KR20120119560A - Enhancing method for transduction of fusion protein by adding pergolide - Google Patents

Enhancing method for transduction of fusion protein by adding pergolide Download PDF

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KR20120119560A
KR20120119560A KR1020110037569A KR20110037569A KR20120119560A KR 20120119560 A KR20120119560 A KR 20120119560A KR 1020110037569 A KR1020110037569 A KR 1020110037569A KR 20110037569 A KR20110037569 A KR 20110037569A KR 20120119560 A KR20120119560 A KR 20120119560A
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최수영
김대원
박진서
음원식
황현숙
김덕수
한규형
임순성
강태천
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한림대학교 산학협력단
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Abstract

PURPOSE: An increase method of fused protein into living cells or organs by adding pergolide to fused protein in which protein transport domains and therapeutic protein are covalently bonded is provided to drastically enhance infiltration efficiency of the fused protein. CONSTITUTION: An increase method of fused protein into living cells or organs is processed by adding pergolide to fused protein in which protein transport domains and therapeutic protein are covalently bonded. The pergolide is primarily processed in the living cells or organs than the fused protein. A protein transport domain is Tat, PEP-1, oligolysine, oligo arginine or oligo(lysine and arginine). The pergolide is pergolide mesylate. The therapeutic protein is an enzyme which removes the active oxygen species. The therapeutic protein is catalase, superoxide dismutase or glutathione peroxidase. A composition for preventing and treating disease due to oxidative stress and inflammatory diseases comprises the fused protein and pergolide of the enzyme having the active oxygen species.

Description

단백질 수송 도메인과 치료용 단백질이 공유결합한 융합 단백질에 퍼골라이드를 가하여 융합 단백질의 생체 세포 또는 조직 내 도입을 증가시키는 방법 {Enhancing method for transduction of fusion protein by adding pergolide}A method for increasing the introduction of a fusion protein into a living cell or tissue by adding a pergolide to a fusion protein covalently bonded to a protein transport domain and a therapeutic protein. {Enhancing method for transduction of fusion protein by adding pergolide}

본 발명은 단백질 수송 도메인과 치료용 단백질이 공유결합한 융합 단백질에 퍼골라이드를 가하여 융합 단백질의 생체 세포 또는 조직 내 도입을 증가시키는 방법 및 단백질 수송 도메인과 공유결합한 활성산소종 제거 활성을 가진 효소의 융합 단백질 및 퍼골라이드를 포함하는 산화적 스트레스에 의한 질환 및 염증성 질환 예방 및 치료제 조성물에 관한 것이다. The present invention provides a method for increasing the introduction of a fusion protein into a living cell or tissue by adding a pergolide to a fusion protein covalently bonded to a protein transport domain and a therapeutic protein, and fusion of an enzyme having an active oxygen species removal activity covalently bound to the protein transport domain. It relates to a composition for preventing and treating diseases caused by oxidative stress and inflammatory diseases including protein and pergolide.

수산화 라디칼, 수퍼옥사이드 음이온 및 과산화수소와 같은 활성산소종은 다양한 보통 세포 과정으로부터 생성되며 세포 손상, 세포 사멸 및 결과적으로 염증, 종양, 알러지 및 허혈과 같은 질병에 이르게 된다 [D.R. Bickers, M. Athar, J. Invest. Dermatol. 126 (2006) 2565-2575, J.M. Mates, Toxicol. 83 (2000) 83-104, R.A. Floyd, FASEB J. 4 (1990) 2587-2597]. 수퍼옥사이드 디스뮤테이즈, 카탈레이즈 및 글루타치온 퍼옥시데이즈와 같은 항산화 효소들은 과생성된 활성산소종을 감소시키고 산화 손상을 일으키는 다양한 원인들에 맞서 세포를 보호하고 산화 환원 상태의 균형을 유지시키는 기능을 수행한다 [B.A. Freeman et al., J. Biol. Chem. 258 (1983) 12534-12542, V.R. Muzykantov, J. Control Release 71 (2001) 1-21, D.V. Sakharov et al., Biochim. Biophys. Acta 930 (1987) 140-144, D. St. Clair et al., Biomed. Pharmacother. 59 (2005) 209-214]. 따라서, 이와 같은 항산화 효소들은 치료 물질로서 유용하다. 그러나, 이와 같은 치료물질은 크기 및 생화학적 특성으로 인해 세포 내로 수송하는 데 문제가 있어서 실제로 세포 내에서 치료 효능을 나타내기에 어려움이 있다 [R.D. Egleton, T. P. Davis, Peptides 18 (1997) 1431-1439].
Free radicals such as hydroxyl radicals, superoxide anions and hydrogen peroxide are produced from a variety of normal cellular processes leading to cell damage, cell death and consequent diseases such as inflammation, tumors, allergies and ischemia. DR Bickers, M. Athar, J. Invest. Dermatol. 126 (2006) 2565-2575, JM Mates, Toxicol. 83 (2000) 83-104, RA Floyd, FASEB J. 4 (1990) 2587-2597. Antioxidant enzymes, such as superoxide dismutase, catalase and glutathione peroxidase, have the ability to reduce over-produced free radical species and protect cells against various causes of oxidative damage and to balance the redox state. [BA Freeman et al., J. Biol. Chem. 258 (1983) 12534-12542, VR Muzykantov, J. Control Release 71 (2001) 1-21, DV Sakharov et al., Biochim. Biophys. Acta 930 (1987) 140-144, D. St. Clair et al., Biomed. Pharmacother. 59 (2005) 209-214. Therefore, such antioxidant enzymes are useful as therapeutic substances. However, such therapeutic agents have difficulty in transporting them due to their size and biochemical properties, which makes them difficult to show therapeutic efficacy in cells [RD Egleton, TP Davis, Peptides 18 (1997) 1431-1439]. .

염증은 손상, 감염, 또는 면역계에 의해 외래 물질이라고 인식된 분자에 대한 체내 반응을 말하는 것이다. 염증은 조직의 통증, 종창 또는 기능 변화를 특징으로 한다. 염증성 질환은 조직 또는 기관에서 면역계가 이상 수준으로 활성화되는 것을 특징으로 하며, 이는 조직 또는 기관에서 기능 이상이나 질환을 유발할 수 있다.Inflammation refers to damage, infection, or reaction in the body to molecules recognized as foreign by the immune system. Inflammation is characterized by pain, swelling, or a change in function of the tissue. Inflammatory diseases are characterized by abnormal activation of the immune system in tissues or organs, which can cause dysfunction or disease in tissues or organs.

염증성 질환은 전세계에 걸쳐 사망의 주요 원인 중 하나이다. 염증성 질환은 여러 기관 및 조직, 예컨대 혈관, 심장, 뇌, 신경, 관절, 피부, 폐, 눈, 위장관, 신장, 갑상선, 부신, 췌장, 간 및 근육에 침범한다. 염증성 질환의 치료는 제약 회사와 연구자들의 관심의 대상이다. 최근 가장 일반적인 염증성 질환의 치료에 대한 접근 방법은 염증성 질환을 일으키는 분자 또는 그 수용체에 결합하는 화합물과 사이토카인의 전-염증성 기능에 관한 것이다. 이 분야에서 최근의 많은 연구에도 불구하고 현재의 염증성 질환에 대한 요법은 비특이적 약물로 증상을 완화시키는 것과 염증을 감소시키는 것, 질병으로 진행하는 것을 늦추는 것 등이지만, 이러한 요법에는 약물 부작용 및 내성이라는 심각한 문제가 있다.
Inflammatory disease is one of the leading causes of death worldwide. Inflammatory diseases affect various organs and tissues such as blood vessels, heart, brain, nerves, joints, skin, lungs, eyes, gastrointestinal tract, kidneys, thyroid, adrenal glands, pancreas, liver and muscles. Treatment of inflammatory diseases is of interest to pharmaceutical companies and researchers. Recently, the most common approach to the treatment of inflammatory diseases relates to the pro-inflammatory function of cytokines and compounds that bind to the molecule or its receptor causing the inflammatory disease. Despite many recent studies in this field, current therapies for inflammatory diseases include alleviating symptoms with non-specific drugs, reducing inflammation, and slowing the progression to disease. There is a serious problem.

생체는 항상성 유지를 위해 산화촉진물질 (prooxidant)과 산화억제물질 (antioxidant)들이 균형을 이루고 있다. 만약 이 균형이 깨져서 산화 촉진 쪽으로 기울게 되면 세포에 유해한 작용을 하게 되는데, 이를 산화적 스트레스라고 한다. 이런 산화적 스트레스는 기본적으로 노화와 밀접한 관련을 갖고 있으며, 체내 DNA 손상, 지질의 과산화로 인한 세포막의 손상, 단백질과 지질의 산화 등을 가져와 동맥경화나 암, 백내장, 노인성 치매, 파킨슨씨병과 같은 질환 등을 유발한다. The living body has a balance of prooxidants and antioxidants to maintain homeostasis. If this balance is broken and tilted towards oxidation, it is harmful to the cell, which is called oxidative stress. These oxidative stresses are fundamentally related to aging, and can lead to DNA damage in the body, damage to cell membranes due to lipid peroxidation, oxidation of proteins and lipids, and the like, such as atherosclerosis, cancer, cataracts, senile dementia and Parkinson's disease Cause disease, etc.

염증반응의 조절은 대단히 복잡한 것으로 알려져 있는데, 이는 생체 내 복구체계의 증강 및 손상을 감소시키기 위한 일련의 활성기전이며, 모든 조직에서 잘 조절되는 것으로 알려져 있다. 그러나 반복되는 조직의 손상이나 재생에 의해 염증반응이 지속되면, 염증관련 세포에서 활성산소종과 활성질소종(Reactive Nitrogen Species; RNS)이 과다 생성되고, 그 결과로 영구적인 유전자의 변형이 야기된다. 이처럼 활성산소종과 활성질소종은 생체 내 여러 가지 세포의 작용을 조절하는 염증 반응과 깊이 관련되어 있다.
The regulation of the inflammatory response is known to be very complex, a series of active mechanisms to reduce the buildup and damage of the repair system in vivo, and is known to be well regulated in all tissues. However, prolonged inflammatory reactions due to repeated tissue damage or regeneration cause excessive production of reactive oxygen species and reactive nitrogen species (RNS) in inflammatory cells, resulting in permanent genetic modification. . As such, reactive oxygen species and reactive nitrogen species are deeply involved in the inflammatory response that regulates the action of various cells in vivo.

많은 연구자들은 치료용 단백질을 Tat이나 PEP-1 같은 단백질 수송 도메인 (protein transduction domains; PTD)과 융합시키면 세포 내로 효율적으로 수송할 수 있음을 보고한바 있다 [G.P. Dietz, Curr. Pharm. Biotechnol. 11 (2010) 167-174, H. Matsui et al., Curr. Protein Pept. Sci. 4 (2003) 151-157, J. Prochiantz, Curr. Opin. Cell Biol. 12 (2000) 400-406, J.B. Rothbard et al., Nat. Med. 6 (2000) 1253-1257, S.R. Schwarze et al., Science 285 (1999) 1569-1572, S.R. Schwarze et al., Trends Cell Biol. 10 (2000) 290-295, J.S. Wadia, S.F. Dowdy, Curr. Opin. Biotechnol. 13 (2002) 52-56, J.S. Wadia, S.F. Dowdy, Curr. Protein Pept. Sci. 4 (2003) 97-104]. 또한, 본 발명자들은 PEP-1-카탈레이즈, PEP-1-rpS3 및 Tat-SOD 융합 단백질 발현 벡터를 구축하였다. PEP-1-카탈레이즈 융합 단백질은 성상세포와 저빌 뇌 내로 빠르게 침투하여 허혈 손상에 대해 신경세포를 보호하였다 [D.W. Kim et al., Free Radic. Biol. Med. 47 (2009) 941-952]. Tat-SOD는 마우스 당뇨 모델에서 췌장 베타세포 내로 침투하여 마우스의 당뇨 증세를 개선하였다 [W.S. Eum et al., Free Radic. Biol. Med. 37 (2004) 339-349]. 또, 마우스 모델에서 세포 내로 침투한 PEP-1-rpS3는 COX-2 (cyclooxygenase-2), 인터류킨-1β, 인터류킨-6 및 종양괴사인자-α와 같은 사이토카인과 염증성 매개자의 발현을 억제함으로써 TPA에 의해 유도된 피부 염증을 저해하였다 [E.H. Ahn et al., Toxicol. 276 (2010) 192-197].Many researchers have reported that fusion of therapeutic proteins with protein transduction domains (PTD), such as Tat or PEP-1, can efficiently transport them into cells [GP Dietz, Curr. Pharm. Biotechnol. 11 (2010) 167-174, H. Matsui et al., Curr. Protein Pept. Sci. 4 (2003) 151-157, J. Prochiantz, Curr. Opin. Cell Biol. 12 (2000) 400-406, JB Rothbard et al., Nat. Med. 6 (2000) 1253-1257, SR Schwarze et al., Science 285 (1999) 1569-1572, SR Schwarze et al., Trends Cell Biol. 10 (2000) 290-295, JS Wadia, SF Dowdy, Curr. Opin. Biotechnol. 13 (2002) 52-56, JS Wadia, SF Dowdy, Curr. Protein Pept. Sci. 4 (2003) 97-104. In addition, we constructed PEP-1-catalase, PEP-1-rpS3 and Tat-SOD fusion protein expression vectors. PEP-1-catalase fusion protein rapidly penetrated into astrocytic and gerbil brains to protect neurons against ischemic damage [DW Kim et al., Free Radic. Biol. Med. 47 (2009) 941-952. Tat-SOD penetrated into pancreatic beta cells in a mouse diabetic model to improve diabetic symptoms in mice [WS Eum et al., Free Radic. Biol. Med . 37 (2004) 339-349. In addition, PEP-1-rpS3 penetrated into cells in mouse models inhibited the expression of cytokines and inflammatory mediators such as COX-2 (cyclooxygenase-2), interleukin-1β, interleukin-6, and tumor necrosis factor-α. Inhibited skin inflammation induced by EH Ahn et al., Toxicol . 276 (2010) 192-197].

도파민 수용체 작동제 (Dopamine receptor agonists)는 에르고라인 (ergolines)과 비에르고라인 (non-ergolines)으로 나뉜다. 에르고라인은 맥각(麥角)의 알칼로이드에서 유래하며, 브로모크립틴 (bromocriptine), 퍼골라이드 (pergolide), 리슈라이드 (lisuride) 및 케이버고라인 (cabergoline)이 이에 포함된다. 이들은 도파민 D2 수용체뿐만 아니라 도파민 D1 수용체, 아드레날린 수용체 및 5-하이드록시트립타민 수용체에도 결합한다. 퍼골라이드 메실레이트 (Pergolide mesylate; PM)는 합성된 에르고라인 유도체이고 파킨슨병 치료에 이용된다 [U. Bonuccelli et al., Parkinsonism Relat. Disord.15s (2009) s44-s53]. 퍼골라이드의 IUPAC 명칭은 (8β)-8-[(methylthio)methyl]-6-propylergoline이다.
Dopamine receptor agonists are divided into ergolines and non-ergolines. Ergoline is derived from ergot alkaloids, including bromocriptine, pergolide, lisuride and cabergoline. They bind to the dopamine D2 receptor as well as the dopamine D1 receptor, the adrenergic receptor and the 5-hydroxytryptamine receptor. Pergolide mesylate (PM) is a synthesized ergoline derivative and is used to treat Parkinson's disease [U. Bonuccelli et al., Parkinsonism Relat. Disord. 15 s (2009) s44-s53]. The IUPAC name for pergolide is (8β) -8-[(methylthio) methyl] -6-propylergoline.

특허 제472938호는 세포 침투 효율을 향상시킨 수송 도메인-목표 단백질-수송 도메인 융합 단백질 및 그 용도에 관한 것으로서, HIV-1 Tat 펩타이드, 올리고라이신, 올리고아르기닌과 같은 단백질 수송 도메인이 목표 단백질의 N-말단 및/또는 C-말단에 공유결합되어 세포 침투성을 향상시킨 융합 단백질에 대해 개시하고 있다.Patent No. 472938 relates to a transport domain-target protein-transport domain fusion protein and its use for improved cell penetration efficiency, wherein protein transport domains such as HIV-1 Tat peptide, oligolysine, oligoarginine, Disclosed is a fusion protein covalently attached to the terminal and / or C-terminus to enhance cell permeability.

또한, 특허 제490362호는 단백질 수송 도메인으로서 올리고라이신이 단백질의 N-말단 및/또는 C-말단에 공유결합되어 세포 침투성을 향상시킨 기술에 대해 개시하고 있다.In addition, Patent No. 493662 discloses a technique in which oligolysine as a protein transport domain is covalently bonded to the N-terminus and / or C-terminus of a protein to improve cell permeability.

상기 특허들을 비롯하여 많은 연구자들의 논문을 통해 HIV-a Tat 펩타이드, 올리고라이신, 올리고아르기닌, 올리고(라이신, 아르기닌) 및 PEP-1 펩타이드 등이 단백질의 세포 침투성을 향상시킨다는 사실이 밝혀졌다.
Many researchers' papers, including those patents, have shown that HIV-a Tat peptide, oligolysine, oligoarginine, oligos (lysine, arginine) and PEP-1 peptides enhance the cellular permeability of proteins.

본 발명은 활성산소종 제거 활성을 나타내며, 염증 치료용으로 사용될 수 있는 단백질에 단백질 수송 도메인을 결합한 융합 단백질을 생체 세포 또는 조직 내로 좀더 원활하게 침투시킬 수 있는 방법을 제공하려는 것을 목적으로 한다.An object of the present invention is to provide a method for more smoothly penetrating into a living cell or tissue a fusion protein that exhibits reactive oxygen species removal activity and binds a protein transport domain to a protein that can be used for the treatment of inflammation.

또한, 본 발명은 종래 염증 치료에 이용 가능한 것으로 알려진 활성산소종 제거 활성을 나타내는 효소의 융합 단백질에 퍼골라이드를 가함으로써 좀더 효과적인 항염증 조성물을 제공하려는 것을 목적으로 한다.
It is also an object of the present invention to provide a more effective anti-inflammatory composition by adding pergolide to a fusion protein of an enzyme that exhibits active oxygen species removal activity known to be available for conventional inflammation treatment.

단백질 수송 도메인은 외래 단백질을 세포 내로 운반하는데 효과적임이 알려져 있다. 본 발명자들은 HaCaT 인간 각질세포 및 마우스 피부로 PEP-1-카탈레이즈의 세포침투에 대한 퍼골라이드 메실레이트 (pergolide mesylate; PM)의 영향 및 TPA에 의해 유도된 염증에 대한 PEP-1-카탈레이즈의 항염증 활성에 대한 퍼골라이드의 영향을 웨스턴 블랏과 조직학적 분석으로 시험하였다. 퍼골라이드는 세포 독성 없이 PEP-1-카탈레이즈를 HaCaT 세포 내로 농도 및 시간 의존적으로 도입시켰다. 마우스 부종 모델에서 PEP-1-카탈레이즈는 TPA에 의해 유도되는 사이클로옥시제네이즈-2(cycloxygenase-2), 유도 가능한 산화질소 합성효소(inducible nitric oxide synthase), 인터류킨-6, 인터류킨-1β 및 종양괴사인사 α와 같은 염증 매개자 및 사이토카인의 발현 증가를 억제하였다. 반면, 퍼골라이드 단독으로는 항염증 효과에 현저한 효과를 나타내지 못하였다. 그러나, PEP-1-카탈레이즈와 퍼골라이드를 함께 처리하면 항염증 효과가 PEP-1-카탈레이즈를 단독 사용할 때보다 현저히 증가하였다. 이러한 결과는 퍼골라이드가 단백질 수송 도메인과 융합한 치료용 융합 단백질을 목표 세포나 목표 조직에 더 잘 침투하도록 작용하며, 다양한 질병에 대한 약제의 치료 효과를 높여주는 것으로 판단된다.
Protein transport domains are known to be effective in transporting foreign proteins into cells. We describe the effect of pergolide mesylate (PM) on cell penetration of PEP-1-catalase into HaCaT human keratinocytes and mouse skin and the effect of PEP-1-catalase on inflammation induced by TPA. The effect of pergolide on anti-inflammatory activity was tested by Western blot and histological analysis. Pergolide introduced PEP-1-catalase in a concentration and time dependent manner into HaCaT cells without cytotoxicity. In mouse edema models, PEP-1-catalase is induced by TPA-induced cycloxygenase-2, inducible nitric oxide synthase, interleukin-6, interleukin-1β and tumors. Inhibition of inflammatory mediators such as necrosis α and increased expression of cytokines. On the other hand, pergolide alone did not show a significant effect on the anti-inflammatory effect. However, treatment with PEP-1-catalase and pergolide together increased the anti-inflammatory effect significantly when compared with PEP-1-catalase alone. These results suggest that pergolide works better to infiltrate therapeutic fusion proteins into the target cells or target tissues with the protein transport domain and enhance the therapeutic effect of drugs against various diseases.

본 발명은 단백질 수송 도메인과 치료용 단백질이 공유결합한 융합 단백질에 퍼골라이드를 가하여 융합 단백질의 생체 세포 또는 조직 내 도입을 증가시키는 방법을 제공한다. 치료용 단백질은 생체의 질병이나 이상 상태를 개선하거나 치료하는 기능을 하는 단백질을 포괄적으로 칭하는 것으로서, 그 범위에 특별한 제한은 없으며, 다양한 효소 등을 포함하는 개념이다. The present invention provides a method of increasing the introduction of a fusion protein into a living cell or tissue by adding a pergolide to a fusion protein covalently bonded to a protein transport domain and a therapeutic protein. The therapeutic protein is a generic term for a protein that functions to ameliorate or treat a disease or abnormal state of a living body, and there is no particular limitation on the scope thereof, and it is a concept including various enzymes.

또한, 본 발명은 상기 퍼골라이드를 융합 단백질보다 먼저 생체 세포 또는 조직에 처리하는 것을 특징으로 한다.In addition, the present invention is characterized in that the pergolide is treated to living cells or tissues before the fusion protein.

또한, 본 발명은 상기 단백질 수송 도메인이 Tat 또는 PEP-1임을 특징으로 한다.In addition, the present invention is characterized in that the protein transport domain is Tat or PEP-1.

또한, 본 발명은 상기 퍼골라이드가 퍼골라이드 메실레이트임을 특징으로 한다.In addition, the present invention is characterized in that the pergolide is a pergolide mesylate.

또한, 본 발명은 상기 치료용 단백질이 활성산소종을 제거하는 효소임을 특징으로 한다.In addition, the present invention is characterized in that the therapeutic protein is an enzyme for removing reactive oxygen species.

또한, 본 발명은 상기 치료용 단백질이 카탈레이즈, 수퍼옥사이드 디스뮤테이즈 및 글루타치온 퍼옥시데이즈 중 선택된 1종임을 특징으로 한다.In addition, the present invention is characterized in that the therapeutic protein is one selected from catalase, superoxide dismutase and glutathione peroxidase.

또한, 본 발명은 단백질 수송 도메인과 공유결합한 활성산소종 제거 활성을 가진 효소의 융합 단백질 및 퍼골라이드를 포함하는 산화적 스트레스에 의한 질환 및 염증성 질환의 예방 및 치료제 조성물을 제공한다.The present invention also provides a composition for preventing and treating inflammatory diseases and inflammatory diseases, including fusion proteins and pergolides of enzymes having reactive oxygen species removal activity covalently bound to the protein transport domain.

또한, 본 발명의 상기 조성물에서 상기 퍼골라이드는 퍼골라이드 메실레이트임을 특징으로 한다.In addition, the pergolide in the composition of the present invention is characterized in that the pergolide mesylate.

또한, 본 발명의 상기 조성물에서 상기 활성산소종 제거 활성을 가진 효소는 카탈레이즈, 수퍼옥사이드 디스뮤테이즈 및 글루타치온 퍼옥시데이즈 중 선택된 1종임을 특징으로 한다.In addition, the enzyme having the active oxygen species removal activity in the composition of the present invention is characterized in that one selected from catalase, superoxide dismutase and glutathione peroxidase.

또한, 본 발명의 상기 조성물에서 상기 단백질 수송 도메인은 Tat 또는 PEP-1임을 특징으로 한다.In addition, the protein transport domain in the composition of the present invention is characterized in that Tat or PEP-1.

또한, 본 발명에서 상기 산화적 스트레스에 의한 질환은 특별히 제한되지는 않으나, 특히 암, 골수염, 후천성 면역결핍증, 심맥관계 질환, 대장직장암, 방광암, 관상동맥질환, 알츠하이머병, 파킨슨병, 헌팅턴병, 만성 신장병, 알콜성 간질환, 폐쇄성 폐질환, 인슐린저항 증후군 또는 당뇨병인 것을 특징으로 한다.In addition, the disease caused by the oxidative stress in the present invention is not particularly limited, in particular cancer, osteomyelitis, acquired immunodeficiency syndrome, cardiovascular disease, colorectal cancer, bladder cancer, coronary artery disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, chronic Kidney disease, alcoholic liver disease, obstructive pulmonary disease, insulin resistance syndrome or diabetes.

또한, 본 발명에서 상기 염증성 질환은 특별히 제한되지는 않으나, 동맥경화증, 알러지 질환, 비염, 천식, 급성통증, 만성통증, 치주염, 치은염, 염증성 장질환, 통풍, 심근경색, 울혈성 심부전, 고혈압, 협심증, 위궤양, 뇌경색, 다운증후군, 다발성 경화증, 비만, 당뇨, 치매, 우울증, 정신분열증, 결핵, 수면장애, 패혈증, 화상, 피부염 또는 췌장염인 것을 특징으로 한다. 이와 같은 염증성 질환은 공통적으로 IL-1β, IL-6 등 염증 매개자의 발현 증가에 의하여 발생한다. In addition, the inflammatory disease in the present invention is not particularly limited, arteriosclerosis, allergic diseases, rhinitis, asthma, acute pain, chronic pain, periodontitis, gingivitis, inflammatory bowel disease, gout, myocardial infarction, congestive heart failure, high blood pressure, Angina pectoris, gastric ulcer, cerebral infarction, Down syndrome, multiple sclerosis, obesity, diabetes, dementia, depression, schizophrenia, tuberculosis, sleep disorders, sepsis, burns, dermatitis or pancreatitis. Such inflammatory diseases are commonly caused by increased expression of inflammatory mediators such as IL-1β and IL-6.

또한, 본 발명은 단백질 수송 도메인과 공유결합한 치료용 단백질의 융합 단백질 및 퍼골라이드를 포함하는 산화적 스트레스에 의한 질환 및 염증성 질환의 예방 및 개선용 피부외용제 조성물에 관한 것이다. The present invention also relates to an external skin composition for the prevention and improvement of diseases caused by oxidative stress and inflammatory diseases, including pergolide and fusion proteins of therapeutic proteins covalently bound to the protein transport domain.

또한, 본 발명은 단백질 수송 도메인과 공유결합한 치료용 단백질의 융합 단백질 및 퍼골라이드를 포함하는 산화적 스트레스에 의한 질환 및 염증성 질환의 예방 및 개선용 화장료 조성물에 관한 것이다.
The present invention also relates to a cosmetic composition for the prevention and improvement of diseases caused by oxidative stress and inflammatory diseases, including pergolide and fusion proteins of therapeutic proteins covalently bound with the protein transport domain.

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.

"융합 단백질"이란 단백질 수송 도메인과 화물 분자를 포함하며, 단백질 수송 도메인과 화물 분자(cargo molecule, 즉 본 발명에서는 카탈레이즈를 비롯한 치료용 단백질을 의미함)의 유전적 융합이나 화학 결합으로 형성된 공유결합 복합체를 의미한다. 본 명세서와 도면에서는 구체적인 일 실시예로서 "PEP-1-카탈레이즈"는 융합 단백질 중 카탈레이즈의 N-말단에 PEP-1 단백질 수송 도메인이 결합된 것을 말한다.A "fusion protein" includes a protein transport domain and a cargo molecule, and is formed by a genetic fusion or chemical bond between the protein transport domain and a cargo molecule (that is, in the present invention, a therapeutic protein, including catalase). Means a binding complex. In the present specification and drawings, as a specific example, "PEP-1-catalase" refers to the binding of the PEP-1 protein transport domain to the N-terminus of the catalase in the fusion protein.

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

또한, "표적 세포"란 수송 도메인에 의해 화물 분자가 전달되는 세포를 의미하는 것으로서 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. "Target cell" also means a cell to which a cargo molecule is delivered by a transport domain, i.e., a target cell is a cell in the body, ie, a cell constituting an organ or tissue of a living animal or human, or a living animal or human It is meant to include the microorganisms found. In addition, target cells are meant to include extracellular cells, ie cultured animal cells, human cells or microorganisms.

본 명세서의 "단백질 수송 도메인"은 화물 분자(목표 단백질) 펩타이드 또는 단백질과 공유결합을 이루어 별도의 수용체나 운반체, 에너지를 필요로 하지 않고 상기 펩타이드나 단백질을 세포 내로 도입시킬 수 있는 것을 말하며, 예를 들면 PEP-1 펩타이드를 말한다.As used herein, the term "protein transport domain" refers to a covalent bond with a cargo molecule (target protein) peptide or protein, so that the peptide or protein can be introduced into a cell without the need for a separate receptor, carrier, or energy. For example, PEP-1 peptide.

본 명세서의 "목표 단백질"은 PEP-1 단백질 수송 도메인과 공유결합을 이루어 세포 내로 도입되어 활성을 나타내는 분자를 의미한다. "화물 분자", "치료용 단백질"과 동일한 의미이다.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. It has the same meaning as "cargo molecule", "therapeutic protein".

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 발명에서 단백질 수송 도메인으로는 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 도메인, 6 내지 12개의 아미노산 잔기로 구성되며 아르기닌 또는 라이신 잔기를 3/4 이상 포함하는 수송도메인, 6 내지 12개의 라이신으로 구성되는 올리고라이신 수송 도메인, 6 내지 12개의 아르기닌으로 구성되는 올리고아르기닌 수송 도메인 또는 6 내지 12개의 라이신 또는 아르기닌으로 구성되는 올리고(라이신,아르기닌) 수송 도메인을 들 수 있다. 또한, 본 발명의 일 실시예에서 목표 단백질(화물 분자)은 치료용 단백질이고, 좀더 구체적으로는 활성산소종을 제거하는 효소를 말한다. 좀더 구체적으로는 카탈레이즈, 수퍼옥사이드 디스뮤테이즈, 글루타치온 퍼옥시데이즈 중 선택된 1종을 말한다. 상기 단백질 수송 도메인 및 목표 단백질은 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 서열간의 일정 범위의 상동성 예컨대 85-100% 범위 내에서 동일?유사한 생물학적 활성을 갖는 절편 또는 이들의 유도체들도 본 발명의 권리범위에 포함된다.In the present invention, the protein transport domain is composed of 15-30 amino acids, a non-hydrophilic domain comprising 5 or more tryptophan, a hydrophilic domain including a large number of lysine and a protein transport domain consisting of a spacer separating the two domains, A transport domain consisting of 6 to 12 amino acid residues and containing at least 3/4 of an arginine or lysine residue, an oligolysine transport domain consisting of 6 to 12 lysines, an oligoarginine transport domain consisting of 6 to 12 arginines, or 6 And oligo (lysine, arginine) transport domains composed of from twelve lysine or arginine. In addition, in one embodiment of the present invention, the target protein (cargo molecule) is a therapeutic protein, and more specifically, refers to an enzyme for removing reactive oxygen species. More specifically, it refers to one selected from catalase, superoxide dismutase and glutathione peroxidase. The protein transport domain and the target protein may be substituted with other amino acid (s) of similar polarity in which one or more amino acids in the sequence are functionally equivalent in response to a silent change. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, 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 present invention are fragments or derivatives thereof having the same or similar biological activity within a range of homology, such as 85-100%, between the fusion protein and amino acid sequence of the present invention.

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

또한, 본 발명은 상기 세포 도입성(cell-transducing) 치료용 융합 단백질 및 퍼골라이드를 유효성분으로 함유하고 약학적으로 허용되는 담체를 포함하는 것을 특징으로 하는 염증성 질환 치료용 약학 조성물에 관한 것이다.In addition, the present invention relates to a pharmaceutical composition for treating inflammatory diseases, comprising a pharmaceutically acceptable carrier containing the fusion protein for cell-transducing therapy and pergolide as an active ingredient.

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

이와 같이 제조된 약제학적 제제는 목적하는 바에 따라 경구로 투여하거나, 비경구 방식 즉, 정맥 내 , 피하, 복강 내 투여 또는 국소적용할 수 있다. 용량은 일일 투여량 0.01㎍~100㎎/㎏을 1 내지 수회에 나누어 투여할 수 있다. 특정 환자에 대한 투여용량 수준은 환자의 체중, 연령, 성별, 건강상태, 투여시간, 투여방법, 배설율, 질환의 중증도 등에 따라 변화될 수 있다.The pharmaceutical preparations thus prepared may be administered orally as desired, or parenterally, ie, intravenously, subcutaneously, intraperitoneally, or topically. The dose may be administered by dividing the daily dose of 0.01 μg-100 mg / kg into one to several times. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

본 발명은 또한, 상기 치료용 단백질의 융합 단백질 및 퍼골라이드를 유효성분으로 하는 다양한 산화적 스트레스에 의한 질환 및 염증성 질환 예방과 개선에 유용한 건강 기능성 식품 조성물을 제공한다.The present invention also provides a health functional food composition useful for the prevention and improvement of diseases caused by various oxidative stress and inflammatory diseases using the fusion protein and pergolide of the therapeutic protein as an active ingredient.

본 발명의 상기 세포 도입성 치료용 융합 단백질 및 퍼골라이드를 첨가할 수 있는 식품으로는, 각종 식품류, 예를 들어, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있으며, 환제, 분말, 과립, 침제, 정제, 캡슐 또는 음료 형태로 사용할 수 있다. 이때, 식품 또는 음료 중의 상기 상기 세포 도입성 치료용 융합 단백질 및 퍼골라이드의 양은, 일반적으로 본 발명의 건강식품 조성물의 경우 전체 식품 중량의 0.01 내지 15 중량%, 바람직하게는 0.2 내지 10중량%로 가할 수 있으며, 건강 음료 조성물의 경우 100㎖를 기준으로 0.1 내지 30g, 바람직하게는 0.2 내지 5g의 비율로 가할 수 있다. 본 발명의 건강 음료 조성물은 지시된 비율로 필수 성분으로서 상기 상기 세포 도입성 치료용 융합 단백질 및 퍼골라이드를 함유하는 외에는 액체성분에는 특별한 제한점은 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예로는 모노사카라이드, 예를 들어, 포도당, 과당 등 디사카라이드, 예를 들어 말토스, 수크로스 등 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등)) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다. 상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함Foods to which the fusion proteins and pergolides for the cell-introduced therapeutic treatment of the present invention may be added include various foods, for example, beverages, gums, teas, vitamin complexes, dietary supplements, pills, powders, It can be used in the form of granules, acupuncture, tablets, capsules or beverages. In this case, the amount of the cell-introduced therapeutic fusion protein and pergolide in food or beverage is generally in the range of 0.01 to 15% by weight, preferably 0.2 to 10% by weight of the total food weight of the health food composition of the present invention. It may be added, in the case of a health beverage composition may be added in a ratio of 0.1 to 30g, preferably 0.2 to 5g based on 100ml. The health beverage composition of the present invention has no specific limitations on the liquid components other than the above-mentioned fusion protein and pergolide for therapeutic introduction into cells as essential ingredients in the indicated ratios, and various flavors or natural carbohydrates, such as ordinary beverages, etc. May be contained as an additional component. Examples of the above-mentioned natural carbohydrates include monosaccharides, for example, disaccharides such as glucose and fructose, polysaccharides such as maltose and sucrose, and conventional sugars such as dextrin, cyclodextrin, and xylitol. Sugar alcohols such as sorbitol and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention. In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compositions of the present invention are pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks

유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 중요하지는 않으나 본 발명의 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.
You can. These components may be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

본 발명은 또한, 상기 산화적 스트레스에 의한 질환 및 염증성 질환 치료용 단백질의 융합 단백질 및 퍼골라이드를 유효성분으로 하는 화장료 조성물 또는 피부 외용제 조성물을 제공한다. 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 산화적 스트레스에 의한 질환 및 염증성 질환 치료용 단백질의 융합 단백질과 퍼골라이드를 함유시키고 여기에 향료, 킬레이트제, 색소, 산화방지제, 방부제 등을 필요에 따라 사용하는 한편, 물성개선을 목적으로 단백질, 미네랄, 비타민 등 합성 또는 천연소재를 병용할 수 있다.
The present invention also provides a cosmetic composition or an external skin composition comprising the fusion protein and pergolide of the protein for treating the disease caused by oxidative stress and inflammatory disease. The coating agent which uses the fusion protein of this invention as an active ingredient can be easily manufactured in any form according to the conventional manufacturing method. As an example, in preparing a cream coating agent, a fusion protein and a pergolide of a protein for treating diseases caused by oxidative stress and inflammatory diseases in a cream base of a general oil-in-water type (O / W) or water-in-oil type (W / O) It may contain a fragrance, a chelating agent, a pigment, an antioxidant, a preservative, and the like, as necessary, while using synthetic or natural materials such as proteins, minerals, vitamins, etc. for the purpose of improving the properties.

본 발명에서 퍼골라이드를 단백질 수송 도메인이 공유결합된 치료용 단백질의 융합 단백질과 함께 HaCaT 세포 또는 마우스 피부에 처리한 경우 융합 단백질의 침투 효율이 현저히 높아졌으며, 또한 마우스 염증 모델에서 염증 개선 효과도 현저해졌다. 퍼골라이드 단독으로는 염증 개선 효과나 염증성 사이토카인 억제 효과가 거의 없으나, 융합 단백질과 함께 처리한 경우 침투 효율 및 염증 개선 효과가 융합 단백질 단독 처리의 경우보다 분명하게 향상되었다.In the present invention, when the pergolide is treated with HaCaT cells or mouse skin together with a fusion protein of a therapeutic protein covalently bound to a protein transport domain, the penetration efficiency of the fusion protein is significantly increased, and the inflammation improvement effect is also remarkable in the mouse inflammation model. Done Pergolide alone has little effect of improving inflammation or inhibiting inflammatory cytokines, but when treated with a fusion protein, the penetration efficiency and inflammation improvement are clearly improved than that of the fusion protein alone.

도 1은 생체 외 (in vitro) 및 생체 내 (in vivo) 조건에서 PEP-1-카탈레이즈 세포침투에 미치는 퍼골라이드 메실레이트의 영향을 나타내는 것이다.
(A) 퍼골라이드 메실레이트 {8β-8-[(methylthio) methyl]-6-propylergoline; PM}의 구조,
(B) PEP-1-카탈레이즈 세포침투가 PM 투여량 의존적인지를 확인하기 위하여 세포를 다양한 농도의 PEP-1-카탈레이즈로 처리하기 한 시간 전 PM(1.0 ㎍/ml)으로 처리한 군과 PM 처리하지 않은 군을 비교하였다. PEP-1-카탈레이즈 세포침투가 PM 처리시간 의존적인지를 확인하기 위하여 0 ~ 60분간 PEP-1-카탈레이즈 (1μM)만 넣고 세포를 배양하거나 또는 PEP-1-카탈레이즈와 PM (1.0 ㎍/ml)를 함께 넣어 세포를 배양하였다. 세포침투된 PEP-1-카탈레이즈는 웨스턴 블랏팅으로 분석하였다.
(C) 마우스 피부에 세포침투된 PEP-1-카탈레이즈의 위치를 촬영한 사진이다. PEP-1-카탈레이즈 (3.0μM)를 마우스 등 피부의 면도한 부위에 60분간 국소도포하였다. 피부 조직은 토끼 항-히스티딘 IgG (1:400)로 면역염색한 후 바이오틴이 결합된 염소 항-토끼 IgG (1:200)로 염색하였다. 3, 3'-다이아미노벤지딘(diaminobenzidine)으로 시각화하여 현미경으로 관찰하였다.
도 2는 HaCaT 인간 각질세포 (keratinocytes)의 생존율에 미치는 PM의 영향을 나타낸다. 세포를 PM (1.0 ㎍/ml)으로 한 시간 미리 처리한 후 PEP-1-카탈레이즈와 함께 한 시간 배양하였다. 세포를 TPA (4.0 ㎍/ml)로 12시간 동안 처리하였다. 세포 생존율은 MTT 어세이로 평가하였고, TPA 처리하지 않은 대조군에 대한 백분율로 나타내었다.
도 3은 PM 존재시 TPA에 의해 유도된 귀 부종에 대한 PEP-1-카탈레이즈의 영향을 나타낸다. 3일 동안 하루 한 번씩 TPA (1.0 ㎍/ear)를 마우스 귀에 처리하여 부종을 형성하였다. 그 후 PM (100 ㎍/ear)을 마우스 귀에 국소도포하고 그 다음 PEP-1-카탈레이즈로 처리하였다.
(A) PEP-1-카탈레이즈 처리 후 귀 두께 및
(B) 귀 중량을 측정하였다.
(C) 마우스 귀 조직을 조직화학적으로 분석하였다.
1, 음성대조군 (Untreated control);
2, TPA-처리 피부;
3, TPA 처리 후 PM 처리한 피부;
4, TPA 처리 후 PEP-1-카탈레이즈 처리한 피부;
5, TPA 처리 후 PM과 PEP-1-카탈레이즈 처리한 피부.
도 4는 TPA에 의해 유도된 마우스 염증 모델에서 친염증성 사이토카인과 매개자 (mediators)의 수준에 대한 PEP-1-카탈레이즈의 저해 효과를 나타낸다. 마우스 귀를 TPA (1.0 ㎍/ear)로 하루 한 번씩 3일간 먼저 처리하였다. 그후 PM을 마우스 귀에 국소 적용한 다음 PEP-1-카탈레이즈로 처리하였다. 귀 생검 (ear biopsies)에서 얻어진 단백질은 10% SDS PAGE 젤로 분리한 다음 나이트로셀룰로즈 막에 이동시켰다. COX-1, iNOS, IL-6, LI-1β, TNF-α 및 β 액틴 발현 정도를 웨스턴 블랏팅으로 분석하였다.
레인 1, 무처리 대조군;
레인 2, TPA-처리 염증 유발 피부;
레인 3, TPA로 염증 유발 후 PM 처리;
레인 4, TPA로 염증 유발 후 PEP-1-카탈레이즈 처리;
레인 5, TPA로 염증 유발 후 PM 및 PEP-1-카탈레이즈 처리.Figure 1 shows the effect of pergolide mesylate on PEP-1-catalase cell infiltration in in vitro and in vivo conditions.
(A) pergolide mesylate {8β-8-[(methylthio) methyl] -6-propylergoline; PM} 's structure,
(B) groups treated with PM (1.0 μg / ml) one hour before treatment with various concentrations of PEP-1-catalase to determine whether PEP-1-catalase cell infiltration was PM dose dependent; Groups not treated with PM were compared. To determine if PEP-1-catalase cell infiltration is PM treatment time dependent, incubate the cells with only PEP-1-catalase (1 μM) for 0 to 60 minutes or PEP-1-catalase and PM (1.0 μg / ml) were put together to culture the cells. Cell penetrated PEP-1-catalase was analyzed by western blotting.
(C) The photograph of the location of PEP-1-catalase which penetrated the mouse skin. PEP-1-catalase (3.0 μM) was applied topically to the shaved areas of the skin such as mice for 60 minutes. Skin tissue was immunostained with rabbit anti-histidine IgG (1: 400) and then stained with goat anti-rabbit IgG (1: 200) bound to biotin. Visualized with 3, 3'-diaminobenzidine and observed under a microscope.
2 shows the effect of PM on the survival of HaCaT human keratinocytes. Cells were pretreated with PM (1.0 μg / ml) one hour before incubating with PEP-1-catalase for one hour. Cells were treated with TPA (4.0 μg / ml) for 12 hours. Cell viability was assessed by MTT assay and expressed as a percentage of the control not treated with TPA.
3, For ear edema induced by TPA in the presence of PM The effect of PEP-1-catalase is shown. Edema was formed by treating mouse ears with TPA (1.0 μg / ear) once daily for three days. PM (100 μg / ear) was then applied topically to mouse ears and then treated with PEP-1-catalase.
(A) Ear thickness after PEP-1-catalase treatment and
(B) Ear weight was measured.
(C) Mouse ear tissue was analyzed histologically.
1, untreated control;
2, TPA-treated skin;
3, PM treated skin after TPA treatment;
4, skin treated with PEP-1-catalase after TPA treatment;
5, PM and PEP-1-catalized skin after TPA treatment.
4 shows the inhibitory effect of PEP-1-catalase on the levels of proinflammatory cytokines and mediators in a mouse inflammation model induced by TPA. Mouse ears were first treated with TPA (1.0 μg / ear) once daily for 3 days. PM was then applied topically to the mouse ears and then treated with PEP-1-catalase. Proteins obtained from ear biopsies were separated on 10% SDS PAGE gels and then transferred to nitrocellulose membranes. COX-1, iNOS, IL-6, LI-1β, TNF-α and β actin expression levels were analyzed by Western blotting.
Lane 1, untreated control;
Lane 2, TPA-treated inflammation causing skin;
Lane 3, PM treatment following induction of inflammation with TPA;
Lane 4, PEP-1-catalase treatment after induction with TPA;
Lane 5, PM and PEP-1-catalase treatment after inflammation induced by TPA.

이하, 실시예를 들어 본 발명의 구성을 좀더 자세히 설명한다. 특별히, 본 발명의 실시예에서 융합 단백질의 일 실시예로 PEP-1을 단백질 수송 도메인으로 선택하고, 치료용 단백질로 카탈레이즈를 선택하여 PEP-1-카탈레이즈 융합 단백질에 대하여 설명하였으나, Tat, 올리고라이신, 올리고아르기닌 등 다양한 단백질 수송 도메인을 치료용 단백질의 N-말단 및/또는 C-말단에 결합시킨 융합 단백질에 대해서도 본 발명의 명세서 및 도면에 제시된 결과와 유사한 세포침투 증대, 세포사멸 저해 등의 결과를 거둘 수 있었으며, 본 발명의 범위가 실시예의 기재 범위 내로 한정되는 것이 아님은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 자명하다. 또한, 치료용 단백질의 예시로서 실시예에서는 카탈레이즈와 수퍼옥사이드 디스뮤테이즈를 선택하였으나, 생체 기능의 이상 및 질병을 개선시키는 효소를 비롯한 다양한 단백질을 치료용 단백질로 선택할 수 있음은 당업자에게 자명하다.
Hereinafter, the configuration of the present invention will be described in more detail with reference to examples. Particularly, in the embodiment of the present invention, PEP-1-catalase fusion protein was described by selecting PEP-1 as the protein transport domain and catalase as the therapeutic protein in one embodiment of the fusion protein. As for the fusion protein in which various protein transport domains such as oligolysine and oligoarginine are bound to the N-terminus and / or C-terminus of the therapeutic protein, cell penetration enhancement and inhibition of apoptosis similar to those shown in the specification and drawings of the present invention, etc. It is apparent to those skilled in the art that the scope of the present invention is not limited to the scope of the embodiments of the present invention. In addition, in the embodiment, catalase and superoxide dismutase were selected as examples of therapeutic proteins, but it is apparent to those skilled in the art that various proteins including enzymes for improving abnormalities and diseases of biological functions can be selected as therapeutic proteins. .

재료material

퍼골라이드 메실레이트는 Sigma-Aldrich (St. Louis, MO, USA)에서 구입하였고, COX-2, iNOS, IL-6, IL-1β 및 β 액틴 각각에 대한 1차 항체는 Santa Cruz Biotechnology (Santa Cruz, CA, USA)에서 구입하였다. DNA 서브클로닝 (subcolning)에 사용한 택 폴리머라아제 (Tag polymerase), T4 리가아제 (ligase), 제한효소 (restriction enzyme) 등은 Takara에서 구입하였고, Tat, PEP-1은 Gibco BRL custom primer(USA)에서 합성하였다. IPTG는 Duchefa(Netherland)에서 구입하였다. 재조합 단백질 정제를 위한 히스티딘 결합 금속 킬레이팅 수지와 컬럼은 Novagen에서 구입하였다. 그리고 니켈 설페이트(nikel sulfate)와 이미다졸 등은 Sigma사에서 구입하였다. 박테리아 형질전환에 이용한 대장균 균주는 본 실험실에서 보유하고 있던 것이며, 단백질 발현을 위해 대장균 JM109(DE3) 균주를 사용하였으며 미생물 배지의 박토트립톤(Bacto tryptone), 효모추출물(yeast extract), 한천 등은 Difco 제품을 사용하였다. 세포 배양에 필요한 배지는 Gibco 제품을 사용하였고 인간 카탈레이즈 DNA, 인간 수퍼옥사이드 디스뮤테이즈는 PCR 방법으로 인간 간 cDNA 라이브러리에서 증폭시켰다. 기타 약품은 특별한 언급이 없는 경우 Sigma-Aldrich (MO, USA)에서 구입하였다.
Pergolide mesylate was purchased from Sigma-Aldrich (St. Louis, MO, USA), and the primary antibodies against COX-2, iNOS, IL-6, IL-1β and β actin, respectively, were selected from Santa Cruz Biotechnology (Santa Cruz). , CA, USA). Tag polymerase, T4 ligase, restriction enzyme, etc. used for DNA subcloning were purchased from Takara, and Tat and PEP-1 were used for Gibco BRL custom primer (USA). Synthesis at IPTG was purchased from Duchefa (Netherland). Histidine-bound metal chelating resins and columns for recombinant protein purification were purchased from Novagen. Nickel sulfate and imidazole were purchased from Sigma. E. coli strains used for bacterial transformation were possessed by the laboratory, and E. coli JM109 (DE3) strain was used for protein expression. Bacto tryptone, yeast extract and agar of microbial medium were used. Difco product was used. As a medium for cell culture, Gibco products were used and human catalase DNA and human superoxide dismutase were amplified in human liver cDNA library by PCR method. Other drugs were purchased from Sigma-Aldrich (MO, USA) unless otherwise noted.

실시예 1: 발현백터의 제조 및 형질전환Example 1 Preparation and Transformation of Expression Vectors

PEP-1에 해당하는 두 종류의 올리고뉴클레오타이드를 NdeⅠ-XhoⅠ 제한효소로 자른 pET15b에 연결(ligation)하여 삽입하였다. 이어 인간 신장(kidney) 카탈레이즈의 cDNA의 서열을 기본으로 하여 2종류의 올리고뉴클레오타이드를 합성하였다.Two kinds of oligonucleotides corresponding to PEP-1 were inserted by ligation to pET15b cut with NdeI-XhoI restriction enzyme. Subsequently, two kinds of oligonucleotides were synthesized based on the sequence of cDNA of human kidney catalase.

정방향 프라이머는 5'-ctcgagatggctgacagccgggatcccgcc-3'로 XhoⅠ제한부위를 지니고 있으며 역방향 프라이머의 서열은 5'-ggatcctcacagatttgccrrctcccttgcc-3'로 BamHⅠ 제한부위를 가지고 있다.The forward primer has a XhoI restriction site of 5'-ctcgagatggctgacagccgggatcccgcc-3 'and the reverse primer sequence has a BamHI restriction site of 5'-ggatcctcacagatttgccrrctcccttgcc-3'.

중합효소 연쇄반응(PCR)은 thermal cycler(Perkin-Elmer, model 9600)에서 수행하였으며 반응 혼합액을 50㎕ 실리콘 튜브 (siliconized reaction tube)에 넣고 94℃에서 5분간 가열하였다. PCR 반응은 94℃에서 40초간 30회 연장 (extension), 61℃에서 40초간 변성 (denaturation), 72℃에서 10분, 37℃에서 1분간 최종 연장을 유도하였다. 그리고 아가로즈 젤 전기이동 (agarose gel electrophoresis)으로 분리하여 반응물을 분리하고 이것을 pGEM-T에 연결(ligation)시켰다. 그리고 이 벡터를 형질전환용 세포 (competent cell)에 형질전환(transformation)시키고 플라스미드를 분리하여 인간 카탈레이즈 cDNA가 포함된 벡터를 XhoⅠ과 BamHⅠ으로 절단한 다음 pET-15b 및 pET-15b-Tat (Arg, Lys, PEP-1)발현 벡터에 삽입하였다. 벡터의 발현은 T7 프로모터와 lacO-오퍼레이터의 조절 하에 있다.Polymerase chain reaction (PCR) was performed in a thermal cycler (Perkin-Elmer, model 9600) and the reaction mixture was placed in a 50 µl silicon tube (siliconized reaction tube) and heated at 94 ° C for 5 minutes. PCR reactions led to 30 extensions for 40 seconds at 94 ° C, denaturation for 40 seconds at 61 ° C, 10 minutes at 72 ° C, and 1 minute at 37 ° C. The reactants were separated by agarose gel electrophoresis and ligation was carried out to pGEM-T. The vector was transformed into a transformant cell and transformed into plasmids. The vector containing human catalysed cDNA was cleaved with XhoI and BamHI and then pET-15b and pET-15b-Tat (Arg). , Lys, PEP-1) was inserted into the expression vector. Expression of the vector is under the control of the T7 promoter and lacO-operator.

상기 방법과 동일한 방법으로 녹색 형광 단백질의 융합 단백질 즉, PEP-1-녹색 형광 단백질 발현 벡터 (pPEP-1-GFP)를 제조하였다.In the same manner as the above method, a fusion protein of green fluorescent protein, that is, PEP-1-green fluorescent protein expression vector (pPEP-1-GFP) was prepared.

또한, PEP-1-수퍼옥사이드 디스뮤테이즈 융합 단백질은 위 방법을 변형하여 제조하였다. Also, PEP-1-superoxide dismutase fusion protein was prepared by modifying the above method.

또한, 단백질 수송 도메인으로 Tat을 선택하여 제조한 Tat-카탈레이즈 융합 단백질, Tat-수퍼옥사이드 디스뮤테이즈는 공개특허 제10-2002-10445호에 개시된 방법에 따라 제조하였다. In addition, the Tat-catalase fusion protein, Tat- superoxide dismutase, prepared by selecting Tat as the protein transport domain, was prepared according to the method disclosed in Publication 10-2002-10445.

뿐만 아니라, 올리고라이신 또는 올리고아르기닌을 단백질 수송 도메인으로 한 융합 단백질은 특허 제490362호의 방법에 따라 또는 이 방법을 응용하여 제조하였다.
In addition, a fusion protein using oligolysine or oligoarginine as the protein transport domain was prepared according to the method of Patent No. 493662 or by applying this method.

실시예 2: 재조합 카탈레이즈 융합단백질의 제조 및 정제Example 2: Preparation and Purification of Recombinant Catalase Fusion Protein

융합단백질 발현벡터 (pPEP-1-CAT)가 포함되어 있는 E. coli JM109 (DE3) 세포를 앰피실린이 포함된 LB 배지에 넣고 37℃에서 250 rpm으로 교반하며 배양하였다. 이 발현벡터는 사람 카탈레이즈, 단백질 수송 도메인 (PEP-1) 그리고 아미노산 말단부분에 6개의 히스티딘 잔기를 발현시킬 수 있는 cDNA를 포함하고 있다. 배양액 내의 박테리아 농도가 O.D600nm에서 0.6?1.0이 될 때 IPTG를 최종농도가 1.0mM되게 배지에 첨가한 다음 4시간 동안 단백질을 과대발현시켰다.E. coli JM109 (DE3) cells containing the fusion protein expression vector (pPEP-1-CAT) were placed in LB medium containing ampicillin and incubated at 37 ° C. at 250 rpm. The expression vector contains human catalase, protein transport domain (PEP-1) and cDNA capable of expressing six histidine residues at the amino acid terminal. When the bacterial concentration in the culture solution was 0.6-1.0 at O.D600 nm, IPTG was added to the medium at a final concentration of 1.0 mM, and the protein was overexpressed for 4 hours.

배양한 세포를 원심분리하여 모은 뒤 결합완충액 5ml (5mM 이미다졸, 500mM NaCl, 20mM Tris-HCl, pH 7.9)를 넣고 프렌치 프레스 (french pressure)로 세포를 용균 (lysis)시키고 재조합 단백질을 정제하였다.The cultured cells were collected by centrifugation, 5 ml of binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9) was added, and the cells were lysed by French press to purify the recombinant protein.

한편, 히스티딘 결합 수지를 조심스럽게 섞어서 폴리프로필렌 컬럼에 팩킹한 후 D.D.W 3배 부피를 흘려 수지를 닦아낸다. 그리고 5배 부피의 charge buffer (50mM NiSO4)와 3배 부피의 결합 완충액을 차례로 흘려 수지를 활성화시켰다.Meanwhile, the histidine-binding resin is carefully mixed and packed in a polypropylene column, followed by a 3-fold volume of DDW to wipe off the resin. The resin was activated by flowing a 5-fold charge buffer (50mM NiSO 4 ) and a 3-fold binding buffer.

변성시킨 재조합 단백질을 원심분리하여 상층액을 즉시 수지에 부하하고 10배 부피의 결합완충액과 6배 부피의 세척 완충액 (60mM 이미다졸, 500mM NaCl, 20mM Tris-HCl, pH 7.9)을 차례로 흘려주어 정제하고자 하는 단백질 외의 다른 단백질을 제거하였다. 완충액이 수지에서 완전히 빠진 후 3배 부피의 용출 완충액 (1M 이미다졸, 500mM NaCl, 20mM Tris-HCl, pH 7.9)로 융합단백질을 용출하였다. 그리고 융합단백질이 포함된 분획들을 모아 세파덱스 G-25 컬럼 크로마토그래피를 수행하여 분획 중에 포함된 염분을 제거하였다.
The denatured recombinant protein was centrifuged to immediately load the supernatant onto the resin, and purified by flowing 10-fold binding buffer and 6-fold wash buffer (60 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9) in that order. Proteins other than the protein to be removed were removed. The fusion protein was eluted with three volumes of elution buffer (1 M imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9) after the buffer had completely drained from the resin. The fractions containing the fusion protein were collected and Sephadex G-25 column chromatography was performed to remove the salts contained in the fractions.

실시예 3: 세포 배양과 MTT 어세이Example 3: Cell Culture and MTT Assays

HaCaT 인간 각질세포 (keratinocytes)를 10% 가열 불활성화 우태혈청 및 항생제 (100 ㎍/ml 스트렙토마이신 및 100 U/ml 페니실린)가 포함된 DMEM (Dulbecco's modified Eagle's medium)에 가하여 95% 공기, 5% CO2 의 습한 조건으로 37℃에서 배양하였다. 세포를 6-웰 플레이트에 넣고 70% 성장할 때까지 배양하여 세포 생존율을 평가하였다. 세포를 먼저 PM (1.0 ㎍/ml)로 한 시간 동안 처리하였다. PM을 제거한 다음 세포를 다양한 농도의 PEP-1-카탈레이즈 (0.5-3 μM)로 한 시간 동안 처리하였다. 세포를 DPBS (Dulbecco's phosphate buffered saline)로 세척한 다음 TPA (12-O-tetradecanoylphorbol-13-acetate) (4.0 ㎍/ml)로 열두 시간 동안 처리하였다. 세포 생존율 평가는 MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide} 어세이 프로토콜에 따라 수행하였다 [D.T. Vistica et al., Cancer Res. 51 (1991) 2515-2520]. 세포 생존율은 무처리 대조군에 대한 백분율로 나타내었다.
HaCaT human keratinocytes were added to Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum and antibiotics (100 μg / ml streptomycin and 100 U / ml penicillin) in 95% air, 5% CO Incubated at 37 ° C. under 2 moist conditions. Cell viability was assessed by placing cells in 6-well plates and culturing until 70% growth. Cells were first treated with PM (1.0 μg / ml) for one hour. After removal of PM, cells were treated with various concentrations of PEP-1-catalase (0.5-3 μM) for one hour. Cells were washed with Dulbecco's phosphate buffered saline (DPBS) and then treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) (4.0 μg / ml) for 12 hours. Cell viability assessment was performed according to the MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-dipheyltetrazolium bromide} assay protocol [DT Vistica et al., Cancer Res . 51 (1991) 2515-2520. Cell viability is expressed as a percentage of untreated control.

실시예 4: HaCaT 세포 내로 PEP-1-카탈레이즈 융합 단백질 도입Example 4: Incorporation of PEP-1-catalase fusion protein into HaCaT cells

PEP-1-카탈레이즈는 앞선 논문의 방법에 따라 제조 및 정제되었다 [D.W. Kim et al., Free Radic. Biol. Med. 47 (2009) 941-952 참조]. PEP-1-카탈레이즈 세포침투의 농도 의존성을 평가하기 위하여 세포를 6-웰 플레이트에서 70%가 되도록 배양한 다음 PM (1.0 ㎍/ml)으로 한 시간 동안 미리 처리하였고, 이후 다양한 농도의 PEP-1-카탈레이즈 (0.5~3 μM)를 가하여 한 시간 더 배양하였다. 세포침투의 시간 의존성을 평가하시 위하여 세포에 1 μM의 PEP-1-카탈레이즈를 가하고 배양 시간 (10~60 분)을 달리 하여 배양하였다. 세포를 하비스트하고 세포 추출액을 웨스턴 블랏으로 분석하였다.
PEP-1-catalase was prepared and purified according to the method of the previous paper [DW Kim et al., Free Radic. Biol. Med. 47 (2009) 941-952]. To assess the concentration dependence of PEP-1-catalase cell infiltration, cells were cultured to 70% in 6-well plates and then pretreated with PM (1.0 μg / ml) for one hour, followed by various concentrations of PEP- 1-catalase (0.5-3 μM) was added and incubated for another hour. To evaluate the time dependence of cell infiltration, 1 μM of PEP-1-catalase was added to the cells and cultured with different incubation times (10 to 60 minutes). The cells were harvested and cell extracts analyzed by Western blot.

실시예 5: 웨스턴 블랏 분석Example 5: Western Blot Analysis

세포 용균액 및 귀 생검 균질파쇄액 중의 단백질을 10% SDS-폴리아크릴아마이드 젤 전기영동 (SDS-PAGE)으로 분리한 후 나이트로셀룰로스 막에 전이시켰다. 막은 PBS에 용해시킨 5% 탈지유로 블로킹한 후 1차 항체로 4℃에서 오버나잇 배양하였다. 호스래디쉬 퍼옥시데이즈가 결합된 2차 항체로 막을 배양한 후 매뉴얼에 따라 ECL (enhanced chemiluminescence)로 시각화하였다 (Amersham, Piscataway, NJ, USA).
Proteins in cell lysate and ear biopsy homogenate were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membrane. The membranes were blocked with 5% skim milk dissolved in PBS and then incubated overnight at 4 ° C. with primary antibodies. Membranes were incubated with horseradish peroxidase bound secondary antibodies and visualized by enhanced chemiluminescence (ECL) according to the manual (Amersham, Piscataway, NJ, USA).

실시예 6: 마우스 염증 모델Example 6: Mouse Inflammation Model

6주령 ICR 마우스를 한림대 실험동물센터에서 구입하였다. 동물은 일정 온도 (23 ℃)와 60%의 상대습도로 12시간 명암 사이클로 배양하였고, 물과 사료를 자유롭게 섭취하였다. 동물과 관련된 모든 실험 절차와 관리는 국립수의과학검역원 (National Veterinary Research & Quarantine Service of Korea)의 실험동물 보호 및 이용에 관한 가이드를 따랐고, 이를 한림대학교 의과대학 동물 보호 및 이용 위원회가 입증한다. Six-week-old ICR mice were purchased from Hallym University Experimental Animal Center. Animals were incubated in a 12 hour light and dark cycle at a constant temperature (23 ° C.) and 60% relative humidity. Water and feed were freely consumed. All experimental procedures and controls involving animals have followed the National Veterinary Research & Quarantine Service of Korea's Guide to the Protection and Use of Laboratory Animals, which is evidenced by the Hallym University College of Medicine Animal Care and Use Committee.

피부 염증을 유발하기 위하여 각 마우스 (n=5)의 오른 쪽 귀를 20 ㎕의 아세톤에 녹인 TPA (1.0 μM)로 처리하였다. PM (100 ㎍/ear)을 동일한 피부 표면에 처리한 후 한 시간이 지난 다음 PEP-1-카탈레이즈 (3 μM)를 국소 도포하였다. 3일 동안 위의 처리를 반복하였다. 4일째에 오른 쪽 귀를 직경 5㎜로 뚫어 각 그룹 귀의 두께와 중량을 측정하였다. 조직학적 분석을 위해 귀 생검은 4% 파라포름알데하이드로 고정하고, 파라핀에 끼워 넣고, 5 ㎛ 두께로 절편을 만들어 헤마토자일린과 에오신으로 염색하였다. 웨스턴 블랏 분석을 위해 귀 생검은 단백질 분해효소 저해제 칵테일 (Sigma-Aldrich)이 함유된 조직 단백질 추출용 완충액에 넣고 균질화하여 0.1% 트리톤 X-100 존재 하에 15 분간 얼음에 방치하였다. 각 균질액은 10,000 x g로 10 분간 원심분리하였다.
To induce skin inflammation, the right ear of each mouse (n = 5) was treated with TPA (1.0 μM) dissolved in 20 μl of acetone. PEP-1-catalase (3 μM) was applied topically one hour after PM (100 μg / ear) was treated on the same skin surface. The above treatment was repeated for 3 days. On day 4, the right ear was drilled with a diameter of 5 mm to measure the thickness and weight of each group ear. For histological analysis, ear biopsies were fixed with 4% paraformaldehyde, embedded in paraffin, slices 5 μm thick, and stained with hematozain and eosin. For Western blot analysis, ear biopsies were placed in tissue protein extraction buffer containing protease inhibitor cocktail (Sigma-Aldrich), homogenized and left on ice for 15 minutes in the presence of 0.1% Triton X-100. Each homogenate was centrifuged at 10,000 x g for 10 minutes.

결과 1: 생체 외(Result 1: In vitro ( in vitroin vitro ) 및 생체 내() And in vivo ( in vivoin vivo ) 조건에서 PEP-1-카탈레이즈 세포침투에 미치는 퍼골라이드 메실레이트의 영향Effect of Pergolide Mesylate on PEP-1-catalase Infiltration

Tat과 PEP-1 같은 단백질 수송 도메인 (Protein transduction domains; PTDs)은 외생 고분자를 세포 또는 동물 체내로 운반할 수 있다 [G.P. Dietz, Curr. Pharm. Biotechol. 11 (2010) 167-174, J.S. Wadia et al., Curr. Opin. Biotechnol. 13 (2002) 52-56, J.S. Wadia et al., Curr. Protein Pept. Sci. 4 (2003) 97-104, M.C. Morris et al., Nat. Biotechonol. 19 (2001) 1173-1176]. 최근에는 단백질 수송 도메인과 융합된 분자를 다양한 질병의 치료제로 이용할 수 있다는 연구 결과들이 발표되고 있다. Protein transduction domains (PTDs) such as Tat and PEP-1 can carry exogenous polymers into cells or animals [GP Dietz, Curr. Pharm. Biotechol . 11 (2010) 167-174, JS Wadia et al., Curr. Opin. Biotechnol. 13 (2002) 52-56, JS Wadia et al., Curr. Protein Pept. Sci. 4 (2003) 97-104, MC Morris et al., Nat. Biotechonol. 19 (2001) 1173-1176. Recently, studies have shown that molecules fused with protein transport domains can be used as therapeutic agents for various diseases.

슈퍼옥사이드 디스뮤테이즈, 카탈레이즈 및 글루타치온 퍼옥시데이즈와 같은 항산화 효소들은 세포 내 산화 환원 균형을 유지시키며, 세포 방어메카니즘으로 작용하는 것으로 알려져 있다. 앞선 연구에서 본 발명자들은 PEP-1-rpS3, Tat-SOD 및 PEP-1-카탈레이즈가 세포 및 동물 체내로 효과적으로 도입되며, 이러한 세포 도입이 항산화 활성을 강화시킴을 밝힌바 있다 [D.W. Kim et al., Free Radic. Biol. Med. 47 (2009) 941-952, E.H. Ahn et al., Toxicol. 276 (2010) 192-197, S.H. Choi et al., FEBS Lett. 580 (2006) 6755-6762, I.K. Hwang et al., J. Neurosci. Res. 86 (2008) 1823-1835]. Antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidases are known to maintain intracellular redox balance and act as cell defense mechanisms. In previous studies, we have found that PEP-1-rpS3, Tat-SOD and PEP-1-catalase are effectively introduced into cells and animals, and that cell introduction enhances antioxidant activity [DW Kim et al. ., Free Radic. Biol. Med. 47 (2009) 941-952, EH Ahn et al., Toxicol. 276 (2010) 192-197, SH Choi et al., FEBS Lett . 580 (2006) 6755-6762, IK Hwang et al., J. Neurosci. Res. 86 (2008) 1823-1835.

퍼골라이드 메실레이트(PM)는 도파민 작동제(dopaminergic agonist)이고, 파킨슨병 치료제로서보다는 증세개선제로서 알려져 있다. 최근의 연구는 퍼골라이드 메실레이트가 체외(in vitro) 및 체내(in vivo) 동물 모델에서 신경보호기능을 나타낸다고 밝히고 있다 [U. Bonuccelli et al., Parkinsonism Relat. Disord.15s (2009) s44-s53, D. Uberti et al., Eur. J. Pharmacol. 434 (2002) 17-20, D. Uberti et al., Biochem. Pharmacol. 67 (2004) 1743-1750, D. Uberti et al., Neurochem. Res. 32 (2007) 1726-1729, Y. Ihara et al., J. Neurol. Sci. 170 (1999) 90-95]. 퍼골라이드 메실레이트의 구조는 도 1의 A와 같다. Pergolide mesylate (PM) is a dopaminergic agonist and is known as a symptom enhancer rather than as a treatment for Parkinson's disease. Recent studies have shown that pergolide mesylate exhibits neuroprotective functions in in vitro and in vivo animal models [U. Bonuccelli et al., Parkinsonism Relat. Disord. 15s (2009) s44-s53, D. Uberti et al., Eur. J. Pharmacol . 434 (2002) 17-20, D. Uberti et al., Biochem. Pharmacol. 67 (2004) 1743-1750, D. Uberti et al., Neurochem. Res . 32 (2007) 1726-1729, Y. Ihara et al., J. Neurol. Sci. 170 (1999) 90-95]. The structure of the pergolide mesylate is the same as A in FIG.

본 발명자들은 퍼골라이드 메실레이트가 HaCaT 각질세포와 마우스 피부로 들어가는 단백질 수송 도메인을 결합한 융합 단백질의 세포도입 효과를 증가시키는지를 웨스턴 블랏과 조직학적 분석으로 시험하였다. 도 1B와 같이, PEP-1-카탈레이즈는 농도 및 시간 의존적으로 세포에 도입되었다. 퍼골라이드 메실레이트 존재 하에 PEP-1-카탈레이즈의 세포 도입은 PM을 처리하지 않은 무처리 대조군과 비교할 때 현저히 증가하였다. 또한, PM이 다른 종류의 PEP-1-융합 단백질의 세포 도입률을 증가시키는지를 확인하기 위하여 한 시간 동안 PM으로 처리한 세포에 PEP-1-녹색형광단백질(green fluorescent protein) (PEP-1-GFP)을 처리하였다. PEP-1-GFP는 PEP-1-카탈레이즈와 유사한 결과를 나타내었다 (데이타 나타내지 않음). 따라서, 퍼골라이드 메실레이트는 인비트로에서 PEP-1-융합 단백질의 세포 도입을 촉진시키는 것으로 사료된다.We tested by Western blot and histological analysis whether pergolide mesylate increases the transduction effect of a fusion protein that binds HaCaT keratinocytes and protein transport domains entering the mouse skin. As shown in FIG. 1B, PEP-1-catalase was introduced into cells in a concentration and time dependent manner. Cell introduction of PEP-1-catalase in the presence of pergolide mesylate was significantly increased as compared to the untreated control without PM. In addition, PEP-1-green fluorescent protein (PEP-1-GFP) was applied to cells treated with PM for one hour to confirm whether PM increased the cellular introduction rate of other types of PEP-1-fusion proteins. ) Was treated. PEP-1-GFP gave similar results to PEP-1-catalase (data not shown). Thus, pergolide mesylate is thought to promote cellular introduction of PEP-1-fusion proteins in vitro.

PEP-1-카탈레이즈의 생체 내 (in vivo) 마우스 피부로의 세포도입에 대한 PM의 영향을 평가하기 위하여, PM (100 ㎍/ear)을 마우스 피부에 한 시간 동안 국소 도포한 다음 PEP-1-카탈레이즈 (3.0 μM)에 노출시킨 다음 마우스 피부로 PEP-1-카탈레이즈의 도입 정도를 면역조직화학적 분석으로 비교하였다. 도 1C와 같이, PEP-1-카탈레이즈는 PM이 없을 때보다 PM이 있을 때 마우스 피부 진피 내로 더 깊이 침투하였다. 이와 같은 결과는 PM이 인비트로뿐만 아니라 인비보에서도 PEP-1-카탈레이즈의 도입을 촉진시킨다는 것을 말해준다.
To assess the effect of PM on the introduction of PEP-1-catalase into cell skin in vivo , PM (100 μg / ear) was applied topically to mouse skin for one hour followed by PEP-1 Exposure to catalase (3.0 μM) followed by introduction of PEP-1-catalase into mouse skin was compared by immunohistochemical analysis. As shown in FIG. 1C, PEP-1-catalase penetrated deeper into mouse dermal dermis with PM than without PM. These results indicate that PM promotes the introduction of PEP-1-catalase not only in vitro but also in vivo.

결과 2: PM의 세포 독성 평가Outcome 2: Assessing Cytotoxicity of PM

본 발명자들은 HaCaT 세포의 생존율에 미치는 PM의 영향을 평가하였다. TPA (4.0 ㎍/ml)는 무처리 세포와 비교하여 50%의 세포 독성을 나타내었다. 1.0 ㎍/ml 농도의 PM은 세포독성을 나타내지 않았으므로 세포 생존율의 변화는 관찰되지 않았다 (도 2). PEP-1-카탈레이즈 (0.5-3 μM)를 처리하면 대조군에 비하여 세포 생존율이 농도 의존적으로 증가하였다. 뿐만 아니라, PEP-1-카탈레이즈 (0.5-3 μM)와 PM (1.0 ㎍/ml)을 함께 처리한 경우 PEP-1-카탈레이즈 단독 처리한 경우보다 현저히 세포 생존율이 증가하였다. 이와 같은 결과는 PM이 세포 생존율에 직접적인 효과는 없으나, TPA에 의하여 유도되는 세포 독성에 대한 PEP-1-카탈레이즈의 보호 효과를 증대시킴을 나타낸다.
We evaluated the effect of PM on the viability of HaCaT cells. TPA (4.0 μg / ml) showed 50% cytotoxicity compared to untreated cells. PM at 1.0 μg / ml did not show cytotoxicity, so no change in cell viability was observed (FIG. 2). Treatment with PEP-1-catalase (0.5-3 μM) resulted in a concentration dependent increase in cell viability compared to the control. In addition, treatment with PEP-1-catalase (0.5-3 μM) and PM (1.0 μg / ml) significantly increased cell viability compared to treatment with PEP-1-catalase alone. These results indicate that PM has no direct effect on cell viability but enhances the protective effect of PEP-1-catalase on cytotoxicity induced by TPA.

결과 3: PM 존재시 PAP-1-카탈레이즈의 항염증 활성 증가Outcome 3: Increased anti-inflammatory activity of PAP-1-catalase in the presence of PM

TPA는 활성산소종 생성을 유도하고, 활성산소종은 지방, DNA, RNA 및 단백질과 같은 많은 세포내 고분자의 산화를 일으킴으로써 결과적으로 종양 유발뿐만 아니라 염증성 사이토카인 (TNF-α, IL-1β 및 IL-6)과 효소 (COX-2, iNOS) 발현을 포함하여 염증 반응을 일으킨다 [H.Y. Song et al., Biochem. Pharmacol. 75 (2008) 1348-1357, J.Z. Qin et al., J. Biol. Chem. 274 (1999) 37957-37964, H. Zhou et al., J. Biol. Chem . 275 (2000) 22868-22875]. 마우스 부종 모델에서 TPA 적용 후 귀 중량 및 두께 증가는 유도된 염증의 분명한 증거이다 [H.Y. Song et al., Biochem. Pharmacol. 75 (2008) 1348-1357, P.L. Stanley et al., Skin Pharmacol. 4 (1991) 262-271]. 본 발명자들은 TPA로 염증을 유발한 다음 PM 처리와 PEP-1-카탈레이즈의 항염증 활성 간의 상관관계를 연구하였다. PEP-1-카탈레이즈 국소 처리는 TPA에 의해 유도된 귀 두께와 중량 증가를 현저히 저해시켰다 (도 3A 및 3B). 특히, PM과 PEP-1-카탈레이즈를 함께 처리하면 PEP-1-카탈레이즈 단독 처리한 군과 비교할 때 좀더 현저한 귀 두께 및 중량 감소효과를 거두었다. 반면, PM 단독 처리는 TPA에 의해 유도된 귀 부종을 약간만 감소시키는 것으로 나타났다. 조직학적 결과는 귀 중량과 두께를 측정한 결과와 일치하였다 (도 3C). 이러한 결과는 PM이 PEP-1-카탈레이즈의 항염증 활성을 명백히 증대시킴을 의미한다.TPA induces the production of reactive oxygen species, which cause oxidation of many intracellular polymers such as fat, DNA, RNA and proteins, resulting in tumor-causing as well as inflammatory cytokines (TNF-α, IL-1β and Inflammatory response, including IL-6) and enzyme (COX-2, iNOS) expression [HY Song et al., Biochem. Pharmacol . 75 (2008) 1348-1357, JZ Qin et al., J. Biol. Chem . 274 (1999) 37957-37964, H. Zhou et al., J. Biol. Chem . 275 (2000) 22868-22875. Increasing ear weight and thickness after TPA in a mouse edema model is clear evidence of induced inflammation [HY Song et al., Biochem. Pharmacol . 75 (2008) 1348-1357, PL Stanley et al., Skin Pharmacol. 4 (1991) 262-271. We studied the correlation between PM treatment and anti-inflammatory activity of PEP-1-catalase after inducing inflammation with TPA. PEP-1-catalase topical treatment significantly inhibited ear thickness and weight gain induced by TPA (FIGS. 3A and 3B). In particular, treatment with PM and PEP-1-catalase resulted in more significant ear thickness and weight reduction effects compared to the group treated with PEP-1-catalase alone. PM alone, on the other hand, was shown to slightly reduce ear edema induced by TPA. Histological results were consistent with the results of ear weight and thickness measurements (FIG. 3C). These results indicate that PM clearly increases the anti-inflammatory activity of PEP-1-catalase.

항염 매개자 및 사이토카인의 발현을 감소시키는 PEP-1-카탈레이즈의 항염증 활성에 대한 PM의 효과를 시험하기 위하여 TPA 국소도포 및 PEP-1-카탈레이즈 국소도포 이후 웨스턴 블랏 분석으로 COX-2, iNOS, IL-6, IL-1β 및 TNF-α의 발현수준을 측정하였다. 도 4와 같이, TPA 도포는 분명히 항염증 매개자와 사이토카인의 발현을 유도하였다 (레인 2). 반면, PM 처리군은 항염증 매개자와 사이토카인의 발현이 저해되었고 (레인 3), PEP-1-카탈레이즈 처리군은 항염증 매개자와 사이토카인의 발현이 분명히 억제되었다 (레인 4). 뿐만 아니라, PEP-1-카탈레이즈와 PM 동시처리 군에서는 PEP-1-카탈레이즈 단독 처리군에 비하여 항염증 매개자와 사이토카인의 발현이 좀더 현저히 감소하였다 (레인 5). 이와 같은 결과는 PM으로 마우스 피부를 선처리한 경우 PEP-1-카탈레이즈의 항염증 효과를 증대시키는 것으로 보인다. To test the effect of PM on the anti-inflammatory activity of PEP-1-catalase that reduces the expression of anti-inflammatory mediators and cytokines, COB-2, TPA topical and PEP-1-catalase topical post-blot western blot analysis Expression levels of iNOS, IL-6, IL-1β and TNF-α were measured. As shown in FIG. 4, TPA application clearly induced the expression of anti-inflammatory mediators and cytokines (lane 2). In contrast, the PM-treated group inhibited the expression of anti-inflammatory mediators and cytokines (lane 3), whereas the PEP-1-catalase treated group clearly inhibited the expression of anti-inflammatory mediators and cytokines (lane 4). In addition, in the PEP-1-catalase and PM treatment group, anti-inflammatory mediator and cytokine expression was significantly reduced compared to the PEP-1-catalase treatment group (lane 5). These results appear to increase the anti-inflammatory effect of PEP-1-catalase when pretreated mouse skin with PM.

PM은 도파민 작동제이고, 원래의 약학적 활성은 도파민 결핍에 의한 신경퇴행 질환인 파킨슨병의 신경보호 또는 치료작용이 아니라 증세완화작용인 것으로 알려져 있다. 그러나, 몇몇 연구에서는 PM이 자유 라디칼 생산을 감소시키고 지방의 과산화를 방지하는 것을 포함하여 신경보호기능을 한다고 보고되어 있다 [U. Bonuccelli et al., Parkinsonism Relat. Disord.15s (2009) s44-s53, D. Uberti et al., Neurochem. Res. 32 (2007) 1726-1729]. SH-SY5Y 신경세포를 H2O2로 처리하면 글루타치온, NF-kB 핵 전이, p53 및 p53-타겟 유전자 및 세포사멸이 감소하는데, 이는 PM에 의해 저해된다. 또한, 이와 같은 퍼골라이드의 보호 효과는 (-)-설피라이드 (sulpiride), SCH23390, 페녹시벤자민 (phenoxybenzamine) 및 스피페론 (spiperone)과 같은 도파민 수용체 저해제 (dopamine receptor antagonists)에 의해 저해되지 않기 때문에, PM의 이러한 효과는 도파민 수용체 활성과는 독립적이다 [D. Uberti et al., Eur. J. Pharmacol. 434 (2002) 17-20, D. Uberti et al., Biochem. Pharmacol. 67 (2004) 1743-1750, M. Grilli et al., Cell Death Differ. 6 (1999) 22-27, M. Grilli et al., Biochem. Pharmacol. 57 (1999) 1-7]. PM is a dopamine agonist and its original pharmacological activity is known to be a symptomatic relief, not a neuroprotective or therapeutic action of Parkinson's disease, a neurodegenerative disease caused by dopamine deficiency. However, several studies have reported that PM functions neuroprotective functions, including reducing free radical production and preventing fat peroxidation [U. Bonuccelli et al., Parkinsonism Relat. Disord. 15 s (2009) s44-s53, D. Uberti et al., Neurochem. Res . 32 (2007) 1726-1729. Treatment of SH-SY5Y neurons with H 2 O 2 reduces glutathione, NF-kB nuclear metastasis, p53 and p53-target genes and apoptosis, which are inhibited by PM. In addition, the protective effect of such pergolides is not inhibited by dopamine receptor antagonists such as (-)-sulpiride, SCH23390, phenoxybenzamine and spyrone This effect of PM is independent of dopamine receptor activity [D. Uberti et al., Eur. J. Pharmacol . 434 (2002) 17-20, D. Uberti et al., Biochem. Pharmacol. 67 (2004) 1743-1750, M. Grilli et al., Cell Death Differ . 6 (1999) 22-27, M. Grilli et al., Biochem. Pharmacol . 57 (1999) 1-7.

웨스턴 블랏과 조직학적 분석에 기초하여 우리는 SH-SY5Y 신경세포에서 PM의 신경보호활성과 반대로 PM 단독으로는 TPA에 의해 유도된 HaCaT 인간 각질세포 염증에 대한 염증성 매개자와 사이토카인의 발현을 직접적으로 억제하지는 못함을 밝혔다.Based on Western blot and histological analysis, we directly direct the expression of inflammatory mediators and cytokines for TPA-induced HaCaT human keratinocyte inflammation, as opposed to PM's neuroprotective activity in SH-SY5Y neurons. It was not suppressed.

그렇지만, PM은 PEP-1-카탈레이즈의 각질세포 및 마우스 피부로의 도입을 증가시키고, 결과적으로 TPA-유도 염증에 대한 PEP-1-카탈레이즈의 항염증 효과를 증대시켰다.However, PM increased the introduction of PEP-1-catalase into keratinocytes and mouse skin and consequently increased the anti-inflammatory effect of PEP-1-catalase on TPA-induced inflammation.

결론적으로, PM이 PEP-1-융합 단백질의 세포 도입을 증대시키는 정확한 메카니즘은 분명하지 않지만, PM이 파킨슨병 치료제로서뿐만 아니라 단백질 수송 도메인과 융합한 융합 단백질의 목표 세포 또는 목표 조직 내 도입을 증대시키는 목적으로 유용하다.
In conclusion, although the exact mechanism by which PM enhances the cellular introduction of PEP-1-fusion proteins is not clear, PM may enhance the introduction of fusion proteins into target cells or target tissues fused with protein transport domains as well as Parkinson's disease treatment. Useful for the purpose.

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

카탈레이즈 융합 단백질 20 ㎎Catalase Fusion Protein 20 mg

퍼골라이드 메실레이트 20 ㎎Pergolide Mesylate 20 mg

옥수수전분 100 ㎎Corn starch 100 mg

유당 100 ㎎Lactose 100 mg

스테아린산 마그네슘 2 mgMagnesium stearate 2 mg

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

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

카탈레이즈 융합 단백질 20 ㎎Catalase Fusion Protein 20 mg

퍼골라이드 메실레이트 20 ㎎Pergolide Mesylate 20 mg

옥수수전분 100 ㎎Corn starch 100 mg

유당 100 ㎎Lactose 100 mg

스테아린산 마그네슘 2 ㎎2 mg magnesium stearate

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

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

카탈레이즈 융합 단백질 0.2 ㎎Catalase Fusion Protein 0.2 mg

퍼골라이드 메실레이트 0.25 ㎎Pergolide Mesylate 0.25 mg

주사용 멸균 증류수 적량Sterile sterilized water for injection

pH 조절제 적량pH adjuster

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

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

카탈레이즈 융합 단백질 10 ㎎Catalase Fusion Protein 10 mg

퍼골라이드 메실레이트 12 ㎎Pergolide Mesylate 12 mg

이성화당 10 g10 g per isomer

만니톨 5 g5 g mannitol

정제수 적량Purified water quantity

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

<제형예 5: 스프레이제 제조><Formulation Example 5 Preparation of Spray Agent>

카탈레이즈 융합 단백질 10 w/v%(1 mg/ml)Catalase Fusion Protein 10 w / v% (1 mg / ml)

퍼골라이드 메실레이트 10 w/v%(1 ㎍/ml)Pergolide mesylate 10 w / v% (1 μg / ml)

점증제 0.05 w/v%Thickener 0.05 w / v%

AT ECO-cide(천연방부제) 0.7 w/v%AT ECO-cide 0.7 w / v%

정제수 적량Purified water quantity

통상의 스프레이제 제조방법에 따라 위 성분을 혼합하여 스프레이제를 제조하였다.
A spraying agent was prepared by mixing the above components according to a conventional spraying method.

<제형예 6: 로션타입 피부외용제 제조><Formulation Example 6: Manufacture of Lotion Type Skin Exterior Agent>

수퍼옥사이드 디스뮤테이즈 융합 단백질 10 w/v%(1 mg/ml)Superoxide Dismutase Fusion Protein 10 w / v% (1 mg / ml)

퍼골라이드 메실레이트 10 w/v%(1 ㎍/ml)Pergolide mesylate 10 w / v% (1 μg / ml)

Olivem 600 0.6 w/v%Olivem 600 0.6 w / v%

세틸 알코올 (Cetyl alcohol) 0.07 w/v%Cetyl alcohol 0.07 w / v%

AT ECO-cide (천연방부제) 1.2 w/v%AT ECO-cide 1.2 w / v%

정제수 적량Purified water quantity

통상의 로션타입 피부외용제 제조방법에 따라 위 성분을 혼합하여 제조하였다.
And was prepared by mixing the above ingredients according to a conventional lotion type external preparation for skin preparation.

<제형예 7: 크림타입 피부외용제 제조><Formulation Example 7: Manufacture of external cream type skin preparation>

수퍼옥사이드 디스뮤테이즈 융합 단백질 10 w/v% (1 mg/ml)Superoxide Dismutase Fusion Protein 10 w / v% (1 mg / ml)

퍼골라이드 메실레이트 10 w/v% (1 ㎍/ml)Pergolide mesylate 10 w / v% (1 μg / ml)

Olivem 600 0.6 w/v%Olivem 600 0.6 w / v%

세틸 알코올 0.06 w/v%Cetyl alcohol0.06 w / v%

AT ECO-cide (천연방부제) 1.2 w/v%AT ECO-cide 1.2 w / v%

정제수 적량Purified water quantity

통상의 크림타입 피부외용제 제조방법에 따라 위 성분을 혼합하여 제조하였다.
The above ingredients were mixed according to a conventional cream type skin external agent preparation method.

<제형예 8: 연고타입 피부외용제 제조><Formulation example 8: Manufacture of ointment type skin external preparation>

카탈레이즈 융합 단백질 10 w/v% (1 mg/ml)Catalase Fusion Protein 10 w / v% (1 mg / ml)

퍼골라이드 메실레이트 10 w/v% (1 ㎍/ml)Pergolide mesylate 10 w / v% (1 μg / ml)

Motanov 68 : 0.7 w/v%Motanov 68: 0.7 w / v%

Motanov L : 0.7 w/v%Motanov L: 0.7 w / v%

Montanox 60 : 0.2 w/v%Montanox 60: 0.2 w / v%

세토스 KD : 0.5 w/v%Setos KD: 0.5 w / v%

AT ECO-cide (천연방부제) : 1.2 w/v%AT ECO-cide: 1.2 w / v%

정제수 적량Purified water quantity

통상의 연고타입 피부외용제 제조방법에 따라 위 성분을 혼합하여 제조하였다.It was prepared by mixing the above components according to the conventional ointment type skin external preparation method.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Enhancing method for transduction of fusion protein by adding pergolide <130> hallym-sychoi-pergolide <160> 30 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ctcgagatgg ctgacagccg ggatcccgcc 30 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggatcctcac agatttgccr rctcccttgc c 31 <210> 3 <211> 1671 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for Tat-catalase fusion protein <400> 3 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggctgac agccgggatc ccgccagcga ccagatgcag 120 cactggaagg agcagcgggc cgcgcagaaa gctgatgtcc tgaccactgg agctggtaac 180 ccagtaggag acaaacttaa tgttattaca gtagggcccc gtgggcccct tcttgttcag 240 gatgtggttt tcactgatga aatggctcat tttgaccgag agagaattcc tgagagagtt 300 gtgcatgcta aaggagcagg ggcctttggc tactttgagg tcacacatga cattaccaaa 360 tactccaagg caaaggtatt tgagcatatt ggaaagaaga ctcccatcgc agttcggttc 420 tccactgttg ctggagaatc gggttcagct gacacagttc gggaccctcg tgggtttgca 480 gtgaaatttt acacagaaga tggtaactgg gatctcgttg gaaataacac ccccattttc 540 ttcatcaggg atcccatatt gtttccatct tttatccaca gccaaaagag aaatcctcag 600 acacatctga aggatccgga catggtctgg gacttctgga gcctacgtcc tgagtctctg 660 catcaggttt ctttcttgtt cagtgatcgg gggattccag atggacatcg ccacatgaat 720 ggatatggat cacatacttt caagctggtt aatgcaaatg gggaggcagt ttattgcaaa 780 ttccattata agactgacca gggcatcaaa aacctttctg ttgaagatgc ggcgagactt 840 tcccaggaag atcctgacta tggcatccgg gatcttttta acgccattgc cacaggaaag 900 tacccctcct ggacttttta catccaggtc atgacattta atcaggcaga aacttttcca 960 tttaatccat tcgatctcac caaggtttgg cctcacaagg actaccctct catcccagtt 1020 ggtaaactgg tcttaaaccg gaatccagtt aattactttg ctgaggttga acagatagcc 1080 ttcgacccaa gcaacatgcc acctggcatt gaggccagtc ctgacaaaat gcttcagggc 1140 cgcctttttg cctatcctga cactcaccgc catcgcctgg gacccaatta tcttcatata 1200 cctgtgaact gtccctaccg tgctcgagtg gccaactacc agcgtgacgg cccgatgtgc 1260 atgcaggaca atcagggtgg tgctccaaat tactacccca acagctttgg tgctccggaa 1320 caacagcctt ctgccctgga gcacagcatc caatattctg gagaagtgcg gagattcaac 1380 actgccaatg atgataacgt tactcaggtg cgggcattct atgtgaacgt gctgaatgag 1440 gaacagagga aacgtctgtg tgagaacatt gccggccacc tgaaggatgc acaaattttc 1500 atccagaaga aagcggtcaa gaacttcact gaggtccacc ctgactacgg gagccacatc 1560 caggctcttc tggacaagta caatgctgag aagcctaaga atgcgattca cacctttgtg 1620 cagtccggat ctcacttggc ggcaagggag aaggcaaatc tgtgaggatc c 1671 <210> 4 <211> 554 <212> PRT <213> Artificial Sequence <220> <223> Tat-catalase fusion protein <400> 4 His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His 1 5 10 15 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp Ser Arg 20 25 30 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala 35 40 45 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp 50 55 60 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln 65 70 75 80 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile 85 90 95 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe 100 105 110 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu 115 120 125 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala 130 135 140 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala 145 150 155 160 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn 165 170 175 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile 180 185 190 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met 195 200 205 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser 210 215 220 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn 225 230 235 240 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala 245 250 255 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu 260 265 270 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly 275 280 285 Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp 290 295 300 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro 305 310 315 320 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro 325 330 335 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr 340 345 350 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro 355 360 365 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala 370 375 380 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile 385 390 395 400 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp 405 410 415 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr 420 425 430 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His 435 440 445 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp 450 455 460 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu 465 470 475 480 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp 485 490 495 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val 500 505 510 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn 515 520 525 Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser 530 535 540 His Leu Ala Ala Arg Glu Lys Ala Asn Leu 545 550 <210> 5 <211> 1670 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for catalase-Tat fusion protein <400> 5 catcatcatc atcatcacag cagcggcctg gtgccggcgg cagccactcg agatggctga 60 cagccgggat cccgccagcg accagatgca gcactggaag gagcagcggg ccgcgcagaa 120 agctgatgtc ctgaccactg gagctggtaa cccagtagga gacaaactta atgttattac 180 agtagggccc cgtgggcccc ttcttgttca ggatgtggtt ttcactgatg aaatggctca 240 ttttgaccga gagagaattc ctgagagagt tgtgcatgct aaaggagcag gggcctttgg 300 ctactttgag gtcacacatg acattaccaa atactccaag gcaaaggtat ttgagcatat 360 tggaaagaag actcccatcg cagttcggtt ctccactgtt gctggagaat cgggttcagc 420 tgacacagtt cgggaccctc gtgggtttgc agtgaaattt tacacagaag atggtaactg 480 ggatctcgtt ggaaataaca cccccatttt cttcatcagg gatcccatat tgtttccatc 540 ttttatccac agccaaaaga gaaatcctca gacacatctg aaggatccgg acatggtctg 600 ggacttctgg agcctacgtc ctgagtctct gcatcaggtt tctttcttgt tcagtgatcg 660 ggggattcca gatggacatc gccacatgaa tggatatgga tcacatactt tcaagctggt 720 taatgcaaat ggggaggcag tttattgcaa attccattat aagactgacc agggcatcaa 780 aaacctttct gttgaagatg cggcgagact ttcccaggaa gatcctgact atggcatccg 840 ggatcttttt aacgccattg ccacaggaaa gtacccctcc tggacttttt acatccaggt 900 catgacattt aatcaggcag aaacttttcc atttaatcca ttcgatctca ccaaggtttg 960 gcctcacaag gactaccctc tcatcccagt tggtaaactg gtcttaaacc ggaatccagt 1020 taattacttt gctgaggttg aacagatagc cttcgaccca agcaacatgc cacctggcat 1080 tgaggccagt cctgacaaaa tgcttcaggg ccgccttttt gcctatcctg acactcaccg 1140 ccatcgcctg ggacccaatt atcttcatat acctgtgaac tgtccctacc gtgctcgagt 1200 ggccaactac cagcgtgacg gcccgatgtg catgcaggac aatcagggtg gtgctccaaa 1260 ttactacccc aacagctttg gtgctccgga acaacagcct tctgccctgg agcacagcat 1320 ccaatattct ggagaagtgc ggagattcaa cactgccaat gatgataacg ttactcaggt 1380 gcgggcattc tatgtgaacg tgctgaatga ggaacagagg aaacgtctgt gtgagaacat 1440 tgccggccac ctgaaggatg cacaaatttt catccagaag aaagcggtca agaacttcac 1500 tgaggtccac cctgactacg ggagccacat ccaggctctt ctggacaagt acaatgctga 1560 gaagcctaag aatgcgattc acacctttgt gcagtccgga tctcacttgg cggcaaggga 1620 gaaggcaaat ctgggatcct aggaagaagc ggagacagcg acgaagatag 1670 <210> 6 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> catalase-Tat fusion protein <400> 6 Met Ala Asp Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys 1 5 10 15 Glu Gln Arg Ala Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly 20 25 30 Asn Pro Val Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly 35 40 45 Pro Leu Leu Val Gln Asp Val Val Phe Thr Asp Glu Met Ala His Phe 50 55 60 Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly 65 70 75 80 Ala Phe Gly Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys 85 90 95 Ala Lys Val Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg 100 105 110 Phe Ser Thr Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp 115 120 125 Pro Arg Gly Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp 130 135 140 Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu 145 150 155 160 Phe Pro Ser Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu 165 170 175 Lys Asp Pro Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser 180 185 190 Leu His Gln Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly 195 200 205 His Arg His Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn 210 215 220 Ala Asn Gly Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln 225 230 235 240 Gly Ile Lys Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu 245 250 255 Asp Pro Asp Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly 260 265 270 Lys Tyr Pro Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln 275 280 285 Ala Glu Thr Phe Pro Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro 290 295 300 His Lys Asp Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg 305 310 315 320 Asn Pro Val Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro 325 330 335 Ser Asn Met Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln 340 345 350 Gly Arg Leu Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro 355 360 365 Asn Tyr Leu His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala 370 375 380 Asn Tyr Gln Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly 385 390 395 400 Ala Pro Asn Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro 405 410 415 Ser Ala Leu Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe 420 425 430 Asn Thr Ala Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val 435 440 445 Asn Val Leu Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala 450 455 460 Gly His Leu Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys 465 470 475 480 Asn Phe Thr Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu 485 490 495 Leu Asp Lys Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe 500 505 510 Val Gln Ser Gly Ser His Leu Ala Ala Arg Glu Lys Ala Asn Leu Arg 515 520 525 Lys Lys Arg Arg Gln Arg Arg Arg 530 535 <210> 7 <211> 1699 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for Tat-catalase-Tat fusion protein <400> 7 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggctgac agccgggatc ccgccagcga ccagatgcag 120 cactggaagg agcagcgggc cgcgcagaaa gctgatgtcc tgaccactgg agctggtaac 180 ccagtaggag acaaacttaa tgttattaca gtagggcccc gtgggcccct tcttgttcag 240 gatgtggttt tcactgatga aatggctcat tttgaccgag agagaattcc tgagagagtt 300 gtgcatgcta aaggagcagg ggcctttggc tactttgagg tcacacatga cattaccaaa 360 tactccaagg caaaggtatt tgagcatatt ggaaagaaga ctcccatcgc agttcggttc 420 tccactgttg ctggagaatc gggttcagct gacacagttc gggaccctcg tgggtttgca 480 gtgaaatttt acacagaaga tggtaactgg gatctcgttg gaaataacac ccccattttc 540 ttcatcaggg atcccatatt gtttccatct tttatccaca gccaaaagag aaatcctcag 600 acacatctga aggatccgga catggtctgg gacttctgga gcctacgtcc tgagtctctg 660 catcaggttt ctttcttgtt cagtgatcgg gggattccag atggacatcg ccacatgaat 720 ggatatggat cacatacttt caagctggtt aatgcaaatg gggaggcagt ttattgcaaa 780 ttccattata agactgacca gggcatcaaa aacctttctg ttgaagatgc ggcgagactt 840 tcccaggaag atcctgacta tggcatccgg gatcttttta acgccattgc cacaggaaag 900 tacccctcct ggacttttta catccaggtc atgacattta atcaggcaga aacttttcca 960 tttaatccat tcgatctcac caaggtttgg cctcacaagg actaccctct catcccagtt 1020 ggtaaactgg tcttaaaccg gaatccagtt aattactttg ctgaggttga acagatagcc 1080 ttcgacccaa gcaacatgcc acctggcatt gaggccagtc ctgacaaaat gcttcagggc 1140 cgcctttttg cctatcctga cactcaccgc catcgcctgg gacccaatta tcttcatata 1200 cctgtgaact gtccctaccg tgctcgagtg gccaactacc agcgtgacgg cccgatgtgc 1260 atgcaggaca atcagggtgg tgctccaaat tactacccca acagctttgg tgctccggaa 1320 caacagcctt ctgccctgga gcacagcatc caatattctg gagaagtgcg gagattcaac 1380 actgccaatg atgataacgt tactcaggtg cgggcattct atgtgaacgt gctgaatgag 1440 gaacagagga aacgtctgtg tgagaacatt gccggccacc tgaaggatgc acaaattttc 1500 atccagaaga aagcggtcaa gaacttcact gaggtccacc ctgactacgg gagccacatc 1560 caggctcttc tggacaagta caatgctgag aagcctaaga atgcgattca cacctttgtg 1620 cagtccggat ctcacttggc ggcaagggag aaggcaaatc tgggatccta ggaagaagcg 1680 gagacagcga cgaagatag 1699 <210> 8 <211> 549 <212> PRT <213> Artificial Sequence <220> <223> Tat-catalase-Tat fusion protein <400> 8 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp Ser Arg 1 5 10 15 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala 20 25 30 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp 35 40 45 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln 50 55 60 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile 65 70 75 80 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe 85 90 95 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu 100 105 110 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala 115 120 125 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala 130 135 140 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn 145 150 155 160 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile 165 170 175 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met 180 185 190 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser 195 200 205 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn 210 215 220 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala 225 230 235 240 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu 245 250 255 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly 260 265 270 Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp 275 280 285 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro 290 295 300 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro 305 310 315 320 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr 325 330 335 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro 340 345 350 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala 355 360 365 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile 370 375 380 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp 385 390 395 400 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr 405 410 415 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His 420 425 430 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp 435 440 445 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu 450 455 460 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp 465 470 475 480 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val 485 490 495 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn 500 505 510 Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser 515 520 525 His Leu Ala Ala Arg Glu Lys Ala Asn Leu Gly Ser Arg Lys Lys Arg 530 535 540 Arg Gln Arg Arg Arg 545 <210> 9 <211> 1708 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1-catalase fusion protein <400> 9 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccataa aagaaacctg 60 gtgggaaacc tggtggaccg aatggtctca gccgaaaaaa aaacgtaaag tgctcgagat 120 ggctgacagc cgggatcccg ccagcgacca gatgcagcac tggaaggagc agcgggccgc 180 gcagaaagct gatgtcctga ccactggagc tggtaaccca gtaggagaca aacttaatgt 240 tattacagta gggccccgtg ggccccttct tgttcaggat gtggttttca ctgatgaaat 300 ggctcatttt gaccgagaga gaattcctga gagagttgtg catgctaaag gagcaggggc 360 ctttggctac tttgaggtca cacatgacat taccaaatac tccaaggcaa aggtatttga 420 gcatattgga aagaagactc ccatcgcagt tcggttctcc actgttgctg gagaatcggg 480 ttcagctgac acagttcggg accctcgtgg gtttgcagtg aaattttaca cagaagatgg 540 taactgggat ctcgttggaa ataacacccc cattttcttc atcagggatc ccatattgtt 600 tccatctttt atccacagcc aaaagagaaa tcctcagaca catctgaagg atccggacat 660 ggtctgggac ttctggagcc tacgtcctga gtctctgcat caggtttctt tcttgttcag 720 tgatcggggg attccagatg gacatcgcca catgaatgga tatggatcac atactttcaa 780 gctggttaat gcaaatgggg aggcagttta ttgcaaattc cattataaga ctgaccaggg 840 catcaaaaac ctttctgttg aagatgcggc gagactttcc caggaagatc ctgactatgg 900 catccgggat ctttttaacg ccattgccac aggaaagtac ccctcctgga ctttttacat 960 ccaggtcatg acatttaatc aggcagaaac ttttccattt aatccattcg atctcaccaa 1020 ggtttggcct cacaaggact accctctcat cccagttggt aaactggtct taaaccggaa 1080 tccagttaat tactttgctg aggttgaaca gatagccttc gacccaagca acatgccacc 1140 tggcattgag gccagtcctg acaaaatgct tcagggccgc ctttttgcct atcctgacac 1200 tcaccgccat cgcctgggac ccaattatct tcatatacct gtgaactgtc cctaccgtgc 1260 tcgagtggcc aactaccagc gtgacggccc gatgtgcatg caggacaatc agggtggtgc 1320 tccaaattac taccccaaca gctttggtgc tccggaacaa cagccttctg ccctggagca 1380 cagcatccaa tattctggag aagtgcggag attcaacact gccaatgatg ataacgttac 1440 tcaggtgcgg gcattctatg tgaacgtgct gaatgaggaa cagaggaaac gtctgtgtga 1500 gaacattgcc ggccacctga aggatgcaca aattttcatc cagaagaaag cggtcaagaa 1560 cttcactgag gtccaccctg actacgggag ccacatccag gctcttctgg acaagtacaa 1620 tgctgagaag cctaagaatg cgattcacac ctttgtgcag tccggatctc acttggcggc 1680 aagggagaag gcaaatctgt gaggatcc 1708 <210> 10 <211> 550 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-catalase fusion protein <400> 10 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Asp Ser Arg Asp Pro Ala Ser 20 25 30 Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala Gln Lys Ala Asp 35 40 45 Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp Lys Leu Asn Val 50 55 60 Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln Asp Val Val Phe 65 70 75 80 Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile Pro Glu Arg Val 85 90 95 Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe Glu Val Thr His 100 105 110 Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu His Ile Gly Lys 115 120 125 Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala Gly Glu Ser Gly 130 135 140 Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala Val Lys Phe Tyr 145 150 155 160 Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn Thr Pro Ile Phe 165 170 175 Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile His Ser Gln Lys 180 185 190 Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met Val Trp Asp Phe 195 200 205 Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser Phe Leu Phe Ser 210 215 220 Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn Gly Tyr Gly Ser 225 230 235 240 His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala Val Tyr Cys Lys 245 250 255 Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu Ser Val Glu Asp 260 265 270 Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly Ile Arg Asp Leu 275 280 285 Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp Thr Phe Tyr Ile 290 295 300 Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro Phe Asn Pro Phe 305 310 315 320 Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro Leu Ile Pro Val 325 330 335 Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr Phe Ala Glu Val 340 345 350 Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro Gly Ile Glu Ala 355 360 365 Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala Tyr Pro Asp Thr 370 375 380 His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile Pro Val Asn Cys 385 390 395 400 Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp Gly Pro Met Cys 405 410 415 Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr Pro Asn Ser Phe 420 425 430 Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His Ser Ile Gln Tyr 435 440 445 Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp Asp Asn Val Thr 450 455 460 Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu Glu Gln Arg Lys 465 470 475 480 Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp Ala Gln Ile Phe 485 490 495 Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val His Pro Asp Tyr 500 505 510 Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn Ala Glu Lys Pro 515 520 525 Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser His Leu Ala Ala 530 535 540 Arg Glu Lys Ala Asn Leu 545 550 <210> 11 <211> 1707 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for catalase-PEP-1 fusion protein <400> 11 catcatcatc atcatcacag cagcggcctg gtgccggcgg cagccactcg agatggctga 60 cagccgggat cccgccagcg accagatgca gcactggaag gagcagcggg ccgcgcagaa 120 agctgatgtc ctgaccactg gagctggtaa cccagtagga gacaaactta atgttattac 180 agtagggccc cgtgggcccc ttcttgttca ggatgtggtt ttcactgatg aaatggctca 240 ttttgaccga gagagaattc ctgagagagt tgtgcatgct aaaggagcag gggcctttgg 300 ctactttgag gtcacacatg acattaccaa atactccaag gcaaaggtat ttgagcatat 360 tggaaagaag actcccatcg cagttcggtt ctccactgtt gctggagaat cgggttcagc 420 tgacacagtt cgggaccctc gtgggtttgc agtgaaattt tacacagaag atggtaactg 480 ggatctcgtt ggaaataaca cccccatttt cttcatcagg gatcccatat tgtttccatc 540 ttttatccac agccaaaaga gaaatcctca gacacatctg aaggatccgg acatggtctg 600 ggacttctgg agcctacgtc ctgagtctct gcatcaggtt tctttcttgt tcagtgatcg 660 ggggattcca gatggacatc gccacatgaa tggatatgga tcacatactt tcaagctggt 720 taatgcaaat ggggaggcag tttattgcaa attccattat aagactgacc agggcatcaa 780 aaacctttct gttgaagatg cggcgagact ttcccaggaa gatcctgact atggcatccg 840 ggatcttttt aacgccattg ccacaggaaa gtacccctcc tggacttttt acatccaggt 900 catgacattt aatcaggcag aaacttttcc atttaatcca ttcgatctca ccaaggtttg 960 gcctcacaag gactaccctc tcatcccagt tggtaaactg gtcttaaacc ggaatccagt 1020 taattacttt gctgaggttg aacagatagc cttcgaccca agcaacatgc cacctggcat 1080 tgaggccagt cctgacaaaa tgcttcaggg ccgccttttt gcctatcctg acactcaccg 1140 ccatcgcctg ggacccaatt atcttcatat acctgtgaac tgtccctacc gtgctcgagt 1200 ggccaactac cagcgtgacg gcccgatgtg catgcaggac aatcagggtg gtgctccaaa 1260 ttactacccc aacagctttg gtgctccgga acaacagcct tctgccctgg agcacagcat 1320 ccaatattct ggagaagtgc ggagattcaa cactgccaat gatgataacg ttactcaggt 1380 gcgggcattc tatgtgaacg tgctgaatga ggaacagagg aaacgtctgt gtgagaacat 1440 tgccggccac ctgaaggatg cacaaatttt catccagaag aaagcggtca agaacttcac 1500 tgaggtccac cctgactacg ggagccacat ccaggctctt ctggacaagt acaatgctga 1560 gaagcctaag aatgcgattc acacctttgt gcagtccgga tctcacttgg cggcaaggga 1620 gaaggcaaat ctgggatcct aaaagaaacc tggtgggaaa cctggtggac cgaatggtct 1680 cagccgaaaa aaaaacgtaa agtgtag 1707 <210> 12 <211> 566 <212> PRT <213> Artificial Sequence <220> <223> catalase-PEP-1 fusion protein <400> 12 His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His 1 5 10 15 Leu Glu Met Ala Asp Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His 20 25 30 Trp Lys Glu Gln Arg Ala Ala Gln Lys Ala Asp Val Leu Thr Thr Gly 35 40 45 Ala Gly Asn Pro Val Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro 50 55 60 Arg Gly Pro Leu Leu Val Gln Asp Val Val Phe Thr Asp Glu Met Ala 65 70 75 80 His Phe Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Lys Gly 85 90 95 Ala Gly Ala Phe Gly Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr 100 105 110 Ser Lys Ala Lys Val Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala 115 120 125 Val Arg Phe Ser Thr Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val 130 135 140 Arg Asp Pro Arg Gly Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn 145 150 155 160 Trp Asp Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro 165 170 175 Ile Leu Phe Pro Ser Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr 180 185 190 His Leu Lys Asp Pro Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro 195 200 205 Glu Ser Leu His Gln Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro 210 215 220 Asp Gly His Arg His Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu 225 230 235 240 Val Asn Ala Asn Gly Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr 245 250 255 Asp Gln Gly Ile Lys Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser 260 265 270 Gln Glu Asp Pro Asp Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala 275 280 285 Thr Gly Lys Tyr Pro Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe 290 295 300 Asn Gln Ala Glu Thr Phe Pro Phe Asn Pro Phe Asp Leu Thr Lys Val 305 310 315 320 Trp Pro His Lys Asp Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu 325 330 335 Asn Arg Asn Pro Val Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe 340 345 350 Asp Pro Ser Asn Met Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met 355 360 365 Leu Gln Gly Arg Leu Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu 370 375 380 Gly Pro Asn Tyr Leu His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg 385 390 395 400 Val Ala Asn Tyr Gln Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln 405 410 415 Gly Gly Ala Pro Asn Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln 420 425 430 Gln Pro Ser Ala Leu Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg 435 440 445 Arg Phe Asn Thr Ala Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe 450 455 460 Tyr Val Asn Val Leu Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn 465 470 475 480 Ile Ala Gly His Leu Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala 485 490 495 Val Lys Asn Phe Thr Glu Val His Pro Asp Tyr Gly Ser His Ile Gln 500 505 510 Ala Leu Leu Asp Lys Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His 515 520 525 Thr Phe Val Gln Ser Gly Ser His Leu Ala Ala Arg Glu Lys Ala Asn 530 535 540 Leu Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro 545 550 555 560 Lys Lys Lys Arg Lys Val 565 <210> 13 <211> 1773 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1-catalase-PEP-1 fusion protein <400> 13 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccataa aagaaacctg 60 gtgggaaacc tggtggaccg aatggtctca gccgaaaaaa aaacgtaaag tgctcgagat 120 ggctgacagc cgggatcccg ccagcgacca gatgcagcac tggaaggagc agcgggccgc 180 gcagaaagct gatgtcctga ccactggagc tggtaaccca gtaggagaca aacttaatgt 240 tattacagta gggccccgtg ggccccttct tgttcaggat gtggttttca ctgatgaaat 300 ggctcatttt gaccgagaga gaattcctga gagagttgtg catgctaaag gagcaggggc 360 ctttggctac tttgaggtca cacatgacat taccaaatac tccaaggcaa aggtatttga 420 gcatattgga aagaagactc ccatcgcagt tcggttctcc actgttgctg gagaatcggg 480 ttcagctgac acagttcggg accctcgtgg gtttgcagtg aaattttaca cagaagatgg 540 taactgggat ctcgttggaa ataacacccc cattttcttc atcagggatc ccatattgtt 600 tccatctttt atccacagcc aaaagagaaa tcctcagaca catctgaagg atccggacat 660 ggtctgggac ttctggagcc tacgtcctga gtctctgcat caggtttctt tcttgttcag 720 tgatcggggg attccagatg gacatcgcca catgaatgga tatggatcac atactttcaa 780 gctggttaat gcaaatgggg aggcagttta ttgcaaattc cattataaga ctgaccaggg 840 catcaaaaac ctttctgttg aagatgcggc gagactttcc caggaagatc ctgactatgg 900 catccgggat ctttttaacg ccattgccac aggaaagtac ccctcctgga ctttttacat 960 ccaggtcatg acatttaatc aggcagaaac ttttccattt aatccattcg atctcaccaa 1020 ggtttggcct cacaaggact accctctcat cccagttggt aaactggtct taaaccggaa 1080 tccagttaat tactttgctg aggttgaaca gatagccttc gacccaagca acatgccacc 1140 tggcattgag gccagtcctg acaaaatgct tcagggccgc ctttttgcct atcctgacac 1200 tcaccgccat cgcctgggac ccaattatct tcatatacct gtgaactgtc cctaccgtgc 1260 tcgagtggcc aactaccagc gtgacggccc gatgtgcatg caggacaatc agggtggtgc 1320 tccaaattac taccccaaca gctttggtgc tccggaacaa cagccttctg ccctggagca 1380 cagcatccaa tattctggag aagtgcggag attcaacact gccaatgatg ataacgttac 1440 tcaggtgcgg gcattctatg tgaacgtgct gaatgaggaa cagaggaaac gtctgtgtga 1500 gaacattgcc ggccacctga aggatgcaca aattttcatc cagaagaaag cggtcaagaa 1560 cttcactgag gtccaccctg actacgggag ccacatccag gctcttctgg acaagtacaa 1620 tgctgagaag cctaagaatg cgattcacac ctttgtgcag tccggatctc acttggcggc 1680 aagggagaag gcaaatctgg gatcctaaaa gaaacctggt gggaaacctg gtggaccgaa 1740 tggtctcagc cgaaaaaaaa acgtaaagtg tag 1773 <210> 14 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-catalase-PEP-1 fusion protein <400> 14 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Asp Ser Arg Asp Pro Ala Ser 20 25 30 Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala Gln Lys Ala Asp 35 40 45 Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp Lys Leu Asn Val 50 55 60 Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln Asp Val Val Phe 65 70 75 80 Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile Pro Glu Arg Val 85 90 95 Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe Glu Val Thr His 100 105 110 Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu His Ile Gly Lys 115 120 125 Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala Gly Glu Ser Gly 130 135 140 Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala Val Lys Phe Tyr 145 150 155 160 Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn Thr Pro Ile Phe 165 170 175 Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile His Ser Gln Lys 180 185 190 Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met Val Trp Asp Phe 195 200 205 Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser Phe Leu Phe Ser 210 215 220 Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn Gly Tyr Gly Ser 225 230 235 240 His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala Val Tyr Cys Lys 245 250 255 Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu Ser Val Glu Asp 260 265 270 Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly Ile Arg Asp Leu 275 280 285 Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp Thr Phe Tyr Ile 290 295 300 Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro Phe Asn Pro Phe 305 310 315 320 Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro Leu Ile Pro Val 325 330 335 Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr Phe Ala Glu Val 340 345 350 Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro Gly Ile Glu Ala 355 360 365 Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala Tyr Pro Asp Thr 370 375 380 His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile Pro Val Asn Cys 385 390 395 400 Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp Gly Pro Met Cys 405 410 415 Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr Pro Asn Ser Phe 420 425 430 Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His Ser Ile Gln Tyr 435 440 445 Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp Asp Asn Val Thr 450 455 460 Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu Glu Gln Arg Lys 465 470 475 480 Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp Ala Gln Ile Phe 485 490 495 Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val His Pro Asp Tyr 500 505 510 Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn Ala Glu Lys Pro 515 520 525 Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser His Leu Ala Ala 530 535 540 Arg Glu Lys Ala Asn Leu Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp 545 550 555 560 Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 565 570 <210> 15 <211> 1664 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for olygolysine-catalase fusion protein <400> 15 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaactcgag atggctgaca gccgggatcc cgccagcgac cagatgcagc 120 actggaagga gcagcgggcc gcgcagaaag ctgatgtcct gaccactgga gctggtaacc 180 cagtaggaga caaacttaat gttattacag tagggccccg tgggcccctt cttgttcagg 240 atgtggtttt cactgatgaa atggctcatt ttgaccgaga gagaattcct gagagagttg 300 tgcatgctaa aggagcaggg gcctttggct actttgaggt cacacatgac attaccaaat 360 actccaaggc aaaggtattt gagcatattg gaaagaagac tcccatcgca gttcggttct 420 ccactgttgc tggagaatcg ggttcagctg acacagttcg ggaccctcgt gggtttgcag 480 tgaaatttta cacagaagat ggtaactggg atctcgttgg aaataacacc cccattttct 540 tcatcaggga tcccatattg tttccatctt ttatccacag ccaaaagaga aatcctcaga 600 cacatctgaa ggatccggac atggtctggg acttctggag cctacgtcct gagtctctgc 660 atcaggtttc tttcttgttc agtgatcggg ggattccaga tggacatcgc cacatgaatg 720 gatatggatc acatactttc aagctggtta atgcaaatgg ggaggcagtt tattgcaaat 780 tccattataa gactgaccag ggcatcaaaa acctttctgt tgaagatgcg gcgagacttt 840 cccaggaaga tcctgactat ggcatccggg atctttttaa cgccattgcc acaggaaagt 900 acccctcctg gactttttac atccaggtca tgacatttaa tcaggcagaa acttttccat 960 ttaatccatt cgatctcacc aaggtttggc ctcacaagga ctaccctctc atcccagttg 1020 gtaaactggt cttaaaccgg aatccagtta attactttgc tgaggttgaa cagatagcct 1080 tcgacccaag caacatgcca cctggcattg aggccagtcc tgacaaaatg cttcagggcc 1140 gcctttttgc ctatcctgac actcaccgcc atcgcctggg acccaattat cttcatatac 1200 ctgtgaactg tccctaccgt gctcgagtgg ccaactacca gcgtgacggc ccgatgtgca 1260 tgcaggacaa tcagggtggt gctccaaatt actaccccaa cagctttggt gctccggaac 1320 aacagccttc tgccctggag cacagcatcc aatattctgg agaagtgcgg agattcaaca 1380 ctgccaatga tgataacgtt actcaggtgc gggcattcta tgtgaacgtg ctgaatgagg 1440 aacagaggaa acgtctgtgt gagaacattg ccggccacct gaaggatgca caaattttca 1500 tccagaagaa agcggtcaag aacttcactg aggtccaccc tgactacggg agccacatcc 1560 aggctcttct ggacaagtac aatgctgaga agcctaagaa tgcgattcac acctttgtgc 1620 agtccggatc tcacttggcg gcaagggaga aggcaaatct gtga 1664 <210> 16 <211> 538 <212> PRT <213> Artificial Sequence <220> <223> oligolysine-catalase fusion protein <400> 16 Lys Lys Lys Lys Lys Lys Lys Lys Lys Leu Glu Met Ala Asp Ser Arg 1 5 10 15 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala 20 25 30 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp 35 40 45 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln 50 55 60 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile 65 70 75 80 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe 85 90 95 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu 100 105 110 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala 115 120 125 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala 130 135 140 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn 145 150 155 160 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile 165 170 175 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met 180 185 190 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser 195 200 205 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn 210 215 220 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala 225 230 235 240 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu 245 250 255 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly 260 265 270 Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp 275 280 285 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro 290 295 300 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro 305 310 315 320 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr 325 330 335 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro 340 345 350 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala 355 360 365 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile 370 375 380 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp 385 390 395 400 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr 405 410 415 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His 420 425 430 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp 435 440 445 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu 450 455 460 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp 465 470 475 480 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val 485 490 495 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn 500 505 510 Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser 515 520 525 His Leu Ala Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 17 <211> 1664 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for oligoarginine-catalase fusion protein <400> 17 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaga agaagaagaa 60 gaagaagaag aagactcgag atggctgaca gccgggatcc cgccagcgac cagatgcagc 120 actggaagga gcagcgggcc gcgcagaaag ctgatgtcct gaccactgga gctggtaacc 180 cagtaggaga caaacttaat gttattacag tagggccccg tgggcccctt cttgttcagg 240 atgtggtttt cactgatgaa atggctcatt ttgaccgaga gagaattcct gagagagttg 300 tgcatgctaa aggagcaggg gcctttggct actttgaggt cacacatgac attaccaaat 360 actccaaggc aaaggtattt gagcatattg gaaagaagac tcccatcgca gttcggttct 420 ccactgttgc tggagaatcg ggttcagctg acacagttcg ggaccctcgt gggtttgcag 480 tgaaatttta cacagaagat ggtaactggg atctcgttgg aaataacacc cccattttct 540 tcatcaggga tcccatattg tttccatctt ttatccacag ccaaaagaga aatcctcaga 600 cacatctgaa ggatccggac atggtctggg acttctggag cctacgtcct gagtctctgc 660 atcaggtttc tttcttgttc agtgatcggg ggattccaga tggacatcgc cacatgaatg 720 gatatggatc acatactttc aagctggtta atgcaaatgg ggaggcagtt tattgcaaat 780 tccattataa gactgaccag ggcatcaaaa acctttctgt tgaagatgcg gcgagacttt 840 cccaggaaga tcctgactat ggcatccggg atctttttaa cgccattgcc acaggaaagt 900 acccctcctg gactttttac atccaggtca tgacatttaa tcaggcagaa acttttccat 960 ttaatccatt cgatctcacc aaggtttggc ctcacaagga ctaccctctc atcccagttg 1020 gtaaactggt cttaaaccgg aatccagtta attactttgc tgaggttgaa cagatagcct 1080 tcgacccaag caacatgcca cctggcattg aggccagtcc tgacaaaatg cttcagggcc 1140 gcctttttgc ctatcctgac actcaccgcc atcgcctggg acccaattat cttcatatac 1200 ctgtgaactg tccctaccgt gctcgagtgg ccaactacca gcgtgacggc ccgatgtgca 1260 tgcaggacaa tcagggtggt gctccaaatt actaccccaa cagctttggt gctccggaac 1320 aacagccttc tgccctggag cacagcatcc aatattctgg agaagtgcgg agattcaaca 1380 ctgccaatga tgataacgtt actcaggtgc gggcattcta tgtgaacgtg ctgaatgagg 1440 aacagaggaa acgtctgtgt gagaacattg ccggccacct gaaggatgca caaattttca 1500 tccagaagaa agcggtcaag aacttcactg aggtccaccc tgactacggg agccacatcc 1560 aggctcttct ggacaagtac aatgctgaga agcctaagaa tgcgattcac acctttgtgc 1620 agtccggatc tcacttggcg gcaagggaga aggcaaatct gtga 1664 <210> 18 <211> 538 <212> PRT <213> Artificial Sequence <220> <223> oligoarginine-catalase fusion protein <400> 18 Arg Arg Arg Arg Arg Arg Arg Arg Arg Leu Glu Met Ala Asp Ser Arg 1 5 10 15 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala 20 25 30 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp 35 40 45 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln 50 55 60 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile 65 70 75 80 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe 85 90 95 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu 100 105 110 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala 115 120 125 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala 130 135 140 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn 145 150 155 160 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile 165 170 175 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met 180 185 190 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser 195 200 205 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn 210 215 220 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala 225 230 235 240 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu 245 250 255 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly 260 265 270 Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp 275 280 285 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro 290 295 300 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro 305 310 315 320 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr 325 330 335 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro 340 345 350 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala 355 360 365 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile 370 375 380 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp 385 390 395 400 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr 405 410 415 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His 420 425 430 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp 435 440 445 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu 450 455 460 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp 465 470 475 480 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val 485 490 495 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn 500 505 510 Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser 515 520 525 His Leu Ala Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 19 <211> 536 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1-SOD fusion protein <400> 19 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatggcgacg aaggccgtgt gcgtgctgaa gggcgacggc ccagtgcagg 120 gcatcatcaa tttcgagcag aaggaaagta atggaccagt gaaggtgtgg ggaagcatta 180 aaggactgac tgaaggcctg catggattcc atgttcatga gtttggagat aatacagcag 240 gctgtaccag tgcaggtcct cactttaatc ctctatccag aaaacacggt gggccaaagg 300 atgaagagag gcatgttgga gacttgggca atgtgactgc tgacaaagat ggtgtggccg 360 atgtgtctat tgaagattct gtgatctcac tctcaggaga ccattgcatc attggccgca 420 cactggtggt ccatgaaaaa gcagatgact tgggcaaagg tggaaatgaa gaaagtacaa 480 agacaggaaa cgctggaagt cgtttggctt gtggtgtaat tgggatcgcc caataa 536 <210> 20 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-SOD fusion protein <400> 20 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Thr Lys Ala Val Cys Val Leu 20 25 30 Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu 35 40 45 Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu 50 55 60 Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr Ala Gly 65 70 75 80 Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser Arg Lys His Gly 85 90 95 Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu Gly Asn Val Thr 100 105 110 Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser Val Ile 115 120 125 Ser Leu Ser Gly Asp His Cys Ile Ile Gly Arg Thr Leu Val Val His 130 135 140 Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys 145 150 155 160 Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly Ile Ala 165 170 175 Gln <210> 21 <211> 536 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for SOD-PEP-1 fusion protein <400> 21 atggcgacga aggccgtgtg cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 60 ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact 120 gaaggcctgc atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt 180 gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg 240 catgttggag acttgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt 300 gaagattctg tgatctcact ctcaggagac cattgcatca ttggccgcac actggtggtc 360 catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacaggaaac 420 gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aaggatccta aaagaaacct 480 ggtgggaaac ctggtggacc gaatggtctc agccgaaaaa aaaacgtaaa gtgtag 536 <210> 22 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> SOD-PEP-1 fusion protein <400> 22 Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln 1 5 10 15 Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val 20 25 30 Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val 35 40 45 His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His 50 55 60 Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg 65 70 75 80 His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala 85 90 95 Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys 100 105 110 Ile Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly 115 120 125 Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg 130 135 140 Leu Ala Cys Gly Val Ile Gly Ile Ala Gln Lys Glu Thr Trp Trp Glu 145 150 155 160 Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 165 170 175 <210> 23 <211> 607 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1-SOD-PEP-1 fusion protein <400> 23 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatggcgacg aaggccgtgt gcgtgctgaa gggcgacggc ccagtgcagg 120 gcatcatcaa tttcgagcag aaggaaagta atggaccagt gaaggtgtgg ggaagcatta 180 aaggactgac tgaaggcctg catggattcc atgttcatga gtttggagat aatacagcag 240 gctgtaccag tgcaggtcct cactttaatc ctctatccag aaaacacggt gggccaaagg 300 atgaagagag gcatgttgga gacttgggca atgtgactgc tgacaaagat ggtgtggccg 360 atgtgtctat tgaagattct gtgatctcac tctcaggaga ccattgcatc attggccgca 420 cactggtggt ccatgaaaaa gcagatgact tgggcaaagg tggaaatgaa gaaagtacaa 480 agacaggaaa cgctggaagt cgtttggctt gtggtgtaat tgggatcgcc caaggatcct 540 aaaagaaacc tggtgggaaa cctggtggac cgaatggtct cagccgaaaa aaaaacgtaa 600 agtgtag 607 <210> 24 <211> 200 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-SOD-PEP-1 fusion protein <400> 24 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Thr Lys Ala Val Cys Val Leu 20 25 30 Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu 35 40 45 Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu 50 55 60 Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr Ala Gly 65 70 75 80 Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser Arg Lys His Gly 85 90 95 Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu Gly Asn Val Thr 100 105 110 Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser Val Ile 115 120 125 Ser Leu Ser Gly Asp His Cys Ile Ile Gly Arg Thr Leu Val Val His 130 135 140 Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys 145 150 155 160 Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly Ile Ala 165 170 175 Gln Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser 180 185 190 Gln Pro Lys Lys Lys Arg Lys Val 195 200 <210> 25 <211> 499 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for Tat-SOD fusion protein <400> 25 taggaagaag cggagacagc gacgaagact cgagatggcg acgaaggccg tgtgcgtgct 60 gaagggcgac ggcccagtgc agggcatcat caatttcgag cagaaggaaa gtaatggacc 120 agtgaaggtg tggggaagca ttaaaggact gactgaaggc ctgcatggat tccatgttca 180 tgagtttgga gataatacag caggctgtac cagtgcaggt cctcacttta atcctctatc 240 cagaaaacac ggtgggccaa aggatgaaga gaggcatgtt ggagacttgg gcaatgtgac 300 tgctgacaaa gatggtgtgg ccgatgtgtc tattgaagat tctgtgatct cactctcagg 360 agaccattgc atcattggcc gcacactggt ggtccatgaa aaagcagatg acttgggcaa 420 aggtggaaat gaagaaagta caaagacagg aaacgctgga agtcgtttgg cttgtggtgt 480 aattgggatc gcccaataa 499 <210> 26 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> Tat-SOD fusion protein <400> 26 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Thr Lys Ala 1 5 10 15 Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe 20 25 30 Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys 35 40 45 Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp 50 55 60 Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser 65 70 75 80 Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu 85 90 95 Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu 100 105 110 Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile Ile Gly Arg Thr 115 120 125 Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu 130 135 140 Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val 145 150 155 160 Ile Gly Ile Ala Gln 165 <210> 27 <211> 499 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for SOD-Tat fusion protein <400> 27 atggcgacga aggccgtgtg cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 60 ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact 120 gaaggcctgc atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt 180 gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg 240 catgttggag acttgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt 300 gaagattctg tgatctcact ctcaggagac cattgcatca ttggccgcac actggtggtc 360 catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacaggaaac 420 gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aaggatccta ggaagaagcg 480 gagacagcga cgaagatag 499 <210> 28 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> SOD-Tat fusion protein <400> 28 Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln 1 5 10 15 Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val 20 25 30 Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Gly 35 40 45 Ser Ile Lys Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro 50 55 60 His Phe Asn Pro Leu Ser Arg Lys His His Gly Phe Pro Lys Asp His 65 70 75 80 Gly Ser Arg His Gly Ser Gly Thr Glu Gly Leu Asn Gly Ser Thr Gly 85 90 95 Leu Thr Lys Asp Gly Val Gly Leu Thr Val Ser Ile Glu Leu Thr Ser 100 105 110 Pro His Phe Ser Leu Ser Gly Thr Glu Ser Asn Gly Pro Val Lys Val 115 120 125 Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Gly 130 135 140 Ser Ile Lys Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro 145 150 155 160 His Phe Asn Pro Leu Ser Arg Lys Lys Arg Arg Gln Arg Arg Arg 165 170 175 <210> 29 <211> 533 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for Tat-SOD-Tat fusion protein <400> 29 taggaagaag cggagacagc gacgaagact cgagatggcg acgaaggccg tgtgcgtgct 60 gaagggcgac ggcccagtgc agggcatcat caatttcgag cagaaggaaa gtaatggacc 120 agtgaaggtg tggggaagca ttaaaggact gactgaaggc ctgcatggat tccatgttca 180 tgagtttgga gataatacag caggctgtac cagtgcaggt cctcacttta atcctctatc 240 cagaaaacac ggtgggccaa aggatgaaga gaggcatgtt ggagacttgg gcaatgtgac 300 tgctgacaaa gatggtgtgg ccgatgtgtc tattgaagat tctgtgatct cactctcagg 360 agaccattgc atcattggcc gcacactggt ggtccatgaa aaagcagatg acttgggcaa 420 aggtggaaat gaagaaagta caaagacagg aaacgctgga agtcgtttgg cttgtggtgt 480 aattgggatc gcccaaggat cctaggaaga agcggagaca gcgacgaaga tag 533 <210> 30 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> Tat-SOD-Tat fusion protein <400> 30 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Thr Lys Ala 1 5 10 15 Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe 20 25 30 Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys 35 40 45 Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp 50 55 60 Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser 65 70 75 80 Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu 85 90 95 Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu 100 105 110 Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile Ile Gly Arg Thr 115 120 125 Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu 130 135 140 Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val 145 150 155 160 Ile Gly Ile Ala Gln Gly Ser Arg Lys Lys Arg Arg Gln Arg Arg Arg 165 170 175 <110> Industry Academic Cooperation Foundation, Hallym University <120> Enhancing method for transduction of fusion protein by adding          pergolide <130> hallym-sychoi-pergolide <160> 30 <170> Kopatentin 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ctcgagatgg ctgacagccg ggatcccgcc 30 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggatcctcac agatttgccr rctcccttgc c 31 <210> 3 <211> 1671 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for Tat-catalase fusion protein <400> 3 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggctgac agccgggatc ccgccagcga ccagatgcag 120 cactggaagg agcagcgggc cgcgcagaaa gctgatgtcc tgaccactgg agctggtaac 180 ccagtaggag acaaacttaa tgttattaca gtagggcccc gtgggcccct tcttgttcag 240 gatgtggttt tcactgatga aatggctcat tttgaccgag agagaattcc tgagagagtt 300 gtgcatgcta aaggagcagg ggcctttggc tactttgagg tcacacatga cattaccaaa 360 tactccaagg caaaggtatt tgagcatatt ggaaagaaga ctcccatcgc agttcggttc 420 tccactgttg ctggagaatc gggttcagct gacacagttc gggaccctcg tgggtttgca 480 gtgaaatttt acacagaaga tggtaactgg gatctcgttg gaaataacac ccccattttc 540 ttcatcaggg atcccatatt gtttccatct tttatccaca gccaaaagag aaatcctcag 600 acacatctga aggatccgga catggtctgg gacttctgga gcctacgtcc tgagtctctg 660 catcaggttt ctttcttgtt cagtgatcgg gggattccag atggacatcg ccacatgaat 720 ggatatggat cacatacttt caagctggtt aatgcaaatg gggaggcagt ttattgcaaa 780 ttccattata agactgacca gggcatcaaa aacctttctg ttgaagatgc ggcgagactt 840 tcccaggaag atcctgacta tggcatccgg gatcttttta acgccattgc cacaggaaag 900 tacccctcct ggacttttta catccaggtc atgacattta atcaggcaga aacttttcca 960 tttaatccat tcgatctcac caaggtttgg cctcacaagg actaccctct catcccagtt 1020 ggtaaactgg tcttaaaccg gaatccagtt aattactttg ctgaggttga acagatagcc 1080 ttcgacccaa gcaacatgcc acctggcatt gaggccagtc ctgacaaaat gcttcagggc 1140 cgcctttttg cctatcctga cactcaccgc catcgcctgg gacccaatta tcttcatata 1200 cctgtgaact gtccctaccg tgctcgagtg gccaactacc agcgtgacgg cccgatgtgc 1260 atgcaggaca atcagggtgg tgctccaaat tactacccca acagctttgg tgctccggaa 1320 caacagcctt ctgccctgga gcacagcatc caatattctg gagaagtgcg gagattcaac 1380 actgccaatg atgataacgt tactcaggtg cgggcattct atgtgaacgt gctgaatgag 1440 gaacagagga aacgtctgtg tgagaacatt gccggccacc tgaaggatgc acaaattttc 1500 atccagaaga aagcggtcaa gaacttcact gaggtccacc ctgactacgg gagccacatc 1560 caggctcttc tggacaagta caatgctgag aagcctaaga atgcgattca cacctttgtg 1620 cagtccggat ctcacttggc ggcaagggag aaggcaaatc tgtgaggatc c 1671 <210> 4 <211> 554 <212> PRT <213> Artificial Sequence <220> <223> Tat-catalase fusion protein <400> 4 His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His   1 5 10 15 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp Ser Arg              20 25 30 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala          35 40 45 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp      50 55 60 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln  65 70 75 80 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile                  85 90 95 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe             100 105 110 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu         115 120 125 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala     130 135 140 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala 145 150 155 160 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn                 165 170 175 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile             180 185 190 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met         195 200 205 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser     210 215 220 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn 225 230 235 240 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala                 245 250 255 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu             260 265 270 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly         275 280 285 Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp     290 295 300 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro 305 310 315 320 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro                 325 330 335 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr             340 345 350 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro         355 360 365 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala     370 375 380 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile 385 390 395 400 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp                 405 410 415 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr             420 425 430 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His         435 440 445 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp     450 455 460 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu 465 470 475 480 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp                 485 490 495 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val             500 505 510 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn         515 520 525 Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser     530 535 540 His Leu Ala Ala Arg Glu Lys Ala Asn Leu 545 550 <210> 5 <211> 1670 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for catalase-Tat fusion protein <400> 5 catcatcatc atcatcacag cagcggcctg gtgccggcgg cagccactcg agatggctga 60 cagccgggat cccgccagcg accagatgca gcactggaag gagcagcggg ccgcgcagaa 120 agctgatgtc ctgaccactg gagctggtaa cccagtagga gacaaactta atgttattac 180 agtagggccc cgtgggcccc ttcttgttca ggatgtggtt ttcactgatg aaatggctca 240 ttttgaccga gagagaattc ctgagagagt tgtgcatgct aaaggagcag gggcctttgg 300 ctactttgag gtcacacatg acattaccaa atactccaag gcaaaggtat ttgagcatat 360 tggaaagaag actcccatcg cagttcggtt ctccactgtt gctggagaat cgggttcagc 420 tgacacagtt cgggaccctc gtgggtttgc agtgaaattt tacacagaag atggtaactg 480 ggatctcgtt ggaaataaca cccccatttt cttcatcagg gatcccatat tgtttccatc 540 ttttatccac agccaaaaga gaaatcctca gacacatctg aaggatccgg acatggtctg 600 ggacttctgg agcctacgtc ctgagtctct gcatcaggtt tctttcttgt tcagtgatcg 660 ggggattcca gatggacatc gccacatgaa tggatatgga tcacatactt tcaagctggt 720 taatgcaaat ggggaggcag tttattgcaa attccattat aagactgacc agggcatcaa 780 aaacctttct gttgaagatg cggcgagact ttcccaggaa gatcctgact atggcatccg 840 ggatcttttt aacgccattg ccacaggaaa gtacccctcc tggacttttt acatccaggt 900 catgacattt aatcaggcag aaacttttcc atttaatcca ttcgatctca ccaaggtttg 960 gcctcacaag gactaccctc tcatcccagt tggtaaactg gtcttaaacc ggaatccagt 1020 taattacttt gctgaggttg aacagatagc cttcgaccca agcaacatgc cacctggcat 1080 tgaggccagt cctgacaaaa tgcttcaggg ccgccttttt gcctatcctg acactcaccg 1140 ccatcgcctg ggacccaatt atcttcatat acctgtgaac tgtccctacc gtgctcgagt 1200 ggccaactac cagcgtgacg gcccgatgtg catgcaggac aatcagggtg gtgctccaaa 1260 ttactacccc aacagctttg gtgctccgga acaacagcct tctgccctgg agcacagcat 1320 ccaatattct ggagaagtgc ggagattcaa cactgccaat gatgataacg ttactcaggt 1380 gcgggcattc tatgtgaacg tgctgaatga ggaacagagg aaacgtctgt gtgagaacat 1440 tgccggccac ctgaaggatg cacaaatttt catccagaag aaagcggtca agaacttcac 1500 tgaggtccac cctgactacg ggagccacat ccaggctctt ctggacaagt acaatgctga 1560 gaagcctaag aatgcgattc acacctttgt gcagtccgga tctcacttgg cggcaaggga 1620 gaaggcaaat ctgggatcct aggaagaagc ggagacagcg acgaagatag 1670 <210> 6 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> catalase-Tat fusion protein <400> 6 Met Ala Asp Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys   1 5 10 15 Glu Gln Arg Ala Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly              20 25 30 Asn Pro Val Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly          35 40 45 Pro Leu Leu Val Gln Asp Val Val Phe Thr Asp Glu Met Ala His Phe      50 55 60 Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly  65 70 75 80 Ala Phe Gly Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys                  85 90 95 Ala Lys Val Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg             100 105 110 Phe Ser Thr Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp         115 120 125 Pro Arg Gly Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp     130 135 140 Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu 145 150 155 160 Phe Pro Ser Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu                 165 170 175 Lys Asp Pro Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser             180 185 190 Leu His Gln Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly         195 200 205 His Arg His Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn     210 215 220 Ala Asn Gly Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln 225 230 235 240 Gly Ile Lys Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu                 245 250 255 Asp Pro Asp Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly             260 265 270 Lys Tyr Pro Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln         275 280 285 Ala Glu Thr Phe Pro Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro     290 295 300 His Lys Asp Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg 305 310 315 320 Asn Pro Val Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro                 325 330 335 Ser Asn Met Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln             340 345 350 Gly Arg Leu Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro         355 360 365 Asn Tyr Leu His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala     370 375 380 Asn Tyr Gln Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly 385 390 395 400 Ala Pro Asn Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro                 405 410 415 Ser Ala Leu Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe             420 425 430 Asn Thr Ala Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val         435 440 445 Asn Val Leu Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala     450 455 460 Gly His Leu Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys 465 470 475 480 Asn Phe Thr Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu                 485 490 495 Leu Asp Lys Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe             500 505 510 Val Gln Ser Gly Ser His Leu Ala Ala Arg Glu Lys Ala Asn Leu Arg         515 520 525 Lys Lys Arg Arg Gln Arg Arg Arg     530 535 <210> 7 <211> 1699 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for Tat-catalase-Tat fusion protein <400> 7 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggctgac agccgggatc ccgccagcga ccagatgcag 120 cactggaagg agcagcgggc cgcgcagaaa gctgatgtcc tgaccactgg agctggtaac 180 ccagtaggag acaaacttaa tgttattaca gtagggcccc gtgggcccct tcttgttcag 240 gatgtggttt tcactgatga aatggctcat tttgaccgag agagaattcc tgagagagtt 300 gtgcatgcta aaggagcagg ggcctttggc tactttgagg tcacacatga cattaccaaa 360 tactccaagg caaaggtatt tgagcatatt ggaaagaaga ctcccatcgc agttcggttc 420 tccactgttg ctggagaatc gggttcagct gacacagttc gggaccctcg tgggtttgca 480 gtgaaatttt acacagaaga tggtaactgg gatctcgttg gaaataacac ccccattttc 540 ttcatcaggg atcccatatt gtttccatct tttatccaca gccaaaagag aaatcctcag 600 acacatctga aggatccgga catggtctgg gacttctgga gcctacgtcc tgagtctctg 660 catcaggttt ctttcttgtt cagtgatcgg gggattccag atggacatcg ccacatgaat 720 ggatatggat cacatacttt caagctggtt aatgcaaatg gggaggcagt ttattgcaaa 780 ttccattata agactgacca gggcatcaaa aacctttctg ttgaagatgc ggcgagactt 840 tcccaggaag atcctgacta tggcatccgg gatcttttta acgccattgc cacaggaaag 900 tacccctcct ggacttttta catccaggtc atgacattta atcaggcaga aacttttcca 960 tttaatccat tcgatctcac caaggtttgg cctcacaagg actaccctct catcccagtt 1020 ggtaaactgg tcttaaaccg gaatccagtt aattactttg ctgaggttga acagatagcc 1080 ttcgacccaa gcaacatgcc acctggcatt gaggccagtc ctgacaaaat gcttcagggc 1140 cgcctttttg cctatcctga cactcaccgc catcgcctgg gacccaatta tcttcatata 1200 cctgtgaact gtccctaccg tgctcgagtg gccaactacc agcgtgacgg cccgatgtgc 1260 atgcaggaca atcagggtgg tgctccaaat tactacccca acagctttgg tgctccggaa 1320 caacagcctt ctgccctgga gcacagcatc caatattctg gagaagtgcg gagattcaac 1380 actgccaatg atgataacgt tactcaggtg cgggcattct atgtgaacgt gctgaatgag 1440 gaacagagga aacgtctgtg tgagaacatt gccggccacc tgaaggatgc acaaattttc 1500 atccagaaga aagcggtcaa gaacttcact gaggtccacc ctgactacgg gagccacatc 1560 caggctcttc tggacaagta caatgctgag aagcctaaga atgcgattca cacctttgtg 1620 cagtccggat ctcacttggc ggcaagggag aaggcaaatc tgggatccta ggaagaagcg 1680 gagacagcga cgaagatag 1699 <210> 8 <211> 549 <212> PRT <213> Artificial Sequence <220> <223> Tat-catalase-Tat fusion protein <400> 8 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp Ser Arg   1 5 10 15 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala              20 25 30 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp          35 40 45 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln      50 55 60 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile  65 70 75 80 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe                  85 90 95 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu             100 105 110 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala         115 120 125 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala     130 135 140 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn 145 150 155 160 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile                 165 170 175 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met             180 185 190 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser         195 200 205 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn     210 215 220 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala 225 230 235 240 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu                 245 250 255 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly             260 265 270 Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp         275 280 285 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro     290 295 300 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro 305 310 315 320 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr                 325 330 335 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro             340 345 350 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala         355 360 365 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile     370 375 380 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp 385 390 395 400 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr                 405 410 415 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His             420 425 430 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp         435 440 445 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu     450 455 460 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp 465 470 475 480 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val                 485 490 495 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn             500 505 510 Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser         515 520 525 His Leu Ala Ala Arg Glu Lys Ala Asn Leu Gly Ser Arg Lys Lys Arg     530 535 540 Arg Gln Arg Arg Arg 545 <210> 9 <211> 1708 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1-catalase fusion protein <400> 9 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccataa aagaaacctg 60 gtgggaaacc tggtggaccg aatggtctca gccgaaaaaa aaacgtaaag tgctcgagat 120 ggctgacagc cgggatcccg ccagcgacca gatgcagcac tggaaggagc agcgggccgc 180 gcagaaagct gatgtcctga ccactggagc tggtaaccca gtaggagaca aacttaatgt 240 tattacagta gggccccgtg ggccccttct tgttcaggat gtggttttca ctgatgaaat 300 ggctcatttt gaccgagaga gaattcctga gagagttgtg catgctaaag gagcaggggc 360 ctttggctac tttgaggtca cacatgacat taccaaatac tccaaggcaa aggtatttga 420 gcatattgga aagaagactc ccatcgcagt tcggttctcc actgttgctg gagaatcggg 480 ttcagctgac acagttcggg accctcgtgg gtttgcagtg aaattttaca cagaagatgg 540 taactgggat ctcgttggaa ataacacccc cattttcttc atcagggatc ccatattgtt 600 tccatctttt atccacagcc aaaagagaaa tcctcagaca catctgaagg atccggacat 660 ggtctgggac ttctggagcc tacgtcctga gtctctgcat caggtttctt tcttgttcag 720 tgatcggggg attccagatg gacatcgcca catgaatgga tatggatcac atactttcaa 780 gctggttaat gcaaatgggg aggcagttta ttgcaaattc cattataaga ctgaccaggg 840 catcaaaaac ctttctgttg aagatgcggc gagactttcc caggaagatc ctgactatgg 900 catccgggat ctttttaacg ccattgccac aggaaagtac ccctcctgga ctttttacat 960 ccaggtcatg acatttaatc aggcagaaac ttttccattt aatccattcg atctcaccaa 1020 ggtttggcct cacaaggact accctctcat cccagttggt aaactggtct taaaccggaa 1080 tccagttaat tactttgctg aggttgaaca gatagccttc gacccaagca acatgccacc 1140 tggcattgag gccagtcctg acaaaatgct tcagggccgc ctttttgcct atcctgacac 1200 tcaccgccat cgcctgggac ccaattatct tcatatacct gtgaactgtc cctaccgtgc 1260 tcgagtggcc aactaccagc gtgacggccc gatgtgcatg caggacaatc agggtggtgc 1320 tccaaattac taccccaaca gctttggtgc tccggaacaa cagccttctg ccctggagca 1380 cagcatccaa tattctggag aagtgcggag attcaacact gccaatgatg ataacgttac 1440 tcaggtgcgg gcattctatg tgaacgtgct gaatgaggaa cagaggaaac gtctgtgtga 1500 gaacattgcc ggccacctga aggatgcaca aattttcatc cagaagaaag cggtcaagaa 1560 cttcactgag gtccaccctg actacgggag ccacatccag gctcttctgg acaagtacaa 1620 tgctgagaag cctaagaatg cgattcacac ctttgtgcag tccggatctc acttggcggc 1680 aagggagaag gcaaatctgt gaggatcc 1708 <210> 10 <211> 550 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-catalase fusion protein <400> 10 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Asp Ser Arg Asp Pro Ala Ser              20 25 30 Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala Gln Lys Ala Asp          35 40 45 Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp Lys Leu Asn Val      50 55 60 Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln Asp Val Val Phe  65 70 75 80 Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile Pro Glu Arg Val                  85 90 95 Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe Glu Val Thr His             100 105 110 Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu His Ile Gly Lys         115 120 125 Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala Gly Glu Ser Gly     130 135 140 Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala Val Lys Phe Tyr 145 150 155 160 Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn Thr Pro Ile Phe                 165 170 175 Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile His Ser Gln Lys             180 185 190 Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met Val Trp Asp Phe         195 200 205 Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser Phe Leu Phe Ser     210 215 220 Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn Gly Tyr Gly Ser 225 230 235 240 His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala Val Tyr Cys Lys                 245 250 255 Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu Ser Val Glu Asp             260 265 270 Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly Ile Arg Asp Leu         275 280 285 Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp Thr Phe Tyr Ile     290 295 300 Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro Phe Asn Pro Phe 305 310 315 320 Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro Leu Ile Pro Val                 325 330 335 Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr Phe Ala Glu Val             340 345 350 Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro Gly Ile Glu Ala         355 360 365 Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala Tyr Pro Asp Thr     370 375 380 His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile Pro Val Asn Cys 385 390 395 400 Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp Gly Pro Met Cys                 405 410 415 Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr Pro Asn Ser Phe             420 425 430 Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His Ser Ile Gln Tyr         435 440 445 Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp Asp Asn Val Thr     450 455 460 Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu Glu Gln Arg Lys 465 470 475 480 Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp Ala Gln Ile Phe                 485 490 495 Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val His Pro Asp Tyr             500 505 510 Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn Ala Glu Lys Pro         515 520 525 Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser His Leu Ala Ala     530 535 540 Arg Glu Lys Ala Asn Leu 545 550 <210> 11 <211> 1707 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for catalase-PEP-1 fusion protein <400> 11 catcatcatc atcatcacag cagcggcctg gtgccggcgg cagccactcg agatggctga 60 cagccgggat cccgccagcg accagatgca gcactggaag gagcagcggg ccgcgcagaa 120 agctgatgtc ctgaccactg gagctggtaa cccagtagga gacaaactta atgttattac 180 agtagggccc cgtgggcccc ttcttgttca ggatgtggtt ttcactgatg aaatggctca 240 ttttgaccga gagagaattc ctgagagagt tgtgcatgct aaaggagcag gggcctttgg 300 ctactttgag gtcacacatg acattaccaa atactccaag gcaaaggtat ttgagcatat 360 tggaaagaag actcccatcg cagttcggtt ctccactgtt gctggagaat cgggttcagc 420 tgacacagtt cgggaccctc gtgggtttgc agtgaaattt tacacagaag atggtaactg 480 ggatctcgtt ggaaataaca cccccatttt cttcatcagg gatcccatat tgtttccatc 540 ttttatccac agccaaaaga gaaatcctca gacacatctg aaggatccgg acatggtctg 600 ggacttctgg agcctacgtc ctgagtctct gcatcaggtt tctttcttgt tcagtgatcg 660 ggggattcca gatggacatc gccacatgaa tggatatgga tcacatactt tcaagctggt 720 taatgcaaat ggggaggcag tttattgcaa attccattat aagactgacc agggcatcaa 780 aaacctttct gttgaagatg cggcgagact ttcccaggaa gatcctgact atggcatccg 840 ggatcttttt aacgccattg ccacaggaaa gtacccctcc tggacttttt acatccaggt 900 catgacattt aatcaggcag aaacttttcc atttaatcca ttcgatctca ccaaggtttg 960 gcctcacaag gactaccctc tcatcccagt tggtaaactg gtcttaaacc ggaatccagt 1020 taattacttt gctgaggttg aacagatagc cttcgaccca agcaacatgc cacctggcat 1080 tgaggccagt cctgacaaaa tgcttcaggg ccgccttttt gcctatcctg acactcaccg 1140 ccatcgcctg ggacccaatt atcttcatat acctgtgaac tgtccctacc gtgctcgagt 1200 ggccaactac cagcgtgacg gcccgatgtg catgcaggac aatcagggtg gtgctccaaa 1260 ttactacccc aacagctttg gtgctccgga acaacagcct tctgccctgg agcacagcat 1320 ccaatattct ggagaagtgc ggagattcaa cactgccaat gatgataacg ttactcaggt 1380 gcgggcattc tatgtgaacg tgctgaatga ggaacagagg aaacgtctgt gtgagaacat 1440 tgccggccac ctgaaggatg cacaaatttt catccagaag aaagcggtca agaacttcac 1500 tgaggtccac cctgactacg ggagccacat ccaggctctt ctggacaagt acaatgctga 1560 gaagcctaag aatgcgattc acacctttgt gcagtccgga tctcacttgg cggcaaggga 1620 gaaggcaaat ctgggatcct aaaagaaacc tggtgggaaa cctggtggac cgaatggtct 1680 cagccgaaaa aaaaacgtaa agtgtag 1707 <210> 12 <211> 566 <212> PRT <213> Artificial Sequence <220> <223> catalase-PEP-1 fusion protein <400> 12 His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His   1 5 10 15 Leu Glu Met Ala Asp Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His              20 25 30 Trp Lys Glu Gln Arg Ala Ala Gln Lys Ala Asp Val Leu Thr Thr Gly          35 40 45 Ala Gly Asn Pro Val Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro      50 55 60 Arg Gly Pro Leu Leu Val Gln Asp Val Val Phe Thr Asp Glu Met Ala  65 70 75 80 His Phe Asp Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Lys Gly                  85 90 95 Ala Gly Ala Phe Gly Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr             100 105 110 Ser Lys Ala Lys Val Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala         115 120 125 Val Arg Phe Ser Thr Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val     130 135 140 Arg Asp Pro Arg Gly Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn 145 150 155 160 Trp Asp Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro                 165 170 175 Ile Leu Phe Pro Ser Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr             180 185 190 His Leu Lys Asp Pro Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro         195 200 205 Glu Ser Leu His Gln Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro     210 215 220 Asp Gly His Arg His Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu 225 230 235 240 Val Asn Ala Asn Gly Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr                 245 250 255 Asp Gln Gly Ile Lys Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser             260 265 270 Gln Glu Asp Pro Asp Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala         275 280 285 Thr Gly Lys Tyr Pro Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe     290 295 300 Asn Gln Ala Glu Thr Phe Pro Phe Asn Pro Phe Asp Leu Thr Lys Val 305 310 315 320 Trp Pro His Lys Asp Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu                 325 330 335 Asn Arg Asn Pro Val Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe             340 345 350 Asp Pro Ser Asn Met Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met         355 360 365 Leu Gln Gly Arg Leu Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu     370 375 380 Gly Pro Asn Tyr Leu His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg 385 390 395 400 Val Ala Asn Tyr Gln Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln                 405 410 415 Gly Gly Ala Pro Asn Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln             420 425 430 Gln Pro Ser Ala Leu Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg         435 440 445 Arg Phe Asn Thr Ala Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe     450 455 460 Tyr Val Asn Val Leu Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn 465 470 475 480 Ile Ala Gly His Leu Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala                 485 490 495 Val Lys Asn Phe Thr Glu Val His Pro Asp Tyr Gly Ser His Ile Gln             500 505 510 Ala Leu Leu Asp Lys Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His         515 520 525 Thr Phe Val Gln Ser Gly Ser His Leu Ala Ala Arg Glu Lys Ala Asn     530 535 540 Leu Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro 545 550 555 560 Lys Lys Lys Arg Lys Val                 565 <210> 13 <211> 1773 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1-catalase-PEP-1 fusion protein <400> 13 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccataa aagaaacctg 60 gtgggaaacc tggtggaccg aatggtctca gccgaaaaaa aaacgtaaag tgctcgagat 120 ggctgacagc cgggatcccg ccagcgacca gatgcagcac tggaaggagc agcgggccgc 180 gcagaaagct gatgtcctga ccactggagc tggtaaccca gtaggagaca aacttaatgt 240 tattacagta gggccccgtg ggccccttct tgttcaggat gtggttttca ctgatgaaat 300 ggctcatttt gaccgagaga gaattcctga gagagttgtg catgctaaag gagcaggggc 360 ctttggctac tttgaggtca cacatgacat taccaaatac tccaaggcaa aggtatttga 420 gcatattgga aagaagactc ccatcgcagt tcggttctcc actgttgctg gagaatcggg 480 ttcagctgac acagttcggg accctcgtgg gtttgcagtg aaattttaca cagaagatgg 540 taactgggat ctcgttggaa ataacacccc cattttcttc atcagggatc ccatattgtt 600 tccatctttt atccacagcc aaaagagaaa tcctcagaca catctgaagg atccggacat 660 ggtctgggac ttctggagcc tacgtcctga gtctctgcat caggtttctt tcttgttcag 720 tgatcggggg attccagatg gacatcgcca catgaatgga tatggatcac atactttcaa 780 gctggttaat gcaaatgggg aggcagttta ttgcaaattc cattataaga ctgaccaggg 840 catcaaaaac ctttctgttg aagatgcggc gagactttcc caggaagatc ctgactatgg 900 catccgggat ctttttaacg ccattgccac aggaaagtac ccctcctgga ctttttacat 960 ccaggtcatg acatttaatc aggcagaaac ttttccattt aatccattcg atctcaccaa 1020 ggtttggcct cacaaggact accctctcat cccagttggt aaactggtct taaaccggaa 1080 tccagttaat tactttgctg aggttgaaca gatagccttc gacccaagca acatgccacc 1140 tggcattgag gccagtcctg acaaaatgct tcagggccgc ctttttgcct atcctgacac 1200 tcaccgccat cgcctgggac ccaattatct tcatatacct gtgaactgtc cctaccgtgc 1260 tcgagtggcc aactaccagc gtgacggccc gatgtgcatg caggacaatc agggtggtgc 1320 tccaaattac taccccaaca gctttggtgc tccggaacaa cagccttctg ccctggagca 1380 cagcatccaa tattctggag aagtgcggag attcaacact gccaatgatg ataacgttac 1440 tcaggtgcgg gcattctatg tgaacgtgct gaatgaggaa cagaggaaac gtctgtgtga 1500 gaacattgcc ggccacctga aggatgcaca aattttcatc cagaagaaag cggtcaagaa 1560 cttcactgag gtccaccctg actacgggag ccacatccag gctcttctgg acaagtacaa 1620 tgctgagaag cctaagaatg cgattcacac ctttgtgcag tccggatctc acttggcggc 1680 aagggagaag gcaaatctgg gatcctaaaa gaaacctggt gggaaacctg gtggaccgaa 1740 tggtctcagc cgaaaaaaaa acgtaaagtg tag 1773 <210> 14 <211> 573 <212> PRT <213> Artificial Sequence <220> PEP-1-catalase-PEP-1 fusion protein <400> 14 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Asp Ser Arg Asp Pro Ala Ser              20 25 30 Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala Gln Lys Ala Asp          35 40 45 Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp Lys Leu Asn Val      50 55 60 Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln Asp Val Val Phe  65 70 75 80 Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile Pro Glu Arg Val                  85 90 95 Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe Glu Val Thr His             100 105 110 Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu His Ile Gly Lys         115 120 125 Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala Gly Glu Ser Gly     130 135 140 Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala Val Lys Phe Tyr 145 150 155 160 Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn Thr Pro Ile Phe                 165 170 175 Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile His Ser Gln Lys             180 185 190 Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met Val Trp Asp Phe         195 200 205 Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser Phe Leu Phe Ser     210 215 220 Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn Gly Tyr Gly Ser 225 230 235 240 His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala Val Tyr Cys Lys                 245 250 255 Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu Ser Val Glu Asp             260 265 270 Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly Ile Arg Asp Leu         275 280 285 Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp Thr Phe Tyr Ile     290 295 300 Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro Phe Asn Pro Phe 305 310 315 320 Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro Leu Ile Pro Val                 325 330 335 Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr Phe Ala Glu Val             340 345 350 Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro Gly Ile Glu Ala         355 360 365 Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala Tyr Pro Asp Thr     370 375 380 His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile Pro Val Asn Cys 385 390 395 400 Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp Gly Pro Met Cys                 405 410 415 Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr Pro Asn Ser Phe             420 425 430 Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His Ser Ile Gln Tyr         435 440 445 Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp Asp Asn Val Thr     450 455 460 Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu Glu Gln Arg Lys 465 470 475 480 Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp Ala Gln Ile Phe                 485 490 495 Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val His Pro Asp Tyr             500 505 510 Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn Ala Glu Lys Pro         515 520 525 Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser His Leu Ala Ala     530 535 540 Arg Glu Lys Ala Asn Leu Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp 545 550 555 560 Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val                 565 570 <210> 15 <211> 1664 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for olygolysine-catalase fusion protein <400> 15 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaactcgag atggctgaca gccgggatcc cgccagcgac cagatgcagc 120 actggaagga gcagcgggcc gcgcagaaag ctgatgtcct gaccactgga gctggtaacc 180 cagtaggaga caaacttaat gttattacag tagggccccg tgggcccctt cttgttcagg 240 atgtggtttt cactgatgaa atggctcatt ttgaccgaga gagaattcct gagagagttg 300 tgcatgctaa aggagcaggg gcctttggct actttgaggt cacacatgac attaccaaat 360 actccaaggc aaaggtattt gagcatattg gaaagaagac tcccatcgca gttcggttct 420 ccactgttgc tggagaatcg ggttcagctg acacagttcg ggaccctcgt gggtttgcag 480 tgaaatttta cacagaagat ggtaactggg atctcgttgg aaataacacc cccattttct 540 tcatcaggga tcccatattg tttccatctt ttatccacag ccaaaagaga aatcctcaga 600 cacatctgaa ggatccggac atggtctggg acttctggag cctacgtcct gagtctctgc 660 atcaggtttc tttcttgttc agtgatcggg ggattccaga tggacatcgc cacatgaatg 720 gatatggatc acatactttc aagctggtta atgcaaatgg ggaggcagtt tattgcaaat 780 tccattataa gactgaccag ggcatcaaaa acctttctgt tgaagatgcg gcgagacttt 840 cccaggaaga tcctgactat ggcatccggg atctttttaa cgccattgcc acaggaaagt 900 acccctcctg gactttttac atccaggtca tgacatttaa tcaggcagaa acttttccat 960 ttaatccatt cgatctcacc aaggtttggc ctcacaagga ctaccctctc atcccagttg 1020 gtaaactggt cttaaaccgg aatccagtta attactttgc tgaggttgaa cagatagcct 1080 tcgacccaag caacatgcca cctggcattg aggccagtcc tgacaaaatg cttcagggcc 1140 gcctttttgc ctatcctgac actcaccgcc atcgcctggg acccaattat cttcatatac 1200 ctgtgaactg tccctaccgt gctcgagtgg ccaactacca gcgtgacggc ccgatgtgca 1260 tgcaggacaa tcagggtggt gctccaaatt actaccccaa cagctttggt gctccggaac 1320 aacagccttc tgccctggag cacagcatcc aatattctgg agaagtgcgg agattcaaca 1380 ctgccaatga tgataacgtt actcaggtgc gggcattcta tgtgaacgtg ctgaatgagg 1440 aacagaggaa acgtctgtgt gagaacattg ccggccacct gaaggatgca caaattttca 1500 tccagaagaa agcggtcaag aacttcactg aggtccaccc tgactacggg agccacatcc 1560 aggctcttct ggacaagtac aatgctgaga agcctaagaa tgcgattcac acctttgtgc 1620 agtccggatc tcacttggcg gcaagggaga aggcaaatct gtga 1664 <210> 16 <211> 538 <212> PRT <213> Artificial Sequence <220> <223> oligolysine-catalase fusion protein <400> 16 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Leu Glu Met Ala Asp Ser Arg   1 5 10 15 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala              20 25 30 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp          35 40 45 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln      50 55 60 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile  65 70 75 80 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe                  85 90 95 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu             100 105 110 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala         115 120 125 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala     130 135 140 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn 145 150 155 160 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile                 165 170 175 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met             180 185 190 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser         195 200 205 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn     210 215 220 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala 225 230 235 240 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu                 245 250 255 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly             260 265 270 Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp         275 280 285 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro     290 295 300 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro 305 310 315 320 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr                 325 330 335 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro             340 345 350 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala         355 360 365 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile     370 375 380 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp 385 390 395 400 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr                 405 410 415 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His             420 425 430 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp         435 440 445 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu     450 455 460 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp 465 470 475 480 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val                 485 490 495 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn             500 505 510 Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser         515 520 525 His Leu Ala Ala Arg Glu Lys Ala Asn Leu     530 535 <210> 17 <211> 1664 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for oligoarginine-catalase fusion protein <400> 17 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaga agaagaagaa 60 gaagaagaag aagactcgag atggctgaca gccgggatcc cgccagcgac cagatgcagc 120 actggaagga gcagcgggcc gcgcagaaag ctgatgtcct gaccactgga gctggtaacc 180 cagtaggaga caaacttaat gttattacag tagggccccg tgggcccctt cttgttcagg 240 atgtggtttt cactgatgaa atggctcatt ttgaccgaga gagaattcct gagagagttg 300 tgcatgctaa aggagcaggg gcctttggct actttgaggt cacacatgac attaccaaat 360 actccaaggc aaaggtattt gagcatattg gaaagaagac tcccatcgca gttcggttct 420 ccactgttgc tggagaatcg ggttcagctg acacagttcg ggaccctcgt gggtttgcag 480 tgaaatttta cacagaagat ggtaactggg atctcgttgg aaataacacc cccattttct 540 tcatcaggga tcccatattg tttccatctt ttatccacag ccaaaagaga aatcctcaga 600 cacatctgaa ggatccggac atggtctggg acttctggag cctacgtcct gagtctctgc 660 atcaggtttc tttcttgttc agtgatcggg ggattccaga tggacatcgc cacatgaatg 720 gatatggatc acatactttc aagctggtta atgcaaatgg ggaggcagtt tattgcaaat 780 tccattataa gactgaccag ggcatcaaaa acctttctgt tgaagatgcg gcgagacttt 840 cccaggaaga tcctgactat ggcatccggg atctttttaa cgccattgcc acaggaaagt 900 acccctcctg gactttttac atccaggtca tgacatttaa tcaggcagaa acttttccat 960 ttaatccatt cgatctcacc aaggtttggc ctcacaagga ctaccctctc atcccagttg 1020 gtaaactggt cttaaaccgg aatccagtta attactttgc tgaggttgaa cagatagcct 1080 tcgacccaag caacatgcca cctggcattg aggccagtcc tgacaaaatg cttcagggcc 1140 gcctttttgc ctatcctgac actcaccgcc atcgcctggg acccaattat cttcatatac 1200 ctgtgaactg tccctaccgt gctcgagtgg ccaactacca gcgtgacggc ccgatgtgca 1260 tgcaggacaa tcagggtggt gctccaaatt actaccccaa cagctttggt gctccggaac 1320 aacagccttc tgccctggag cacagcatcc aatattctgg agaagtgcgg agattcaaca 1380 ctgccaatga tgataacgtt actcaggtgc gggcattcta tgtgaacgtg ctgaatgagg 1440 aacagaggaa acgtctgtgt gagaacattg ccggccacct gaaggatgca caaattttca 1500 tccagaagaa agcggtcaag aacttcactg aggtccaccc tgactacggg agccacatcc 1560 aggctcttct ggacaagtac aatgctgaga agcctaagaa tgcgattcac acctttgtgc 1620 agtccggatc tcacttggcg gcaagggaga aggcaaatct gtga 1664 <210> 18 <211> 538 <212> PRT <213> Artificial Sequence <220> <223> oligoarginine-catalase fusion protein <400> 18 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Leu Glu Met Ala Asp Ser Arg   1 5 10 15 Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg Ala Ala              20 25 30 Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val Gly Asp          35 40 45 Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu Val Gln      50 55 60 Asp Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu Arg Ile  65 70 75 80 Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly Tyr Phe                  85 90 95 Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val Phe Glu             100 105 110 His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr Val Ala         115 120 125 Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly Phe Ala     130 135 140 Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly Asn Asn 145 150 155 160 Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser Phe Ile                 165 170 175 His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro Asp Met             180 185 190 Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln Val Ser         195 200 205 Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His Met Asn     210 215 220 Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly Glu Ala 225 230 235 240 Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gln Gly Ile Lys Asn Leu                 245 250 255 Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp Tyr Gly             260 265 270 Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Tyr Pro Ser Trp         275 280 285 Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr Phe Pro     290 295 300 Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro His Lys Asp Tyr Pro 305 310 315 320 Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val Asn Tyr                 325 330 335 Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met Pro Pro             340 345 350 Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu Phe Ala         355 360 365 Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu His Ile     370 375 380 Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln Arg Asp 385 390 395 400 Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn Tyr Tyr                 405 410 415 Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu Glu His             420 425 430 Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala Asn Asp         435 440 445 Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu Asn Glu     450 455 460 Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu Lys Asp 465 470 475 480 Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr Glu Val                 485 490 495 His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys Tyr Asn             500 505 510 Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Gln Ser Gly Ser         515 520 525 His Leu Ala Ala Arg Glu Lys Ala Asn Leu     530 535 <210> 19 <211> 536 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1-SOD fusion protein <400> 19 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatggcgacg aaggccgtgt gcgtgctgaa gggcgacggc ccagtgcagg 120 gcatcatcaa tttcgagcag aaggaaagta atggaccagt gaaggtgtgg ggaagcatta 180 aaggactgac tgaaggcctg catggattcc atgttcatga gtttggagat aatacagcag 240 gctgtaccag tgcaggtcct cactttaatc ctctatccag aaaacacggt gggccaaagg 300 atgaagagag gcatgttgga gacttgggca atgtgactgc tgacaaagat ggtgtggccg 360 atgtgtctat tgaagattct gtgatctcac tctcaggaga ccattgcatc attggccgca 420 cactggtggt ccatgaaaaa gcagatgact tgggcaaagg tggaaatgaa gaaagtacaa 480 agacaggaaa cgctggaagt cgtttggctt gtggtgtaat tgggatcgcc caataa 536 <210> 20 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-SOD fusion protein <400> 20 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Thr Lys Ala Val Cys Val Leu              20 25 30 Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu          35 40 45 Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu      50 55 60 Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr Ala Gly  65 70 75 80 Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser Arg Lys His Gly                  85 90 95 Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu Gly Asn Val Thr             100 105 110 Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser Val Ile         115 120 125 Ser Leu Ser Gly Asp His Cys Ile Gly Arg Thr Leu Val Val His     130 135 140 Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys 145 150 155 160 Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly Ile Ala                 165 170 175 Gln     <210> 21 <211> 536 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for SOD-PEP-1 fusion protein <400> 21 atggcgacga aggccgtgtg cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 60 ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact 120 gaaggcctgc atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt 180 gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg 240 catgttggag acttgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt 300 gaagattctg tgatctcact ctcaggagac cattgcatca ttggccgcac actggtggtc 360 catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacaggaaac 420 gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aaggatccta aaagaaacct 480 ggtgggaaac ctggtggacc gaatggtctc agccgaaaaa aaaacgtaaa gtgtag 536 <210> 22 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> SOD-PEP-1 fusion protein <400> 22 Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln   1 5 10 15 Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val              20 25 30 Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val          35 40 45 His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His      50 55 60 Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg  65 70 75 80 His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala                  85 90 95 Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys             100 105 110 Ile Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly         115 120 125 Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg     130 135 140 Leu Ala Cys Gly Val Ile Gly Ile Ala Gln Lys Glu Thr Trp Trp Glu 145 150 155 160 Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val                 165 170 175 <210> 23 <211> 607 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for PEP-1-SOD-PEP-1 fusion protein <400> 23 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatggcgacg aaggccgtgt gcgtgctgaa gggcgacggc ccagtgcagg 120 gcatcatcaa tttcgagcag aaggaaagta atggaccagt gaaggtgtgg ggaagcatta 180 aaggactgac tgaaggcctg catggattcc atgttcatga gtttggagat aatacagcag 240 gctgtaccag tgcaggtcct cactttaatc ctctatccag aaaacacggt gggccaaagg 300 atgaagagag gcatgttgga gacttgggca atgtgactgc tgacaaagat ggtgtggccg 360 atgtgtctat tgaagattct gtgatctcac tctcaggaga ccattgcatc attggccgca 420 cactggtggt ccatgaaaaa gcagatgact tgggcaaagg tggaaatgaa gaaagtacaa 480 agacaggaaa cgctggaagt cgtttggctt gtggtgtaat tgggatcgcc caaggatcct 540 aaaagaaacc tggtgggaaa cctggtggac cgaatggtct cagccgaaaa aaaaacgtaa 600 agtgtag 607 <210> 24 <211> 200 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-SOD-PEP-1 fusion protein <400> 24 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Thr Lys Ala Val Cys Val Leu              20 25 30 Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu          35 40 45 Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu      50 55 60 Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr Ala Gly  65 70 75 80 Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser Arg Lys His Gly                  85 90 95 Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu Gly Asn Val Thr             100 105 110 Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser Val Ile         115 120 125 Ser Leu Ser Gly Asp His Cys Ile Gly Arg Thr Leu Val Val His     130 135 140 Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys 145 150 155 160 Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly Ile Ala                 165 170 175 Gln Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser             180 185 190 Gln Pro Lys Lys Lys Arg Lys Val         195 200 <210> 25 <211> 499 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for Tat-SOD fusion protein <400> 25 taggaagaag cggagacagc gacgaagact cgagatggcg acgaaggccg tgtgcgtgct 60 gaagggcgac ggcccagtgc agggcatcat caatttcgag cagaaggaaa gtaatggacc 120 agtgaaggtg tggggaagca ttaaaggact gactgaaggc ctgcatggat tccatgttca 180 tgagtttgga gataatacag caggctgtac cagtgcaggt cctcacttta atcctctatc 240 cagaaaacac ggtgggccaa aggatgaaga gaggcatgtt ggagacttgg gcaatgtgac 300 tgctgacaaa gatggtgtgg ccgatgtgtc tattgaagat tctgtgatct cactctcagg 360 agaccattgc atcattggcc gcacactggt ggtccatgaa aaagcagatg acttgggcaa 420 aggtggaaat gaagaaagta caaagacagg aaacgctgga agtcgtttgg cttgtggtgt 480 aattgggatc gcccaataa 499 <210> 26 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> Tat-SOD fusion protein <400> 26 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Thr Lys Ala   1 5 10 15 Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe              20 25 30 Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys          35 40 45 Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp      50 55 60 Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser  65 70 75 80 Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu                  85 90 95 Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu             100 105 110 Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile Ile Gly Arg Thr         115 120 125 Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu     130 135 140 Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val 145 150 155 160 Ile Gly Ile Ala Gln                 165 <210> 27 <211> 499 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for SOD-Tat fusion protein <400> 27 atggcgacga aggccgtgtg cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 60 ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact 120 gaaggcctgc atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt 180 gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg 240 catgttggag acttgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt 300 gaagattctg tgatctcact ctcaggagac cattgcatca ttggccgcac actggtggtc 360 catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacaggaaac 420 gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aaggatccta ggaagaagcg 480 gagacagcga cgaagatag 499 <210> 28 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> SOD-Tat fusion protein <400> 28 Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln   1 5 10 15 Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val              20 25 30 Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Gly          35 40 45 Ser Ile Lys Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro      50 55 60 His Phe Asn Pro Leu Ser Arg Lys His His Gly Phe Pro Lys Asp His  65 70 75 80 Gly Ser Arg His Gly Ser Gly Thr Glu Gly Leu Asn Gly Ser Thr Gly                  85 90 95 Leu Thr Lys Asp Gly Val Gly Leu Thr Val Ser Ile Glu Leu Thr Ser             100 105 110 Pro His Phe Ser Leu Ser Gly Thr Glu Ser Asn Gly Pro Val Lys Val         115 120 125 Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Gly     130 135 140 Ser Ile Lys Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro 145 150 155 160 His Phe Asn Pro Leu Ser Arg Lys Lys Arg Arg Gln Arg Arg Arg                 165 170 175 <210> 29 <211> 533 <212> DNA <213> Artificial Sequence <220> <223> coding sequence for Tat-SOD-Tat fusion protein <400> 29 taggaagaag cggagacagc gacgaagact cgagatggcg acgaaggccg tgtgcgtgct 60 gaagggcgac ggcccagtgc agggcatcat caatttcgag cagaaggaaa gtaatggacc 120 agtgaaggtg tggggaagca ttaaaggact gactgaaggc ctgcatggat tccatgttca 180 tgagtttgga gataatacag caggctgtac cagtgcaggt cctcacttta atcctctatc 240 cagaaaacac ggtgggccaa aggatgaaga gaggcatgtt ggagacttgg gcaatgtgac 300 tgctgacaaa gatggtgtgg ccgatgtgtc tattgaagat tctgtgatct cactctcagg 360 agaccattgc atcattggcc gcacactggt ggtccatgaa aaagcagatg acttgggcaa 420 aggtggaaat gaagaaagta caaagacagg aaacgctgga agtcgtttgg cttgtggtgt 480 aattgggatc gcccaaggat cctaggaaga agcggagaca gcgacgaaga tag 533 <210> 30 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> Tat-SOD-Tat fusion protein <400> 30 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Thr Lys Ala   1 5 10 15 Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe              20 25 30 Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys          35 40 45 Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp      50 55 60 Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser  65 70 75 80 Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu                  85 90 95 Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu             100 105 110 Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile Ile Gly Arg Thr         115 120 125 Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu     130 135 140 Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val 145 150 155 160 Ile Gly Ile Ala Gln Gly Ser Arg Lys Lys Arg Arg Gln Arg Arg Arg                 165 170 175

Claims (15)

생체 세포 또는 조직에 퍼골라이드와 단백질 수송 도메인과 치료용 단백질이 공유결합한 융합 단백질을 가하여 융합 단백질의 생체 세포 또는 조직 내 도입을 증가시키는 방법.
A method of increasing the introduction of a fusion protein into a living cell or tissue by adding a fusion protein covalently bonded to a pergolide, a protein transport domain, and a therapeutic protein to the living cell or tissue.
제1항에 있어서,
상기 퍼골라이드는 융합 단백질보다 먼저 생체 세포 또는 조직에 처리하는 것을 특징으로 하는 방법.
The method of claim 1,
Wherein said pergolide is treated in living cells or tissue prior to fusion protein.
제1항에 있어서,
상기 단백질 수송 도메인은 Tat, PEP-1, 올리고라이신, 올리고아르기닌 또는 올리고(라이신, 아르기닌)임을 특징으로 하는 방법.
The method of claim 1,
The protein transport domain is Tat, PEP-1, oligolysine, oligoarginine or oligo (lysine, arginine).
제1항에 있어서,
상기 퍼골라이드는 퍼골라이드 메실레이트임을 특징으로 하는 방법.
The method of claim 1,
The pergolide is a pergolide mesylate.
제1항에 있어서,
상기 치료용 단백질은 활성산소종을 제거하는 효소임을 특징으로 하는 방법.The method of claim 1,
The therapeutic protein is an enzyme that removes free radical species. 제5항에 있어서,
상기 치료용 단백질은 카탈레이즈, 수퍼옥사이드 디스뮤테이즈 및 글루타치온 퍼옥시데이즈 중 선택된 1종임을 특징으로 하는 방법.
The method of claim 5,
Wherein said therapeutic protein is one selected from catalase, superoxide dismutase, and glutathione peroxidase.
단백질 수송 도메인과 공유결합한 활성산소종 제거 활성을 가진 효소의 융합 단백질 및 퍼골라이드를 포함하는 산화적 스트레스에 의한 질환 및 염증성 질환의 예방 및 치료용 조성물.
A composition for the prevention and treatment of diseases caused by oxidative stress and inflammatory diseases comprising a fusion protein and pergolide of an enzyme having an active oxygen species removal activity covalently bound to a protein transport domain.
제7항에 있어서,
상기 퍼골라이드는 퍼골라이드 메실레이트임을 특징으로 하는 조성물.
The method of claim 7, wherein
The pergolide is a composition characterized in that the pergolide mesylate.
제7항에 있어서,
상기 활성산소종 제거 활성을 가진 효소는 카탈레이즈, 수퍼옥사이드 디스뮤테이즈 및 글루타치온 퍼옥시데이즈 중 선택된 1종임을 특징으로 하는 조성물.
The method of claim 7, wherein
The enzyme having the reactive oxygen species removal activity is a composition characterized in that one selected from catalase, superoxide dismutase and glutathione peroxidase.
제7항에 있어서,
상기 단백질 수송 도메인은 Tat, PEP-1, 올리고라이신, 올리고아르기닌 또는 올리고(라이신, 아르기닌)임을 특징으로 하는 조성물.
The method of claim 7, wherein
Wherein said protein transport domain is Tat, PEP-1, oligolysine, oligoarginine or oligo (lysine, arginine).
제7항에 있어서,
상기 산화적 스트레스에 의한 질환은 암, 골수염, 후천성 면역결핍증, 심맥관계 질환, 대장직장암, 방광암, 관상동맥질환, 알츠하이머병, 파킨슨병, 헌팅턴병, 만성 신장병, 알콜성 간질환, 폐쇄성 폐질환, 인슐린저항 증후군 또는 당뇨병인 것을 특징으로 하는 조성물.
The method of claim 7, wherein
The disease caused by oxidative stress is cancer, osteomyelitis, acquired immunodeficiency syndrome, cardiovascular disease, colorectal cancer, bladder cancer, coronary artery disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, chronic kidney disease, alcoholic liver disease, obstructive pulmonary disease, insulin A composition characterized in that it is resistance syndrome or diabetes.
제7항에 있어서,
상기 염증성 질환은 동맥경화증, 알러지 질환, 비염, 천식, 급성통증, 만성통증, 치주염, 치은염, 염증성 장질환, 통풍, 심근경색, 울혈성 심부전, 고혈압, 협심증, 위궤양, 뇌경색, 다운증후군, 다발성 경화증, 비만, 당뇨, 치매, 우울증, 정신분열증, 결핵, 수면장애, 패혈증, 화상, 피부염 또는 췌장염인 것을 특징으로 하는 조성물.
The method of claim 7, wherein
The inflammatory diseases include atherosclerosis, allergic diseases, rhinitis, asthma, acute pain, chronic pain, periodontitis, gingivitis, inflammatory bowel disease, gout, myocardial infarction, congestive heart failure, hypertension, angina pectoris, gastric ulcer, cerebral infarction, Down syndrome, multiple sclerosis , Obesity, diabetes, dementia, depression, schizophrenia, tuberculosis, sleep disorders, sepsis, burns, dermatitis or pancreatitis.
단백질 수송 도메인과 공유결합한 활성산소종 제거 활성을 가진 효소의 융합 단백질 및 퍼골라이드를 포함하는 산화적 스트레스에 의한 기능 이상 및 염증에 의한 기능 이상의 예방 및 개선용 피부외용제 조성물.
A skin external preparation composition for preventing and improving dysfunction due to oxidative stress and dysfunction due to inflammation, including a fusion protein and pergolide of an enzyme having an active oxygen species removal activity covalently bound to a protein transport domain.
단백질 수송 도메인과 공유결합한 활성산소종 제거 활성을 가진 효소의 융합 단백질 및 퍼골라이드를 포함하는 화장료 조성물.
A cosmetic composition comprising a pergolide and a fusion protein of an enzyme having reactive oxygen species removal activity covalently bound to a protein transport domain.
단백질 수송 도메인과 공유결합한 활성산소종 제거 활성을 가진 효소의 융합 단백질 및 퍼골라이드를 포함하는 산화적 스트레스에 의한 기능 이상 및 염증에 의한 기능 이상의 예방 및 개선용 건강 기능성 식품 조성물.
A health functional food composition for preventing and improving dysfunction by oxidative stress and dysfunction by inflammation, including a fusion protein and pergolide of an enzyme having an active oxygen species removal activity covalently bound to a protein transport domain.
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* Cited by examiner, † Cited by third party
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KR20200012587A (en) * 2018-07-27 2020-02-05 강원대학교산학협력단 Composition for prevention or treatment of chronic obstructive pulmonary disease comprising PTD-bFGF or incresed exosome by PTD-bFGF
WO2024025299A1 (en) * 2022-07-25 2024-02-01 충남대학교 산학협력단 Recombinant expression vector for catalase secretion and escherichia coli strain transformed therewith

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US4797405A (en) * 1987-10-26 1989-01-10 Eli Lilly And Company Stabilized pergolide compositions
KR20090026838A (en) * 2007-09-11 2009-03-16 한림대학교 산학협력단 3-O-[beta-D-GLUCOPYRANOSYL(1->4)-alpha-L-ARABINOPYRANOSYL] -HEDERAGENIN INCREASING TRANSDUCING ACTIVITY OF CELL TRANSDUCING FUSION PROTEIN
KR101090749B1 (en) * 2009-10-14 2011-12-08 한림대학교 산학협력단 Vaccinium uliginosum L. extract which enhances cell transducibility of superoxide dismutase fusion protein

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200012587A (en) * 2018-07-27 2020-02-05 강원대학교산학협력단 Composition for prevention or treatment of chronic obstructive pulmonary disease comprising PTD-bFGF or incresed exosome by PTD-bFGF
WO2024025299A1 (en) * 2022-07-25 2024-02-01 충남대학교 산학협력단 Recombinant expression vector for catalase secretion and escherichia coli strain transformed therewith

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