly Gly Leu Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp 145 150 155 160 Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser                 165 170 175 Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro             180 185 190 Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser         195 200 205 Val Pro Val Trp Gly Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser     210 215 220 Asp Glu Glu Asn Gly Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala 225 230 235 240 Ala Ser Met Arg Ala Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val                 245 250 255 Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys             260 265 270 Leu Lys Arg Lys Tyr Gly Ser Arg Arg Arg Arg Arg Arg Arg Arg Arg         275 280 285

Claims (3)

A PRAS40 fusion protein consisting of 9 to 15 amino acid residues and having a transporting domain comprising 3/4 or more of arginine or lysine residues covalently bonded to at least one terminal of PRAS40 (Proline-rich Akt substrate 40) A pharmaceutical composition for preventing or treating autosomal dominant polycystic kidney disease.
The method of claim 1, wherein the transport domain is at least one of HIV Tat 49-57 residue, oligo lysine, oligoarginine, or oligo (lysine, arginine) Or a pharmaceutical composition for therapeutic use.
The fusion protein according to claim 1, wherein the PRAS40 fusion protein has the amino acid sequence of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, Or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of autosomal dominant polycystic kidney disease.

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