KR20090026382A

KR20090026382A - Cell-transducing pten fusion protein

Info

Publication number: KR20090026382A
Application number: KR1020070091301A
Authority: KR
Inventors: 최수영; 음원식; 박진서; 김대원; 최진희; 장상호
Original assignee: 한림대학교 산학협력단
Priority date: 2007-09-10
Filing date: 2007-09-10
Publication date: 2009-03-13

Abstract

A cell introduction PTEN fusion protein is provided to improve cell permeation of PTEN(Phosphatase and tensin homologue deleted on chromosome 10) and to treat cancer, death of skin cell and atopy. A cell introduction PTEN fusion protein has amino acid sequences of a sequence number 7, a sequence number 9 or a sequence number 11 and is formed by covalent bonding PEP-1 protein transport domain of a sequence number 3 at one-side end of PTEN(Phosphatase and tensin homologue deleted on chromosome 10) protein. The recombinant polynucleotide has nucleotide sequence of a sequence number 6, a sequence number 8 or a sequence number 10. The cell introduction PTEN fusion protein is coded from the recombinant polynucleotide.

Description

Cell-transducing PTEN fusion protein

본 발명은 단백질의 세포 도입에 관한 것으로서, 좀더 구체적으로는 PTEN 단백질의 말단에 단백질 수송 도메인이 공유결합된 상태의 융합단백질에 관한 것이다.The present invention relates to the introduction of proteins into cells, and more particularly, to a fusion protein in a state in which a protein transport domain is covalently bonded to a terminal of a PTEN protein.

피부는 자외선(UV), 산화성 공기오염원 등의 유해한 환경에 계속 노출된다. 활성산소종(Reactive Oxygen Species)은 염증성 피부질환, 피부암, 피부 자가면역질환, 광독성, 감광성, 피부노화 등에 관여하고 있다고 알려져 있다[Cross, C.C., Halliwell, B. and Borish, E.T. (1987) Ann. Intern. Med., Davis Conference 107, 526-545].Skin is constantly exposed to harmful environments such as ultraviolet (UV) light and oxidizing air pollutants. Reactive Oxygen Species are known to be involved in inflammatory skin diseases, skin cancer, skin autoimmune diseases, phototoxicity, photosensitivity, and skin aging [Cross, C.C., Halliwell, B. and Borish, E.T. (1987) Ann. Intern. Med., Davis Conference 107, 526-545.

활성산소종은 산소를 이용하여 에너지를 얻는 모든 생명체에서, 여러 세포 내 대사과정의 부산물로 필연적으로 생성된다. 이러한 활성산소종은 세포 내의 단백질, 핵산 및 지방과 같은 생체고분자에 손상을 입히며, 인체의 여러 질병의 진행과정과 깊게 관련되어 있다. 특히, 발암과정, 뇌졸중, 관절염, 동맥 경화, 방사선 손상 및 염증반응에 관여하며 정상적인 노화과정에서도 노화를 촉진하는 중요한 요인으로 작용한다[Floyd, R.A.(1990) FASEB J. 4, 2587-2597.Anderson, W. F.(1998) Nature 392, 25-30, Halliwell B. and Gutteridge J.M.C. (1999) Free radicals in biology and medicine, Oxford University Press, Oxford].Free radicals are inevitably produced as by-products of many intracellular metabolic processes in all living things that use oxygen for energy. These reactive oxygen species damage biopolymers such as proteins, nucleic acids and fats in cells and are deeply involved in the progression of various diseases in the human body. In particular, it is involved in carcinogenesis, stroke, arthritis, arteriosclerosis, radiation damage, and inflammatory reactions, and plays an important role in promoting aging even during normal aging [Floyd, RA (1990) FASEB J. 4, 2587-2597.Anderson , WF (1998) Nature 392, 25-30, Halliwell B. and Gutteridge JMC (1999) Free radicals in biology and medicine, Oxford University Press, Oxford.

크로모좀 10 상의 인산화효소 및 결손된 텐신 유사체(Phosphatase and tensin homologue deleted on chromosome 10; 이하 발명의 상세한 설명, 청구범위 및 도면에서 "PTEN"으로 약칭함)은 MMAC-1(mutated multiple advanced cancers) 또는 TEP-1(TGF-β-regulated and epithelial cell-enriched phosphatase)으로 불린다. 사람의 10번 염색체에 존재하는 PTEN 단백질은 암 세포의 이동, 생존 및 세포사멸에 중요한 역할을 하는 종양억제 단백질로 잘 알려져 있다. 최근 종양억제 단백질인 PTEN이 항암 작용뿐 아니라 염증반응을 억제한다는 사실이 밝혀지고 있다[Li, J. et al.(1997) Science 275, 1943-1947, Steck, P.A. et al.(1997) Nat. Genet. 15, 356-362, Yamada, K.M. et al.(2001) J. Cell Sci. 114, 2375-2382, Lee, K.S. et al.(2006) Mol. Pharmacol. 69, 1829-1839, Kwak, Y.G. et al.(2003) J. Clin. Invest. 111, 1083-1092, Lee. Y.C. et al.(2004) Arch. Immunol. Ther. Exp. 52, 250-254.].Phosphatase and tensin homologue deleted on chromosome 10 (abbreviated as "PTEN" in the description, claims and drawings below) of chromosome 10 is MMAC-1 (mutated multiple advanced cancers) or It is called TEP-1 (TGF-β-regulated and epithelial cell-enriched phosphatase). PTEN protein present in human chromosome 10 is well known as a tumor suppressor protein that plays an important role in the migration, survival and apoptosis of cancer cells. Recently, tumor suppressor protein PTEN has been shown to inhibit inflammatory responses as well as anticancer activity [Li, J. et al. (1997) Science 275, 1943-1947, Steck, P.A. et al. (1997) Nat. Genet. 15, 356-362, Yamada, K.M. et al. (2001) J. Cell Sci. 114, 2375-2382, Lee, K.S. et al. (2006) Mol. Pharmacol. 69, 1829-1839, Kwak, Y.G. et al. (2003) J. Clin. Invest. 111, 1083-1092, Lee. Y.C. et al. (2004) Arch. Immunol. Ther. Exp. 52, 250-254.].

생체 내 고분자들 특히 피부는 이러한 염증 및 자유라디칼의 해로운 작용에 항상 노출되어 있기 때문에 다양한 피부질환에서 PTEN을 치료목적으로 이용하려는 관심이 매우 높아지고 있다. 현재 가장 주목을 받고 있는 것은 유전자 치료이다. 그러나 유전자 치료는 유전자의 운반방법이 용이하지 않고, 표적세포에서의 발현이 낮고, 세포에서 단백질이 발현되는 기간이 짧고, 인위적으로 표적세포에서 발현되는 단백질의 양을 인위적으로 조절하기가 매우 어려운 점 등 여러 가지 문제점이 있다[Del Zoppo, G.J., Wagner, S. and Tagaya, M. (1997) Drugs 54, 9-38].In vivo polymers, especially the skin, are always exposed to such harmful effects of inflammation and free radicals, and there is a great interest in using PTEN for various skin diseases. At the moment the most attention is on gene therapy. However, gene therapy is not easy to carry genes, low expression in target cells, short duration of protein expression in cells, and very difficult to artificially control the amount of protein expressed in target cells. And several problems (Del Zoppo, GJ, Wagner, S. and Tagaya, M. (1997) Drugs 54, 9-38).

치료를 위한 약물이나 단백질을 세포 내로 이동시키는 데 있어 목표 단백질을 세포막을 거쳐 직접 전달하는 방법을 생각할 수 있다. 그러나 단백질은 크기나 여러 생화학적 성질 때문에 세포막을 통과하기가 매우 어렵다. 일반적으로 분자량 600달톤 이상의 물질은 세포막을 통과하기가 거의 불가능한 것으로 알려져 있다.In moving drugs or proteins for treatment into cells, one can think of direct delivery of target proteins across cell membranes. However, proteins are difficult to cross through cell membranes due to their size and biochemical properties. In general, it is known that a substance having a molecular weight of 600 Daltons or more is almost impossible to pass through the cell membrane.

최근, 단백질의 운반 방법 중 하나로 PEP-1 펩타이드를 이용하여 자연상태의 이형단백질을 세포 내로 운반할 수 있다는 것이 밝혀졌다[Morris, M.C., Depollier, J., Mery, J., Heitz, F. and Divita, G. (2001) Nat. Biotech. 19, 1173-1176]. PEP-1 펩타이드는 21개의 아미노산(KETWWETWWTEW SQP KKKRKV)으로 이루어졌고, 3개의 도메인{소수성(hydrophobic), 스페이서(spacer), 친수성(hydrophilic) 도메인}을 갖고 있다. 지금까지, PEP-1 펩타이드를 이용한 연구에서는 PEP-1 펩타이드와 외부 단백질을 동시에 세포에 투여하였을 경우 자연상태로 단백질을 세포 내로 운반할 수 있다는 것만 밝혀졌다. 또한, PEP-1 펩타이드는 HIV-1 Tat 단백질에 비해 단백질 치료제로서의 여러 가지 장점 즉, 생리학적 완충액에서의 안정성, 독성의 결여, 혈청에 대한 민감성의 결여 등을 나타낸다. 그러나, PEP-1 펩타이드는 녹색형광단백질이나 β-갈락토시데이즈와 같은 외부 단백질과 일정한 비율로 맞추어 투여해야만 세포 내에 효과적으로 단백질이 운반되는 것으로 확인되었다. 그러나, 현재까지도 PEP-1 펩타이드가 치료 단백질이나 모든 단 백질을 세포 내로 운반할 수 있는지는 아직까지 밝혀지지 않았다. 본 발명자들의 2007년 연구결과에서 PEP-1-α-락트알부민(α-lactalbumin) 융합단백질을 제조 및 정제하여 피부 세포 및 조직 내 침투 효율을 측정한 결과, PEP-1-α-락트알부민 융합단백질은 배양한 피부세포에는 빠른 시간 내에 효과적으로 잘 침투되나, 실제 피부조직으로의 침투는 기존의 다른 단백질(예컨대, 수퍼옥사이드 디스뮤테이즈, 카탈레이즈 등)에서 보여준 것처럼 높게 나타나지 않고 피부 조직 내 침투효율이 현저히 떨어지는 것을 확인한바 있다.Recently, it has been found that PEP-1 peptides can be used to transport naturally occurring heterologous proteins into cells [Morris, MC, Depollier, J., Mery, J., Heitz, F. and Divita, G. (2001) Nat. Biotech. 19, 1173-1176. The PEP-1 peptide consists of 21 amino acids (KETWWETWWTEW SQP KKKRKV) and has three domains (hydrophobic, spacer, hydrophilic domain). So far, studies using PEP-1 peptides have shown that when PEP-1 peptides and foreign proteins are administered to cells at the same time, the proteins can be transported naturally into the cells. In addition, PEP-1 peptides exhibit several advantages as protein therapeutics over HIV-1 Tat protein: stability in physiological buffers, lack of toxicity, lack of sensitivity to serum, and the like. However, PEP-1 peptides have been found to be effectively transported into cells only when administered in proportion to external proteins such as green fluorescent protein or β-galactosidase. However, it is not yet known whether PEP-1 peptides can transport therapeutic proteins or all proteins into cells. As a result of the 2007 study of the present inventors, PEP-1-α-lactalbumin fusion protein was prepared and purified to measure penetration efficiency in skin cells and tissues. As a result, PEP-1-α-lactalbumin fusion protein was measured. Silver penetrated skin cells can be effectively penetrated quickly and effectively, but the penetration into skin tissues does not appear as high as those of other proteins (e.g. superoxide dismutase, catalase, etc.). I have noticed a significant drop.

본 발명의 목적은 PTEN의 세포 침투성을 높인 융합 단백질을 제공하려는 것이다.It is an object of the present invention to provide a fusion protein with enhanced cell permeability of PTEN.

또한, 본 발명의 목적은 PEP-1을 포함하여 15∼30개의 아미노산으로 구성되고, 5개 이상의 트립토판을 포함하는 비친수성 도메인, 라이신을 다수 포함하는 친수성 도메인 및 상기 두 도메인을 분리시켜 주는 스페이서로 구성된 단백질 수송 도메인을 PTEN의 적어도 일측 말단과 공유결합시킴으로써 세포 침투성을 극대화하려는 것이다.In addition, an object of the present invention is a non-hydrophilic domain consisting of 15 to 30 amino acids, including PEP-1, including at least five tryptophan, a hydrophilic domain including a large number of lysine and a spacer separating the two domains The protein transport domain is intended to maximize cell permeability by covalently binding at least one end of the PTEN.

그리하여, 본 발명자들은 자연상태의 단백질을 세포 내로 운반하는 PEP-1 펩타이드를 외부 단백질인 사람 PTEN에 융합시켰고, 융합 단백질을 대장균에서 과대발현시켰으며, 금속 킬레이팅 친화 크로마토그래피로 쉽고 편리하게 정제하였다. 정제된 융합 단백질이 효과적으로 생물학적인 활성을 지니고 세포 내로 운반되는 것을 배양된 피부 세포 및 조직실험을 통하여 확인하였다. 그리고, PEP-1-PTEN(이하 본 발명의 상세한 설명, 청구범위 및 도면에서 "PEP-PTEN"과 혼용함) 융합 단백질이 피부 세포 내로 운반되며 피부 세포사를 효과적으로 보호함을 밝혀냈다.Thus, the present inventors fused the PEP-1 peptide carrying the natural protein into the cell to an external protein, human PTEN, overexpressed the fusion protein in Escherichia coli, and purified easily and conveniently by metal chelating affinity chromatography. . Purified fusion proteins were effectively transported into cells with biological activity through cultured skin cells and tissue experiments. And it has been found that PEP-1-PTEN (hereinafter, interchangeably with “PEP-PTEN” in the description, claims and figures) of the fusion protein is carried into skin cells and effectively protects skin cell death.

본 발명은 사람 PTEN(Phosphatase and tensin homologue deleted on chromosome 10)을 효과적인 방법으로 단백질 수준에서 세포 내로 직접 투과시키는 기술을 제공한다.The present invention provides a technique for the direct penetration of human PTEN (Phosphatase and tensin homologue deleted on chromosome 10) into the cell at the protein level in an effective manner.

본 발명에 따르면, 아토피 피부 염증을 회복하는데 있어 PTEN을 자연상태로 세포 내 투여함으로써 피부질환을 치료하는 단백질 치료와 피부암에 PEP-1-PTEN이 효과적으로 활용될 수 있을 것으로 사료된다.According to the present invention, it is thought that PEP-1-PTEN may be effectively used for protein treatment and skin cancer to treat skin diseases by intracellular administration of PTEN in a natural state to restore atopic dermatitis.

본 발명을 바탕으로 PEP-1-PTEN을 세포 내로 직접 전달하여 아토피 피부염으로부터 피부세포 및 조직을 보호할 수 있기 때문에 질병 이외에도 항암제 및 기능성 화장품 산업 등 더욱 효과적으로 본 발명이 광범위하게 활용될 수 있다.Based on the present invention, since PEP-1-PTEN can be delivered directly into cells to protect skin cells and tissues from atopic dermatitis, the present invention can be widely used more effectively in addition to diseases such as anticancer drugs and functional cosmetics industries.

PEP-1 펩타이드가 이형 단백질을 자연상태 그대로 세포 내로 이동시키는데 운반체로서 이용될 수 있다는 것이 밝혀졌다. 그러나, 아직까지 PEP-1 펩타이드를 이용한 실험은 매우 드문 실정이다. 따라서, 본 발명자들은 PEP-1 펩타이드를 비롯한 단백질 수송 도메인이 종양억제 단백질로 잘 알려진 PTEN 단백질을 세포 내로 운반시킬 수 있는지, 운반된 PEP-1-PTEN 단백질이 세포 내에서 단백질의 기능(항염증 기능)을 나타내는지를 알아보고자 연구를 수행하였다.It has been found that PEP-1 peptides can be used as carriers to transport heterologous proteins into cells in their natural state. However, experiments with PEP-1 peptides are still very rare. Therefore, the inventors have found that protein transport domains, including PEP-1 peptides, can transport PTEN proteins, known as tumor suppressor proteins, into cells, and that the delivered PEP-1-PTEN proteins function within the cells (anti-inflammatory function). The study was performed to see if

먼저, 본 발명자들은 PEP-1-PTEN, PTEN-PEP-1 및 PEP-1-PTEN-PEP-1 융합단백질을 과대발현시키고 쉽게 정제할 수 있는 발현벡터를 개발하였다. 이 발현벡터는 인간 PTEN cDNA, PEP 펩타이드 (21 아미노산) 그리고 6개의 히스티딘이 연속적으로 연결되어 있다. 이 발현벡터를 이용하여 PEP-1-PTEN, PTEN-PEP-1 및 PEP-1- PTEN-PEP-1 융합단백질을 대장균에서 과대발현시켜 자연상태로 Ni² ⁺-친화 크로마토그래피 컬럼을 이용하여 정제하였다. PEP-1-PTEN, PTEN-PEP-1 및 PEP-1-PTEN-PEP-1의 과대발현은 상당히 높게 나타났으며, 이런 결과로 정제된 단백질의 양도 높게 나타났다. 웨스턴 블랏 분석으로 배양된 피부 세포에 정제된 PTEN 융합단백질이 농도 및 시간 의존적으로 세포에 운반되는 것을 확인하였다. 세포 내로 투과된 PTEN 융합단백질은 최소 세포 내에서 48시간 지속적으로 유지되었으며 PTEN 융합단백질은 배양된 세포뿐 아니라, 마우스의 피부조직에도 효율적으로 투과되었다. 또한 세포 내로 투과된 PTEN 융합단백질은 LPS(Lipopolysaccharide)에 의한 세포사 및 COX-2(Cyclooxygenase-2) 발현을 효과적으로 억제하는 것을 알 수 있었다.First, the inventors have developed an expression vector that can overexpress and easily purify PEP-1-PTEN, PTEN-PEP-1 and PEP-1-PTEN-PEP-1 fusion proteins. This expression vector is a human PTEN cDNA, PEP peptide (21 amino acids) and six histidines are connected in series. Using this expression vector, by over-expression of the PEP-1-PTEN, PTEN- PEP-1 and PEP-1- PTEN-PEP-1 fusion protein in E. coli Ni ² ⁺ in the natural state - purified using affinity chromatography column It was. The overexpression of PEP-1-PTEN, PTEN-PEP-1 and PEP-1-PTEN-PEP-1 was quite high, resulting in a high amount of purified protein. Western blot analysis confirmed that PTEN fusion proteins purified on cultured skin cells were delivered to the cells in a concentration and time dependent manner. The PTEN fusion protein permeated into cells was maintained for 48 hours in a minimum of cells, and the PTEN fusion protein was efficiently permeated not only in cultured cells but also in skin tissues of mice. In addition, the PTEN fusion protein permeated into cells was found to effectively inhibit cell death and COX-2 (Cyclooxygenase-2) expression by LPS (Lipopolysaccharide).

이러한 결과는 PTEN 융합단백질이 세포 내로 투과가 잘 일어나고, 세포 내에서 PTEN 기능을 잘 나타내고 있음을 의미한다. 따라서 이러한 PTEN 융합단백질은 피부암 및 아토피 피부염 등 피부 질환 치료 분야에 다양하게 응용될 가능성을 제시해 준다.These results indicate that the PTEN fusion protein is well permeated into cells and shows well PTEN function in cells. Therefore, this PTEN fusion protein suggests the possibility of various applications in the treatment of skin diseases such as skin cancer and atopic dermatitis.

PTEN 융합 단백질을 유효성분으로 함유하는 약제학적 조성물은 약제학적 분야에서 통상적으로 허용되는 담체와 함께 배합하여 통상적인 방법에 의해 주사형태 또는 도포제로 제형화할 수 있다. 주사용 조성물은 등장성 수용액 또는 현탁액이 바람직하고, 언급한 조성물은 멸균되고/되거나 보조제(예: 방부제, 안정화제, 습윤제 또는 유화제 용액 촉진제, 삼투압 조절을 위한 염/또는 완충제)를 함유한다. 또 한, 이들은 기타 치료적으로 유용한 물질을 함유할 수 있다. PTEN 융합단백질을 함유한 약제학적 조성물은 암치료제로서 유용하며 특히 피부암 치료제, 아토피성 염증 치료제, 녹내장 여과 수술 후 섬유화 반흔 형성 억제제로서 유용하다.Pharmaceutical compositions containing a PTEN fusion protein as an active ingredient may be formulated in an injectable form or a coating by conventional methods in combination with a carrier that is conventionally acceptable in the pharmaceutical art. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries (eg, preservatives, stabilizers, wetting or emulsifier solution promoters, salts or buffers for controlling osmotic pressure). In addition, they may contain other therapeutically valuable substances. Pharmaceutical compositions containing PTEN fusion proteins are useful as cancer therapeutic agents, in particular as skin cancer treatment agents, atopic inflammatory agents, and inhibitors of fibrotic scar formation after glaucoma filtration surgery.

이와 같이 제조된 약제학적 제제는 목적하는 바에 따라 비경구 방식 즉, 피하 투여, 근육 투여 또는 국소적용(topical application)할 수 있으며, 용량은 일일 투여량이 0.001㎍~10㎎/㎏의 양을 1 내지 수회에 나누어 투여할 수 있다. 특정 환자에 대한 투여용량 수준은 환자의 체중, 연령, 성별, 건강상태, 투여시간, 투여방법, 질환의 중증도 등에 따라 변화될 수 있다.The pharmaceutical preparations thus prepared may be parenterally, ie, subcutaneously, intramuscularly, or topically, as desired. The dosage may range from 1 mg to 10 mg / kg in a daily dosage of 1 to 10 mg / kg. It can be divided into several doses. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, severity of the disease, and the like.

또한, 본 발명의 융합 단백질을 유효성분으로 하는 도포제는 통상적인 제조방법에 따라 어떤 형태로든 용이하게 제조할 수 있다. 일례로서 크림형 도포제를 제조함에 있어서는 일반적인 수중유형(O/W) 또는 유중수형(W/O)의 크림 베이스에 본 발명의 PTEN 융합 단백질을 함유시키고 여기에 향료, 킬레이트제, 색소, 산화방지제, 방부제 등을 필요에 따라 사용하는 한편, 물성개선을 목적으로 단백질, 미네랄, 비타민 등 합성 또는 천연소재를 병용할 수 있다.In addition, the coating agent containing the fusion protein of the present invention as an active ingredient can be easily prepared in any form according to a conventional manufacturing method. As an example, in the preparation of cream coating agents, the PTEN fusion protein of the present invention is contained in a general oil-in-water (O / W) or water-in-oil (W / O) cream base, and includes perfumes, chelating agents, pigments, antioxidants, While preservatives and the like are used as necessary, synthetic or natural materials such as proteins, minerals and vitamins can be used in combination for the purpose of improving the properties.

뿐만 아니라, 본 발명의 융합 단백질은 화장료로서 이용할 수 있는데, pH 조절제, 향료, 유화제, 방부제 등을 필요에 따라 부가하여 통상의 화장료 제조 방법으로 화장수, 젤, 수용성 파우더, 지용성 파우더, 수용성 리퀴드, 크림 또는 에센스 등으로 제형화될 수 있다.In addition, the fusion protein of the present invention can be used as a cosmetic, by adding a pH adjuster, fragrance, emulsifier, preservatives, etc. as needed, in the usual cosmetic preparation method, lotion, gel, water-soluble powder, fat-soluble powder, water-soluble liquid, cream Or an essence or the like.

본 발명자들은 PTEN 융합 단백질을 마우스의 피부에 침투실험한 결과 진피층까지 원활하게 침투하는 것을 확인할 수 있었다. 따라서, PTEN 융합 단백질을 약제 조성물 및/또는 도포제(본 명세서에서 "피부 외용제"와 동일한 의미로 사용함) 조성물의 주요성분으로 이용할 수 있음을 밝혔다.The present inventors have confirmed that the PTEN fusion protein penetrates into the skin of the mouse and penetrates smoothly to the dermis layer. Accordingly, it has been found that PTEN fusion proteins can be used as the principal component of pharmaceutical compositions and / or coatings (used herein in the same sense as "skin external preparations") compositions.

본 발명은 PTEN 단백질을 세포 내 또는 피부 내부로 효율적으로 전달하기 위한 방법을 제공한다. 본 발명에 따른 PTEN 단백질 분자의 세포 내 전달은 PTEN에 15∼30개의 아미노산으로 구성되고, 5개 이상의 트립토판을 포함하는 비친수성 도메인(hydrophobic domain), 라이신을 4개 이상 다수 포함하는 친수성 도메인(hydrophilic domain) 및 상기 두 도메인을 분리시켜 주는 스페이서(spacer)로 구성된 수송 도메인이 공유결합된 세포투과성 수송도메인이 공유결합된 형태의 융합 단백질을 구성하여 수행된다. 세포투과성 수송도메인은 PTEN 단백질의 양 말단 즉, N- 말단 및/또는 C- 말단에 결합시켰다. 본 발명의 상기 수송도메인의 일례로는 21개의 아미노산으로 구성되고, 서열번호 3과 같은 아미노산 서열로 이루어진 PEP-1 펩타이드를 들 수 있다. 그러나, 본 발명의 단백질 수송 도메인이 서열번호 3의 PEP-1 펩타이드로만 한정되는 것은 아니고, PEP-1의 아미노산 서열 일부 치환이나 부가, 결여로 PEP-1 펩타이드와 유사한 기능을 하는 펩타이드를 제조하는 것이 본 발명이 속하는 분야에서 통상의 지식을 가진 당업자에게는 용이하므로, 15∼30개의 아미노산으로 구성되고, 5개 이상의 트립토판을 포함하는 비친수성 도메인(hydrophobic domain), 라이신을 4개 이상 다수 포함하는 친수성 도메인(hydrophilic domain) 및 상기 두 도메인을 분리시켜 주는 스페이서(spacer)로 구성된 단백질 수송 도메인과 이로부터 아미노산 일부 치환으로 동일·유사한 단백 질 수송기능을 수행하는 단백질 수송 도메인을 이용한 융합 단백질도 본 발명의 범위에 속함은 자명하다고 할 것이다.The present invention provides a method for efficiently delivering PTEN protein into cells or into the skin. Intracellular delivery of PTEN protein molecules according to the present invention consists of 15-30 amino acids in PTEN, a hydrophobic domain comprising 5 or more tryptophans, and a hydrophilic domain comprising 4 or more lysines. and a transport domain composed of a spacer separating the two domains and a covalently bonded cell-permeable transport domain. The cell permeable transport domain was bound to both ends of the PTEN protein, ie the N- and / or C-terminus. An example of the transport domain of the present invention includes a PEP-1 peptide consisting of 21 amino acids and consisting of an amino acid sequence such as SEQ ID NO: 3. However, the protein transport domain of the present invention is not limited only to the PEP-1 peptide of SEQ ID NO: 3, and the production of a peptide having a function similar to that of the PEP-1 peptide by partial replacement, addition or lack of the amino acid sequence of PEP-1 Since it is easy for those skilled in the art to which the present invention pertains, a hydrophilic domain consisting of 15 to 30 amino acids, including 5 or more tryptophans, and a hydrophilic domain including 4 or more of lysine Fusion protein using a protein transport domain consisting of a hydrophilic domain and a spacer separating the two domains and a protein transport domain that performs the same or similar protein transport function by substituting a partial amino acid from the scope of the present invention Belonging will be self-evident.

구체적으로, 본 발명은 PTEN 융합 단백질, 이 융합 단백질을 제조하기 위한 재조합 뉴클레오타이드와 벡터, 융합 단백질을 포함하는 치료, 예방 목적의 약학 조성물, 피부 외용제 조성물 등에 관한 것이다.Specifically, the present invention relates to a PTEN fusion protein, a recombinant nucleotide and vector for producing the fusion protein, a therapeutic composition comprising the fusion protein, a pharmaceutical composition for prevention, a composition for external application for skin, 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.

"PTEN 융합 단백질"이란 단백질 수송 도메인과 PTEN을 포함하며, 수송 도메인과 화물 분자(cargo molecule, 즉 본 발명에서는 PTEN을 의미함)의 유전적 융합이나 화학 결합으로 형성된 공유결합 복합체를 의미한다. 본 명세서에서는 PEP-1 수송도메인이 결합된 방향에 따라 "PEP-1-PTEN"("PEP-PTEN"과 혼용함), "PTEN-PEP-1" 또는 "PEP-1-PTEN-PEP-1" 융합 단백질을 제조하여 실험하였다."PTEN fusion protein" refers to a covalent complex comprising a protein transport domain and a PTEN, formed by genetic fusion or chemical bonding of the transport domain with a cargo molecule (ie, PTEN in the present invention). In this specification, "PEP-1-PTEN" (mixed with "PEP-PTEN"), "PTEN-PEP-1" or "PEP-1-PTEN-PEP-1" depending on the direction in which the PEP-1 transport domain is bound. Fusion protein was prepared and tested.

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 수송 도메인에 의해 화물 분자가 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 동물 세포, 인체 세포 또는 미생물을 포함하는 의미이다.In addition, "target cell" means a cell to which a cargo molecule 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 명세서의 "단백질 수송 도메인"은 펩타이드, 단백질과 공유결합을 이루어 별도의 수용체나 운반체, 에너지를 필요로 하지 않고 상기 펩타이드나 단백질을 세포 내로 도입시킬 수 있는 것을 말하며, 예를 들면 PEP-1 펩타이드(서열번호 3)를 말한다.As used herein, the term "protein transport domain" refers to a covalent bond with a peptide or a protein, which may introduce the peptide or protein into a cell without the need for a separate receptor, carrier, or energy. For example, a PEP-1 peptide (SEQ ID NO: 3).

본 명세서의 "목표 단백질"은 PEP-1 단백질 수송 도메인과 공유결합을 이루어 세포 내로 도입되어 활성을 나타내는 분자를 의미한다.As used herein, “target protein” refers to a molecule that is covalently bound to a PEP-1 protein transport domain and introduced into a cell to exhibit activity.

또한, 본 명세서에서는 단백질 또는 펩타이드를 세포 내로 "도입"시키는 것에 대하여 "형질도입", "운반", "침투", "수송", "전달", "투과", "통과"한다는 표현들과 혼용하였다.In addition, the present specification is interchangeable with the expressions "transduction", "transport", "penetration", "transport", "transfer", "transmission", "pass" for "introducing" a protein or peptide into a cell. It was.

본 발명은 15∼30개의 아미노산으로 구성되고, 5개 이상의 트립토판을 포함하는 비친수성 도메인, 라이신을 다수 포함하는 친수성 도메인 및 상기 두 도메인을 분리시켜 주는 스페이서로 구성된 단백질 수송 도메인이 PTEN 단백질의 적어도 일측 말단에 공유결합된 세포 도입성(cell-transducing) PTEN 융합 단백질에 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. 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. 음성전하를 띤 산성 아미노산은 아스파르트산 및 글루탐산을 포함한다. 또한, 본 발명의 융합 단백질과 아미노산 서열 간의 일정 범위의 상동성 예컨대 85-100% 범위 내의 동일 유사한 생물학적 활성을 갖는 절편 또는 이들의 유도체들도 본 발명의 권리범위에 포함된다.At least one side of the PTEN protein is a protein transport domain consisting of 15-30 amino acids, a non-hydrophilic domain including 5 or more tryptophan, a hydrophilic domain including a large number of lysines, and a spacer separating the two domains. It relates to a cell-transducing PTEN fusion protein covalently bound to the end. 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. 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. Negatively charged acidic amino acids include aspartic acid and glutamic acid. Also included within the scope of the invention are fragments or derivatives thereof having the same similar biological activity within a range of homology between the fusion protein and amino acid sequence of the invention, such as 85-100%.

또한, 본 발명은 상기 세포 도입성 PTEN 융합 단백질이 서열번호 7, 서열번호 9 또는 서열번호 11의 아미노산 서열을 갖는 것을 특징으로 한다.In addition, the present invention is characterized in that the cell-introducing PTEN fusion protein has an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.

또한, 본 발명은 상기 단백질 수송도메인(protein transducing domain; "PTD")이 PTEN 단백질의 카복시 말단과 아미노 말단의 일측 또는 양측에 한 개 이상 공유결합된 것을 특징으로 한다.In addition, the present invention is characterized in that the protein transducing domain (PTD) is one or more covalently bonded to one or both sides of the carboxy terminal and the amino terminal of the PTEN protein.

또한, 본 발명은 상기 세포 도입성(cell-transducing) PTEN 융합 단백질을 유효성분으로 함유하는 것을 특징으로 하는 피부 외용제 조성물에 관한 것이다.The present invention also relates to an external composition for skin, comprising the cell-transducing PTEN fusion protein as an active ingredient.

또한, 본 발명은 상기 세포 도입성 PTEN 융합 단백질을 유효성분으로 함유하고 약학적으로 허용되는 담체를 포함하는 것을 특징으로 하는 약제학적 조성물에 관한 것이다.In addition, the present invention relates to a pharmaceutical composition comprising the cell-introducing PTEN fusion protein as an active ingredient and comprising a pharmaceutically acceptable carrier.

또한, 본 발명은 PTEN cDNA에 단백질 수송도메인 펩타이드 코딩 DNA 서열이 결합되어 상기 세포도입성 융합 단백질을 코딩하는 서열번호 6번, 8번, 10번을 비롯한 재조합 폴리뉴클레오타이드에 관한 것이다. 본 발명의 범위는 서열번호 6번, 8번, 10번의 재조합 폴리뉴클레오타이드뿐만 아니라 유전암호의 codon degeneracy에 의한 서열을 갖는 핵산분자들에도 미친다.In addition, the present invention relates to a recombinant polynucleotide including SEQ ID NOs: 6, 8, 10, which binds a protein transport domain peptide coding DNA sequence to PTEN cDNA and encodes the cell-inducible fusion protein. The scope of the present invention extends not only to recombinant polynucleotides of SEQ ID NOs 6, 8 and 10, but also to nucleic acid molecules having a sequence by codon degeneracy of the genetic code.

뿐만 아니라, 본 발명은 상기 융합 단백질을 발현시키기 위하여 상기 재조합 폴리뉴클레오타이드를 포함하여 구성되는 것을 특징으로 하는, 세포 도입성 융합 단백질을 발현시키는 벡터에 관한 것이다.In addition, the present invention relates to a vector expressing a cell transfusion fusion protein, characterized in that comprising the recombinant polynucleotide to express the fusion protein.

아래에서는 실시예를 통하여 본 발명의 구성을 좀더 자세히 설명한다. 그러나, 본 발명의 범위가 실시예의 기재에 한정되는 것은 아니다. 특별히 본 실시예에서는 PTEN 융합단백질 중 "PEP-1-PTEN" 융합단백질을 예로 들어 설명하였으나, "PTEN-PEP-1" 및 "PEP-1-PTEN-PEP-1" 융합단백질 또한 PEP-1-PTEN과 동일하거나 유사한 발현 양상, 세포 침투성, 안정성, 시간 및 농도 의존적 침투성, 피부 조직 침투성을 나타내었음을 밝힌다.Hereinafter, the configuration of the present invention through the embodiment in more detail. However, the scope of the present invention is not limited to the description of the examples. In particular, in the present embodiment, the "PEP-1-PTEN" fusion protein of the PTEN fusion protein has been described as an example, but the "PTEN-PEP-1" and "PEP-1-PTEN-PEP-1" fusion proteins are also described as PEP-1- The expression pattern, cell permeability, stability, time and concentration-dependent permeability, and skin tissue permeability are shown.

<재료><Material>

제한 효소와 T4 DNA 라이게이즈(ligase)는 Promega(USA)에서 구입하였고, Pfu 폴리머라아제(polymerase)는 stratagene(USA)에서 구입하였다. Tat 올리고뉴클레오타이드는 Gibco BRL custom primer(USA)에서 합성하였다. IPTG는 Duchefa(Netherland)에서 구입하였다. pET-15b와 BL21(DE3) 플라스미드는 Novagen(USA)에서 구입하였고, Ni-나이트릴로트리아세트산 세파로즈 수퍼플로우(nitrilo- triactic acid sepharose superflow)는 Qiagen(Germany)에서 구입하였다. 사람 PTEN cDNA는 PCR 방법으로 사람 간 cDNA 라이브러리에서 분리하였다. 이외 모든 시약은 특급 제품을 이용하였다.Restriction enzymes and T4 DNA ligase were purchased from Promega (USA) and Pfu polymerase was purchased from stratagene (USA). Tat oligonucleotides were synthesized in Gibco BRL custom primer (USA). IPTG was purchased from Duchefa (Netherland). pET-15b and BL21 (DE3) plasmids were purchased from Novagen (USA) and Ni-nitrilotriactic acid sepharose superflow was purchased from Qiagen (Germany). Human PTEN cDNA was isolated from human liver cDNA library by PCR method. All other reagents were used as express products.

실시예Example 1: 재조합 1: recombination PEPPEP -1--One- PTENPTEN 융합단백질의Fusion protein 발현벡터 제조 및 형질변환 Expression vector preparation and transformation

기능을 가진 단백질을 세포 내로 침투시키는 기술을 개발하기 위하여 세포 내로 목표 단백질을 전달할 수 있는 융합 단백질 발현벡터를 제조하였고, PEP-1 펩타이드가 단백질을 세포 내로 전달하는 능력을 용이하게 분석하기 위하여 인간 PTEN 단백질을 선택하였다.In order to develop a technique for infiltrating a protein having a function into a cell, a fusion protein expression vector capable of delivering a target protein into a cell was prepared, and a human PTEN was easily analyzed to easily analyze the ability of the PEP-1 peptide to deliver the protein into the cell. Protein was selected.

먼저, PEP-1-PTEN 융합단백질을 생산하기 위해 PEP-1 펩타이드(KETWWE TWWTEW SQP KKKRKV; 21 아미노산)가 포함된 pET-PEP 발현벡터를 제조하였다. PEP-1 펩타이드에 해당하는 두 종류의 올리고뉴클레오타이드(상위 쇄, 5'-TATGAAAGAAACCTGGTGGGAAACCTGGTGGACCGAATGGTCTCAGCCGAAAAAAAAACGTAAAGTGC-3'; 하위 쇄, 5'-TCGAGCACTTTACGTTTTTTTTTCGGCTGACACCATTCGGTCCACCAGGTTTCCCACCAGGTTTCTTTCC-3')를 Nde Ⅰ-Xho Ⅰ 제한효소로 자른 pET-15b에 결찰(ligation)하여 삽입하였다. 이어, 사람 PTEN의 cDNA의 서열을 기본으로 하여 두 종류의 올리고뉴클레오타이드를 합성하였다. 정방향 프라이머는 5’-CTCGAGATGGCAGCCATCATCAAAGAGATC-3'로 XhoⅠ 제한부위를 지니고 있으며, 역방향 프라이머는 5’-GGATCCTCAGACTTTTGTAATTTGTGTATG-3'로 BamH Ⅰ 제한부위를 갖고 있다.First, PEP-1-PTEN To produce a fusion protein, a pET-PEP expression vector containing a PEP-1 peptide (KETWWE TWWTEW SQP KKKRKV; 21 amino acids) was prepared. Up of the two types for the PEP-1 peptide nucleotides; the (top strand, 5'-TATGAAAGAAACCTGGTGGGAAACCTGGTGGACCGAATGGTCTCAGCCGAAAAAAAAACGTAAAGTGC-3 'Sub-chain, 5'-TCGAGCACTTTACGTTTTTTTTTCGGCTGACACCATTCGGTCCACCAGGTTTCCCACCAGGTTTCTTTCC-3') Nde Ⅰ - cut by restriction enzymes Xho Ⅰ ligated to pET-15b Inserted by ligation. Subsequently, two kinds of oligonucleotides were synthesized based on the sequence of the cDNA of human PTEN. The forward primer has a Xho I restriction site with 5'-CTCGAGATGGCAGCCATCATCAAAGAGATC-3 'and the reverse primer has a BamH I restriction site with 5'-GGATCCTCAGACTTTTGTAATTTGTGTATG-3'.

중합효소 연쇄반응(Polymerase Chain Reaction)은 온열 순환 반응기(Perkin-Elmer, model 9600)에서 수행하였다. 반응 혼합액을 50㎕ 실리콘 튜브에 넣고 94℃에서 5분간 가열하였다. PCR 반응을 수행하였다. PCR 수행 후 아가로즈 젤 전기영동으로 반응물을 분리하고 이것을 TA 클로닝 벡터(Invitrogen, Sandiego, USA)에 결찰(ligation)한 다음, 컴피턴트 세포(competent cell)에 형질변환시키고 형질변환된 박테리아로부터 플라스미드를 알칼리 용균법으로 분리하였다. 사람 PTEN cDNA가 포함된 TA 벡터를 Xho Ⅰ과 BamH Ⅰ로 절단한 다음 PEP 발현 벡터에 삽입하였다. PEP-1-PTEN으로 형질변환된 E. coli BL21 (DE3)를 선택한 다음, 콜로니를 100㎖ LB 배지에 접종하고 IPTG(isopropyl-beta-D-thiogalactopyranoside) 0.5mM를 배지 내에 첨가하여 재조합된 PEP-1-PTEN의 과대발현을 유도하였다. 과대발현된 PEP-1-PTEN은 SDS-폴리아크릴아마이드 젤 전기영동과 웨스턴 블랏 분석으로 확인하였다.Polymerase chain reaction was carried out in a thermal cycle reactor (Perkin-Elmer, model 9600). The reaction mixture was placed in a 50 μl silicon tube and heated at 94 ° C. for 5 minutes. PCR reactions were performed. After PCR, the reaction was isolated by agarose gel electrophoresis, which was ligated to a TA cloning vector (Invitrogen, Sandiego, USA), transformed into competent cells, and plasmids from the transformed bacteria. It was separated by alkaline lysis method. TA vectors containing human PTEN cDNA were identified by Xho I and Cleavage with BamH I and insertion into the PEP expression vector. E. coli BL21 (DE3) transformed with PEP-1-PTEN was selected, and the colonies were inoculated in 100 ml LB medium and 0.5 mM of isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to the medium to recombine the PEP-. Overexpression of 1-PTEN was induced. Overexpressed PEP-1-PTEN was confirmed by SDS-polyacrylamide gel electrophoresis and Western blot analysis.

실시예Example 2: 2: PEPPEP -1--One- PTENPTEN 융합 단백질의 발현 및 정제 Expression and Purification of Fusion Proteins

본 연구실에서 제조한 인간 PTEN cDNA가 포함되어 있는 E. coli BL21 (DE3) 세포 (PEP-1-PTEN)를 암피실린이 포함된 LB 배지에 넣고 37℃에서 200 rpm으로 교반하며 배양하였다. 배양액 내의 박테리아 농도가 O.D₆₀₀ = 0.5~1.0을 나타낼 때 IPTG를 배지 내에 첨가하여 최종농도가 0.5mM와 1mM가 되게 한 다음 30℃에서 12 시간을 더 배양하였다. 배양한 세포를 원심분리하여 모은 뒤 5㎖ 결합 완충액(5mM 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)을 넣고 초음파 분쇄기로 분쇄(sonication)하였다. 원심분리하여 상층액을 즉시 Ni² ⁺-니트릴로 트리아세트산 세파로즈 슈퍼 플로우(Ni² ⁺-nitrilotriacetic acid sepharose super flow) 컬럼에 부하하고 10배 부피의 결합 완충액과 6배 부피의 세척 완충액(60mM 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)으로 세척한 다음 용출 완충액(1M 이미다졸, 0.5M NaCl, 20mM Tris-HCl, pH 7.9)으로 융합 단백질을 용출하였다. 이어, 융합 단백질이 포함된 분획들을 모아 PD-10 컬럼 크로마토그래피를 수행하여 분획 중에 포함된 염분을 제거하였다. E. coli BL21 (DE3) cells (PEP-1-PTEN) containing human PTEN cDNA prepared in this laboratory were placed in LB medium containing ampicillin and incubated at 37 ° C. at 200 rpm. When the concentration of bacteria in the culture medium was OD ₆₀₀ = 0.5-1.0, IPTG was added into the medium so that the final concentration was 0.5 mM and 1 mM, and then further incubated for 12 hours at 30 ° C. The cultured cells were collected by centrifugation, 5 ml binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) was added, and pulverized with an ultrasonic grinder. The supernatant was immediately centrifuged Ni ² ⁺ - acrylonitrile tree ethyl Sepharose Super flow ^{^{(Ni 2 + -nitrilotriacetic acid sepharose super}} flow) load the column in binding buffer and 10-fold volume of the washing buffer 6 times the volume of (60mM already The fusion protein was washed with dozol, 0.5M NaCl, 20mM Tris-HCl, pH 7.9) followed by elution buffer (1M imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9). Subsequently, the fractions containing the fusion protein were collected and PD-10 column chromatography was performed to remove salts contained in the fractions.

정제된 단백질 농도는 우혈청 알부민을 표준물질로 사용하여 브래드포드 방법으로 측정하였다.Purified protein concentration was determined by Bradford method using bovine serum albumin as a standard.

실시예Example 3: 피부 세포배양 및 3: skin cell culture and PEPPEP -1--One- PTENPTEN 융합 단백질의 세포 투과 Cell Permeation of Fusion Proteins

피부세포는 37℃에서 95% 공기와 5% CO₂를 공급해주며 20mM HEPES/NaOH (pH 7.4), 5mM NaHCO₃, 10% FBS(fetal bovine serum) 및 항생제(100㎍/㎖ 스트렙토마이신, 100U/㎖ 페니실린)가 포함된 DMEM(Dulbecco's Modified Eagle's Medium)에서 배양하였다.Dermal cells provide 95% air and 5% CO ₂ at 37 ° C with 20 mM HEPES / NaOH (pH 7.4), 5 mM NaHCO ₃ , 10% fetal bovine serum (FBS) and antibiotics (100 μg / ml streptomycin, 100 U / Cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing ml penicillin).

PEP-1-PTEN 융합 단백질의 세포 내 투과를 관찰하기 위하여, 피부 세포를 6-웰 플레이트에서 4~6시간 동안 키운 뒤 10% FBS가 포함된 신선한 DMEM 배양액으로 교체하고, 재조합 PEP-1-PTEN을 농도 및 시간별로 배양액 내에 처리하였다. 처리 후 세포를 트립신-EDTA(Gibco BRL)로 처리하고 인산 완충액 생리식염수(phosphate buffered saline; "PBS")로 충분히 세척하고 세포를 분쇄한 다음 세포 내로 투과된 PTEN의 양을 웨스턴 블랏팅으로 측정하였다.To observe intracellular penetration of PEP-1-PTEN fusion protein, skin cells were grown for 4-6 hours in 6-well plates, replaced with fresh DMEM culture containing 10% FBS, and recombinant PEP-1-PTEN Was treated in the culture by concentration and time. After treatment, the cells were treated with trypsin-EDTA (Gibco BRL), thoroughly washed with phosphate buffered saline ("PBS"), the cells were ground and the amount of PTEN penetrated into the cells was measured by Western blotting. .

실시예Example 4: 4: 웨스턴Weston 블랏Blot 분석 analysis

PEP-1-PTEN의 세포 내 투과를 확인하기 위하여 웨스턴 블랏 방법을 수행하였다. 먼저 단백질은 자연상태로 정제하였다. 준비된 피부 세포에 PEP-1-PTEN(3μM)의 융합 단백질을 처리한 다음 1시간 후, 세포들만 모아서 웨스턴 블랏을 수행하였다. 세포 분쇄액 내의 단백질을 12% SDS 폴리아크릴아마이드 젤로 분리한 다음 젤에 있는 단백질을 니트로셀룰로스 멤브레인(nitrocellulose membrane; Amersham, UK)으로 전기이동시켰다. 단백질이 이동된 니트로셀룰로스 멤브레인을 5% 탈분유(non-dry milk)가 들어 있는 PBS로 블로킹하였다. 이어 멤브레인은 래빗 항-히스티딘 폴리클론 항체(Santacruze, USA, 1:1,000)로 1시간 처리하였다. 세척 후, 호스래디쉬 퍼옥시다아제-결합된 마우스 항-래빗 IgG 항체(1:10,000 희석)와 한 시간 반응시켰다. 최종적으로 ECL 킷트(ECL; Amersham) 을 이용하여 사람 PTEN 모노클론 항체에 반응하는 단백질 띠를 확인하였다.Western blot methods were performed to confirm intracellular penetration of PEP-1-PTEN. First, the protein was purified in its natural state. 1 hour after the fusion protein of PEP-1-PTEN (3 μM) was treated to the prepared skin cells, only cells were collected and Western blot was performed. Proteins in the cell mill were separated with a 12% SDS polyacrylamide gel and then the proteins in the gel were electrophoresed to a nitrocellulose membrane (Amersham, UK). The protein-shifted nitrocellulose membrane was blocked with PBS containing 5% non-dry milk. The membrane was then treated with rabbit anti-histidine polyclonal antibody (Santacruze, USA, 1: 1,000) for 1 hour. After washing, one hour of reaction with horseradish peroxidase-bound mouse anti-rabbit IgG antibody (1: 10,000 dilution) was performed. Finally, an ECL kit (ECL; Amersham) was used to identify protein bands that respond to human PTEN monoclonal antibodies.

실시예Example 5: 5: 피부세포로Into skin cells 투과된 Transmitted PEPPEP -1--One- PTENPTEN 융합 단백질의 안정성 Stability of Fusion Proteins

세포 내로 투과된 PEP-1-PTEN 융합 단백질의 세포 내 안정성을 측정하기 위하여, 피부세포를 6-웰 플레이트에서 4~6시간 동안 키운 뒤 FBS가 포함되지 않은 1㎖의 신선한 배양액으로 교체하고 재조합 PEP-1-PTEN 융합 단백질을 배양액 내에 처리하였다. 처리 한 시간 후 세포를 트립신-EDTA로 처리하고 PBS로 충분히 세척하였다. 이어 FBS가 포함된 배지를 넣고 계속 배양하면서 시간별로 세포를 모아 세포 내에 남아 있는 융합 단백질의 양을 웨스턴 블랏팅으로 측정하였다.To measure the intracellular stability of the PEP-1-PTEN fusion protein permeated into cells, skin cells were grown in 6-well plates for 4-6 hours and then replaced with 1 ml fresh culture medium without FBS and recombinant PEP. -1-PTEN fusion protein was treated in culture. One hour after treatment cells were treated with trypsin-EDTA and washed well with PBS. Then, the medium containing the FBS was added, and the cells were continuously cultured over time, and the amount of fusion protein remaining in the cells was measured by Western blotting.

실시예Example 6: 면역반응 억제 효과 6: inhibitory effect of immune response

LPS(Lipopolysaccharide)로 유도한 면역억제 효과를 측정하기 위해서 피부세포를 6-웰 플레이트에서 키운 뒤 재조합 PEP-1-PTEN 융합 단백질을 배양액 내에 1시간 전에 처리한 후, LPS를 투여하였다. 투여 후 COX(Cyclooxygenase)-2의 발현과 세포사멸을 웨스턴 블랏팅과 MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] 분석을 통하여 확인하였다.In order to measure the immunosuppressive effect induced by lipopolysaccharide (LPS), skin cells were grown in 6-well plates and treated with recombinant PEP-1-PTEN fusion protein 1 hour before in the culture medium, followed by administration of LPS. Expression and apoptosis of COX (Cyclooxygenase) -2 after administration were confirmed by Western blotting and MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] analysis.

실시예Example 7: 면역조직염색( 7: Immune tissue staining ImmunohistochemistryImmunohistochemistry ))

PEP-1-PTEN 융합 단백질이 피부 조직 내로 투과되는지를 확인하기 위해서 면역조직염색을 실시하였다. 실험동물은 수컷 ICR 마우스를 사용하였다. 실험동물은 질소와 산소 혼합가스에 들어있는 3% 이소플루란스(isoflurance)로 마취시켰으며, 피부에 있는 털을 제거한 다음 PEP-1-PTEN 융합 단백질을 피부에 발라 한 시간 동안 처리하였다. 그 후 피부 조직을 냉동조직 절편으로 만들었다.Immunohistostaining was performed to confirm that PEP-1-PTEN fusion protein was permeated into skin tissue. Experimental animals used male ICR mice. The experimental animals were anesthetized with 3% isoflurance contained in a nitrogen and oxygen mixed gas. After removing the hair on the skin, PEP-1-PTEN fusion protein was applied to the skin and treated for one hour. The skin tissue was then made into frozen tissue sections.

면역조직염색에 앞서 냉동조직 절편을 상온에서 30분간 건조한 후 37℃에서 2시간 이상 완전히 말린 다음 실험을 시작하였다. 조직 절편은 0.01M PBS로 10분간 완충시킨 후 다시 증류수로 10분간 세척하였다. 이후 7분씩 2회 PBS로 세척한 다음 조직 내의 내인성 퍼옥시다제를 제거하기 위해서 0.5% 과산화수소가 함유된 PBS 용액에서 20분간 반응시켰다. 이후 PBS로 7분씩 2회 세척한 후 비-특이적 반응을 방지하기 위하여 조직을 PBS에 들어있는 5% 정상 염소 혈청과 30분간 반응시켰다. 이후 과정은 통상적인 스트렙타비딘-바이오틴 퍼옥시다제(Streptavidin- biotin peroxidase) 법을 이용한 면역조직화학반응을 실시하였다.Prior to immunohistostaining, frozen tissue sections were dried at room temperature for 30 minutes and then completely dried at 37 ° C. for at least 2 hours before starting the experiment. Tissue sections were buffered with 0.01 M PBS for 10 minutes and washed again with distilled water for 10 minutes. After washing twice with PBS for 7 minutes each, and then reacted for 20 minutes in a PBS solution containing 0.5% hydrogen peroxide to remove endogenous peroxidase in the tissue. After washing twice with PBS for 7 minutes, the tissues were reacted with 5% normal goat serum in PBS for 30 minutes to prevent non-specific reactions. Subsequently, the immunohistochemical reaction was performed using a conventional streptavidin-biotin peroxidase method.

1차 항체는 토끼 항-마우스 히스티딘(Santa cruz, USA)을 사용하였다. 면역조직화학반응에 이용된 모든 항체는 2% 정상 염소 혈청 및 0.1% triton X-100이 함유된 0.1M PBS(pH 7.4)에 희석하여 사용하였다. 피부 조직은 1차 항체를 1:500으로 희석하여 4℃에서 48시간 동안 반응시켰다. 이후 2차 항체로 바이오틴 결합된 토끼 항-마우스 IgG(Vector, USA)를 1:200으로 희석하여 상온에서 2 시간 반응시킨 후 1:200으로 희석한 퍼옥시다제-결합된 스트렙타비딘(Vector, USA)으로 상온에서 2시간 반응시켰다. 이상 각 단계의 반응 후에는 PBS 용액으로 4~5차례 세척하였다.The primary antibody was rabbit anti-mouse histidine (Santa cruz, USA). All antibodies used for immunohistochemistry were diluted in 0.1M PBS (pH 7.4) containing 2% normal goat serum and 0.1% triton X-100. The skin tissue was diluted 1: 500 primary antibody and reacted at 4 ° C. for 48 hours. Thereafter, the rabbit anti-mouse IgG (Vector, USA) conjugated with a secondary antibody was diluted 1: 200 and reacted at room temperature for 2 hours, followed by a peroxidase-linked streptavidin (Vector, diluted 1: 200). USA) at room temperature for 2 hours. After the reaction of each step was washed 4-5 times with PBS solution.

항원항체반응이 끝난 조직은 0.03% 과산화수소와 0.05% DAB(3,3´- diaminobenzidine tetrachloride; Sigma, USA)가 함유된 트리스 완충액(pH 7.4)에서 1~5분간 발색하였다. 발색반응이 끝난 조직은 수세 후 수용성 봉입제인 크리스탈 마운트(crystal mount; Biomeda, USA)를 이용하여 봉입한 다음 현미경 관찰 후 사진촬영(Axioscope microscope; Carl Zeiss, Germany)을 하였다.Antigen-reacted tissues were developed in Tris buffer (pH 7.4) containing 0.03% hydrogen peroxide and 0.05% DAB (3,3'-diaminobenzidine tetrachloride; Sigma, USA) for 1-5 minutes. After completion of the color reaction, the tissue was encapsulated using a crystal mount (Biomeda, USA), which is a water-soluble encapsulant, and then photographed after a microscope observation (Axioscope microscope; Carl Zeiss, Germany).

결과 1: Result 1: PEPPEP -1--One- PTENPTEN 융합 단백질의 과대발현 및 정제 Overexpression and Purification of Fusion Proteins

PEP-1-PTEN 융합 단백질을 과대발현시키기 위해 사람 PTEN cDNA, PEP 펩타이드(KETWWETWWTEW SQP KKKRKV; 21아미노산) 및 6개의 히스티딘이 연속적으로 포함되어 있는 PEP-1-PTEN 발현 벡터를 개발하였다(도 1).To overexpress the PEP-1-PTEN fusion protein, a PEP-1-PTEN expression vector was developed containing human PTEN cDNA, PEP peptide (KETWWETWWTEW SQP KKKRKV; 21 amino acids) and six histidines in succession (FIG. 1). .

IPTG로 융합 단백질의 과대발현을 유도한 대장균 세포를 4℃에서 초음파 분쇄기로 파쇄한 다음 원심분리하여 상층액의 단백질을 12% SDS-폴리아크릴아마이드 젤 전기영동으로 분리하였다. 도 2는 PEP-1-PTEN의 과대발현과 정제된 단백질을 쿠마시 브릴리언트 블루로 염색한 단백질 띠(도 2A)와 웨스턴 블랏으로 확인한 결과이다(도 2B). 도 2A의 레인 2는 0.5mM IPTG를 처리하여 과대발현된 융합단백질을 나타내고 있다.Escherichia coli cells, which induced overexpression of the fusion protein with IPTG, were disrupted by an ultrasonic grinder at 4 ° C. and centrifuged to separate supernatant proteins by 12% SDS-polyacrylamide gel electrophoresis. FIG. 2 shows the results of overexpression of PEP-1-PTEN and protein blots stained with Coomassie Brilliant Blue (FIG. 2A) and Western blot (FIG. 2B). Lane 2 in FIG. 2A shows the overexpressed fusion protein by treatment with 0.5 mM IPTG.

PEP-1-PTEN 융합 단백질은 N-말단에 6개의 히스티딘을 포함하고 있기 때문에 고정 금속-킬레이트 친화 크로마토그래피(immobilized metal-chelate affinity chromatography) 단일 단계로 융합 단백질을 자연상태로 순수하게 정제하였다. 또한 정제된 PEP-1-PTEN 융합 단백질을 쿠마시 브릴리언트 블루로 염색하여 확인한 결과이다.Since the PEP-1-PTEN fusion protein contained six histidines at the N-terminus, the fusion protein was purified in nature purely in a single step using immobilized metal-chelate affinity chromatography. In addition, the purified PEP-1-PTEN fusion protein was confirmed by staining with Coomassie brilliant blue.

과대발현 및 정제된 PEP-1-PTEN 융합 단백질을 웨스턴 블랏팅으로 다시 한번 확인하였다. 도 2B에 나타낸 바와 같이 6x 히스티딘 항체에 반응하는 PEP-1-PEN 띠는 도 2A의 단백질 띠와 동일한 위치에서 관찰되었다.Overexpressed and purified PEP-1-PTEN fusion protein was once again confirmed by western blotting. As shown in FIG. 2B, the PEP-1-PEN band responding to the 6x histidine antibody was observed at the same position as the protein band of FIG. 2A.

결과result 2: 2: PEPPEP -1--One- PTENPTEN 융합 단백질의 Of fusion proteins 피부세포Skin cells 투과 Penetration

자연 상태에서 정제한 PEP-1-PTEN 융합 단백질의 피부 세포 투과가 시간 및 농도에 따라 어떻게 변화되는지를 관찰하였다. 도 3에 나타낸 것처럼 자연 상태의 PEP-1-PTEN는 시간 및 농도 의존적으로 피부 세포 내로 투과되는 것을 웨스턴 블랏팅으로 확인하였다. 도 3A는 3μM PEP-1-PTEN을 시간에 따라, 도 3B는 농도에 따라 PEP-1-PTEN을 1시간 동안 세포배양액 내에 처리하였을 때의 결과이다. PEP-1-PTEN의 세포 내 투과 정도를 농도별로 관찰했을 때, 농도 의존적으로 세포 내로 투 과된 PEP-1-PTEN의 양이 증가되는 것을 확인하였다. 또한, 시간별로 관찰하였을 경우 처리 시간에 비례하여 세포 내로 투과된 PEP-1-PTEN의 양이 증가함을 확인하였다. 이러한 결과는 PEP-1-PTEN 융합 단백질이 시간 및 농도 의존적으로 세포 내 투과가 일어남을 말해준다.It was observed how the skin cell permeation of the purified PEP-1-PTEN fusion protein in nature changed with time and concentration. As shown in FIG. 3, Western blotting confirmed that natural PEP-1-PTEN penetrated into skin cells in a time and concentration-dependent manner. FIG. 3A shows the results when 3 μM PEP-1-PTEN was treated with time, and FIG. 3B was treated with PEP-1-PTEN in the cell culture solution for 1 hour according to the concentration. When observing the degree of intracellular penetration of PEP-1-PTEN by concentration, it was confirmed that the amount of PEP-1-PTEN permeated into cells was increased in a concentration-dependent manner. In addition, when observed over time, it was confirmed that the amount of PEP-1-PTEN permeated into the cells increased in proportion to the treatment time. These results indicate that the PEP-1-PTEN fusion protein undergoes time and concentration dependent intracellular penetration.

피부 세포 내로 투과된 PEP-1-PTEN은 상당한 기간 동안 세포 내에서 안정성을 유지해야만 효과적으로 단백질 치료에 응용할 수 있다. 도 3C에 나타낸 바와 같이 세포 내로 투과된 PEP-1-PTEN은 시간에 따라 분해되어 단백질의 양이 점차 감소하였으나, 최소 24시간 동안 세포 내에서 존재함을 알 수 있었다. 따라서, 세포 내로 투과된 PEP-1-PTEN은 최소 24시간은 안정성을 유지하고 있기 때문에 유용하게 단백질치료에 응용할 수 있으리라 사료된다.PEP-1-PTEN permeated into skin cells must remain stable in the cells for a significant period of time before they can be effectively applied to protein therapy. As shown in FIG. 3C, PEP-1-PTEN penetrated into cells was degraded with time, and the amount of protein gradually decreased, but it was found to exist in cells for at least 24 hours. Therefore, PEP-1-PTEN penetrated into cells is useful for protein treatment because it maintains stability for at least 24 hours.

결과result 3: 3: PEPPEP -1--One- PTENPTEN 융합 단백질의 마우스 피부로 투과Permeation of Fusion Proteins into Mouse Skin

마우스 피부에도 PEP-1-PTEN이 효과적으로 투과가 일어나는지 알아보기 위해서 면역조직염색을 이용하여 확인하였다. 도 4는 마우스 피부 조직을 염색한 결과로 50㎍ PEP-1-PTEN을 1시간 피부에 발라주고 염색하였을 경우 PEP-1-PTEN이 피부조직 안으로 투과된 모습을 잘 보여주고 있다. 투과된 PEP-1-PTEN은 피부조직의 상피세포 및 진피세포까지 투과함을 확인하였다.PEP-1-PTEN was also confirmed using immunohistostaining to determine whether permeation effectively occurs in mouse skin. Figure 4 shows the appearance of PEP-1-PTEN penetrated into the skin tissue when stained with 50 ㎍ PEP-1-PTEN skin for 1 hour as a result of staining mouse skin tissue. Permeated PEP-1-PTEN was confirmed to penetrate the epithelial and dermal cells of the skin tissue.

결과result 4:4: PEPPEP -1--One- PTENPTEN 융합 단백질의 생물학적 기능Biological Functions of Fusion Proteins

피부 세포 내로 투과된 융합 단백질은 그 고유한 활성을 유지해야만 이를 단 백질 치료에 응용할 수 있다. 따라서, 세포 내로 투과된 융합단백질이 어느 정도 생물학적인 활성을 지니는지는 매우 중요한 문제이다. 따라서 정제된 PEP-1-PTEN 단백질의 생물학적 기능을 알아보기 위해 LPS에 의해 유발되는 COX-2 발현과 세포사를 MTT 분석을 이용하여 확인하였다. 정제한 PEP-1-PTEN 재조합 단백질은 농도별로 LPS에 의한 세포사 및 COX-2 발현을 억제함을 확인하였다(도 5, 도 6). 세포 내로 투과된 PEP-1-PTEN 융합 단백질이 효과적으로 작용하고 있음을 알 수 있었다. 따라서, 상기 결과 3 및 결과 4로부터 PTEN 단백질이 PTEN 단백질의 효능으로 잘 알려진 항암제로서의 기능, 염증 억제 기능 등이 생체 내에서 원활히 수행될 수 있을 것으로 예측된다.Fusion proteins permeated into skin cells must maintain their inherent activity before they can be applied to protein treatment. Therefore, the degree of biological activity of the fusion protein penetrated into the cell is a very important problem. Therefore, in order to examine the biological function of purified PEP-1-PTEN protein, COX-2 expression and cell death induced by LPS were confirmed using MTT analysis. Purified PEP-1-PTEN recombinant protein was confirmed to inhibit cell death and COX-2 expression by LPS by concentration (Fig. 5, Fig. 6). It was found that the PEP-1-PTEN fusion protein penetrated into the cell is effective. Therefore, it is expected from the results 3 and 4 that the PTEN protein can be performed smoothly in vivo as a function of an anticancer agent, an inflammation inhibitory function and the like, well known for the efficacy of the PTEN protein.

도 1은 PEP-1-PTEN 융합 단백질 발현 벡터의 개략도. PEP-1-PTEN 융합 단백질 발현 벡터는 벡터 pET-15b를 바탕으로 하여 구성되었다. 합성 PEP-1 올리고머는 pET-15b의 Nde Ⅰ, Xho Ⅰ 제한부위에, 그리고 사람 PTEN cDNA는 pET-15b의 Xho Ⅰ, BamH Ⅰ 제한부위에 삽입되었다. 발현은 IPTG를 가하여 유도되었다.1 is a schematic representation of a PEP-1-PTEN fusion protein expression vector. PEP-1-PTEN fusion protein expression vector was constructed based on vector pET-15b. Synthetic PEP-1 oligomers were inserted at the Nde I, Xho I restriction sites of pET-15b, and human PTEN cDNA was inserted at the Xho I, BamH I restriction sites of pET-15b. Expression was induced by addition of IPTG.

도 2는 PEP-1-PTEN 융합 단백질의 발현과 정제에 관한 사진이다. 세포의 단백질 추출물과 정제된 융합 단백질이 12% SDS-PAGE(A)로 분석되었고 항-래빗 폴리히스티딘 항체로 웨스턴 블랏 분석되었다(B).Figure 2 is a photograph of the expression and purification of PEP-1-PTEN fusion protein. Protein extracts of cells and purified fusion proteins were analyzed by 12% SDS-PAGE (A) and Western blot analysis with anti-rabbit polyhistidine antibodies (B).

레인 1: 비유도(non-induced) PEP-1-PTENLane 1: non-induced PEP-1-PTEN

레인 2: 유도된 PEP-1-PTENLane 2: Induced PEP-1-PTEN

레인 3: 정제된 PEP-1-PTENLane 3: purified PEP-1-PTEN

도 3은 PEP-1-PTEN 융합 단백질의 세포 도입을 웨스턴 블랏팅한 사진이다. 3 is a photograph of Western blotting of cell introduction of PEP-1-PTEN fusion protein.

(A) 3μM의 PEP-1-PTEN 융합 단백질을 배양 배지에 10~60분간 가했다.(A) 3 μM of PEP-1-PTEN fusion protein was added to the culture medium for 10 to 60 minutes.

(B) 0.5~3μM PEP-1-PTEN 융합 단백질을 배양 배지에 60분간 가했다.(B) 0.5-3 μM PEP-1-PTEN fusion protein was added to the culture medium for 60 minutes.

(C) 3μM PEP-1-PTEN 융합 단백질로 1~48시간 전처리한 세포.(C) Cells pretreated with 3 μM PEP-1-PTEN fusion protein for 1-48 hours.

도 4는 PEP-1-PTEN 융합 단백질로 세포도입한 동물 피부를 조직화학 분석한 것이다. 50㎍의 PEP-1-PTEN 융합 단백질을 마우스 등 피부를 면도한 곳에 도포하고 60분간 처리하였다. 피부 조직의 동결 절편을 토끼 항-히스티딘 IgG(1:400)으로 면역염색한 후 바이오틴이 결합된 염소 항-토끼 IgG(1:200)로 염색하였다. 절편은 3,3'-다이아미노벤지딘(diaminobenzidine)으로 염색하여 액시오스코프 현미경으로 관찰하였다.Figure 4 is a histochemical analysis of animal skin introduced into the PEP-1-PTEN fusion protein. 50 μg of PEP-1-PTEN fusion protein was applied to the shaved skin of the mouse and treated for 60 minutes. Frozen sections of skin tissue were immunostained with rabbit anti-histidine IgG (1: 400) and then stained with biotin-bound goat anti-rabbit IgG (1: 200). Sections were stained with 3,3'-diaminobenzidine and observed under an axioscope microscope.

도 5는 세포로 투과된 PEP-1-PTEN 융합 단백질이 LPS로 유도된 COX-2의 발현을 억제하는 것을 나타내는 결과이다. 피부 세포를 3μM의 PEP-1-PTEN을 넣고 또는 넣지 않고 LPS에 노출시켰다.5 is a result showing that the PEP-1-PTEN fusion protein permeated into cells inhibits the expression of COX-2 induced by LPS. Skin cells were exposed to LPS with or without 3 μM of PEP-1-PTEN.

도 6은 피부 세포의 생존률에 대한 세포투과 PEP-1-PTEN 융합 단백질의 효과를 나타낸다. 한 시간 동안 0.5~3μM의 PEP-1-PTEN 융합 단백질로 전처리한 피부 세포를 LPS에 노출시켰다. 세포 생존률은 MTT를 이용하여 발색 분석으로 측정하였다.Figure 6 shows the effect of cell permeation PEP-1-PTEN fusion protein on the survival rate of skin cells. Skin cells pretreated with 0.5-3 μM PEP-1-PTEN fusion protein were exposed to LPS for one hour. Cell viability was determined by color analysis using MTT.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Cell-transducing PTEN fusion protein <130> hallymU-PEP-PTEN <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> top strand oligonucleotide coding PEP-1 <400> 1 tatgaaagaa acctggtggg aaacctggtg gaccgaatgg tctcagccga aaaaaaaacg 60 taaagtgc 68 <210> 2 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> bottom strand oligonucleotide coding PEP-1 <400> 2 tcgagcactt tacgtttttt tttcggctga caccattcgg tccaccaggt ttcccaccag 60 gtttctttcc 70 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> protein transducing domain called PEP-1 <400> 3 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forwaed primer <400> 4 ctcgagatgg cagccatcat caaagagatc 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 5 ggatcctcag acttttgtaa tttgtgtatg 30 <210> 6 <211> 1287 <212> DNA <213> Artificial Sequence <220> <223> recombinant polynucleotide coding PEP-1-PTEN fusion protein <220> <221> CDS <222> (1)..(1278) <400> 6 atg aaa gaa acc tgg tgg gaa acc tgg tgg acc gaa tct cag ccg aaa 48 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys 1 5 10 15 aaa aaa cgt aaa gtg ctc gag atg aca gcc atc atc aaa gag atc gtt 96 Lys Lys Arg Lys Val Leu Glu Met Thr Ala Ile Ile Lys Glu Ile Val 20 25 30 agc aga aac aaa agg aga tat caa gag gat gga ttc gac tta gac ttg 144 Ser Arg Asn Lys Arg Arg Tyr Gln Glu Asp Gly Phe Asp Leu Asp Leu 35 40 45 acc tat att tat cca aac att att gct atg gga ttt cct gca gaa aga 192 Thr Tyr Ile Tyr Pro Asn Ile Ile Ala Met Gly Phe Pro Ala Glu Arg 50 55 60 ctt gaa ggc gta tac agg aac aat att gat gat gta gta agg ttt ttg 240 Leu Glu Gly Val Tyr Arg Asn Asn Ile Asp Asp Val Val Arg Phe Leu 65 70 75 80 gat tca aag cat aaa aac cat tac aag ata tac aat ctt tgt gct gaa 288 Asp Ser Lys His Lys Asn His Tyr Lys Ile Tyr Asn Leu Cys Ala Glu 85 90 95 aga cat tat gac acc gcc aaa ttt aat tgc aga gtt gca caa tat cct 336 Arg His Tyr Asp Thr Ala Lys Phe Asn Cys Arg Val Ala Gln Tyr Pro 100 105 110 ttt gaa gac cat aac cca cca cag cta gaa ctt atc aaa ccc ttt tgt 384 Phe Glu Asp His Asn Pro Pro Gln Leu Glu Leu Ile Lys Pro Phe Cys 115 120 125 gaa gat ctt gac caa tgg cta agt gaa gat gac aat cat gtt gca gca 432 Glu Asp Leu Asp Gln Trp Leu Ser Glu Asp Asp Asn His Val Ala Ala 130 135 140 att cac tgt aaa gct gga aag gga cga act ggt gta atg ata tgt gca 480 Ile His Cys Lys Ala Gly Lys Gly Arg Thr Gly Val Met Ile Cys Ala 145 150 155 160 tat tta tta cat cgg ggc aaa ttt tta aag gca caa gag gcc cta gat 528 Tyr Leu Leu His Arg Gly Lys Phe Leu Lys Ala Gln Glu Ala Leu Asp 165 170 175 ttc tat ggg gaa gta agg acc aga gac aaa aag gga gta act att ccc 576 Phe Tyr Gly Glu Val Arg Thr Arg Asp Lys Lys Gly Val Thr Ile Pro 180 185 190 agt cag agg cgc tat gtg tat tat tat agc tac ctg tta aag aat cat 624 Ser Gln Arg Arg Tyr Val Tyr Tyr Tyr Ser Tyr Leu Leu Lys Asn His 195 200 205 ctg gat tat aga cca gtg gca ctg ttg ttt cac aag atg atg ttt gaa 672 Leu Asp Tyr Arg Pro Val Ala Leu Leu Phe His Lys Met Met Phe Glu 210 215 220 act att cca atg ttc agt ggc gga act tgc aat cct cag ttt gtg gtc 720 Thr Ile Pro Met Phe Ser Gly Gly Thr Cys Asn Pro Gln Phe Val Val 225 230 235 240 tgc cag cta aag gtg aag ata tat tcc tcc aat tca gga ccc aca cga 768 Cys Gln Leu Lys Val Lys Ile Tyr Ser Ser Asn Ser Gly Pro Thr Arg 245 250 255 cgg gaa gac aag ttc atg tac ttt gag ttc cct cag ccg tta cct gtg 816 Arg Glu Asp Lys Phe Met Tyr Phe Glu Phe Pro Gln Pro Leu Pro Val 260 265 270 tgt ggt gat atc aaa gta gag ttc ttc cac aaa cag aac aag atg cta 864 Cys Gly Asp Ile Lys Val Glu Phe Phe His Lys Gln Asn Lys Met Leu 275 280 285 aaa aag gac aaa atg ttt cac ttt tgg gta aat aca ttc ttc ata cca 912 Lys Lys Asp Lys Met Phe His Phe Trp Val Asn Thr Phe Phe Ile Pro 290 295 300 gga cca gag gaa acc tca gaa aaa gta gaa aat gga agt cta tgt gat 960 Gly Pro Glu Glu Thr Ser Glu Lys Val Glu Asn Gly Ser Leu Cys Asp 305 310 315 320 caa gaa atc gat agc att tgc agt ata gag cgt gca gat aat gac aag 1008 Gln Glu Ile Asp Ser Ile Cys Ser Ile Glu Arg Ala Asp Asn Asp Lys 325 330 335 gaa tat cta gta ctt act tta aca aaa aat gat ctt gac aaa gca aat 1056 Glu Tyr Leu Val Leu Thr Leu Thr Lys Asn Asp Leu Asp Lys Ala Asn 340 345 350 aaa gac aaa gcc aac cga tac ttt tct cca aat ttt aag gtg aag ctg 1104 Lys Asp Lys Ala Asn Arg Tyr Phe Ser Pro Asn Phe Lys Val Lys Leu 355 360 365 tac ttc aca aaa aca gta gag gag ccg tca aat cca gag gct agc agt 1152 Tyr Phe Thr Lys Thr Val Glu Glu Pro Ser Asn Pro Glu Ala Ser Ser 370 375 380 tca act tct gta aca cca gat gtt agt gac aat gaa cct gat cat tat 1200 Ser Thr Ser Val Thr Pro Asp Val Ser Asp Asn Glu Pro Asp His Tyr 385 390 395 400 aga tat tct gac acc act gac tct gat cca gag aat gaa cct ttt gat 1248 Arg Tyr Ser Asp Thr Thr Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp 405 410 415 gaa gat cag cat aca caa att aca aaa gtc tg aggatcc 1287 Glu Asp Gln His Thr Gln Ile Thr Lys Val 420 425 <210> 7 <211> 426 <212> PRT <213> Artificial Sequence <400> 7 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Thr Ala Ile Ile Lys Glu Ile Val 20 25 30 Ser Arg Asn Lys Arg Arg Tyr Gln Glu Asp Gly Phe Asp Leu Asp Leu 35 40 45 Thr Tyr Ile Tyr Pro Asn Ile Ile Ala Met Gly Phe Pro Ala Glu Arg 50 55 60 Leu Glu Gly Val Tyr Arg Asn Asn Ile Asp Asp Val Val Arg Phe Leu 65 70 75 80 Asp Ser Lys His Lys Asn His Tyr Lys Ile Tyr Asn Leu Cys Ala Glu 85 90 95 Arg His Tyr Asp Thr Ala Lys Phe Asn Cys Arg Val Ala Gln Tyr Pro 100 105 110 Phe Glu Asp His Asn Pro Pro Gln Leu Glu Leu Ile Lys Pro Phe Cys 115 120 125 Glu Asp Leu Asp Gln Trp Leu Ser Glu Asp Asp Asn His Val Ala Ala 130 135 140 Ile His Cys Lys Ala Gly Lys Gly Arg Thr Gly Val Met Ile Cys Ala 145 150 155 160 Tyr Leu Leu His Arg Gly Lys Phe Leu Lys Ala Gln Glu Ala Leu Asp 165 170 175 Phe Tyr Gly Glu Val Arg Thr Arg Asp Lys Lys Gly Val Thr Ile Pro 180 185 190 Ser Gln Arg Arg Tyr Val Tyr Tyr Tyr Ser Tyr Leu Leu Lys Asn His 195 200 205 Leu Asp Tyr Arg Pro Val Ala Leu Leu Phe His Lys Met Met Phe Glu 210 215 220 Thr Ile Pro Met Phe Ser Gly Gly Thr Cys Asn Pro Gln Phe Val Val 225 230 235 240 Cys Gln Leu Lys Val Lys Ile Tyr Ser Ser Asn Ser Gly Pro Thr Arg 245 250 255 Arg Glu Asp Lys Phe Met Tyr Phe Glu Phe Pro Gln Pro Leu Pro Val 260 265 270 Cys Gly Asp Ile Lys Val Glu Phe Phe His Lys Gln Asn Lys Met Leu 275 280 285 Lys Lys Asp Lys Met Phe His Phe Trp Val Asn Thr Phe Phe Ile Pro 290 295 300 Gly Pro Glu Glu Thr Ser Glu Lys Val Glu Asn Gly Ser Leu Cys Asp 305 310 315 320 Gln Glu Ile Asp Ser Ile Cys Ser Ile Glu Arg Ala Asp Asn Asp Lys 325 330 335 Glu Tyr Leu Val Leu Thr Leu Thr Lys Asn Asp Leu Asp Lys Ala Asn 340 345 350 Lys Asp Lys Ala Asn Arg Tyr Phe Ser Pro Asn Phe Lys Val Lys Leu 355 360 365 Tyr Phe Thr Lys Thr Val Glu Glu Pro Ser Asn Pro Glu Ala Ser Ser 370 375 380 Ser Thr Ser Val Thr Pro Asp Val Ser Asp Asn Glu Pro Asp His Tyr 385 390 395 400 Arg Tyr Ser Asp Thr Thr Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp 405 410 415 Glu Asp Gln His Thr Gln Ile Thr Lys Val 420 425 <210> 8 <211> 1281 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide for PTEN-PEP-1 fusion protein <220> <221> CDS <222> (1)..(1278) <400> 8 atg aca gcc atc atc aaa gag atc gtt agc aga aac aaa agg aga tat 48 Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr 1 5 10 15 caa gag gat gga ttc gac tta gac ttg acc tat att tat cca aac att 96 Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30 att gct atg gga ttt cct gca gaa aga ctt gaa ggc gta tac agg aac 144 Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35 40 45 aat att gat gat gta gta agg ttt ttg gat tca aag cat aaa aac cat 192 Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His 50 55 60 tac aag ata tac aat ctt tgt gct gaa aga cat tat gac acc gcc aaa 240 Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys 65 70 75 80 ttt aat tgc aga gtt gca caa tat cct ttt gaa gac cat aac cca cca 288 Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro 85 90 95 cag cta gaa ctt atc aaa ccc ttt tgt gaa gat ctt gac caa tgg cta 336 Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu 100 105 110 agt gaa gat gac aat cat gtt gca gca att cac tgt aaa gct gga aag 384 Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys 115 120 125 gga cga act ggt gta atg ata tgt gca tat tta tta cat cgg ggc aaa 432 Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys 130 135 140 ttt tta aag gca caa gag gcc cta gat ttc tat ggg gaa gta agg acc 480 Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 aga gac aaa aag gga gta act att ccc agt cag agg cgc tat gtg tat 528 Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr 165 170 175 tat tat agc tac ctg tta aag aat cat ctg gat tat aga cca gtg gca 576 Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala 180 185 190 ctg ttg ttt cac aag atg atg ttt gaa act att cca atg ttc agt ggc 624 Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly 195 200 205 gga act tgc aat cct cag ttt gtg gtc tgc cag cta aag gtg aag ata 672 Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile 210 215 220 tat tcc tcc aat tca gga ccc aca cga cgg gaa gac aag ttc atg tac 720 Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr 225 230 235 240 ttt gag ttc cct cag ccg tta cct gtg tgt ggt gat atc aaa gta gag 768 Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu 245 250 255 ttc ttc cac aaa cag aac aag atg cta aaa aag gac aaa atg ttt cac 816 Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His 260 265 270 ttt tgg gta aat aca ttc ttc ata cca gga cca gag gaa acc tca gaa 864 Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu 275 280 285 aaa gta gaa aat gga agt cta tgt gat caa gaa atc gat agc att tgc 912 Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys 290 295 300 agt ata gag cgt gca gat aat gac aag gaa tat cta gta ctt act tta 960 Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu 305 310 315 320 aca aaa aat gat ctt gac aaa gca aat aaa gac aaa gcc aac cga tac 1008 Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr 325 330 335 ttt tct cca aat ttt aag gtg aag ctg tac ttc aca aaa aca gta gag 1056 Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu 340 345 350 gag ccg tca aat cca gag gct agc agt tca act tct gta aca cca gat 1104 Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp 355 360 365 gtt agt gac aat gaa cct gat cat tat aga tat tct gac acc act gac 1152 Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp 370 375 380 tct gat cca gag aat gaa cct ttt gat gaa gat cag cat aca caa att 1200 Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile 385 390 395 400 aca aaa gtc gga tcc atg aaa gaa acc tgg tgg gaa acc tgg tgg acc 1248 Thr Lys Val Gly Ser Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr 405 410 415 gaa tct cag ccg aaa aaa aaa cgt aaa gtg ta g 1281 Glu Ser Gln Pro Lys Lys Lys Arg Lys Val 420 425 <210> 9 <211> 426 <212> PRT <213> Artificial Sequence <400> 9 Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr 1 5 10 15 Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30 Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35 40 45 Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His 50 55 60 Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys 65 70 75 80 Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro 85 90 95 Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu 100 105 110 Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys 115 120 125 Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys 130 135 140 Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr 165 170 175 Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala 180 185 190 Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly 195 200 205 Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile 210 215 220 Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr 225 230 235 240 Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu 245 250 255 Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His 260 265 270 Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu 275 280 285 Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys 290 295 300 Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu 305 310 315 320 Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr 325 330 335 Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu 340 345 350 Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp 355 360 365 Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp 370 375 380 Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile 385 390 395 400 Thr Lys Val Gly Ser Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr 405 410 415 Glu Ser Gln Pro Lys Lys Lys Arg Lys Val 420 425 <210> 10 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide for PEP-PTEN-PEP fusion protein <220> <221> CDS <222> (1)..(1347) <400> 10 atg aaa gaa acc tgg tgg gaa acc tgg tgg acc gaa tct cag ccg aaa 48 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys 1 5 10 15 aaa aaa cgt aaa gtg ctc gag atg aca gcc atc atc aaa gag atc gtt 96 Lys Lys Arg Lys Val Leu Glu Met Thr Ala Ile Ile Lys Glu Ile Val 20 25 30 agc aga aac aaa agg aga tat caa gag gat gga ttc gac tta gac ttg 144 Ser Arg Asn Lys Arg Arg Tyr Gln Glu Asp Gly Phe Asp Leu Asp Leu 35 40 45 acc tat att tat cca aac att att gct atg gga ttt cct gca gaa aga 192 Thr Tyr Ile Tyr Pro Asn Ile Ile Ala Met Gly Phe Pro Ala Glu Arg 50 55 60 ctt gaa ggc gta tac agg aac aat att gat gat gta gta agg ttt ttg 240 Leu Glu Gly Val Tyr Arg Asn Asn Ile Asp Asp Val Val Arg Phe Leu 65 70 75 80 gat tca aag cat aaa aac cat tac aag ata tac aat ctt tgt gct gaa 288 Asp Ser Lys His Lys Asn His Tyr Lys Ile Tyr Asn Leu Cys Ala Glu 85 90 95 aga cat tat gac acc gcc aaa ttt aat tgc aga gtt gca caa tat cct 336 Arg His Tyr Asp Thr Ala Lys Phe Asn Cys Arg Val Ala Gln Tyr Pro 100 105 110 ttt gaa gac cat aac cca cca cag cta gaa ctt atc aaa ccc ttt tgt 384 Phe Glu Asp His Asn Pro Pro Gln Leu Glu Leu Ile Lys Pro Phe Cys 115 120 125 gaa gat ctt gac caa tgg cta agt gaa gat gac aat cat gtt gca gca 432 Glu Asp Leu Asp Gln Trp Leu Ser Glu Asp Asp Asn His Val Ala Ala 130 135 140 att cac tgt aaa gct gga aag gga cga act ggt gta atg ata tgt gca 480 Ile His Cys Lys Ala Gly Lys Gly Arg Thr Gly Val Met Ile Cys Ala 145 150 155 160 tat tta tta cat cgg ggc aaa ttt tta aag gca caa gag gcc cta gat 528 Tyr Leu Leu His Arg Gly Lys Phe Leu Lys Ala Gln Glu Ala Leu Asp 165 170 175 ttc tat ggg gaa gta agg acc aga gac aaa aag gga gta act att ccc 576 Phe Tyr Gly Glu Val Arg Thr Arg Asp Lys Lys Gly Val Thr Ile Pro 180 185 190 agt cag agg cgc tat gtg tat tat tat agc tac ctg tta aag aat cat 624 Ser Gln Arg Arg Tyr Val Tyr Tyr Tyr Ser Tyr Leu Leu Lys Asn His 195 200 205 ctg gat tat aga cca gtg gca ctg ttg ttt cac aag atg atg ttt gaa 672 Leu Asp Tyr Arg Pro Val Ala Leu Leu Phe His Lys Met Met Phe Glu 210 215 220 act att cca atg ttc agt ggc gga act tgc aat cct cag ttt gtg gtc 720 Thr Ile Pro Met Phe Ser Gly Gly Thr Cys Asn Pro Gln Phe Val Val 225 230 235 240 tgc cag cta aag gtg aag ata tat tcc tcc aat tca gga ccc aca cga 768 Cys Gln Leu Lys Val Lys Ile Tyr Ser Ser Asn Ser Gly Pro Thr Arg 245 250 255 cgg gaa gac aag ttc atg tac ttt gag ttc cct cag ccg tta cct gtg 816 Arg Glu Asp Lys Phe Met Tyr Phe Glu Phe Pro Gln Pro Leu Pro Val 260 265 270 tgt ggt gat atc aaa gta gag ttc ttc cac aaa cag aac aag atg cta 864 Cys Gly Asp Ile Lys Val Glu Phe Phe His Lys Gln Asn Lys Met Leu 275 280 285 aaa aag gac aaa atg ttt cac ttt tgg gta aat aca ttc ttc ata cca 912 Lys Lys Asp Lys Met Phe His Phe Trp Val Asn Thr Phe Phe Ile Pro 290 295 300 gga cca gag gaa acc tca gaa aaa gta gaa aat gga agt cta tgt gat 960 Gly Pro Glu Glu Thr Ser Glu Lys Val Glu Asn Gly Ser Leu Cys Asp 305 310 315 320 caa gaa atc gat agc att tgc agt ata gag cgt gca gat aat gac aag 1008 Gln Glu Ile Asp Ser Ile Cys Ser Ile Glu Arg Ala Asp Asn Asp Lys 325 330 335 gaa tat cta gta ctt act tta aca aaa aat gat ctt gac aaa gca aat 1056 Glu Tyr Leu Val Leu Thr Leu Thr Lys Asn Asp Leu Asp Lys Ala Asn 340 345 350 aaa gac aaa gcc aac cga tac ttt tct cca aat ttt aag gtg aag ctg 1104 Lys Asp Lys Ala Asn Arg Tyr Phe Ser Pro Asn Phe Lys Val Lys Leu 355 360 365 tac ttc aca aaa aca gta gag gag ccg tca aat cca gag gct agc agt 1152 Tyr Phe Thr Lys Thr Val Glu Glu Pro Ser Asn Pro Glu Ala Ser Ser 370 375 380 tca act tct gta aca cca gat gtt agt gac aat gaa cct gat cat tat 1200 Ser Thr Ser Val Thr Pro Asp Val Ser Asp Asn Glu Pro Asp His Tyr 385 390 395 400 aga tat tct gac acc act gac tct gat cca gag aat gaa cct ttt gat 1248 Arg Tyr Ser Asp Thr Thr Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp 405 410 415 gaa gat cag cat aca caa att aca aaa gtc gga tcc atg aaa gaa acc 1296 Glu Asp Gln His Thr Gln Ile Thr Lys Val Gly Ser Met Lys Glu Thr 420 425 430 tgg tgg gaa acc tgg tgg acc gaa tct cag ccg aaa aaa aaa cgt aaa 1344 Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys Lys Lys Arg Lys 435 440 445 gtg tag 1350 Val <210> 11 <211> 449 <212> PRT <213> Artificial Sequence <400> 11 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Thr Ala Ile Ile Lys Glu Ile Val 20 25 30 Ser Arg Asn Lys Arg Arg Tyr Gln Glu Asp Gly Phe Asp Leu Asp Leu 35 40 45 Thr Tyr Ile Tyr Pro Asn Ile Ile Ala Met Gly Phe Pro Ala Glu Arg 50 55 60 Leu Glu Gly Val Tyr Arg Asn Asn Ile Asp Asp Val Val Arg Phe Leu 65 70 75 80 Asp Ser Lys His Lys Asn His Tyr Lys Ile Tyr Asn Leu Cys Ala Glu 85 90 95 Arg His Tyr Asp Thr Ala Lys Phe Asn Cys Arg Val Ala Gln Tyr Pro 100 105 110 Phe Glu Asp His Asn Pro Pro Gln Leu Glu Leu Ile Lys Pro Phe Cys 115 120 125 Glu Asp Leu Asp Gln Trp Leu Ser Glu Asp Asp Asn His Val Ala Ala 130 135 140 Ile His Cys Lys Ala Gly Lys Gly Arg Thr Gly Val Met Ile Cys Ala 145 150 155 160 Tyr Leu Leu His Arg Gly Lys Phe Leu Lys Ala Gln Glu Ala Leu Asp 165 170 175 Phe Tyr Gly Glu Val Arg Thr Arg Asp Lys Lys Gly Val Thr Ile Pro 180 185 190 Ser Gln Arg Arg Tyr Val Tyr Tyr Tyr Ser Tyr Leu Leu Lys Asn His 195 200 205 Leu Asp Tyr Arg Pro Val Ala Leu Leu Phe His Lys Met Met Phe Glu 210 215 220 Thr Ile Pro Met Phe Ser Gly Gly Thr Cys Asn Pro Gln Phe Val Val 225 230 235 240 Cys Gln Leu Lys Val Lys Ile Tyr Ser Ser Asn Ser Gly Pro Thr Arg 245 250 255 Arg Glu Asp Lys Phe Met Tyr Phe Glu Phe Pro Gln Pro Leu Pro Val 260 265 270 Cys Gly Asp Ile Lys Val Glu Phe Phe His Lys Gln Asn Lys Met Leu 275 280 285 Lys Lys Asp Lys Met Phe His Phe Trp Val Asn Thr Phe Phe Ile Pro 290 295 300 Gly Pro Glu Glu Thr Ser Glu Lys Val Glu Asn Gly Ser Leu Cys Asp 305 310 315 320 Gln Glu Ile Asp Ser Ile Cys Ser Ile Glu Arg Ala Asp Asn Asp Lys 325 330 335 Glu Tyr Leu Val Leu Thr Leu Thr Lys Asn Asp Leu Asp Lys Ala Asn 340 345 350 Lys Asp Lys Ala Asn Arg Tyr Phe Ser Pro Asn Phe Lys Val Lys Leu 355 360 365 Tyr Phe Thr Lys Thr Val Glu Glu Pro Ser Asn Pro Glu Ala Ser Ser 370 375 380 Ser Thr Ser Val Thr Pro Asp Val Ser Asp Asn Glu Pro Asp His Tyr 385 390 395 400 Arg Tyr Ser Asp Thr Thr Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp 405 410 415 Glu Asp Gln His Thr Gln Ile Thr Lys Val Gly Ser Met Lys Glu Thr 420 425 430 Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys Lys Lys Arg Lys 435 440 445 Val <110> Industry Academic Cooperation Foundation, Hallym University <120> Cell-transducing PTEN fusion protein <130> hallymU-PEP-PTEN <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> top strand oligonucleotide coding PEP-1 <400> 1 tatgaaagaa acctggtggg aaacctggtg gaccgaatgg tctcagccga aaaaaaaacg 60 taaagtgc 68 <210> 2 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> bottom strand oligonucleotide coding PEP-1 <400> 2 tcgagcactt tacgtttttt tttcggctga caccattcgg tccaccaggt ttcccaccag 60 gtttctttcc 70 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> protein transducing domain called PEP-1 <400> 3 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forwaed primer <400> 4 ctcgagatgg cagccatcat caaagagatc 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 5 ggatcctcag acttttgtaa tttgtgtatg 30 <210> 6 <211> 1287 <212> DNA <213> Artificial Sequence <220> <223> recombinant polynucleotide coding PEP-1-PTEN fusion protein <220> <221> CDS (222) (1) .. (1278) <400> 6 atg aaa gaa acc tgg tgg gaa acc tgg tgg acc gaa tct cag ccg aaa 48 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys 1 5 10 15 aaa aaa cgt aaa gtg ctc gag atg aca gcc atc atc aaa gag atc gtt 96 Lys Lys Arg Lys Val Leu Glu Met Thr Ala Ile Ile Lys Glu Ile Val 20 25 30 agc aga aac aaa agg aga tat caa gag gat gga ttc gac tta gac ttg 144 Ser Arg Asn Lys Arg Arg Tyr Gln Glu Asp Gly Phe Asp Leu Asp Leu 35 40 45 acc tat att tat cca aac att att gct atg gga ttt cct gca gaa aga 192 Thr Tyr Ile Tyr Pro Asn Ile Ila Ala Met Gly Phe Pro Ala Glu Arg 50 55 60 ctt gaa ggc gta tac agg aac aat att gat gat gta gta agg ttt ttg 240 Leu Glu Gly Val Tyr Arg Asn Asn Ile Asp Asp Val Val Arg Phe Leu 65 70 75 80 gat tca aag cat aaa aac cat tac aag ata tac aat ctt tgt gct gaa 288 Asp Ser Lys His Lys Asn His Tyr Lys Ile Tyr Asn Leu Cys Ala Glu 85 90 95 aga cat tat gac acc gcc aaa ttt aat tgc aga gtt gca caa tat cct 336 Arg His Tyr Asp Thr Ala Lys Phe Asn Cys Arg Val Ala Gln Tyr Pro 100 105 110 ttt gaa gac cat aac cca cca cag cta gaa ctt atc aaa ccc ttt tgt 384 Phe Glu Asp His Asn Pro Pro Gln Leu Glu Leu Ile Lys Pro Phe Cys 115 120 125 gaa gat ctt gac caa tgg cta agt gaa gat gac aat cat gtt gca gca 432 Glu Asp Leu Asp Gln Trp Leu Ser Glu Asp Asp Asn His Val Ala Ala 130 135 140 att cac tgt aaa gct gga aag gga cga act ggt gta atg ata tgt gca 480 Ile His Cys Lys Ala Gly Lys Gly Arg Thr Gly Val Met Ile Cys Ala 145 150 155 160 tat tta tta cat cgg ggc aaa ttt tta aag gca caa gag gcc cta gat 528 Tyr Leu Leu His Arg Gly Lys Phe Leu Lys Ala Gln Glu Ala Leu Asp 165 170 175 ttc tat ggg gaa gta agg acc aga gac aaa aag gga gta act att ccc 576 Phe Tyr Gly Glu Val Arg Thr Arg Asp Lys Lys Gly Val Thr Ile Pro 180 185 190 agt cag agg cgc tat gtg tat tat tat agc tac ctg tta aag aat cat 624 Ser Gln Arg Arg Tyr Val Tyr Tyr Tyr Ser Tyr Leu Leu Lys Asn His 195 200 205 ctg gat tat aga cca gtg gca ctg ttg ttt cac aag atg atg ttt gaa 672 Leu Asp Tyr Arg Pro Val Ala Leu Leu Phe His Lys Met Met Phe Glu 210 215 220 act att cca atg ttc agt ggc gga act tgc aat cct cag ttt gtg gtc 720 Thr Ile Pro Met Phe Ser Gly Gly Thr Cys Asn Pro Gln Phe Val Val 225 230 235 240 tgc cag cta aag gtg aag ata tat tcc tcc aat tca gga ccc aca cga 768 Cys Gln Leu Lys Val Lys Ile Tyr Ser Ser Asn Ser Gly Pro Thr Arg 245 250 255 cgg gaa gac aag ttc atg tac ttt gag ttc cct cag ccg tta cct gtg 816 Arg Glu Asp Lys Phe Met Tyr Phe Glu Phe Pro Gln Pro Leu Pro Val 260 265 270 tgt ggt gat atc aaa gta gag ttc ttc cac aaa cag aac aag atg cta 864 Cys Gly Asp Ile Lys Val Glu Phe Phe His Lys Gln Asn Lys Met Leu 275 280 285 aaa aag gac aaa atg ttt cac ttt tgg gta aat aca ttc ttc ata cca 912 Lys Lys Asp Lys Met Phe His Phe Trp Val Asn Thr Phe Phe Ile Pro 290 295 300 gga cca gag gaa acc tca gaa aaa gta gaa aat gga agt cta tgt gat 960 Gly Pro Glu Glu Thr Ser Glu Lys Val Glu Asn Gly Ser Leu Cys Asp 305 310 315 320 caa gaa atc gat agc att tgc agt ata gag cgt gca gat aat gac aag 1008 Gln Glu Ile Asp Ser Ile Cys Ser Ile Glu Arg Ala Asp Asn Asp Lys 325 330 335 gaa tat cta gta ctt act tta aca aaa aat gat ctt gac aaa gca aat 1056 Glu Tyr Leu Val Leu Thr Leu Thr Lys Asn Asp Leu Asp Lys Ala Asn 340 345 350 aaa gac aaa gcc aac cga tac ttt tct cca aat ttt aag gtg aag ctg 1104 Lys Asp Lys Ala Asn Arg Tyr Phe Ser Pro Asn Phe Lys Val Lys Leu 355 360 365 tac ttc aca aaa aca gta gag gag ccg tca aat cca gag gct agc agt 1152 Tyr Phe Thr Lys Thr Val Glu Glu Pro Ser Asn Pro Glu Ala Ser Ser 370 375 380 tca act tct gta aca cca gat gtt agt gac aat gaa cct gat cat tat 1200 Ser Thr Ser Val Thr Pro Asp Val Ser Asp Asn Glu Pro Asp His Tyr 385 390 395 400 aga tat tct gac acc act gac tct gat cca gag aat gaa cct ttt gat 1248 Arg Tyr Ser Asp Thr Thr Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp 405 410 415 gaa gat cag cat aca caa att aca aaa gtc tg aggatcc 1287 Glu Asp Gln His Thr Gln Ile Thr Lys Val 420 425 <210> 7 <211> 426 <212> PRT <213> Artificial Sequence <400> 7 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Thr Ala Ile Ile Lys Glu Ile Val 20 25 30 Ser Arg Asn Lys Arg Arg Tyr Gln Glu Asp Gly Phe Asp Leu Asp Leu 35 40 45 Thr Tyr Ile Tyr Pro Asn Ile Ila Ala Met Gly Phe Pro Ala Glu Arg 50 55 60 Leu Glu Gly Val Tyr Arg Asn Asn Ile Asp Asp Val Val Arg Phe Leu 65 70 75 80 Asp Ser Lys His Lys Asn His Tyr Lys Ile Tyr Asn Leu Cys Ala Glu 85 90 95 Arg His Tyr Asp Thr Ala Lys Phe Asn Cys Arg Val Ala Gln Tyr Pro 100 105 110 Phe Glu Asp His Asn Pro Pro Gln Leu Glu Leu Ile Lys Pro Phe Cys 115 120 125 Glu Asp Leu Asp Gln Trp Leu Ser Glu Asp Asp Asn His Val Ala Ala 130 135 140 Ile His Cys Lys Ala Gly Lys Gly Arg Thr Gly Val Met Ile Cys Ala 145 150 155 160 Tyr Leu Leu His Arg Gly Lys Phe Leu Lys Ala Gln Glu Ala Leu Asp 165 170 175 Phe Tyr Gly Glu Val Arg Thr Arg Asp Lys Lys Gly Val Thr Ile Pro 180 185 190 Ser Gln Arg Arg Tyr Val Tyr Tyr Tyr Ser Tyr Leu Leu Lys Asn His 195 200 205 Leu Asp Tyr Arg Pro Val Ala Leu Leu Phe His Lys Met Met Phe Glu 210 215 220 Thr Ile Pro Met Phe Ser Gly Gly Thr Cys Asn Pro Gln Phe Val Val 225 230 235 240 Cys Gln Leu Lys Val Lys Ile Tyr Ser Ser Asn Ser Gly Pro Thr Arg 245 250 255 Arg Glu Asp Lys Phe Met Tyr Phe Glu Phe Pro Gln Pro Leu Pro Val 260 265 270 Cys Gly Asp Ile Lys Val Glu Phe Phe His Lys Gln Asn Lys Met Leu 275 280 285 Lys Lys Asp Lys Met Phe His Phe Trp Val Asn Thr Phe Phe Ile Pro 290 295 300 Gly Pro Glu Glu Thr Ser Glu Lys Val Glu Asn Gly Ser Leu Cys Asp 305 310 315 320 Gln Glu Ile Asp Ser Ile Cys Ser Ile Glu Arg Ala Asp Asn Asp Lys 325 330 335 Glu Tyr Leu Val Leu Thr Leu Thr Lys Asn Asp Leu Asp Lys Ala Asn 340 345 350 Lys Asp Lys Ala Asn Arg Tyr Phe Ser Pro Asn Phe Lys Val Lys Leu 355 360 365 Tyr Phe Thr Lys Thr Val Glu Glu Pro Ser Asn Pro Glu Ala Ser Ser 370 375 380 Ser Thr Ser Val Thr Pro Asp Val Ser Asp Asn Glu Pro Asp His Tyr 385 390 395 400 Arg Tyr Ser Asp Thr Thr Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp 405 410 415 Glu Asp Gln His Thr Gln Ile Thr Lys Val 420 425 <210> 8 <211> 1281 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide for PTEN-PEP-1 fusion protein <220> <221> CDS (222) (1) .. (1278) <400> 8 atg aca gcc atc atc ac aaa gag atc gtt agc aga aac aaa agg aga tat 48 Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr 1 5 10 15 caa gag gat gga ttc gac tta gac ttg acc tat att tat cca aac att 96 Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30 att gct atg gga ttt cct gca gaa aga ctt gaa ggc gta tac agg aac 144 Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35 40 45 aat att gat gat gta gta agg ttt ttg gat tca aag cat aaa aac cat 192 Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His 50 55 60 tac aag ata tac aat ctt tgt gct gaa aga cat tat gac acc gcc aaa 240 Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys 65 70 75 80 ttt aat tgc aga gtt gca caa tat cct ttt gaa gac cat aac cca cca 288 Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro 85 90 95 cag cta gaa ctt atc aaa ccc ttt tgt gaa gat ctt gac caa tgg cta 336 Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu 100 105 110 agt gaa gat gac aat cat gtt gca gca att cac tgt aaa gct gga aag 384 Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys 115 120 125 gga cga act ggt gta atg ata tgt gca tat tta tta cat cgg ggc aaa 432 Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys 130 135 140 ttt tta aag gca caa gag gcc cta gat ttc tat ggg gaa gta agg acc 480 Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 aga gac aaa aag gga gta act att ccc agt cag agg cgc tat gtg tat 528 Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr 165 170 175 tat tat agc tac ctg tta aag aat cat ctg gat tat aga cca gtg gca 576 Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala 180 185 190 ctg ttg ttt cac aag atg atg ttt gaa act att cca atg ttc agt ggc 624 Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly 195 200 205 gga act tgc aat cct cag ttt gtg gtc tgc cag cta aag gtg aag ata 672 Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile 210 215 220 tat tcc tcc aat tca gga ccc aca cga cgg gaa gac aag ttc atg tac 720 Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr 225 230 235 240 ttt gag ttc cct cag ccg tta cct gtg tgt ggt gat atc aaa gta gag 768 Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu 245 250 255 ttc ttc cac aaa cag aac aag atg cta aaa aag gac aaa atg ttt cac 816 Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His 260 265 270 ttt tgg gta aat aca ttc ttc ata cca gga cca gag gaa acc tca gaa 864 Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu 275 280 285 aaa gta gaa aat gga agt cta tgt gat caa gaa atc gat agc att tgc 912 Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys 290 295 300 agt ata gag cgt gca gat aat gac aag gaa tat cta gta ctt act tta 960 Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu 305 310 315 320 aca aaa aat gat ctt gac aaa gca aat aaa gac aaa gcc aac cga tac 1008 Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr 325 330 335 ttt tct cca aat ttt aag gtg aag ctg tac ttc aca aaa aca gta gag 1056 Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu 340 345 350 gag ccg tca aat cca gag gct agc agt tca act tct gta aca cca gat 1104 Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp 355 360 365 gtt agt gac aat gaa cct gat cat tat aga tat tct gac acc act gac 1152 Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp 370 375 380 tct gat cca gag aat gaa cct ttt gat gaa gat cag cat aca caa att 1200 Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile 385 390 395 400 aca aaa gtc gga tcc atg aaa gaa acc tgg tgg gaa acc tgg tgg acc 1248 Thr Lys Val Gly Ser Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr 405 410 415 gaa tct cag ccg aaa aaa aaa cgt aaa gtg ta g 1281 Glu Ser Gln Pro Lys Lys Lys Arg Lys Val 420 425 <210> 9 <211> 426 <212> PRT <213> Artificial Sequence <400> 9 Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr 1 5 10 15 Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30 Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35 40 45 Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His 50 55 60 Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys 65 70 75 80 Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro 85 90 95 Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu 100 105 110 Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys 115 120 125 Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys 130 135 140 Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr 165 170 175 Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala 180 185 190 Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly 195 200 205 Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile 210 215 220 Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr 225 230 235 240 Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu 245 250 255 Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His 260 265 270 Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu 275 280 285 Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys 290 295 300 Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu 305 310 315 320 Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr 325 330 335 Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu 340 345 350 Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp 355 360 365 Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp 370 375 380 Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile 385 390 395 400 Thr Lys Val Gly Ser Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr 405 410 415 Glu Ser Gln Pro Lys Lys Lys Arg Lys Val 420 425 <210> 10 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide for PEP-PTEN-PEP fusion protein <220> <221> CDS (222) (1) .. (1347) <400> 10 atg aaa gaa acc tgg tgg gaa acc tgg tgg acc gaa tct cag ccg aaa 48 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys 1 5 10 15 aaa aaa cgt aaa gtg ctc gag atg aca gcc atc atc aaa gag atc gtt 96 Lys Lys Arg Lys Val Leu Glu Met Thr Ala Ile Ile Lys Glu Ile Val 20 25 30 agc aga aac aaa agg aga tat caa gag gat gga ttc gac tta gac ttg 144 Ser Arg Asn Lys Arg Arg Tyr Gln Glu Asp Gly Phe Asp Leu Asp Leu 35 40 45 acc tat att tat cca aac att att gct atg gga ttt cct gca gaa aga 192 Thr Tyr Ile Tyr Pro Asn Ile Ila Ala Met Gly Phe Pro Ala Glu Arg 50 55 60 ctt gaa ggc gta tac agg aac aat att gat gat gta gta agg ttt ttg 240 Leu Glu Gly Val Tyr Arg Asn Asn Ile Asp Asp Val Val Arg Phe Leu 65 70 75 80 gat tca aag cat aaa aac cat tac aag ata tac aat ctt tgt gct gaa 288 Asp Ser Lys His Lys Asn His Tyr Lys Ile Tyr Asn Leu Cys Ala Glu 85 90 95 aga cat tat gac acc gcc aaa ttt aat tgc aga gtt gca caa tat cct 336 Arg His Tyr Asp Thr Ala Lys Phe Asn Cys Arg Val Ala Gln Tyr Pro 100 105 110 ttt gaa gac cat aac cca cca cag cta gaa ctt atc aaa ccc ttt tgt 384 Phe Glu Asp His Asn Pro Pro Gln Leu Glu Leu Ile Lys Pro Phe Cys 115 120 125 gaa gat ctt gac caa tgg cta agt gaa gat gac aat cat gtt gca gca 432 Glu Asp Leu Asp Gln Trp Leu Ser Glu Asp Asp Asn His Val Ala Ala 130 135 140 att cac tgt aaa gct gga aag gga cga act ggt gta atg ata tgt gca 480 Ile His Cys Lys Ala Gly Lys Gly Arg Thr Gly Val Met Ile Cys Ala 145 150 155 160 tat tta tta cat cgg ggc aaa ttt tta aag gca caa gag gcc cta gat 528 Tyr Leu Leu His Arg Gly Lys Phe Leu Lys Ala Gln Glu Ala Leu Asp 165 170 175 ttc tat ggg gaa gta agg acc aga gac aaa aag gga gta act att ccc 576 Phe Tyr Gly Glu Val Arg Thr Arg Asp Lys Lys Gly Val Thr Ile Pro 180 185 190 agt cag agg cgc tat gtg tat tat tat agc tac ctg tta aag aat cat 624 Ser Gln Arg Arg Tyr Val Tyr Tyr Tyr Ser Tyr Leu Leu Lys Asn His 195 200 205 ctg gat tat aga cca gtg gca ctg ttg ttt cac aag atg atg ttt gaa 672 Leu Asp Tyr Arg Pro Val Ala Leu Leu Phe His Lys Met Met Phe Glu 210 215 220 act att cca atg ttc agt ggc gga act tgc aat cct cag ttt gtg gtc 720 Thr Ile Pro Met Phe Ser Gly Gly Thr Cys Asn Pro Gln Phe Val Val 225 230 235 240 tgc cag cta aag gtg aag ata tat tcc tcc aat tca gga ccc aca cga 768 Cys Gln Leu Lys Val Lys Ile Tyr Ser Ser Asn Ser Gly Pro Thr Arg 245 250 255 cgg gaa gac aag ttc atg tac ttt gag ttc cct cag ccg tta cct gtg 816 Arg Glu Asp Lys Phe Met Tyr Phe Glu Phe Pro Gln Pro Leu Pro Val 260 265 270 tgt ggt gat atc aaa gta gag ttc ttc cac aaa cag aac aag atg cta 864 Cys Gly Asp Ile Lys Val Glu Phe Phe His Lys Gln Asn Lys Met Leu 275 280 285 aaa aag gac aaa atg ttt cac ttt tgg gta aat aca ttc ttc ata cca 912 Lys Lys Asp Lys Met Phe His Phe Trp Val Asn Thr Phe Phe Ile Pro 290 295 300 gga cca gag gaa acc tca gaa aaa gta gaa aat gga agt cta tgt gat 960 Gly Pro Glu Glu Thr Ser Glu Lys Val Glu Asn Gly Ser Leu Cys Asp 305 310 315 320 caa gaa atc gat agc att tgc agt ata gag cgt gca gat aat gac aag 1008 Gln Glu Ile Asp Ser Ile Cys Ser Ile Glu Arg Ala Asp Asn Asp Lys 325 330 335 gaa tat cta gta ctt act tta aca aaa aat gat ctt gac aaa gca aat 1056 Glu Tyr Leu Val Leu Thr Leu Thr Lys Asn Asp Leu Asp Lys Ala Asn 340 345 350 aaa gac aaa gcc aac cga tac ttt tct cca aat ttt aag gtg aag ctg 1104 Lys Asp Lys Ala Asn Arg Tyr Phe Ser Pro Asn Phe Lys Val Lys Leu 355 360 365 tac ttc aca aaa aca gta gag gag ccg tca aat cca gag gct agc agt 1152 Tyr Phe Thr Lys Thr Val Glu Glu Pro Ser Asn Pro Glu Ala Ser Ser 370 375 380 tca act tct gta aca cca gat gtt agt gac aat gaa cct gat cat tat 1200 Ser Thr Ser Val Thr Pro Asp Val Ser Asp Asn Glu Pro Asp His Tyr 385 390 395 400 aga tat tct gac acc act gac tct gat cca gag aat gaa cct ttt gat 1248 Arg Tyr Ser Asp Thr Thr Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp 405 410 415 gaa gat cag cat aca caa att aca aaa gtc gga tcc atg aaa gaa acc 1296 Glu Asp Gln His Thr Gln Ile Thr Lys Val Gly Ser Met Lys Glu Thr 420 425 430 tgg tgg gaa acc tgg tgg acc gaa tct cag ccg aaa aaa aaa cgt aaa 1344 Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys Lys Lys Arg Lys 435 440 445 gtg tag 1350 Val <210> 11 <211> 449 <212> PRT <213> Artificial Sequence <400> 11 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Thr Ala Ile Ile Lys Glu Ile Val 20 25 30 Ser Arg Asn Lys Arg Arg Tyr Gln Glu Asp Gly Phe Asp Leu Asp Leu 35 40 45 Thr Tyr Ile Tyr Pro Asn Ile Ila Ala Met Gly Phe Pro Ala Glu Arg 50 55 60 Leu Glu Gly Val Tyr Arg Asn Asn Ile Asp Asp Val Val Arg Phe Leu 65 70 75 80 Asp Ser Lys His Lys Asn His Tyr Lys Ile Tyr Asn Leu Cys Ala Glu 85 90 95 Arg His Tyr Asp Thr Ala Lys Phe Asn Cys Arg Val Ala Gln Tyr Pro 100 105 110 Phe Glu Asp His Asn Pro Pro Gln Leu Glu Leu Ile Lys Pro Phe Cys 115 120 125 Glu Asp Leu Asp Gln Trp Leu Ser Glu Asp Asp Asn His Val Ala Ala 130 135 140 Ile His Cys Lys Ala Gly Lys Gly Arg Thr Gly Val Met Ile Cys Ala 145 150 155 160 Tyr Leu Leu His Arg Gly Lys Phe Leu Lys Ala Gln Glu Ala Leu Asp 165 170 175 Phe Tyr Gly Glu Val Arg Thr Arg Asp Lys Lys Gly Val Thr Ile Pro 180 185 190 Ser Gln Arg Arg Tyr Val Tyr Tyr Tyr Ser Tyr Leu Leu Lys Asn His 195 200 205 Leu Asp Tyr Arg Pro Val Ala Leu Leu Phe His Lys Met Met Phe Glu 210 215 220 Thr Ile Pro Met Phe Ser Gly Gly Thr Cys Asn Pro Gln Phe Val Val 225 230 235 240 Cys Gln Leu Lys Val Lys Ile Tyr Ser Ser Asn Ser Gly Pro Thr Arg 245 250 255 Arg Glu Asp Lys Phe Met Tyr Phe Glu Phe Pro Gln Pro Leu Pro Val 260 265 270 Cys Gly Asp Ile Lys Val Glu Phe Phe His Lys Gln Asn Lys Met Leu 275 280 285 Lys Lys Asp Lys Met Phe His Phe Trp Val Asn Thr Phe Phe Ile Pro 290 295 300 Gly Pro Glu Glu Thr Ser Glu Lys Val Glu Asn Gly Ser Leu Cys Asp 305 310 315 320 Gln Glu Ile Asp Ser Ile Cys Ser Ile Glu Arg Ala Asp Asn Asp Lys 325 330 335 Glu Tyr Leu Val Leu Thr Leu Thr Lys Asn Asp Leu Asp Lys Ala Asn 340 345 350 Lys Asp Lys Ala Asn Arg Tyr Phe Ser Pro Asn Phe Lys Val Lys Leu 355 360 365 Tyr Phe Thr Lys Thr Val Glu Glu Pro Ser Asn Pro Glu Ala Ser Ser 370 375 380 Ser Thr Ser Val Thr Pro Asp Val Ser Asp Asn Glu Pro Asp His Tyr 385 390 395 400 Arg Tyr Ser Asp Thr Thr Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp 405 410 415 Glu Asp Gln His Thr Gln Ile Thr Lys Val Gly Ser Met Lys Glu Thr 420 425 430 Trp Trp Glu Thr Trp Trp Thr Glu Ser Gln Pro Lys Lys Lys Arg Lys 435 440 445 Val

Claims

A PEP-1 protein transport domain of SEQ ID NO: 3 is covalently linked to at least one end of a Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) protein and has an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 Cell-transducing PTEN fusion protein.

A cosmetic composition comprising the cell-introducing PTEN fusion protein of claim 1 as an active ingredient.

Fibrotic scars after cancer treatment, skin cancer treatment, skin cell death inhibition, atopic inflammation treatment and glaucoma filtration surgery, comprising the cell-introducing PTEN fusion protein of claim 1 as an active ingredient and comprising a pharmaceutically acceptable carrier. Pharmaceutical composition for inhibiting formation.

Recombinant polynucleotide, characterized in that it has a nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 to which the protein transport domain peptide coding DNA sequence is coupled to the PTEN cDNA encoding the cell transmissive PTEN fusion protein of claim 1 .

The vector expressing the cell-introducing PTEN fusion protein, characterized in that comprising the recombinant polynucleotide of claim 4 to express the fusion protein of claim 1.