KR101578494B1 - Antiaging cosmetic composition containing cell-transducible carbonyl fusion protein and pozzolan - Google Patents

Antiaging cosmetic composition containing cell-transducible carbonyl fusion protein and pozzolan Download PDF

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KR101578494B1
KR101578494B1 KR1020140060339A KR20140060339A KR101578494B1 KR 101578494 B1 KR101578494 B1 KR 101578494B1 KR 1020140060339 A KR1020140060339 A KR 1020140060339A KR 20140060339 A KR20140060339 A KR 20140060339A KR 101578494 B1 KR101578494 B1 KR 101578494B1
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fusion protein
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최수영
박진서
유동환
이영승
김대원
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한림대학교 산학협력단
주식회사 지한
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
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Abstract

인간 카보닐기 환원효소 1 (CBR1)은 NADPH-의존적 짧은 사슬 탈수소효소/환원효소 수퍼패밀리의 구성원이며, 이 패밀리는 항산화 기능을 통하여 신경세포 생존에 중요한 역할을 수행하는 것으로 알려져 있다. 본 발명자들은 세포투과성 PEP-1-CBR1 융합단백질을 제조하여 세포 및 조직 내로 투과하여 LPS 또는 과산화수소로 유도되는 염증 및 세포사멸에 보호작용을 나타내는지를 시험하였다. 그 결과, PEP-1-CBR1 융합단백질은 효과적으로 HaCaT 세포 내로 투과하여 과산화수소로 유도되는 염증 및 세포사멸에 대해 현저한 세포 보호작용을 나타내었다. 뿐만 아니라, 포졸란을 전처리하였을 경우 세포 사멸기전에 대한 보호효과가 더 월등함을 알 수 있었다. PEP-1-CBR1 융합단백질 및 포졸란이 항산화 기능성 화장료로 이용 가능함을 밝혔다.Human Carbonyl Reductase 1 (CBR1) is a member of the NADPH-dependent short chain dehydrogenase / reductase superfamily, which is known to play an important role in nerve cell survival through antioxidant function. The present inventors prepared a cell permeable PEP-1-CBR1 fusion protein, and examined whether it exhibited protection against inflammation and apoptosis induced by LPS or hydrogen peroxide by permeating into cells and tissues. As a result, the PEP-1-CBR1 fusion protein effectively permeated into HaCaT cells and showed remarkable cytoprotective action against hydrogen peroxide induced inflammation and apoptosis. In addition, the pre - treatment of pozzolan showed a superior protective effect against apoptosis mechanism. PEP-1-CBR1 fusion protein and pozzolan are available as antioxidant functional cosmetics.

Description

세포투과성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물{Antiaging cosmetic composition containing cell-transducible carbonyl fusion protein and pozzolan}The present invention relates to an anti-aging functional cosmetic composition containing cell-permeable carbonyl group reducing enzyme fusion protein and pozzolan,

본 발명은 세포침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물에 관한 것으로, 카보닐기 환원효소의 C-말단 및/또는 N-말단에 단백질 수송도메인이 공유결합된 융합단백질과 포졸란이 세포사멸 보호효과를 나타냄을 통하여 세포투과성 카보닐기 환원효소 융합단백질이 항노화 기능성 화장료 용도로 이용 가능함을 밝혔다.The present invention relates to an anti-aging functional cosmetic composition comprising a cell permeable carbonyl group reductase fusion protein and a pozzolan, and is characterized by comprising a fusion protein in which a protein transport domain is covalently bonded to the C-terminal and / or N-terminal of a carbonyl group- Pozzolan shows protective effect on apoptosis, suggesting that the cell permeable carbonyl group reductase fusion protein can be used as an anti-aging functional cosmetic.

광노화는 피부가 자외선 등의 광에 반복적으로 노출되어 피부의 외양 또는 기능이 변화되는 것을 의미한다[Ridder GM등: J. Am. Acad. Dermatol., 25, 751-760(1991)]. 좀더 구체적으로 말하자면, 광노화는 자외선에 의해 피부의 주요 구성물질인 지질, 단백질, 다당류 및 핵산 등이 손상되어 피부세포 및 조직이 파괴되고, 결국 피부노화 현상이 생겨나는 것이다. 특히 피부의 결합조직인 콜라겐, 히알루론산, 엘라스틴, 프로테오글라이칸, 피브로넥틴 등이 절단되어 피부 탄력에 지장을 주고 광노화의 주요 현상인 깊은 주름을 형성하게 되고 이것이 더 심해질 경우 DNA 변이에 의해 돌연변이, 암 유발, 면역기능 저하의 사태에 이르게 된다.Photoaging means that the skin is repeatedly exposed to light such as ultraviolet light to change the appearance or function of the skin [Ridder GM et al .: J. Am. Acad. Dermatol., 25, 751-760 (1991)]. More specifically, photoaging is the damage of lipids, proteins, polysaccharides and nucleic acids which are the main constituents of the skin due to ultraviolet rays, resulting in the destruction of skin cells and tissues, resulting in skin aging. In particular, collagen, hyaluronic acid, elastin, proteoglycan, and fibronectin, which are the connective tissues of the skin, are severed to interfere with skin elasticity and form deep wrinkles, which are the main phenomenon of photoaging. When this becomes worse, mutations, And the immune function is reduced.

생체 내에서 콜라겐과 같은 세포외기질의 합성과 분해는 적절하게 조절되지만 노화가 진행되면서 특히 자외선에 만성적으로 노출되는 부위에서는 그 합성이 감소하고 분해는 촉진되는데, 자외선 등에 의해 콜라겐을 분해하는 효소인 기질금속단백질 분해효소(matrix metalloproteinase, 이하 ‘MMP’라 칭함) 발현이 촉진되어 피부 탄력이 저하되고 주름이 형성된다.
Although the synthesis and degradation of extracellular matrix such as collagen in vivo is appropriately controlled, the synthesis is reduced and the decomposition is promoted at a site where chronic exposure to ultraviolet rays is particularly evident as the aging proceeds. The degradation is promoted by ultraviolet rays or the like, Expression of matrix metalloproteinase (hereinafter referred to as 'MMP') is promoted, and skin elasticity is lowered and wrinkles are formed.

인간 카보닐기 환원효소 1 (carbonyl reductase 1; CBR1)은 NADPH-의존적이고, 모노머이며, 짧은 사슬 탈수소효소/환원효소 수퍼패밀리에 속하는 세포질효소이다. CBR1은 간, 신장 및 신경세포를 포함하는 다양한 인간 조직에 분포한다 [Forrest, G. L.; Gonzalez, B. Carbonyl reductase. Chem . Biol . Interact . 129: 21-40; 2000, Rashid, M. A.; Lee, S.; Tak, E.; Lee, J.; Choi, T. G.; Lee, J. W.; Kim, J. B.; Youn, J. H.; Kang, I.; Ha, J.; Kim, S. S. Carbonyl reductase 1 protects pancreatic β-cells against oxidative stress-induced apoptosis in glucotoxicity and glucolipotoxicity. Free Radic . Biol . Med . 49: 1522-1533; 2010]. 몇몇 연구결과는 CBR1이 산화 스트레스, 신경퇴화 및 세포자기사멸에서 보호 역할을 수행함을 보여준다 [Ismail, E.; Al-Mulla, F.; Tsuchida, S.; Suto, K.; Motley, P.; Harrison, P.R.; Birnie, G. D. Carbonyl reductase: a novel metastasis-modulating function. Cancer Res . 60: 1173-1176; 2000, Botella, J. A.; Ulschmid, J. K.; Gruenewald, C.; Moehle, C.; Kretzschmar, D.; Becker, K.; Schneuwly, S. The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration. Curr . Biol . 14: 782-786; 2004]. 뿐만 아니라, CBR1은 세포에서 산화스트레스 동안 지방 알데하이드를 불활성화한다. 따라서, CBR1은 산화 스트레스에서 유래하는 세포손상에 관한 보호역할을 수행하는 것으로 보인다 [Doorn, J. A.; Master, E.; Blum, A.; Claffey, D.J.; Petersen, D. R. Human carbonyl reductase catalyzes reduction of 4-oxonon-2-enal. Biochem . 43: 13106-13114; 2004, Maser, E. Neuroprotective role for carbonyl reductase. Biochem. Biophys . Res . Commun . 340: 1019-1022; 2006]. 그러나, 노화현상에서 CBR1 단백질의 생물학적 기능과 기작은 거의 알려지지 않았다. Human carbonyl reductase 1 (CBR1) is a NADPH-dependent, monomeric, cytoplasmic enzyme belonging to the short chain dehydrogenase / reductase superfamily. CBR1 is distributed in a variety of human tissues including liver, kidney and nerve cells [Forrest, GL; Gonzalez, B. Carbonyl reductase. Chem . Biol . Interact . 129 : 21-40; 2000, Rashid, MA; Lee, S .; Tak, E .; Lee, J .; Choi, TG; Lee, JW; Kim, JB; Youn, JH; Kang, I .; Ha, J .; Kim, SS Carbonyl reductase 1 protects pancreatic β-cells against oxidative stress-induced apoptosis in glucotoxicity and glucolipotoxicity. Free Radic . Biol . Med . 49 : 1522-1533; 2010]. Several studies have shown that CBR1 plays a protective role in oxidative stress, neurodegeneration, and apoptosis [Ismail, E .; Al-Mulla, F .; Tsuchida, S .; Suto, K .; Motley, P .; Harrison, PR; Birnie, GD Carbonyl reductase: a novel metastasis-modulating function. Cancer Res . 60 : 1173-1176; 2000, Botella, JA; Ulschmid, JK; Gruenewald, C .; Moehle, C .; Kretzschmar, D .; Becker, K .; Schneuwly, S. The Drosophila carbonyl reductase inhibits oxidative stress-induced neurodegeneration. Curr . Biol . 14 : 782-786; 2004]. In addition, CBR1 inactivates fatty aldehydes during oxidative stress in cells. Thus, CBR1 appears to play a protective role for cellular damage resulting from oxidative stress [Doorn, JA; Master, E .; Blum, A .; Claffey, DJ; Petersen, DR Human carbonyl reductase catalyzes reduction of 4-oxonon-2-enal. Biochem . 43 : 13106-13114; 2004, Maser, E. Neuroprotective role for carbonyl reductase. Biochem. Biophys . Res . Commun . 340 : 1019-1022; 2006]. However, little is known about the biological function and mechanism of CBR1 protein in aging.

외부 거대분자들이 내부 세포막을 투과하기는 어렵다. 아미노산 서열 짧은 사슬로 이루어진 단백질 수송 도메인은 외부 거대분자들이 포유류 세포 내로 투과되는 것을 용이하게 해준다 [Wadia, J. S.; Dowdy, S. F. Protein transduction technology. Curr . Opin . Biotechnol . 13: 52-56; 2002, Schwarze, S.; Ho, A.; Vocero-Akbani, A.; Dowdy, S. In vivo protein transduction: Delivery of a biologically active protein into the mouse. Science 285: 1569-1572; 1999]. 본 발명자들은 인 비트로와 인 비보에서 단백질 수송도메인과 융합된 다양한 융합단백질들을 세포 및 조직 내로 투과하는 것을 보고한바 있고, 이들 융합단백질들은 세포사멸에 대응하여 세포를 효과적으로 보호하였다 [An, J. J. et al., FEBS J. 275: 1296-1308; 2008, Ahn, E. H. et al., Toxicol . 276: 192-197; 2010, Kim, S. Y. et al., J. Invest . Dermatol . 131: 1477-1485; 2011, Eum, W. S. et al., Free Radic. Biol . Med . 37: 1656-1669; 2004, Ahn, E. H. et al., Free Radic . Biol . Med. 55: 36-45; 2013, Sohn, E. J. et al., Biochim . Biophys . Acta 1820: 1647-1655; 2012, Kim, D. W. et al., BMB Rep . 46: 124-129; 2013, Kim, M. J. et al., Free Radic . Biol . Med . 63: 432-445; 2013]. It is difficult for external macromolecules to penetrate the inner cell membrane. Amino acid sequences Short chain protein translocation domains facilitate the permeation of foreign macromolecules into mammalian cells [Wadia, JS; Dowdy, SF Protein transduction technology. Curr . Opin . Biotechnol . 13 : 52-56; 2002, Schwarze, S .; Ho, A .; Vocero-Akbani, A .; Dowdy, S. In vivo protein transduction: Delivery of a biologically active protein into the mouse. Science 285 : 1569-1572; 1999]. The present inventors have reported transmission of various fusion proteins fused with protein transport domains into cells and tissues in in vitro and in vivo, and these fusion proteins effectively protected cells in response to apoptosis [An, JJ et al ., FEBS J. 275 : 1296-1308; 2008, Ahn, EH et al., Toxicol . 276 : 192-197; 2010, Kim, SY et al., J. Invest . Dermatol . 131 : 1477-1485; 2011, Eum, WS et al., Free Radic. Biol . Med . 37 : 1656-1669; 2004, Ahn, EH et al., Free Radic . Biol . Med. 55 : 36-45; 2013, Sohn, EJ et al., Biochim . Biophys . Acta 1820 : 1647-1655; 2012, Kim, DW et al., BMB Rep . 46 : 124-129; 2013, Kim, MJ et al., Free Radic . Biol . Med . 63 : 432-445; 2013].

지다당 (Lipopolysaccharide; LPS)은 그람 음성균 외부 막 구성요소로 잘 알려져 있고, 이는 염증반응과 COX-2 (cyclooxygenase-2), 사이토카인 (인터루킨-1β; IL-1 및 IL-6), 종양괴사인자-α (TNF-α) 및 활성산소종 생산을 유도한다. 이러한 염증성 매개자들은 다양한 염증성 질환의 발병과 밀접하게 관련되어 있다 [Goraca A, Huk-Kolega H, Kleniewska P, Piechota-Polanczyk A, Skibska B. (2013) Pharmacol Rep 65: 179-186, Hewett JA, Roth RA. (1993) Pharmacol Rev 45: 382-411, Yoshino S, Sasatomi E, Ohsawa M. (2000) Immunobiol 99: 607-614, Bertolini A, Ottani A, Sandrini M. (2002) Curr Med Chem 9: 1033-1043]. 또한, 생성된 활성산소종은 세포의 구조와 기능을 변화시켜 세포사멸에 기여한다 [Halliwell B, Gutteridge JMC. (1999) Free radicals in biology and medicine. Oxford University Press, Oxford, Hald A, Lotharius J. (2005) Exp Neurol 193: 279-290].Lipopolysaccharide (LPS) is well known as an outer membrane component of Gram-negative bacteria, which is involved in inflammatory responses and COX-2 (cyclooxygenase-2), cytokines (interleukin-1β, IL-1 and IL- Induce factor-alpha (TNF-a) and active oxygen species production. These inflammatory mediators are closely related to the onset of a variety of inflammatory diseases (Goraca A, Huk-Kolega H, Kleniewska P, Piechota-Polanczyk A, Skibska B. (2013) Pharmacol Rep 65: 179-186, Hewett JA, Roth RA. (1993) Pharmacol Rev 45: 382-411, Yoshino S, Sasatomi E, Ohsawa M. (2000) Immunobiol 99: 607-614, Bertolini A, Ottanie, Sandrini M. (2002) Curr Med Chem 9: 1033-1043 ]. In addition, the generated reactive oxygen species change cell structure and function and contribute to apoptosis (Halliwell B, Gutteridge JMC. (1999) Free radicals in biology and medicine. Oxford University Press, Oxford, Halde, Lotharius J. (2005) Exp Neurol 193: 279-290].

단백질을 치료제로 사용하는 것은 분자량, 낮은 투과성 및 생화학적 특성으로 인해 한계가 있었다 [van den Berg A, Dowdy SF. (2011) Curr Opin Biotechnol 22: 888-893, Zhao B, Guo Y, Fu A. (2012) Appl Biochem Biotechnol 166: 1463-1471]. 그러나, 많은 연구자들이 단백질 수송 도메인을 이용하여 치료용 단백질을 세포 및 조직 내로 전달하는 것이 가능하며 의료용으로 이용할 수 있음을 밝혔다 [van den Berg A, Dowdy SF. (2011) Curr Opin Biotechnol 22: 888-893, Zhao B, Guo Y, Fu A. (2012) Appl Biochem Biotechnol 166: 1463-1471, Wadia JS, Dowdy SF. (2002) Curr Opin Biotechnol 13: 52-56, Morris MC et al. (2001) Nat Biotechnol 19: 1173-1176, Dietz GP. (2010) Curr Pharm Biotechnol 11: 167-174, Embury J et al. (2001) Diabetes 50: 1706-1713, Kubo E et al. (2008) Am J Physiol Cell Physiol 294: C842-C855]. 본 발명자들은 단백질 수송 도메인과 융합된 단백질이 세포 및 조직 내로 잘 투과되며, 뿐만 아니라, 항노화에 효과가 있음을 밝혔다 [Eum WS et al. (2004) Free Radic Biol Med 37: 1656-1669, Sohn EJ et al. (2012) Biochim Biophys Acta 1820: 1647-1655, Lee YP et al. (2012) FEBS J 279: 1929-1942, Ahn EH et al. (2013) Free Radic Biol Med 55: 36-45, Kim SY et al. (2011) J Invest Dermatol 131: 1477-1485, Kim MJ et al. (2013) Free Radic Biol Med 63: 432-445, Kim DW et al. (2013) BMB Rep 46: 124-129, Kim SY et al. (2011) Immunobiol 216: 771-781, Kim DS et al. (2012) BMB Rep 45: 532-537].The use of proteins as therapeutic agents has been limited by their molecular weight, low permeability and biochemical properties [van den Berg A, Dowdy SF. (2011) Curr Opin Biotechnol 22: 888-893, Zhao B, Guo Y, Fu A. (2012) Appl Biochem Biotechnol 166: 1463-1471]. However, many researchers have shown that it is possible to transfer therapeutic proteins into cells and tissues using protein transport domains and to be used for medical applications [van den Berg A, Dowdy SF. (2011) Curr Opin Biotechnol 22: 888-893, Zhao B, Guo Y, Fu A. (2012) Appl Biochem Biotechnol 166: 1463-1471, Wadia JS, Dowdy SF. (2002) Curr Opin Biotechnol 13: 52-56, Morris MC et al. (2001) Nat Biotechnol 19: 1173-1176, Dietz GP. (2010) Curr Pharm Biotechnol 11: 167-174, Embury J et al. (2001) Diabetes 50: 1706-1713, Kubo E et al. (2008) Am J Physiol Cell Physiol 294: C842-C855]. We have shown that proteins fused with protein transport domains are well permeated into cells and tissues, as well as being effective in anti-aging [Eum WS et al. (2004) Free Radic Biol Med 37: 1656-1669, Sohn EJ et al. (2012) Biochim Biophys Acta 1820: 1647-1655, Lee YP et al. (2012) FEBS J 279: 1929-1942, Ahn EH et al. (2013) Free Radic Biol Med 55: 36-45, Kim SY et al. (2011) J Invest Dermatol 131: 1477-1485, Kim MJ et al. (2013) Free Radic Biol Med 63: 432-445, Kim DW et al. (2013) BMB Rep 46: 124-129, Kim SY et al. (2011) Immunobiol 216: 771-781, Kim DS et al. (2012) BMB Rep 45: 532-537].

본 발명은 CBR1 융합단백질 및 포졸란을 화장품 원료로 이용하여 상용성이 우수한 항노화 기능성 화장료 조성물을 제공하려는 것이다. An object of the present invention is to provide an anti-aging functional cosmetic composition having excellent compatibility by using CBR1 fusion protein and pozzolan as raw materials for cosmetics.

본 발명자들은 CBR1 융합단백질이 세포 및 조직 내로 잘 투과하여 LPS 또는 과산화수소로 유도되는 염증 및 세포사멸에 보호작용을 나타내는지를 시험하였다. 그 결과, Tat-CBR1 융합단백질은 효과적으로 HaCaT 세포 내로 투과하여 과산화수소로 유도되는 염증 및 세포사멸에 대해 현저한 세포 보호작용을 나타내었다. 뿐만 아니라, 포졸란을 전처리하였을 경우 세포사멸 기전에 대한 보호효과가 더 월등함을 알 수 있었고, CBR1 융합단백질 및 포졸란이 항노화 기능성 화장료로 이용 가능함을 밝혔다.
The present inventors have examined whether the CBR1 fusion protein is well permeated into cells and tissues and shows protection against inflammation and apoptosis induced by LPS or hydrogen peroxide. As a result, the Tat-CBR1 fusion protein effectively permeated into HaCaT cells and showed a significant cytoprotective effect on hydrogen peroxide-induced inflammation and apoptosis. In addition, the pre - treatment of pozzolan showed better protective effect against apoptosis mechanism, and CBR1 fusion protein and pozzolan could be used as anti - aging functional cosmetic.

포졸란(pozzolan) 광물은 화산회로 이루어진 것으로서 납석의 일종으로 알려져 있다. 이는 백악기시대에 생성된 고아물질로 알려져 있으며, 주요 산지로는 이태리의 바리섬 인근 포죠리 마을에서 발견되어 유래된 광물로서 서기 2세기경 로마시대에 이미 유명한 콜로세움 경기장을 비롯하여 판테온 등의 건축물에 시멘트 대신에 사용되었다. 이러한 건축물들이 오늘날까지 균열 없고 부식 없이 오랜 세월 원형 그대로 보존되고 있는 것은 포졸란이 쓰였기 때문으로 학자들에 의해 밝혀진 바 있다. 그외 분포지역으로는 미국의 파마운티지역, 말레이시아, 및 인도 등 극히 제한된 지역국가에서만 생산되고 있고 특이한 것은 이태리와 한국산 이외는 게르마늄 성분이나 원적외선 방출효과가 전무하다는 사실 또한 특이하며, 아시아권에서는 광활한 중국의 평원 지역마저 동일 광맥이 전혀 존재하지 않는다.Pozzolan minerals are made of volcanic rocks and are known to be a kind of pyrophyllite. It is known as an orphan material created during the Cretaceous period and its major origin is the minerals found in the town of Pozzoli near Bari Island in Italy. It is known as the Colosseum Stadium in the 2nd century AD, It was used instead. These buildings have been preserved in their original form for many years without cracks and corrosion to this day, as the pozzolans have been used by scholars. Other distribution areas are produced only in extremely limited regions such as the United States of America, Malaysia, and India. Unusual is the fact that there is no germanium or infrared emission effect except for Italy and Korea. In Asia, Even plains do not have the same vein at all.

콘크리트 제조시 기본재료에 첨가되어 콘크리트에 특수한 성능을 부여하거나 성질을 개선하기 위한 혼화재료로 사용되었으며, 폐기물 고형화를 위한 무기성 고화재료는 대개 시멘트 반응성을 보유하여 적절한 함수율의 무기성슬러지(중금속류 함유된 것)의 고형화에 적합하다. 또한, 시멘트와 친화성이 좋은 포졸란(pozzolan)은 슬러지 함수율 조절과 시멘트 대체 고화보조제로 이용할 수 있고, 비용이 비교적 저렴하다는 장점 등으로 가장 많이 이용되고 있다.
The inorganic solidification material for the solidification of waste is usually cement reactive and is used as an inorganic sludge with a proper water content (heavy metals Lt; / RTI > In addition, pozzolan, which has good affinity with cement, is most widely used because it can be used as a sludge moisture content control and cement substitute solidification aid, and its cost is relatively low.

본 발명은 세포 침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물에 관한 것이다.The present invention relates to an anti-aging functional cosmetic composition comprising a cell-permeable carbonyl group reducing enzyme fusion protein and a pozzolan.

또한, 본 발명은 상기 세포 침투성 카보닐기 환원효소 융합단백질은 카보닐기 환원효소의 C-말단과 N-말단 중 어느 한 곳 이상에 단백질 수송도메인이 공유결합된 것을 특징으로 하는 세포 침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물에 관한 것이다.The cell permeable carbonyl group reductase fusion protein of the present invention is characterized in that a protein transport domain is covalently bonded to at least one of a C-terminal and an N-terminal of a carbonyl group reductase, A fusion protein, and a pozzolan.

또한, 본 발명은 상기 단백질 수송 도메인은 Tat 펩타이드 또는 HIV-1 Tat 펩타이드임을 특징으로 하는 세포 침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물에 관한 것이다.The present invention also relates to an anti-aging functional cosmetic composition comprising a cell permeable carbonyl group reducing enzyme fusion protein and a pozzolan, wherein the protein transport domain is a Tat peptide or an HIV-1 Tat peptide.

또한, 본 발명은 상기 세포 침투성 카보닐기 환원효소 융합단백질은 서열번호 4, 6, 8, 10, 12 및 14 중 선택된 1종임을 특징으로 하는 세포 침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물에 관한 것이다.The cell permeable carbonyl group reductase fusion protein of the present invention is one selected from the group consisting of SEQ ID NOS: 4, 6, 8, 10, 12 and 14, and the cell permeable carbonyl group reductase fusion protein To an aging functional cosmetic composition.

또한, 본 발명은 상기 천연 포졸란은 5~20㎛ 파장에서 90~97%의 원적외선을 방출하는 것을 특징으로 하는 세포 침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물에 관한 것이다.The present invention also relates to an anti-aging functional cosmetic composition comprising the cell permeable carbonyl group reducing enzyme fusion protein and the pozzolan, wherein the natural pozzolan emits 90 to 97% far infrared rays at a wavelength of 5 to 20 μm.

또한, 본 발명은 상기 화장료 조성물이 화장수, 젤, 수용성 리퀴드, 크림, 연고, 에센스, 샴푸, 헤어린스, 헤어트리트먼트, 헤어무스, 립스틱, 수중유(O/W)형 및 유중수(W/O)형의 제형 중에서 선택되는 것을 특징으로 한다.The present invention also relates to the cosmetic composition as described above, wherein the cosmetic composition is at least one selected from the group consisting of lotion, gel, water-soluble liquid, cream, ointment, essence, shampoo, hair rinse, hair treatment, hair mousse, lipstick, oil / O) type. ≪ / RTI >

또한, 본 발명의 조성물은 캡슐제, 액상제, 연고제, 첩부제 또는 약학적으로 허용되는 담체를 이용한 서방화 제제 등의 약학 조성물로 제제화할 수 있으며, 약리학적으로 허용되는 기제, 운반제, 부형제, 결합제(예: 전분, 트래거캔스 고무, 젤라틴, 당밀, 폴리비닐 알콜, 폴리비닐에테르, 폴리비닐 피롤리돈, 히드록시프로필 셀룰로오스, 메틸 셀룰로오스, 에틸 셀룰로오스 및 카르복시메틸 셀룰로오스), 분쇄제(예: 한천, 전분, 벨라틴 가루, 카르복시메틸 셀룰로오스 나트륨, 카르복시메틸 셀룰로오스 칼슘, 결정 셀룰로오스, 탄산칼슘, 탄산수소나트륨 및 알긴산 나트륨), 윤활제(예: 스테아린산 마그네슘, 활석, 수첨 식물유) 및 착색제 등을 포함한다. 상기 운반제 및 부형제로는 젖당, 글루코오스, 수크로오스, 만니톨, 감자녹말, 옥수수녹말, 탄산칼슘, 인산칼슘 및 셀룰로오스 등을 사용한다.In addition, the composition of the present invention can be formulated into pharmaceutical compositions such as capsules, liquid preparations, ointments, patches or sustained release preparations using pharmaceutically acceptable carriers, and can be formulated into pharmacologically acceptable bases, carriers, , Binders (such as starch, tragacanth gum, gelatin, molasses, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, ethylcellulose and carboxymethylcellulose) (Such as agar, starch, bellatin powder, carboxymethylcellulose sodium, carboxymethylcellulose calcium, crystalline cellulose, calcium carbonate, sodium bicarbonate and sodium alginate), lubricants such as magnesium stearate, talc, hydrogenated vegetable oils, do. Examples of the carriers and excipients include lactose, glucose, sucrose, mannitol, potato starch, cornstarch, calcium carbonate, calcium phosphate and cellulose.

상기 외에도 안정화제, 용해보조제, 경피흡수 촉진제 등의 보조제 방향제, 방부제 등의 첨가제가 더 첨가될 수 있다.In addition to the above, additives such as stabilizers, solubilizers, perfume fragrances such as transdermal absorption accelerators, and preservatives may be further added.

이와 같이 제조되는 약학 조성물은 증상에 따라 일일 1회 내지 수회 피부에 적용할 수 있으며 증상의 호전 정도에 따라 적용을 조절할 수 있다.
The pharmaceutical composition thus prepared can be applied to the skin once to several times a day depending on the symptoms, and the application can be controlled according to the degree of symptom improvement.

본 발명에 따른 CBR1 단백질 분자의 세포 내 전달은 CBR1에 15∼30개의 아미노산으로 구성되고, 5개 이상의 트립토판을 포함하는 비친수성 도메인(hydrophobic domain), 라이신을 4개 이상 다수 포함하는 친수성 도메인(hydrophilic domain) 및 상기 두 도메인을 분리시켜 주는 스페이서(spacer)로 구성된 수송 도메인이 공유결합된, Pep-1을 비롯한 세포투과성 수송도메인 또는 HIV Tat 세포투과성 도메인이 목표 단백질인 CBR1의 N-말단 및/또는 C-말단과 공유결합된 형태의 융합단백질을 구성하여 수행된다. 본 발명의 상기 수송도메인의 일례로는 21개의 아미노산으로 구성되고 서열번호 3과 같은 아미노산 서열로 이루어진 PEP-1 펩타이드, HIV Tat 49~57 펩타이드를 들 수 있다. 그러나, 본 발명의 단백질 수송 도메인이 서열번호 3의 PEP-1 펩타이드, HIV Tat 49~57 잔기 펩타이드로만 한정되는 것은 아니고, PEP-1 또는 HIV Tat의 아미노산 서열 일부 치환이나 부가, 결여로 PEP-1 펩타이드 또는 HIV Tat 펩타이드와 유사한 기능을 하는 펩타이드를 제조하는 것이 본 발명이 속하는 분야에서 통상의 지식을 가진 당업자에게는 용이하므로, 15∼30개의 아미노산으로 구성되고, 5개 이상의 트립토판을 포함하는 비친수성 도메인(hydrophobic domain), 라이신을 4개 이상 다수 포함하는 친수성 도메인(hydrophilic domain) 및 상기 두 도메인을 분리시켜 주는 스페이서(spacer)로 구성된 단백질 수송 도메인과 이로부터 아미노산 일부 치환으로 동일·유사한 단백질 수송기능을 수행하는 단백질 수송 도메인, 또는 6 내지 12개의 아미노산 잔기로 구성되며 아르기닌 또는 라이신 잔기를 3/4 이상 포함하는 수송도메인, 6 내지 12개의 라이신으로 구성되는 올리고라이신 수송 도메인, 6 내지 12개의 아르기닌으로 구성되는 올리고아르기닌 수송 도메인 또는 6 내지 12개의 라이신 또는 아르기닌으로 구성되는 올리고(라이신,아르기닌) 수송 도메인도 발명의 범위에 속함은 자명하다고 할 것이다.
The intracellular delivery of the CBR1 protein molecule according to the present invention comprises 15-30 amino acids in CBR1, a hydrophobic domain containing 5 or more tryptophan, a hydrophilic domain containing 4 or more lysines domain, and a spacer that separates the two domains is covalently linked to the N-terminus of CBR1, the target protein, and / or the HIV Tat cell permeable domain, including Pep-1, Terminus and a covalently linked form of the fusion protein with the C-terminus. Examples of the transport domain of the present invention include a PEP-1 peptide consisting of 21 amino acids and an amino acid sequence as shown in SEQ ID NO: 3, HIV Tat 49 to 57 peptide. However, the protein transport domain of the present invention is not limited to PEP-1 peptide or HIV Tat 49 to 57 residue peptide of SEQ ID NO: 3, but may be substituted with PEP-1 It is easy for those skilled in the art to make peptides having similar functions to peptides or HIV Tat peptides, and thus it is possible to use peptides having 15-30 amino acids and having 5 or more tryptophan, a protein transport domain composed of a hydrophilic domain having a hydrophobic domain, a hydrophilic domain containing four or more lysines, and a spacer separating the two domains, A protein transport domain to be performed, or 6 to 12 amino acid residues, and arginine or An oligolysine transport domain consisting of 6 to 12 lysines, an oligosaccharide transport domain consisting of 6 to 12 arginines or an oligoarginine transport domain consisting of 6 to 12 lysines or arginines Lysine, arginine) transport domains belong to the scope of the invention.

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

"CBR1 융합단백질"이란 단백질 수송 도메인과 CBR1 단백질을 포함하며, 단백질 수송 도메인과 화물 분자(cargo molecule, 즉 본 발명에서는 CBR1을 의미함)의 유전적 융합이나 화학 결합으로 형성된 공유결합 복합체를 의미한다. 본 명세서와 도면에서 구체적인 일 실시예로서 "PEP-1-CBR1"은 CBR1 융합단백질 중 CBR1의 N-말단에 PEP-1 단백질 수송 도메인이 결합된 것을 말한다."CBR1 fusion protein" means a covalent complex formed by genetic fusion or chemical bonding between a protein transport domain and a CBR1 protein, and a protein transport domain and a cargo molecule (i.e., CBR1 in the present invention) . As a specific example in the present specification and drawings, "PEP-1-CBR1" refers to a PEP-1 protein transport domain bound to the N-terminus of CBR1 among CBR1 fusion proteins.

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

또한, "표적 세포"란 수송 도메인에 의해 화물 분자가 전달되는 세포를 의미하는 것으로서 즉, 표적 세포는 체내 세포, 다시 말하여 살아있는 동물 또는 인간의 장기 또는 조직을 구성하는 세포 또는 살아있는 동물 또는 인간에서 발견되는 미생물을 포함하는 의미이다. 또한, 표적 세포는 체외 세포, 즉 배양된 동물 세포, 인체 세포 또는 미생물을 포함하는 의미이다. 구체적으로 본 명세서에서 표적 세포는 피부세포를 의미한다.The term "target cell" refers to a cell to which a cargo molecule is delivered by a transport domain. That is, the target cell is a cell, that is, a living organism or a cell or organ It is meant to include microorganisms that are found. In addition, the target cell means an extracellular cell, that is, a cultured animal cell, a human cell or a microorganism. Specifically, in the present specification, the target cell refers to a skin cell.

본 명세서의 "단백질 수송 도메인"은 화물 분자(목표 단백질) 펩타이드 또는 단백질과 공유결합을 이루어 별도의 수용체나 운반체, 에너지를 필요로 하지 않고 상기 펩타이드나 단백질을 세포 내로 도입시킬 수 있는 것을 말하며, 예를 들면 PEP-1 펩타이드를 말한다.As used herein, the term "protein transport domain" refers to a peptide or protein that is covalently bonded to a cargo molecule (target protein) peptide or protein and can introduce the peptide or protein into cells without requiring additional receptor, carrier, energy, For example, it refers to PEP-1 peptide.

본 명세서의 "목표 단백질"은 PEP-1 단백질 수송 도메인과 공유결합을 이루어 세포 내로 도입되어 활성을 나타내는 분자를 의미한다. "화물 분자"와 동일한 의미이다.As used herein, the term "target protein" refers to a molecule that is covalently bound to a PEP-1 protein transduction domain and introduced into cells to exhibit activity. It is synonymous with "cargo molecule".

또한, 본 명세서에서는 단백질 또는 펩타이드를 세포 내로 "도입"시키는 것에 대하여 "형질도입", "운반", "침투", "수송", "전달", "투과", "통과"한다는 표현들과 혼용하였다.
Also, in the present specification, the term "introduction", "transport", "penetration", "transport", "transfer", "permeation", "pass" Respectively.

본 발명에서 단백질 수송 도메인으로는 15∼30개의 아미노산으로 구성되고, 5개 이상의 트립토판을 포함하는 비친수성 도메인, 라이신을 다수 포함하는 친수성 도메인 및 상기 두 도메인을 분리시켜 주는 스페이서로 구성된 단백질 수송 도메인, 6 내지 12개의 아미노산 잔기로 구성되며 아르기닌 또는 라이신 잔기를 3/4 이상 포함하는 수송도메인, 6 내지 12개의 라이신으로 구성되는 올리고라이신 수송 도메인, 6 내지 12개의 아르기닌으로 구성되는 올리고아르기닌 수송 도메인 또는 6 내지 12개의 라이신 또는 아르기닌으로 구성되는 올리고(라이신,아르기닌) 수송 도메인을 들 수 있다. 또한, 목표 단백질(화물 분자)는 CBR1이다. 상기 단백질 수송 도메인 및 목표 단백질은 silent change에 따라 서열 내에서 하나 이상의 아미노산이 기능적으로 동등하게 작용하는 유사한 극성의 다른 아미노산(들)로 치환될 수 있다. 서열 내 아미노산 치환은 그 아미노산이 속하는 클래스의 다른 구성원들로부터 선택될 수 있다. 예컨대, 소수성 아미노산 분류는 알라닌, 발린, 류이신, 이소류이신, 페닐알라닌, 발린, 트립토판, 프롤린 및 메티오닌을 포함한다. 극성 중성 아미노산은 글리신, 세린, 트레오닌, 시스테인, 티로신, 아스파라긴 및 글루타민을 포함한다. 양성 염기성 아미노산은 아르기닌, 라이신 및 히스티딘을 포함한다. 음성전하를 띤 산성 아미노산은 아스파르트산 및 글루탐산을 포함한다. 또한, 본 발명의 융합단백질과 아미노산 서열간의 일정 범위의 상동성 예컨대 85-100% 범위 내에서 동일·유사한 생물학적 활성을 갖는 절편 또는 이들의 유도체들도 본 발명의 권리범위에 포함된다.In the present invention, the protein transport domain is a protein transport domain composed of 15 to 30 amino acids and composed of a non-hydrophilic domain containing 5 or more tryptophan, a hydrophilic domain containing many lysines, and a spacer separating the two domains, A transport domain comprising 6 to 12 amino acid residues and comprising ¾ or more arginine or a lysine residue, an oligolysine transport domain consisting of 6 to 12 lysines, an oligoroginalin transport domain consisting of 6 to 12 arginines, Or an oligo (lysine, arginine) transport domain consisting of 12 lysine or arginine. In addition, the target protein (cargo molecule) is CBR1. The protein transport domain and the target protein may be replaced by other amino acid (s) of similar polarity in which one or more amino acids functionally equivalently function in the sequence according to the silent change. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, the hydrophobic amino acid class includes alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positive basic amino acids include arginine, lysine and histidine. Acidic amino acids with negative charge include aspartic acid and glutamic acid. In addition, fragments or derivatives thereof having the same or similar biological activity within a certain range of homology, such as 85-100%, between the fusion protein of the present invention and the amino acid sequence are included in the scope of the present invention.

또한, 본 발명은 상기 세포 도입성 CBR1 융합단백질이 서열번호 4, 서열번호 6, 서열번호 8, 서열번호 10, 서열번호 12 또는 서열번호 14의 아미노산 서열을 갖는 것을 특징으로 한다.In addition, the present invention is characterized in that the cell-inducing CBR1 fusion protein has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO:

또한, 본 발명은 상기 단백질 수송도메인(protein transducing domain; "PTD")이 CBR1 단백질의 카복시 말단과 아미노 말단의 일측 또는 양측에 공유결합된 것을 특징으로 한다.Also, the present invention is characterized in that the protein transducing domain ("PTD") is covalently bonded to one or both of the carboxy terminal and the amino terminal of the CBR1 protein.

본 발명의 CBR1 융합단백질은 투여량 및 처리시간 의존적으로 세포 내로 투과하였고, 투과된 CBR1 융합단백질은 세포 내에서 36시간 동안 현저한 수준을 나타내었다. The CBR1 fusion protein of the present invention was permeabilized intracellularly in a dose and treatment time period, and the permeated CBR1 fusion protein showed a remarkable level in the cells for 36 hours.

또한, 본 발명의 CBR1 융합단백질은 COX-2와 iNOS 단백질 발현 수준을 현저히 감소시켰다.In addition, the CBR1 fusion protein of the present invention significantly reduced COX-2 and iNOS protein expression levels.

또한, 본 발명의 CBR1 융합단백질은 MAPKs와 NF-kB 활성화에 대해 세포 보호효과를 나타내었다. CBR1 융합단백질 처리세포에서는 인산화된 ERK1/2, p38 및 JNK 발현수준이 현저히 감소하였다. 또한, 투과된 CBR1 융합단백질은 세포 내 p65와 IkBα 인산화 수준을 현저히 저해하였다.Also, CBR1 fusion proteins of the present invention showed a cytoprotective effect on the MAPKs and NF- k B activation. In CBR1 fusion protein treated cells, phosphorylated ERK1 / 2, p38 and JNK expression levels were significantly decreased. Further, the transmitted CBR1 fusion protein was significantly inhibited in the p65 and I k Bα phosphorylation level cell.

또한, 본 발명의 CBR1 융합단백질은 세포로 투과되어 H2O2 처리한 세포에서 활성산소종 수준을 감소시킴으로써 세포 보호효과를 나타내었다.In addition, the CBR1 fusion protein of the present invention showed cytoprotective effect by reducing the level of reactive oxygen species in cells permeated into H 2 O 2 permeable to cells.

뿐만 아니라, H2O2로 처리한 세포에서 인산화된 Akt와 p53 수준이 현저히 증가한 반면, CBR1 융합단백질 처리세포에서는 현저히 감소함을 밝혔다. 투과된 PEP-1-CBR1 융합단백질이 세포자기사멸 경로를 저해함으로써 H2O2로 유도되는 세포사멸로부터 세포를 보호함을 말해준다.In addition, the levels of phosphorylated Akt and p53 were significantly increased in H 2 O 2 treated cells, while those in CBR1 fusion protein treated cells were significantly decreased. The permeable PEP-1-CBR1 fusion protein inhibits the cell apoptotic pathway, thereby protecting the cell from H 2 O 2 -induced cell death.

뿐만 아니라, CBR1 융합단백질은 TPA로 유도되는 동물 염증모델에서 COX-2 발현 수준과 TNF-α, IL-1β 및 IL-6 생성을 현저히 감소시켰고, TPA로 유도되는 피부염증 동물모델에서 TPA로 유도되는 p38, ERK1/2 및 JNK 인산화와 p65와 IkBα인산화를 저해하였다.In addition, the CBR1 fusion protein significantly reduced COX-2 expression levels, TNF-α, IL-1β and IL-6 production in TPA-induced animal inflammation models and induced TPA-induced skin inflammation in animal models of TPA a p38, ERK1 / 2 and JNK phosphorylation and p65 and I k Bα phosphorylation is inhibited.

또한, CBR1 융합단백질을 처리하기 전 포졸란을 전처리하였을 경우, COX-2와 iNOS 단백질 발현 수준 감소, ERK1/2, p38 및 JNK 발현수준 감소 등 세포사멸로부터 보호효과가 더욱 높아졌다.In addition, pretreatment with pozzolan before treatment with CBR1 fusion protein resulted in a further protective effect against apoptosis, such as decreased levels of COX-2 and iNOS protein expression, ERK1 / 2, p38 and JNK expression levels.

도 1은 PEP-1-CBR1 융합단백질, CBR1 단백질을 HaCaT 세포에 침투시키고, 포졸란을 전 처리한 후 PEP-1-CBR1 융합단백질, CBR1 단백질을 HaCaT 세포에 침투시켜 웨스턴 블랏 분석하여, 시간 및 농도 의존적으로 침투됨을 확인하였다.
도 2는 PEP-1-CBR1 융합단백질, 포졸란 전 처리+PEP-1-CBR1 융합단백질의 HaCaT 세포 침투 후 단백질 안전성을 평가하였다.
도 3은 PEP-1-CBR1 융합단백질, 포졸란 전 처리+PEP-1-CBR1 융합단백질의 HaCaT 세포 침투 후 세포 내 Baw와 Bcl-2 수준을 웨스턴 블랏 분석으로 확인하였다.
도 4은 PEP-1-CBR1 융합단백질, 포졸란 전 처리+PEP-1-CBR1 융합단백질의 HaCaT 세포 침투 후 세포 내 Akt 수준을 웨스턴 블랏 분석으로 확인하였다.
도 5는 PEP-1-CBR1 융합단백질, 포졸란 전 처리+PEP-1-CBR1 융합단백질의 HaCaT 세포 침투 후 세포 내 JNK, ERK, P38 수준을 웨스턴 블랏 분석으로 확인하였다.
도 6는 PEP-1-CBR1 융합단백질, 포졸란 전 처리+PEP-1-CBR1 융합단백질의 HaCaT 세포 침투 후 세포 내 p65, Iκ-Bα 수준을 웨스턴 블랏 분석으로 확인하였다.
도 7는 PEP-1-CBR1 융합단백질, 포졸란 전 처리+PEP-1-CBR1 융합단백질의 HaCaT 세포 침투 후 세포 내 COX2, iNOS, IL-6, IL-1β, TNF-α 수준을 웨스턴 블랏 분석으로 확인하였다.
도 8은 PEP-1-CBR1 융합단백질, 포졸란 전 처리+PEP-1-CBR1 융합단백질의 HaCaT 세포 침투 후 세포 내 COX2, iNOS, IL-6, IL-1β, TNF-α 수준을 RT-PCR 분석으로 확인하였다. FIG. 1 shows the results of Western blot analysis of PEP-1-CBR1 fusion protein and CBR1 protein infiltrated into HaCaT cells, and PEP-1-CBR1 fusion protein and CBR1 protein were permeated into HaCaT cells after pre- And it is confirmed that it is infiltrated dependently.
FIG. 2 evaluates protein safety of PEP-1-CBR1 fusion protein, pozzolanic pretreatment + PEP-1-CBR1 fusion protein after HaCaT cell infiltration.
FIG. 3 shows the results of western blot analysis of intracellular Baw and Bcl-2 levels after HaCaT cell infiltration of PEP-1-CBR1 fusion protein and pozzolana pretreatment + PEP-1-CBR1 fusion protein.
FIG. 4 shows the intracellular Akt level after HaCaT cell infiltration of PEP-1-CBR1 fusion protein, pozzolana pretreatment + PEP-1-CBR1 fusion protein by Western blot analysis.
FIG. 5 shows the levels of JNK, ERK and P38 in cells after HaCaT cell infiltration of PEP-1-CBR1 fusion protein, pozzolana pretreatment + PEP-1-CBR1 fusion protein by Western blot analysis.
FIG. 6 shows the levels of p65 and Iκ-Bα after HaCaT cell infiltration of PEP-1-CBR1 fusion protein, pozzolanic pretreatment + PEP-1-CBR1 fusion protein by Western blot analysis.
FIG. 7 shows Western blot analysis of intracellular COX2, iNOS, IL-6, IL-1β and TNF-α levels after HaCaT cell infiltration of PEP-1-CBR1 fusion protein and pozzolana pretreatment + PEP-1-CBR1 fusion protein Respectively.
FIG. 8 shows RT-PCR analysis of intracellular COX2, iNOS, IL-6, IL-1β and TNF-α levels after HaCaT cell infiltration of PEP-1-CBR1 fusion protein and pozzolana pretreatment + PEP-1-CBR1 fusion protein Respectively.

아래에서는 구체적인 실시예를 들어 본 발명의 구성을 좀 더 자세히 설명한다. 그러나, 본 발명의 범위가 실시예의 기재범위 내로 한정되는 것이 아님은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 자명하다. 특히, 본 발명의 구체적인 실시예에서는 PEP-1-CBR1 융합단백질에 대하여만 기술하였으나, Tat-CBR1 융합단백질, CBR1-Tat 융합단백질, Tat-CBR1-Tat 융합단백질, CBR1-PEP-1 융합단백질 및 PEP-1-CBR1-PEP-1 융합단백질도 PEP-1-CBR1 융합단백질과 같이 세포 투과성을 나타내며, 세포실험 등에서 PEP-1-CBR1 융합단백질과 유사한 정도의 결과를 나타낸다.
Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the scope of the embodiments. In particular, although the PEP-1-CBR1 fusion protein was only described in the specific examples of the present invention, the fusion protein of Tat-CBR1 fusion protein, CBR1-Tat fusion protein, Tat-CBR1-Tat fusion protein, CBR1- PEP-1-CBR1-PEP-1 fusion protein also shows cell permeability like PEP-1-CBR1 fusion protein and shows similar results to PEP-1-CBR1 fusion protein in cell experiments.

재료material

12-O-TPA(tetradecanoylphorbol-13-acetate)는 Sigma-Aldrich (St. Louis, MO, USA)에서 구입하였고, Ni2 + 나이트릴로삼초산 세파로즈 수퍼플로우 (Ni2 +-nitrilotri-acetic acid Sepharose superflow)는 Qiagen (Valencia, CA, USA)에서 구입하였다. DMEM(Dulbecco''s modified Eagle's medium)은 Lonza (Walkersville, MD, USA)에서 구입하였고, FBS와 항생제는 Gibco BRL에서 구입하였다. 합성 Tat 펩타이드는 Peptron (Daejeon, Korea)에서 구입하였고, 기본 항체와 액틴은 각각 Cell Signaling Technology (Beverly MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA)에서 구입하였다. 다른 화학물질 및 시약들은 입수 가능한 최상급의 제품을 이용하였다.
 12- O -TPA (tetradecanoylphorbol-13- acetate) was purchased from (St. Louis, MO, USA) Sigma-Aldrich, Ni 2 + Night reels three seconds acid Sepharose Super flow (Ni 2 + - nitrilotri-acetic acid Sepharose superflow) were purchased from Qiagen (Valencia, CA, USA). DMEM (Dulbecco's modified Eagle's medium) was purchased from Lonza (Walkersville, MD, USA), and FBS and antibiotics were purchased from Gibco BRL. The synthetic Tat peptide was purchased from Peptron (Daejeon, Korea), and the primary antibody and actin were purchased from Cell Signaling Technology (Beverly MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other chemicals and reagents used the highest quality products available.

CBR1CBR1 융합단백질Fusion protein 발현 및 정제 Expression and purification

PEP-1 발현벡터는 종래기술과 같이 제조하였다[27]. CBR1은 HepG2 세포주에서 올리고-dT 프라이머를 이용하여 역전사 PCR로 얻은 cDNA로 서브클론하였다. CBR1은 PCR을 통해 센스 프라이머 5'-CCG CTC GAG TCG TCC GGC ATC-3'과 안티센스 프라이머 5'-CGG GAT CCT TAT CAC CAC TGT TCA AC-3'을 이용하여 증폭하였다. PCR로 얻은 CBR1 산물은 TA 클로닝벡터로 E. coli 내에서 증폭하였고, Tat 발현벡터에 삽입하였다. PEP-1-CBR1 단백질을 발현하고 정제하기 위해, 플라스미드는 BL21 (DE3)에 형질변환되었고, 0.1 mM의 IPTG (isopropyl-β-D-thio-galactoside) (Duchefa, Haarlem, Netherlands)를 이용하여 18 ℃에서 8시간 동안 과발현하였다. 하비스트한 세포는 초음파 처리로 용균한 다음, Ni2 +-니트릴로삼초산 세파로즈 친화컬럼과 PD-10 컬럼 크로마토그래피 (Amersham, Brauncschweig, Germany)를 이용하여 제조자의 지시대로 PEP-1-CBR1 융합단백질을 정제하였다. 정제된 단백질 농도는 우혈청 알부민을 표준으로 하여 브래드포드 방법으로 측정하였다[28].
The PEP-1 expression vector was prepared as in the prior art [27]. CBR1 was subcloned into cDNA obtained by reverse transcription PCR using oligo-dT primer in HepG2 cell line. CBR1 was amplified by PCR using sense primer 5'-CCG CTC GAG TCG TCC GGC ATC-3 'and antisense primer 5'-CGG GAT CCT TAT CAC CAC TGT TCA AC-3'. The CBR1 product obtained by PCR was amplified in E. coli as TA cloning vector and inserted into Tat expression vector. To express and purify the PEP-1-CBR1 protein, the plasmid was transformed into BL21 (DE3) and transformed with 0.1 mM IPTG (isopropyl- beta -D-thio-galactoside) (Duchefa, Haarlem, ≪ / RTI > for 8 hours. Harvest the cells, and then, Ni 2 + lysed by sonication-acrylonitrile three seconds acid Sepharose affinity column and PD-10 column chromatography (Amersham, Brauncschweig, Germany) PEP -1-CBR1 fusion as directed by the manufacturer using a The protein was purified. The purified protein concentration was measured by the Bradford method using bovine serum albumin as the standard [28].

HaCaTHaCaT 세포 내로  Into the cell PEPPEP -1--One- CBR1CBR1 융합단백질의Of the fusion protein 투과 Permeation

HaCaT 세포는 20 mM Hepes/NaOH (pH 7.4), 5 mM NaHCO3, 10% FBS 및 항생제 (100 ㎍/ml 스트렙토마이신, 100 U/ml 페니실린)를 함유하는 DMEM (Dulbecco's minimum essential medium; Lonza BioWhittaker, MD, USA) 내에서 95% 공기 및 5% CO2의 습한 조건 하에서 37 ℃로 배양하였다. PEP-1-CBR1 융합단백질의 세포투과가 투여량 및 농도 의존적임을 평가하기 위해, 다양한 농도의 PEP-1-CBR1 융합단백질 (0.1~1 μM)로 세포를 한 시간 동안 처리하였다. PEP-1-CBR1 융합단백질 처리 후 세포는 트립신-EDTA로 처리하고 PBS (phosphate-buffered saline)로 세척한 후 세포를 하비스트하고, 세포추출물은 웨스턴 블랏 분석에 이용하였다.
HaCaT cells were cultured in DMEM (Dulbecco's minimum essential medium; Lonza BioWhittaker, MD) containing 20 mM Hepes / NaOH (pH 7.4), 5 mM NaHCO3, 10% FBS and antibiotics (100 ug / ml streptomycin, 100 U / ml penicillin) , under humid conditions, 95% in the USA) air and 5% CO 2 and incubated with 37 ℃. Cells were treated with various concentrations of PEP-1-CBR1 fusion protein (0.1-1 μM) for one hour to assess the dose and concentration dependence of the cell permeability of the PEP-1-CBR1 fusion protein. After treatment with PEP-1-CBR1 fusion protein, the cells were treated with trypsin-EDTA, washed with PBS (phosphate-buffered saline) and harvested. Cell extracts were used for Western blot analysis.

웨스턴Western 블랏Blat 분석 analysis

용균된 단백질은 SDS-PAGE로 분리한 다음 나이트로셀룰로스 막 또는 PVDF 막으로 옮겼다. 막은 5% 저지방유와 0.1% Tween 20이 함유된 TBS 완충액 (TBST)으로 블로킹하였다. 블로킹된 막은 지시된 일차항체와 HRP 결합된 이차항체로 면역 블랏팅하였다. 타겟 단백질은 제조자 (Amersham, Franklin Lakes, NJ, USA)의 지시에 따라 화학발광으로 탐지하였다[27].CBR1 단백질을 발현하고 정제하기 위해, 플라스미드는 BL21 (DE3)에 형질변환되었고, 0.1 mM의 IPTG (isopropyl-β-D-thio-galactoside) (Duchefa, Haarlem, Netherlands)를 이용하여 18 ℃에서 8시간 동안 과발현하였다. 하비스트한 세포는 초음파 처리로 용균한 다음, Ni2 +-니트릴로삼초산 세파로즈 친화컬럼과 PD-10 컬럼 크로마토그래피 (Amersham, Brauncschweig, Germany)를 이용하여 제조자의 지시대로 PEP-1-CBR1 융합단백질을 정제하였다. 정제된 단백질 농도는 우혈청 알부민을 표준으로 하여 브래드포드 방법으로 측정하였다[28].
The lysed proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane or PVDF membrane. The membranes were blocked with TBS buffer (TBST) containing 5% low fat fat and 0.1% Tween 20. The blocked membrane was immunoblotted with the indicated primary antibody and HRP-conjugated secondary antibody. The target protein was detected by chemiluminescence according to the manufacturer's instructions (Amersham, Franklin Lakes, NJ, USA). [27] To express and purify the CBR1 protein, the plasmid was transformed into BL21 (DE3) Was overexpressed at 18 ° C for 8 hours using IPTG (isopropyl-β-D-thio-galactoside) (Duchefa, Haarlem, Netherlands). Harvest the cells, and then, Ni 2 + lysed by sonication-acrylonitrile three seconds acid Sepharose affinity column and PD-10 column chromatography (Amersham, Brauncschweig, Germany) PEP -1-CBR1 fusion as directed by the manufacturer using a The protein was purified. The purified protein concentration was measured by the Bradford method using bovine serum albumin as the standard [28].

결과 1: Result 1: CBR1CBR1 융합단백질과Fusion protein 포졸란의Pozzolana 침투 효과 Penetration effect

CBR1 단백질과 CBR1 융합단백질을 피부세포인 HaCaT 세포에 시간 의존적, 농도 의존적으로 처리하여 CBR1 융합단백질이 세포 내로 시간 의존적, 농도 의존적으로 침투됨을 웨스턴 블랏 분석을 통해 확인하였다. 또한 단백질을 처리하기 전 포졸란(10/㎖)을 세포에 처리한 후 1시간 동안 배양하여 워싱하고, 단백질을 처리한 결과 CBR1 융합단백질의 침투성 변화는 거의 없는 것으로 확인되었다. 이는 포졸란이 CBR1 융합단백질의 침투성에는 영향을 미치지 않음을 확인할 수 있었다(도 1).
CBR1 protein and CBR1 fusion protein were treated in time-dependent and concentration-dependent manner on HaCaT cells, which are skin cells, to confirm that CBR1 fusion protein infiltrated into cells in a time-dependent and concentration-dependent manner by Western blot analysis. In addition, it was confirmed that the permeability change of CBR1 fusion protein was almost not changed by treating the cells with pozzolan (10 / ml) before treating with the protein, washing and washing the cells for 1 hour. It was confirmed that the pozzolan does not affect the permeability of the CBR1 fusion protein (Fig. 1).

결과 2: Result 2: CBR1CBR1 융합단백질과Fusion protein 포졸란의Pozzolana HaCaTHaCaT 세포 침투 후 단백질 안정성 Protein stability after cell infiltration

단백질을 처리하기 전 포졸란(10/㎖)을 세포에 처리한 후 1시간 동안 배양하여 워싱하고, CBR1 융합단백질을 1시간 동안 처리하여 워싱한 후 침투된 단백질의 안전성을 확인하였다. 그 결과, 포졸란을 전처리한 군에 침투된 CBR1 융합단백질이 더 안정적으로 유지됨을 확인하였다(도 2).
Before the protein treatment, the cells were treated with pozzolan (10 / ml) for 1 hour, washed and treated with CBR1 fusion protein for 1 hour to confirm the safety of the infiltrated protein. As a result, it was confirmed that the CBR1 fusion protein infiltrated into the group pretreated with pozzolan was maintained more stably (Fig. 2).

결과 3: Result 3: CBR1CBR1 융합단백질과Fusion protein 포졸란의Pozzolana HaCaTHaCaT 세포 침투 후  After cell infiltration BaxBax 와 Wow BclBcl -2 기전 연구-2 Mechanism Research

피부 세포인 HaCaT 세포에 H2O2을 1시간 동안 처리한 후(100mM) Bax와 Bcl-2를 웨스턴 블랏 분석을 통해 확인하였다. 그 결과, H2O2을 처리한 후 HaCaT 세포에 Bax와 Bcl2 신호에 의한 세포사멸 기전을 확인하였고, CBR1 융합단백질을 처리하고 H2O2에 의한 세포사멸 보호 효과를 확인하였다. 또한 포졸란을 전처리하였을 경우(위와 같은 방법으로 배양) 세포사멸 기전에 대한 보호가 상승효과를 나타냄을 확인할 수 있었다(도 3). (1 : 대조군, 2 : H2O2 , 3 : H2O2 + CBR1, 4 : H2O2 + PEP-1-CBR1 융합단백질)
The skin cells, HaCaT cells, were treated with H 2 O 2 for 1 hour (100 mM) and then Bax and Bcl-2 were confirmed by Western blot analysis. As a result, after treatment with H 2 O 2 , the cell death mechanism by Bax and Bcl2 signal was confirmed in HaCaT cells, and the effect of CBR1 fusion protein treatment was confirmed by H 2 O 2 . In addition, it was confirmed that when pozzolan was pretreated (cultured by the above method), the protection against apoptosis mechanism was synergistic (FIG. 3). (1: control, 2: H 2 O 2, 3: H 2 O 2 + CBR1, 4: H 2 O 2 + PEP-1-CBR1 fusion protein)

결과 4: Result 4: CBR1CBR1 융합단백질과Fusion protein 포졸란의Pozzolana HaCaTHaCaT 세포 침투 후  After cell infiltration AktAkt 기전 연구 Mechanism Research

피부 세포인 HaCaT 세포에 H2O2을 1시간 동안 처리 후(100mM) Akt를 웨스턴 블랏 분석을 통해 확인하였다. 그 결과 H2O2을 처리한 후 HaCaT 세포에서 Akt 신호에 의한 세포사멸 기전을 확인하였고, CBR1 융합단백질을 처리하자 H2O2에 의한 세포사멸 보호 효과를 확인하였다. 또한 포졸란을 전처리하였을 경우(위와 같은 방법으로 배양) 세포사멸 기전에 대한 보호가 상승효과를 나타냄을 확인할 수 있었다(도 4). (1 : 대조군, 2 : H2O2 , 3 : H2O2 + CBR1, 4 : H2O2 + PEP-1-CBR1 융합단백질)
HaCaT cells, which are skin cells, were treated with H 2 O 2 for 1 hour (100 mM) and then confirmed by Western blot analysis. As a result after processing the H 2 O 2 was confirmed cell death mechanism by Akt signal from HaCaT cells, let handle CBR1 fusion protein was confirmed that cell death protective effect of H 2 O 2. It was also confirmed that when pozzolans were pretreated (cultured in the same manner as above), the protection against apoptosis was synergistic (FIG. 4). (1: control, 2: H 2 O 2, 3: H 2 O 2 + CBR1, 4: H 2 O 2 + PEP-1-CBR1 fusion protein)

결과 5: Result 5: CBR1CBR1 융합단백질과Fusion protein 포졸란의Pozzolana HaCaTHaCaT 세포 침투 후  After cell infiltration JNKJNK , , ERKERK , , P38P38 기전 연구 Mechanism Research

피부 세포인 HaCaT 세포에 H2O2을 1시간 동안 처리한 후(100mM) JNK, ERK, P38을 웨스턴 블랏 분석을 통해 확인하였다. 그 결과 H2O2을 처리한 후 HaCaT 세포에서 JNK, ERK, P38 신호에 의한 세포사멸 기전을 확인하였고, CBR1 융합단백질을 처리하자 H2O2의 의한 세포사멸의 보호 효과를 확인하였다. 또한 포졸란을 전처리하였을 경우(위와 같은 방법으로 배양) 세포사멸 기전에 대한 보호가 특히 p-P38에서 상승작용을 나타냄을 확인할 수 있었다(도 5). (1 : 대조군, 2 : H2O2 , 3 : H2O2 + CBR1, 4 : H2O2 + PEP-1-CBR1 융합단백질)
HaCaT cells were treated with H 2 O 2 for 1 hour (100 mM), and then JNK, ERK and P38 were analyzed by Western blot analysis. The result was then treated with H 2 O 2 in HaCaT cells confirm the cell death mechanism by JNK, ERK, P38 signal, let's handle CBR1 fusion protein was confirmed the protective effect of cell death by the H 2 O 2. In addition, it was confirmed that when pozzolan was pretreated (cultured by the above method), the protection against the apoptosis mechanism was synergistic, especially in p-P38 (FIG. 5). (1: control, 2: H 2 O 2, 3: H 2 O 2 + CBR1, 4: H 2 O 2 + PEP-1-CBR1 fusion protein)

결과 6: Result 6: CBR1CBR1 융합단백질과Fusion protein 포졸란의Pozzolana HaCaTHaCaT 세포 침투 후  After cell infiltration p65p65 , Iκ-Bα 기전 연구, Mechanism of Iκ-Bα

피부 세포인 HaCaT 세포에 H2O2을 1시간 동안 처리한 후(100mM) p65, Iκ-Bα를 웨스턴 블랏 분석을 통해 확인하였다. 그 결과 H2O2을 처리한 후 HaCaT 세포에서 p65, Iκ-Bα 신호에 의한 세포사멸 기전을 확인하였고, CBR1 융합단백질을 처리하자 H2O2 에 의한 세포사멸의 보호 효과를 확인하였다. 또한 포졸란을 전처리하였을 경우(위와 같은 방법으로 배양) 세포사멸 기전에 대한 보호효과가 상승작용을 나타냄을 확인할 수 있었다(도 6). (1 : 대조군, 2 : H2O2 , 3 : H2O2 + CBR1, 4 : H2O2 + PEP-1-CBR1 융합단백질)
HaCaT cells, which are skin cells, were treated with H 2 O 2 for 1 hour (100 mM), and p65 and Iκ-Bα were confirmed by Western blot analysis. As a result, after treatment with H 2 O 2 , the apoptotic mechanism of p65 and Iκ-Bα signal was confirmed in HaCaT cells, and the CBR1 fusion protein was treated with H 2 O 2 To protect against apoptosis. Also, it was confirmed that when pozzolan was pretreated (cultured in the same manner as above), the protective effect against the apoptosis mechanism was synergistic (FIG. 6). (1: control, 2: H 2 O 2, 3: H 2 O 2 + CBR1, 4: H 2 O 2 + PEP-1-CBR1 fusion protein)

결과 7: Result 7: CBR1CBR1 융합단백질과Fusion protein 포졸란의Pozzolana HaCaTHaCaT 세포 침투 후  After cell infiltration COX2COX2 , , iNOSiNOS , IL-6, , IL-6, ILIL -1β, -1β, TNFTNF -α 기전 연구-α mechanism research

피부 세포인 HaCaT 세포에 H2O2을 1시간 동안 처리한 후(100mM) COX2, iNOS, IL-6, IL-1β, TNF-α를 웨스턴 블랏 분석을 통해 확인하였다. 그 결과 H2O2을 처리한 후 HaCaT 세포에 COX2, iNOS, IL-6, IL-1β, TNF-α 신호에 의한 세포사멸 기전을 확인하였고, CBR1 융합단백질을 처리하자 H2O2에 의한 세포사멸을 보호 효과를 확인하였다. 또한 포졸란을 전처리하였을 경우(위와 같은 방법으로 배양) 세포사멸 기전에 대한 보호효과가 시너직하게 나타남을 확인할 수 있었다(도 7). (IL-1β, TNF-α제외), (1 : 대조군, 2 : H2O2 , 3 : H2O2 + CBR1, 4 : H2O2 + PEP-1-CBR1).HACaT cells were treated with H 2 O 2 for 1 hour (100 mM), and then COX 2, iNOS, IL-6, IL-1β and TNF-α were analyzed by Western blot analysis. The result was confirmed after the treatment with H 2 O 2 COX2, iNOS in HaCaT cells, IL-6, IL-1β , cell death mechanism by TNF-α signal, let's handle CBR1 fusion proteins by H 2 O 2 The protective effect of apoptosis was confirmed. In addition, it was confirmed that the protective effect on the apoptosis mechanism of the pozzolana was precisely observed (Fig. 7). (Except for IL-1β and TNF-α), (1: control group, 2: H 2 O 2 , 3: H 2 O 2 + CBR1, 4: H 2 O 2 + PEP-1-CBR1).

또한, RT-PCR을 실험하였다. 그 결과 웨스턴 블랏 분석과 같은 결과를 얻었으며, 특히 IL-6, TNF-α에서 탁월한 효능을 확인하였다(도 8).
RT-PCR was also performed. As a result, the same results as the Western blot analysis were obtained, and particularly, excellent efficacy was confirmed in IL-6 and TNF-α (FIG. 8).

실시예Example 1: 화장수 제조 1: Lotion production

95% 에탄올 8g에 폴리피로리돈 0.05g, 올레일알콜 0.1g, 폴리옥시에틸렌모노올레이트 0.2g, 향료 0.2g, 파라옥시안식향산메틸에스테르 0.1g, 소량의 산화방지제, 소량의 색소를 혼합 용해한다. 상기에서 수득한 CBR1 융합단백질 500㎎, 포졸란 1g, 글리세린 5g을 정제수 85.35g에 용해한 것에 상기 혼합액을 첨가한 후 교반하여 항노화 효과가 있는 화장수를 얻었다.
0.05 g of polypyrrolidone, 0.1 g of oleyl alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of p-hydroxybenzoic acid methyl ester, a small amount of antioxidant and a small amount of pigment are mixed and dissolved in 8 g of 95% ethanol . 500 mg of the CBR1 fusion protein obtained above, 1 g of pozzolan and 5 g of glycerin were dissolved in 85.35 g of purified water. The mixture was added to the mixture, followed by stirring to obtain a lotion having anti-aging effect.

실시예Example 2: 유액 제조 2: Milk production

세틸알콜 1.2g, 스쿠알란 10g, 바세린 2g, 파라옥시안식향산에틸에스테르 0.2g, 글리세린모노에스테아레이드 1g, 폴리옥시에틸렌(20몰부가)모노올레이트 1g 및 향료 0.1g을 70℃에서 가열, 혼합용해하고, CBR1 융합단백질 500㎎, 포졸란 1g, 디프로필렌글리콜 5g, 폴리에틸렌글리콜-1500 2g, 트리에탄올아민 0.2g, 정제수 76.2g을 75℃로 가열해서 용해시킨다. 양자를 혼합하여 유화시킨 후 냉각하여 수중유(O/W)형의 항노화 효과가 있는 유액을 얻었다.
10 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of p-hydroxybenzoic acid ethyl ester, 1 g of glycerin monoestearylate, 1 g of polyoxyethylene (20 mol added) monooleate and 0.1 g of perfume were heated, 500 mg of the CBR1 fusion protein, 1 g of pozzolan, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1,500, 0.2 g of triethanolamine and 76.2 g of purified water were dissolved by heating at 75 캜. The two were mixed and emulsified and then cooled to obtain oil-in-water (O / W) type anti-aging oil.

실시예Example 3:  3: 미용액Serum 제조 Produce

95% 에틸알콜 5g에 폴리옥시에틸렌솔비탄모노올레이트 1.2g, 키툴로오즈 0.3g, 히야론산나트륨 0.2g, 비타민 E-아세테이트 0.2g, 감초산 나트륨 0.2g, 파라옥시안식향산에틸에스테르 0.1g, CBR1 융합단백질 500㎎, 포졸란 1g 및 적량의 색소를 혼합하여 항노화 효과가 있는 미용액을 얻었다.To 5 g of 95% ethyl alcohol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of quitrose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium permanganate, 0.1 g of paraoxybenzoic acid ethyl ester, 500 mg of the CBR1 fusion protein, 1 g of pozzolan and an appropriate amount of pigment were mixed to obtain a serum having an anti-aging effect.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Antiaging cometic composition containing cell-trasnducible carbonyl reductase fusion protein and pozzolan <130> HallymU-sychoi-CBR1-antiagingcosmetic <160> 14 <170> KopatentIn 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ccgctcgagt cgtccggcat c 21 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cgggatcctt atcaccactg ttcaac 26 <210> 3 <211> 868 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding Tat-Carbonyl Reductase 1 fusion protein <400> 3 taggaagaag cggagacagc gacgaagact cgagatgtcg tccggcatcc atgtagcgct 60 ggtgactgga ggcaacaagg gcatcggctt ggccatcgtg cgcgacctgt gccggctgtt 120 ctcgggggac gtggtgctca cggcgcggga cgtgacgcgg ggccaggcgg ccgtacagca 180 gctgcaggcg gagggcctga gcccgcgctt ccaccagctg gacatcgacg atctgcagag 240 catccgcgcc ctgcgcgact tcctgcgcaa ggagtacggg ggcctggacg tgctggtcaa 300 caacgcgggc atcgccttca aggttgctga tcccacaccc tttcatattc aagctgaagt 360 gacgatgaaa acaaatttct ttggtacccg agatgtgtgc acagaattac tccctctaat 420 aaaaccccaa gggagagtgg tgaacgtatc tagcatcatg agcgtcagag cccttaaaag 480 ctgcagccca gagctgcagc agaagttccg cagtgagacc atcactgagg aggagctggt 540 ggggctcatg aacaagtttg tggaggatac aaagaaggga gtgcaccaga aggagggctg 600 gcccagcagc gcatacgggg tgacgaagat tggcgtcacc gttctgtcca ggatccacgc 660 caggaaactg agtgagcaga ggaaagggga caagatcctc ctgaatgcct gctgcccagg 720 gtgggtgaga actgacatgg cgggacccaa ggccaccaag agcccagaag aaggtgcaga 780 gacccctgtg tacttggccc ttttgccccc agatgctgag ggtccccatg gacaatttgt 840 ttcagagaag agagttgaac agtggtga 868 <210> 4 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Tat-Carbonyl Reductase 1 fusion protein <400> 4 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ser Ser Gly Ile 1 5 10 15 His Val Ala Leu Val Thr Gly Gly Asn Lys Gly Ile Gly Leu Ala Ile 20 25 30 Val Arg Asp Leu Cys Arg Leu Phe Ser Gly Asp Val Val Leu Thr Ala 35 40 45 Arg Asp Val Thr Arg Gly Gln Ala Ala Val Gln Gln Leu Gln Ala Glu 50 55 60 Gly Leu Ser Pro Arg Phe His Gln Leu Asp Ile Asp Asp Leu Gln Ser 65 70 75 80 Ile Arg Ala Leu Arg Asp Phe Leu Arg Lys Glu Tyr Gly Gly Leu Asp 85 90 95 Val Leu Val Asn Asn Ala Gly Ile Ala Phe Lys Val Ala Asp Pro Thr 100 105 110 Pro Phe His Ile Gln Ala Glu Val Thr Met Lys Thr Asn Phe Phe Gly 115 120 125 Thr Arg Asp Val Cys Thr Glu Leu Leu Pro Leu Ile Lys Pro Gln Gly 130 135 140 Arg Val Val Asn Val Ser Ser Ile Met Ser Val Arg Ala Leu Lys Ser 145 150 155 160 Cys Ser Pro Glu Leu Gln Gln Lys Phe Arg Ser Glu Thr Ile Thr Glu 165 170 175 Glu Glu Leu Val Gly Leu Met Asn Lys Phe Val Glu Asp Thr Lys Lys 180 185 190 Gly Val His Gln Lys Glu Gly Trp Pro Ser Ser Ala Tyr Gly Val Thr 195 200 205 Lys Ile Gly Val Thr Val Leu Ser Arg Ile His Ala Arg Lys Leu Ser 210 215 220 Glu Gln Arg Lys Gly Asp Lys Ile Leu Leu Asn Ala Cys Cys Pro Gly 225 230 235 240 Trp Val Arg Thr Asp Met Ala Gly Pro Lys Ala Thr Lys Ser Pro Glu 245 250 255 Glu Gly Ala Glu Thr Pro Val Tyr Leu Ala Leu Leu Pro Pro Asp Ala 260 265 270 Glu Gly Pro His Gly Gln Phe Val Ser Glu Lys Arg Val Glu Gln Trp 275 280 285 <210> 5 <211> 871 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Carbonyl Reductase 1-Tat fusion protein <400> 5 atgtcgtccg gcatccatgt agcgctggtg actggaggca acaagggcat cggcttggcc 60 atcgtgcgcg acctgtgccg gctgttctcg ggggacgtgg tgctcacggc gcgggacgtg 120 acgcggggcc aggcggccgt acagcagctg caggcggagg gcctgagccc gcgcttccac 180 cagctggaca tcgacgatct gcagagcatc cgcgccctgc gcgacttcct gcgcaaggag 240 tacgggggcc tggacgtgct ggtcaacaac gcgggcatcg ccttcaaggt tgctgatccc 300 acaccctttc atattcaagc tgaagtgacg atgaaaacaa atttctttgg tacccgagat 360 gtgtgcacag aattactccc tctaataaaa ccccaaggga gagtggtgaa cgtatctagc 420 atcatgagcg tcagagccct taaaagctgc agcccagagc tgcagcagaa gttccgcagt 480 gagaccatca ctgaggagga gctggtgggg ctcatgaaca agtttgtgga ggatacaaag 540 aagggagtgc accagaagga gggctggccc agcagcgcat acggggtgac gaagattggc 600 gtcaccgttc tgtccaggat ccacgccagg aaactgagtg agcagaggaa aggggacaag 660 atcctcctga atgcctgctg cccagggtgg gtgagaactg acatggcggg acccaaggcc 720 accaagagcc cagaagaagg tgcagagacc cctgtgtact tggccctttt gcccccagat 780 gctgagggtc cccatggaca atttgtttca gagaagagag ttgaacagtg gtgaggatcc 840 taggaagaag cggagacagc gacgaagata g 871 <210> 6 <211> 286 <212> PRT <213> Artificial Sequence <220> <223> Carbonyl Reductase 1-Tat fusion protein <400> 6 Met Ser Ser Gly Ile His Val Ala Leu Val Thr Gly Gly Asn Lys Gly 1 5 10 15 Ile Gly Leu Ala Ile Val Arg Asp Leu Cys Arg Leu Phe Ser Gly Asp 20 25 30 Val Val Leu Thr Ala Arg Asp Val Thr Arg Gly Gln Ala Ala Val Gln 35 40 45 Gln Leu Gln Ala Glu Gly Leu Ser Pro Arg Phe His Gln Leu Asp Ile 50 55 60 Asp Asp Leu Gln Ser Ile Arg Ala Leu Arg Asp Phe Leu Arg Lys Glu 65 70 75 80 Tyr Gly Gly Leu Asp Val Leu Val Asn Asn Ala Gly Ile Ala Phe Lys 85 90 95 Val Ala Asp Pro Thr Pro Phe His Ile Gln Ala Glu Val Thr Met Lys 100 105 110 Thr Asn Phe Phe Gly Thr Arg Asp Val Cys Thr Glu Leu Leu Pro Leu 115 120 125 Ile Lys Pro Gln Gly Arg Val Val Asn Val Ser Ser Ile Met Ser Val 130 135 140 Arg Ala Leu Lys Ser Cys Ser Pro Glu Leu Gln Gln Lys Phe Arg Ser 145 150 155 160 Glu Thr Ile Thr Glu Glu Glu Leu Val Gly Leu Met Asn Lys Phe Val 165 170 175 Glu Asp Thr Lys Lys Gly Val His Gln Lys Glu Gly Trp Pro Ser Ser 180 185 190 Ala Tyr Gly Val Thr Lys Ile Gly Val Thr Val Leu Ser Arg Ile His 195 200 205 Ala Arg Lys Leu Ser Glu Gln Arg Lys Gly Asp Lys Ile Leu Leu Asn 210 215 220 Ala Cys Cys Pro Gly Trp Val Arg Thr Asp Met Ala Gly Pro Lys Ala 225 230 235 240 Thr Lys Ser Pro Glu Glu Gly Ala Glu Thr Pro Val Tyr Leu Ala Leu 245 250 255 Leu Pro Pro Asp Ala Glu Gly Pro His Gly Gln Phe Val Ser Glu Lys 260 265 270 Arg Val Glu Gln Trp Arg Lys Lys Arg Arg Gln Arg Arg Arg 275 280 285 <210> 7 <211> 905 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Tat-Carbonyl Reductase 1-Tat fusion protein <400> 7 taggaagaag cggagacagc gacgaagact cgagatgtcg tccggcatcc atgtagcgct 60 ggtgactgga ggcaacaagg gcatcggctt ggccatcgtg cgcgacctgt gccggctgtt 120 ctcgggggac gtggtgctca cggcgcggga cgtgacgcgg ggccaggcgg ccgtacagca 180 gctgcaggcg gagggcctga gcccgcgctt ccaccagctg gacatcgacg atctgcagag 240 catccgcgcc ctgcgcgact tcctgcgcaa ggagtacggg ggcctggacg tgctggtcaa 300 caacgcgggc atcgccttca aggttgctga tcccacaccc tttcatattc aagctgaagt 360 gacgatgaaa acaaatttct ttggtacccg agatgtgtgc acagaattac tccctctaat 420 aaaaccccaa gggagagtgg tgaacgtatc tagcatcatg agcgtcagag cccttaaaag 480 ctgcagccca gagctgcagc agaagttccg cagtgagacc atcactgagg aggagctggt 540 ggggctcatg aacaagtttg tggaggatac aaagaaggga gtgcaccaga aggagggctg 600 gcccagcagc gcatacgggg tgacgaagat tggcgtcacc gttctgtcca ggatccacgc 660 caggaaactg agtgagcaga ggaaagggga caagatcctc ctgaatgcct gctgcccagg 720 gtgggtgaga actgacatgg cgggacccaa ggccaccaag agcccagaag aaggtgcaga 780 gacccctgtg tacttggccc ttttgccccc agatgctgag ggtccccatg gacaatttgt 840 ttcagagaag agagttgaac agtggtgagg atcctaggaa gaagcggaga cagcgacgaa 900 gatag 905 <210> 8 <211> 299 <212> PRT <213> Artificial Sequence <220> <223> Tat-Carbonyl Reductase 1-Tat fusion protein <400> 8 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ser Ser Gly Ile 1 5 10 15 His Val Ala Leu Val Thr Gly Gly Asn Lys Gly Ile Gly Leu Ala Ile 20 25 30 Val Arg Asp Leu Cys Arg Leu Phe Ser Gly Asp Val Val Leu Thr Ala 35 40 45 Arg Asp Val Thr Arg Gly Gln Ala Ala Val Gln Gln Leu Gln Ala Glu 50 55 60 Gly Leu Ser Pro Arg Phe His Gln Leu Asp Ile Asp Asp Leu Gln Ser 65 70 75 80 Ile Arg Ala Leu Arg Asp Phe Leu Arg Lys Glu Tyr Gly Gly Leu Asp 85 90 95 Val Leu Val Asn Asn Ala Gly Ile Ala Phe Lys Val Ala Asp Pro Thr 100 105 110 Pro Phe His Ile Gln Ala Glu Val Thr Met Lys Thr Asn Phe Phe Gly 115 120 125 Thr Arg Asp Val Cys Thr Glu Leu Leu Pro Leu Ile Lys Pro Gln Gly 130 135 140 Arg Val Val Asn Val Ser Ser Ile Met Ser Val Arg Ala Leu Lys Ser 145 150 155 160 Cys Ser Pro Glu Leu Gln Gln Lys Phe Arg Ser Glu Thr Ile Thr Glu 165 170 175 Glu Glu Leu Val Gly Leu Met Asn Lys Phe Val Glu Asp Thr Lys Lys 180 185 190 Gly Val His Gln Lys Glu Gly Trp Pro Ser Ser Ala Tyr Gly Val Thr 195 200 205 Lys Ile Gly Val Thr Val Leu Ser Arg Ile His Ala Arg Lys Leu Ser 210 215 220 Glu Gln Arg Lys Gly Asp Lys Ile Leu Leu Asn Ala Cys Cys Pro Gly 225 230 235 240 Trp Val Arg Thr Asp Met Ala Gly Pro Lys Ala Thr Lys Ser Pro Glu 245 250 255 Glu Gly Ala Glu Thr Pro Val Tyr Leu Ala Leu Leu Pro Pro Asp Ala 260 265 270 Glu Gly Pro His Gly Gln Phe Val Ser Glu Lys Arg Val Glu Gln Trp 275 280 285 Gly Ser Arg Lys Lys Arg Arg Gln Arg Arg Arg 290 295 <210> 9 <211> 905 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding PEP-Carbonyl Reductase 1 fusion protein <400> 9 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatgtcgtcc ggcatccatg tagcgctggt gactggaggc aacaagggca 120 tcggcttggc catcgtgcgc gacctgtgcc ggctgttctc gggggacgtg gtgctcacgg 180 cgcgggacgt gacgcggggc caggcggccg tacagcagct gcaggcggag ggcctgagcc 240 cgcgcttcca ccagctggac atcgacgatc tgcagagcat ccgcgccctg cgcgacttcc 300 tgcgcaagga gtacgggggc ctggacgtgc tggtcaacaa cgcgggcatc gccttcaagg 360 ttgctgatcc cacacccttt catattcaag ctgaagtgac gatgaaaaca aatttctttg 420 gtacccgaga tgtgtgcaca gaattactcc ctctaataaa accccaaggg agagtggtga 480 acgtatctag catcatgagc gtcagagccc ttaaaagctg cagcccagag ctgcagcaga 540 agttccgcag tgagaccatc actgaggagg agctggtggg gctcatgaac aagtttgtgg 600 aggatacaaa gaagggagtg caccagaagg agggctggcc cagcagcgca tacggggtga 660 cgaagattgg cgtcaccgtt ctgtccagga tccacgccag gaaactgagt gagcagagga 720 aaggggacaa gatcctcctg aatgcctgct gcccagggtg ggtgagaact gacatggcgg 780 gacccaaggc caccaagagc ccagaagaag gtgcagagac ccctgtgtac ttggcccttt 840 tgcccccaga tgctgagggt ccccatggac aatttgtttc agagaagaga gttgaacagt 900 ggtga 905 <210> 10 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> PEP-Carbonyl Reductase 1 fusion protein <400> 10 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ser Ser Gly Ile His Val Ala Leu 20 25 30 Val Thr Gly Gly Asn Lys Gly Ile Gly Leu Ala Ile Val Arg Asp Leu 35 40 45 Cys Arg Leu Phe Ser Gly Asp Val Val Leu Thr Ala Arg Asp Val Thr 50 55 60 Arg Gly Gln Ala Ala Val Gln Gln Leu Gln Ala Glu Gly Leu Ser Pro 65 70 75 80 Arg Phe His Gln Leu Asp Ile Asp Asp Leu Gln Ser Ile Arg Ala Leu 85 90 95 Arg Asp Phe Leu Arg Lys Glu Tyr Gly Gly Leu Asp Val Leu Val Asn 100 105 110 Asn Ala Gly Ile Ala Phe Lys Val Ala Asp Pro Thr Pro Phe His Ile 115 120 125 Gln Ala Glu Val Thr Met Lys Thr Asn Phe Phe Gly Thr Arg Asp Val 130 135 140 Cys Thr Glu Leu Leu Pro Leu Ile Lys Pro Gln Gly Arg Val Val Asn 145 150 155 160 Val Ser Ser Ile Met Ser Val Arg Ala Leu Lys Ser Cys Ser Pro Glu 165 170 175 Leu Gln Gln Lys Phe Arg Ser Glu Thr Ile Thr Glu Glu Glu Leu Val 180 185 190 Gly Leu Met Asn Lys Phe Val Glu Asp Thr Lys Lys Gly Val His Gln 195 200 205 Lys Glu Gly Trp Pro Ser Ser Ala Tyr Gly Val Thr Lys Ile Gly Val 210 215 220 Thr Val Leu Ser Arg Ile His Ala Arg Lys Leu Ser Glu Gln Arg Lys 225 230 235 240 Gly Asp Lys Ile Leu Leu Asn Ala Cys Cys Pro Gly Trp Val Arg Thr 245 250 255 Asp Met Ala Gly Pro Lys Ala Thr Lys Ser Pro Glu Glu Gly Ala Glu 260 265 270 Thr Pro Val Tyr Leu Ala Leu Leu Pro Pro Asp Ala Glu Gly Pro His 275 280 285 Gly Gln Phe Val Ser Glu Lys Arg Val Glu Gln Trp 290 295 300 <210> 11 <211> 905 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Carbonyl Reductase 1-PEP-1 fusion protein <400> 11 atgtcgtccg gcatccatgt agcgctggtg actggaggca acaagggcat cggcttggcc 60 atcgtgcgcg acctgtgccg gctgttctcg ggggacgtgg tgctcacggc gcgggacgtg 120 acgcggggcc aggcggccgt acagcagctg caggcggagg gcctgagccc gcgcttccac 180 cagctggaca tcgacgatct gcagagcatc cgcgccctgc gcgacttcct gcgcaaggag 240 tacgggggcc tggacgtgct ggtcaacaac gcgggcatcg ccttcaaggt tgctgatccc 300 acaccctttc atattcaagc tgaagtgacg atgaaaacaa atttctttgg tacccgagat 360 gtgtgcacag aattactccc tctaataaaa ccccaaggga gagtggtgaa cgtatctagc 420 atcatgagcg tcagagccct taaaagctgc agcccagagc tgcagcagaa gttccgcagt 480 gagaccatca ctgaggagga gctggtgggg ctcatgaaca agtttgtgga ggatacaaag 540 aagggagtgc accagaagga gggctggccc agcagcgcat acggggtgac gaagattggc 600 gtcaccgttc tgtccaggat ccacgccagg aaactgagtg agcagaggaa aggggacaag 660 atcctcctga atgcctgctg cccagggtgg gtgagaactg acatggcggg acccaaggcc 720 accaagagcc cagaagaagg tgcagagacc cctgtgtact tggccctttt gcccccagat 780 gctgagggtc cccatggaca atttgtttca gagaagagag ttgaacagtg gtgaggatcc 840 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 900 aagtg 905 <210> 12 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> Carbonyl Reductase 1-PEP-1 fusion protein <400> 12 Met Ser Ser Gly Ile His Val Ala Leu Val Thr Gly Gly Asn Lys Gly 1 5 10 15 Ile Gly Leu Ala Ile Val Arg Asp Leu Cys Arg Leu Phe Ser Gly Asp 20 25 30 Val Val Leu Thr Ala Arg Asp Val Thr Arg Gly Gln Ala Ala Val Gln 35 40 45 Gln Leu Gln Ala Glu Gly Leu Ser Pro Arg Phe His Gln Leu Asp Ile 50 55 60 Asp Asp Leu Gln Ser Ile Arg Ala Leu Arg Asp Phe Leu Arg Lys Glu 65 70 75 80 Tyr Gly Gly Leu Asp Val Leu Val Asn Asn Ala Gly Ile Ala Phe Lys 85 90 95 Val Ala Asp Pro Thr Pro Phe His Ile Gln Ala Glu Val Thr Met Lys 100 105 110 Thr Asn Phe Phe Gly Thr Arg Asp Val Cys Thr Glu Leu Leu Pro Leu 115 120 125 Ile Lys Pro Gln Gly Arg Val Val Asn Val Ser Ser Ile Met Ser Val 130 135 140 Arg Ala Leu Lys Ser Cys Ser Pro Glu Leu Gln Gln Lys Phe Arg Ser 145 150 155 160 Glu Thr Ile Thr Glu Glu Glu Leu Val Gly Leu Met Asn Lys Phe Val 165 170 175 Glu Asp Thr Lys Lys Gly Val His Gln Lys Glu Gly Trp Pro Ser Ser 180 185 190 Ala Tyr Gly Val Thr Lys Ile Gly Val Thr Val Leu Ser Arg Ile His 195 200 205 Ala Arg Lys Leu Ser Glu Gln Arg Lys Gly Asp Lys Ile Leu Leu Asn 210 215 220 Ala Cys Cys Pro Gly Trp Val Arg Thr Asp Met Ala Gly Pro Lys Ala 225 230 235 240 Thr Lys Ser Pro Glu Glu Gly Ala Glu Thr Pro Val Tyr Leu Ala Leu 245 250 255 Leu Pro Pro Asp Ala Glu Gly Pro His Gly Gln Phe Val Ser Glu Lys 260 265 270 Arg Val Glu Gln Trp Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp Trp 275 280 285 Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 290 295 300 <210> 13 <211> 976 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding PEP-1-Carbonyl Reductase 1-PEP-1 fusion protein <400> 13 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatgtcgtcc ggcatccatg tagcgctggt gactggaggc aacaagggca 120 tcggcttggc catcgtgcgc gacctgtgcc ggctgttctc gggggacgtg gtgctcacgg 180 cgcgggacgt gacgcggggc caggcggccg tacagcagct gcaggcggag ggcctgagcc 240 cgcgcttcca ccagctggac atcgacgatc tgcagagcat ccgcgccctg cgcgacttcc 300 tgcgcaagga gtacgggggc ctggacgtgc tggtcaacaa cgcgggcatc gccttcaagg 360 ttgctgatcc cacacccttt catattcaag ctgaagtgac gatgaaaaca aatttctttg 420 gtacccgaga tgtgtgcaca gaattactcc ctctaataaa accccaaggg agagtggtga 480 acgtatctag catcatgagc gtcagagccc ttaaaagctg cagcccagag ctgcagcaga 540 agttccgcag tgagaccatc actgaggagg agctggtggg gctcatgaac aagtttgtgg 600 aggatacaaa gaagggagtg caccagaagg agggctggcc cagcagcgca tacggggtga 660 cgaagattgg cgtcaccgtt ctgtccagga tccacgccag gaaactgagt gagcagagga 720 aaggggacaa gatcctcctg aatgcctgct gcccagggtg ggtgagaact gacatggcgg 780 gacccaaggc caccaagagc ccagaagaag gtgcagagac ccctgtgtac ttggcccttt 840 tgcccccaga tgctgagggt ccccatggac aatttgtttc agagaagaga gttgaacagt 900 ggtgaggatc ctaaaagaaa cctggtggga aacctggtgg accgaatggt ctcagccgaa 960 aaaaaaacgt aaagtg 976 <210> 14 <211> 323 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-Carbonyl Reductase 1-PEP-1 fusion protein <400> 14 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ser Ser Gly Ile His Val Ala Leu 20 25 30 Val Thr Gly Gly Asn Lys Gly Ile Gly Leu Ala Ile Val Arg Asp Leu 35 40 45 Cys Arg Leu Phe Ser Gly Asp Val Val Leu Thr Ala Arg Asp Val Thr 50 55 60 Arg Gly Gln Ala Ala Val Gln Gln Leu Gln Ala Glu Gly Leu Ser Pro 65 70 75 80 Arg Phe His Gln Leu Asp Ile Asp Asp Leu Gln Ser Ile Arg Ala Leu 85 90 95 Arg Asp Phe Leu Arg Lys Glu Tyr Gly Gly Leu Asp Val Leu Val Asn 100 105 110 Asn Ala Gly Ile Ala Phe Lys Val Ala Asp Pro Thr Pro Phe His Ile 115 120 125 Gln Ala Glu Val Thr Met Lys Thr Asn Phe Phe Gly Thr Arg Asp Val 130 135 140 Cys Thr Glu Leu Leu Pro Leu Ile Lys Pro Gln Gly Arg Val Val Asn 145 150 155 160 Val Ser Ser Ile Met Ser Val Arg Ala Leu Lys Ser Cys Ser Pro Glu 165 170 175 Leu Gln Gln Lys Phe Arg Ser Glu Thr Ile Thr Glu Glu Glu Leu Val 180 185 190 Gly Leu Met Asn Lys Phe Val Glu Asp Thr Lys Lys Gly Val His Gln 195 200 205 Lys Glu Gly Trp Pro Ser Ser Ala Tyr Gly Val Thr Lys Ile Gly Val 210 215 220 Thr Val Leu Ser Arg Ile His Ala Arg Lys Leu Ser Glu Gln Arg Lys 225 230 235 240 Gly Asp Lys Ile Leu Leu Asn Ala Cys Cys Pro Gly Trp Val Arg Thr 245 250 255 Asp Met Ala Gly Pro Lys Ala Thr Lys Ser Pro Glu Glu Gly Ala Glu 260 265 270 Thr Pro Val Tyr Leu Ala Leu Leu Pro Pro Asp Ala Glu Gly Pro His 275 280 285 Gly Gln Phe Val Ser Glu Lys Arg Val Glu Gln Trp Gly Ser Lys Glu 290 295 300 Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys 305 310 315 320 Arg Lys Val <110> Industry Academic Cooperation Foundation, Hallym University <120> Antiaging cometic composition containing cell-trasnducible          carbonyl reductase fusion protein and pozzolan <130> HallymU-sychoi-CBR1-antiagingcosmetic <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ccgctcgagt cgtccggcat c 21 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cgggatcctt atcaccactg ttcaac 26 <210> 3 <211> 868 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Tat-Carbonyl Reductase 1 fusion protein <400> 3 taggaagaag cggagacagc gacgaagact cgagatgtcg tccggcatcc atgtagcgct 60 ggtgactgga ggcaacaagg gcatcggctt ggccatcgtg cgcgacctgt gccggctgtt 120 ctcgggggac gtggtgctca cggcgcggga cgtgacgcgg ggccaggcgg ccgtacagca 180 gctgcaggcg gagggcctga gcccgcgctt ccaccagctg gacatcgacg atctgcagag 240 catccgcgcc ctgcgcgact tcctgcgcaa ggagtacggg ggcctggacg tgctggtcaa 300 caacgcgggc atcgccttca aggttgctga tcccacaccc tttcatattc aagctgaagt 360 gacgatgaaa acaaatttct ttggtacccg agatgtgtgc acagaattac tccctctaat 420 aaaaccccaa gggagagtgg tgaacgtatc tagcatcatg agcgtcagag cccttaaaag 480 ctgcagccca gagctgcagc agaagttccg cagtgagacc atcactgagg aggagctggt 540 ggggctcatg aacaagtttg tggaggatac aaagaaggga gtgcaccaga aggagggctg 600 gcccagcagc gcatacgggg tgacgaagat tggcgtcacc gttctgtcca ggatccacgc 660 caggaaactg agtgagcaga ggaaagggga caagatcctc ctgaatgcct gctgcccagg 720 gtgggtgaga actgacatgg cgggacccaa ggccaccaag agcccagaag aaggtgcaga 780 gacccctgtg tacttggccc ttttgccccc agatgctgag ggtccccatg gacaatttgt 840 ttcagagaag agagttgaac agtggtga 868 <210> 4 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Tat-Carbonyl Reductase 1 fusion protein <400> 4 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ser Ser Gly Ile   1 5 10 15 His Val Ala Leu Val Thr Gly Gly Asn Lys Gly Ile Gly Leu Ala Ile              20 25 30 Val Arg Asp Leu Cys Arg Leu Phe Ser Gly Asp Val Val Leu Thr Ala          35 40 45 Arg Asp Val Thr Arg Gly Gln Ala Ala Val Gln Gln Leu Gln Ala Glu      50 55 60 Gly Leu Ser Pro Arg Phe His Gln Leu Asp Ile Asp Asp Leu Gln Ser  65 70 75 80 Ile Arg Ala Leu Arg Asp Phe Leu Arg Lys Glu Tyr Gly Gly Leu Asp                  85 90 95 Val Leu Val Asn Asn Ala Gly Ile Ala Phe Lys Val Ala Asp Pro Thr             100 105 110 Pro Phe His Ile Gln Ala Glu Val Thr Met Lys Thr Asn Phe Phe Gly         115 120 125 Thr Arg Asp Val Cys Thr Glu Leu Leu Pro Leu Ile Lys Pro Gln Gly     130 135 140 Arg Val Val Asn Val Ser Ser Ile Met Ser Val Arg Ala Leu Lys Ser 145 150 155 160 Cys Ser Pro Glu Leu Gln Gln Lys Phe Arg Ser Glu Thr Ile Thr Glu                 165 170 175 Glu Glu Leu Val Gly Leu Met Asn Lys Phe Val Glu Asp Thr Lys Lys             180 185 190 Gly Val His Gln Lys Glu Gly Trp Pro Ser Ser Ala Tyr Gly Val Thr         195 200 205 Lys Ile Gly Val Thr Val Leu Ser Arg Ile His Ala Arg Lys Leu Ser     210 215 220 Glu Gln Arg Lys Gly Asp Lys Ile Leu Leu Asn Ala Cys Cys Pro Gly 225 230 235 240 Trp Val Arg Thr Asp Met Ala Gly Pro Lys Ala Thr Lys Ser Pro Glu                 245 250 255 Glu Gly Ala Glu Thr Pro Val Tyr Leu Ala Leu Leu Pro Pro Asp Ala             260 265 270 Glu Gly Pro His Gly Gln Phe Val Ser Glu Lys Arg Val Glu Gln Trp         275 280 285 <210> 5 <211> 871 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Carbonyl Reductase 1-Tat fusion protein <400> 5 atgtcgtccg gcatccatgt agcgctggtg actggaggca acaagggcat cggcttggcc 60 atcgtgcgcg acctgtgccg gctgttctcg ggggacgtgg tgctcacggc gcgggacgtg 120 acgcggggcc aggcggccgt acagcagctg caggcggagg gcctgagccc gcgcttccac 180 cagctggaca tcgacgatct gcagagcatc cgcgccctgc gcgacttcct gcgcaaggag 240 tacgggggcc tggacgtgct ggtcaacaac gcgggcatcg ccttcaaggt tgctgatccc 300 acaccctttc atattcaagc tgaagtgacg atgaaaacaa atttctttgg tacccgagat 360 gtgtgcacag aattactccc tctaataaaa ccccaaggga gagtggtgaa cgtatctagc 420 atcatgagcg tcagagccct taaaagctgc agcccagagc tgcagcagaa gttccgcagt 480 gagaccatca ctgaggagga gctggtgggg ctcatgaaca agtttgtgga ggatacaaag 540 aagggagtgc accagaagga gggctggccc agcagcgcat acggggtgac gaagattggc 600 gtcaccgttc tgtccaggat ccacgccagg aaactgagtg agcagaggaa aggggacaag 660 atcctcctga atgcctgctg cccagggtgg gtgagaactg acatggcggg acccaaggcc 720 accaagagcc cagaagaagg tgcagagacc cctgtgtact tggccctttt gcccccagat 780 gctgagggtc cccatggaca atttgtttca gagaagagag ttgaacagtg gtgaggatcc 840 taggaagaag cggagacagc gacgaagata g 871 <210> 6 <211> 286 <212> PRT <213> Artificial Sequence <220> <223> Carbonyl Reductase 1-Tat fusion protein <400> 6 Met Ser Ser Gly Ile His Val Ala Leu Val Thr Gly Gly Asn Lys Gly   1 5 10 15 Ile Gly Leu Ala Ile Val Arg Asp Leu Cys Arg Leu Phe Ser Gly Asp              20 25 30 Val Val Leu Thr Ala Arg Asp Val Thr Arg Gly Gln Ala Ala Val Gln          35 40 45 Gln Leu Gln Ala Glu Gly Leu Ser Pro Arg Phe His Gln Leu Asp Ile      50 55 60 Asp Asp Leu Gln Ser Ile Arg Ala Leu Arg Asp Phe Leu Arg Lys Glu  65 70 75 80 Tyr Gly Gly Leu Asp Val Leu Val Asn Asn Ala Gly Ile Ala Phe Lys                  85 90 95 Val Ala Asp Pro Thr Pro Phe His Ile Gln Ala Glu Val Thr Met Lys             100 105 110 Thr Asn Phe Phe Gly Thr Arg Asp Val Cys Thr Glu Leu Leu Pro Leu         115 120 125 Ile Lys Pro Gln Gly Arg Val Val Asn Val Ser Ser Ile Met Ser Val     130 135 140 Arg Ala Leu Lys Ser Cys Ser Pro Glu Leu Gln Gln Lys Phe Arg Ser 145 150 155 160 Glu Thr Ile Thr Glu Glu Glu Leu Val Gly Leu Met Asn Lys Phe Val                 165 170 175 Glu Asp Thr Lys Lys Gly Val His Gln Lys Glu Gly Trp Pro Ser Ser             180 185 190 Ala Tyr Gly Val Thr Lys Ile Gly Val Thr Val Leu Ser Arg Ile His         195 200 205 Ala Arg Lys Leu Ser Glu Gln Arg Lys Gly Asp Lys Ile Leu Leu Asn     210 215 220 Ala Cys Cys Pro Gly Trp Val Arg Thr Asp Met Ala Gly Pro Lys Ala 225 230 235 240 Thr Lys Ser Pro Glu Glu Gly Ala Glu Thr Pro Val Tyr Leu Ala Leu                 245 250 255 Leu Pro Pro Asp Ala Glu Gly Pro His Gly Gln Phe Val Ser Glu Lys             260 265 270 Arg Val Glu Gln Trp Arg Lys Lys Arg Arg Gln Arg Arg Arg         275 280 285 <210> 7 <211> 905 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Tat-Carbonyl Reductase 1-Tat fusion          protein <400> 7 taggaagaag cggagacagc gacgaagact cgagatgtcg tccggcatcc atgtagcgct 60 ggtgactgga ggcaacaagg gcatcggctt ggccatcgtg cgcgacctgt gccggctgtt 120 ctcgggggac gtggtgctca cggcgcggga cgtgacgcgg ggccaggcgg ccgtacagca 180 gctgcaggcg gagggcctga gcccgcgctt ccaccagctg gacatcgacg atctgcagag 240 catccgcgcc ctgcgcgact tcctgcgcaa ggagtacggg ggcctggacg tgctggtcaa 300 caacgcgggc atcgccttca aggttgctga tcccacaccc tttcatattc aagctgaagt 360 gacgatgaaa acaaatttct ttggtacccg agatgtgtgc acagaattac tccctctaat 420 aaaaccccaa gggagagtgg tgaacgtatc tagcatcatg agcgtcagag cccttaaaag 480 ctgcagccca gagctgcagc agaagttccg cagtgagacc atcactgagg aggagctggt 540 ggggctcatg aacaagtttg tggaggatac aaagaaggga gtgcaccaga aggagggctg 600 gcccagcagc gcatacgggg tgacgaagat tggcgtcacc gttctgtcca ggatccacgc 660 caggaaactg agtgagcaga ggaaagggga caagatcctc ctgaatgcct gctgcccagg 720 gtgggtgaga actgacatgg cgggacccaa ggccaccaag agcccagaag aaggtgcaga 780 gacccctgtg tacttggccc ttttgccccc agatgctgag ggtccccatg gacaatttgt 840 ttcagagaag agagttgaac agtggtgagg atcctaggaa gaagcggaga cagcgacgaa 900 gatag 905 <210> 8 <211> 299 <212> PRT <213> Artificial Sequence <220> <223> Tat-Carbonyl Reductase 1-Tat fusion protein <400> 8 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ser Ser Gly Ile   1 5 10 15 His Val Ala Leu Val Thr Gly Gly Asn Lys Gly Ile Gly Leu Ala Ile              20 25 30 Val Arg Asp Leu Cys Arg Leu Phe Ser Gly Asp Val Val Leu Thr Ala          35 40 45 Arg Asp Val Thr Arg Gly Gln Ala Ala Val Gln Gln Leu Gln Ala Glu      50 55 60 Gly Leu Ser Pro Arg Phe His Gln Leu Asp Ile Asp Asp Leu Gln Ser  65 70 75 80 Ile Arg Ala Leu Arg Asp Phe Leu Arg Lys Glu Tyr Gly Gly Leu Asp                  85 90 95 Val Leu Val Asn Asn Ala Gly Ile Ala Phe Lys Val Ala Asp Pro Thr             100 105 110 Pro Phe His Ile Gln Ala Glu Val Thr Met Lys Thr Asn Phe Phe Gly         115 120 125 Thr Arg Asp Val Cys Thr Glu Leu Leu Pro Leu Ile Lys Pro Gln Gly     130 135 140 Arg Val Val Asn Val Ser Ser Ile Met Ser Val Arg Ala Leu Lys Ser 145 150 155 160 Cys Ser Pro Glu Leu Gln Gln Lys Phe Arg Ser Glu Thr Ile Thr Glu                 165 170 175 Glu Glu Leu Val Gly Leu Met Asn Lys Phe Val Glu Asp Thr Lys Lys             180 185 190 Gly Val His Gln Lys Glu Gly Trp Pro Ser Ser Ala Tyr Gly Val Thr         195 200 205 Lys Ile Gly Val Thr Val Leu Ser Arg Ile His Ala Arg Lys Leu Ser     210 215 220 Glu Gln Arg Lys Gly Asp Lys Ile Leu Leu Asn Ala Cys Cys Pro Gly 225 230 235 240 Trp Val Arg Thr Asp Met Ala Gly Pro Lys Ala Thr Lys Ser Pro Glu                 245 250 255 Glu Gly Ala Glu Thr Pro Val Tyr Leu Ala Leu Leu Pro Pro Asp Ala             260 265 270 Glu Gly Pro His Gly Gln Phe Val Ser Glu Lys Arg Val Glu Gln Trp         275 280 285 Gly Ser Arg Lys Lys Arg Arg Gln Arg Arg Arg     290 295 <210> 9 <211> 905 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding PEP-Carbonyl Reductase 1 fusion protein <400> 9 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatgtcgtcc ggcatccatg tagcgctggt gactggaggc aacaagggca 120 tcggcttggc catcgtgcgc gacctgtgcc ggctgttctc gggggacgtg gtgctcacgg 180 cgcgggacgt gacgcggggc caggcggccg tacagcagct gcaggcggag ggcctgagcc 240 cgcgcttcca ccagctggac atcgacgatc tgcagagcat ccgcgccctg cgcgacttcc 300 tgcgcaagga gtacgggggc ctggacgtgc tggtcaacaa cgcgggcatc gccttcaagg 360 ttgctgatcc cacacccttt catattcaag ctgaagtgac gatgaaaaca aatttctttg 420 gtacccgaga tgtgtgcaca gaattactcc ctctaataaa accccaaggg agagtggtga 480 acgtatctag catcatgagc gtcagagccc ttaaaagctg cagcccagag ctgcagcaga 540 agttccgcag tgagaccatc actgaggagg agctggtggg gctcatgaac aagtttgtgg 600 aggatacaaa gaagggagtg caccagaagg agggctggcc cagcagcgca tacggggtga 660 cgaagattgg cgtcaccgtt ctgtccagga tccacgccag gaaactgagt gagcagagga 720 aaggggacaa gatcctcctg aatgcctgct gcccagggtg ggtgagaact gacatggcgg 780 gacccaaggc caccaagagc ccagaagaag gtgcagagac ccctgtgtac ttggcccttt 840 tgcccccaga tgctgagggt ccccatggac aatttgtttc agagaagaga gtggaacagt 900 ggtga 905 <210> 10 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> PEP-Carbonyl Reductase 1 fusion protein <400> 10 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ser Ser Gly Ile His Val Ala Leu              20 25 30 Val Thr Gly Gly Asn Lys Gly Ile Gly Leu Ala Ile Val Arg Asp Leu          35 40 45 Cys Arg Leu Phe Ser Gly Asp Val Val Leu Thr Ala Arg Asp Val Thr      50 55 60 Arg Gly Gln Ala Ala Val Gln Gln Leu Gln Ala Glu Gly Leu Ser Pro  65 70 75 80 Arg Phe His Gln Leu Asp Ile Asp Asp Leu Gln Ser Ile Arg Ala Leu                  85 90 95 Arg Asp Phe Leu Arg Lys Glu Tyr Gly Gly Leu Asp Val Leu Val Asn             100 105 110 Asn Ala Gly Ile Ala Phe Lys Val Ala Asp Pro Thr Pro Phe His Ile         115 120 125 Gln Ala Glu Val Thr Met Lys Thr Asn Phe Phe Gly Thr Arg Asp Val     130 135 140 Cys Thr Glu Leu Leu Pro Leu Ile Lys Pro Gln Gly Arg Val Val Asn 145 150 155 160 Val Ser Ser Ile Met Ser Val Arg Ala Leu Lys Ser Cys Ser Pro Glu                 165 170 175 Leu Gln Gln Lys Phe Arg Ser Glu Thr Ile Thr Glu Glu Glu Leu Val             180 185 190 Gly Leu Met Asn Lys Phe Val Glu Asp Thr Lys Lys Gly Val His Gln         195 200 205 Lys Glu Gly Trp Pro Ser Ser Ala Tyr Gly Val Thr Lys Ile Gly Val     210 215 220 Thr Val Leu Ser Arg Ile His Ala Arg Lys Leu Ser Glu Gln Arg Lys 225 230 235 240 Gly Asp Lys Ile Leu Leu Asn Ala Cys Cys Pro Gly Trp Val Arg Thr                 245 250 255 Asp Met Ala Gly Pro Lys Ala Thr Lys Ser Pro Glu Glu Gly Ala Glu             260 265 270 Thr Pro Val Tyr Leu Ala Leu Leu Pro Pro Asp Ala Glu Gly Pro His         275 280 285 Gly Gln Phe Val Ser Glu Lys Arg Val Glu Gln Trp     290 295 300 <210> 11 <211> 905 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Carbonyl Reductase 1-PEP-1 fusion protein <400> 11 atgtcgtccg gcatccatgt agcgctggtg actggaggca acaagggcat cggcttggcc 60 atcgtgcgcg acctgtgccg gctgttctcg ggggacgtgg tgctcacggc gcgggacgtg 120 acgcggggcc aggcggccgt acagcagctg caggcggagg gcctgagccc gcgcttccac 180 cagctggaca tcgacgatct gcagagcatc cgcgccctgc gcgacttcct gcgcaaggag 240 tacgggggcc tggacgtgct ggtcaacaac gcgggcatcg ccttcaaggt tgctgatccc 300 acaccctttc atattcaagc tgaagtgacg atgaaaacaa atttctttgg tacccgagat 360 gtgtgcacag aattactccc tctaataaaa ccccaaggga gagtggtgaa cgtatctagc 420 atcatgagcg tcagagccct taaaagctgc agcccagagc tgcagcagaa gttccgcagt 480 gagaccatca ctgaggagga gctggtgggg ctcatgaaca agtttgtgga ggatacaaag 540 aagggagtgc accagaagga gggctggccc agcagcgcat acggggtgac gaagattggc 600 gtcaccgttc tgtccaggat ccacgccagg aaactgagtg agcagaggaa aggggacaag 660 atcctcctga atgcctgctg cccagggtgg gtgagaactg acatggcggg acccaaggcc 720 accaagagcc cagaagaagg tgcagagacc cctgtgtact tggccctttt gcccccagat 780 gctgagggtc cccatggaca atttgtttca gagaagagag ttgaacagtg gtgaggatcc 840 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 900 aagtg 905 <210> 12 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> Carbonyl Reductase 1-PEP-1 fusion protein <400> 12 Met Ser Ser Gly Ile His Val Ala Leu Val Thr Gly Gly Asn Lys Gly   1 5 10 15 Ile Gly Leu Ala Ile Val Arg Asp Leu Cys Arg Leu Phe Ser Gly Asp              20 25 30 Val Val Leu Thr Ala Arg Asp Val Thr Arg Gly Gln Ala Ala Val Gln          35 40 45 Gln Leu Gln Ala Glu Gly Leu Ser Pro Arg Phe His Gln Leu Asp Ile      50 55 60 Asp Asp Leu Gln Ser Ile Arg Ala Leu Arg Asp Phe Leu Arg Lys Glu  65 70 75 80 Tyr Gly Gly Leu Asp Val Leu Val Asn Asn Ala Gly Ile Ala Phe Lys                  85 90 95 Val Ala Asp Pro Thr Pro Phe His Ile Gln Ala Glu Val Thr Met Lys             100 105 110 Thr Asn Phe Phe Gly Thr Arg Asp Val Cys Thr Glu Leu Leu Pro Leu         115 120 125 Ile Lys Pro Gln Gly Arg Val Val Asn Val Ser Ser Ile Met Ser Val     130 135 140 Arg Ala Leu Lys Ser Cys Ser Pro Glu Leu Gln Gln Lys Phe Arg Ser 145 150 155 160 Glu Thr Ile Thr Glu Glu Glu Leu Val Gly Leu Met Asn Lys Phe Val                 165 170 175 Glu Asp Thr Lys Lys Gly Val His Gln Lys Glu Gly Trp Pro Ser Ser             180 185 190 Ala Tyr Gly Val Thr Lys Ile Gly Val Thr Val Leu Ser Arg Ile His         195 200 205 Ala Arg Lys Leu Ser Glu Gln Arg Lys Gly Asp Lys Ile Leu Leu Asn     210 215 220 Ala Cys Cys Pro Gly Trp Val Arg Thr Asp Met Ala Gly Pro Lys Ala 225 230 235 240 Thr Lys Ser Pro Glu Glu Gly Ala Glu Thr Pro Val Tyr Leu Ala Leu                 245 250 255 Leu Pro Pro Asp Ala Glu Gly Pro His Gly Gln Phe Val Ser Glu Lys             260 265 270 Arg Val Glu Gln Trp Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp Trp         275 280 285 Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val     290 295 300 <210> 13 <211> 976 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding PEP-1-Carbonyl Reductase 1-PEP-1 fusion          protein <400> 13 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatgtcgtcc ggcatccatg tagcgctggt gactggaggc aacaagggca 120 tcggcttggc catcgtgcgc gacctgtgcc ggctgttctc gggggacgtg gtgctcacgg 180 cgcgggacgt gacgcggggc caggcggccg tacagcagct gcaggcggag ggcctgagcc 240 cgcgcttcca ccagctggac atcgacgatc tgcagagcat ccgcgccctg cgcgacttcc 300 tgcgcaagga gtacgggggc ctggacgtgc tggtcaacaa cgcgggcatc gccttcaagg 360 ttgctgatcc cacacccttt catattcaag ctgaagtgac gatgaaaaca aatttctttg 420 gtacccgaga tgtgtgcaca gaattactcc ctctaataaa accccaaggg agagtggtga 480 acgtatctag catcatgagc gtcagagccc ttaaaagctg cagcccagag ctgcagcaga 540 agttccgcag tgagaccatc actgaggagg agctggtggg gctcatgaac aagtttgtgg 600 aggatacaaa gaagggagtg caccagaagg agggctggcc cagcagcgca tacggggtga 660 cgaagattgg cgtcaccgtt ctgtccagga tccacgccag gaaactgagt gagcagagga 720 aaggggacaa gatcctcctg aatgcctgct gcccagggtg ggtgagaact gacatggcgg 780 gacccaaggc caccaagagc ccagaagaag gtgcagagac ccctgtgtac ttggcccttt 840 tgcccccaga tgctgagggt ccccatggac aatttgtttc agagaagaga gtggaacagt 900 ggtgaggatc ctaaaagaaa cctggtggga aacctggtgg accgaatggt ctcagccgaa 960 aaaaaaacgt aaagtg 976 <210> 14 <211> 323 <212> PRT <213> Artificial Sequence <220> PEP-1-Carbonyl Reductase 1-PEP-1 fusion protein <400> 14 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ser Ser Gly Ile His Val Ala Leu              20 25 30 Val Thr Gly Gly Asn Lys Gly Ile Gly Leu Ala Ile Val Arg Asp Leu          35 40 45 Cys Arg Leu Phe Ser Gly Asp Val Val Leu Thr Ala Arg Asp Val Thr      50 55 60 Arg Gly Gln Ala Ala Val Gln Gln Leu Gln Ala Glu Gly Leu Ser Pro  65 70 75 80 Arg Phe His Gln Leu Asp Ile Asp Asp Leu Gln Ser Ile Arg Ala Leu                  85 90 95 Arg Asp Phe Leu Arg Lys Glu Tyr Gly Gly Leu Asp Val Leu Val Asn             100 105 110 Asn Ala Gly Ile Ala Phe Lys Val Ala Asp Pro Thr Pro Phe His Ile         115 120 125 Gln Ala Glu Val Thr Met Lys Thr Asn Phe Phe Gly Thr Arg Asp Val     130 135 140 Cys Thr Glu Leu Leu Pro Leu Ile Lys Pro Gln Gly Arg Val Val Asn 145 150 155 160 Val Ser Ser Ile Met Ser Val Arg Ala Leu Lys Ser Cys Ser Pro Glu                 165 170 175 Leu Gln Gln Lys Phe Arg Ser Glu Thr Ile Thr Glu Glu Glu Leu Val             180 185 190 Gly Leu Met Asn Lys Phe Val Glu Asp Thr Lys Lys Gly Val His Gln         195 200 205 Lys Glu Gly Trp Pro Ser Ser Ala Tyr Gly Val Thr Lys Ile Gly Val     210 215 220 Thr Val Leu Ser Arg Ile His Ala Arg Lys Leu Ser Glu Gln Arg Lys 225 230 235 240 Gly Asp Lys Ile Leu Leu Asn Ala Cys Cys Pro Gly Trp Val Arg Thr                 245 250 255 Asp Met Ala Gly Pro Lys Ala Thr Lys Ser Pro Glu Glu Gly Ala Glu             260 265 270 Thr Pro Val Tyr Leu Ala Leu Leu Pro Pro Asp Ala Glu Gly Pro His         275 280 285 Gly Gln Phe Val Ser Glu Lys Arg Val Glu Gln Trp Gly Ser Lys Glu     290 295 300 Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys 305 310 315 320 Arg Lys Val            

Claims (5)

세포 침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물.
An anti-aging functional cosmetic composition comprising a cell permeable carbonyl group reducing enzyme fusion protein and a pozzolan.
청구항 1에 있어서,
상기 세포 침투성 카보닐기 환원효소 융합단백질은 카보닐기 환원효소의 C-말단과 N-말단 중 어느 한 곳 이상에 단백질 수송도메인이 공유결합된 것을 특징으로 하는 세포 침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물.
The method according to claim 1,
The cell permeable carbonyl group reducing enzyme fusion protein is characterized in that a protein-transporting domain is covalently bonded to at least one of the C-terminus and the N-terminus of a carbonyl group reductase, and a cell permeable carbonyl group reductase fusion protein and a pozzolan Wherein the anti-aging functional cosmetic composition comprises:
청구항 1에 있어서,
상기 단백질 수송 도메인은 Tat 펩타이드 또는 HIV-1 Tat 펩타이드임을 특징으로 하는 세포 침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물.
The method according to claim 1,
Wherein the protein transport domain is a Tat peptide or an HIV-1 Tat peptide, and a cell permeable carbonyl group reductase fusion protein and a pozzolan.
청구항 1에 있어서,
상기 세포 침투성 카보닐기 환원효소 융합단백질은 서열번호 4, 6, 8, 10, 12 및 14 중 선택된 1종 이상임을 특징으로 하는 세포 침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물.
The method according to claim 1,
Wherein the cell permeable carbonyl group reducing enzyme fusion protein is at least one selected from SEQ ID NOs: 4, 6, 8, 10, 12 and 14, and an anti-aging functional cosmetic composition comprising a pozzolan .
청구항 1에 있어서,
상기 포졸란은 5~20㎛ 파장에서 90~97%의 원적외선을 방출하는 것을 특징으로 하는 세포 침투성 카보닐기 환원효소 융합단백질 및 포졸란을 포함하는 항노화 기능성 화장료 조성물.The method according to claim 1,
Wherein the pozzolan emits 90 to 97% far infrared rays at a wavelength of 5 to 20 占 퐉, wherein the cell permeable carbonyl group reducing enzyme fusion protein and the pozzolan.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200052773A (en) 2018-11-07 2020-05-15 (주)하랑 Mask pack compositions comprising pozzolan as active ingredient

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200052773A (en) 2018-11-07 2020-05-15 (주)하랑 Mask pack compositions comprising pozzolan as active ingredient

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