KR20130003243A - Cell-transducible pras40 fusion protein - Google Patents

Cell-transducible pras40 fusion protein Download PDF

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KR20130003243A
KR20130003243A KR1020110064429A KR20110064429A KR20130003243A KR 20130003243 A KR20130003243 A KR 20130003243A KR 1020110064429 A KR1020110064429 A KR 1020110064429A KR 20110064429 A KR20110064429 A KR 20110064429A KR 20130003243 A KR20130003243 A KR 20130003243A
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pras40
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최수영
황현숙
음원식
김대원
박진서
김덕수
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한림대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Abstract

PURPOSE: A PRAS40 fusion protein with improved cell penetration efficiency is provided to effectively suppress reactive oxygen species(ROS) generation and to be used s a therapeutic agent for treating Parkinson`s disease. CONSTITUTION: A PRAS40 fusion protein contains 9-15 amino acid residues and improves cell penetration efficiency by covalent bond of a transport domain with at least one end of PRAS40(proline-rich Akt substrate 40). The transport domain is HIV Tat 49-57 residue, oligolysine, oligoarginine, or oligo(lysine,arginine). A pharmaceutical composition for preventing and treating Parkinson`s disease, Huntingtun`s disease, Alzheimer`s disease, cerebral ischemia contains the PRAS40 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

Description

세포침투 효율이 향상된 ＰＲＡＳ40 융합단백질 {Cell-transducible PRAS40 fusion protein}Cell-transducible PRAS40 fusion protein with improved cell penetration efficiency

본 발명은 PRAS40 융합단백질을 유효성분으로 포함하는 파킨슨병, 헌팅턴병, 알츠하이머병, 뇌허혈을 포함하는 뇌신경질환의 예방 및 치료용 약제학적 조성물 및 신경질환 예방 및 개선용 건강기능식품 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention and treatment of cerebral neurological diseases, including Parkinson's disease, Huntington's disease, Alzheimer's disease, cerebral ischemia, and a nutraceutical composition for preventing and improving neurological diseases.

활성산소종은 다양한 생리적 조건에서 생성되며, 세포내 거대분자에 손상을 초래한다. 뇌는 총 체중의 2~3%만을 구성하고 있지만, 신체에 사용되는 산소의 약 20%를 소비한다. 따라서, 산화 스트레스로 인해 유발된 손상된 생체분자들이 뇌에 축적되어 알츠하이머병, 파킨슨병 및 헌팅턴병과 같은 신경퇴행 질병을 일으킬 수 있다는 가능성이 제기되고 있다 [Chinta, S. J.; Anderson, J. K. Biochim. Biophys. Acta 1780:1362-1367; 2008, de Moura, M. B. et al. Environ. Mol. Mutagen. 51:391-405; 2010, Mates, J. M. Toxicol. 83:83-104; 2000]. 특히 파킨슨병은 흑색질 치밀부 (substantia nigra, pars compacta)에 도파민 뉴런이 파괴되고 유비퀴틴 및 α-시누클라인과 같은 단백질로 이루어진 루이체 (Lewy bodies)가 축적되는 것이 특징인데, 이것이 진전, 운동완만증, 강직 및 자세 불안정과 같은 증세를 유발한다 [Dawson, T. M.; Dawson, V. L. Science 302:819-822; 2003, Eriksen, J. L. et al. Arch. Neurol. 62:353-357; 2005, Forno, L. S. J. Neuropathol. Exp. Neurol. 55:259-272; 1996]. 현재까지 파킨슨병의 정확한 발병 메카니즘은 밝혀지지 않았지만, 유전인자, 노화, 제초제 및 신경독성물질 등이 파킨슨병과 관련이 있다 [Miller, R. L. et al. Neurochem. Res. 34:55-65; 2009]. Free radicals are produced under various physiological conditions and cause damage to intracellular macromolecules. The brain makes up only 2-3% of your total body weight, but consumes about 20% of the body's oxygen. Thus, the possibility that damaged biomolecules induced by oxidative stress can accumulate in the brain and cause neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease [Chinta, SJ; Anderson, JK Biochim. Biophys. Acta 1780 : 1362-1367; 2008, de Moura, MB et al. Environ. Mol. Mutagen . 51 : 391-405; 2010, Mates, JM Toxicol. 83 : 83-104; 2000]. In particular, Parkinson's disease is characterized by the destruction of dopamine neurons and accumulation of Lewy bodies made up of proteins such as ubiquitin and α-synucline in the substantia nigra (pars compacta). Causes symptoms such as stiffness and postural instability [Dawson, TM; Dawson, VL Science 302 : 819-822; 2003, Eriksen, JL et al. Arch. Neurol . 62 : 353-357; 2005, Forno, LS J. Neuropathol. Exp. Neurol . 55 : 259-272; 1996]. To date, the exact pathogenesis of Parkinson's disease has not been determined, but genetic factors, aging, herbicides and neurotoxics have been linked to Parkinson's disease [Miller, RL et al. Neurochem. Res . 34 : 55-65; 2009].

파킨슨 환자의 도파민 뉴런 손실은 화학물질 및 농약 노출과 관련이 있다. 친유성 물질인 MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)는 혈관-뇌 장벽을 쉽게 통과하는데, 이것은 아교세포 (glial cells)에서 모노아민 산화효소 B에 의해 MPP+ (1-methyl-4-phenylpyridinium)로 전환된다. 결과적으로 MPP+는 뇌 흑색질 내에서 미토콘드리아에서 활성산소종을 생성하고, 전자전달계의 복합체 Ⅰ (complex I)을 차단하며, 세포사멸 일련반응을 활성화시키고 도파민 뉴런을 파괴한다 [Yokoyama, H. et al. Neurol. Sci. 29:293-301; 2008, Eberhardt, O.; Schulz, J. B. Toxicol. Lett. 139:135-151; 2003, Fukuda, T. Neuropathology 21:323-332; 2001, Kalivendi, S. V. et al. Biochem. J. 371:151-164; 2003]. 자유 라디칼과 퀴닌 (quinine)으로 산화되는 신경독성물질인 6-OHDA (6-Hydroxydopamine)는 MPP+와 함께 파킨슨병 실험모델에 이용되어 왔다 [Miller, R. L. et al. Neurochem. Res. 34:55-65; 2009]. 그러나, MPTP 또는 6-OHDA에 노출시키면 파킨슨병의 마크인 루이체가 생성되지 않는다. 다른 연구결과들은 로테논 (rotenone) 및 파라쿠앗 (paraquat: PQ)이 산화 스트레스를 증가시키고, 도파민 뉴런의 퇴화를 유도하여 마침내 동물 모델에서 파킨슨병을 유발한다는 증거를 제시하였다 [Miller, R. L. et al. Neurochem. Res. 34:55-65; 2009, Synder, S. H. et al. Nature 317:198-199; 1985].Dopamine neuronal loss in Parkinson's patients is associated with exposure to chemicals and pesticides. The lipophilic substance MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) easily crosses the vascular-brain barrier, which is expressed by MPP by monoamine oxidase B in glial cells. + (1-methyl-4-phenylpyridinium). As a result, MPP + produces reactive oxygen species in the mitochondria within the brain melanoma, blocks the complex I of the electron transport system, activates apoptosis serial reactions and destroys dopamine neurons [Yokoyama, H. et al. . Neurol. Sci. 29 : 293-301; 2008, Eberhardt, O .; Schulz, JB Toxicol. Lett . 139 : 135-151; 2003, Fukuda, T. Neuropathology 21 : 323-332; 2001, Kalivendi, SV et al. Biochem. J. 371 : 151-164; 2003]. 6-OHDA (6-Hydroxydopamine), a neurotoxic substance oxidized with free radicals and quinine, has been used in Parkinson's disease experimental models with MPP + [Miller, RL et al. Neurochem. Res . 34 : 55-65; 2009]. However, exposure to MPTP or 6-OHDA does not produce Lewy bodies, a mark of Parkinson's disease. Other studies provide evidence that rotenone and paraquat (PQ) increase oxidative stress, induce degeneration of dopamine neurons, and finally cause Parkinson's disease in animal models [Miller, RL et al. . Neurochem. Res . 34 : 55-65; 2009, Synder, SH et al. Nature 317 : 198-199; 1985].

mTOR (Mammalian target of rapamycin)는 PI3K (phosphoinositide 3-kinase) 관련 카이네이즈 군에 속하는데, 세포 에너지, 아미노산과 같은 영양분 및 인슐린, 인슐린-유사 성장인자와 같은 동화작용 성장인자의 결핍에 반응하여 단백질 합성 조절, 세포 성장 및 세포사멸의 조절에 중추적인 역할을 수행한다 [Dunlop, E. A.; Tee, A. R. Cell. Signal. 21:827-835; 2009, Vander Haar, E, et al. Nat. Cell Biol. 9:316-323; 2007]. 또한, 많은 종양에서 mTOR는 구조적으로 활성화되는데, 이는 종양유전자 활성화 조절 및 종양억제 유전자의 손실 실패로 인한 것이다 [Garcia, J. A.; Danielpour, D. Mol. Cancer Ther. 7:1347-1354; 2008, Madhunapantula, S. V. et al. Cancer Res. 67:3626-3636; 2007]. mTORC1 (mTOR complexes containing Raptor) 또는 mTORC2 (mTOR complexes containing Rictor)는 두 가지 구별되는 기능을 발휘하며, 다른 메카니즘에 의해 조절된다. mTORC1은 PI3K/AKT 경로를 통해 활성화되며, S6K (S6 kinase) 및 4E-BP (eIF-4E binding protein)을 포함하는 다양한 기질을 인산화하여, 단백질 합성, 리보솜 생합성, 세포 증식 및 성장을 조절한다 [Dunlop, E. A.; Tee, A. R. Cell. Signal. 21:827-835; 2009, Sancak, Y. et al. Mol. Cell 25:903-915; 2007, Dey, N. et al. J Cell Physiol. 225: 27-41; 2010]. 특히, 몇몇 연구결과는 AKT에 의해 활성화되는 기질로 밝혀진 PRAS40이 Raptor와 결합을 통해 mTORC1과 결합할 수 있고 mTORC1 활성화에 관여하는 것으로 나타났다 [Kovacina, K.S. et al. J. Biol. Chem. 278:10189; 2003, Yu, F. et al. J. Cereb. Blood Flow Metab. 28:44-52; 2008, Fonseca, B. D. et al. J. Biol. Chem. 282:24514-24524; 2007]. PRAS40 siRNA를 이용한 다른 연구결과들은 mTORC1 결합에 필요한 TOR-신호 (TOS) 모티프를 포함하는 PRAS40이 mTORC1의 기질이 될 수 있다고 제시하였다 [Oshiro, N. et al. J. Biol. Chem. 282:20329-20339; 2007, Wang, L. et al. J. Biol. Chem. 283:15619-15627; 2008]. 뿐만 아니라, PRAS40은 세포 대사에 다양한 역할을 수행하는 14-3-3-sigma (stratifin)과 결합한다 [Pozuelo Rubio, M. et al. Biochem. J. 379:395-408; 2004]. The mTOR (Mammalian target of rapamycin) belongs to a group of phosphoinositide 3-kinase (PI3K) -related kinases, which synthesize protein in response to cellular energy, nutrients such as amino acids, and anabolic growth factors such as insulin and insulin-like growth factors. Plays a pivotal role in the regulation, regulation of cell growth and apoptosis [Dunlop, EA; Tee, AR Cell. Signal . 21 : 827-835; 2009, Vander Haar, E, et al. Nat. Cell Biol . 9 : 316-323; 2007]. In addition, mTOR is structurally activated in many tumors due to oncogene activation regulation and failure to lose tumor suppressor genes [Garcia, JA; Danielpour, D. Mol. Cancer Ther. 7 : 1347-1354; 2008, Madhunapantula, SV et al. Cancer Res. 67 : 3626-3636; 2007]. mTORC1 (mTOR complexes containing Raptor) or mTORC2 (mTOR complexes containing Rictor) exerts two distinct functions and is regulated by different mechanisms. mTORC1 is activated through the PI3K / AKT pathway and phosphorylates a variety of substrates including S6K (S6 kinase) and 4E-BP (eIF-4E binding protein) to regulate protein synthesis, ribosomal biosynthesis, cell proliferation and growth [ Dunlop, EA; Tee, AR Cell. Signal . 21 : 827-835; 2009, Sancak, Y. et al. Mol. Cell 25: 903-915; 2007, Dey, N. et al. J Cell Physiol . 225 : 27-41; 2010]. In particular, several studies have shown that PRAS40, which is identified as a substrate activated by AKT, can bind mTORC1 through Raptor binding and is involved in mTORC1 activation [Kovacina, KS et al. J. Biol. Chem . 278 : 10189; 2003, Yu, F. et al. J. Cereb. Blood Flow Metab . 28 : 44-52; 2008, Fonseca, BD et al. J. Biol. Chem . 282 : 24514-24524; 2007]. Other studies using PRAS40 siRNA suggested that PRAS40, which contains the TOR-signaling (TOS) motif required for mTORC1 binding, could be a substrate for mTORC1 [Oshiro, N. et al. J. Biol. Chem . 282 : 20329-20339; 2007, Wang, L. et al. J. Biol. Chem. 283 : 15619-15627; 2008]. In addition, PRAS40 binds to 14-3-3-sigma (stratifin), which plays various roles in cellular metabolism [Pozuelo Rubio, M. et al. Biochem. J. 379 : 395-408; 2004].

몇몇 펩타이드 및 단백질 약제는 치료 가능성을 갖고 있지만, 분자 크기, 친수성 및 생화학적 특성으로 인해 타겟 세포 및 조직에 효과적으로 전달되지 못한다. 특히 뇌의 항상성을 유지시키는데 중추적인 역할을 수행하는 혈관-뇌 장벽의 존재는 치료제가 뇌로 확산되는 것을 제한하여 치료 효과를 낮춘다 [Egleton, R. D.; Davis, T. P. Peptides 18:1431-1439; 1997]. 많은 연구자들은 약제를 화학적으로 변형하는 방법 그리고 전달자와 결합시키는 방법 등의 전략이 약제의 생체 내 활용성을 증대시킬 수 있음을 제안하였다 [Egleton, R. D.; Davis, T. P. Peptides 18:1431-1439; 1997, Dietz, G. P. Curr. Pharm. Biotechol. 11:167-174; 2010, Wadia, J.; Dowdy, S. F. Curr. Opin. Biotechnol. 13:52-56; 2002].
Some peptide and protein agents have therapeutic potential but are not effectively delivered to target cells and tissues due to their molecular size, hydrophilicity, and biochemical properties. In particular, the presence of the vascular-brain barrier, which plays a pivotal role in maintaining homeostasis of the brain, limits the spread of the therapeutic agent to the brain and lowers the therapeutic effect [Egleton, RD; Davis, TP Peptides 18 : 1431-1439; 1997]. Many researchers have suggested that strategies such as chemically modifying a drug and combining it with a carrier can increase the bioavailability of the drug [Egleton, RD; Davis, TP Peptides 18 : 1431-1439; 1997, Dietz, GP Curr. Pharm. Biotechol. 11 : 167-174; 2010, Wadia, J .; Dowdy, SF Curr. Opin. Biotechnol . 13 : 52-56; 2002].

본 발명은 파킨슨병을 비롯한 뇌신경질환의 효과적인 치료제를 제공하는 것을 목적으로 한다.
An object of the present invention is to provide an effective therapeutic agent for cerebral neurological diseases including Parkinson's disease.

상기 목적을 달성하기 위하여 본 발명자들은 PRAS40이 단백질 수송 도메인과 융합되면 혈관-뇌 장벽을 넘어 마우스 모델에서 파킨슨병 발병을 방지할 수 있는지를 연구하였다.
In order to achieve the above object, the present inventors studied whether PRAS40 can be prevented from developing Parkinson's disease in a mouse model by crossing the vascular-brain barrier when fused with a protein transport domain.

사람 면역결핍 바이러스(HIV-1)의 Tat 단백질을 비롯한 단백질 수송 도메인 (Protein Transducing Domain)이 이형 단백질을 세포 내로 이동시키는데 운반체로써 이용될 수 있다는 것이 여러 연구자들에 의하여 밝혀졌다. 따라서 본 발명에서는 이러한 단백질 수송 도메인이 PRAS40을 세포 내로 운반시킬 수 있는지, 운반된 PRAS40 융합단백질이 세포 내에서 단백질의 기능을 나타내는지를 알아보고자 수행하였다.Protein transducing domains, including the Tat protein of human immunodeficiency virus (HIV-1), have been found to be used as carriers to transport heterologous proteins into cells. Therefore, the present invention was performed to determine whether the protein transport domain can transport PRAS40 into cells, and whether the transported PRAS40 fusion protein exhibits the function of proteins in the cells.

본 발명에서는 단백질을 세포 및 조직 내로 운반하는 단백질 수송 도메인을 외부 단백질인 PRAS40에 융합시켰고, 융합단백질을 대장균에서 과대발현시켰으며, 금속 킬레이팅 친화 크로마토그래피로 쉽고 편리하게 정제하였다. In the present invention, the protein transport domain that carries the protein into cells and tissues was fused to the external protein PRAS40, the fusion protein was overexpressed in E. coli, and purified easily and conveniently by metal chelating affinity chromatography.

본 발명의 구체적인 일 실시예에서는, PRAS40 융합단백질을 과대발현시키고 쉽게 정제할 수 있는 PRAS40 발현벡터를 개발하였다. 이 발현벡터는 인간 PRAS40 cDNA (서열번호 8), 단백질 수송 도메인 그리고 6개의 히스티딘이 연속적으로 연결되어 있다. 이 발현벡터를 이용하여 PRAS40 융합단백질을 대장균에서 과대발현시켜 Ni2+-친화 크로마토그래피 컬럼을 이용하여 정제하였다. PRAS40 융합단백질의 과대발현은 상당히 높게 나타났으며, 이 결과로 정제된 단백질의 양도 높게 나타났다.In a specific embodiment of the present invention, PRAS40 that can overexpress and easily purify the PRAS40 fusion protein Expression vectors were developed. This expression vector is a continuous linkage of human PRAS40 cDNA (SEQ ID NO: 8), protein transport domain and six histidines. Using this expression vector, PRAS40 fusion protein was overexpressed in E. coli and purified using a Ni 2+ -affinity chromatography column. The overexpression of the PRAS40 fusion protein was significantly higher, resulting in a higher amount of purified protein.

또한, 본 발명자들은 정제된 융합단백질이 효과적으로 생물학적인 활성을 지니고 있으며, 세포 내로 운반된다는 것을 배양된 신경세포 및 동물실험을 통하여 확인하였다. 그리고, PRAS40 융합단백질이 신경 세포 내로 운반되며 신경 세포사를 효과적으로 보호함을 밝혀냈다. 즉, 배양된 신경세포에 정제된 PRAS40 융합단백질이 농도 및 시간 의존적으로 세포에 운반되는 것을 웨스턴 블랏 방법으로 확인하였다. 또한, 세포 내로 투과된 PRAS40 융합단백질은 세포 내에서 최소 24시간 지속적으로 유지되었다. In addition, the inventors have confirmed through cultured neurons and animal experiments that the purified fusion protein has an effective biological activity and is transported into cells. In addition, it has been found that PRAS40 fusion protein is transported into nerve cells and effectively protects nerve cell death. That is, it was confirmed by Western blot method that the PRAS40 fusion protein purified in cultured neurons is transported to the cells in a concentration and time-dependent manner. In addition, PRAS40 fusion protein permeated into cells was maintained for at least 24 hours in the cells.

본 발명자들은 인 비트로와 인 비보에서 PRAS40 융합단백질이 MPP+에 의해 유도된 산화 스트레스로부터 도파민 뉴런 세포를 보호할 수 있는지를 시험하였고, 그 결과 도파민 뉴런 세포의 생존율이 증가하였다. 세포도입된 PRAS40 융합단백질은 MPP+에 의해 유도된 활성산소종 생산을 효과적으로 억제하였고, SH-SY5Y 세포에서 세포사멸을 상당한 정도로 약화시켰다. 또한, MPTP에 의해 유도된 마우스 파킨슨병 모델에서 크레실 바이올렛 염색 및 타이로신 수산화효소 면역염색은 PRAS40 융합단백질이 뇌혈관장벽을 효율적으로 가로질러 흑색질에서 도파민 뉴런세포를 보호할 수 있음을 보여주었다. 파킨슨병 외에도 헌팅턴병, 알츠하이머병, 뇌허혈과 같은 뇌신경질환은 신경세포 사멸과 밀접한 관련을 가지므로, PRAS40 융합단백질의 위와 같은 신경세포 사멸 저해효과는 PRAS40 융합단백질을 함유한 조성물을 이들 뇌신경질환의 예방 및 치료, 개선 용도로 이용할 수 있음을 말해준다.We tested whether the PRAS40 fusion protein could protect dopamine neuron cells from oxidative stress induced by MPP + in vitro and in vivo, resulting in increased dopamine neuron cell survival. Transduced PRAS40 fusion protein effectively inhibited the production of free radical species induced by MPP + and significantly attenuated apoptosis in SH-SY5Y cells. In addition, cresyl violet staining and tyrosine hydroxylase immunostaining in the MPTP-induced mouse Parkinson's disease model showed that PRAS40 fusion protein can efficiently protect dopamine neurons from melanocytes across the cerebrovascular barrier. In addition to Parkinson's disease, neurological diseases such as Huntington's disease, Alzheimer's disease, and cerebral ischemia are closely related to neuronal cell death. It can be used for treatment or improvement.

본 발명의 구성을 좀더 자세히 설명하면, 본 발명의 일 실시예에서 본 발명자들은 먼저 Tat-PRAS40 융합단백질을 과대 발현시키고 쉽게 정제할 수 있는 Tat-PRAS40 발현 벡터를 개발하였다. 이 발현 벡터는 인간 PRAS40, Tat 형질도입 부위의 9개 아미노산(Tat 49-57), 그리고 아미노산 말단부분에 6개의 히스티딘 잔기를 발현시킬 수 있는 cDNA를 포함하고 있다.In more detail the configuration of the present invention, in one embodiment of the present invention we first developed a Tat-PRAS40 expression vector that can overexpress and easily purify the Tat-PRAS40 fusion protein. The 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 acid terminal.

이 발현벡터를 이용하여 Tat-PRAS40 융합단백질을 대장균에서 과대 발현시켰으며 Ni-친화 크로마토그래피를 이용하여 정제하였다. 배양된 세포에 Tat-PRAS40 융합단백질이 시간 및 농도 의존적으로 세포에 운반되는 것을 웨스턴 블랏으로 확인하였다. 세포 내로 투과된 Tat-PRAS40 융합단백질은 세포 내에서 최소 24시간 지속적으로 유지되었으며, 산화 스트레스에 의한 세포사멸을 억제하였다.Using this expression vector, the Tat-PRAS40 fusion protein was overexpressed in E. coli and purified using Ni-affinity chromatography. Western blot confirmed that the Tat-PRAS40 fusion protein was delivered to the cells in time and concentration-dependently in cultured cells. Tat-PRAS40 fusion protein permeated into cells was maintained for at least 24 hours in cells and inhibited cell death by oxidative stress.

이러한 결과는 Tat-PRAS40 융합단백질이 세포 내로 투과가 잘 일어나고, 세포 내에서 PRAS40의 기능을 잘 나타내고 있음을 의미한다. 따라서 이러한 Tat-PRAS40 융합단백질은 활성산소종과 관련된 증세 또는 파킨슨병 및 뇌허혈 등의 뇌신경질환에 응용할 수 있는 가능성을 제시해 준다.
These results indicate that the Tat-PRAS40 fusion protein permeates well into cells and well represents the function of PRAS40 in cells. Therefore, this Tat-PRAS40 fusion protein suggests the possibility of application to symptoms related to reactive oxygen species or neurological diseases such as Parkinson's disease and cerebral ischemia.

PRAS40 융합단백질을 포함하여 수송도메인 융합단백질을 유효성분으로 함유하는 약제학적 조성물은 약제학적 분야에서 통상적으로 허용되는 담체와 함께 배합하여 통상적인 방법에 의해 경구 또는 주사 형태 등으로 제형화할 수 있다. 경구용 조성물로는 예를 들면 정제 및 젤라틴 캡슐이 있으며, 이들은 활성 성분 이외에도 희석제(예: 락토스, 덱스트로스, 수크로스, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활탁제(예: 실리카, 탤크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/또는 폴리에틸렌 글리콜)을 함유하고, 정제는 또한 결합제(예: 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 메틸셀룰로스, 나트륨 카복시메틸셀룰로스 및/또는 폴리비닐피롤리돈)를 함유하며, 경우에 따라서 붕해제(예: 전분, 한천, 알긴산 또는 그의 나트륨염) 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제 및 감미제를 함유하는 것이 바람직하다. 주사용 조성물은 등장성 수용액 또는 현탁액이 바람직하고, 언급한 조성물은 멸균되고/되거나 보조제(예: 방부제, 안정화제, 습윤제 또는 유화제 용액 촉진제, 삼투압 조절을 위함 염/또는 완충제)를 함유한다. 또한 이들은 기타 치료적으로 유용한 물질을 함유할 수 있다.Pharmaceutical compositions containing a transport domain fusion protein as an active ingredient, including the PRAS40 fusion protein, may be formulated in oral or injectable form and the like by a conventional method by combining with a carrier generally acceptable in the pharmaceutical field. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, may contain diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants (e.g. silica, talc) , Stearic acid and its magnesium or calcium salts and / or polyethylene glycols, the tablets also contain binders (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone). ), And optionally a disintegrant (eg starch, agar, alginic acid or its sodium salt) or boiling mixture and / or absorbent, colorant, flavor and sweetener. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries such as preservatives, stabilizers, wetting or emulsifier solution promoters, salts or buffers for controlling osmotic pressure. They may also contain other therapeutically valuable substances.

이와 같이 제조된 약제학적 제제는 목적하는 바에 따라 경구로 투여하거나, 비경구 방식 즉, 정맥 내 , 피하, 복강 내 투여 또는 국소적용할 수 있으며, 천식에 적용하는 경우에는 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자들에게 널리 알려진 바와 같이 흡입 방식이나 스프레이 방식의 제제로 제형화할 수 있다. 용량은 일일 투여량 0.0001~100㎎/㎏을 1 내지 수회에 나누어 투여할 수 있다. 특정 환자에 대한 투여용량 수준은 환자의 체중, 연령, 성별, 건강상태, 투여시간, 투여방법, 배설율, 질환의 중증도 등에 따라 변화될 수 있다.The pharmaceutical preparations thus prepared may be administered orally as desired, or parenterally, i.e., intravenously, subcutaneously, intraperitoneally, or topically, and when applied to asthma in the art to which this invention pertains. As is well known to those skilled in the art, it may be formulated into inhaled or sprayed formulations. The dose may be administered by dividing the daily dose between 0.0001 and 100 mg / kg in one to several times. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

나아가, 본 발명은 상기 PRAS40 융합단백질을 유효성분으로 하고 약학적으로 허용되는 담체를 포함하는 것을 특징으로 하는, 파킨슨병, 루게릭병, 헌팅턴병, 알츠하이머병과 같은 신경 퇴행성 질환, 뇌허혈과 같은 신경질환의 예방과 치료에 유용한 약제학적 조성물을 제공한다.Furthermore, the present invention is characterized in that the PRAS40 fusion protein as an active ingredient and comprising a pharmaceutically acceptable carrier, neurodegenerative diseases such as Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Alzheimer's disease, prevention of neurological diseases such as cerebral ischemia Provided are pharmaceutical compositions useful for treatment.

본 발명은 또한, 상기 PRAS40 융합단백질을 유효성분으로 하며, 파킨슨병, 루게릭병, 헌팅턴병, 알츠하이머병, 뇌허혈과 같은 뇌신경질환 개선용 건강 기능성 식품 조성물을 제공한다.
The present invention also provides the PRAS40 fusion protein as an active ingredient, and provides a functional food composition for improving neurological diseases such as Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Alzheimer's disease, and cerebral ischemia.

본 발명은 또한 PRAS40 단백질을 세포 내로 효율적으로 전달하기 위한 방법을 제공한다. 본 발명에 따른 PRAS40 단백질 분자의 세포 내 전달은 HIV Tat을 비롯한 단백질 수송 도메인이 공유결합된 형태의 융합단백질을 구성하여 수행된다. 본 발명의 상기 수송도메인의 일례로는 aa 49-57의 아홉 개의 아미노산으로 구성되고, 서열번호 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 또는 33과 같은 아미노산 서열로 이루어진 HIV Tat 펩타이드를 들 수 있다. 그러나, 본 발명의 단백질 수송 도메인이 서열번호 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 또는 33의 Tat 펩타이드로만 한정되는 것은 아니고, Tat의 아미노산 서열 일부 치환이나 부가, 결여로 Tat 펩타이드와 유사한 기능을 하는 펩타이드를 제조하는 것이 본 발명이 속하는 분야에서 통상의 지식을 가진 당업자에게는 용이하므로, 7~15개의 아미노산으로 구성되고, 라이신 또는 아르기닌을 4개 이상 다수 포함하는 단백질 수송 도메인과 이로부터 아미노산 일부 치환으로 동일·유사한 단백질 수송기능을 수행하는 단백질 수송 도메인을 이용한 융합단백질도 본 발명의 범위에 속함은 자명하다고 할 것이다.The present invention also provides a method for efficiently delivering 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 the form of a covalently bonded protein transport domain including HIV Tat. One example of the transport domain of the present invention consists of nine amino acids of aa 49-57 and amino acids such as SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33 And HIV Tat peptides consisting of sequences. However, the protein transport domain of the present invention is not limited to the Tat peptide of SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, and partially substituted with the amino acid sequence of Tat In addition, it is easy for those skilled in the art to manufacture a peptide having a function similar to that of the Tat peptide by lacking, and is composed of 7 to 15 amino acids and includes a plurality of lysine or arginine. It will be apparent that a fusion protein using a protein transport domain and a protein transport domain that performs the same or similar protein transport function by substituting a partial amino acid therefrom are also included in the scope of the present invention.

구체적으로, 본 발명은 PRAS40 융합단백질, 이 융합단백질을 제조하기 위한 재조합 뉴클레오타이드와 벡터, 이 융합단백질을 포함하는 치료, 예방 목적의 약학 조성물, 건강 기능성 식품 등에 관한 것이다.Specifically, the present invention relates to a PRAS40 fusion protein, a recombinant nucleotide and a vector for producing the fusion protein, a treatment comprising the fusion protein, a pharmaceutical composition for the purpose of prevention, a health functional food, and the like.

본 발명의 상세한 설명 등에서 사용되는 주요 용어의 정의는 다음과 같다.Definitions of main terms used in the detailed description of the present invention are as follows.

"PRAS40 융합단백질"이란 단백질 수송 도메인과 PRAS40을 포함하며, 수송 도메인과 목표 단백질(즉, 본 발명에서는 PRAS40을 의미함)의 유전적 융합이나 화학 결합으로 형성된 공유결합 복합체를 의미한다. 본 명세서에서는 "Tat-PRAS40", "PRAS40 융합단백질" 등과 혼용하였다."PRAS40 fusion protein" refers to a covalent complex comprising a protein transport domain and PRAS40, formed by genetic fusion or chemical bonding of a transport domain with a target protein (ie, PRAS40 in the present invention). In the present specification, "Tat-PRAS40", "PRAS40 fusion protein" and the like are mixed.

"목표 단백질"이란 본래 표적 세포로 들어갈 수 없거나, 본래 유용한 속도로 표적 세포로 들어갈 수 없는 수송도메인 또는 이의 단편이 아닌 분자로서, 수송도메인과 융합되기 전의 분자 그 자체 또는 수송도메인-목표 단백질 복합체의 목표 단백질 부분을 의미한다. 목표 단백질로서는 폴리펩티드, 단백질, 펩타이드를 포함하며, 본 발명에서는 PRAS40을 의미한다.A “target protein” is a molecule that is not originally a transport domain or fragment thereof that cannot enter the target cell or is not able to enter the target cell at its original useful rate, and is a molecule of the transport domain or of the transport domain-target protein complex prior to fusion with the transport domain. Refers to the target protein portion. The target protein includes a polypeptide, a protein, a peptide, and means PRAS40 in the present invention.

"융합단백질"이란 수송도메인 및 한 개 이상의 목표 단백질 부분을 포함하며, 수송도메인과 목표 단백질의 유전적 융합이나 화학 결합으로 형성된 복합체를 의미한다."Fused protein" means a complex comprising a transport domain and one or more target protein moieties and formed by genetic fusion or chemical bonding of the transport domain and the target protein.

또한, 상기 "유전적 융합"이란 단백질을 코딩하는 DNA 서열의 유전적 발현을 통해서 형성된 선형, 공유결합으로 이루어진 연결을 의미한다.In addition, the "genetic fusion" means a linear, covalent linkage formed through the genetic expression of the DNA sequence encoding the protein.

또한, "표적 세포"란 수송도메인에 의해 목표 단백질이 전달되는 세포를 의미하는 것으로서, 표적 세포는 체내 또는 체외의 세포를 말한다. 즉, 표적 세포는 체내 세포, 다시 말하여 살아있는 동물 또는 인간의 장기 또는 조직을 구성하는 세포 또는 살아있는 동물 또는 인간에서 발견되는 미생물을 포함하는 의미이다. 또한, 표적 세포는 체외 세포, 즉 배양된 동물세포, 인체 세포 또는 미생물을 포함하는 의미이다.In addition, "target cell" means a cell to which a target protein is delivered by a transport domain, and a target cell refers to a cell in or outside the body. That is, a target cell is meant to include cells in the body, that is, cells constituting organs or tissues of a living animal or human, or microorganisms found in a living animal or human. In addition, target cells are meant to include extracellular cells, ie cultured animal cells, human cells or microorganisms.

본 발명에서의 "단백질 수송 도메인"은 고분자 유기화합물, 예컨대 올리고뉴클레오타이드, 펩타이드, 단백질, 올리고당 또는 다당류 등과 공유결합을 이루어 별도의 수용체나 운반체, 에너지를 필요로 하지 않고 상기 유기화합물들을 세포 내로 도입시킬 수 있는 것을 말한다.The "protein transport domain" in the present invention forms a covalent bond with a high molecular organic compound such as oligonucleotide, peptide, protein, oligosaccharide or polysaccharide to introduce the organic compound into cells without requiring a separate receptor, carrier or energy. Say what you can.

또한, 본 명세서에서는 단백질, 펩타이드, 유기화합물을 세포 내로 "도입"시키는 것에 대하여 "운반", "침투", "수송", "전달", "투과", "통과"한다는 표현들과 혼용하였다.
In addition, the specification is used interchangeably with the expressions "transport", "penetration", "transport", "delivery", "transmission", "pass" for "introducing" a protein, peptide, organic compound into a cell.

본 발명은 9 내지 15개의 아미노산 잔기로 구성되며 아르기닌 또는 라이신 잔기를 3/4 이상 포함하는 수송도메인이 PRAS40 (Proline-rich Akt substrate 40)의 최소한 일측 말단에 공유결합되어 세포침투 효율이 향상된 PRAS40 융합단백질을 제공한다.The present invention is composed of 9 to 15 amino acid residues, and the PRAS40 fusion improved cell penetration efficiency by covalently bonded to at least one end of the PRAS40 (Proline-rich Akt substrate 40) containing at least 3/4 arginine or lysine residues Provide protein.

또한, 본 발명은 상기 수송도메인이 HIV Tat 49-57 잔기, Pep-1 펩타이드, 올리고라이신, 올리고아르기닌 또는 올리고(라이신,아르기닌) 중의 1종 이상인 것을 특징으로 한다.In addition, the present invention is characterized in that the transport domain is at least one of HIV Tat 49-57 residue, Pep-1 peptide, oligolysine, oligoarginine or oligo (lysine, arginine).

또한, 본 발명은 상기 PRAS40 융합단백질의 아미노산 서열이 서열번호 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 또는 33과 같은 것을 특징으로 한다. PRAS40 융합단백질 제조에서 제한부위 서열의 선택 등에 따라 다양한 서열의 융합단백질을 얻을 수 있으며, 이는 본 발명이 속하는 기술분야에서 보통의 지식을 가진 사람에게 자명하다. 위 아미노산 서열은 예시적인 것일 뿐 PRAS40 융합단백질 아미노산 서열이 위에 나열된 서열로 한정되는 것이 아님은 자명하다.In addition, the present invention is characterized in that the amino acid sequence of the PRAS40 fusion protein is SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33. In the production of PRAS40 fusion protein, it is possible to obtain a fusion protein of various sequences according to the selection of restriction region sequences, which is obvious to those skilled in the art. It is apparent that the above amino acid sequences are exemplary only, and the PRAS40 fusion protein amino acid sequence is not limited to the sequences listed above.

또한, 본 발명은 PRAS40 코딩 cDNA의 최소한 일측 말단에 상기 수송 도메인 코딩 올리고뉴클레오타이드 서열이 결합되어 상기 PRAS40 융합단백질을 코딩하는 재조합 폴리뉴클레오타이드를 제공한다.The present invention also provides a recombinant polynucleotide encoding the PRAS40 fusion protein by binding the transport domain coding oligonucleotide sequence to at least one end of the PRAS40 coding cDNA.

또한, 본 발명은 상기 재조합 폴리뉴클레오타이드의 염기 서열이 서열번호 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 또는 32와 같은 것을 특징으로 한다. PRAS40 융합단백질 제조를 위한 폴리뉴클레오타이드는 제한부위 서열의 선택 등에 따라 다양한 서열의 폴리뉴클레오타이드를 얻을 수 있으며, 이는 본 발명이 속하는 기술분야에서 보통의 지식을 가진 사람에게 자명하다. 위 폴리뉴클레오타이드 서열은 예시적인 것일 뿐 PRAS40 융합단백질 폴리뉴클레오타이드 서열이 위에 나열된 서열로 한정되는 것이 아님은 자명하다.In addition, the present invention is characterized in that the base sequence of the recombinant polynucleotide is SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32. Polynucleotides for the production of PRAS40 fusion protein can obtain a polynucleotide of various sequences according to the selection of restriction region sequences, which is obvious to those skilled in the art. It is apparent that the above polynucleotide sequences are exemplary only and that the PRAS40 fusion protein polynucleotide sequence is not limited to the sequences listed above.

또한, 본 발명은 상기 융합단백질을 발현시키기 위하여 PRAS40 코딩 cDNA의 최소한 일측 말단에 수송 도메인 코딩 올리고뉴클레오타이드 서열이 결합되어 PRAS40 융합단백질을 코딩하는 재조합 폴리뉴클레오타이드를 포함하는 PRAS40 융합단백질 발현벡터를 제공한다.The present invention also provides a PRAS40 fusion protein expression vector comprising a recombinant polynucleotide encoding a PRAS40 fusion protein by binding to a transport domain coding oligonucleotide sequence at least one end of the PRAS40 coding cDNA to express the fusion protein.

또한, 본 발명은 상기 PRAS40 융합단백질을 유효성분으로 하고 약학적으로 허용되는 담체를 포함하며, 파킨슨병, 헌팅턴병, 알츠하이머병, 뇌허혈을 포함하는 뇌신경질환의 예방 및 치료용 약제학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prevention and treatment of cerebral neurological diseases, including Parkinson's disease, Huntington's disease, Alzheimer's disease, and cerebral ischemia, comprising the PRAS40 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

또한, 본 발명은 상기 PRAS40 융합단백질을 유효성분으로 하며, 파킨슨병, 헌팅턴병, 알츠하이머병, 뇌허혈을 포함하는 뇌신경질환의 예방 및 개선 효과가 있는 건강기능식품 조성물을 제공한다.
In another aspect, the present invention provides a health functional food composition having the PRAS40 fusion protein as an active ingredient, Parkinson's disease, Huntington's disease, Alzheimer's disease, cerebral neurological diseases, including cerebral ischemia.

본 발명은 9~15개의 아미노산으로 구성되고, 라이신 또는 아르기닌을 4개 이상 포함하는 단백질 수송 도메인이 PRAS40 단백질의 적어도 일측 말단에 공유결합된 세포도입성(cell-transducing) PRAS40 융합단백질에 관한 것이다. 또한, silent change에 따라 서열 내에서 하나 이상의 아미노산이 기능적으로 동등하게 작용하는 유사한 극성의 다른 아미노산(들)로 치환될 수 있다. 서열 내 아미노산 치환은 그 아미노산이 속하는 클래스의 다른 구성원들로부터 선택될 수 있다.The present invention relates to a cell-transducing PRAS40 fusion protein consisting of 9 to 15 amino acids, wherein a protein transport domain comprising four or more lysine or arginine covalently attached to at least one end of the PRAS40 protein. In addition, silent changes may result in one or more amino acids in the sequence being substituted with other amino acid (s) of similar polarity that function functionally equivalently. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs.

예컨대, 소수성 아미노산 분류는 알라닌, 발린, 류이신, 이소류이신, 페닐알라닌, 발린, 트립토판, 프롤린 및 메티오닌을 포함한다. 극성 중성 아미노산은 글리신, 세린, 트레오닌, 시스테인, 티로신, 아스파라긴 및 글루타민을 포함한다. 양성 염기성 아미노산은 아르기닌, 라이신 및 히스티딘을 포함한다. 음성 전하를 띤 산성 아미노산은 아스파르트산 및 글루탐산을 포함한다. 또한, 본 발명의 융합단백질과 아미노산 서열 간의 일정 범위의 상동성 예컨대 85-100% 범위 내의 동일 유사한 생물학적 활성을 갖는 절편 또는 이들의 유도체들도 본 발명의 권리범위에 포함된다.For example, hydrophobic amino acid classifications include 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. Negative charged acidic amino acids include aspartic acid and glutamic acid. Also included within the scope of the present invention are fragments or derivatives thereof having the same similar biological activity within a range of homology between the fusion protein of the present invention and the amino acid sequence such as in the range of 85-100%.

또한, 본 발명은 PRAS40 cDNA에 단백질 수송도메인 펩타이드 코딩 DNA 서열이 결합되어 상기 세포도입성 융합단백질을 코딩하는 서열번호 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 또는 32번을 비롯한 재조합 폴리뉴클레오타이드에 관한 것이다. 본 발명의 범위는 서열번호 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 또는 32번의 재조합 폴리뉴클레오타이드뿐만 아니라 유전암호의 codon degeneracy에 의한 서열을 갖는 핵산분자들에도 미친다.
In addition, the present invention is SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 is coupled to a protein transport domain peptide coding DNA sequence to PRAS40 cDNA coding the cell-introduced fusion protein Or recombinant polynucleotides, including 32. The scope of the present invention is not only for recombinant polynucleotides of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, but also nucleic acid molecules having a sequence by the codon degeneracy of the genetic code. Crazy

본 발명의 PRAS40 융합단백질은 인 비보 및 인 비트로에서 신경세포에 원활히 도입되었다.The PRAS40 fusion protein of the present invention was smoothly introduced into neurons in vivo and in vitro.

또한, 본 발명의 PRAS40 융합단백질은 산화 스트레스에 의한 세포사멸로부터 신경세포를 보호하였다. In addition, the PRAS40 fusion protein of the present invention protected neurons from cell death by oxidative stress.

또한, 본 발명의 PRAS40 융합단백질은 동물 파킨슨병 모델에서 산화 스트레스로 인한 파킨슨병 발병을 억제하였다.In addition, the PRAS40 fusion protein of the present invention inhibited the development of Parkinson's disease due to oxidative stress in the animal Parkinson's disease model.

따라서, 본 발명의 PRAS40 융합단백질은 산화 스트레스로 인한 세포사멸과 밀접한 관련이 있는 알츠하이머병, 파킨슨병, 헌팅턴병, 뇌허혈 등의 뇌신경질환 예방 및 치료에 유용하다.
Therefore, the PRAS40 fusion protein of the present invention is useful for preventing and treating cerebral neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and cerebral ischemia which are closely related to cell death due to oxidative stress.

도 1은 PRAS40 및 Tat-PRAS40 융합단백질 정제를 나타낸다. (A) Tat-PRAS40 융합단백질 및 PRAS40 서열 모식도이다. (B) 정제된 PRAS40 및 Tat-PRAS40 융합단백질을 10% SDS-PAGE로 확인하였다. (C) PRAS40 및 Tat-PRAS40 융합단백질을 항-토끼 폴리히스티딘 항체로 웨스턴 블랏분석하여 확인하였다. M: 마커 (EBM-1035, Elpisbiotech, Korea); 레인 1, 정제된 Tat-PRAS40 융합단백질; 레인 2, PRAS40.
도 2는 Tat-PRAS40 융합단백질이 SH-SY5Y 세포로 효과적으로 도입됨을 나타낸다. (A) Tat-PRAS40 융합단백질을 농도 (0.1~5 μM)를 달리 하여 한 시간 동안 처리하여 투여량 의존적으로 도입됨을 확인하였다. (B) 3 μM의 Tat-PRAS40 융합단백질 처리 시간 (10~120분)을 달리 하여 시간 의존적으로 도입됨을 확인하였다. (C) 3 μM의 Tat-PRAS40 융합단백질을 한 시간 처리하여 SH-SY5Y 세포에 도입된 융합단백질의 안정성을 48시간 동안 웨스턴 블랏 분석으로 측정하였다. (D) 한 시간 동안 각 단백질을 3 μM 농도로 처리하고 토끼 항-히스티딘 1차 항체 및 Alexa488 결합된 항-토끼 2차 항체로 시각화한 다음 공촛점 형광현미경으로 관찰하였다.
C: 무처리 대조군;
PRAS40: PRAS40 단백질로 처리한 세포;
Tat-PRAS40: Tat-PRAS40 융합단백질로 처리한 세포.
도 3은 인 비트로에서 Tat-PRAS40 융합단백질의 신경세포 보호기능을 나타낸다. (A) Tat-PRAS40 (0.5~3 μM), PRAS40 (3 μM) 및 Tat-GFP (3 μM)로 한 시간 동안 처리한 다음 MPP+ (5 mM)로 18시간 동안 처리하여 세포독성을 유도하여 MTT 평가법으로 무처리 대조군과 비교하여 세포 생존율을 백분율로 나타내었다. (B) SH-SY5Y 세포에서 Tat-PRAS40 융합단백질이 활성산소종 생성에 미치는 영향을 나타낸다. 각 단백질과 함께 배양한 세포를 MPP+로 처리한 다음 배양하였다. 세포를 형광 염료, DCF-DA로 염색한 후 형광현미경으로 관찰하였다. (C) 세포 내 활성산소종 수준을 ELISA 판독기로 측정하였다. 평균 형광강도는 세 번의 관찰에서 얻은 값이다. 별표는 MPP+ 처리군과 다른 군 간의 통계학적으로 유의한 차이를 나타낸다 (* P <0.01).
도 4는 SH-SY5Y 세포에서 Tat-PRAS40 융합단백질이 세포사멸을 억제함을 나타낸다. 세포를 Tat-PRAS40 융합단백질, PRAS40, Tat-GFP 및 스타우로스포린으로 한 시간 동안 미리 처리하고 MPP+ (5 mM)를 24시간 동안 처리하여 세포사멸을 유도하였다. (A) 절단된 캐스페이즈-3 수준, (B) Bax 및 Bcl-2 수준을 웨스턴 블랏 분석으로 나타내고 밀도계로 측정하였다. β-액틴 분석 결과를 내부 대조군 (internal control)으로 보여준다.
레인 1, 대조군; 레인 2, MPP+; 레인 3, MPP+ 및 Tat-PRAS40 융합단백질 (0.5 μM); 레인 4, MPP+ 및 Tat-PRAS40 융합단백질 (1 μM); 레인 5, MPP+ 및 Tat-PRAS40 융합단백질 (3 μM); 레인 6, MPP+ 및 PRAS40 (3 μM); 레인 7, MPP+ 및 Tat-GFP (3 μM); 레인 8, 스타우로스포린 (1 μM).
도 5는 Tat-PRAS40 융합단백질이 SH-SY5Y 세포사멸의 DNA 단편화를 보호함을 나타낸다. (A) DNA 단편화를 TUNEL 평가법으로 탐지하였다. (B) 각각의 형광강도를 밀도계로 측정하였고, 상대적인 형광강도를 대조군에 대한 백분율로 나타내었다. 각 군 투여량은 다음과 같다: Tat-PRAS40 융합단백질, 0.5~3 μM; PRAS40, 3 μM. 별표는 MPP+ 처리군과 다른 군 간의 통계학적으로 유의한 차이를 나타낸다 (* P <0.01).
도 6은 Tat-PRAS40 융합단백질을 복강주사 방법으로 마우스 뇌에 전달할 수 있음을 나타낸다. Tat-PRAS40 융합단백질 및 PRAS40 2 mg/kg씩을 복강주사하였다. 뇌 조직을 토끼 항-히스티딘 항체 (1:400)로 면역염색한 후 바이오틴 결합된 염소 항-래빗 2차 항체 (1:200)로 염색하였다. 절편은 DAB로 시각화하여 현미경으로 관찰하였다.
도 7은 세포도입된 Tat-PRAS40 융합단백질이 파킨슨병 마우스 모델에서 신경보호효과를 나타냄을 보여준다. Tat-PRAS40 융합단백질 및 PRAS40 2 mg/kg씩 복강주사하였다. 그 후 MPTP (20 mg/kg)를 4회 복강주사하고 7일 후 마우스를 희생시켰다. 흑색질에서 MPP+로 세포독성을 유도한 후 (A)는 크레실 바이올렛 염색결과이고, (B)는 이중염색 (크레실 바이올렛 및 TH 면역조직화학 염색) 결과이다. (C) 흑색질에서 TH 양성 세포 수를 정량하였다. 별표는 MPP+ 처리군과 다른 군 간의 통계학적으로 유의한 차이를 나타낸다 (* P <0.01). 1 shows PRAS40 and Tat-PRAS40 fusion protein purification. (A) Schematic diagram of the Tat-PRAS40 fusion protein and PRAS40 sequence. (B) Purified PRAS40 and Tat-PRAS40 fusion proteins were identified by 10% SDS-PAGE. (C) PRAS40 and Tat-PRAS40 fusion proteins were confirmed by Western blot analysis with anti-rabbit polyhistidine antibodies. M: marker (EBM-1035, Elpisbiotech, Korea); Lane 1, purified Tat-PRAS40 fusion protein; Lane 2, PRAS40.
2 shows that Tat-PRAS40 fusion protein is effectively introduced into SH-SY5Y cells. (A) Tat-PRAS40 fusion protein was treated for one hour at different concentrations (0.1-5 μM) and confirmed to be dose-dependently introduced. (B) 3 μM Tat-PRAS40 fusion protein treatment time (10 ~ 120 minutes) by varying the time-dependent introduction was confirmed. (C) Stability of the fusion protein introduced into SH-SY5Y cells by one hour treatment of 3 μM Tat-PRAS40 fusion protein was measured by Western blot analysis for 48 hours. (D) Each protein was treated at 3 μM concentration for one hour and visualized with rabbit anti-histidine primary antibody and Alexa488 bound anti-rabbit secondary antibody and observed by confocal fluorescence microscopy.
C: untreated control;
PRAS40: cells treated with PRAS40 protein;
Tat-PRAS40: Cells treated with Tat-PRAS40 fusion protein.
Figure 3 shows the neuronal protective function of Tat-PRAS40 fusion protein in vitro. (A) treatment with Tat-PRAS40 (0.5-3 μM), PRAS40 (3 μM) and Tat-GFP (3 μM) for 1 hour followed by MPP + (5 mM) for 18 hours to induce cytotoxicity. MTT assay showed cell viability as a percentage compared to untreated control. (B) The effect of Tat-PRAS40 fusion protein on the production of reactive oxygen species in SH-SY5Y cells. Cells incubated with each protein were treated with MPP + and then cultured. Cells were stained with a fluorescent dye, DCF-DA, and observed with a fluorescence microscope. (C) Intracellular reactive oxygen species levels were measured with an ELISA reader. The mean fluorescence intensity was obtained from three observations. Asterisks indicate a statistically significant difference between the MPP + treatment group and the other groups ( * P < 0.01).
4 shows that Tat-PRAS40 fusion protein inhibits apoptosis in SH-SY5Y cells. Cells were pretreated with Tat-PRAS40 fusion protein, PRAS40, Tat-GFP and staurosporin for one hour and MPP + (5 mM) for 24 hours to induce apoptosis. (A) Cleaved caspase-3 levels, (B) Bax and Bcl-2 levels were shown by Western blot analysis and measured by density meter. The β-actin assay results are shown as internal control.
Lane 1, control; Lane 2, MPP + ; Lane 3, MPP + and Tat-PRAS40 fusion protein (0.5 μM); Lane 4, MPP + and Tat-PRAS40 fusion protein (1 μM); Lane 5, MPP + and Tat-PRAS40 fusion protein (3 μΜ); Lane 6, MPP + and PRAS40 (3 μΜ); Lane 7, MPP + and Tat-GFP (3 μΜ); Lane 8, staurosporin (1 μM).
5 shows that Tat-PRAS40 fusion protein protects DNA fragmentation of SH-SY5Y apoptosis. (A) DNA fragmentation was detected by TUNEL assay. (B) Each fluorescence intensity was measured with a density meter, and the relative fluorescence intensity was expressed as a percentage of the control group. Each group dose is as follows: Tat-PRAS40 fusion protein, 0.5-3 μM; PRAS40, 3 μΜ. Asterisks indicate a statistically significant difference between the MPP + treatment group and the other groups ( * P < 0.01).
Figure 6 shows that the Tat-PRAS40 fusion protein can be delivered to the mouse brain by the intraperitoneal injection method. Tat-PRAS40 fusion protein and PRAS40 2 mg / kg each were intraperitoneally injected. Brain tissue was immunostained with rabbit anti-histidine antibody (1: 400) and then stained with biotin-bound goat anti-rabbit secondary antibody (1: 200). Sections were visualized with DAB and observed under a microscope.
Figure 7 shows that the transduced Tat-PRAS40 fusion protein shows a neuroprotective effect in Parkinson's disease mouse model. Tat-PRAS40 fusion protein and PRAS40 were injected intraperitoneally at 2 mg / kg. MPTP (20 mg / kg) was then intraperitoneally injected four times and mice were sacrificed 7 days later. Induction of cytotoxicity with MPP + in the black matter (A) is the result of cresyl violet staining, (B) is the result of double staining (cresyl violet and TH immunohistochemical staining). (C) The number of TH positive cells in the black matter was quantified. Asterisks indicate a statistically significant difference between the MPP + treatment group and the other groups ( * P < 0.01).

이하, 구체적인 실시예를 들어 본 발명의 구성을 좀더 자세히 설명한다. 그러나, 본 발명의 범위가 실시예의 기재에만 한정되는 것이 아님은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 자명하다. 특히, 각 실험예의 결과로서 구체적인 실시예에서 제조한 여러 가지 융합단백질 중 Tat-PRAS40 융합단백질을 시료로 한 데이터를 기재하였으나, 그 외의 융합단백질들 또한 Tat-PRAS40 융합단백질을 시료로 한 결과와 유사한 정도(71~105%)의 결과를 나타내었음을 밝힌다.
Hereinafter, the configuration of the present invention in more detail with reference to specific examples. However, it is obvious to those skilled in the art that the scope of the present invention is not limited only to the description of the embodiments. Particularly, as a result of each experimental example, data of a sample of Tat-PRAS40 fusion protein was described among various fusion proteins prepared in specific examples, but other fusion proteins were also similar to those of Tat-PRAS40 fusion protein. Indicate that the results were accurate (71-105%).

재료material

제한 엔도뉴클레이즈 (Restriction endonuclease) 및 T4 DNA 연결효소는 Promega (Madison, WI, USA)에서 구입하였다. 플라스미드 pET-15b 및 Escherichia coli strain BL21 (DE3)는 Novagen (Hilden, Germany)에서 구입하였다. 올리고뉴클레오타이드는 Bioneer (Dajeon, Korea)에서 합성하였다. Ni2+-니트릴로삼초산 세파로즈 슈퍼플로우 컬럼은 Qiagen (Valencia, CA, USA)에서 입수하였다. 우태혈청과 항생제는 Gibco BRL (Grand Island, NY, USA)에서 구입하였다. 다른 화학약품은 특별한 언급이 없으면 Sigma-Aldrich (St. Louis, MO, USA)에서 구입한 것이고 입수 가능한 최상의 분석등급 제품을 사용하였다.
Restriction endonuclease and T4 DNA ligase were purchased from Promega (Madison, WI, 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+ -nitrilotriacetic acid Sepharose superflow column was obtained from Qiagen (Valencia, CA, USA). Fetal bovine 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 used the best analytical grade product available.

PRAS40 융합단백질 발현과 정제PRAS40 Fusion Protein Expression and Purification

PRAS40 및 Tat-PRAS40 융합단백질 발현 벡터를 구축하였다: 센스 프라이머 서열은 5'-CTCGAGCGCAGCCCC-3' (서열번호 6)이고 XhoI 제한부위를 포함하며, 안티센스 프라이머 서열은 5'-GGATCCTCAATATTTCCGCTTCAG-3' (서열번호 7)이고 BamHI 제한부위를 포함한다. PRAS40 프라이머들과 cDNA 주형을 이용하여 중합효소 연쇄반응 (PCR)을 수행하였으며, PCR 산물은 TA 클로닝 벡터에 삽입되었다. PRAS40 cDNA를 포함하는 TA 벡터는 XhoI 및 BamHI으로 절단하여 절단된 PRAS40 cDNA와 함께 두 종류의 pET-15b 벡터에 연결시켰다. 한 벡터는 Tat-PRAS40 발현 벡터로서 Tat 서열과 6개의 연속 히스티딘 서열을 포함한다. 다른 벡터는 PRAS40 발현 벡터로서 여섯 개의 연속 히스티딘 서열만을 포함한다.PRAS40 and Tat-PRAS40 fusion protein expression vectors were constructed: the sense primer sequence was 5'-CTCGAGCGCAGCCCC-3 '(SEQ ID NO: 6) and included Xho I restriction sites, and the antisense primer sequence was 5'-GGATCCTCAATATTTCCGCTTCAG-3' ( SEQ ID NO: 7) and includes the Bam HI restriction site. Polymerase chain reaction (PCR) was performed using PRAS40 primers and cDNA template, and PCR product was inserted into TA cloning vector. The TA vector containing the PRAS40 cDNA was linked to two types of pET-15b vector with the PRAS40 cDNA digested with Xho I and Bam HI. One vector is a Tat-PRAS40 expression vector comprising a Tat sequence and six consecutive histidine sequences. Another vector contains only six consecutive histidine sequences as a PRAS40 expression vector.

PRAS40과 Tat-PRAS40 융합단백질을 생산하기 위하여 각 벡터 구축물로 E. coli BL21 (DE3) 세포를 형질전환시켰다. 형질전환된 세균은 100 ㎖의 LB 배지에서 37 ℃로 600 ㎚의 광학밀도가 0.5가 될 때까지 배양한 다음 0.5 mM IPTG (isopropyl-β-D-thiogalactoside)를 가하여 37 ℃에서 4시간 동안 유도하였다. 세포를 모으고 결합완충액 (10 mM 이미다졸, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) 내에서 4℃ 조건으로 초음파분해하여 용균시키고 원심분리하였다. 상층액을 2.0 ㎖ Ni2+-니트릴로삼초산 세파로즈 슈퍼플로우 컬럼에 가하였다. 컬럼을 10배의 결합완충액 및 7배의 세척완충액 (60 mM 이미다졸, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9)으로 세척하였다. PRAS40과 Tat-PRAS40 융합단백질은 용출완충액 (280 mM 이미다졸, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9)으로 용출시켰다. 정제된 단백질에 포함된 염은 PD-10 탈염 컬럼 (Amersham, Braunschweig, Germany)으로 제거하였다. 단백질 농도는 브래드포드 방법으로 우혈청 알부민을 표준물질로 이용하여 결정하였다.
 E. coli BL21 (DE3) cells were transformed with each vector construct to produce PRAS40 and Tat-PRAS40 fusion proteins. The transformed bacteria were incubated in 100 ml of LB medium at 37 ° C. until the optical density of 600 nm was 0.5 and then 0.5 mM IPTG (isopropyl-β-D-thiogalactoside) was added for 4 hours at 37 ° C. . Cells were collected and solubilized by sonication at 4 ° C. in binding buffer (10 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and lysed and centrifuged. The supernatant was added to a 2.0 mL Ni 2+ -nitrilotriacetic acid Sepharose superflow column. The column was washed with 10-fold binding buffer and 7-fold 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). Salts contained in the purified protein were removed by PD-10 desalting column (Amersham, Braunschweig, Germany). Protein concentration was determined using the bovine serum albumin as a standard by the Bradford method.

세포 배양 및 PRAS40 융합단백질 도입Cell culture and introduction of PRAS40 fusion protein

SH-SY5Y 인간 신경아세포종 (neuroblastoma) 세포주는 10% 우태혈청 (FBS) 및 항생제 (100 ㎍/ml 스트렙토마이신, 100 U/ml 페니실린)를 포함하는 EMEM (Eagle's Minimum Essential Medium) 배지에 37 ℃, 95% 공기 및 5% CO2의 습한 조건에서 보관하였다.SH-SY5Y human neuroblastoma cell line at 37 ° C., 95 in EMEM (Eagle's Minimum Essential Medium) medium containing 10% fetal bovine serum (FBS) and antibiotics (100 μg / ml streptomycin, 100 U / ml penicillin) Stored in humid conditions with% air and 5% CO 2 .

PRAS40과 PRAS40 융합단백질이 SH-SY5Y 세포에 투여량 의존적으로 도입되는지를 확인하기 위해 세포를 지름 60 ㎜ 접시에 충분히 자라도록 하였고, 한 시간 동안 다양한 농도 (0.1~5 μM)의 PRAS40 융합단백질과 함께 배양하였다. To ensure that PRAS40 and PRAS40 fusion proteins were dose-dependently introduced into SH-SY5Y cells, the cells were allowed to grow sufficiently in 60 mm diameter plates, and with various concentrations of PRAS40 fusion protein (0.1-5 μM) for one hour. Incubated.

각 단백질이 시간 의존적으로 세포도입되는지를 알아보기 위해 세포를 다양한 시간 (10~120분)동안 PRAS40 융합단백질 (3 μM)로 처리하였다. Cells were treated with PRAS40 fusion protein (3 μM) for various times (10-120 minutes) to determine if each protein was time-dependently introduced.

세포는 하비스트하여 세포 추출물을 제조한 후 웨스턴블랏 분석하였다. 또, SH-SY5Y 세포에 신경독성을 유도하기 위해 세포를 MPP+ (1-methyl-4-phenylpyridinium )(5 mM)로 처리하였다.
The cells were harvested to prepare cell extracts and then subjected to Western blot analysis. In addition, cells were treated with MPP + (1-methyl-4-phenylpyridinium) (5 mM) in order to induce neurotoxicity in SH-SY5Y cells.

웨스턴블랏 분석Western blot analysis

각 세포 용균액으로부터 얻은 β-액틴 표준화된 동량의 단백질을 10% SDS-PAGE로 분석하였다. 분석된 단백질은 5% 탈지유를 포함하는 TBS-T 완충액 (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5)으로 블로킹시킨 나이트로셀룰로스 막으로 전기이동시켰다. 막은 항-히스티딘 다클론항체 또는 다양한 항체 (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA)로 탐침한 다음, 호스래디쉬 퍼옥시데이즈-결합된 염소 항-토끼 면역글로블린 (1:10,000; Sigma-Aldrich)으로 탐지하였다. 결합된 항체 복합체는 제조자의 지시 (Immobilon Western Chemiluminescent AP Substrate; Millipore, Billerica, MA, USA)대로 강화 화학발광제로 시각화하였다. 띠의 강도는 밀도계로 측정하였고 이미지 J 소프트웨어 (NIH, Bethesda, MD, USA)로 분석하였다.
Β-actin normalized equivalent amounts of protein from each cell lysate were analyzed by 10% SDS-PAGE. Proteins analyzed were electrophoresed with nitrocellulose membranes blocked with TBS-T buffer containing 5% skim milk (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5). The membrane was probed with an anti-histidine polyclonal antibody or various antibodies (1: 10,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by horseradish peroxidase-coupled goat anti-rabbit immunoglobulin (1: 10,000) Sigma-Aldrich). Bound antibody complexes were visualized with enhanced chemiluminescent agents as directed by the manufacturer (Immobilon Western Chemiluminescent AP Substrate; Millipore, Billerica, Mass., USA). Band strength was measured with a density meter and analyzed with Image J software (NIH, Bethesda, MD, USA).

공촛점 형광현미경Confocal Fluorescence Microscope

SH-SY5Y 세포를 유리 커버슬립에 올려놓고 PRAS40 및 Tat-PRAS40 융합단백질 (3 μM)과 함께 37 ℃에서 한 시간 동안 배양하였다. PBS로 두 번 세척한 다음 세포를 4% 파라포름알데하이드로 실온에서 10분간 고정시켰다. 항-히스티딘 1차 항체를 1:2,000으로 희석하고 각 시료와 함께 실온에서 한 시간 동안 배양하였다. Alexa fluor 488이 결합된 2차 항체 (Invitrogen, Carlsbad, CA, USA)를 1:15,000으로 희석한 다음 암조건으로 실온에서 45분간 배양하였다. 핵은 1 ㎍/㎖의 4'6-다이아미디노-2-페닐인돌 (4'6-diamidino-2-phenylindole : Roche Applied Science, Basel, Switzerland)로 30분간 염색하였다. 형광은 Olympus FV-300 공촛점 형광 현미경 (Olympus, Tokyo, Japan)으로 분석하였다.
SH-SY5Y cells were placed on glass coverslips and incubated with PRAS40 and Tat-PRAS40 fusion proteins (3 μM) at 37 ° C. for one hour. After washing twice with PBS, the cells were fixed for 10 minutes at room temperature with 4% paraformaldehyde. The anti-histidine primary antibody was diluted 1: 2,000 and incubated with each sample for one hour at room temperature. Alexa fluor 488 conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) was diluted 1: 15,000 and incubated for 45 minutes at room temperature under dark conditions. The nuclei were stained with 1 μg / ml of 4'6-diamidino-2-phenylindole (4'6-diamidino-2-phenylindole: Roche Applied Science, Basel, Switzerland) for 30 minutes. Fluorescence was analyzed by Olympus FV-300 confocal fluorescence microscopy (Olympus, Tokyo, Japan).

생존율 평가Survival rate

An established assay [34] based on MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide}를 기반으로 한 확립된 평가방법을 이용하여 6웰 플레이트에 시드되어 70% 배양된 SH-SY5Y 세포의 생존율을 평가하였다. 세포는 다양한 농도의 각 단백질로 한 시간 동안 처리하였다. DPBS (Dulbecco's phosphate buffered saline)로 세척한 다음 세포를 MPP+ (5 mM)에 18시간 동안 노출하였다. 세포 생존율은 MPP+ 를 처리하지 않은 대조군과 비교하여 백분율로 나타내었다.
An established assay [34] based on MTT 70% seeded in 6-well plates using established assays based on {3- (4,5-dimethylthiazol-2-yl) -2,5-dipheyltetrazolium bromide} The viability of the cultured SH-SY5Y cells was evaluated. Cells were treated with various concentrations of each protein for one hour. After washing with Dulbecco's phosphate buffered saline (DPBS), the cells were exposed to MPP + (5 mM) for 18 hours. Cell viability is expressed as a percentage compared to the control not treated with MPP + .

산화스트레스 측정Oxidation Stress Measurement

세포내 활성산소종 수준은 활성산소종에 민감한 염료 DCF-DA (2'7'-dichlorofluorescein diacetate)로 측정하였다. 이 염료는 활성산소종에 의해 산화되어 형광을 내는 DCF (2'7'-dichlorofluorescein)이 된다 [Takanashi, T. et al. Invest. Ophthalmol. Vis. Sci. 38:2721-2728; 1997]. SH-SY5Y 세포는 Tat-PRAS40 융합단백질 (0.5~3 μM)와 함께 한 시간 동안 배양한 다음 MPP+ (5 mM)로 한 시간 동안 처리하였다. 세포를 DPBS로 두 번 세척한 다음 DCF-DA를 최종 농도 20 μM이 되도록 가하여 한 시간 동안 처리하였고, DPBS로 두 번 세척하여 제거하였다. 각 시료에서 DCF 형광의 수준은 485 nm (여기) 및 538 nm (방출)에서 ELISA 플레이트 판독기 (Labsystems Oy, Helsinki, Finland)로 모니터하였다.
Intracellular reactive oxygen species level was determined by dye DCF-DA (2'7'-dichlorofluorescein diacetate) sensitive to reactive oxygen species. This dye becomes DCF (2'7'-dichlorofluorescein) that is oxidized by reactive oxygen species to fluoresce [Takanashi, T. et al. Invest. Ophthalmol. Vis. Sci . 38 : 2721-2728; 1997]. SH-SY5Y cells were incubated with Tat-PRAS40 fusion protein (0.5-3 μM) for one hour and then treated with MPP + (5 mM) for one hour. Cells were washed twice with DPBS and then treated with DCF-DA to a final concentration of 20 μM for one hour and washed twice with DPBS to remove. The level of DCF fluorescence in each sample was monitored by an ELISA plate reader (Labsystems Oy, Helsinki, Finland) at 485 nm (excitation) and 538 nm (emission).

TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling) 평가방법TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling)

SH-SY5Y 세포를 유리 커버슬립에 올려놓고 형광 단백질 (0.5~3 μM)과 함께 37 ℃에서 한 시간 동안 배양하였다. 배지를 제거한 후 세포를 DPBS로 두 번 세척하고 MPP+ (5 mM)로 네 시간 동안 처리하였다. TUNEL 염색은 세포 사멸 탐지 키트 (Roche Applied Science, Basel, Switzerland)로 수행하였다. Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan)을 이용하여 형광현미경 사진을 촬영하였다.
SH-SY5Y cells were placed on glass coverslips and incubated with fluorescent proteins (0.5-3 μM) at 37 ° C. for one hour. After removing the medium the cells were washed twice with DPBS and treated with MPP + (5 mM) for 4 hours. TUNEL staining was performed with a cell death detection kit (Roche Applied Science, Basel, Switzerland). Fluorescence micrographs were taken using an Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan).

동물모델연구Animal Model Research

8주령 수컷 C57BL/6 마우스를 한림대학교 실험동물센터에서 구입하였다. 동물은 일정 온도 (23 ℃)와 일정한 상대습도 (60%) 조건에서 명암을 12시간씩 주며 사육하였다. 음식과 물은 자유롭게 접근 가능한 조건이었다. 동물 및 동물 사육을 포함한 모든 실험 절차는 한국의 국립수의과학검역원의 실험동물사용과 주의지침서에 따랐고 한림대학교 의과대학 실험동물운영위원회에 의해 승인되었다. Eight-week-old male C57BL / 6 mice were purchased from Hallym University Experimental Animal Center. Animals were bred for 12 h at constant temperature (23 ° C) and constant relative humidity (60%). Food and water were freely accessible. All experimental procedures, including animals and animal breeding, were in accordance with the Guidelines for the Use and Use of Laboratory Animals by the National Veterinary Research and Quarantine Service of Korea and were approved by the Laboratory Animal Steering Committee of Hallym University Medical School.

마우스 조직으로 PRAS40 융합단백질과 PRAS40의 인 비보 세포도입을 시험하기 위해 각 단백질을 1회 2 mg/kg 양으로 복강주사하였다. 뇌 조직을 제거한 다음 토끼 항-히스티딘 다클론 항체 (1:400) 및 바이오틴 결합된 염소 항-토끼 2차 항체 (1:200)로 면역염색하였다. 또한, PRAS40 융합단백질이 파킨슨병을 막는지를 확인하기 위해 PRAS40 융합단백질을 마우스 체중에 대해 1회 2 mg/kg로 복강주사하였다. 다음 날 신경독성물질 (MPTP; 20 mg/kg)을 두 시간 간격으로 네 번 복강주사하였다. 마우스는 7일 뒤 희생되었다.
Each protein was intraperitoneally injected at a dose of 2 mg / kg once to test the in vivo cell transduction of PRAS40 fusion protein and PRAS40 into mouse tissue. Brain tissue was removed and then immunostained with rabbit anti-histidine polyclonal antibody (1: 400) and biotin-bound goat anti-rabbit secondary antibody (1: 200). In addition, to determine whether PRAS40 fusion protein prevents Parkinson's disease, PRAS40 fusion protein was intraperitoneally injected at 2 mg / kg once per mouse body weight. The next day neurotoxic (MPTP; 20 mg / kg) was intraperitoneally injected four times at two hour intervals. Mice were sacrificed after 7 days.

흑색질에서 타이로신 하이드록실레이즈 발현을 위한 조직 처리와 면역조직화학Tissue Treatment and Immunohistochemistry for Tyrosine Hydroxylase Expression in Melanoma

조직 처리와 면역염색을 수행하였다 [Kim, D. S. et al. J. Comp. Neurol. 511:581-598; 2008, Kim, J. E. et al. Epilepsia 50:654-663; 2009, Shin, J. et al. J. Neurosci. 29:15375-15385; 2009]. 간단히 설명하면, 동물에 펜토바비탈 (100 mg/kg 체중)을 복강주사하여 마취시키고 4% 파라포름알데하이드로 심장을 관통하여 (transcardially) 고정하였다. 뇌를 제거한 후 같은 고정제로 네 시간 동안 후고정하였고, 크리오스타트로 30 ㎛ 두께로 동결 및 절단하였다. 절편들을 3% 우혈청 알부민이 포함된 PBS로 30분간 실온으로 블로킹한 다음 토끼 항-TH IgG 일차 항체 (1:500, Santa Cruz Biotechnology) 및 0.3% Triton X-100를 포함하는 PBS와 함께 실온에서 배양하였다. PBS로 10분씩 세 번 세척한 다음 절편을 바이오틴 결합된 항-토끼 IgG (Vector, Burlingame, CA, USA)와 함께 배양하였다. 니슬소체 (Nissl bodies)에 대한 크레실 바이올렛 역염색을 수행하였다. 그 후 절편을 DAB (3,3'-diaminobenzidine)이 포함된 0.1 M 트리스 완충액으로 시각화한 다음 젤라틴 코팅된 슬라이드에 올려놓았다. Olympus DP72 디지탈 카메라 및 DP2-BSW 현미경 디지탈 카메라 소프트웨어로 영상을 캡쳐하고 분석하였다. 도면은 Adobe Photoshop 7.0 (San Jose, CA, USA)로 제작하였다. 영상 조작은 전체 영상에 대해 밝기를 조절하였다.
Tissue treatment and immunostaining were performed [Kim, DS et al. J. Comp. Neurol. 511 : 581-598; 2008, Kim, JE et al. Epilepsia 50 : 654-663; 2009, Shin, J. et al. J. Neurosci. 29 : 15375-15385; 2009]. Briefly, animals were anesthetized with pentobarbital (100 mg / kg body weight) intraperitoneally and fixed by transcardially with 4% paraformaldehyde through the heart. The brain was removed and postfixed for 4 hours with the same fixative, and frozen and cut to 30 μm thickness with cryostat. Sections were blocked for 30 minutes at room temperature with PBS containing 3% bovine serum albumin and then at room temperature with PBS containing rabbit anti-TH IgG primary antibody (1: 500, Santa Cruz Biotechnology) and 0.3% Triton X-100. Incubated. After washing three times with PBS for 10 minutes, the sections were incubated with biotin-bound anti-rabbit IgG (Vector, Burlingame, CA, USA). Cresyl violet reverse staining was performed on Nissl bodies. Sections were then visualized with 0.1 M Tris buffer containing DAB (3,3'-diaminobenzidine) and then placed on gelatin coated slides. Images were captured and analyzed with Olympus DP72 digital cameras and DP2-BSW microscope digital camera software. The drawings were made with Adobe Photoshop 7.0 (San Jose, CA, USA). Image manipulation adjusted the brightness over the entire image.

통계 분석Statistical analysis

데이타는 평균 ± 표준편차로 나타내었다. 각 군 간의 비교는 Student's t test에 의해 수행되었다. P < 0.01 값은 통계적으로 유의함을 나타낸다.
Data is expressed as mean ± standard deviation. Comparisons between groups were performed by Student's t test. A value of P <0.01 indicates that it is statistically significant.

결과 1: PRAS40 융합단백질 발현과 정제Result 1: PRAS40 Fusion Protein Expression and Purification

PRAS40 및 PRAS40 융합단백질 발현 벡터를 위 기재와 같이 제조하였다. 도 1A와 같이, Tat-PRAS40 융합단백질은 N-말단에 6개의 연속되는 히스티딘과 Tat 도메인 서열이 연속되며, PRAS40는 6개의 연속되는 히스티딘 서열만을 포함한다. PRAS40 및 PRAS40 융합단백질 모두 과발현되었고, SDS-PAGE와 웨스턴블랏 분석으로 확인하였다 (도 1B 및 1C). 정제된 PRAS40 및 PRAS40 융합단백질의 분자량은 각각 약 40 및 43kDa이었다.
PRAS40 and PRAS40 fusion protein expression vectors were prepared as described above. As shown in FIG. 1A, the Tat-PRAS40 fusion protein is contiguous with six consecutive histidine and Tat domain sequences at the N-terminus, and PRAS40 contains only six consecutive histidine sequences. Both PRAS40 and PRAS40 fusion proteins were overexpressed and confirmed by SDS-PAGE and Western blot analysis (FIGS. 1B and 1C). The molecular weights of the purified PRAS40 and PRAS40 fusion proteins were about 40 and 43 kDa, respectively.

결과 2: PRAS40 융합단백질은 Outcome 2: PRAS40 fusion protein SH-SY5YSH-SY5Y 세포에 효과적으로 도입되었다. Effectively introduced into the cells.

PRAS40 융합단백질이 SH-SY5Y 세포에 효과적으로 전달되는지를 확인하기 위하여 도입되는 PRAS40 융합단백질의 양을 측정하여 PRAS40 단백질의 도입량과 비교하였다 (도 2A 및 2B). PRAS40 융합단백질은 시간 및 투여량 의존적으로 세포 내 도입되었다. 반면, PRAS40 단백질은 세포 내로 도입되지 않았다. 세포 내에서 PRAS40 융합단백질의 안정성을 평가하는 시험에서 PRAS40 융합단백질은 3 μM로 한 시간 동안 처리한 결과 36시간 가까이 세포 내에서 안정적인 것으로 나타났다 (도 2C). SH-SY5Y 세포 내에서 PRAS40 융합단백질의 분포를 알아보기 위해 공촛점 형광현미경을 이용하였다. PRAS40 융합단백질은 분명히 세포질 내에 존재하였다 (도 2D). 이러한 결과는 PRAS40이 단백질 수송 도메인과 융합함으로써 SH-SY5Y 세포 내로 전달될 수 있고 세포 내에서 비교적 긴 시간 동안 유지될 수 있음을 말해준다.
In order to confirm that PRAS40 fusion protein is effectively delivered to SH-SY5Y cells, the amount of PRAS40 fusion protein introduced was measured and compared with the amount of PRAS40 protein introduced (FIGS. 2A and 2B). PRAS40 fusion proteins were introduced into cells in a time and dose dependent manner. In contrast, PRAS40 protein was not introduced into the cell. In a test evaluating the stability of the PRAS40 fusion protein in the cell, the PRAS40 fusion protein was treated with 3 μM for one hour and was found to be stable in the cell for 36 hours (FIG. 2C). Confocal fluorescence microscopy was used to determine the distribution of PRAS40 fusion protein in SH-SY5Y cells. PRAS40 fusion protein was clearly present in the cytoplasm (FIG. 2D). These results indicate that PRAS40 can be delivered into SH-SY5Y cells by fusion with the protein transport domain and maintained for a relatively long time in the cells.

결과 3: 세포도입된 PRAS40 융합단백질은 산화 스트레스에서 Outcome 3: Transduced PRAS40 fusion protein SH-SY5Y 세포를SH-SY5Y cells 보호한다.Protect.

MPP+는 도파민 뉴런 세포 사멸을 유도하는 약제로 널리 알려져 있다 [Miller, R. L. et al. Neurochem. Res. 34:55-65; 2009]. PRAS40 융합단백질이 산화 스트레스 유도된 독성으로부터 신경 세포를 보호할 수 있는지를 확인하기 위해 SH-SY5Y 세포를 PRAS40 융합단백질 (0.5~3 μM) 및 PRAS40 단백질 (3 μM)로 한 시간 동안 처리한 다음 MPP+에 18시간 동안 노출시켜 세포독성을 유도하였다.MPP + is widely known as a drug for inducing dopamine neuronal cell death [Miller, RL et al. Neurochem. Res . 34 : 55-65; 2009]. SH-SY5Y cells were treated with PRAS40 fusion protein (0.5-3 μM) and PRAS40 protein (3 μM) for one hour to determine if PRAS40 fusion protein could protect neurons from oxidative stress-induced toxicity, followed by MPP Cytotoxicity was induced by exposure to + for 18 hours.

도 3A와 같이, MPP+는 대조군의 약 50%로 세포 생존율을 감소시켰다. 음성 대조군으로 PRAS40 및 Tat-녹색형광단백질 (Tat-GFP)로 처리한 군은 산화 스트레스로부터 신경세포를 보호하지 못했다. 반면, PRAS40 융합단백질은 SH-SY5Y 세포 생존율을 투여량 의존적으로 현저히 증대시켰다 (MPP+ 단독 처리와 비교하여 P < 0.01). 또한, DCF-DA 염색은 PRAS40 융합단백질 처리가 MPP+ 처리에 의한 활성산소종 생성을 투여량 의존적으로 감소시킴을 보여준다 (도 3B 및 3C). PRAS40 융합단백질은 모든 농도에서 활성산소종 생성을 현저히 억제하였다. 특히 PRAS40 융합단백질 (3 μM) 처리한 세포의 형광 강도는 무처리 대조군과 거의 유사하였다. 반면, MPP+를 단독 처리한 세포와 MPP+ 및 PRAS40을 함께 처리한 세포에서 생성되는 활성산소종의 양은 차이가 없었다. PRAS40은 활성산소종 생성 저해능이 없는 것으로 나타났다. 세포생존시험에 따르면, 세포내 활성산소종 수준 측정에서 일관된 결과가 나타났다. 따라서, 이러한 결과는 PRAS40 융합단백질이 그 자체의 세포막 투과능에 힘입어 SH-SY5Y 세포로 전달되어 MPP+ 처리에 의하여 유발된 활성산소종 생성을 현저히 억제함을 말한다.
As shown in Figure 3A, MPP + reduced cell viability to about 50% of the control. Negative controls treated with PRAS40 and Tat-Green Fluorescent Protein (Tat-GFP) did not protect neurons from oxidative stress. In contrast, PRAS40 fusion protein significantly increased SH-SY5Y cell viability in a dose dependent manner ( P <0.01 compared to MPP + alone). In addition, DCF-DA staining showed that PRAS40 fusion protein treatment prevented the generation of reactive oxygen species by MPP + treatment. Dose dependent reduction is shown (FIGS. 3B and 3C). PRAS40 fusion protein significantly inhibited the generation of reactive oxygen species at all concentrations. In particular, the fluorescence intensity of cells treated with PRAS40 fusion protein (3 μM) was almost similar to the untreated control. In contrast, cells treated with MPP + alone There was no difference in the amount of reactive oxygen species produced in the cells treated with MPP + and PRAS40. PRAS40 was shown to be free of inhibitors of reactive oxygen species production. Cell survival studies have shown consistent results in measuring intracellular reactive oxygen species levels. Thus, these results indicate that PRAS40 fusion protein is delivered to SH-SY5Y cells due to its own membrane permeability, which significantly inhibits the generation of reactive oxygen species induced by MPP + treatment.

결과 4: MPPResult 4: MPP ++ -유도된 세포사멸 일련반응 활성화 및 DNA 단편화는 PRAS40 융합단백질에 의해 저해된다.Induced apoptosis serial activation and DNA fragmentation are inhibited by PRAS40 fusion protein.

MPP+는 전자전달사슬 복합체를 저해하고 세포사멸 일련반응을 유발하여 세포 사멸을 일으킨다 [Eberhardt, O.; Schulz, J. B. Toxicol. Lett. 139:135-151; 2003]. 따라서, 세포사멸에서 PRAS40 융합단백질의 역할을 시험하기 위해 MPP+로 세포사멸을 유발한 다음 세포사멸 일련반응에서 몇 가지 마커의 수준을 측정하였다. 도 4A 및 4B와 같이, PRAS40 융합단백질은 절단된 캐스페이즈-3 (caspase-3) 및 Bax와 같은 친세포사멸 단백질의 수준을 투여량 의존적으로 감소시켰고, 역으로 Bcl-2와 같은 항세포사멸 단백질 (anti-apoptotic protein)의 수준은 증가시켰다. 그러나, PRAS40 또는 Tat-GFP를 처리한 경우는 MPP+로 처리한 세포에서 친세포사멸 및 친생존 (pro-survival) 단백질 간의 균형이 세포사멸 유도제인 스타우로스포린 (staurosporine)을 처리한 경우와 유사하였다. 또한, DNA 단편화가 세포사멸의 특징으로 널리 알려져 있기 때문에 각 시료에서 DNA 단편화를 조사하여 TUNEL 평가방법을 이용하여 정량하였으며, 이를 무처리 대조군과 비교하였다. 세포사멸 세포는 녹색 형광을 나타내었다. 대부분의 세포사멸 핵은 주로 MPP+ 또는 MPP+ + PRAS40에 노출된 시료에서 관찰되었고, 반면 PRAS40 융합단백질 처리한 시료에서는 세포사멸된 세포의 수가 투여량 의존적으로 감소하였다 (도 5A 및 5B). 특히, PRAS40 융합단백질을 처리한 SH-SY5Y 세포는 무처리 대조군과 비슷한 정도로 세포를 사멸로부터 보호하였다. 이러한 결과는 PRAS40 융합단백질이 MPP+에 의해 자극된 세포사멸을 억제하여 SH-SY5Y 세포의 생존을 증대시킬 수 있음을 의미한다.
MPP + inhibits electron transport chain complexes and induces apoptosis serial reactions resulting in cell death [Eberhardt, O .; Schulz, JB Toxicol. Lett . 139 : 135-151; 2003]. Therefore, in order to test the role of PRAS40 fusion protein in apoptosis, apoptosis was induced with MPP + and the levels of several markers were measured in apoptosis serial reactions. As shown in Figures 4A and 4B, PRAS40 fusion protein dose-dependently reduced levels of apoptotic proteins such as cleaved caspase-3 and Bax, and conversely anti-apoptotic cells such as Bcl-2. The level of anti-apoptotic protein was increased. However, treatment with PRAS40 or Tat-GFP showed a similar balance between proliferative and pro-survival proteins in MPP + -treated cells, as compared with staurosporine, an apoptosis inducer. It was. In addition, since DNA fragmentation is widely known as a feature of apoptosis, DNA fragmentation was examined in each sample and quantified using the TUNEL evaluation method, and compared with the untreated control group. Apoptotic cells showed green fluorescence. Most of the apoptosis nuclei were observed mainly in samples exposed to MPP + or MPP + + PRAS40, whereas in PRAS40 fusion protein treated samples the number of apoptotic cells was dose dependently reduced (FIGS. 5A and 5B). In particular, SH-SY5Y cells treated with PRAS40 fusion protein protected cells from death to a similar extent as untreated controls. These results indicate that PRAS40 fusion protein can inhibit the apoptosis stimulated by MPP + to increase the survival of SH-SY5Y cells.

결과 5: PRAS40 융합단백질은 복강주사로 마우스 뇌에 전달된다.Result 5: PRAS40 fusion protein is delivered to the mouse brain by intraperitoneal injection.

PRAS40 융합단백질의 인 비트로 세포도입 효과를 조사하기 위해 PRAS40 융합단백질 및 PRAS40을 복강주사로 마우스에 투여하였고, 뇌 조직으로의 도입을 항-히스티딘 항체로 면역조직화학 분석하여 비교하였다. 무처리 대조군과 비교할 때 PRAS40 융합단백질은 마우스 뇌 흑색질에서 선명하게 관찰된 반면, PRAS40은 뇌 조직에 전달되지 않았다 (도 6). 이러한 결과는 PRAS40 융합단백질이 인 비트로뿐만 아니라 인 비보에서도 효과적으로 도입될 수 있음을 제시한다.
To investigate the in vitro cell introduction effect of PRAS40 fusion protein, PRAS40 fusion protein and PRAS40 were administered to mice by intraperitoneal injection, and their introduction into brain tissue was compared by immunohistochemical analysis with anti-histidine antibody. Untreated control In comparison, PRAS40 fusion protein was clearly observed in mouse brain melanoma, whereas PRAS40 was not delivered to brain tissue (FIG. 6). These results suggest that PRAS40 fusion protein can be effectively introduced in vivo as well as in vitro.

결과 5: 도입된 PRAS40 융합단백질은 파킨슨병 모델에서 신경보호활성을 나타낸다.Results 5: The introduced PRAS40 fusion protein showed neuroprotective activity in the Parkinson's disease model.

다음으로 본 발명자들은 PRAS40 융합단백질이 MPTP/MPP+로 인한 산화적 손상 후 인 비보에서 파킨슨병 발병을 보호할 수 있는지를 마우스 파킨슨병 모델에서 조사하였다. 크레실 바이올렛 염색 데이타 (도 7A)는 흑색질에서 MPTP 처리가 세포사멸을 현저히 증가시킴을 보여준다. PRAS40 처리는 MPTP로 유도된 세포 사멸에 아무런 영향을 미치지 않았다. 그러나, PRAS40 융합단백질로 미리 처리한 마우스의 흑질에서는 대조군과 비교하여 세포가 사멸로부터 보호되는 현상이 관찰되었다. 또한, 도파민 뉴런 손실이 파킨슨병의 분명한 증거이므로 사멸 뉴런의 종류를 확인할 필요가 있다. 그리하여 본 발명자들은 도파민 뉴런 특이적 항-TH 항체를 이용한 면역조직화학 및 크레실 바이올렛 염색으로 이중염색을 수행하였다. 도파민 뉴런을 의미하는 TH-양성 세포는 무처리 대조군 및 MPTP + PRAS40 융합단백질 처리군에서 탐지되었다 (도 7B). 반면, MPTP + PRAS40 군은 MPTP 단독처리군과 마찬가지로 TH 면역반응 세포가 거의 없었다 (도 7C). 대조군과 비교할 때 MPTP 처리군에서 흑색질의 TH-양성 세포수는 현저히 감소하였다. 그러나, PRAS40 융합단백질 처리군은 MPTP에 의해 유발된 도파민 뉴런 사멸에 대해 세포 보호효과를 나타내었다. 종합하여 보면 이러한 결과들은 PRAS40 융합단백질이 MPTP 또는 MPP+와 같은 산화 스트레스에 의해 유도된 세포사멸로부터 흑색질의 도파민 뉴런 세포를 현저히 보호함을 말해준다.
Next, we investigated whether PRAS40 fusion protein can protect against the development of Parkinson's disease in vivo after oxidative damage caused by MPTP / MPP + in a mouse Parkinson's disease model. Cresyl violet staining data (FIG. 7A) show that MPTP treatment significantly increases apoptosis in melanocytes. PRAS40 treatment had no effect on MPTP induced cell death. However, in the black matter of mice previously treated with PRAS40 fusion protein, the cells were protected from death compared to the control group. In addition, dopamine neuron loss is a clear evidence of Parkinson's disease, so it is necessary to confirm the type of killed neuron. Thus we performed dual staining with immunohistochemistry and cresyl violet staining with dopamine neuron specific anti-TH antibodies. TH-positive cells, meaning dopamine neurons, were untreated control and MPTP + Detected in PRAS40 fusion protein treated group (FIG. 7B). MPTP, on the other hand, + PRAS40 group had almost no TH immune response cells as in MPTP alone (FIG. 7C). Compared with the control group, the number of TH-positive cells in the black matter was significantly decreased in the MPTP treated group. However, PRAS40 fusion protein treatment group showed a cytoprotective effect against dopamine neuron killing induced by MPTP. Taken together, these results indicate that PRAS40 fusion protein significantly protects black dopamine neuron cells from apoptosis induced by oxidative stress such as MPTP or MPP + .

<제형예 1: 정제의 제조>Formulation Example 1: Preparation of Tablet

PRAS40 융합단백질 10 ㎎PRAS40 fusion protein 10 mg

옥수수전분 100 ㎎Corn starch 100 mg

유당 100 ㎎Lactose 100 mg

스테아린산 마그네슘 2 mgMagnesium stearate 2 mg

상기 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.
After mixing the above components, tablets were prepared by tableting according to a conventional preparation method of tablets.

<제형예 2: 캡슐제의 제조>Formulation Example 2: Preparation of Capsule

PRAS40 융합단백질 5 ㎎PRAS40 fusion protein 5 mg

옥수수전분 100 ㎎Corn starch 100 mg

유당 100 ㎎Lactose 100 mg

스테아린산 마그네슘 2 ㎎2 mg magnesium stearate

통상의 캡슐제 제조방법에 따라 상기 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.
The above components were mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

<제형예 3: 주사제의 제조>Formulation Example 3: Preparation of Injection

PRAS40 융합단백질 1 ㎎PRAS40 fusion protein 1 mg

주사용 멸균 증류수 적량Sterile sterilized water for injection

pH 조절제 적량pH adjuster

통상의 주사제 제조방법에 따라 1 앰플당(2㎖) 상기의 성분 함량으로 제조하였다.
(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

<제형예 4: 액제의 제조>Formulation Example 4: Preparation of Liquid

PRAS40 융합단백질 0.5 ㎎PRAS40 fusion protein 0.5 mg

이성화당 10 g10 g per isomer

만니톨 5 g5 g mannitol

정제수 적량Purified water quantity

통상의 액제 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 100㎖로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조하였다.
Each component was added to purified water in accordance with a conventional liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto to adjust the whole volume to 100 ml. The resulting solution was filled in a brown bottle and sterilized to prepare a liquid preparation Respectively.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Cell-transducible PRAS40 fusion protein <130> hallym-PRAS40 <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 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 Ala Glu Arg Phe Arg 20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro 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 Pro Ala Pro 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 Pro 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 accacagcca cccagtccca cacccagccc accccggcct accctggcca gagaggacaa 360 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 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 Ala Glu Arg Phe Arg 20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro 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 Pro Ala Pro 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 Pro 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 Ala Pro 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 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 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 accacagcca cccagtccca cacccagccc accccggcct accctggcca gagaggacaa 360 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 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 Ala Pro 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 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 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 Leu Glu Met Ala Ser Gly Arg 1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Ala Glu Arg Phe Arg 20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro 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 Pro Ala Pro 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 Pro 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 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 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 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 Leu Ala Glu 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 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 Arg Leu Glu Met Ala Ser Gly Arg 1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Ala Glu Arg Phe Arg 20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro 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 Pro Ala Pro 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 Pro 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 PRAS40-9Arg 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> PRAS40-9Arg 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 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 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 Leu Ala Glu 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 <110> Industry Academic Cooperation Foundation, Hallym University <120> Cell-transducible PRAS40 fusion protein <130> hallym-PRAS40 <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 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 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 Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro 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 Pro Ala Pro 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 Pro 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 accacagcca cccagtccca cacccagccc accccggcct accctggcca gagaggacaa 360 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 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 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 Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro 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 Pro Ala Pro 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 Pro 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 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 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 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 accacagcca cccagtccca cacccagccc accccggcct accctggcca gagaggacaa 360 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 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 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 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 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 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 Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro 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 Pro Ala Pro 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 Pro 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 PRAS40-9 Lys 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> PRAS40-9 Lys 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 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 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             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 Arg Pro Gly Pro      50 55 60 Cys Ala Tyr Ala Ala His Gly Arg Gly Ala Leu Ala Glu 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         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 Arg Arg Leu Glu Met Ala Ser Gly Arg   1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro 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 Pro Ala Pro 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 Pro 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 PRAS40-9Arg 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> PRAS40-9Arg 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 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 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 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 Arg Pro Gly Pro      50 55 60 Cys Ala Tyr Ala Ala His Gly Arg Gly Ala Leu Ala Glu 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 Arg         275 280 285

Claims (8)

9 내지 15개의 아미노산 잔기로 구성되며 아르기닌 또는 라이신 잔기를 3/4 이상 포함하는 수송도메인이 PRAS40 (Proline-rich Akt substrate 40)의 최소한 일측 말단에 공유결합되어 세포침투 효율이 향상된 PRAS40 융합단백질.
A PRAS40 fusion protein having 9 to 15 amino acid residues, wherein a transport domain comprising 3/4 or more of arginine or lysine residues is covalently attached to at least one end of PRAS40 (Proline-rich Akt substrate 40), thereby improving cell penetration efficiency.
제1항에 있어서, 상기 수송도메인은 HIV Tat 49-57 잔기, 올리고라이신, 올리고아르기닌 또는 올리고(라이신,아르기닌) 중의 1종 이상인 것을 특징으로 하는 PRAS40 융합단백질.
The PRAS40 fusion protein according to claim 1, wherein said transport domain is at least one of HIV Tat 49-57 residues, oligolysine, oligoarginine or oligo (lysine, arginine).
제1항에 있어서, 상기 PRAS40 융합단백질은 그 아미노산 서열이 서열번호 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 또는 33과 같은 것을 특징으로 하는 PRAS40 융합단백질.
According to claim 1, wherein the PRAS40 fusion protein PRAS40 fusion protein, characterized in that the amino acid sequence of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33.
PRAS40 코딩 cDNA의 최소한 일측 말단에 상기 수송 도메인 코딩 올리고뉴클레오타이드 서열이 결합되어 상기 제1항 내지 제3항 중 어느 한 항의 PRAS40 융합단백질을 코딩하는 재조합 폴리뉴클레오타이드.
A recombinant polynucleotide encoding the PRAS40 fusion protein of any one of claims 1 to 3 by binding the transport domain coding oligonucleotide sequence to at least one end of a PRAS40 coding cDNA.
제4항에 있어서, 상기 재조합 폴리뉴클레오타이드는 그 염기 서열이 서열번호 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 또는 32와 같은 것을 특징으로 하는, PRAS40 융합단백질을 코딩하는 재조합 폴리뉴클레오타이드.
The PRAS40 fusion protein according to claim 4, wherein the recombinant polynucleotide has a nucleotide sequence such as SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. Polynucleotide encoding a protein.
9 내지 15개의 아미노산 잔기로 구성되며 아르기닌 또는 라이신 잔기를 3/4 이상 포함하는 수송도메인이 PRAS40 (Proline-rich Akt substrate 40)의 최소한 일측 말단에 공유결합되어 세포침투 효율이 향상된 PRAS40 융합단백질을 발현시키기 위하여 PRAS40 코딩 cDNA의 최소한 일측 말단에 수송 도메인 코딩 올리고뉴클레오타이드 서열이 결합되어 PRAS40 융합단백질을 코딩하는 청구항 4 또는 청구항 5의 재조합 폴리뉴클레오타이드를 포함하는 PRAS40 융합단백질 발현벡터.
A transport domain consisting of 9 to 15 amino acid residues and containing at least 3/4 of arginine or lysine residues is covalently linked to at least one end of PRAS40 (Proline-rich Akt substrate 40) to express PRAS40 fusion protein with improved cell penetration efficiency. A PRAS40 fusion protein expression vector comprising a recombinant polynucleotide of claim 4 or 5 wherein a transport domain coding oligonucleotide sequence is coupled to at least one end of a PRAS40 coding cDNA to encode a PRAS40 fusion protein.
제1항 내지 제3항 중 어느 한 항의 PRAS40 융합단백질을 유효성분으로 하고 약학적으로 허용되는 담체를 포함하며, 파킨슨병, 헌팅턴병, 알츠하이머병, 뇌허혈을 포함하는 뇌신경질환의 예방 및 치료용 약제학적 조성물.
A pharmaceutical composition for the prevention and treatment of cerebral neurological diseases, including Parkinson's disease, Huntington's disease, Alzheimer's disease and cerebral ischemia, comprising the PRAS40 fusion protein of any one of claims 1 to 3 as an active ingredient and a pharmaceutically acceptable carrier. Composition.
제1항 내지 제3항 중 어느 한 항의 PRAS40 융합단백질을 유효성분으로 하며, 파킨슨병, 루게릭병, 헌팅턴병, 알츠하이머병, 뇌허혈을 포함하는 뇌신경질환의 예방 및 개선 효과가 있는 건강기능식품 조성물.
Claims 1 to 3 of the PRAS40 fusion protein as an active ingredient, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Alzheimer's disease, a healthy functional food composition having an effect of preventing and improving brain neurological diseases including cerebral ischemia.
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