KR20230093890A - Elastin domain derived polypeptide - Google Patents
Elastin domain derived polypeptide Download PDFInfo
- Publication number
- KR20230093890A KR20230093890A KR1020210182882A KR20210182882A KR20230093890A KR 20230093890 A KR20230093890 A KR 20230093890A KR 1020210182882 A KR1020210182882 A KR 1020210182882A KR 20210182882 A KR20210182882 A KR 20210182882A KR 20230093890 A KR20230093890 A KR 20230093890A
- Authority
- KR
- South Korea
- Prior art keywords
- exon
- gly
- val
- ala
- domain
- Prior art date
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 60
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 53
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 48
- 108010014258 Elastin Proteins 0.000 title claims abstract description 40
- 102000016942 Elastin Human genes 0.000 title claims abstract description 37
- 229920002549 elastin Polymers 0.000 title claims abstract description 37
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 10
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims abstract description 8
- 239000004472 Lysine Substances 0.000 claims abstract description 8
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 8
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 4
- 235000004279 alanine Nutrition 0.000 claims abstract description 4
- 150000001413 amino acids Chemical class 0.000 claims description 25
- 235000001014 amino acid Nutrition 0.000 claims description 14
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 2
- 101000851054 Homo sapiens Elastin Proteins 0.000 abstract description 12
- 102000054289 human ELN Human genes 0.000 abstract description 12
- 230000007704 transition Effects 0.000 abstract description 10
- 239000000463 material Substances 0.000 abstract 1
- 108010029020 prolylglycine Proteins 0.000 description 28
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 25
- 108090000623 proteins and genes Proteins 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 20
- 235000018102 proteins Nutrition 0.000 description 19
- CLNJSLSHKJECME-BQBZGAKWSA-N Pro-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1 CLNJSLSHKJECME-BQBZGAKWSA-N 0.000 description 17
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 14
- 108010037335 tyrosyl-prolyl-glycyl-glycine Proteins 0.000 description 13
- 108010047495 alanylglycine Proteins 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- OLPPXYMMIARYAL-QMMMGPOBSA-N Gly-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)CN OLPPXYMMIARYAL-QMMMGPOBSA-N 0.000 description 10
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 description 10
- SJRUJQFQVLMZFW-WPRPVWTQSA-N Val-Pro-Gly Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SJRUJQFQVLMZFW-WPRPVWTQSA-N 0.000 description 10
- LMFXXZPPZDCPTA-ZKWXMUAHSA-N Ala-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N LMFXXZPPZDCPTA-ZKWXMUAHSA-N 0.000 description 9
- UGVQELHRNUDMAA-BYPYZUCNSA-N Gly-Ala-Gly Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)NCC([O-])=O UGVQELHRNUDMAA-BYPYZUCNSA-N 0.000 description 9
- 108010079364 N-glycylalanine Proteins 0.000 description 8
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 8
- FJVAQLJNTSUQPY-CIUDSAMLSA-N Ala-Ala-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN FJVAQLJNTSUQPY-CIUDSAMLSA-N 0.000 description 7
- SMCGQGDVTPFXKB-XPUUQOCRSA-N Ala-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N SMCGQGDVTPFXKB-XPUUQOCRSA-N 0.000 description 7
- KSOBNUBCYHGUKH-UWVGGRQHSA-N Gly-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN KSOBNUBCYHGUKH-UWVGGRQHSA-N 0.000 description 7
- JSOXWWFKRJKTMT-WOPDTQHZSA-N Val-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N JSOXWWFKRJKTMT-WOPDTQHZSA-N 0.000 description 7
- 108010028939 alanyl-alanyl-lysyl-alanine Proteins 0.000 description 7
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 6
- ZVFVBBGVOILKPO-WHFBIAKZSA-N Ala-Gly-Ala Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O ZVFVBBGVOILKPO-WHFBIAKZSA-N 0.000 description 6
- FQCILXROGNOZON-YUMQZZPRSA-N Gln-Pro-Gly Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O FQCILXROGNOZON-YUMQZZPRSA-N 0.000 description 6
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- XXROXFHCMVXETG-UWVGGRQHSA-N Val-Gly-Val Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXROXFHCMVXETG-UWVGGRQHSA-N 0.000 description 6
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- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 5
- GQMNEJMFMCJJTD-NHCYSSNCSA-N Val-Pro-Gln Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O GQMNEJMFMCJJTD-NHCYSSNCSA-N 0.000 description 5
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- 230000000704 physical effect Effects 0.000 description 5
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- UKPGFKQVRITNFM-KBPBESRZSA-N 2-[[2-[[(2s)-1-[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]acetic acid Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(=O)NCC(O)=O)C1=CC=C(O)C=C1 UKPGFKQVRITNFM-KBPBESRZSA-N 0.000 description 3
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- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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Abstract
Description
본 발명은 종래 엘라스틴 도메인 유래 폴리펩타이드와 상이한 새로운 형태의 엘라스틴 도메인 유래 폴리펩타이드에 관한 것으로, 인간 엘라스티과 유사한 온도에서 상전이가 일어나는 엘라스틴 도메인 유래 폴리펩타이드에 관한 것이다. The present invention relates to a new type of elastin domain-derived polypeptide different from the conventional elastin domain-derived polypeptide, and relates to an elastin domain-derived polypeptide in which a phase transition occurs at a temperature similar to that of human elastin.
엘라스틴은 세포외 기질의 핵심 단백질로 결합 조직에 존재하는 매우 탄력적인 성분으로, 이는 신체의 많은 조직이 늘어나고 수축을 하면서도 본래의 형태를 유지하는 특징을 부여한다. 조직에 이러한 유연성, 신축성, 팽창성 등을 부여하는 엘라스틴 및 엘라스틴 유래 단백질은 다양한 생물학적 매트릭스와 결합하여 형태학적, 물리학적 및 생물학적 특성을 조절한다. Elastin is a key protein of the extracellular matrix and is a very elastic component present in connective tissue, which gives many tissues in the body the characteristic of maintaining their original shape while stretching and contracting. Elastin and elastin-derived proteins, which impart such flexibility, elasticity, and expansibility to tissues, bind to various biological matrices to control morphological, physical, and biological properties.
엘라스틴은 소수성 아미노산과 높은 분자간 결합을 가지며 이러한 특징으로 인해 분해가 잘 되지 않는 특징을 가진다. 한편, 재조합 트로포엘라스틴과 엘라스틴 유사 단백질은 엘라스틴 기반 생체 재료의 구성에 주로 사용된다. 펩타이드 합성 및 재조합 단백질 제조 기술에 기초하여 엘라스틴으로부터의 선택적인 영역을 모방하려는 시도가 이루어지고 있다. 엘라스틴 유사 단백질은 반복적인 힘에 의해 구조적인 변형이 일어나게 되었을 때 다시 원래 형태를 유지하는 복원력을 가져야 하고, 생체 내에서 분해가 되지 않고 오랫동안 생체 내에서 형태를 유지해야 하며, 세포 성장 및 주화성을 가져야 한다.Elastin has a high intermolecular bond with hydrophobic amino acids, and due to these characteristics, it has a characteristic that it is not easily decomposed. On the other hand, recombinant tropoelastin and elastin-like proteins are mainly used for the construction of elastin-based biomaterials. Attempts have been made to mimic selective regions from elastin based on peptide synthesis and recombinant protein production techniques. Elastin-like proteins must have resilience to maintain their original shape when structural deformation occurs due to repetitive force, must maintain their shape in vivo for a long time without decomposition in vivo, and have cell growth and chemotaxis should have
종래의 엘라스틴 유사 단백질의 설계는 'VPGXG'이나 'GXXGPG'와 같은 엘라스틴의 모티프를 반복적으로 나열함으로써 수행되었다. 이러한 설계의 경우 자연 상태의 엘라스틴 보다 뛰어난 탄성 혹은 물리적 강도를 가질 수 있으나, 단순한 모티프의 반복으로 인해 자연 엘라스틴이 가지는 복합적인 물성, 생체 내 유지성, 그리고 세포 친화성을 모두 충족시키기 어렵다. Conventional elastin-like proteins have been designed by repetitively listing elastin motifs such as 'VPGXG' or 'GXXGPG'. In the case of such a design, it may have elasticity or physical strength superior to natural elastin, but it is difficult to satisfy all of the complex physical properties, in vivo retention, and cell compatibility of natural elastin due to the repetition of simple motifs.
따라서, 기존 인간 엘라스틴의 구조적인 특징을 그대로 가져올 수 있어서 인간 엘라스틴의 특성과 유사한 특성을 나타내며, 또한 세포와의 상호 작용 능력이 우수한 엘라스틴 유사 펩타이드 또는 단백질이 개발되는 경우 관련 분야에서 널리 적용될 수 있을 것으로 기대된다. Therefore, it is expected that if an elastin-like peptide or protein with excellent cell interaction ability is developed, it can be widely applied in the related field because it can bring the structural characteristics of existing human elastin as it is, so it shows properties similar to those of human elastin. It is expected.
본 발명의 한 측면에 의하면 인간 엘라스티과 유사한 온도에서 상전이가 일어나며 인장력 등의 물성이 우수한 엘라스틴 도메인 유래 폴리펩타이드를 제공하는 것이다. According to one aspect of the present invention, a phase transition occurs at a temperature similar to that of human elastin and an elastin domain-derived polypeptide having excellent physical properties such as tensile strength is provided.
이에 본 발명의 일 견지에 의하면, 엑손-9, 엑손-16, 엑손-18, 엑손-20, 엑손-22, 엑손-24, 및 엑손-26으로 이루어진 그룹으로부터 선택된 적어도 하나의 프롤린이 풍부한 소수성 제1 도메인; 엑손-6, 엑손-15, 엑손-17, 엑손-19, 엑손-21, 엑손-23, 엑손-25, 엑손-27, 엑손-29, 및 엑손-31로 이루어진 그룹으로부터 선택된 라이신 및 알라닌을 포함하는 제2 도메인; 엑손-4, 엑손-8, 엑손-10, 엑손-12, 및 엑손-13으로 이루어진 그룹으로부터 선택된 라이신 및 프롤린을 포함하는 제3 도메인; 및 엑손-36을 포함하는, 엘라스틴 도메인 유래 폴리펩타이드가 제공된다.Accordingly, according to one aspect of the present invention, at least one proline-rich hydrophobic agent selected from the group consisting of exon-9, exon-16, exon-18, exon-20, exon-22, exon-24, and exon-26 1 domain; lysine and alanine selected from the group consisting of exon-6, exon-15, exon-17, exon-19, exon-21, exon-23, exon-25, exon-27, exon-29, and exon-31 a second domain that does; a third domain comprising lysine and proline selected from the group consisting of exon-4, exon-8, exon-10, exon-12, and exon-13; and an elastin domain-derived polypeptide comprising exon-36.
본 발명에 의한 엘라스틴 도메인 유래 폴리펩타이드는 인간 엘라스틴과 유사한 형태 및 특성을가지며, 보다 상세하게는 인체의 자연 엘라스틴와 비슷한 상전이 온도를 가져 이로 인해 인간의 체온 하 생체 환경에서 적용될 수 있다. 또한 인간 엘라스틴이 가지는 세포와의 상호작용하는 도메인을 포함하여 인간 자연 엘라스틴과 유사한 세포 상호 작용을 발현할 수 있다. The elastin domain-derived polypeptide according to the present invention has a shape and characteristics similar to human elastin, and more specifically, has a phase transition temperature similar to that of human elastin, and thus can be applied in a biological environment under human body temperature. In addition, it can express cell interactions similar to human natural elastin, including a domain that interacts with cells of human elastin.
도 1(a)은 EDDP-242 그리고 도 1(b)는 EDDP-424의 유전자 서열이 각각 삽입된 벡터 pET-22b(+)의 지도를 나타낸 것이다.
도 2는 펩타이드 EDDP-242 및 EDDP-424의 온도 별 탁도를 측정하여 상전이 온도를 확인한 결과를 나타낸 것이다.
도 3은 펩타이드 EDDP-242 및 EDDP-424의 탄성 특성(인장력 시험, Tensile test)을 확인한 결과를 나타내는 그래프이다.
도 4는 EDDP-242와 EDDP-424의 도메인 서열을 도식적으로 나타낸 것이다.FIG. 1(a) is a map of the vector pET-22b(+) into which the gene sequences of EDDP-242 and FIG. 1(b) are inserted, respectively.
Figure 2 shows the results of confirming the phase transition temperature by measuring the turbidity of the peptides EDDP-242 and EDDP-424 by temperature.
Figure 3 is a graph showing the results of confirming the elastic properties (tensile test, Tensile test) of the peptides EDDP-242 and EDDP-424.
Figure 4 schematically shows the domain sequences of EDDP-242 and EDDP-424.
이하, 첨부된 도면을 참조하여 본 발명의 바람직한 실시 형태를 설명한다. 그러나, 본 발명의 실시 형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시 형태로 한정되는 것은 아니다. Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified in various forms, and the scope of the present invention is not limited to the embodiments described below.
본 발명에 의하면 자연의 인간 엘라스틴의 구조적인 특성을 구현하기 위하여 엘라스틴에서 각 특징을 가지는 4가지 도메인을 구분하고, 이들 도메인을 각각 조합한 엘라스틴 도메인 유래 폴리펩타이드가 제공된다. 한편, 본 발명에 있어서 단백질과 (폴리)펩타이드는 상호호환적으로 사용될 수 있다.According to the present invention, in order to realize the structural characteristics of natural human elastin, elastin is divided into four domains having each characteristic, and an elastin domain-derived polypeptide is provided in which these domains are combined. Meanwhile, in the present invention, proteins and (poly)peptides may be used interchangeably.
보다 상세하게 본 발명의 폴리펩타이드는 엑손-9, 엑손-16, 엑손-18, 엑손-20, 엑손-22, 엑손-24, 및 엑손-26으로 이루어진 그룹으로부터 선택된 적어도 하나의 프롤린이 풍부한 소수성 제1 도메인; 엑손-6, 엑손-15, 엑손-17, 엑손-19, 엑손-21, 엑손-23, 엑손-25, 엑손-27, 엑손-29, 및 엑손-31로 이루어진 그룹으로부터 선택된 라이신 및 알라닌을 포함하는 제2 도메인; 엑손-4, 엑손-8, 엑손-10, 엑손-12, 및 엑손-13으로 이루어진 그룹으로부터 선택된 라이신 및 프롤린을 포함하는 제3 도메인; 및 엑손-36(제4 도메인)을 포함하는 것이다. More specifically, the polypeptide of the present invention is at least one proline-rich hydrophobic agent selected from the group consisting of exon-9, exon-16, exon-18, exon-20, exon-22, exon-24, and exon-26 1 domain; lysine and alanine selected from the group consisting of exon-6, exon-15, exon-17, exon-19, exon-21, exon-23, exon-25, exon-27, exon-29, and exon-31 a second domain that does; a third domain comprising lysine and proline selected from the group consisting of exon-4, exon-8, exon-10, exon-12, and exon-13; and exon-36 (fourth domain).
본 발명은 글라이신이 풍부한 소수성 도메인으로 분류되는 엑손-2, 엑손-3, 엑손-5, 엑손-7, 엑손-11, 엑손-14, 엑손-28, 엑손-30, 엑손-32, 및 엑손-33으로 이루어진 그룹으로부터 선택된 아미노산 서열을 포함하지 않을 수 있다. Exon-2, exon-3, exon-5, exon-7, exon-11, exon-14, exon-28, exon-30, exon-32, and exon-3, which are classified as glycine-rich hydrophobic domains. and may not contain an amino acid sequence selected from the group consisting of 33.
세포 표면의 글리코사미노글리칸(GAGs)은 토로포엘라스틴의 C-말단과 상호작용한다. 트로포엘라스틴은 다른 많은 세포 결합 단백질에서 발견되는 RGD 의존성 인테그린에 대한 일반적인 Arg-Gly-Asp (RGD) 결합 서열을 포함하지 않으며, 그 대신 인테그린에 대한 결합 부위인 αvβ3가 트로포엘라스틴의 C-말단에서 확인된다. 이러한 인테그린 결합 부위는 엑손-36에 의해 암호화된 영역에서 단백질의 극단 C-말단에서 GRKRK로 좁혀지고(Gly-Arg-Lys-Arg-Lys) 트로포엘라스틴에 대한 진피섬유아세포 접착을 지원한다. 이러한 세포 표면 수용체는 세포와 엘라스틴 사이의 상호작용을 중재하고, 이는 차례로 세포 행동을 수정하는 여러 세포 사이의 신호 전달 캐스케이드를 활성화시킨다. 본 발명의 펩타이드는 이러한 특성을 가지는 엑손-36을 포함함으로써 엘라스틴의 중요한 특징인 세포와의 상호 작용을 발현할 수 있다. Cell surface glycosaminoglycans (GAGs) interact with the C-terminus of toropoelastin. Tropoelastin does not contain the common Arg-Gly-Asp (RGD) binding sequence for RGD-dependent integrins found in many other cell-associated proteins, but instead the α v β 3 binding site for integrin is the C- confirmed at the end. This integrin binding site is narrowed to GRKRK at the extreme C-terminus of the protein in the region encoded by exon-36 (Gly-Arg-Lys-Arg-Lys) and supports dermal fibroblast adhesion to tropoelastin. These cell surface receptors mediate the interaction between the cell and elastin, which in turn activates a signaling cascade between multiple cells that modifies cell behavior. By including exon-36 having these characteristics, the peptide of the present invention can express the interaction with cells, which is an important feature of elastin.
예를 들어 본 발명의 상기 폴리펩타이드는 [제1 도메인-제3 도메인] 블럭, [제1 도메인-제3 도메인- 제1 도메인] 블럭, 또는 [제3 도메인-제1 도메인] 블록으로 이루어지는 그룹으로부터 선택된 적어도 하나의 블럭을 포함하는 것일 수 있으며, 이들 블럭은 적어도 한번 이상 반복하여 포함될 수 있으며, 각 블록은 서로 인접하거나 또는 블록 사이에 다른 아미노산 서열이 존재하도록 이격되어 포함될 수 있다. 한편, 상기 블럭 내에서 도메인의 위치는 변경될 수 있다. 한편, 상기 각 블럭 내에서의 도메인의 위치는 변경될 수 있다.For example, the polypeptide of the present invention is a group consisting of a [first domain-third domain] block, a [first domain-third domain-first domain] block, or a [third domain-first domain] block. It may include at least one block selected from, and these blocks may be included repeatedly at least once, and each block may be included adjacent to each other or spaced apart so that another amino acid sequence exists between the blocks. Meanwhile, the position of the domain within the block may be changed. Meanwhile, the location of the domain within each block may be changed.
바람직하게 본 발명에서 소수성 도메인으로 엑손-9 및 엑손-18 중 적어도 하나를 선택할 수 있으며, 가교 친수성 도메인으로 엑손-8 및 엑손-19 중 적어도 하나를 선택할 수 있다. Preferably, in the present invention, at least one of exon-9 and exon-18 may be selected as the hydrophobic domain, and at least one of exon-8 and exon-19 may be selected as the bridging hydrophilic domain.
상기 소수성 도메인인 엑손-9 및 엑손-18의 도메인에는 두가지 유형인 -PG- 와 -GG-를 포함할 수 있으며, 이들은 β-턴(turn)을 유발하고, 또한 이 도메인에서 PPII(poly-proline type Ⅱ helix)를 가진다. 이 두 도메인 중 엑손-9의 경우 60%의 타입(type) II β-턴, 20%의 타입(type) I β-턴, 및 나머지의 20%의 질서가 없는 형태를 가질 수 있다. 엑손-9 및 엑손-18 등에서 발견되는 β-턴은 엘라스틴에 존재하는 주요한 2차 구조이다.The domains of exon-9 and exon-18, which are hydrophobic domains, may include two types of -PG- and -GG-, which induce a β-turn, and also produce poly-proline (PPII) in this domain. type II helix). Of these two domains, exon-9 may have 60% type II β-turns, 20% type I β-turns, and the remaining 20% disordered conformation. The β-turn found in exon-9 and exon-18 is the main secondary structure present in elastin.
상기 폴리펩타이드는 엑손-8, 엑손-9, 엑손-19, 및 엑손-36을 포함하는 것일 수 있으며, 나아가 상기 폴리펩타이드는 엑손-18을 추가로 포함하는 것일 수 있다.The polypeptide may include exon-8, exon-9, exon-19, and exon-36, and may further include exon-18.
엑손-8의 서열은 GAVVPQPGAGVKPGKVP로 17개의 아미노산으로 이루어지고, 엑손-9의 서열은 GVGLPGVYPGGVLPGA로 16개의 아미노산으로 이루어지고, 엑손-18의 서열은 GAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVP로 49개의 아미노산으로 이루어지고, 엑손-19의 서열은 GVVSPEAAAKAAAKAAKY로 18개의 아미노산으로 이루어지며, 엑손-36의 경우 GGACLGKACGRKRK로 14개의 아미노산으로 이루어질 수 있는 것으로, 본 발명의 엘라스틴 도메인 유래 폴리펩타이드는 엑손-8은 서열번호 1, 엑손-9는 서열번호 2, 엑손-19는 서열번호 4, 및 엑손-36은 서열번호 5의 아미노산 서열을 포함하는 것일 수 있으며, 또한 엑손-18은 서열번호 3의 아미노산 서열을 갖는 것일 수 있다. The sequence of exon-8 consists of 17 amino acids as GAVVPQPGAGVKPGKVP, the sequence of exon-9 consists of 16 amino acids as GVGLPGVYPGGVLPGA, the sequence of exon-18 consists of 49 amino acids as GAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVP, and the sequence of exon-19 is composed of 18 amino acids as GVVSPEAAAKAAAKAAKY, and in the case of exon-36, it may consist of 14 amino acids as GGACLGKACGRKRK, and the elastin domain-derived polypeptide of the present invention has SEQ ID NO: 1 for exon-8 and SEQ ID NO: 2 for exon-9 , Exon-19 may include the amino acid sequence of SEQ ID NO: 4, and exon-36 may include the amino acid sequence of SEQ ID NO: 5, and exon-18 may have the amino acid sequence of SEQ ID NO: 3.
본 발명의 폴리펩타이드는 예를 들어 인간 엘라스틴과 구조적 유사성을 위해 도메인의 서열을 엑손-9, 엑손-8, 엑손-18, 엑손-19, 엑손-9, 엑손-8 순으로 포함할 수 있다. For structural similarity to human elastin, the polypeptide of the present invention may include domain sequences in the order of exon-9, exon-8, exon-18, exon-19, exon-9, and exon-8.
나아가, 정제 및 단백질 확인을 위해서 His-tag 등의 서열을 폴리펩타이드에 추가할 수 있다.Furthermore, a sequence such as a His-tag may be added to the polypeptide for purification and protein identification.
한편, 본 발명에 있어서 상기 제2 도메인은 N 말단에 위치하지 않으며, 엑손-36은 C 말단에 위치하는 것이 바람직하다. Meanwhile, in the present invention, the second domain is not located at the N-terminus, and exon-36 is preferably located at the C-terminus.
본 발명의 엘라스틴 도메인 유래 폴리펩타이드의 길이는 특히 제한되는 것은 아니지만, 예를 들어 상기 폴리펩타이드는 400 내지 450개, 예를 들어 410 내지 430개의 아미노산을 포함하는 것일 수 있다..The length of the elastin domain-derived polypeptide of the present invention is not particularly limited, but may include, for example, 400 to 450 amino acids, for example, 410 to 430 amino acids.
예시적인 본 발명의 상기 폴리펩타이드는 서열번호 6 또는 서열번호 7의 아미노산 서열을 포함하는 것일 수 있다. 본 발명에 있어서 서열번호 6의 폴리펩타이드는 'EDDP-424'로 지칭될 수 있으며, 서열번호 7 의 폴리펩타이드는 EDDP-242로 지칭될 수 있다.The exemplary polypeptide of the present invention may include the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7. In the present invention, the polypeptide of SEQ ID NO: 6 may be referred to as 'EDDP-424', and the polypeptide of SEQ ID NO: 7 may be referred to as EDDP-242.
이하, 구체적인 실시예를 통해 본 발명을 보다 구체적으로 설명한다. 하기 실시예는 본 발명의 이해를 돕기 위한 예시에 불과하며, 본 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely examples to aid understanding of the present invention, and the scope of the present invention is not limited thereto.
실시예Example
1. 엘라스틴 도메인 유래 폴리펩타이드 설계1. Elastin domain-derived polypeptide design
본 발명의 엘라스틴 도메인 유래 폴리펩타이드는 인간 엘라스틴의 도메인인 엑손-8, 엑손-9, 엑손-19 및 엑손-36의 도메인 조합, 또는 이에 엑손-18를 추가한 도메인의 조합을 통해 제조하였다. 이때 각 엑손의 유전자 서열은 다음과 같다. The elastin domain-derived polypeptide of the present invention was prepared by combining human elastin domains, exon-8, exon-9, exon-19, and exon-36, or a combination of domains to which exon-18 was added. At this time, the gene sequence of each exon is as follows.
엑손-8의 서열은 서열번호 1과 같이 GAVVPQPGAGVKPGKVP의 17개의 아미노산으로 이루어져 있으며, 엑손-9의 서열은 서열번호 2와 같이 GVGLPGVYPGGVLPGA의 16개의 아미노산으로 이루어져 있고, 엑손-18의 서열은 서열번호 3과 같이 GAAA GLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVP의 49개의 아미노산으로 이루어져 있으며, 엑손-19의 서열은 서열번호 4와 같이 GVVSPEAAAKAAAKAAKY의 18개의 아미노산으로 이루어져 있으며, 마지막으로 엑손-36의 경우 서열번호 5와 같이 GGACLGKACGRKRK의 14개의 아미노산으로 이루어져 있다. The sequence of exon-8 consists of 17 amino acids of GAVVPQPGAGVKPGKVP as shown in SEQ ID NO: 1, the sequence of exon-9 consists of 16 amino acids of GVGLPGVYPGGVLPGA as of SEQ ID NO: 2, and the sequence of exon-18 has SEQ ID NO: 3 and Similarly, GAAA GLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVP consists of 49 amino acids, the sequence of exon-19 consists of 18 amino acids of GVVSPEAAAKAAAKAKY as shown in SEQ ID NO: 4, and finally, exon-36 consists of 14 amino acids of GGACLGKACGRKRK as shown in SEQ ID NO: 5 there is.
엑손-8, 엑손-9, 엑손-19 및 엑손-36을 포함하는 펩타이드 및 이에 엑손-18를 추가로 포함하는 펩타이드를 제조하였으며, 이들을 각각 EDDP-424 및 EDDP-242로 지칭한다. EDDP-424의 서열은 서열번호 6과 같으며, EDDP-242의 서열은 서열번호 7과 같다. EDDP-242와 EDDP-424의 도메인 서열은 하기 및 도 4에 나타낸 바와 같이 구성되었으며, 엑손 9의 경우 베타턴을 하는 단백질 서열인 Pro-Gly 서열이 풍부하여 단백질의 물성에 큰 영향을 주며, 중앙의 엑손 18과 19의 서열 중 Ala-Ala-Ala-Lys-Ala-Ala-Lys-Tyr-Gly-Ala-Ala-Ala-Gly-Leu 서열은 세포와의 접착에 영향을 크게 준다. 이렇게 펩타이드 서열의 앞 및 뒤에 엑손 9와 엑손 8를 배치하고, 펩타이드 서열의 중앙에 엑손 18와 엑손 19의 반복 서열을 배치할 수 있으며, 이와 같은 이들의 기본적인 배치를 유지하면서 필요에 따라 반복 횟수 등을 변화시킴으로써 단백질의 물성과 세포상호작용의 변화를 조절할 수 있다. 한편, 서열번호 6 및 7는 펩타이드의 C 말단에 정제 및 확인을 위한 His-tag 서열을 포함한다.Peptides including exon-8, exon-9, exon-19 and exon-36 and peptides further including exon-18 were prepared, and they are referred to as EDDP-424 and EDDP-242, respectively. The sequence of EDDP-424 is shown in SEQ ID NO: 6, and the sequence of EDDP-242 is shown in SEQ ID NO: 7. The domain sequences of EDDP-242 and EDDP-424 were constructed as shown below and in FIG. 4, and in the case of
- EDDP(Elastin domain derived protein)-242 아미노산 서열- EDDP (Elastin domain derived protein)-242 amino acid sequence
GVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGVGVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGV
KPGKVPGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPKPGKVPGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSP
EAAAKAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAEAAAKAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAA
VPGVVSPEAAAKAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGVPGVVSPEAAAKAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPG
AGIPGAAVPGVVSPEAAAKAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAAGIPGAAVPGVVSPEAAAKAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGA
GIPVVPGAGIPGAAVPGVVSPEAAAKAAAKAAKYGVGLPGVYPGGVLPGAGAVVPQPGAGGIPVVPGAGIPGAAVPGVVSPEAAAKAAAKAAKYGVGLPGVYPGGVLPGAGAVVPQPGAG
VKPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGGACLGKACGRKRKHHHHHHVKPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGGACLGKACGRKRKHHHHHH
- EDDP(Elastin domain derived protein)-424 아미노산 서열- EDDP (Elastin domain derived protein)-424 amino acid sequence
GVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGVGVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGV
KPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGAGAVVPKPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGAGAVVP
QPGAGVKPGKVPGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVQPGAGVKPGKVPGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAV
PGVVSPEAAAKAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAPGVVSPEAAAKAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGA
GIPGAAVPGVVSPEAAAKAAAKAAKYGVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGGIPGAAVPGVVSPEAAAKAAAKAAKYGVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPG
VGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGVKVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGVK
PGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGGACLGKACGRKRKHHHHHHPGKVPGVGLPGVYPGGVLPGAGAVVPQPGAGVKPGKVPGGACLGKACGRKRKHHHHHH
한편, 상기 EDDP-424 및 EDDP-242에서 각 C 말단에 엑손-36을 위치하도록 하였으며, 그 결과 인간 엘라스틴에서의 세포와의 상호 작용 특성을 발현할 수 있다. On the other hand, in EDDP-424 and EDDP-242, exon-36 was located at each C-terminus, and as a result, human elastin's interaction characteristics with cells can be expressed.
2. 엘라스틴 도메인 유래 폴리펩타이드 제조2. Production of Elastin Domain-Derived Polypeptides
상기 EDDP-424 및 EDDP-242를 코딩하는 유전자를 벡터 pET-22b(+)에 각각 삽입하여 도 1(a) 및 도 1(b)와 같은 발현 벡터를 제조하였다. Expression vectors as shown in FIGS. 1(a) and 1(b) were prepared by inserting the genes encoding EDDP-424 and EDDP-242 into the vector pET-22b(+), respectively.
이들을 각각 다른 균주인 BL21(DE3), BLR(DE3) 및 BLR(DE3)-pLysS에 삽입하였으며, 상기 유전자가 삽입된 균주들을 이용하여 SDS-PAGE와 웨스턴 블롯(Western-blot)을 통해 각 균주 별 단백질 발현량을 확인하였고, 발현량이 가장 많은 BLR(DE3)을 선택하였다. These were inserted into different strains, BL21(DE3), BLR(DE3) and BLR(DE3)-pLysS, and each strain was analyzed through SDS-PAGE and Western-blot using the strains into which the gene was inserted. The protein expression level was confirmed, and BLR (DE3) with the highest expression level was selected.
이후 발현된 폴리펩타이드의 용해성을 확인하기 위해 배양된 균주를 파쇄하여 용해성 부분과 불용성 부분을 각각 채취한 후 SDS-PAGE와 웨스턴 블롯을 통해 확인 하였으며, 그 결과 단백질이 용해성임을 확인하였다.Then, in order to confirm the solubility of the expressed polypeptide, the cultured strain was disrupted, and the soluble and insoluble parts were collected, respectively, and confirmed by SDS-PAGE and Western blotting, and as a result, it was confirmed that the protein was soluble.
순수한 결과물을 얻기 위해서 단백질의 정제를 진행하였으며, 먼저 파쇄된 세포가 들어있는 용액에 폴리에테르이미드 10% 용액을 파쇄된 세포가 들어있는 용액의 최종 부피가 4%가 되도록 넣어주어 용액 내 유전자 오염물을 제거하였다. 그 후 온도를 50℃로 상승시켜서 온도에 취약한 다른 오염 단백질을 변성시켜 오염 단백질을 제거하였다. 다음으로 용액 내에 NaCl 2.5M을 넣어 EDDP-242 및 EDDP-424를 석출하고 이 보다 더 용해도가 높은 오염물을 제거하였다. 마지막으로 증류수를 이용하여 용해되지 않는 오염물을 제거하고, 이때 오염물이 석출이 되지 않을 때까지 반복하여 정제된 폴리펩타이드를 획득하였다.In order to obtain a pure product, the protein was purified. First, a 10% polyetherimide solution was added to the solution containing the disrupted cells so that the final volume of the solution containing the disrupted cells was 4%, thereby eliminating genetic contaminants in the solution. Removed. Thereafter, the temperature was raised to 50° C. to denature other contaminating proteins vulnerable to temperature, thereby removing contaminating proteins. Next, 2.5M NaCl was added into the solution to precipitate EDDP-242 and EDDP-424, and contaminants with higher solubility were removed. Finally, insoluble contaminants were removed using distilled water, and at this time, the purified polypeptide was obtained repeatedly until the contaminants did not precipitate.
3. 엘라스틴 도메인 유래 폴리펩타이드의 특성 확인3. Confirmation of the characteristics of the elastin domain-derived polypeptide
(1) 상전이 온도(1) Phase transition temperature
상기 2.에서 획득된 엘라스틴 도메인 유래 폴리펩타이드의 상전이가 일어나는 온도를 확인하기 위해서, 폴리펩타이드 EDDP-242 및 EDDP-424의 온도 별 탁도를 측정하였다. 이때 각 폴리펩타이드는 PBS 7.4pH 용액에 각 펩타이드를 0.1wt%으로 녹여서 용액을 준비하였으며, 25℃에서 75℃까지의 온도에서 파장 600nm에서 O.D. 값을 측정하였다. In order to confirm the temperature at which the phase transition of the elastin domain-derived polypeptide obtained in 2. above occurs, the turbidity of the polypeptides EDDP-242 and EDDP-424 according to temperature was measured. At this time, each polypeptide was prepared by dissolving 0.1 wt% of each peptide in a PBS 7.4 pH solution, and O.D. values were measured.
그 결과 도 2에서 확인할 수 있는 바와 같이 폴리펩타이드 EDDP-242 은 약 50℃ 내지 60℃에서 상전이가 일어나며 EDDP-424의 경우 약 60℃ 내지 70℃에서 상전이가 일어나는 것을 확인할 수 있었다. 이러한 상전이 온도는 체내 온도인 약 37℃ 보다 더 높기 때문에 생체 적용에 적합하다.As a result, as can be seen in FIG. 2, it was confirmed that the phase transition of the polypeptide EDDP-242 occurred at about 50° C. to 60° C., and the phase transition of EDDP-424 occurred at about 60° C. to 70° C. Since this phase transition temperature is higher than the body temperature of about 37° C., it is suitable for biological applications.
(2) 탄성 특성(2) elastic properties
본 발명 폴리펩타이드의 탄성 특성을 측정하기 위해서 각 폴리펩타이드 EDDP-242 및 EDDP-424를 각각 PBS 7.4pH 용액에 15wt%가 되도록 용해시켜 용액을 제조하고, 상기 용액에 Genpin 30mM를 넣어 12시간 가교시켜 젤을 형성하여 인장 특성 확인을 위한 실험을 진행하였다. In order to measure the elastic properties of the polypeptides of the present invention, each of the polypeptides EDDP-242 and EDDP-424 was dissolved in a PBS 7.4 pH solution to a concentration of 15 wt% to prepare a solution, and Genpin 30mM was added to the solution to crosslink for 12 hours. An experiment was conducted to confirm the tensile properties by forming a gel.
그 결과 도 3 및 하기 표 1에서 확인할 수 있는 바와 같이 EDDP-242의 탄성성(Tensile strain)은 0.75±0.15 (mm/mm), 그리고 EDDP-424의 경우 탄성성은 0.73±0.26이며, EDDP-242의 탄성한계점(Tensile stress)은 11.76±3.5 kPa 그리고 EDDP-424의 경우 탄성한계점은 17.03±2.6, 또한 EDDP-242의 탄성률(Elastic modulus)은 16.14±4.3 kPa, 그리고 EDDP-424의 경우 탄성률은 25.44±6.7인 것으로 확인하였다. As a result, as can be seen in Figure 3 and Table 1 below, the elasticity (tensile strain) of EDDP-242 is 0.75 ± 0.15 (mm / mm), and in the case of EDDP-424, the elasticity is 0.73 ± 0.26, EDDP-242 Tensile stress is 11.76±3.5 kPa, EDDP-424 has 17.03±2.6, EDDP-242 has 16.14±4.3 kPa, and EDDP-424 has 25.44 elastic modulus. It was found to be ±6.7.
이상에서 본 발명의 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고, 청구범위에 기재된 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 다양한 수정 및 변형이 가능하다는 것은 당 기술분야의 통상의 지식을 가진 자에게는 자명할 것이다.Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention described in the claims. It will be obvious to those skilled in the art.
<110> POSCO <120> Elastin derived polypeptide <130> DPP20212260 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> EXON-8 <400> 1 Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val 1 5 10 15 Pro <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> EXON-9 <400> 2 Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala 1 5 10 15 <210> 3 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> EXON-18 <400> 3 Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro Gly Phe Gly Pro Gly 1 5 10 15 Val Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 20 25 30 Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile Pro Gly Ala Ala Val 35 40 45 Pro <210> 4 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> EXON-19 <400> 4 Gly Val Val Ser Pro Glu Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala 1 5 10 15 Lys Tyr <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> EXON-36 <400> 5 Gly Gly Ala Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg Lys 1 5 10 <210> 6 <211> 420 <212> PRT <213> Artificial Sequence <220> <223> EDDP-242 <400> 6 Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala 1 5 10 15 Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val 20 25 30 Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly 35 40 45 Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys 50 55 60 Val Pro Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro Gly Phe Gly 65 70 75 80 Pro Gly Val Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 85 90 95 Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile Pro Gly Ala 100 105 110 Ala Val Pro Gly Val Val Ser Pro Glu Ala Ala Ala Lys Ala Ala Ala 115 120 125 Lys Ala Ala Lys Tyr Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro 130 135 140 Gly Phe Gly Pro Gly Val Val Gly Val Pro Gly Ala Gly Val Pro Gly 145 150 155 160 Val Gly Val Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile 165 170 175 Pro Gly Ala Ala Val Pro Gly Val Val Ser Pro Glu Ala Ala Ala Lys 180 185 190 Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Ala Gly Leu Val Pro 195 200 205 Gly Gly Pro Gly Phe Gly Pro Gly Val Val Gly Val Pro Gly Ala Gly 210 215 220 Val Pro Gly Val Gly Val Pro Gly Ala Gly Ile Pro Val Val Pro Gly 225 230 235 240 Ala Gly Ile Pro Gly Ala Ala Val Pro Gly Val Val Ser Pro Glu Ala 245 250 255 Ala Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Ala Gly 260 265 270 Leu Val Pro Gly Gly Pro Gly Phe Gly Pro Gly Val Val Gly Val Pro 275 280 285 Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ile Pro Val 290 295 300 Val Pro Gly Ala Gly Ile Pro Gly Ala Ala Val Pro Gly Val Val Ser 305 310 315 320 Pro Glu Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr Gly Val 325 330 335 Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly Ala 340 345 350 Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly 355 360 365 Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly 370 375 380 Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro 385 390 395 400 Gly Gly Ala Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg Lys His His 405 410 415 His His His His 420 <210> 7 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> EDDP-424 <400> 7 Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala 1 5 10 15 Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val 20 25 30 Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly 35 40 45 Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys 50 55 60 Val Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro 65 70 75 80 Gly Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly 85 90 95 Lys Val Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu 100 105 110 Pro Gly Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro 115 120 125 Gly Lys Val Pro Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro Gly 130 135 140 Phe Gly Pro Gly Val Val Gly Val Pro Gly Ala Gly Val Pro Gly Val 145 150 155 160 Gly Val Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile Pro 165 170 175 Gly Ala Ala Val Pro Gly Val Val Ser Pro Glu Ala Ala Ala Lys Ala 180 185 190 Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Ala Gly Leu Val Pro Gly 195 200 205 Gly Pro Gly Phe Gly Pro Gly Val Val Gly Val Pro Gly Ala Gly Val 210 215 220 Pro Gly Val Gly Val Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala 225 230 235 240 Gly Ile Pro Gly Ala Ala Val Pro Gly Val Val Ser Pro Glu Ala Ala 245 250 255 Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr Gly Val Gly Leu Pro Gly 260 265 270 Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly Ala Val Val Pro Gln 275 280 285 Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly Val Gly Leu Pro 290 295 300 Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly Ala Val Val Pro 305 310 315 320 Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly Val Gly Leu 325 330 335 Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly Ala Val Val 340 345 350 Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly Val Gly 355 360 365 Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly Ala Val 370 375 380 Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly Gly 385 390 395 400 Ala Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg Lys His His His His 405 410 415 His His <110> POSCO <120> Elastin derived polypeptide <130> DPP20212260 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> PRT <213> artificial sequence <220> <223> EXON-8 <400> 1 Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val 1 5 10 15 Pro <210> 2 <211> 16 <212> PRT <213> artificial sequence <220> <223> EXON-9 <400> 2 Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala 1 5 10 15 <210> 3 <211> 49 <212> PRT <213> artificial sequence <220> <223> EXON-18 <400> 3 Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro Gly Phe Gly Pro Gly 1 5 10 15 Val Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 20 25 30 Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile Pro Gly Ala Ala Val 35 40 45 Pro <210> 4 <211> 18 <212> PRT <213> artificial sequence <220> <223> EXON-19 <400> 4 Gly Val Val Ser Pro Glu Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala 1 5 10 15 Lys Tyr <210> 5 <211> 14 <212> PRT <213> artificial sequence <220> <223> EXON-36 <400> 5 Gly Gly Ala Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg Lys 1 5 10 <210> 6 <211> 420 <212> PRT <213> artificial sequence <220> <223> EDDP-242 <400> 6 Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala 1 5 10 15 Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val 20 25 30 Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly 35 40 45 Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys 50 55 60 Val Pro Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro Gly Phe Gly 65 70 75 80 Pro Gly Val Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 85 90 95 Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile Pro Gly Ala 100 105 110 Ala Val Pro Gly Val Val Ser Pro Glu Ala Ala Ala Lys Ala Ala Ala 115 120 125 Lys Ala Ala Lys Tyr Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro 130 135 140 Gly Phe Gly Pro Gly Val Val Gly Val Pro Gly Ala Gly Val Pro Gly 145 150 155 160 Val Gly Val Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile 165 170 175 Pro Gly Ala Ala Val Pro Gly Val Val Ser Pro Glu Ala Ala Ala Lys 180 185 190 Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Ala Gly Leu Val Pro 195 200 205 Gly Gly Pro Gly Phe Gly Pro Gly Val Val Gly Val Pro Gly Ala Gly 210 215 220 Val Pro Gly Val Gly Val Pro Gly Ala Gly Ile Pro Val Val Pro Gly 225 230 235 240 Ala Gly Ile Pro Gly Ala Ala Val Pro Gly Val Val Ser Pro Glu Ala 245 250 255 Ala Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Ala Gly 260 265 270 Leu Val Pro Gly Gly Pro Gly Phe Gly Pro Gly Val Val Gly Val Pro 275 280 285 Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ile Pro Val 290 295 300 Val Pro Gly Ala Gly Ile Pro Gly Ala Ala Val Pro Gly Val Val Ser 305 310 315 320 Pro Glu Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr Gly Val 325 330 335 Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly Ala 340 345 350 Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly 355 360 365 Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly 370 375 380 Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro 385 390 395 400 Gly Gly Ala Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg Lys His His 405 410 415 His His His His 420 <210> 7 <211> 418 <212> PRT <213> artificial sequence <220> <223> EDDP-424 <400> 7 Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala 1 5 10 15 Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val 20 25 30 Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly 35 40 45 Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys 50 55 60 Val Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro 65 70 75 80 Gly Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly 85 90 95 Lys Val Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu 100 105 110 Pro Gly Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro 115 120 125 Gly Lys Val Pro Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro Gly 130 135 140 Phe Gly Pro Gly Val Val Gly Val Pro Gly Ala Gly Val Pro Gly Val 145 150 155 160 Gly Val Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile Pro 165 170 175 Gly Ala Ala Val Pro Gly Val Val Ser Pro Glu Ala Ala Ala Lys Ala 180 185 190 Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Ala Gly Leu Val Pro Gly 195 200 205 Gly Pro Gly Phe Gly Pro Gly Val Val Gly Val Pro Gly Ala Gly Val 210 215 220 Pro Gly Val Gly Val Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala 225 230 235 240 Gly Ile Pro Gly Ala Ala Val Pro Gly Val Val Ser Pro Glu Ala Ala 245 250 255 Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr Gly Val Gly Leu Pro Gly 260 265 270 Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly Ala Val Val Pro Gln 275 280 285 Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly Val Gly Leu Pro 290 295 300 Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly Ala Val Val Pro 305 310 315 320 Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly Val Gly Leu 325 330 335 Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly Ala Val Val 340 345 350 Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly Val Gly 355 360 365 Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Gly Ala Val 370 375 380 Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly Gly 385 390 395 400 Ala Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg Lys His His His His 405 410 415 His His
Claims (9)
엑손-6, 엑손-15, 엑손-17, 엑손-19, 엑손-21, 엑손-23, 엑손-25, 엑손-27, 엑손-29, 및 엑손-31로 이루어진 그룹으로부터 선택된 라이신 및 알라닌을 포함하는 제2 도메인;
엑손-4, 엑손-8, 엑손-10, 엑손-12, 및 엑손-13으로 이루어진 그룹으로부터 선택된 라이신 및 프롤린을 포함하는 제3 도메인; 및
엑손-36을 포함하는,
엘라스틴 도메인 유래 폴리펩타이드
at least one proline-rich hydrophobic first domain selected from the group consisting of exon-9, exon-16, exon-18, exon-20, exon-22, exon-24, and exon-26;
lysine and alanine selected from the group consisting of exon-6, exon-15, exon-17, exon-19, exon-21, exon-23, exon-25, exon-27, exon-29, and exon-31 a second domain that does;
a third domain comprising lysine and proline selected from the group consisting of exon-4, exon-8, exon-10, exon-12, and exon-13; and
Including exon-36,
Elastin domain-derived polypeptide
The method of claim 1, wherein the polypeptide is a group consisting of a [first domain-third domain] block, a [first domain-third domain-first domain] block, or a [third domain-first domain] block. An elastin domain-derived polypeptide comprising at least one block selected from.
The polypeptide according to claim 2, wherein the second domain is not located at the N-terminus and exon-36 is located at the C-terminus.
The polypeptide of claim 1, wherein the polypeptide comprises 400 to 450 amino acids.
The polypeptide of claim 1, wherein the polypeptide includes exon-8, exon-9, exon-19, and exon-36.
The polypeptide of claim 5, wherein the polypeptide further comprises exon-18.
The polypeptide according to claim 5, wherein exon-8 has the amino acid sequence of SEQ ID NO: 1, exon-9 of SEQ ID NO: 2, exon-19 of SEQ ID NO: 4, and exon-36 of SEQ ID NO: 5.
The polypeptide according to claim 6, wherein exon-18 has the amino acid sequence of SEQ ID NO: 3.
The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7.
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