KR100495139B1 - Cell-transducible catalase fusion protein and the use thereof - Google Patents
Cell-transducible catalase fusion protein and the use thereof Download PDFInfo
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Abstract
본 발명은 세포침투성 카탈라제 융합단백질 및 그 용도에 관한 것으로서, 좀더 자세히는 본 발명은 세포내 또는 세포핵 내로 침투하기 어려운 카탈라제에 단백질 형질도입 부위(protein transducing domain; PTD)를 공유결합시킨 카탈라제 융합단백질을 제조함으로써 세포 내로 원활히 침투하여 세포 내에서 해로운 활성산소종을 제거하는 활성을 나타내도록 하는 기술에 관한 것이다.The present invention relates to a cell permeable catalase fusion protein and a use thereof. More specifically, the present invention relates to a cell permeable catalase fusion protein which is covalently bound to a protein transducing domain (PTD) The present invention relates to a technique for allowing a cell to smoothly penetrate into a cell to produce an activity of removing harmful active oxygen species in the cell.
Description
본 발명은 세포침투성 카탈라제 융합단백질 및 그 용도에 관한 것으로서, 좀더 자세히는 본 발명은 세포내 또는 세포핵 내로 침투하기 어려운 카탈라제에 단백질 형질도입 부위(protein transducing domain; PTD)를 공유결합시킨 융합단백질을 제조함으로써 세포 내로 원활히 침투하여 세포 내에서 해로운 활성산소종을 제거하는 활성을 나타내도록 하는 기술에 관한 것이다.The present invention relates to a cell-permeable catalase fusion protein and a use thereof. More specifically, the present invention relates to a cell-permeable catalase fusion protein which is produced by covalently bonding a protein transducing domain (PTD) to a catalase which is difficult to permeate into cells or nuclei. To thereby smoothly penetrate into the cell, thereby exhibiting an activity of removing harmful active oxygen species in the cell.
본 명세서 및 특허청구범위에서 "단백질 형질도입 부위"라 함은 세포 또는 세포핵 내로 도입되기 어려운 유기화합물들과 공유결합하여 이들을 세포 또는 세포핵 내로 운반시켜 주는 역할을 수행하는 수개 내지 십수개의 아미노산으로 이루어진 올리고펩타이드를 말하는 것이다.In the present specification and claims, the term "protein transduction site" refers to an oligopeptide consisting of several to ten amino acids, which plays a role of covalently bonding with organic compounds which are difficult to introduce into cells or nuclei, It is a peptide.
본 명세서 및 특허청구범위에서는 카탈라제를 세포 내로 "도입"시키는 것에 대하여 "운반", "침투", "수송", "전달", "투과", "통과"한다는 표현들을 혼용하였다.In the present specification and claims, the expressions "transport", "penetration", "transport", "transfer", "permeate", "pass" are mixed with respect to "introducing" catalase into a cell.
호기적인 상태에서 인간을 비롯한 모든 생물체들은 환원력이 높은 산소에 의한 산화를 받는다. 에너지를 생성하는 미토콘드리아에서 소비하는 산소의 약 2% 정도는 호흡사슬과정을 통하여 자유 라디칼(free radical)을 생성하며, 이 자유 라디칼은 생체 내에서 이차적인 산화과정을 거쳐 다양한 형태의 활성산소종(reactive oxygen species, ROS)을 생성하게 된다.In the aerobic state, all organisms, including humans, are oxidized by oxygen with high reducing power. Approximately 2% of the oxygen consumed by energy-producing mitochondria produces free radicals through the respiratory chain process, which undergoes a secondary oxidation process in vivo to produce various forms of reactive oxygen species reactive oxygen species (ROS).
공지된 활성산소종은 1O2, OH, O2, H2O2 등이 있으며 이들은 생체 내에서 각종 효소반응에 의해 생성되며 각종 생리활성 물질의 생합성, 면역기능, 약물의 대사 등에서 매우 중요한 역할을 하지만 외부로부터의 방사선, 자외선, 공해 및 각종 스트레스 등에 의해 과잉생성될 경우 오히려 생체에 손상을 가져올 수 있기 때문에 생체는 방어기능으로 슈퍼옥사이드 디스뮤테이스, 카탈라제, 퍼옥시다제 (Peroxidase) 등의 효소를 갖고 있는데 노화가 시작되면 피부의 균형이 깨어지게 되고 각종 활성산소종에 대한 피부보호능력이 떨어지게 된다.Known active oxygen species include 1 O 2 , OH, O 2 , and H 2 O 2 , which are produced by various enzyme reactions in vivo and play a very important role in biosynthesis of various physiologically active substances, immune function, However, in case of over production due to radiation from the outside, ultraviolet rays, pollution, various stresses, etc., the living body may damage the living body. Therefore, the living body has a defense function such as superoxide dismutase, catalase, peroxidase It has enzymes that when the aging starts, the balance of the skin breaks and the skin protection ability against various active oxygen species becomes poor.
카탈라제는 Cu,Zn-슈퍼옥사이드 디스뮤테이스(Cu,Zn-superoxide dismutase)와 함께 이러한 자유라디칼의 해독과 산소의 손상으로부터 세포를 보호해주는 중요한 방어 효소이다[Chance, B. (1979) Physiol. Rev. 59, 527-540]. 모든 생체 내 고분자들은 이러한 자유라디칼의 해로운 작용에 항상 노출되어 있기 때문에 여러 질환에서 카탈라제와 Cu,Zn-슈퍼옥사이드 디스뮤테이스를 치료 목적으로 이용하려는 관심도가 매우 높아지고 있다.Catalase is an important protective enzyme that protects cells from detoxification of free radicals and damage to oxygen with Cu, Zn-superoxide dismutase (Chance, B. (1979) Physiol. Rev. 59, 527-540. Because all in vivo polymers are exposed to the harmful effects of these free radicals, there is a growing interest in using catalase and Cu, Zn-superoxide dismutase for therapeutic purposes in various diseases.
카탈라제는 호모단백질(homoprotein)로서 한 분자당 3가 철이온(Fe3+)이 4개 있으며 효소 활성시에는 산화 상태로 존재한다. 그리고 과산화수소를 산화해서 2분자의 물과 한 분자의 산소로 만든다.Catalase is a homoprotein with four trivalent iron ions (Fe 3+ ) per molecule and is present in the oxidized state during enzyme activation. It oxidizes the hydrogen peroxide into two molecules of water and one molecule of oxygen.
외래 거대분자(exogenous macromolecules)를 살아 있는 세포로 주입하는 연구는 80년대 후반에 HIV-1 바이러스의 조절 단백질인 Tat이 배지에서 살아있는 세포로 침투(internalization)한다는 사실이 처음으로 보고된 이후 시작되었다(Green and Lowenstein. 1988; Fankel and Pabo, 1988). 이러한 단백질의 세포침투는 세포막 인지질 이중층을 통과할 수 있는 단백질 형질도입 부위(protein transduction domain)를 통해 이루어진다.Research into injecting exogenous macromolecules into living cells began after the first reports of the late 1980s that Tat, the regulatory protein of the HIV-1 virus, internalized from the media into living cells Green and Lowenstein, 1988; Fankel and Pabo, 1988). The cellular penetration of these proteins is via the protein transduction domain, which can pass through the cell membrane phospholipid bilayer.
또한 2000년초, 양이온성 아미노산인 아르기닌(arginine) 9개를 연결시킨 올리고펩타이드가 세포 내로 침투되는 신호(signal)로 작용한다는 내용이 보고된 바 있다(Robert, 2000). HIV Tat은 6개의 아르기닌, 2개의 라이신, 1개의 글루타민으로 구성된다는 점에서 양전하(positive charge)가 단백질을 세포 내로 주입시키는 신호로서 역할하고 있음을 추측할 수 있다.In the early 2000s, it was reported that oligopeptides linked with nine arginines, which are cationic amino acids, act as signals that penetrate into cells (Robert, 2000). HIV Tat is composed of six arginines, two lysines, and one glutamine, and it can be assumed that a positive charge acts as a signal to inject the protein into the cell.
이것을 이용하여 본 연구실에서는 HIV-1 Tat의 단백질 형질도입 부위와 결합된 SOD(superoxide dismutase) 융합단백질이 효율적으로 세포질 내로 쉽게 이동한다는 것을 밝혀냈다[Kwon, H.Y. et al,(2000) FEBS Letters. 485, 163-167]. 이러한 기능은 Tat 단백질의 중간부위인 단백질 형질도입 부위의 특성 때문에 나타나며 아직 그 정확한 기전은 알려지지 않은 상태이다. 그러나, Tat 단백질의 세포막 통과에는 특정 수용체나 운반체가 관여하지 않는 것으로 보이며 단백질 형질도입부위가 직접 막의 지질 이중층과 작용함으로써 일어나는 것으로 이해하고 있다[Vives, E., Brodin, P. and Lebleu, B. (1997) J. Biol. Chem. 272, 16010-16017; Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G. and Prochiantz, A. (1996) J. Biol. Chem. 271, 18188-18193]. 이것은 Tat 단백질이 세포막을 통과하여 여러 종류의 세포 내로 투과할 수 있음을 의미한다.Using this, we have found that the superoxide dismutase (SOD) fusion protein bound to the protein transduction site of HIV-1 Tat can be efficiently transferred into the cytoplasm [Kwon, H.Y. et al., (2000) FEBS Letters. 485, 163-167. This function is due to the nature of the protein transduction site, which is an intermediate site of the Tat protein, and its exact mechanism is still unknown. However, it appears that Tat does not involve specific receptors or transporters in the cell membrane passage, and that the protein transduction site occurs directly by acting with the lipid bilayer of the membrane [Vives, E., Brodin, P. and Lebleu, B. (1997) J. Biol. Chem. 272, 16010-16017; Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G. and Prochiantz, A. (1996) J. Biol. Chem. 271, 18188-18193]. This means that the Tat protein can penetrate the cell membrane and penetrate into various types of cells.
본 발명은 카탈라제를 세포 내로 도입하거나 발현시킴에 있어서 종래 기술의 문제점을 해결하고 고효율로 세포 내로 도입시키고 세포 내에서 활성을 가지도록 하려는 것이다.The present invention aims at solving the problems of the prior art in introducing or expressing catalase into cells, introducing them into cells with high efficiency and having activity in cells.
상기 목적을 달성하기 위하여 본 발명자들은 단백질 형질도입 부위인 HIV-1 Tat(49-57) 및 그로부터 N-, C-말단이 수개 결여된 변형체, 아르기닌 잔기가 수개 내지 십수개 결합된 올리고아르기닌, 라이신 잔기가 수개 내지 십수개 결합된 올리고라이신에 카탈라제가 공유결합된 형태의 융합단백질이 효과적으로 세포 내로 투과되는지를 HeLa 세포 및 PC12 세포를 이용한 실험에서 확인하였다. 또한, 단백질 형질도입 부위와 공유결합시킨 카탈라제 융합단백질을 대량 제조하고 순수 정제하기 위한 기술을 개발하였다. 본 연구 결과 재조합된 카탈라제 융합단백질을 대장균에서 과대 발현시켰으며, 금속-킬레이트 친화 크로마토그래피(metal-chelating affinity chromatography) 법으로 쉽고 편리하게 정제하였다. 이렇게 정제된 융합단백질은 배양된 HeLa 세포, PC12 세포 및 피부조직에 침투되었으며, 이와 동시에 세포 및 피부조직 내의 카탈라제 효소 활성도 또한 유의하게 증가함을 관찰하였다. In order to achieve the above object, the present inventors have found that a protein transduction site, HIV-1 Tat (49-57) and variants in which several N- and C-termini are lacking therefrom, oligoarginine in which several to ten arginine residues are combined, It was confirmed by experiments using HeLa cells and PC12 cells that the fusion protein in which the catalase was covalently bound to oligo lysine bound several to ten residues was effectively permeated into the cells. In addition, a technique for mass-producing and purifying a catalase fusion protein covalently bound to a protein transduction site has been developed. The recombinant catalase fusion protein was overexpressed in E. coli and easily and conveniently purified by metal-chelating affinity chromatography. The purified fusion protein was infiltrated into cultured HeLa cells, PC12 cells and skin tissue, and at the same time, catalase enzyme activity in cells and skin tissues was also significantly increased.
본 발명에서는 생체의 방어기전에서 중요한 역할을 담당하고 있는 항산화효소인 카탈라제를 세포 내로 직접 침투시키고 세포 내에서 활성을 가지도록 하기 위하여 단백질 형질도입 부위와 공유결합된 카탈라제 융합단백질, 이 카탈라제 융합단백질을 제조하는 벡터, 이 벡터를 이용하여 카탈라제 융합단백질을 생산하는 방법을 제공한다. 또한, 본 발명은 카탈라제 융합단백질을 이용한 약제 조성물 및 화장료 조성물을 제공한다.In the present invention, in order to directly penetrate the antioxidant enzyme, an antioxidant enzyme that plays an important role in the defense of the body, and to have activity in the cell, a catalase fusion protein covalently bonded to the protein transduction site, And a method for producing a catalase fusion protein using the vector. The present invention also provides a pharmaceutical composition and a cosmetic composition using the catalase fusion protein.
이를 위하여, 세포투과성을 지닌 융합단백질을 발현시킬 수 있는 발현벡터(pTat-CAT, pArg-CAT, pLys-CAT)를 제조하여 대장균에서 과대 발현시켰으며, 이 융합단백질을 효율적으로 정제하였다.For this purpose, expression vectors (pTat-CAT, pArg-CAT and pLys-CAT) capable of expressing fusion proteins having cell permeability were prepared and overexpressed in E. coli, and this fusion protein was efficiently purified.
이 발현벡터는 사람 카탈라제, 형질도입부위(Tat 9개 아미노산 (Tat 49-57), 아르기닌 잔기 9개, 라이신 잔기 9개), 그리고 아미노산 말단부분에 6개의 히스티딘 잔기를 발현시킬 수 있는 cDNA를 포함하고 있다. 이 발현벡터를 이용하여 융합단백질을 대장균에서 과대 발현시켰으며, 변성상태에서 Ni-친화 컬럼(affinity column)을 이용하여 효율적으로 정제하였다.This expression vector contains human catalase, a transduction site (Tat 9 amino acids (Tat 49-57), 9 arginine residues, 9 lysine residues), and cDNA capable of expressing 6 histidine residues at the amino acid terminal . Using this expression vector, the fusion protein was overexpressed in E. coli and efficiently purified using a Ni-affinity column in the denatured state.
정제한 재조합 단백질은 투여한 농도에 비례하여 HeLa 세포 및 PC12 세포 내로 투과됨을 웨스턴 블랏과 형광면역염색 방법으로 확인할 수 있었다. 반면, 대조단백질인 카탈라제는 세포내로 투과되지 않음을 알 수 있었다. 또한, 세포 내로 투과된 재조합 단백질은 세포 내의 카탈라제 활성도를 유의하게 증가시켰으며, 세포 내에서 높은 안정성을 나타내었다.The purified recombinant protein was confirmed to be penetrated into HeLa cells and PC12 cells in proportion to the concentration of the purified recombinant protein by Western blotting and fluorescent immunostaining. On the other hand, the control protein, catalase, was not penetrated into the cells. In addition, the recombinant protein permeated into the cells significantly increased the activity of the catalase in the cells and showed high stability in the cells.
이러한 결과들은 재조합 단백질이 카탈라제와 관련된 인체 질환의 치료를 위한 단백질 치료에 효과적으로 이용될 수 있다는 가능성을 높게 제시해 준다.These results strongly suggest that recombinant proteins can be effectively used for protein therapy for the treatment of human diseases associated with catalase.
본 발명은 단백질 형질도입 부위와 공유결합되어 세포 또는 세포핵 내로 침투 가능한 세포 침투성 카탈라제 융합단백질에 관한 것이다.The present invention relates to a cell permeable catalase fusion protein capable of covalently binding to a protein transduction site and penetrating into a cell or nucleus.
본 발명은 또한 상기 단백질 형질도입 부위가 HIV-1 Tat(49-57), Tat(51-57), Tat(50-56), Tat(50-57), Tat(50-55), 5∼12개의 아르기닌 잔기로 구성되는 올리고 아르기닌, 5∼12개의 라이신 잔기로 구성되는 올리고라이신, 5∼12개의 아르기닌 또는 라이신 잔기로 구성되는 올리고펩타이드 중의 하나인 것을 특징으로 하는 세포 침투성 카탈라제 융합단백질에 관한 것이다.The present invention also relates to the use of the above protein transduction site for the production of HIV-1 Tat (49-57), Tat (51-57), Tat (50-56), Tat (50-57) Oligoarginine consisting of 12 arginine residues, oligo lysine consisting of 5 to 12 lysine residues, and oligopeptide consisting of 5 to 12 arginines or lysine residues .
본 발명은 상기 융합단백질이 서열번호 4, 6, 8번의 아미노산 서열을 갖는 것임을 특징으로 하는 세포 침투성 카탈라제 융합단백질에 관한 것이다.The present invention relates to a cell permeable catalase fusion protein characterized in that the fusion protein has an amino acid sequence of SEQ ID NOS: 4, 6 and 8.
본 발명은 카탈라제 cDNA 또는 그의 등가물(degenerate form)의 5' 말단에 HIV-1 Tat(49-57), Tat(51-57), Tat(50-56), Tat(50-57), Tat(50-55), 5∼12개의 아르기닌 잔기로 구성되는 올리고 아르기닌, 5∼12개의 라이신 잔기로 구성되는 올리고라이신, 5∼12개의 아르기닌 또는 라이신 잔기로 구성되는 올리고펩타이드 중 1종의 단백질 형질도입 부위를 코딩하는 DNA 서열이 결합되어 상기 세포 침투성 카탈라제 융합단백질을 코딩하는 재조합 폴리뉴클레오타이드에 관한 것이다.The present invention relates to a method for the detection of HIV-1 Tat (49-57), Tat (51-57), Tat (50-56), Tat (50-57), Tat 50-55), oligoarginine composed of 5 to 12 arginine residues, oligo lysine composed of 5 to 12 lysine residues, oligo peptide composed of 5 to 12 arginines or lysine residues, To a recombinant polynucleotide encoding the cell permeable catalase fusion protein.
본 발명은 단백질 형질도입 부위를 코딩하는 DNA 서열의 5'측과 작동 유전자(operator) 사이에 6개의 히스티딘 잔기를 코딩하는 DNA 서열이 결합되어 있는 것을 특징으로 하는 상기 세포 침투성 카탈라제 융합단백질을 코딩하는 재조합 폴리뉴클레오타이드에 관한 것이다.The present invention relates to a method for producing a cell-penetrating catalase fusion protein, which comprises the step of binding a DNA sequence coding for 6 histidine residues between the 5 'side of a DNA sequence coding for a protein transduction site and an operator, To recombinant polynucleotides.
또한, 본 발명은 재조합 폴리뉴클레오타이드가 서열번호 3, 5, 7번의 뉴클레오타이드 서열 또는 그의 등가물과 같이 구성된 것을 특징으로 하는 세포 침투성 카탈라제 융합 단백질을 코딩하는 재조합 폴리뉴클레오타이드에 관한 것이다.The present invention also relates to a recombinant polynucleotide encoding a cell permeable catalase fusion protein, wherein the recombinant polynucleotide is constructed as a nucleotide sequence of SEQ ID NO: 3, 5 or 7 or an equivalent thereof.
또한, 본 발명은 상기 세포 침투성 카탈라제 융합단백질을 발현시키기 위하여 상기 재조합 폴리뉴클레오타이드를 포함하여 구성되는 세포 침투성 카탈라제 융합단백질을 발현시키는 벡터에 관한 것이다.The present invention also relates to a vector expressing a cell permeable catalase fusion protein comprising the recombinant polynucleotide for expressing the cell permeable catalase fusion protein.
나아가, 본 발명은 발현벡터가 도 1A의 제한지도와 같이 구성되는 것을 특징으로 하는 세포 침투성 카탈라제 융합단백질을 발현시키는 벡터에 관한 것이다.Further, the present invention relates to a vector expressing a cell permeable catalase fusion protein characterized in that the expression vector is constructed as the restriction map of FIG. 1A.
또한, 본 발명은 상기 발현벡터를 미생물에서 발현시키는 단계; 발현된 카탈라제 융합단백질을 정제하는 단계; 정제된 카탈라제 융합단백질을 세포에 가하는 단계;로 구성되는 세포 침투성 카탈라제 융합단백질을 세포 내로 도입시키는 방법에 관한 것이다.The present invention also relates to a method for producing a microorganism, comprising: expressing the expression vector in a microorganism; Purifying the expressed catalase fusion protein; Adding a purified catalase fusion protein to the cell, and introducing the cell permeable catalase fusion protein into the cell.
또한, 본 발명은 상기 정제 단계에서 카탈라제 융합 단백질을 우레아를 이용하여 변성상태로 되게 하여 정제하는 것을 특징으로 하는 세포침투성 카탈라제 융합단백질을 세포 내로 도입시키는 방법에 관한 것이다.The present invention also relates to a method for introducing a cell permeable catalase fusion protein into a cell, characterized in that in the purification step, the catalase fusion protein is transformed into a denatured state by using urea.
또한, 본 발명은 상기 세포침투성 카탈라제 융합단백질을 유효성분으로 하고 활성산소종을 제거하는 기능을 가지는 것을 특징으로 하는 화장료 조성물에 관한 것이다.The present invention also relates to a cosmetic composition characterized by having the function of removing the active oxygen species with the cell permeable catalase fusion protein as an active ingredient.
또한, 본 발명은 상기 화장료 조성물이 화장수, 젤, 수용성 리퀴드, 수중유(O/W)형 및 유중수(W/O)형인 것을 특징으로 하는 화장료 조성물에 관한 것이다.Further, the present invention relates to a cosmetic composition characterized in that the cosmetic composition is a lotion, gel, water-soluble liquid, oil-in-water (O / W) type and water-in-oil (W / O) type.
뿐만 아니라, 본 발명은 상기 세포침투성 카탈라제 융합단백질을 유효성분으로 하고 약학적으로 허용되는 담체를 포함하는 것을 특징으로 하는 약제학적 조성물에 관한 것이다.In addition, the present invention relates to a pharmaceutical composition comprising the cell-permeable catalase fusion protein as an active ingredient and a pharmaceutically acceptable carrier.
또한, 본 발명은 상기 약제학적 조성물이 사구체신염, 맥관염, 자기면역성 질환, 졸중, 심근경색, 부정맥, 협심증, 특발성 헤모크로마토시스, 방사선 치료에 의한 질병, 조로증, 질병관련 노화, 겸상적혈구증, 말라리아, 폐기종, 심근증, 자기면역 신증후군, 베텔넷-관련 구강암, 고압산소증, 알쯔하이머병, 파킨슨병, 백내장 등과 같은 활성산소종 관련 질환의 치료제로서 사용되는 것을 특징으로 하는 약제학적 조성물에 관한 것이다.The present invention also relates to the use of the above pharmaceutical composition for the manufacture of a medicament for the treatment or prophylaxis of glomerulonephritis, vasculitis, autoimmune diseases, stroke, myocardial infarction, arrhythmia, angina pectoris, idiopathic hemochromatosis, Related diseases such as malaria, emphysema, cardiomyopathy, autoimmune nephrotic syndrome, Bettelet-related oral cancer, hypertension, Alzheimer's disease, Parkinson's disease, cataract and the like.
나아가, 본 발명은 상기 카탈라제 융합단백질 또는 상기 재조합 폴리뉴클레오타이드를 이용하여 활성산소종에 의한 질환을 예방 또는 치료하는 방법에 관한 것이다.Further, the present invention relates to a method for preventing or treating diseases caused by reactive oxygen species using the catalase fusion protein or the recombinant polynucleotide.
뿐만 아니라, 본 발명은 상기 발현벡터로 형질전환시킨 미생물을 이용하여 활성산소종에 의한 질환을 예방 또는 치료하는 방법에 관한 것이다.In addition, the present invention relates to a method for preventing or treating a disease caused by reactive oxygen species using a microorganism transformed with the expression vector.
또한, 본 발명은 상기 세포침투성 카탈라제 융합단백질을 세포 내로 도입시키는 방법을 이용하여 활성산소종에 의한 질환을 예방 또는 치료하는 방법에 관한 것이다.The present invention also relates to a method for preventing or treating diseases caused by reactive oxygen species by introducing the cell permeable catalase fusion protein into cells.
나아가, 본 발명은 상기 활성산소종에 의한 질환이 사구체신염, 맥관염, 자기면역성 질환, 졸중, 심근경색, 부정맥, 협심증, 특발성 헤모크로마토시스, 방사선 치료에 의한 질병, 조로증, 질병관련 노화, 겸상적혈구증, 말라리아, 폐기종, 심근증, 자기면역 신증후군, 베텔넷-관련 구강암, 고압산소증, 알쯔하이머병, 파킨슨병, 백내장인 것을 특징으로 하는 활성산소종에 의한 질환을 예방 또는 치료하는 방법에 관한 것이다.Furthermore, the present invention relates to a pharmaceutical composition for preventing or treating diseases caused by the above-mentioned active oxygen species, wherein the disease is selected from the group consisting of glomerulonephritis, vasculitis, autoimmune disease, stroke, myocardial infarction, arrhythmia, angina pectoris, idiopathic hemochromatosis, The present invention relates to a method for preventing or treating a disease caused by reactive oxygen species characterized by erythropoiesis, malaria, emphysema, cardiomyopathy, autoimmune nephrotic syndrome, Betein-related oral cancer, hypertension, Alzheimer's disease, Parkinson's disease and cataract .
본 발명의 카탈라제 융합 단백질을 유효성분으로 함유하는 약제학적 조성물은 약제학적 분야에서 통상적으로 허용되는 담체와 함께 배합하여 통상적인 방법에 의해 경구 또는 주사형태로 제형할 수 있다. 경구용 조성물로는, 예를 들면 정제 및 젤라틴 캡슐이 있으며, 이들은 활성 성분 이외에도 희석제(예: 락토스, 텍스트로스, 수크로스, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활탁제(예: 실리카, 탤크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/또는 폴리에틸렌 글리콜)를 함유하고, 정제는 또한 결합제(예: 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 메틸셀루로스, 나트륨 카복시메틸셀룰로스 및/또는 폴리비닐피롤리돈)를 함유하며, 경우에 따라서 붕해제(예: 전분, 한천, 알긴산 또는 그의 나트륨 염) 또는 비등 혼합물 및/또는 흡수제, 착색제, 항미제 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 위한 염/또는 완충제)를 함유한다. 또한, 이들은 기타 치료적으로 유용한 물질을 함유할 수 있다.The pharmaceutical composition containing the catalase fusion protein of the present invention as an active ingredient can be formulated in an oral or injection form by a conventional method in combination with a carrier that is conventionally acceptable in the pharmaceutical field. Oral compositions include, for example, tablets and gelatin capsules, which may contain, in addition to the active ingredient, a diluent such as lactose, textol, sucrose, mannitol, sorbitol, cellulose and / or glycine, Wherein the tablets also contain a binder (e.g., magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone) It is preferable to contain a disintegrant (for example, starch, agar, alginic acid or a sodium salt thereof) or a boiling mixture and / or an absorbent, a colorant, an antifogging agent and a sweetening agent. The injectable compositions are preferably isotonic aqueous solutions or suspensions, and the compositions mentioned are sterile and / or contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agent accelerators, salts for controlling osmotic pressure, or buffers. They may also contain other therapeutically valuable substances.
이와 같이 제조된 약제학적 제제는 목적하는 바에 따라 경구로 투여하거나, 비경구 방식 즉, 정맥내, 피하, 복강내 투여 또는 국소적용할 수 있으며, 용량은 일일 투여량이 0.0001~100㎎/㎏의 양을 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 or parenterally, that is, intravenously, subcutaneously, intraperitoneally or topically, depending on the purpose, and the dose may be a dose of 0.0001 to 100 mg / kg per day May be administered in one to several divided doses. The dosage level for a particular patient may vary depending on the patient's body weight, age, sex, health condition, time of administration, method of administration, excretion rate, severity of disease, and the like.
또한, 본 발명의 화장료 조성물은 크림, 연고, 젤, 로션, 화장수 등으로 제형화되어 사용될 수 있으며, 어떤 제형으로 제조하여 사용하는가는 본 발명의 속하는 기술분야의 당업자에 의해 극히 용이하게 결정될 수 있다.In addition, the cosmetic composition of the present invention can be formulated into a cream, an ointment, a gel, a lotion, a lotion, and the like, and it can be extremely easily determined by a person skilled in the art to which the present invention is applied .
또한, 본 발명의 카탈라제 융합단백질을 유효성분으로 하고 활성산소종을 제거하는 기능을 가지는 것을 특징으로 하는 로션, 젤, 에센스, 크림, 화장수 등은 각각 통상적인 제조방법에 따라 어떤 형태로든 용이하게 제조할 수 있으며, 기초 화장품 등에 편리하게 첨가하여 피부노화방지 및 개선제로서 사용될 수 있다.In addition, the lotion, gel, essence, cream, lotion, etc. which are characterized by having the function of removing the active oxygen species with the catalase fusion protein of the present invention as an active ingredient can be easily produced in any form And can be conveniently added to foundation cosmetics or the like to be used as an agent for preventing and improving skin aging.
일례로서 크림을 제조함에 있어서는 일반적인 수중유형(O/W) 또는 유중수형(W/O)의 크림 베이스에 본 발명의 카탈라제 융합단백질을 함유시키고 여기에 향료, 킬레이트제, 색소, 산화방지제, 방부제 등을 사용하는 한편, 물성개선을 목적으로 단백질, 미네랄, 비타민 등 합성 또는 천연소재를 병용할 수 있다.For example, in the case of producing a cream, the creamy base of the general water type (O / W) or water-in-water type (W / O) contains the catalase fusion protein of the present invention and the flavor, chelating agent, colorant, antioxidant, And synthetic or natural materials such as proteins, minerals, and vitamins can be used in combination for the purpose of improving physical properties.
아래에서는 구체적인 실시예를 통하여 본 발명의 구성을 상세히 설명한다. 그러나, 본 발명의 범위가 아래 실시예의 기재에만 한정되는 것은 아니다.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to specific embodiments. However, the scope of the present invention is not limited to the description of the embodiments below.
실험 재료Experimental material
DNA 서브클로닝(subcolning)에 사용한 택 폴리머라아제(Tag polymerase), T4 리가아제(ligase), 제한효소(restriction enzyme) 등은 Takara에서 구입하였고, Tat, 아르기닌 올리고뉴클레오타이드, 라이신 올리고뉴클레오타이드는 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.Tag polymerase, T4 ligase and restriction enzyme used for DNA subcloning were purchased from Takara, Tat, arginine oligonucleotide, and lysine oligonucleotide were purchased from Gibco BRL custom primer (USA). IPTG was purchased from Duchefa (Netherland). Histidine-binding metal chelating resins and columns for recombinant protein purification were purchased from Novagen. And nikel sulfate and imidazole were purchased from Sigma.
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 카탈라제 DNA는 PCR 방법으로 인간 간 cDNA 라이브러리에서 증폭시켰다. 그리고 이외의 모든 시약은 특급 제품을 사용하였다.Escherichia coli strain JM109 (DE3) was used for the expression of proteins. Bacto tryptone, yeast extract, agar, etc., of the microorganism culture medium were used for bacterial transformation. Difco product was used. The culture medium required for cell culture was Gibco product and human catalase DNA was amplified from human liver cDNA library by PCR method. All other reagents were made using high-grade products.
실시예 1: 발현백터의 제조 및 형질전환Example 1: Preparation and transformation of an expression vector
Tat 단백질, 아르기닌 잔기 9개, 라이신 잔기 9개에 해당하는 두 종류의 올리고뉴클레오타이드를 NdeⅠ-XhoⅠ 제한효소로 자른 pET15b에 연결(ligation)하여 삽입하였다. 이어 인간 신장(kidney) 카탈라제의 cDNA의 서열을 기본으로 하여 2종류의 올리고뉴클레오타이드를 합성하였다.Two kinds of oligonucleotides corresponding to Tat protein, 9 arginine residues and 9 lysine residues were inserted into pET15b cut with NdeI-XhoI restriction enzyme. Two kinds of oligonucleotides were synthesized based on the cDNA sequence of human kidney catalase.
정방향의 프라이머는 5'-ctcgagatggctgacagccgggatcccgcc-3'로 XhoⅠ제한부위를 지니고 있으며 역방향프라이머의 서열은 5'-ggatcctcacagatttgccrrctcccttgcc-3'로 BamHⅠ 제한부위를 가지고 있다.The forward primer is "XhoⅠ has a restriction site, and a sequence of the reverse primer was 5'-ggatcctcacagatttgccrrctcccttgcc-3 '5'- ctcgagatggctgacagccgggatcccgcc-3 has a restriction site in BamHⅠ.
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 카탈라제 cDNA가 포함된 벡터를 XhoⅠ과 BamHⅠ으로 절단한 다음 pET-15b 및 pET-15b-Tat(Arg, Lys)발현 벡터에 삽입하였다(그림 1 A). 벡터의 발현은 T7 프로모터와 lacO-오퍼레이터의 조절 하에 있다.Polymerase chain reaction (PCR) was performed in a thermal cycler (Perkin-Elmer, model 9600). The reaction mixture was placed in a 50 μl siliconized reaction tube and heated at 94 ° C for 5 minutes. The PCR reaction was conducted by extension 30 times at 94 ° C for 40 seconds, denaturation at 61 ° C for 40 seconds, final extension at 72 ° C for 10 minutes and at 37 ° C for 1 minute. And separated by agarose gel electrophoresis to separate the reaction product and ligate it to pGEM-T. And transformation (transformation) to the cells (competent cell) for the transformation of the vector, and cutting the vector to remove the plasmid that contains the human catalase cDNA as XhoⅠ and BamHⅠ following pET-15b and pET-15b-Tat (Arg, Lys) expression vector (Fig. 1A). Expression of the vector is under the control of the T7 promoter and lacO-operator.
실시예 2: 재조합 Tat, Arg, Lys-카탈라제 융합단백질의 제조 및 정제Example 2: Preparation and purification of recombinant Tat, Arg, Lys-catalase fusion proteins
융합단백질 발현벡터(pTat, pArg, pLys-CAT)가 포함되어 있는 E. coli JM109(DE3) 세포를 앰피실린이 포함된 LB 배지에 넣고 37℃에서 250 rpm으로 교반하며 배양하였다. 이 발현벡터는 사람 카탈라제, 형질도입부위(Tat 단백질, 9개 라이신, 9개 아르기닌) 그리고 아미노산 말단부분에 6개의 히스티딘 잔기를 발현시킬 수 있는 cDNA를 포함하고 있다(그림 1). 배양액 내의 박테리아 농도가 O.D600nm에서 0.6∼1.0이 될 때 IPTG를 최종농도가 1.0mM되게 배지에 첨가한 다음 4시간동안 단백질을 과대발현시켰다. E. coli JM109 (DE3) cells containing the fusion protein expression vectors (pTat, pArg, pLys-CAT) were added to LB medium containing ampicillin and incubated at 37 ° C with stirring at 250 rpm. This expression vector contains human catalase, the transduction site (Tat protein, 9 lysines, 9 arginines), and cDNA capable of expressing 6 histidine residues at the amino terminal end (Figure 1). When the bacterial concentration in the culture medium was 0.6 to 1.0 at OD 600 nm , IPTG was added to the medium to a final concentration of 1.0 mM, followed by overexpression of the protein for 4 hours.
배양한 세포를 원심분리하여 모은 뒤 100㎖당 2M 우레아가 포함된 결합 완충액 5ml(5mM 이미다졸, 500mM NaCl, 20mM Tris-HCl, pH 7.9)를 넣고 프렌치 프레스(french pressure)로 세포를 용출(lysis)시키고 변성상태(denaturing condition)에서 재조합 단백질을 정제하였다. The cultured cells were harvested by centrifugation, and 5 ml of binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9) containing 2 M urea per 100 ml was added and the cells were lysed by french pressure ) And the recombinant protein was purified in a denaturing condition.
한편, 히스티딘 결합 수지(Hisㆍbind resin)를 조심스럽게 섞어서 폴리프로필렌 컬럼(polypropylene column)에 팩킹(packing)시킨 후 D.D.W 3배 부피를 흘려 수지를 닦아낸다. 그리고 5배 부피의 charge buffer(50mM NiSO4)와 3배 부피의 결합 완충액을 차례로 흘려 수지를 활성화시킨다.On the other hand, the histidine binding resin is carefully mixed and packed in a polypropylene column, and then the resin is wiped by flowing a volume of DDW three times. Then, 5 times volume of charge buffer (50 mM NiSO 4 ) and 3 times volume of binding buffer are flowed in turn to activate the resin.
변성시킨 재조합 단백질을 원심분리하여 상층액을 즉시 수지(resin)에 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 제거한다. 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, and the supernatant was immediately loaded on the resin, and 10-fold volume of binding buffer and 6-fold volume of wash buffer (60 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9) Remove the protein other than the protein to be purified by flowing. After the buffer completely disappeared from the resin, the fusion protein was eluted with three times the volume of elution buffer (1M imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9). The fractions containing the fusion protein were collected and subjected to Sephadex G-25 column chromatography to remove the salt contained in the fraction.
분획의 단백질 농도는 우혈청 알부민을 표준물질로 사용하여 브래드포드(Bradford) 방법으로 측정하였다[Bradford, M.A. (1976) Anal. Biochem. 72, 248-254].The protein concentration of the fraction was determined by the Bradford method using bovine serum albumin as a standard (Bradford, M.A. (1976) Anal. Biochem. 72, 248-254].
실시예 3: HeLa, PC12 세포배양 및 재조합 단백질의 세포 내 투과Example 3: Intracellular permeation of HeLa, PC12 cell culture and recombinant protein
세포배양에 사용되는 배지는 HeLa 세포의 경우 DMEM(Dulbecco's Modified Eagle's Medium)에 10% FBS(fetal bovine serum )을 첨가하여 사용하였고 PC12 세포의 경우 RPMI 1640에 5% FBS와 10% 말 혈청(horse serum)을 첨가하여 사용하였다. 그리고 각 배지 모두 20mM HEPES/NaOH(pH 7.4), 5mM NaHCO3, 항생제(10,000㎍/㎖ 스트렙토마이신, 10,000 U/㎖ 페니실린)을 첨가하여 37℃에서 95% 공기와 5% CO2를 공급해주며 배양하였다.In the case of HeLa cells, 10% fetal bovine serum (FBS) was added to DMEM (Dulbecco's Modified Eagle's Medium). For PC12 cells, RPMI 1640 was supplemented with 5% FBS and 10% horse serum ) Was added and used. And all each medium gives supplying 20mM HEPES / NaOH (pH 7.4) , 5mM NaHCO 3, antibiotics (10,000㎍ / ㎖ streptomycin, 10,000 U / ㎖ penicillin) was added at 37 ℃ 95% air and 5% CO 2 culture Respectively.
융합단백질의 세포 내 투과를 관찰하기 위하여 HeLa 세포를 6-웰 플레이트에서 4∼6시간동안 키운 뒤 FBS가 포함되지 않은 1㎖의 신선한 배양액으로 교체하고 재조합 Tat-카탈라제를 배양액 내에 1시간동안 처리하였다.To observe intracellular permeation of the fusion protein, HeLa cells were grown in 6-well plate for 4 to 6 hours, then replaced with 1 ml of fresh culture medium without FBS, and the recombinant Tat-catalase was treated for 1 hour in the culture medium .
Arg-카탈라제와 Lys-카탈라제는 PC12 세포를 6-웰 플레이트에서 24시간동안 키운 뒤 혈청이 포함되지 않은 신선한 배양액으로 교체하고 Arg-카탈라제는 2uM농도로 2시간, Lys-카탈라제은 시간별, 농도별로 배양액 내에 처리하였다. 그 다음 세포를 트립신-EDTA(Gibco BRL)로 처리하고 PBS(phosphate buffered saline)로 충분히 세척하였다. 세포를 분쇄한 다음 세포 내로 투과된 재조합단백질의 양과 활성도를 측정하였다. 이상의 모든 과정에서는 대조군(control)으로서 PTD가 결합되지 않은 카탈라제를 동시에 세포에 처리하여 비교 실험을 하였다.Arg-catalase and Lys-catalase were grown in 6-well plates for 24 hours and then replaced with fresh serum-free culture. Arg-Catalase was incubated for 2 hours at a concentration of 2 uM and Lys- Respectively. The cells were then treated with trypsin-EDTA (Gibco BRL) and washed thoroughly with PBS (phosphate buffered saline). After the cells were pulverized, the amount and activity of the recombinant proteins permeated into the cells were measured. As a control, PTD-conjugated catalase was treated at the same time for comparative experiments.
실시예 4: 재조합단백질의 효소활성도 측정Example 4: Measurement of enzyme activity of recombinant protein
카탈라제의 활성도는 Aebi의 방법 (1980)에 따라 카탈라제/과산화수소 반응에 의한 과산화수소 분해정도를 분광광도계로 관찰함으로써 측정하였다.The activity of catalase was measured by spectrophotometric measurement of the degree of hydrogen peroxide decomposition by catalase / hydrogen peroxide reaction according to Aebi's method (1980).
표준분석방법은 25℃에서 50 mM 인산칼슘 완충액(potassium phosphate buffer: pH 7.0)에 과산화수소의 최종농도를 10mM 되게 하여 카탈라제에 의한 과산화수소의 분해속도를 240nm에서 측정하였다. 상기 조건 하에서 1분동안 과산화수소를 분해시키는 카탈라제의 양을 1 유니트(unit)로 정의하였다.The standard assay method was to measure the rate of decomposition of hydrogen peroxide by catalase at 240 nm by bringing the final concentration of hydrogen peroxide to 10 mM in 50 mM potassium phosphate buffer (pH 7.0) at 25 ° C. The amount of catalase that decomposes hydrogen peroxide for 1 minute under the above conditions was defined as one unit.
실시예 5: 웨스턴 블랏 분석(Western blot analysis)Example 5: Western blot analysis < RTI ID = 0.0 >
세포 분쇄액 내의 단백질을 10% SDS 폴리아크릴아미드 젤(polyacrylamide gel)로 분리한 다음 젤에 있는 단백질을 PVDF(polyvinylidene fluoride) 멤브레인 (Millipore)으로 전기이동시켰다. 단백질이 이동된 PVDF 멤브레인을 5% 탈지유(non-fat milk) 용액으로 처리하고 사람 카탈라제에 대한 히스티딘 항체로 1시간 처리하였다. 세척 후, 멤브레인을 고우트 항-래빗 IgG(goat anti-rabbit IgG) 항체(Seoulin, 1:10,000 희석)와 1시간동안 반응시켰다. 최종적으로 Enhanced chemiluminescent substrate kit(Amersham, ECL)를 처리하여 사람 카탈라제 항체에 반응하는 단백질 밴드를 확인하였다.The proteins in the cell lysate were separated by 10% SDS polyacrylamide gel, and the proteins in the gel were electrophoresed on PVDF (polyvinylidene fluoride) membrane (Millipore). Protein-transferred PVDF membranes were treated with 5% non-fat milk solution and treated with histidine antibody against human catalase for 1 hour. After washing, the membrane was reacted with a goat anti-rabbit IgG antibody (Seoulin, 1: 10,000 dilution) for 1 hour. Finally, the protein band reacting with the human catalase antibody was confirmed by treatment with an enhanced chemiluminescent substrate kit (Amersham, ECL).
실시예 6: 세포 내로 투과된 융합단백질의 안정성Example 6: Stability of fusion protein permeated into cells
세포 내로 투과된 융합단백질의 세포내 안정성을 측정하기 위하여, 세포를 6-웰 플레이트에서 배양한 뒤 혈청이 포함되지 않은 1㎖의 신선한 배양액으로 교체하였다. 2uM Tat-카탈라제와 4uM Lys-카탈라제를 시간별로 배양액 내에 처리한 후 세포를 트립신-EDTA (Gibco BRL), PBS로 충분히 세척하였다. 그리고 세포를 모아 세포 내의 카탈라제의 양을 웨스턴 블랏으로 분석하고 활성도를 측정하였다. In order to measure the intracellular stability of the fusion protein permeated into the cells, the cells were cultured in 6-well plate and replaced with 1 ml of fresh culture medium containing no serum. 2uM Tat-catalase and 4uM Lys-catalase were treated in the culture medium over time, and the cells were sufficiently washed with trypsin-EDTA (Gibco BRL), PBS. Cells were collected, and the amount of catalase in the cells was analyzed by Western blot and the activity was measured.
실시예 7: 면역형광분석(Immunofluorescence)Example 7: Immunofluorescence < RTI ID = 0.0 >
Tat-카탈라제의 세포 내 투과를 관찰하기 위하여, 24-웰 플레이트에 멸균된 커버 글래스를 넣고 HeLa 세포를 4∼6 시간 동안 키운 뒤 FBS가 포함되지 않은 1㎖의 신선한 배양액으로 교체하고 2μM의 재조합 Tat-카탈라제를 배양액 내에 처리하였다. 1시간후 세포를 트립신-EDTA(Gibco BRL)로 처리하고 PBS로 충분히 세척하였다. 이어 웰에 4% 파라 포름알데히드(paraformaldehyde)를 0.5ml씩 넣고 10분간 배양하여 세포를 고정시킨 후, 차가운 메탄올를 넣고 15초정도 배양하였다. PBS 완충액으로 3회 세척한 후, 카탈라제 항체를 커버 글래스 위에 떨어뜨려 주고 1시간을 상온에서 배양하였다. PBS 완충액으로 카탈라제 항체를 제거하고, FITC-결합된(conjugated) 항체를 PBS에 1:200로 희석하여 커버 글래스 위에 처리하여 상온에서 1시간 배양하였다. 이어 PBS 완충액으로 카탈라제 항체를 제거하고, 커버 글래스를 멸균된 3차 증류수에 세척한 후 공기 중에서 건조시켰다. 슬라이드 글래스 위에 마운팅 용액(mounting solution)과 커버 글래스의 세포가 맞닿도록 덮어주고 커버 글래스를 움직이지 않도록 고정시킨 다음, confocal laser scenning microscope (Bio-Rad, MRC 1024 ES)로 세포 내로 투과된 Tat-카탈라제를 확인하였다. In order to observe intracellular permeation of Tat-catalase, sterilized cover glass was placed in a 24-well plate and HeLa cells were grown for 4 to 6 hours, then replaced with 1 ml of fresh culture medium without FBS, and 2 μM of recombinant Tat - Catalase was treated in culture. After 1 hour, cells were treated with trypsin-EDTA (Gibco BRL) and washed thoroughly with PBS. After 0.5 ml of 4% paraformaldehyde was added to the wells, the cells were incubated for 10 minutes, and then cold methanol was added thereto for 15 seconds. After washing three times with PBS buffer, the catalase antibody was dropped on the cover glass and incubated for 1 hour at room temperature. The catalase antibody was removed with PBS buffer, and the FITC-conjugated antibody was diluted 1: 200 in PBS and incubated on cover glass for 1 hour at room temperature. The catalase antibody was then removed with PBS buffer, the cover glass was washed with sterile distilled water and dried in air. The mounting solution and coverslip cells on the slide glass were held in contact with each other, the cover glass was immobilized on a slide glass, and a Tat-Catalase (Bio-Rad, MRC 1024 ES) Respectively.
실시예 8: 과산화수소에 의한 세포의 생존능력 측정Example 8 Measurement of Cell Viability by Hydrogen Peroxide
PC12 세포를 24-웰 플레이트에 24시간 배양한 다음 혈청이 포함되지 않은 신선한 배지에 재조합 단백질을 농도별로(0.1-4uM) 처리하여 6시간동안 단백질을 세포내로 침투시켰다. 그리고 깨끗이 세척한 세포에 신선한 배지와 과산화화수소를 0.5uM되게 첨가하여 3시간동안 처리하고 배양액을 제거한 다음 MTT(1㎎/㎖ phenol red free medium)용액을 24-웰에 0.5㎖씩 첨가하였다. 3시간 배양시킨 후 MTT 용액을 제거하고 100% 이소프로판올(isopropanol) 0.5㎖씩 넣어 ELISA 측정기(reader)로 570nm에서 흡광도를 측정하여 세포내로 침투된 카탈라제에 의한 세포의 생존능력을 분석하였다. PC12 cells were cultured in a 24-well plate for 24 hours. Then, the recombinant protein was treated at a concentration (0.1-4 uM) in a serum-free fresh medium and permeated into the cells for 6 hours. Then, freshly-washed cells were added with 0.5 uM fresh medium and hydrogen peroxide for 3 hours, the culture solution was removed, and 0.5 ml of MTT (1 mg / ml phenol red free medium) solution was added to the 24-well. After incubation for 3 hours, MTT solution was removed and 0.5 ml of 100% isopropanol was added. The absorbance at 570 nm was measured with an ELISA reader to analyze the viability of the cells by the catalase.
실시예 9: 융합단백질의 피부 내 침투 효과Example 9: Penetration effect of the fusion protein on skin
① 실험동물군의 배정 및 처치 ① Assignment and treatment of experimental animals
체중 200g 내외의 수컷 SD 랫트를 사용하여 실험동물은 대조군(카탈라제 도포)와 실험군(Lys-카탈라제 도포)으로 구분하여 사용하였다. 각 실험동물을 24시간 절식시킨 후, 질소와 산소가 7:3으로 혼합된 개스에 3% 이소플루란(isoflurane)으로 전신마취를 유도한 후에 동일한 개스에 2.5% 이소플루란으로 마취를 유지하면서 복부 피부에 카탈라제와 Lys-카탈라제를 다른 부위에 각각 0.3㎎씩 도포하였다.Male SD rats weighing about 200 g were used, and the experimental animals were divided into a control group (catalase application) and an experimental group (Lys-catalase application). After each animal was fasted for 24 hours, general anesthesia was induced with 3% isoflurane in a 7: 3 mixture of nitrogen and oxygen, followed by anesthesia with 2.5% isoflurane in the same gas Catalase and Lys-Catalase were applied to the abdominal skin at 0.3 mg each at different sites.
② 조직 처리② Organization process
정상군과 각 실험군은 도포 30분, 1시간 또는 2시간 후에 케타민(ketamine) (30 mg/㎏)을 복강내 주사하여 전신마취시킨 후 도살하여 도포부위의 피부를 절취한 후 통상적인 방법으로 10㎛ 냉동조직절편을 제작하였고, 일부 조직은 효소활성 검사에 사용하였다.In the normal group and each experimental group, ketamine (30 mg / kg) was injected intraperitoneally 30 minutes, 1 hour or 2 hours after general anesthesia, and the skin was cut at the application site. ㎛ frozen tissue sections were prepared and some tissues were used for enzyme activity test.
③ 면역조직화학적 염색③ Immunohistochemical staining
제작된 조직절편은 4% 파라포름알데히드로 10분간 고정한 후 0.1M PBS, pH 7.4)으로 10분동안 3회 수세하여 조직 내에 있던 고정액을 제거한 다음 비특이적 면역반응(nonspecific immunoreactivity)을 방지하기 위하여 0.1M PBS(pH 7.4)에 0.3% 트리톤(Triton) X-100, 10% 정상 고우트 혈청(normal goat serum)이 희석된 용액으로 실온에서 1시간동안 반응시켰다. 이후 1차항체인 래빗 항-히스티딘 IgG( rabbit anti-histindine IgG)를 1:500으로 희석하여 실온에서 24시간 반응시켰다. 이후 0.1M PBS로 10분간 3회 세척하여 조직절편 속의 1차항체를 제거한 후 2차항체인 바이오틴이 결합된 고우트 항-래빗 IgG(biotinylated goat anti-rabbit IgG) (Vector Laboratories, USA)를 0.1M PBS로 1:200으로 희석시켜 실온에서 1시간동안 반응시켰다. 반응이 끝난 조직을 0.1M PBS로 10분씩 3회 세척한 후 퍼옥시다아제-결합된 스트렙타비딘(peroxydase-conjugated streptavidin)(Vector Laboratories, USA)을 1:300(in 0.1 M PBS)으로 희석하여 실온에서 1시간 반응시켰다. 면역반응이 끝난 조직절편은 기질액(40㎎ 디아미노벤지딘(diaminobenzidine)/0.045% H2O2 in 100 ㎖ PBS)으로 3분간 발색시켰다. 과도한 발색을 피하기 위하여 광학현미경 하에서 발색 정도를 지속적으로 확인하였다. 발색을 마친 조직 절편을 슬라이드에 부착시킨 다음 증류수와 0.1M PB로 여러 차례 세척하였으며, 통상적인 방법으로 탈수를 거쳐 영구표본으로 작성하였다.The tissue sections were fixed with 4% paraformaldehyde for 10 minutes and then rinsed three times for 10 minutes with 0.1 M PBS, pH 7.4) to remove the fixative in the tissues. Then, to prevent nonspecific immunoreactivity, 0.1 M The reaction was performed at room temperature for 1 hour in a solution of 0.3% Triton X-100 and 10% normal goat serum diluted in PBS (pH 7.4). Then, the primary antibody, rabbit anti-histindine IgG, was diluted 1: 500 and reacted at room temperature for 24 hours. Then, the primary antibody in the tissue section was removed by washing with 0.1 M PBS for 10 minutes for 3 minutes. Then, the secondary antibody, biotinylated goat anti-rabbit IgG (Vector Laboratories, USA) M PBS diluted 1: 200 and reacted at room temperature for 1 hour. The peroxidase-conjugated streptavidin (Vector Laboratories, USA) was diluted with 1: 300 (in 0.1 M PBS) and incubated at room temperature For 1 hour. Immunoreactive tissue sections were stained with substrate solution (40 mg diaminobenzidine / 0.045% H 2 O 2 in 100 ml PBS) for 3 minutes. The degree of color development was continuously confirmed under an optical microscope to avoid excessive coloring. The sections were washed with distilled water and 0.1 M PB several times and dehydrated by conventional methods to prepare permanent samples.
④ 효소활성조사④ Enzyme activity investigation
절취한 피부조직을 생리식염수로 충분히 세척하고 1.5 ㎖의 PBS를 넣어 조직을 가위로 잘게 잘랐다. 조직분쇄기(Polytron)로 조직을 균질분쇄한 다음 원심분리(17,000rpm, 10 min)하여 상청액을 분리하여 카탈라제 활성도를 각각 측정하였다.The cut skin tissue was sufficiently washed with physiological saline and 1.5 ml of PBS was added to cut the tissue into scissors. The tissues were homogenized with a tissue mill (Polytron) and centrifuged (17,000 rpm, 10 min) to separate the supernatant and measure the catalase activity.
상기 실시예에 의한 결과는 다음과 같다.The results of the above embodiment are as follows.
1) 재조합 융합단백질의 발현 및 정제 1) Expression and Purification of Recombinant Fusion Protein
본 발명에서는 도 2에서 나타낸 바와 같이 재조합된 융합단백질을 대장균에서 과대발현시켰으며(도 2A Tat-CAT, 도 2C Lys-CAT), 금속-킬레이팅 친화 크로마토그래피법으로 쉽고 편리하게 정제하였다. 이 융합단백질은 N-말단에 6개의 히스티딘을 포함하고 있기 때문에 고정 금속-킬레이팅 친화 크로마토그래피 (immobilized metal-chelate affinity chromatography) 한 단계로 융합단백질을 거의 순수하게(순도 >90%) 정제하였다(도 2B Tat-CAT, 2D Lys-CAT ).In the present invention, as shown in Fig. 2, the recombinant fusion protein was overexpressed in E. coli (Fig. 2A Tat-CAT, Fig. 2C Lys-CAT) and was easily and conveniently purified by metal-chelating affinity chromatography. Because this fusion protein contains six histidines at the N-terminus, the fusion protein was purified almost pure (purity> 90%) in one step by immobilized metal-chelate affinity chromatography Figure 2B Tat-CAT, 2D Lys-CAT).
정제한 융합단백질은 웨스턴 블랏으로 다시 한번 확인하였다. 도 3A에 나타난 바와 같이 카탈라제 항체에 반응하는 Tat-카탈라제와 카탈라제 밴드를 확인할 수 있었고 Lys-카탈라제(도 3B)도 2D의 단백질 밴드와 동일한 위치에서 각각 관찰되었다.The purified fusion protein was again confirmed by Western blotting. As shown in Fig. 3A, Tat-catalase and catalase band reacting to catalase antibody could be confirmed, and Lys-catalase (Fig. 3B) was also observed at the same position as protein band of 2D.
2) 세포 내로 재조합단백질의 투과 및 활성도 측정2) Measurement of permeability and activity of recombinant protein into cells
정제된 Arg-카탈라제와 카탈라제를 PC12 세포 내로의 투과 효과를 측정하기 위하여 각각 2uM씩 2시간동안 세포에 처리하였는데 Arg-카탈라제만 세포 내로의 침투효과를 관찰할 수 있었고 카탈라제를 처리한 PC12 세포에서는 밴드를 확인할 수 없었다(도 3C).In order to measure the permeation effect of purified Arg-catalase and catalase into PC12 cells, the cells were treated with 2uM for 2 hours each, and the penetration of Arg-Catalase alone into the cells was observed. In the case of PC12 cells treated with catalase, (Fig. 3C).
그리고, 정제된 Tat-카탈라제를 HeLa 세포내로의 투과 정도를 측정하기 위하여 0.5∼2uM의 Tat-카탈라제를 1시간동안 세포배양액 내에 처리하였을 때에 처리한 융합단백질의 농도에 비례하여 세포 내로 투과된 Tat-카탈라제의 양이 증가됨을 웨스턴 블랏팅으로 확인할 수 있었다(도 4A). In order to measure the degree of permeation of purified Tat-catalase into HeLa cells, Tat-catalase, which was treated with 0.5-2 uM of Tat-Catalase in the cell culture for 1 hour, The increased amount of catalase could be confirmed by Western blotting (Fig. 4A).
세포 내로 투과된 융합단백질이 그 고유한 활성을 유지해야만 이를 단백질 치료(protein therapy)에 응용할 수 있다. 따라서 세포 내로 투과된 융합단백질이 어느 정도 효소 활성을 나타내는지는 매우 중요한 문제이다. 본 실험에서 발현시킨 Tat-카탈라제는 도 4B에 나타낸 바와 같이 세포에 처리한 Tat-카탈라제의 양에 비례하여 HeLa 세포 내의 카탈라제 활성도가 증가되었다. 특히 1uM과 2uM의 Tat-카탈라제를 처리했을 때 HeLa 세포 내의 카탈라제 활성도는 융합단백질을 처리하지 않은 세포에 비해 3.2배, 4.9배로 각각 유의하게(p<0.01) 증가되었다.Fusion proteins permeated into cells must maintain their inherent activity before they can be applied to protein therapy. Therefore, it is very important that the fusion protein permeated into the cell exhibits some degree of enzyme activity. As shown in Fig. 4B, Tat-catalase expressed in this experiment increased catalase activity in HeLa cells in proportion to the amount of Tat-catalase treated in the cells. In particular, the catalase activity in HeLa cells treated with 1 uM and 2 uM of Tat-catalase was significantly increased (p <0.01) compared with the untreated cells, 3.2 and 4.9, respectively.
그리고, Lys-카탈라제의 세포 내 투과효과를 관찰하기 위하여 0.5∼4uM의 재조합 단백질을 PC12에 처리하였을 때 역시 농도에 비례하여 세포 내로 침투된 단백질 양이 증가되고(도 5A), 활성도는 단백질을 처리하지 않은 세포에 비해 약 13배 증가되는 것을 관찰할 수 있었다. 카탈라제를 처리한 세포는 대조군과 비슷한 수치를 나타냈다(도 5B). In order to observe the intracellular permeation effect of Lys-catalase, when the recombinant protein of 0.5 to 4 uM was treated with PC12, the amount of the protein penetrated into the cell was also increased in proportion to the concentration (Fig. 5A) Which is about 13 times higher than that of untreated cells. The cells treated with the catalase showed similar values to the control (Fig. 5B).
시간에 의한 세포 내 투과효과는 4uM의 Lys-카탈라제를 0.5∼4시간 PC12 세포에 처리하여 실험하였다. 그 결과 재조합 단백질이 시간에 의해 증가되는 것을 관찰할 수 있었고(도 6A), 30분에서 카탈라제의 활성도는 대조군의 약 4.5배, 4시간에서 약 14.5배 증가된다(도 6B). 그러나 대조 단백질로 사용한 카탈라제를 세포에 처리했을 때는 세포내 카탈라제 활성도 증가를 관찰할 수 없었다.Time-dependent intracellular permeability was measured by treating 4 μM Lys-Catalase for 0.5 to 4 hours with PC12 cells. As a result, it was observed that the recombinant protein was increased by time (Fig. 6A), and the activity of catalase at 30 minutes was increased from about 4.5 times of the control to about 14.5 times from 4 hours (Fig. 6B). However, no increase in intracellular catalase activity was observed when the catalase was used as a control protein.
3) 세포 내로 투과된 재조합 단백질의 안정성3) Stability of recombinant proteins permeated into cells
세포 내로 투과된 융합단백질의 세포 내 안정성을 측정하기 위하여 2uM의 Tat-카탈라제를 1시간 처리한 후 시간별로 분해되지 않고 남아 있는 카탈라제의 양을 웨스턴 블랏으로 확인하였다. 도 7에서 보여주듯이 시간이 경과함에 따라 세포 내로 투과된 Tat-카탈라제는 분해되어 점차적으로 감소되나 그 분해속도는 매우 낮았다. 일단, 세포 내로 투과된 융합단백질은 24시간까지는 거의 분해되지 않고 세포 내에 존재하였으며, 세포 내 투과 후 60시간 이상 세포 내에 존속하는 것으로 나타났다.In order to measure the intracellular stability of the fusion protein permeated into the cells, Tat-Catalase was treated with 2 uM for 1 hour, and the remaining amount of catalase not decomposed by time was confirmed by Western blotting. As shown in FIG. 7, as time passed, Tat-catalase permeated into the cells was degraded and gradually decreased, but the degradation rate was very low. First, the fusion proteins permeated into the cells remained almost intact in the cells until 24 hours, and persisted in the cells for 60 hours or more after intracellular permeation.
그리고 4uM의 Lys-카탈라제를 PC12 세포에 처리하여 시간별로 최대 투과량과 안정성을 웨스턴 블랏으로 확인하였다. 도 8A에서 보여주듯이 6시간까지 단백질이 가장 많이 투과된 것을 관찰할 수 있었고 시간의 경과함에 따라 Tat-카탈라제와 동일한 패턴을 보여 주었다. 그리고 카탈라제의 활성도 또한 세포 내로 투과된 단백질의 양에 비례하여 안정성을 나타냈고 60시간까지 대조군의 약 2배로 나타냈다(도 8B).Then, 4 uM of Lys-catalase was treated with PC12 cells, and the maximum amount of permeation and stability were confirmed by Western blotting with time. As shown in FIG. 8A, it was observed that the protein was most permeated up to 6 hours, and the pattern was the same as that of Tat-catalase over time. The activity of catalase was also stable in proportion to the amount of protein permeated into the cells and was shown to be about twice that of the control group until 60 hours (Fig. 8B).
이러한 결과들은 형질도입부위인 Tat 단백질, 아르기닌 9개, 라이신 9개와 재조합된 단백질이 세포 내로 투과가 잘 일어나고, 세포 내에서의 안정성이 또한 높게 유지되고 있음을 의미하기 때문에 단백질 치료제로서의 가능성을 한 층 더 높게 보여주고 있다. These results indicate that Tat protein, arginine 9, lysine 9 and recombinant protein permeate well into the cell and stability in the cell is maintained at a high level. Therefore, It is showing higher.
4) 형광면역염색4) Fluorescence immunohistochemistry
Tat-카탈라제의 HeLa 세포투과 정도를 다시 한번 형광면역염색으로 확인하였다. 2uM의 Tat-카탈라제를 1시간동안 세포배양액 내에 처리하였을 때에 융합단백질이 세포내 모든 영역으로 투과됨을 형광면역염색법을 이용한 confocal laser scenning microscope (Bio-Rad, MRC 1024 ES)으로 확인할 수 있었다(그림 9). 그러나, 대조 단백질로 사용한 카탈라제를 투여하였을 때에는 세포 내의 형광을 확인할 수 없어 카탈라제가 세포막을 투과하지 못함을 알 수 있었다. The degree of penetration of Tat-catalase in HeLa cells was confirmed by fluorescence immunohistochemistry. When a 2 uM Tat-catalase was treated for 1 hour in cell culture medium, permeation of the fusion protein to all regions of the cell was confirmed by confocal laser scanning microscope (Bio-Rad, MRC 1024 ES) using fluorescence immunohistochemistry ). However, when the catalase used as the control protein was administered, the fluorescence in the cells could not be confirmed, indicating that the catalase could not penetrate the cell membrane.
5) 과산화수소에 의한 세포생존 능력5) Cell viability by hydrogen peroxide
카탈라제는 생체 내에서 과산화수소를 최종적으로 물과 산소로 분해시키는 효소로 오래 전부터 알려져 있다. 과산화수소로 세포독성을 유발하였을 때 세포내로 침투된 Lys-카탈라제는 재조합단백질을 처리하지 않은 세포보다 세포생존율을 40%이상 증가시켰고(4μM 첨가), 대조군 카탈라제는 세포생존율 증가는 관찰되지 않았다(도 10). Catalase has been known for a long time as an enzyme that decomposes hydrogen peroxide into water and oxygen in vivo. When cell toxicity was induced by hydrogen peroxide, Lys-catalase infiltrated into the cells increased cell viability by more than 40% (by adding 4 μM) and did not increase the cell survival rate in the control group of the plasmids (FIG. 10 ).
이러한 결과로부터 세포 내로 투과된 Lys-카탈라제가 과산화수소를 분해시키는 능력이 충분히 있으며 이를 이용한 각종 질병의 치료목적에 사용될 수 있다고 생각된다.These results suggest that Lys-catalase permeated into the cells has sufficient ability to degrade hydrogen peroxide and can be used for the treatment of various diseases using the same.
6) 재조합단백질 피부내 침투 효과6) Effect of penetration of recombinant protein into skin
본 연구결과 카탈라제 대조군의 경우 히스티딘 면역반응은 거의 관찰되지 않았으나(도 11A, C, E), Lys-카탈라제 실험군의 경우 표피층, 진피층에 걸쳐 강한 히스티딘 면역반응이 관찰되었다(도 11 B, D, F). Lys-카탈라제 실험군에서 관찰된 히스티딘 면역반응은 주로 세포핵(nucleus)에서 관찰되었으며 이러한 면역반응이 관찰된 세포는 표피세포, 섬유모세포 및 모낭 및 그 주위의 세포 등에서 고루 관찰되었다. 그리고 피부 조직내로 침투된 Lys-카탈라제의 활성도는 30분에서 대조군에 비해 약 2배 증가되고 시간이 경과함에 따라 4배 가량 증가되는 것을 관찰할 수 있었다(도 12).11A, C and E), whereas in the Lys-catalase group, strong histidine immunoreactivity was observed across the epidermal layer and the dermal layer (Fig. 11B, D, F ). The histidine immunoreactivity observed in the Lys-catalase group was mainly observed in the nucleus, and the cells in which the immune response was observed were observed in the epidermal cells, the fibroblasts, and the surrounding cells. In addition, the activity of Lys-catalase infiltrated into the skin tissue was increased about 2 times as compared to the control group at 30 minutes, and increased about 4 times as time passed (FIG. 12).
본 기술은 인간 카탈라제를 단백질 수준에서 세포 내로 직접 효율적으로 투과시키는 최초의 실험적 보고이다.The technology is the first experimental report to efficiently pass human catalase directly into cells at the protein level.
특히, 세포 내로 투여된 Tat-카탈라제와 Lys-카탈라제는 안정성이 매우 높아 60시간까지 세포 내에 존재할 수 있고 활성도가 있기 때문에 단백질 치료제로서 응용 가능성이 매우 높다. In particular, Tat-catalase and Lys-catalase, which are administered intracellularly, are highly stable and can be present in cells for up to 60 hours and are highly active as protein therapeutics because of their activity.
과산화수소에 의해 유발된 세포독성에서 세포의 생존능력을 증가시키는 것으로 보아 여러 가지 질병의 치료에도 사용될 수 있다. It can be used for the treatment of various diseases as it increases cell viability in the cytotoxicity induced by hydrogen peroxide.
동물실험에서 재조합단백질이 피부 내로 침투된다는 사실을 이용하여 여러 가지 기능성 화장품이나 약품의 개발에 이용할 수 있다.Using the fact that recombinant proteins penetrate into the skin in animal experiments, it can be used to develop various functional cosmetics or drugs.
본 기술을 바탕으로 중요한 항산화 효소의 일종인 카탈라제를 세포 내에 직접 전달하여 우리 인체에 해로운 활성산소종을 제거할 수 있기 때문에 이 효소와 관련된 여러 인체 질환에 응용할 수 있으며 이외에도 화장품 및 건강식품산업 등에 본 기술이 광범위하게 활용될 수 있다.Based on this technology, catalase, which is a kind of important antioxidant enzyme, can be directly transferred into cells to remove reactive oxygen species harmful to human body. Therefore, it can be applied to various human diseases related to this enzyme. Technology can be widely used.
도 1은 Tat-카탈라제의 발현벡터의 제조방법을 도식화한 것이다. A는 pET15b 벡터를 이용한 발현벡터(pTat-CAT, pArg-CAT, pLys-CAT)의 제조방법을 나타내고, B는 발현된 융합단백질 Tat-카탈라제(이하, 본 명세서 및 청구범위, 도면에서 HIV-1 Tat(49-57)과 공유결합된 카탈라제 융합단백질을 의미하며, "Tat-CAT"과 혼용함), Arg-카탈라제(이하, 본 명세서 및 청구범위, 도면에서 9개의 아르기닌 잔기로 구성된 올리고아르기닌과 공유결합된 카탈라제 융합단백질을 의미하며, "Arg-CAT"과 혼용함), Lys-카탈라제(이하, 본 명세서 및 청구범위, 도면에서 9개의 라이신 잔기로 구성된 올리고라이신과 공유결합된 카탈라제 융합단백질을 의미하며, "Lys-CAT"과 혼용함)와 대조 단백질로 사용한 카탈라제의 모식도를 나타낸다.FIG. 1 is a schematic representation of a method for producing an expression vector of Tat-catalase. A represents a method for producing an expression vector (pTat-CAT, pArg-CAT, pLys-CAT) using a pET15b vector and B represents a fusion protein Tat-catalase (hereinafter referred to as HIV- Quot; Tat-CAT "), Arg-catalase (hereinafter referred to as " Tat-CAT "Quot; Arg-CAT "), Lys-catalase (hereinafter referred to as the catalase fusion protein covalently linked to oligo lysine consisting of nine lysine residues in the specification and claims, Quot; Lys-CAT ") and a schematic diagram of catalase used as a control protein.
도 2는 재조합 단백질의 발현 및 정제를 SDS-PAGE로 확인한 것이다. 그림 A는 대장균에서 발현시킨 Tat-카탈라제와 카탈라제를 SDS-PAGE로 확인하였고 그림 B는 정제된 Tat-카탈라제와 카탈라제를 SDS-PAGE로 확인하였다. 그림 C는 대장균에서 발현시킨 Lys-카탈라제와 카탈라제이고 그림 D는 정제한 Lys-카탈라제와 카탈라제 단백질이다.Figure 2 shows the expression and purification of the recombinant protein by SDS-PAGE. Figure A shows that Tat-catalase and catalase expressed in Escherichia coli were detected by SDS-PAGE. Figure B shows purified Tat-catalase and catalase by SDS-PAGE. Figure C is Lys-catalase and catalase expressed in E. coli. Figure D is purified Lys-catalase and catalase proteins.
도 3은 재조합 단백질을 웨스턴 블랏으로 확인한 사진이다. 그림 A는 정제된 Tat-카탈라제와 카탈라제를, 그림 B는 Lys-카탈라제와 카탈라제를 히스티딘 항체를 이용하여 웨스턴 블랏으로 확인하였다. 그림 C는 Arg-카탈라제와 카탈라제를 2uM씩 2시간 처리하여 세포 내로의 침투효과를 확인한 것이다.3 is a photograph showing the recombinant protein by Western blotting. Figure A shows purified Tat-catalase and catalase, and Figure B shows Lys-catalase and catalase by Western blot using histidine antibody. Figure C shows the penetration into the cells by treating Arg-Catalase and Catalase with 2 uM each for 2 hours.
도 4는 Tat-카탈라제의 농도에 따른 세포 투과정도를 웨스턴 블랏으로, 활성도를 특이활성도 그래프로 나타낸 것이다. Tat-카탈라제의 세포 투과성을 알아보기 위해서 0.5∼2μΜ의 Tat-카탈라제를 HeLa 세포배양액 내에 투여하고 1시간 뒤에 세포 내로 투과된 융합단백질의 양을 웨스턴 블랏으로 분석하고(그림 A), 활성도(그림 B)를 측정하였다.FIG. 4 is a graph showing the activity of the cell-permeability according to the concentration of Tat-catalase by Western blot and the specific activity thereof. To investigate the cytotoxicity of Tat-catalase, Tat-catalase was added to the culture medium of HeLa cells in an amount of 0.5 to 2 μM, and after one hour, the amount of the fusion protein permeated into the cells was analyzed by Western blotting (Fig. A) ) Were measured.
도 5는 농도 변화에 따라 PC12 세포 내로 투과된 Lys-카탈라제를 웨스턴 블랏으로, 활성도를 특이활성도 그래프로 나타낸 것이다. 그림 A는 PC12 세포 내로 0.5∼4μM Lys-카탈라제와 카탈라제를 세포배양액 내에 투여하고 3시간 후 세포 내로 투과된 융합단백질을 웨스턴 블랏으로 분석하였고 그림 B는 세포 내로 투과된 재조합 단백질의 활성도를 측정한 것이다.FIG. 5 is a specific activity graph showing the activity of Lys-catalase, which was permeated into PC12 cells according to concentration change, by Western blotting. Figure A shows the results of Western blot analysis of the fusion protein permeated into the cells after 0.5 h to 4 μM Lys-catalase and catalase in the cell culture medium, and Figure 3 shows the activity of the recombinant proteins permeated into the cells .
도 6은 시간별 PC12 세포 내로 투과된 Lys-카탈라제와 활성도를 나타낸 것이다. 그림 A는 4μM의 재조합 단백질을 0.5∼4시간동안 처리하여 세포 내로 투과된 융합단백질에 대해 웨스턴 블랏과 활성도(그림 B)를 측정하였다.Figure 6 shows the activity of Lys-catalase permeated into PC12 cells over time. Figure A shows the western blot and activity (Fig. B) of the fusion proteins permeated into the cells by treating 4 μM of the recombinant protein for 0.5 to 4 hours.
도 7은 HeLa 세포 내로 투과된 Tat-카탈라제의 세포 내 안정성을 웨스턴 블랏으로 나타낸 것이다. 세포 내로 투과된 Tat-카탈라제의 안정성을 측정하기 위하여 2μM의 Tat-카탈라제를 세포배양액 내에 투여하고, 시간별(0-60시간)로 세포 내에 남아 있는 융합단백질의 양을 웨스턴 블랏으로 분석하였다.Figure 7 shows the intracellular stability of Tat-Catalase permeated into HeLa cells in Western blot. In order to measure the stability of Tat-catalase penetrated into cells, 2 μM of Tat-Catalase was added into the cell culture medium and the amount of fusion protein remaining in the cells was analyzed by Western blotting over time (0-60 hours).
도 8은 PC12 세포 내로 투과된 Lys-카탈라제의 세포 내 안정성을 나타낸 것이다. 세포 내로 투과된 Lys-카탈라제의 안정성을 측정하기 위하여 4μM의 Lys-카탈라제를 세포배양액 내에 투여하고 시간별(0∼72시간)로 세포 내에 남아 있는 융합단백질의 양을 웨스턴 블랏(그림 A)과 활성도 측정(그림 B)으로 분석하였다.Fig. 8 shows intracellular stability of Lys-catalase permeated into PC12 cells. To measure the stability of Lys-catalase penetrated into cells, 4 μM of Lys-Catalase was added to the cell culture medium and the amount of fusion protein remaining in the cells (0-72 hours) was measured by Western blotting (Figure B).
도 9는 HeLa 세포 내로 투과된 Tat-카탈라제를 형광면역염색한 것이다. Tat-카탈라제의 세포 투과성을 조직학적으로 확인하기 위하여 2μM의 Tat-카탈라제와 카탈라제를 세포배양액 내에 투여하고 1시간 뒤에 세포 내로 투과된 융합단백질을 형광면역염색법으로 관찰하였다.9 is a fluorescence immunostaining of Tat-Catalase permeated into HeLa cells. To confirm the cytotoxicity of Tat-catalase, 2 μM of Tat-catalase and catalase were added to the cell culture medium. One hour later, the fusion protein permeated into the cells was observed by fluorescence immuno-staining.
도 10은 세포 내로 투과된 Lys-카탈라제에 의한 세포 생존 능력을 그래프로 나타낸 것이다. 세포 내로 투과된 Lys-카탈라제가 외부에서 첨가한 과산화수소에 의한 세포 생존율을 측정하기 위하여 농도별로(0.1-4uM) Lys-카탈라제와 카탈라제를 세포배양액 내에 투여하고 6시간 뒤에 과산화수소로 세포에 독성을 일으킨 후 MTT 어세이를 진행하였다.Fig. 10 is a graph showing cell viability by Lys-catalase permeated into cells. Lys-catalase and catalase (0.1-4 uM) were added to the cell culture medium to measure cell viability by hydrogen peroxide added externally to the cell, and after 6 hours, the cells were poisoned with hydrogen peroxide MTT assay was conducted.
도 11과 12는 Lys-카탈라제의 피부침투 효과를 나타낸 것이다. 복부 피부에 카탈라제와 Lys-카탈라제를 각각 0.3mg씩 도포한 후 시간별(30분, 1시간, 2시간)로 도살하여 조직 내로 투과된 재조합 단백질의 활성도를 측정하였고(도 12) 면역조직화학적 염색실험으로 피부내 침투효과를 분석하였다(도 11).Figures 11 and 12 show the skin penetration effect of Lys-Catalase. Catalase and Lys-catalase were applied to the abdominal skin at a dose of 0.3 mg, respectively, and then slaughtered for 30 minutes, 1 hour, 2 hours, and the activity of the recombinant protein permeated into the tissues was measured (FIG. 12). Immunohistochemical staining (Fig. 11).
도 13은 Tat-카탈라제를 코딩하는 재조합 폴리뉴클레오타이드 서열이다.Figure 13 is a recombinant polynucleotide sequence encoding Tat-catalase.
도 14는 Arg-카탈라제를 코딩하는 재조합 폴리뉴클레오타이드 서열이다.Figure 14 is a recombinant polynucleotide sequence encoding Arg-Catalase.
도 15는 Lys-카탈라제를 코딩하는 재조합 폴리뉴클레오타이드 서열이다.Figure 15 is a recombinant polynucleotide sequence encoding Lys-catalase.
<110> CHOI, SOO YOUNG KWON, HYUK-IL PARK, JIN-SEU KANG, JUNG-HOON WON, MOO-HO KANG, TAE-CHUN LEE, KIL SOO HAN, KYU-HYUNG <120> Cell-transducible catalase fusion protein and the use thereof <130> WJTJ-SHK-CAT <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Homo sapiens <400> 1 ctcgagatgg ctgacagccg ggatcccgcc 30 <210> 2 <211> 31 <212> DNA <213> Homo sapiens <400> 2 ggatcctcac agatttgccr rctcccttgc c 31 <210> 3 <211> 1624 <212> DNA <213> Artificial Sequence <220> <223> recombinant polynucleotide which consisting of a part coding HIV-1 Tat(49-57) and a part coding human liver catalase <220> <221> CDS <222> (2)..(1615) <400> 3 t agg aag aag cgg aga cag cga cga aga ctc gag atg gct gac 43 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp 1 5 10 agc cgg gat ccc gcc agc gac cag atg cag cac tgg aag gag cag cgg 91 Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg 15 20 25 30 gcc gcg cag aaa gct gat gtc ctg acc act gga gct ggt aac cca gta 139 Ala Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val 35 40 45 gga gac aaa ctt aat gtt att aca gta ggg ccc cgt ggg ccc ctt ctt 187 Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu 50 55 60 gtt cag aat gtg gtt ttc act gat gaa atg gct cat ttt gac cga gag 235 Val Gln Asn Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu 65 70 75 aga att cct gag aga gtt gtg cat gct aaa gga gca ggg gcc ttt ggc 283 Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly 80 85 90 tac ttt gag gtc aca cat gac att acc aaa tac tcc aag gca aag gta 331 Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val 95 100 105 110 ttt gag cat att gga aag aag act ccc atc gca gtt cgg ttc tcc act 379 Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr 115 120 125 gtt gct gga gaa tcg ggt tca gct gac aca gtt cgg gac cct cgt ggg 427 Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly 130 135 140 ttt gca gtg aaa ttt tac aca gaa gat ggt aac tgg gat ctc gtt gga 475 Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly 145 150 155 aat aac acc ccc att ttc ttc atc agg gat ccc ata ttg ttt cca tct 523 Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser 160 165 170 ttt atc cac agc caa aag aga aat cct cag aca cat ctg aag gat ccg 571 Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro 175 180 185 190 gac atg gtc tgg gac ttc tgg agc cta cgt cct gag tct ctg cat cag 619 Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln 195 200 205 gtt tct ttc ttg ttc agt gat cgg ggg att cca gat gga cat cgc cac 667 Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His 210 215 220 atg aat gga tat gga tca cat act ttc aag ctg gtt aat gca aat ggg 715 Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly 225 230 235 gag gca gtt tat tgc aaa ttc cat tat aag act ggc cag ggc atc aaa 763 Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Gly Gln Gly Ile Lys 240 245 250 aac ctt tct gtt gaa gat gcg gcg aga ctt tcc cag gaa gat cct gac 811 Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp 255 260 265 270 tat ggc atc cgg gat ctt ttt aac gcc att gcc aca gga aag gac ccc 859 Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Asp Pro 275 280 285 tcc tgg act ttt tac atc cag gtc atg aca ttt aat cag gca gaa act 907 Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr 290 295 300 ttt cca ttt aat cca ttc gat ctc acc agg gtt tgg cct cac aag gac 955 Phe Pro Phe Asn Pro Phe Asp Leu Thr Arg Val Trp Pro His Lys Asp 305 310 315 tac cct ctc atc cca gtt ggt aaa ctg gtc tta aac cgg aat cca gtt 1003 Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val 320 325 330 aat tac ttt gct gag gtt gaa cag ata gcc ttc gac cca agc aac atg 1051 Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met 335 340 345 350 cca cct ggc att gag gcc agt cct gac aaa atg ctt cag ggc cgc ctt 1099 Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu 355 360 365 ttt gcc tat cct gac act cac cgc cat cgc ctg gga ccc aat tat ctt 1147 Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu 370 375 380 cat ata cct gtg aac tgt ccc tac cgt gct cga gtg gcc aac tac cag 1195 His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln 385 390 395 cgt gac ggc ccg atg tgc atg cag gac aat cag ggt ggt gct cca aat 1243 Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn 400 405 410 tac tac ccc aac agc ttt ggt gct ccg gaa caa cag cct tct gcc ctg 1291 Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu 415 420 425 430 gag cac agc atc caa tat tct gga gaa gtg cgg aga ttc aac act gcc 1339 Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala 435 440 445 aat gat gat aac gtt act cag gtg cgg gca ttc tat gtg aac gtg ctg 1387 Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu 450 455 460 aat gag gaa cag agg aaa cgt ctg tgt gag aac att gcc ggc cac ctg 1435 Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu 465 470 475 aag gat gca caa att ttc atc cag aag aaa gcg gtc aag aac ttc act 1483 Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr 480 485 490 gag gtc cac cct gac tac ggg agc cac atc cag gct ctt ctg gac aag 1531 Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys 495 500 505 510 tac aat gct gag aag cct aag aat gcg att cac acc ttt gtg cgg tcc 1579 Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Arg Ser 515 520 525 gga tct cac ttg gtg gca agg gag aag gca aat ctg tgagg atcc 1624 Gly Ser His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 4 <211> 538 <212> PRT <213> Artificial Sequence <400> 4 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 Asn 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 Gly 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 Asp 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 Arg 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 Arg Ser Gly Ser 515 520 525 His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 5 <211> 1624 <212> DNA <213> Artificial Sequence <220> <223> recombinant polynucleotide which consisting of a part coding arginine 9mer and a part coding human liver catalase <220> <221> CDS <222> (2)..(1615) <400> 5 t aga cgc cgt cgc cgt cgc cgt cgc cgt ctc gag atg gct gac 43 Arg Arg Arg Arg Arg Arg Arg Arg Arg Leu Glu Met Ala Asp 1 5 10 agc cgg gat ccc gcc agc gac cag atg cag cac tgg aag gag cag cgg 91 Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg 15 20 25 30 gcc gcg cag aaa gct gat gtc ctg acc act gga gct ggt aac cca gta 139 Ala Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val 35 40 45 gga gac aaa ctt aat gtt att aca gta ggg ccc cgt ggg ccc ctt ctt 187 Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu 50 55 60 gtt cag aat gtg gtt ttc act gat gaa atg gct cat ttt gac cga gag 235 Val Gln Asn Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu 65 70 75 aga att cct gag aga gtt gtg cat gct aaa gga gca ggg gcc ttt ggc 283 Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly 80 85 90 tac ttt gag gtc aca cat gac att acc aaa tac tcc aag gca aag gta 331 Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val 95 100 105 110 ttt gag cat att gga aag aag act ccc atc gca gtt cgg ttc tcc act 379 Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr 115 120 125 gtt gct gga gaa tcg ggt tca gct gac aca gtt cgg gac cct cgt ggg 427 Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly 130 135 140 ttt gca gtg aaa ttt tac aca gaa gat ggt aac tgg gat ctc gtt gga 475 Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly 145 150 155 aat aac acc ccc att ttc ttc atc agg gat ccc ata ttg ttt cca tct 523 Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser 160 165 170 ttt atc cac agc caa aag aga aat cct cag aca cat ctg aag gat ccg 571 Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro 175 180 185 190 gac atg gtc tgg gac ttc tgg agc cta cgt cct gag tct ctg cat cag 619 Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln 195 200 205 gtt tct ttc ttg ttc agt gat cgg ggg att cca gat gga cat cgc cac 667 Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His 210 215 220 atg aat gga tat gga tca cat act ttc aag ctg gtt aat gca aat ggg 715 Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly 225 230 235 gag gca gtt tat tgc aaa ttc cat tat aag act ggc cag ggc atc aaa 763 Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Gly Gln Gly Ile Lys 240 245 250 aac ctt tct gtt gaa gat gcg gcg aga ctt tcc cag gaa gat cct gac 811 Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp 255 260 265 270 tat ggc atc cgg gat ctt ttt aac gcc att gcc aca gga aag gac ccc 859 Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Asp Pro 275 280 285 tcc tgg act ttt tac atc cag gtc atg aca ttt aat cag gca gaa act 907 Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr 290 295 300 ttt cca ttt aat cca ttc gat ctc acc agg gtt tgg cct cac aag gac 955 Phe Pro Phe Asn Pro Phe Asp Leu Thr Arg Val Trp Pro His Lys Asp 305 310 315 tac cct ctc atc cca gtt ggt aaa ctg gtc tta aac cgg aat cca gtt 1003 Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val 320 325 330 aat tac ttt gct gag gtt gaa cag ata gcc ttc gac cca agc aac atg 1051 Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met 335 340 345 350 cca cct ggc att gag gcc agt cct gac aaa atg ctt cag ggc cgc ctt 1099 Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu 355 360 365 ttt gcc tat cct gac act cac cgc cat cgc ctg gga ccc aat tat ctt 1147 Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu 370 375 380 cat ata cct gtg aac tgt ccc tac cgt gct cga gtg gcc aac tac cag 1195 His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln 385 390 395 cgt gac ggc ccg atg tgc atg cag gac aat cag ggt ggt gct cca aat 1243 Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn 400 405 410 tac tac ccc aac agc ttt ggt gct ccg gaa caa cag cct tct gcc ctg 1291 Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu 415 420 425 430 gag cac agc atc caa tat tct gga gaa gtg cgg aga ttc aac act gcc 1339 Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala 435 440 445 aat gat gat aac gtt act cag gtg cgg gca ttc tat gtg aac gtg ctg 1387 Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu 450 455 460 aat gag gaa cag agg aaa cgt ctg tgt gag aac att gcc ggc cac ctg 1435 Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu 465 470 475 aag gat gca caa att ttc atc cag aag aaa gcg gtc aag aac ttc act 1483 Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr 480 485 490 gag gtc cac cct gac tac ggg agc cac atc cag gct ctt ctg gac aag 1531 Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys 495 500 505 510 tac aat gct gag aag cct aag aat gcg att cac acc ttt gtg cgg tcc 1579 Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Arg Ser 515 520 525 gga tct cac ttg gtg gca agg gag aag gca aat ctg tgagg atcc 1624 Gly Ser His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 6 <211> 538 <212> PRT <213> Artificial Sequence <400> 6 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 Asn 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 Gly 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 Asp 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 Arg 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 Arg Ser Gly Ser 515 520 525 His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 7 <211> 1624 <212> DNA <213> Artificial Sequence <220> <223> recombinant polynucleotide which consisting of a part coding lysine 9mer and a part coding human liver catalase <220> <221> CDS <222> (2)..(1615) <400> 7 t aag aag aaa aag aaa aag aaa aag aag ctc gag atg gct gac 43 Lys Lys Lys Lys Lys Lys Lys Lys Lys Leu Glu Met Ala Asp 1 5 10 agc cgg gat ccc gcc agc gac cag atg cag cac tgg aag gag cag cgg 91 Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg 15 20 25 30 gcc gcg cag aaa gct gat gtc ctg acc act gga gct ggt aac cca gta 139 Ala Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val 35 40 45 gga gac aaa ctt aat gtt att aca gta ggg ccc cgt ggg ccc ctt ctt 187 Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu 50 55 60 gtt cag aat gtg gtt ttc act gat gaa atg gct cat ttt gac cga gag 235 Val Gln Asn Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu 65 70 75 aga att cct gag aga gtt gtg cat gct aaa gga gca ggg gcc ttt ggc 283 Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly 80 85 90 tac ttt gag gtc aca cat gac att acc aaa tac tcc aag gca aag gta 331 Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val 95 100 105 110 ttt gag cat att gga aag aag act ccc atc gca gtt cgg ttc tcc act 379 Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr 115 120 125 gtt gct gga gaa tcg ggt tca gct gac aca gtt cgg gac cct cgt ggg 427 Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly 130 135 140 ttt gca gtg aaa ttt tac aca gaa gat ggt aac tgg gat ctc gtt gga 475 Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly 145 150 155 aat aac acc ccc att ttc ttc atc agg gat ccc ata ttg ttt cca tct 523 Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser 160 165 170 ttt atc cac agc caa aag aga aat cct cag aca cat ctg aag gat ccg 571 Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro 175 180 185 190 gac atg gtc tgg gac ttc tgg agc cta cgt cct gag tct ctg cat cag 619 Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln 195 200 205 gtt tct ttc ttg ttc agt gat cgg ggg att cca gat gga cat cgc cac 667 Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His 210 215 220 atg aat gga tat gga tca cat act ttc aag ctg gtt aat gca aat ggg 715 Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly 225 230 235 gag gca gtt tat tgc aaa ttc cat tat aag act ggc cag ggc atc aaa 763 Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Gly Gln Gly Ile Lys 240 245 250 aac ctt tct gtt gaa gat gcg gcg aga ctt tcc cag gaa gat cct gac 811 Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp 255 260 265 270 tat ggc atc cgg gat ctt ttt aac gcc att gcc aca gga aag gac ccc 859 Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Asp Pro 275 280 285 tcc tgg act ttt tac atc cag gtc atg aca ttt aat cag gca gaa act 907 Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr 290 295 300 ttt cca ttt aat cca ttc gat ctc acc agg gtt tgg cct cac aag gac 955 Phe Pro Phe Asn Pro Phe Asp Leu Thr Arg Val Trp Pro His Lys Asp 305 310 315 tac cct ctc atc cca gtt ggt aaa ctg gtc tta aac cgg aat cca gtt 1003 Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val 320 325 330 aat tac ttt gct gag gtt gaa cag ata gcc ttc gac cca agc aac atg 1051 Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met 335 340 345 350 cca cct ggc att gag gcc agt cct gac aaa atg ctt cag ggc cgc ctt 1099 Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu 355 360 365 ttt gcc tat cct gac act cac cgc cat cgc ctg gga ccc aat tat ctt 1147 Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu 370 375 380 cat ata cct gtg aac tgt ccc tac cgt gct cga gtg gcc aac tac cag 1195 His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln 385 390 395 cgt gac ggc ccg atg tgc atg cag gac aat cag ggt ggt gct cca aat 1243 Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn 400 405 410 tac tac ccc aac agc ttt ggt gct ccg gaa caa cag cct tct gcc ctg 1291 Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu 415 420 425 430 gag cac agc atc caa tat tct gga gaa gtg cgg aga ttc aac act gcc 1339 Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala 435 440 445 aat gat gat aac gtt act cag gtg cgg gca ttc tat gtg aac gtg ctg 1387 Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu 450 455 460 aat gag gaa cag agg aaa cgt ctg tgt gag aac att gcc ggc cac ctg 1435 Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu 465 470 475 aag gat gca caa att ttc atc cag aag aaa gcg gtc aag aac ttc act 1483 Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr 480 485 490 gag gtc cac cct gac tac ggg agc cac atc cag gct ctt ctg gac aag 1531 Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys 495 500 505 510 tac aat gct gag aag cct aag aat gcg att cac acc ttt gtg cgg tcc 1579 Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Arg Ser 515 520 525 gga tct cac ttg gtg gca agg gag aag gca aat ctg tgagg atcc 1624 Gly Ser His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 8 <211> 538 <212> PRT <213> Artificial Sequence <400> 8 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 Asn 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 Gly 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 Asp 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 Arg 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 Arg Ser Gly Ser 515 520 525 His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535<110> CHOI, SOO YOUNG KWON, HYUK-IL PARK, JIN-SEU KANG, JUNG-HOON WON, MOO-HO KANG, TAE-CHUN LEE, KIL SOO HAN, KYU-HYUNG <120> Cell-transducible catalase fusion protein and the use thereof <130> WJTJ-SHK-CAT <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 30 <212> DNA <213> Homo sapiens <400> 1 ctcgagatgg ctgacagccg ggatcccgcc 30 <210> 2 <211> 31 <212> DNA <213> Homo sapiens <400> 2 ggatcctcac agatttgccr rctcccttgc c 31 <210> 3 <211> 1624 <212> DNA <213> Artificial Sequence <220> <223> recombinant polynucleotide which consists of a part coding HIV-1 Tat (49-57) and a part coding human liver catalase <220> <221> CDS ≪ 222 > (2) .. (1615) <400> 3 t agg aag aag cgg aga cag cga cga aga ctc gag atg gct gac 43 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp 1 5 10 agc cgg gat ccc gcc agc gac cag atg cag cac tgg aag gag cag cgg 91 Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg 15 20 25 30 gcc gcg cag aaa gct gat gtc ctg acc act gga gct ggt aac cca gta 139 Ala Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val 35 40 45 gga gac aaa ctt aat gtt att aca gta ggg ccc cgt ggg ccc ctt ctt 187 Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu 50 55 60 gtt cag aat gtg gtt ttc act gat gaa atg gct cat ttt gac cga gag 235 Val Gln Asn Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu 65 70 75 aga att cct gag aga gtt gtg cat gct aaa gga gca ggg gcc ttt ggc 283 Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly 80 85 90 tac ttt gag gtc aca cat gac att acc aaa tac tcc aag gca aag gta 331 Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val 95 100 105 110 ttt gag cat att gga aag act ccc atc gca gtt cgg ttc tcc act 379 Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr 115 120 125 gtt gct gga gaa tcg ggt tca gct gac aca gtt cgg gac cct cgt ggg 427 Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly 130 135 140 ttt gca gtg aaa ttt tac aca gaa gat ggt aac tgg gat ctc gtt gga 475 Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly 145 150 155 aat aac acc ccc att ttc ttc atc agg gat ccc ata ttg ttt cca tct 523 Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser 160 165 170 ttt atc cac agc caa aag aga aat cct cag acac cat ctg aag gat ccg 571 Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro 175 180 185 190 gac atg gtc tgg gac ttc tgg agc ct cgt cct gag tct ctg cat cag 619 Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln 195 200 205 gtt tct ttc ttc ttc agt gat cgg ggg att cca gat gga cat cgc cac 667 Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His 210 215 220 atg aat gga tat gga tta cat act ttc aag ctg gtt aat gca aat ggg 715 Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly 225 230 235 gag gca gtt tat tgc aaa ttc cat tat aag act ggc cag ggc atc aaa 763 Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Gly Gln Gly Ile Lys 240 245 250 aac ctt tct gtt gaa gat gcg gcg aga ctt tcc cag gaa gat cct gac 811 Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp 255 260 265 270 tat ggc atc cgg gat ctt ttt aac gcc att gcc aca gga aag gac ccc 859 Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Asp Pro 275 280 285 tcc tgg act ttt tac atc cag gtc atg aca ttt aat cag gca gaa act 907 Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr 290 295 300 ttt cca ttt aat cca ttc gat ctc acc agg gtt tgg cct cac aag gac 955 Phe Pro Phe Asn Pro Phe Asp Leu Thr Arg Val Trp Pro His Lys Asp 305 310 315 tac cct ctc atc cca gtt ggt aaa ctg gtc tta aac cgg aat cca gtt 1003 Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val 320 325 330 aat tac ttt gct gag gta gaa cag ata gcc ttc gac cca agc aac atg 1051 Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met 335 340 345 350 cca cct ggc att gag gcc agt cct gac aaa atg ctt cag ggc cgc ctt 1099 Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu 355 360 365 ttt gcc tat cct gac act cac cgc cat cgc ctg gga ccc aat tat ctt 1147 Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu 370 375 380 cat ata cct gtg aac tgt ccc tac cgt gct cga gtg gcc aac tac cag 1195 His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln 385 390 395 cgt gac ggc ccg atg tgc atg cag gac at cag ggt ggt gct cca aat 1243 Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn 400 405 410 tac tac ccc aac agc ttt ggt gct ccg gaa caa cag cct tct gcc ctg 1291 Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu 415 420 425 430 gag cac agc atc caa tat tct gga gaa gtg cgg aga ttc aac act gcc 1339 Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala 435 440 445 aat gat gat aac gtt act cag gtg cgg gca ttc tat gtg aac gtg ctg 1387 Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu 450 455 460 aat gag gaa cag agg aaa cgt ctg tgt gag aac att gcc ggc cac ctg 1435 Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu 465 470 475 aag gat caa att ttc atc cag aag aaa gcg gtc aag aac ttc act 1483 Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr 480 485 490 gag gtc cac cct gac tac ggg agc cac atc cag gct ctt ctg gac aag 1531 Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys 495 500 505 510 tac aat gct gag aag cct aag aat gcg att cac acc ttt gtg cgg tcc 1579 Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Arg Ser 515 520 525 gga tct cac ttg gtg gca agg gag aag gca aat ctg tgagg atcc 1624 Gly Ser His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 4 <211> 538 <212> PRT <213> Artificial Sequence <400> 4 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 Asn 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 Gly 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 Asp 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 Arg 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 Ale Ile His Thr Phe Val Arg Ser Gly Ser 515 520 525 His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 5 <211> 1624 <212> DNA <213> Artificial Sequence <220> <223> recombinant polynucleotide which consists of a part coding arginine 9mer and a part coding human liver catalase <220> <221> CDS ≪ 222 > (2) .. (1615) <400> 5 t aga cgc cgt cgc cgt cgc cgt cgc cgt ctc gag atg gct gac 43 Arg Arg Arg Arg Arg Arg Arg Arg Leu Glu Met Ala Asp 1 5 10 agc cgg gat ccc gcc agc gac cag atg cag cac tgg aag gag cag cgg 91 Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg 15 20 25 30 gcc gcg cag aaa gct gat gtc ctg acc act gga gct ggt aac cca gta 139 Ala Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val 35 40 45 gga gac aaa ctt aat gtt att aca gta ggg ccc cgt ggg ccc ctt ctt 187 Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu 50 55 60 gtt cag aat gtg gtt ttc act gat gaa atg gct cat ttt gac cga gag 235 Val Gln Asn Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu 65 70 75 aga att cct gag aga gtt gtg cat gct aaa gga gca ggg gcc ttt ggc 283 Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly 80 85 90 tac ttt gag gtc aca cat gac att acc aaa tac tcc aag gca aag gta 331 Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val 95 100 105 110 ttt gag cat att gga aag act ccc atc gca gtt cgg ttc tcc act 379 Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr 115 120 125 gtt gct gga gaa tcg ggt tca gct gac aca gtt cgg gac cct cgt ggg 427 Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly 130 135 140 ttt gca gtg aaa ttt tac aca gaa gat ggt aac tgg gat ctc gtt gga 475 Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly 145 150 155 aat aac acc ccc att ttc ttc atc agg gat ccc ata ttg ttt cca tct 523 Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser 160 165 170 ttt atc cac agc caa aag aga aat cct cag acac cat ctg aag gat ccg 571 Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro 175 180 185 190 gac atg gtc tgg gac ttc tgg agc ct cgt cct gag tct ctg cat cag 619 Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln 195 200 205 gtt tct ttc ttc ttc agt gat cgg ggg att cca gat gga cat cgc cac 667 Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His 210 215 220 atg aat gga tat gga tta cat act ttc aag ctg gtt aat gca aat ggg 715 Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly 225 230 235 gag gca gtt tat tgc aaa ttc cat tat aag act ggc cag ggc atc aaa 763 Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Gly Gln Gly Ile Lys 240 245 250 aac ctt tct gtt gaa gat gcg gcg aga ctt tcc cag gaa gat cct gac 811 Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp 255 260 265 270 tat ggc atc cgg gat ctt ttt aac gcc att gcc aca gga aag gac ccc 859 Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Asp Pro 275 280 285 tcc tgg act ttt tac atc cag gtc atg aca ttt aat cag gca gaa act 907 Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr 290 295 300 ttt cca ttt aat cca ttc gat ctc acc agg gtt tgg cct cac aag gac 955 Phe Pro Phe Asn Pro Phe Asp Leu Thr Arg Val Trp Pro His Lys Asp 305 310 315 tac cct ctc atc cca gtt ggt aaa ctg gtc tta aac cgg aat cca gtt 1003 Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val 320 325 330 aat tac ttt gct gag gta gaa cag ata gcc ttc gac cca agc aac atg 1051 Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met 335 340 345 350 cca cct ggc att gag gcc agt cct gac aaa atg ctt cag ggc cgc ctt 1099 Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu 355 360 365 ttt gcc tat cct gac act cac cgc cat cgc ctg gga ccc aat tat ctt 1147 Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu 370 375 380 cat ata cct gtg aac tgt ccc tac cgt gct cga gtg gcc aac tac cag 1195 His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln 385 390 395 cgt gac ggc ccg atg tgc atg cag gac at cag ggt ggt gct cca aat 1243 Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn 400 405 410 tac tac ccc aac agc ttt ggt gct ccg gaa caa cag cct tct gcc ctg 1291 Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu 415 420 425 430 gag cac agc atc caa tat tct gga gaa gtg cgg aga ttc aac act gcc 1339 Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala 435 440 445 aat gat gat aac gtt act cag gtg cgg gca ttc tat gtg aac gtg ctg 1387 Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu 450 455 460 aat gag gaa cag agg aaa cgt ctg tgt gag aac att gcc ggc cac ctg 1435 Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu 465 470 475 aag gat caa att ttc atc cag aag aaa gcg gtc aag aac ttc act 1483 Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr 480 485 490 gag gtc cac cct gac tac ggg agc cac atc cag gct ctt ctg gac aag 1531 Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys 495 500 505 510 tac aat gct gag aag cct aag aat gcg att cac acc ttt gtg cgg tcc 1579 Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Arg Ser 515 520 525 gga tct cac ttg gtg gca agg gag aag gca aat ctg tgagg atcc 1624 Gly Ser His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 6 <211> 538 <212> PRT <213> Artificial Sequence <400> 6 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 Asn 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 Gly 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 Asp 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 Arg 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 Ale Ile His Thr Phe Val Arg Ser Gly Ser 515 520 525 His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 7 <211> 1624 <212> DNA <213> Artificial Sequence <220> <223> recombinant polynucleotide which consists of a part coding lysine 9mer and a part coding human liver catalase <220> <221> CDS ≪ 222 > (2) .. (1615) <400> 7 t aag aag aaa aag aaa aag aaa aag aag ctc gag atg gct gac 43 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Leu Glu Met Ala Asp 1 5 10 agc cgg gat ccc gcc agc gac cag atg cag cac tgg aag gag cag cgg 91 Ser Arg Asp Pro Ala Ser Asp Gln Met Gln His Trp Lys Glu Gln Arg 15 20 25 30 gcc gcg cag aaa gct gat gtc ctg acc act gga gct ggt aac cca gta 139 Ala Ala Gln Lys Ala Asp Val Leu Thr Thr Gly Ala Gly Asn Pro Val 35 40 45 gga gac aaa ctt aat gtt att aca gta ggg ccc cgt ggg ccc ctt ctt 187 Gly Asp Lys Leu Asn Val Ile Thr Val Gly Pro Arg Gly Pro Leu Leu 50 55 60 gtt cag aat gtg gtt ttc act gat gaa atg gct cat ttt gac cga gag 235 Val Gln Asn Val Val Phe Thr Asp Glu Met Ala His Phe Asp Arg Glu 65 70 75 aga att cct gag aga gtt gtg cat gct aaa gga gca ggg gcc ttt ggc 283 Arg Ile Pro Glu Arg Val Val His Ala Lys Gly Ala Gly Ala Phe Gly 80 85 90 tac ttt gag gtc aca cat gac att acc aaa tac tcc aag gca aag gta 331 Tyr Phe Glu Val Thr His Asp Ile Thr Lys Tyr Ser Lys Ala Lys Val 95 100 105 110 ttt gag cat att gga aag act ccc atc gca gtt cgg ttc tcc act 379 Phe Glu His Ile Gly Lys Lys Thr Pro Ile Ala Val Arg Phe Ser Thr 115 120 125 gtt gct gga gaa tcg ggt tca gct gac aca gtt cgg gac cct cgt ggg 427 Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp Pro Arg Gly 130 135 140 ttt gca gtg aaa ttt tac aca gaa gat ggt aac tgg gat ctc gtt gga 475 Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp Leu Val Gly 145 150 155 aat aac acc ccc att ttc ttc atc agg gat ccc ata ttg ttt cca tct 523 Asn Asn Thr Pro Ile Phe Phe Ile Arg Asp Pro Ile Leu Phe Pro Ser 160 165 170 ttt atc cac agc caa aag aga aat cct cag acac cat ctg aag gat ccg 571 Phe Ile His Ser Gln Lys Arg Asn Pro Gln Thr His Leu Lys Asp Pro 175 180 185 190 gac atg gtc tgg gac ttc tgg agc ct cgt cct gag tct ctg cat cag 619 Asp Met Val Trp Asp Phe Trp Ser Leu Arg Pro Glu Ser Leu His Gln 195 200 205 gtt tct ttc ttc ttc agt gat cgg ggg att cca gat gga cat cgc cac 667 Val Ser Phe Leu Phe Ser Asp Arg Gly Ile Pro Asp Gly His Arg His 210 215 220 atg aat gga tat gga tta cat act ttc aag ctg gtt aat gca aat ggg 715 Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn Ala Asn Gly 225 230 235 gag gca gtt tat tgc aaa ttc cat tat aag act ggc cag ggc atc aaa 763 Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Gly Gln Gly Ile Lys 240 245 250 aac ctt tct gtt gaa gat gcg gcg aga ctt tcc cag gaa gat cct gac 811 Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gln Glu Asp Pro Asp 255 260 265 270 tat ggc atc cgg gat ctt ttt aac gcc att gcc aca gga aag gac ccc 859 Tyr Gly Ile Arg Asp Leu Phe Asn Ala Ile Ala Thr Gly Lys Asp Pro 275 280 285 tcc tgg act ttt tac atc cag gtc atg aca ttt aat cag gca gaa act 907 Ser Trp Thr Phe Tyr Ile Gln Val Met Thr Phe Asn Gln Ala Glu Thr 290 295 300 ttt cca ttt aat cca ttc gat ctc acc agg gtt tgg cct cac aag gac 955 Phe Pro Phe Asn Pro Phe Asp Leu Thr Arg Val Trp Pro His Lys Asp 305 310 315 tac cct ctc atc cca gtt ggt aaa ctg gtc tta aac cgg aat cca gtt 1003 Tyr Pro Leu Ile Pro Val Gly Lys Leu Val Leu Asn Arg Asn Pro Val 320 325 330 aat tac ttt gct gag gta gaa cag ata gcc ttc gac cca agc aac atg 1051 Asn Tyr Phe Ala Glu Val Glu Gln Ile Ala Phe Asp Pro Ser Asn Met 335 340 345 350 cca cct ggc att gag gcc agt cct gac aaa atg ctt cag ggc cgc ctt 1099 Pro Pro Gly Ile Glu Ala Ser Pro Asp Lys Met Leu Gln Gly Arg Leu 355 360 365 ttt gcc tat cct gac act cac cgc cat cgc ctg gga ccc aat tat ctt 1147 Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro Asn Tyr Leu 370 375 380 cat ata cct gtg aac tgt ccc tac cgt gct cga gtg gcc aac tac cag 1195 His Ile Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala Asn Tyr Gln 385 390 395 cgt gac ggc ccg atg tgc atg cag gac at cag ggt ggt gct cca aat 1243 Arg Asp Gly Pro Met Cys Met Gln Asp Asn Gln Gly Gly Ala Pro Asn 400 405 410 tac tac ccc aac agc ttt ggt gct ccg gaa caa cag cct tct gcc ctg 1291 Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gln Gln Pro Ser Ala Leu 415 420 425 430 gag cac agc atc caa tat tct gga gaa gtg cgg aga ttc aac act gcc 1339 Glu His Ser Ile Gln Tyr Ser Gly Glu Val Arg Arg Phe Asn Thr Ala 435 440 445 aat gat gat aac gtt act cag gtg cgg gca ttc tat gtg aac gtg ctg 1387 Asn Asp Asp Asn Val Thr Gln Val Arg Ala Phe Tyr Val Asn Val Leu 450 455 460 aat gag gaa cag agg aaa cgt ctg tgt gag aac att gcc ggc cac ctg 1435 Asn Glu Glu Gln Arg Lys Arg Leu Cys Glu Asn Ile Ala Gly His Leu 465 470 475 aag gat caa att ttc atc cag aag aaa gcg gtc aag aac ttc act 1483 Lys Asp Ala Gln Ile Phe Ile Gln Lys Lys Ala Val Lys Asn Phe Thr 480 485 490 gag gtc cac cct gac tac ggg agc cac atc cag gct ctt ctg gac aag 1531 Glu Val His Pro Asp Tyr Gly Ser His Ile Gln Ala Leu Leu Asp Lys 495 500 505 510 tac aat gct gag aag cct aag aat gcg att cac acc ttt gtg cgg tcc 1579 Tyr Asn Ala Glu Lys Pro Lys Asn Ala Ile His Thr Phe Val Arg Ser 515 520 525 gga tct cac ttg gtg gca agg gag aag gca aat ctg tgagg atcc 1624 Gly Ser His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535 <210> 8 <211> 538 <212> PRT <213> Artificial Sequence <400> 8 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 Asn 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 Gly 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 Asp 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 Arg 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 Ale Ile His Thr Phe Val Arg Ser Gly Ser 515 520 525 His Leu Val Ala Arg Glu Lys Ala Asn Leu 530 535
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KR10-2001-0014147A KR100495139B1 (en) | 2001-03-19 | 2001-03-19 | Cell-transducible catalase fusion protein and the use thereof |
AU2001260729A AU2001260729A1 (en) | 2000-07-26 | 2001-05-21 | Oligolysine transducing domain, oligolysine-cargo molecule complex and uses thereof |
PCT/KR2001/000835 WO2002010219A1 (en) | 2000-07-26 | 2001-05-21 | Oligolysine transducing domain, oligolysine-cargo molecule complex and uses thereof |
US10/333,578 US20040058856A1 (en) | 2000-07-26 | 2001-05-21 | Oligolysine transducing domain, oligolysine-cargo molecule complex and uses thereof |
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KR100495140B1 (en) * | 2003-02-20 | 2005-06-14 | 학교법인 한림대학교 | Cell-transducing transport domain fusion protein and use thereof |
KR100612673B1 (en) * | 2004-08-20 | 2006-08-14 | (주)바이오버드 | Cell-transducing botoxin fusion protein |
KR101962067B1 (en) * | 2018-04-23 | 2019-03-25 | 한림대학교 산학협력단 | Phmaceutical composition for treating Parkinson's disease containing TXNL1 fusion protein |
KR102077762B1 (en) * | 2018-07-27 | 2020-02-17 | 강원대학교산학협력단 | Composition for prevention or treatment of chronic obstructive pulmonary disease comprising PTD-bFGF or incresed exosome by PTD-bFGF |
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