KR102438721B1 - Nanodisc with angiotensin converting enzyme 2 and its usage for disease from angiotensin converting enzyme 2 deficiency - Google Patents

Nanodisc with angiotensin converting enzyme 2 and its usage for disease from angiotensin converting enzyme 2 deficiency Download PDF

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KR102438721B1
KR102438721B1 KR1020220022501A KR20220022501A KR102438721B1 KR 102438721 B1 KR102438721 B1 KR 102438721B1 KR 1020220022501 A KR1020220022501 A KR 1020220022501A KR 20220022501 A KR20220022501 A KR 20220022501A KR 102438721 B1 KR102438721 B1 KR 102438721B1
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nanodisc
converting enzyme
angiotensin converting
ace2
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KR1020220022501A
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권대혁
황재현
김경원
윤정현
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성균관대학교산학협력단
엠브릭스 주식회사
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases, cochleates; Sponge phases
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4813—Exopeptidases (3.4.11. to 3.4.19)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/326—Foods, ingredients or supplements having a functional effect on health having effect on cardiovascular health

Abstract

The present invention relates to a nanodisc containing angiotensin converting enzyme 2 (ACE2) and a membrane scaffold protein (MSP), and use thereof for a disease caused by ACE2 deficiency, and it is confirmed that the substrate conversion ability of ACE2 is improved.

Description

안지오텐신 전환효소 2를 포함하는 나노디스크 및 안지오텐신 전환효소 2 결핍 질환에 대한 이의 용도 {Nanodisc with angiotensin converting enzyme 2 and its usage for disease from angiotensin converting enzyme 2 deficiency}Nanodisc containing angiotensin converting enzyme 2 and its use for angiotensin converting enzyme 2 deficiency disease {Nanodisc with angiotensin converting enzyme 2 and its usage for disease from angiotensin converting enzyme 2 deficiency}

본 발명은 안지오텐신 전환효소 2(Angiotensin converting enzyme 2, ACE2) 와 막구조화 단백질 (membrane scaffold protein, MSP)을 포함하는 나노디스크(nanodisc) 및 ACE2 결핍으로 인한 질환에 대한 이의 용도에 관한 것이다.The present invention relates to a nanodisc containing angiotensin converting enzyme 2 (ACE2) and a membrane scaffold protein (MSP) and its use for a disease caused by ACE2 deficiency.

안지오텐신 전환효소 2(Angiotensin converting enzyme 2, ACE2)는 체내 수분과 혈압을 조절하는 레닌-안지오텐신-알도스테론계(Renin-angiotensin-aldosterone system, RAAS)에서 중요한 역할을 담당한다. Angiotensin converting enzyme 2 (ACE2) plays an important role in the Renin-angiotensin-aldosterone system (RAAS) that regulates water and blood pressure in the body.

레닌-안지오텐신-알도스테론계(RAAS)에서 레닌에 의해 안지오텐시노겐이 안지오텐신 I으로 되고, 안지오텐시전환효소 (angiotensin converting enzyme, ACE)가 안지오텐신 I을 안지오텐신 II로 전환시킨다. 안지오텐신 II는 혈관 내 염증반응, 죽상동맹경화증 촉진 등 심혈관계에 직접적으로 '악영향'을 끼친다. 그런데, 체내의 안지오텐신 II는 상기 ACE2에 의해 안지오텐신(1-7)로 전환되며 그 결과 항상성을 유지할 수 있게 된다. 따라서, ACE2는 체내 항상성 유지에 중요한 효소라 할 수 있다.In the renin-angiotensin-aldosterone system (RAAS), angiotensinogen is converted to angiotensin I by renin, and angiotensin converting enzyme (ACE) converts angiotensin I to angiotensin II. Angiotensin II has a direct 'detrimental effect' on the cardiovascular system, such as inflammatory response in blood vessels and promotion of atherosclerosis. However, angiotensin II in the body is converted to angiotensin (1-7) by the ACE2, and as a result, homeostasis can be maintained. Therefore, ACE2 can be said to be an important enzyme for maintaining homeostasis in the body.

한편, ACE2는 SARS-CoV와 SARS-CoV-2를 포함한 여러 코로나바이러스의 숙주세포 감염 수용체로도 작용한다. 여러 코로나바이러스는 외막의 스파이크 단백질(spike protein)을 숙주 세포 표면에 존재하는 ACE2 수용체에 결합하면서 세포를 감염시킨다. 이때 바이러스 감염으로 인해 ACE2가 소진되면, 체내의 안지오텐신 II 전환율이 낮아진다. 이후 체내에 안지오텐신 II가 안지오텐신 1-7로 전환되지 못하여 양이 증가하면, 숙주의 폐손상 및 심혈관계 손상이라는 결과에 이르게 된다.Meanwhile, ACE2 also acts as a host cell infection receptor for several coronaviruses, including SARS-CoV and SARS-CoV-2. Several coronaviruses infect cells by binding an outer membrane spike protein to the ACE2 receptor on the host cell surface. At this time, when ACE2 is depleted due to viral infection, the conversion rate of angiotensin II in the body is lowered. After that, angiotensin II cannot be converted into angiotensin 1-7 in the body and the amount increases, resulting in host lung damage and cardiovascular damage.

한편, 나노디스크(nanodisc, ND)는 체내의 고밀도 지질단백질(high-density lipoproteins, HDL)의 주요성분인 아포리포단백질 A1 (apolipoprotein A1, Apo-A1)에서 유래한 단백질인 막구조화 단백질 (membrane scaffold protein, MSP)이 인지질 이중막을 원반 형태로 감싸 형성된 구조이다. 나노디스크는 다양한 세포막 단백질의 구조 연구에 주로 쓰인다. 또한, 다양한 생리기능성 물질을 내부에 탑재하여 이를 생체 내로 전달하는 이동체로서 역할을 수행하기도 한다. 나노디스크는 생체 내 유래 물질로서 체내에서 안정적이며 유해한 반응을 유발하지 않아 안전한 장점이 있다.On the other hand, nanodisc (ND) is a protein derived from apolipoprotein A1 (Apo-A1), which is a major component of high-density lipoproteins (HDL) in the body, a membrane scaffold protein (membrane scaffold). Protein, MSP) is a structure formed by enclosing a phospholipid bilayer in the form of a disk. Nanodisc is mainly used to study the structure of various cell membrane proteins. In addition, various physiologically functional substances are loaded therein and serve as a mobile body to deliver them into the living body. Nanodisc is an in vivo-derived material that is stable in the body and does not induce harmful reactions, so it has the advantage of being safe.

대한민국 등록특허 제10-1994279호(2019.06.24)에는, 후각 수용체 단백질을 포함하는 나노디스크 및 그 제조방법이 기재되어 있다.Korean Patent Registration No. 10-1994279 (June 24, 2019) discloses a nanodisc containing an olfactory receptor protein and a method for manufacturing the same. 대한민국 공개특허 제10-2021-0123235호(2021.10.13)에는, 재조합 ACE 단백질을 포함하는 코로나바이러스 감염증-19의 예방 또는 치료용 약학적 조성물이 기재되어 있다.Korean Patent Application Laid-Open No. 10-2021-0123235 (2021.110.13) discloses a pharmaceutical composition for preventing or treating coronavirus infection-19 comprising a recombinant ACE protein.

본 발명은 기존 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2) 보다 기질전환능이 우수하여, ACE2 결핍으로 인한 질환의 개선, 예방 또는 치료 용도로 사용할 수 있는 조성물을 제공하고자 한다.The present invention is to provide a composition that can be used for improvement, prevention or treatment of diseases caused by ACE2 deficiency by having superior substrate converting ability than the existing angiotensin converting enzyme 2 (ACE2).

본 발명은 친수성 부위와 소수성 부위를 갖는 양친매성 지질 분자로부터 형성된 납작한 원반 형태의 이중층 구조로서, 친수성기는 외부로 배향되고, 소수성기는 내부로 배향되어 있는 지질 이중층 (lipid bilayer); 상기 지질 이중층의, '소수성기가 외부로 노출되어 있는 측면'을 둘러싸는 막구조화 단백질 (membrane scaffold protein, MSP); 및 상기 지질 이중층에 안착(anchoring) 또는 결합되어 있는 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)를 포함하는 나노디스크(nanodisc)를 함유하는 것을 특징으로 하는 ACE2 결핍으로 인한 질환의 예방 또는 치료용 약학 조성물을 제공한다. The present invention provides a flat disk-shaped bilayer structure formed from an amphiphilic lipid molecule having a hydrophilic region and a hydrophobic region, wherein the hydrophilic group is oriented outward and the hydrophobic group is oriented inward. A lipid bilayer; Membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer to which the hydrophobic group is exposed'; And angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE2) that is anchored or bound to the lipid bilayer for preventing or treating diseases caused by ACE2 deficiency, characterized in that it contains a nanodisc comprising: A pharmaceutical composition is provided.

또한, 본 발명은 친수성 부위와 소수성 부위를 갖는 양친매성 지질 분자로부터 형성된 납작한 원반 형태의 이중층 구조로서, 친수성기는 외부로 배향되고, 소수성기는 내부로 배향되어 있는 지질 이중층 (lipid bilayer); 상기 지질 이중층의, '소수성기가 외부로 노출되어 있는 측면'을 둘러싸는 막구조화 단백질 (membrane scaffold protein, MSP); 및 상기 지질 이중층에 안착(anchoring) 또는 결합되어 있는 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)를 포함하는 나노디스크(nanodisc)를 함유하는 것을 특징으로 하는 ACE2 결핍으로 인한 질환의 개선용 식품 조성물을 제공한다. In addition, the present invention provides a flat disk-shaped bilayer structure formed from an amphiphilic lipid molecule having a hydrophilic region and a hydrophobic region, wherein the hydrophilic group is oriented outward and the hydrophobic group is oriented inward. A lipid bilayer; Membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer to which the hydrophobic group is exposed'; And Angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE2) that is anchored or bound to the lipid bilayer, characterized in that it contains a nanodisc (nanodisc), characterized in that it contains a food composition for ameliorating a disease caused by ACE2 deficiency provides

상기 본 발명의 조성물에 있어서, 상기 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)는, 바람직하게 C-말단의 소수성을 띄는 트랜스멤브레인 도메인 (transmembrane domain)이 지질 이중층의 소수성 부위에 안착 또는 결합되어 있는 것이 좋다. In the composition of the present invention, the angiotensin converting enzyme 2 (ACE2) preferably has a C-terminal hydrophobic transmembrane domain seated or bound to a hydrophobic region of the lipid bilayer. it's good to be

상기 본 발명의 조성물에 있어서, 상기 친수성 부위와 소수성 부위를 모두 갖는 양친매성 지질 분자는, 바람직하게 인지질인 것이 좋다. 이때, 상기 인지질은, 바람직하게 포스파티딜콜린(phosphatidylcholine), 포스파티딜세린(phosphatidylserine), 포스파티딜에탄올아민(phophatidylethalolamine), 포스파티딜글리세롤(phophatidylglycerol) 및 포스파티딜이노시톨(phophatidylinositol)인 중 선택되는 어느 하나 이상인 것이 좋다. 이때, 상기 포스파티딜콜린(phosphatidylcholine)은, 일 예로 POPC(l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)이고, 상기 포스파티딜세린(phosphatidylserine)은, 일 예로 DOPS(1,2-dioleoyl-sn-glycero-3-phospho-L-serine)일 수 있다. In the composition of the present invention, the amphiphilic lipid molecule having both a hydrophilic moiety and a hydrophobic moiety is preferably a phospholipid. In this case, the phospholipid is preferably phosphatidylcholine, phosphatidylserine, phosphatidylethalolamine, phosphatidylglycerol, and any one or more selected from phosphatidylinositol. In this case, the phosphatidylcholine is, for example, l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and the phosphatidylserine is, for example, DOPS (1,2-dioleoyl-sn). -glycero-3-phospho-L-serine).

상기 본 발명의 조성물에 있어서, 상기 막구조화 단백질은, 바람직하게 헬릭스(helix) 구조를 갖는 양친매성 단백질일 수 있다. 이때, 상기 막구조화 단백질은, 바람직하게 아포리포단백질(apolipoprotein) 또는 아포리포단백질의 '헬릭스 구조 및 양친매성 특성'이 유지된 아포리포단백질의 절편일 수 있다. In the composition of the present invention, the membrane-structured protein may be an amphipathic protein, preferably having a helix structure. In this case, the membrane-structured protein may be preferably an apolipoprotein or a fragment of an apolipoprotein in which the 'helix structure and amphiphilic properties' of the apolipoprotein are maintained.

상기 본 발명의 조성물에 있어서, 상기 안지오텐신 전환효소 2는, 일 예로 수용성 안지오텐신 전환효소 2일 수 있다. In the composition of the present invention, the angiotensin converting enzyme 2 may be, for example, a water-soluble angiotensin converting enzyme 2.

본 발명의 안지오텐신 전환효소 2를 포함하는 나노디스크(nanodisc)는 기질전환능이 우수하다. 따라서, 본 발명의 나노디스크(nanodisc)를 ACE2 결핍으로 인한 질환의 예방 또는 치료용 약학 조성물 및 ACE2 결핍으로 인한 질환의 개선용 식품 조성물로 사용할 수 있다.The nanodisc containing angiotensin converting enzyme 2 of the present invention has excellent substrate conversion ability. Therefore, the nanodisc of the present invention can be used as a pharmaceutical composition for preventing or treating diseases caused by ACE2 deficiency and as a food composition for improving diseases caused by ACE2 deficiency.

도 1은 본 발명의 나노디스크(nanodisc)의 구조를 개략적으로 보여준다.
도 2는 본 발명에서 사용하는 안지오텐신 전환효소 2 (ACE2) 및 수용성 안지오텐신 전환효소 2 (sACE2)의 분자량을 보여준다.
도 3은 본 발명의 나노디스크(NDA) 제조시 사용하는 'ACE2:막구조화 단백질'의 몰 비율 변화에 따른, 본 발명 ACE2 함입 나노디스크의 크로마토그래피(SEC) 정제 결과 변화를 보여준다.
도 4는 본 발명의 나노디스크(NDA)를 동적빛산란(DLS)으로 측정한 결과로, 본 발명의 나노디스크(NDA)의 크기를 보여준다.
도 5는 수용성 안지오텐신 전환효소 2 (sACE2) 또는 '본 발명의 sACE2를 포함하는 나노디스크'의 기질전환능 평가 결과를 보여준다.1 schematically shows the structure of a nanodisc of the present invention.
Figure 2 shows the molecular weights of angiotensin converting enzyme 2 (ACE2) and water-soluble angiotensin converting enzyme 2 (sACE2) used in the present invention.
3 shows a change in the chromatographic (SEC) purification result of the ACE2-containing nanodisk of the present invention according to a change in the molar ratio of 'ACE2:membrane-structured protein' used in manufacturing the nanodisc (NDA) of the present invention.
4 is a result of measuring the nanodisk (NDA) of the present invention by dynamic light scattering (DLS), and shows the size of the nanodisk (NDA) of the present invention.
5 shows the evaluation results of substrate conversion ability of water-soluble angiotensin converting enzyme 2 (sACE2) or 'nanodisc containing sACE2 of the present invention'.

본 발명은 친수성 부위와 소수성 부위를 갖는 양친매성 지질 분자로부터 형성된 납작한 원반 형태의 이중층 구조로서, 친수성기는 외부로 배향되고, 소수성기는 내부로 배향되어 있는 지질 이중층 (lipid bilayer); 상기 지질 이중층의, '소수성기가 외부로 노출되어 있는 측면'을 둘러싸는 막구조화 단백질 (membrane scaffold protein, MSP); 및 상기 지질 이중층에 안착(anchoring) 또는 결합되어 있는 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)를 포함하는 나노디스크(nanodisc)를 함유하는 것을 특징으로 하는 ACE2 결핍으로 인한 질환의 예방 또는 치료용 약학 조성물을 제공한다. The present invention provides a flat disk-shaped bilayer structure formed from an amphiphilic lipid molecule having a hydrophilic region and a hydrophobic region, wherein the hydrophilic group is oriented outward and the hydrophobic group is oriented inward. A lipid bilayer; Membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer to which the hydrophobic group is exposed'; And angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE2) that is anchored or bound to the lipid bilayer for preventing or treating diseases caused by ACE2 deficiency, characterized in that it contains a nanodisc comprising: A pharmaceutical composition is provided.

또한, 본 발명은 친수성 부위와 소수성 부위를 갖는 양친매성 지질 분자로부터 형성된 납작한 원반 형태의 이중층 구조로서, 친수성기는 외부로 배향되고, 소수성기는 내부로 배향되어 있는 지질 이중층 (lipid bilayer); 상기 지질 이중층의, '소수성기가 외부로 노출되어 있는 측면'을 둘러싸는 막구조화 단백질 (membrane scaffold protein, MSP); 및 상기 지질 이중층에 안착(anchoring) 또는 결합되어 있는 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)를 포함하는 나노디스크(nanodisc)를 함유하는 것을 특징으로 하는 ACE2 결핍으로 인한 질환의 개선용 식품 조성물을 제공한다. In addition, the present invention provides a flat disk-shaped bilayer structure formed from an amphiphilic lipid molecule having a hydrophilic region and a hydrophobic region, wherein the hydrophilic group is oriented outward and the hydrophobic group is oriented inward. A lipid bilayer; Membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer to which the hydrophobic group is exposed'; And Angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE2) that is anchored or bound to the lipid bilayer, characterized in that it contains a nanodisc (nanodisc), characterized in that it contains a food composition for ameliorating a disease caused by ACE2 deficiency provides

SARS-CoV와 SARS-CoV-2를 포함한 여러 코로나바이러스는 인체 세포로 침투하기 위해서 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)를 수용체로 이용하는 것으로 알려졌다. 즉, SARS-CoV와 SARS-CoV-2를 포함한 여러 코로나 바이러스가 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)와 결합능이 있는 것으로 알려지고, 코로나 바이러스에 감염되면, 체내의 안지오텐신 전환효소 2가 적절한 기능을 하지 못하게 되어, 폐손상 및 심혈관계 손상으로 이어지게 되는 것으로 알려져 있다. 상기와 같은 점을 이용하여, 안지오텐신 전환효소 2 (ACE2)를 포함하는 바이러스 감염증 치료제의 개발이 시도되고 있으나, 아직 우수한 효능을 보이는 경우는 없었다.Several coronaviruses, including SARS-CoV and SARS-CoV-2, are known to use angiotensin converting enzyme 2 (ACE2) as a receptor to penetrate human cells. That is, several coronaviruses including SARS-CoV and SARS-CoV-2 are known to have binding ability with angiotensin converting enzyme 2 (ACE2). It is known that it does not function properly, leading to lung damage and cardiovascular damage. Using the above points, the development of a therapeutic agent for viral infections including angiotensin converting enzyme 2 (ACE2) has been attempted, but there has been no case showing excellent efficacy yet.

하지만, 본 발명에서 안지오텐신 전환효소 2 (ACE2)를 포함하는 나노디스크(nanodisc)를 제조하였더니 (도 1 참조), 안지오텐신 전환효소 2 (ACE2)의 기질전환능을 현저히 향상시킬 수 있었다. 이는 본 발명의 나노디스크를 이용하는 경우, 안지오텐신 전환효소 2 (ACE2)를 포함하는 치료제의 ACE2 결핍 및 이로 인한 질환의 개선, 예방 또는 치료능을 향상시킬 수 있음을 의미한다.However, in the present invention, when a nanodisc containing angiotensin converting enzyme 2 (ACE2) was prepared (see FIG. 1 ), the substrate conversion ability of angiotensin converting enzyme 2 (ACE2) was remarkably improved. This means that when the nanodisc of the present invention is used, the ACE2 deficiency of a therapeutic agent containing angiotensin converting enzyme 2 (ACE2) and the improvement, prevention or treatment ability thereof can be improved.

한편, 본 발명의 지질 이중층 (lipid bilayer)은 '친수성 부위와 소수성 부위를 갖는 양친매성 지질 분자'로부터 형성된 납작한 원반 형태의 이중층 구조로서, 친수성기는 외부로 배향되고, 소수성기는 내부로 배향되어 있는 것에 특징이 있다 (도 1 참조).On the other hand, the lipid bilayer of the present invention is a flat disc-shaped bilayer structure formed from 'amphiphilic lipid molecules having a hydrophilic region and a hydrophobic region', wherein the hydrophilic group is oriented outward and the hydrophobic group is oriented inward. characteristic (see Fig. 1).

본 발명의 지질 이중층 (lipid bilayer)은 양친매성 지질 (amphipahtic lipid)의 친수성 부위뿐만 아니라, 소수성 부위도 측면을 통해 외부에 노출된 부분을 갖는 원반 형태 (도 1 참조)라는 점에서, 친수성 또는 소수성 부위 중 선택적으로 하나만이 외부에 노출되는 구(sphere) 형태의 리포좀(liposome) 구조상 차이가 있다.The lipid bilayer of the present invention is in the form of a disk having a portion exposed to the outside through the side as well as the hydrophilic portion of the amphiphilic lipid, as well as the hydrophobic portion (see FIG. 1), hydrophilic or hydrophobic There is a difference in the structure of a sphere-shaped liposome in which only one of the sites is selectively exposed to the outside.

상기 '친수성 부위와 소수성 부위를 모두 갖는 양친매성 지질 분자'는 바람직하게 인지질인 것이 좋다. 이때, 상기 인지질은 에로서, 포스파티딜콜린(phosphatidylcholine), 포스파티딜글리세롤(phosphatidylglycerol), 포스파티딜에탄올아민(phosphatidylethanolamine), 포스파티딜세린(phosphatidylserine) 및 포스파티딜이노시톨(phophatidylinositol)로 이루 어진 군에서 선택된 1 종 이상일 수 있으나, 양친매성을 가지고 있어 이중층을 구성할 수 있는 지질이면 반드시 이에 한정되지는 않는다.The 'amphiphilic lipid molecule having both a hydrophilic moiety and a hydrophobic moiety' is preferably a phospholipid. At this time, the phospholipid is Eros, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and phosphatidylserine, but may be one or more selected from the group consisting of phosphatidylinositol, If it is a lipid that has a medium and can constitute a double layer, it is not necessarily limited thereto.

상기 포스파티딜콜린(phosphatidylcholine)은 일 예로 DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine), DLPC(1,2-Dilauroyl-sn-glycero-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycero-3-phosphocholine), DPPC(1,2-Dipalmitoyl-sn-glycero-3-phosphocholine), POPC(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), C13PC, DDPC(1,2-Didecanoyl-sn-glycero-3-phosphocholine), DSPC(1,2-Distearoyl-sn-glycero-3-phosphocholine), DEPC(1,2-Dierucoyl-sn-glycero-3-phosphocholine), DLOPC(1,2-Dilinoleoyl-sn-glycero-3-phosphocholine), EPC(Egg phosphatidylcholine), MSPC( 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine), PMPC(1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), PSPC(1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine), SMPC(1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine) 또는 SPPC(1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine)일 수 있다.The phosphatidylcholine is, for example, DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-Dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-Dimyristoyl- sn-glycero-3-phosphocholine), DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine), POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), C13PC, DDPC (1 ,2-Didecanoyl-sn-glycero-3-phosphocholine), DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine), DEPC (1,2-Dierucoyl-sn-glycero-3-phosphocholine), DLOPC ( 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine), EPC (Egg phosphatidylcholine), MSPC (1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine), PMPC (1-Palmitoyl-2-myristoyl- sn-glycero-3-phosphocholine), PSPC (1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine), SMPC (1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine), or SPPC (1 -Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine).

또한, 상기 포스파티딜글리세롤은 일 예로 DMPG(1,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(1-glycerol)], DPPG(1,2-Dipalmitoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DSPG(1,2-Distearoyl-sn-glycero-3[Phospho-rac-(1-glycerol)), POPG(1-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DEPG(1,2-Dierucoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DLPG(1,2-Dilauroyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DOPG(1,2-Dioleoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]) 또는 DSPG(1,2-Distearoyl-sn-glycero-3[Phospho-rac-(1-glycerol)])일 수 있고, 상기 포스파티딜에탄올아민은, DMPE(1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine), DSPE(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DOPE(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DEPE(1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE(1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine) 또는 POPE(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), 상기 포스파티딜세린은, DOPS(1,2-Dioleoyl-sn-glycero-3-phosphoserine), DLPS(1,2-Dilauroyl-sn-glycero-3-phosphoserine), DMPS(1,2-Dimyristoyl-sn-glycero-3-phosphoserine), DPPS(1,2-Dipalmitoyl-sn-glycero-3-phosphoserine), DSPS(1,2- Distearoyl-sn-glycero-3-phosphoserine) 또는 POPS, 상기 포스파티딜이노시톨(phophatidylinositol)은, 포스파티딜이노시톨-4-인산, 포스파티딜이노시톨-4,5-비스인산, 포스파티딜이노시톨-3,4,5-트리스인산일 수 있다.In addition, the phosphatidylglycerol is, for example, DMPG (1,2-Dimyristoyl-sn-glycero-3 [Phospho-rac- (1-glycerol)], DPPG (1,2-Dipalmitoyl-sn-glycero-3 [Phospho-rac -(1-glycerol)]), DSPG(1,2-Distearoyl-sn-glycero-3[Phospho-rac-(1-glycerol)), POPG(1-Palmitoyl-2-oleoyl-sn-glycero-3[ Phospho-rac-(1-glycerol)]), DEPG(1,2-Dierucoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DLPG(1,2-Dilauroyl-sn-glycero- 3[Phospho-rac-(1-glycerol)]), DOPG (1,2-Dioleoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]) or DSPG (1,2-Distearoyl-sn- It may be glycero-3 [Phospho-rac-(1-glycerol)]), and the phosphatidylethanolamine is DMPE (1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-Dipalmitoyl- sn-glycero-3-phosphoethanolamine), DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DEPE (1,2-Dierucoyl) -sn-glycero-3-phosphoethanolamine), DLPE (1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine) or POPE (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), the phosphatidylserine is , DOPS (1,2-Dioleoyl-sn-glycero-3-phosphoserine), DLPS (1,2-Dil auroyl-sn-glycero-3-phosphoserine), DMPS (1,2-Dimyristoyl-sn-glycero-3-phosphoserine), DPPS (1,2-Dipalmitoyl-sn-glycero-3-phosphoserine), DSPS (1,2 - Distearoyl-sn-glycero-3-phosphoserine) or POPS, the phosphatidylinositol is, phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate, phosphatidylinositol-3,4,5-trisphosphate can be

한편, 본 발명에서는 막구조화 단백질(membrane scaffold protein, MSP)을 사용하는데, 본 발명에서 막구조화 단백질은 본 발명 지질 이중층의 '소수성기가 외부로 노출되어 있는 측면'을 둘러싸는 역할을 수행하여, 본 발명 나노디스크가 안정한 형태로 유지될 수 있게 한다. Meanwhile, in the present invention, a membrane scaffold protein (MSP) is used. In the present invention, the membrane scaffolding protein surrounds the 'side of the lipid bilayer of the present invention to which the hydrophobic group is exposed to the outside'. The invention allows the nanodisk to be maintained in a stable form.

막구조화 단백질은 바람직하게 헬릭스(helix) 구조를 갖는 양친매성 단백질인 것이 좋다. 헬릭스(helix) 구조를 갖는 양친매성 막구조화 단백질(membrane scaffold protein)의 예로는 아포리포단백질(apolipoprotein)이 있다. 아포리포단백질(apolipoprotein)은 혈장지방단백질에 특이적으로 존재하는 단백질로, 지방단백질의 구조를 안정시키고, 지방단백질대사에 관여하는 효소를 활성화하며, 세포표면에 존재하는 지방단백질 수용체에 대한 배위자로서 기능을 하는 것으로 알려져 있다. 상기 아포리포단백질(apolipoprotein)은 일 예로서, 아포리포단백질 A1(ApoA-I), 아포리포단백질 A2(ApoA-2), 아포리포단백질 B(ApoB), 아포리포단백질 C(ApoC) 및 아포리포단백질 E(ApoE), MSP1(Membrane scaffold protein1), MSP1D1, MSP1D2, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSP2, MSP2N1, MSP2N2, MSP2N3, 등이 있다. The membrane-structured protein is preferably an amphiphilic protein having a helix structure. An example of an amphiphilic membrane scaffold protein having a helix structure is apolipoprotein. Apolipoprotein is a protein that is specifically present in plasma lipoproteins, stabilizes the structure of lipoproteins, activates enzymes involved in lipoprotein metabolism, and acts as a ligand for lipoprotein receptors on the cell surface. known to function. The apolipoprotein is an example, apolipoprotein A1 (ApoA-I), apolipoprotein A2 (ApoA-2), apolipoprotein B (ApoB), apolipoprotein C (ApoC) and apolipoprotein Protein E (ApoE), MSP1 (Membrane scaffold protein1), MSP1D1, MSP1D2, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSP2, MSP2N1, MSP2N2, MSP2N3, and the like.

상기에서 일 예로 언급한 ApoA-I은 주로 주변조직으로부터 콜레스테롤을 제거하여 간 또는 다른 리포단백질로 운반하는 직접적인 역할을 수행하는 고밀도 리포단백질(HDL)의 구성요소인 것으로 알려져 있다. Apo-A1은 분자량 28kDa의 243개의 아미노산으로 이루어진 단일 폴리펩타이드로 구성된다. 11개의 아미노산 혹은 22개의 아미노산으로 이루어진 8개의 반복 단위 도메인을 가지며, HDL을 이루는 2차 구조의 알파-헬릭스의 비율이 60 내지 75%인 단백질이다. 또한, ApoE는 ApoA1과 마찬가지로 콜레스테롤의 운반에 관여하는 것으로 알려져 있는데, 33kDa의 299개의 아미노산으로 이루어진 단일 폴리펩타이드로 구성된 단백질이다.ApoA-I mentioned above as an example is known to be a component of high-density lipoprotein (HDL) that plays a direct role in removing cholesterol from surrounding tissues and transporting it to the liver or other lipoproteins. Apo-A1 consists of a single polypeptide consisting of 243 amino acids with a molecular weight of 28 kDa. It is a protein having 8 repeating unit domains consisting of 11 amino acids or 22 amino acids, and having an alpha-helix ratio of 60 to 75% of the secondary structure constituting HDL. Also, ApoE, like ApoA1, is known to be involved in cholesterol transport, and is a protein composed of a single polypeptide consisting of 299 amino acids of 33 kDa.

또한, 본 발명에서는 아포리포단백질의 헬릭스 구조 및 양친매성 특성'이 유지된 아포리포단백질의 절편일 수도 있다. 즉, 상기 아포리포단백질(apolipoprotein)의 '헬릭스 구조 및 양친매성 특성'을 소실하지 않는 범위 내에서, 아포리포단백질 전체가 아닌 그 일부(절편)를 사용할 수도 있는 것이다. In addition, in the present invention, it may be an apolipoprotein fragment in which the helix structure and amphiphilic properties of the apolipoprotein are maintained. That is, as long as the 'helix structure and amphiphilic properties' of the apolipoprotein are not lost, a part (fragment) of the apolipoprotein may be used instead of the whole.

한편, 본 발명에서는 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)를 사용하는데, 상기 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)는 본 발명 지질 이중층에 안착(anchoring) 또는 결합된다. 이때, 상기 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)는 바람직하게 C-말단의 소수성을 띄는 트랜스멤브레인 도메인 (transmembrane domain)이 지질 이중층의 소수성 부위에 안착 또는 결합되어 있는 것이 좋다. 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)는 C-말단에 소수성을 띄는 트랜스멤브레인 도메인 (transmembrane domain)을 가지고 있는데, 이 부위가 지질 이중층 (lipid bilayer)의 소수성 부위와 만나 안착 또는 결합되는 것이다. 안착 또는 결합은 바람직하게 소수성 결합 (hydrophobic interaction)을 통해 이루어 질 수 있다. Meanwhile, in the present invention, angiotensin converting enzyme 2 (ACE2) is used, and the angiotensin converting enzyme 2 (ACE2) is anchored or bound to the lipid bilayer of the present invention. In this case, the angiotensin converting enzyme 2 (ACE2) preferably has a C-terminal hydrophobic transmembrane domain seated or bound to a hydrophobic region of the lipid bilayer. Angiotensin converting enzyme 2 (ACE2) has a hydrophobic transmembrane domain at the C-terminus, which meets the hydrophobic site of the lipid bilayer and is seated or bound . Seating or bonding may preferably be achieved through a hydrophobic interaction.

상기 안지오텐신 전환효소 2는, 바람직하게 수용성 안지오텐신 전환효소 2인 것이 좋다. 기존 안지오텐신 전환효소 2 (ACE2)에는 막관통부위가 있으며, 이를 이용하여 세포막에 함입된 채로 존재한다. 최근 안지오텐신 전환효소 2 (ACE2)의 바이러스와의 결합능이 보고됨에 따라, 이를 이용하여 바이러스의 감염을 예방하거나 감염된 바이러스의 증식을 억제하는 등의 항바이러스제로서의 가능성 대한 연구가 많이 보고되고 있으며, 활용성 및 기능성을 더욱 향상시키기 위해 다양한 유전자 재조합을 통한 수용성 ACE2 (recombinant soluble ACE2, rsACE2)가 개발되고 있다. 본 발명에서 이렇게 개발된 수용성 ACE2를 사용할 수 있는 것이다.The angiotensin converting enzyme 2 is preferably a water-soluble angiotensin converting enzyme 2. Existing angiotensin converting enzyme 2 (ACE2) has a transmembrane site, and using it, it exists while being embedded in the cell membrane. Recently, as the binding ability of angiotensin converting enzyme 2 (ACE2) with a virus has been reported, many studies have been conducted on its potential as an antiviral agent, such as preventing virus infection or inhibiting the proliferation of an infected virus, using it. And in order to further improve the functionality, soluble ACE2 (recombinant soluble ACE2, rsACE2) through various genetic recombination has been developed. In the present invention, the water-soluble ACE2 thus developed can be used.

한편, 본 발명에서는 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)를 나노디스크(nanodisc)에 결합시켰더니, 안지오텐신 전환효소 2의 기질전환능이 그렇지 않은 경우에 비해 현저히 향상됨을 확인할 수 있었다. 상기 ACE2 결핍으로 인한 질환은 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)와 결합능이 있는 바이러스 감염에 의해 유발될 수 있다. 일 예로, SARS-CoV 또는 SARS-CoV-2 바이러스 감염에 의해 유발될 수 있다. SARS-CoV 또는 SARS-CoV-2 바이러스는 세포 침투 시 안지오텐신 전환효소 2를 수용체로 이용하여 침투하는데, 이때 체내의 안지오텐신 전환효소 2가 적절한 기능을 하지 못하게 되어, 장기 손상 일 예로 폐손상 및 심혈관계 손상으로 이어지게 되는 것으로 알려져 있다.On the other hand, in the present invention, when angiotensin converting enzyme 2 (ACE2) was bound to nanodisc, it was confirmed that the substrate converting ability of angiotensin converting enzyme 2 was significantly improved compared to the case where it was not. The disease caused by the ACE2 deficiency may be caused by infection with a virus capable of binding to angiotensin converting enzyme 2 (ACE2). For example, it may be caused by SARS-CoV or SARS-CoV-2 virus infection. SARS-CoV or SARS-CoV-2 virus penetrates using angiotensin-converting enzyme 2 as a receptor when infiltrating cells. At this time, angiotensin-converting enzyme 2 in the body does not function properly. It is known to lead to damage.

한편, 본 발명의 약학 조성물은 약제학적으로 허용 가능한 담체, 희석제 또는 부형제를 더욱 포함할 수 있다. 사용가능한 담체, 부형제 또는 희석제로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유가 있으며, 이중 선택되는 하나 이상을 사용할 수 있다. 또한, 예방 및 치료제가 약제인 경우 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등이 추가적으로 포함될 수 있다.On the other hand, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient. Usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, There are microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and at least one selected from among them may be used. In addition, when the prophylactic and therapeutic agents are pharmaceuticals, fillers, anti-aggregants, lubricants, wetting agents, fragrances, emulsifiers, or preservatives may be additionally included.

한편, 본 발명의 약학 조성물의 제형은 사용 방법에 따라 바람직한 형태로 제조될 수 있으며, 특히 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 채택하여 제형화 하는 것이 좋다. 구체적인 제형의 예로는 경고제(PLASTERS), 과립제(GRANULES), 로션제(LOTIONS), 리니멘트제(LINIMENTS), 리모나데제(LEMONADES), 방향수제(AROMATIC WATERS), 산제(POWDERS), 시럽제(SYRUPS), 안연고제(OPHTALMIC OINTMENTS), 액제(LIQUIDS AND SOLUTIONS), 에어로솔제(AEROSOLS), 엑스제(EXTRACTS), 엘릭실제(ELIXIRS), 연고제(OINTMENTS), 유동엑스제(FLUIDEXTRACTS), 유제(EMULSIONS), 현탁제(SUSPESIONS), 전제(DECOCTIONS), 침제(INFUSIONS), 점안제(OPHTHALMIC SOLUTIONS), 정제(TABLETS), 좌제(SUPPOSITIORIES), 주사제(INJECTIONS), 주정제(SPIRITS), 카타플라스마제(CATAPLSMA), 캅셀제(CAPSULES), 크림제(CREAMS), 트로키제(TROCHES), 틴크제(TINCTURES), 파스타제(PASTES), 환제(PILLS), 연질 또는 경질 젤라틴 캅셀 중 선택되는 어느 하나일 수 있다.On the other hand, the dosage form of the pharmaceutical composition of the present invention may be prepared in a desired form according to the method of use, and in particular, methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal It is better to adopt and formulate it. Examples of specific formulations include PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, syrups ( SYRUPS), OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS ), suspensions (SUSPESIONS), preparations (DECOCTIONS), infusions (INFUSIONS), eye drops (OPHTHALMIC SOLUTIONS), tablets (TABLETS), suppositories (SUPPOSITIORIES), injections (INJECTIONS), alcohol tablets (SPIRITS), CATAPLS ), capsules (CAPSULES), creams (CREAMS), troches (TROCHES), tinctures (TINCTURES), pastas (PASTES), pills (PILLS), it may be any one selected from soft or hard gelatin capsules.

한편, 본 발명의 약학조성물에 있어서, 투여량은 투여방법, 복용자의 연령, 성별 및 체중 및 질환의 중증도 등을 고려하여 결정하는 것이 좋다. 일 예로, 유효성분을 기준으로 하였을 때 1일 0.00001 내지 1,000 mg/kg (체중)으로 1회 이상 경구 투여 가능하다. 다만, 상기의 투여량은 예시하기 위한 일 예에 불과하며, 복용자의 상태와 의사의 처방에 의해 변화될 수 있다.On the other hand, in the pharmaceutical composition of the present invention, the dosage is preferably determined in consideration of the administration method, the age, sex and weight of the user, and the severity of the disease. For example, based on the active ingredient, 0.00001 to 1,000 mg/kg (body weight) per day may be orally administered at least once. However, the above dosage is only an example for illustration, and may be changed by the condition of the user and the prescription of the doctor.

한편, 본 발명은 상기 나노디스크(nanodisc)를 포함하는 것을 특징으로 하는 ACE2 결핍으로 인한 질환 개선용 식품 조성물을 제공한다.On the other hand, the present invention provides a food composition for improving diseases due to ACE2 deficiency, comprising the nanodisc.

본 발명의 식품 조성물은 식품 조성물 전체 중량을 기준으로 본 발명의 나노디스크(nanodisc)를 0.001 내지 99.9 중량%로 포함할 수 있다. 본 발명의 식품 조성물은 일 예로, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 기능성 음료, 건강기능식품 등의 형태일 수 있으며, 식품 제조시 통상적으로 첨가되는 첨가제가 추가될 수 있다. The food composition of the present invention may contain 0.001 to 99.9% by weight of the nanodisc of the present invention based on the total weight of the food composition. The food composition of the present invention may be in the form of, for example, various foods, beverages, gum, tea, vitamin complexes, functional drinks, health functional foods, and the like, and additives commonly added during food production may be added.

이하, 본 발명의 내용을 하기 실시예 및 실험예를 통하여 보다 상세하게 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예 및 실험예에만 한정되는 것은 아니고 그와 등가의 기술적 사상의 변형까지를 포함한다.Hereinafter, the content of the present invention will be described in more detail through the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes modifications of technical ideas equivalent thereto.

[실시예 1: ACE2가 포함된 본 발명의 나노디스크 제작][Example 1: Preparation of nanodisc of the present invention containing ACE2]

1) ACE2 막단백질 정제1) ACE2 membrane protein purification

단백질의 정제를 위해 HEK293(Human embryonic kidney 293) 수용성 부유세포를 37℃, 120 rpm, 8% CO2의 조건하로 배양하여, 1.1×106 cells/mL, 180 mL를 준비하였다. 인간에서 유래한 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2) 유전자 (서열번호 1) 함유 플라스미드 250 μg과 PEI(poly ethylenimine) 750 μg를 배지 20 mL에 혼합한 후, 상기 준비한 180 mL의 세포액과 혼합하여 형질주입(Transfection) 하였다. For protein purification, HEK293 (Human embryonic kidney 293) soluble suspended cells were cultured at 37° C., 120 rpm, and 8% CO 2 conditions to prepare 1.1×10 6 cells/mL, 180 mL. After mixing 250 µg of a plasmid containing human-derived Angiotensin converting enzyme 2 (ACE2) gene (SEQ ID NO: 1) and 750 µg of PEI (poly ethylenimine) in 20 mL of medium, 180 mL of the prepared cell solution and The mixture was transfected.

이후 37℃, 120 rpm, 8% CO2 조건의 인큐베이터에서 세포를 72시간 배양한 후 8000 g에서 10분간 원심분리하여 배지를 제거하고 세포만 얻어내었다. After culturing the cells in an incubator at 37° C., 120 rpm, and 8% CO 2 conditions for 72 hours, centrifugation was performed at 8000 g for 10 minutes to remove the medium and only the cells were obtained.

상기 세포에 1% DDM이 포함된 Tris 완충액을 사용하여 세포를 재부유시키고, 초고속 원심분리기를 이용해 막단백질을 분리 한 후, Ni-NTA 아가로스 비드를 이용해 정제하였다. 이후 0.1% DDM과 저농도의 이미다졸(imidazole)이 포함된 완충액을 이용하여 비드에 결합하지 못한 물질들을 제거하였고, 고농도의 이미다졸을 이용해 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2) (서열번호 2)를 얻어내었다. The cells were resuspended using Tris buffer containing 1% DDM, and membrane proteins were separated using an ultra-high speed centrifuge, followed by purification using Ni-NTA agarose beads. Thereafter, substances that could not be bound to the beads were removed using a buffer containing 0.1% DDM and low concentration of imidazole, and angiotensin converting enzyme 2 (ACE2) (SEQ ID NO: 2) was obtained.

한편, 수용성 안지오텐신 전환효소 2 (Soluble ACE2; sACE2)는 상기의 방법에서 수용성 ACE2 유전자 (서열번호 3) 함유 플라스미드를 이용하여 형질주입(Transfection)을 진행하였으며 37℃, 120 rpm, 8% CO2 조건의 인큐베이터에서 세포를 72시간 배양한 후 8000×g에서 10분간 원심분리하여 세포를 제거하고 상층액을 Ni-NTA 아가로스 비드를 이용해 정제하였다. 이후 저농도의 이미다졸이 포함된 완충액을 이용하여 비드에 결합하지 못한 물질들을 제거하였고, 고농도의 이미다졸을 이용해 수용성 안지오텐신 전환효소 2 (Soluble ACE2; sACE2) (서열번호 4)를 얻어내었다(도 2).On the other hand, water-soluble angiotensin converting enzyme 2 (Soluble ACE2; sACE2) was transfected using a plasmid containing the water-soluble ACE2 gene (SEQ ID NO: 3) in the above method, 37 ° C., 120 rpm, 8% CO 2 conditions After culturing the cells for 72 hours in the incubator of 8000 × g for 10 minutes to remove the cells by centrifugation, and the supernatant was purified using Ni-NTA agarose beads. Thereafter, materials that could not be bound to the beads were removed using a buffer containing imidazole at a low concentration, and water-soluble angiotensin converting enzyme 2 (Soluble ACE2; sACE2) (SEQ ID NO: 4) was obtained using imidazole at a high concentration (Fig. 2). ).

2) ACE2가 포함된 본 발명의 나노디스크(NDA)의 제작 및 정제2) Preparation and purification of nanodisc (NDA) of the present invention containing ACE2

지질로서，POPC(l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)와 DOPS(1,2-dioleoyl-sn-glycero-3-phospho-L-serine)을 클로로포름에 용해시켜 각 25 mg/ml, 10 mg/ml 농도의 지질 용액을 준비하였다. 이후 나노디스크(ND) 완충액(40 mM Tris-Cl, 300 mM NaCl, 0.5 mM EDTA, 50 mM NaC, pH 7.4)으로 녹였을 때 총 지질의 농도가 10 mM, 부피가 1 mL가 되며 동시에 POPC:DOPS의 몰비율이 8:2가 되도록 POPC 용액의 243 μl, DOPS 용액의 65 μl를 유리 튜브에 옮겼다. 이후 질소가스를 가하고, 진공 상태에서 최소 4시간 동안 방치하여 용매를 제거한 후, 리피드 필름을 수득하였다. 상기 수득한 리피드 필름에 상기 ND 완충액 1 mL를 혼합하여 상기 리피드 필름을 수화시키고, 초음파를 55℃에서 30분간 처리하여, 지질이 일정하게 분배된 현탁액을 수득하였다. As lipids, 25 mg each of POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) were dissolved in chloroform A lipid solution of /ml, 10 mg/ml concentration was prepared. Then, when dissolved in nanodisc (ND) buffer (40 mM Tris-Cl, 300 mM NaCl, 0.5 mM EDTA, 50 mM NaC, pH 7.4), the total lipid concentration was 10 mM, the volume was 1 mL, and POPC: 243 μl of POPC solution and 65 μl of DOPS solution were transferred to a glass tube so that the molar ratio of DOPS was 8:2. Thereafter, nitrogen gas was added, and the solvent was removed by leaving it to stand for at least 4 hours in a vacuum state, and then a lipid film was obtained. 1 mL of the ND buffer was mixed with the obtained lipid film to hydrate the lipid film, and ultrasonic waves were treated at 55° C. for 30 minutes to obtain a suspension in which lipids were uniformly distributed.

이후 상기 현탁액에 ACE2(분자량 94.2 kda) 또는 sACE2(분자량 87.0 kDa):막구조화 단백질(membrane scaffold proteins로, 분자량 63 kDa의 ApoA-I:지질의 몰 비율이 0.5:1:120이 되도록 처리하였다. 이후 전체 혼합액과 동일한 양의 바이오비드(bio-beads)를 실온에서 5시간 동안 1회, 4℃에서 16시간 동안 1회, 총 2회 처리하여, 자가조립과정을 통해 ACE2 또는 sACE2가 포함된 나노디스크(NDA)를 제조하였다. Thereafter, the suspension was treated with ACE2 (molecular weight 94.2 kda) or sACE2 (molecular weight 87.0 kDa):membrane scaffold proteins, ApoA-I:lipid having a molecular weight of 63 kDa so that the molar ratio was 0.5:1:120. Afterwards, the same amount of bio-beads as the entire mixture was treated twice, once for 5 hours at room temperature and once for 16 hours at 4°C, and through the self-assembly process, the nano containing ACE2 or sACE2 A disk (NDA) was prepared.

제조된 나노디스크(NDA)를 크기 배제 크로마토그래피(SEC)를 통해 정제하였다. Elution volume 15~16 mL에서 ACE2 또는 sACE2가 함입되지 않은 나노디스크(ND, 분자량 약 200 kDa)가 정제되었으며, Elution volume 13~14 mL에서 ACE2 또는 sACE2가 함입된 나노디스크(NDA, 분자량 약 400 kDa)가 정제되었다 (도 3). The prepared nanodisc (NDA) was purified through size exclusion chromatography (SEC). Nanodisc (ND, molecular weight about 200 kDa) not impregnated with ACE2 or sACE2 was purified from 15 to 16 mL of elution volume, and nanodisk containing ACE2 or sACE2 (NDA, molecular weight about 400 kDa) from 13 to 14 mL of elution volume. ) was purified (Fig. 3).

한편, 도 3은 본 발명의 나노디스크(NDA) 제조시 사용하는 ACE2:막구조화 단백질의 몰 비율 변화에 따른 본 발명 ACE2 함입 나노디스크의 크로마토그래피(SEC) 정제 결과를 보여준다.Meanwhile, FIG. 3 shows the results of chromatography (SEC) purification of the ACE2-containing nanodisc of the present invention according to a change in the molar ratio of ACE2:membrane-structured protein used in manufacturing the nanodisc (NDA) of the present invention.

3) 본 발명의 나노디스크(NDA) 구조 형성 확인3) Confirmation of the formation of the nanodisk (NDA) structure of the present invention

상기 실시예 1의 2)에서 정제된 ND와 NDA를 SDS-PAGE로 분석한 결과, ND에서는 ACE2가 검출되지 않았지만 NDA에서는 ACE2 단백질이 검출되었다(도 4). As a result of analyzing the ND and NDA purified in 2) of Example 1 by SDS-PAGE, ACE2 was not detected in ND, but ACE2 protein was detected in NDA (FIG. 4).

한편, ACE2:막구조화 단백질:지질의 몰비율이 각 0.25:1:120, 0.125:1:120으로 제작 및 정제된 NDA에서는 상기 실시예 1의 2)에서 0.5:1:120의 비율로 제작 및 정제된 NDA보다 ACE2 단백질의 양이 적게 검출되었다. On the other hand, in the NDA prepared and purified in ACE2: membrane-structured protein: lipid molar ratio of 0.25:1:120 and 0.125:1:120, respectively, in 2) of Example 1 above, it was prepared at a ratio of 0.5:1:120 and A lower amount of ACE2 protein than purified NDA was detected.

한편, 상기 실시예 1의 2)에서 정제한 NDA를 동적빛산란(DLS)으로 측정한 결과, NDA의 크기는 ND(약 10.3 nm)의 약 1.5배인 14.9 nm로 측정되었으며, 이를 통해 ACE2가 포함된 나노디스크(NDA)의 구조 형성을 확인할 수 있었다(도 4).On the other hand, as a result of measuring the NDA purified in 2) of Example 1 by dynamic light scattering (DLS), the size of the NDA was measured to be 14.9 nm, which is about 1.5 times the ND (about 10.3 nm), through which ACE2 is included It was confirmed that the structure of the nanodisk (NDA) was formed (FIG. 4).

[실험예 1: ACE2가 포함된 본 발명 나노디스크의 ACE2 결핍으로 인한 질환의 개선, 예방 또는 치료 효능 확인 실험][Experimental Example 1: Experiment to confirm the efficacy of improvement, prevention or treatment of diseases caused by ACE2 deficiency of the present nanodisc containing ACE2]

본 실험에서는 본 발명에서 제작한 ACE2 포함 나노디스크(NDA)가 대조군인 ACE2 대비 효소 역가를 얼마나 잘 보존 또는 발휘하는지를 확인하고자 하였다. 이와 같은 실험을 통해, ACE2 효소의 불활성화또는 결핍로 말미암은 여러 질환의 개선, 예방 또는 치료에 본 발명의 ACE2 포함 나노디스크(NDA)가 유의적절히 사용될 수 있는지를 확인하고자 했다.In this experiment, it was attempted to determine how well the ACE2-containing nanodisc (NDA) prepared in the present invention preserves or exhibits the enzyme titer compared to the control ACE2. Through this experiment, it was attempted to confirm whether the ACE2-containing nanodisc (NDA) of the present invention could be used significantly for the improvement, prevention or treatment of various diseases caused by inactivation or deficiency of the ACE2 enzyme.

실험은 '수용성 ACE2 (sACE2)'를 사용하여 수행하였는데, 하기의 과정으로 진행되었다. sACE2(수용성 ACE2)와 sACE2를 포함하는 나노디스크(NDA)의 기질전환능력을 평가하기 위해 ACE2 activity assay kit를 이용하였다. The experiment was performed using 'water-soluble ACE2 (sACE2)', and the following procedure was performed. ACE2 activity assay kit was used to evaluate the substrate conversion ability of sACE2 (soluble ACE2) and nanodisc (NDA) containing sACE2.

ACE2는 MCA based peptide 기질을 추가하면 이를 절단하여 자유 발형광단 (Free Fluorophore)를 방출하는 능력을 가지는데, 이를 형광 microplate reader로 측정하여 상기 실시예 1의 1)에서 제조한 sACE2와 상기 실시예 1의 2)에서 제조한 sACE2를 포함하는 나노디스크(NDA)의 기질전환능을 측정할 수 있었다.ACE2 has the ability to release a free fluorophore by cleaving the MCA based peptide substrate when it is added. It was possible to measure the substrate conversion ability of the nanodisc (NDA) containing sACE2 prepared in 1 and 2).

구체적으로, sACE2의 농도가 0.125 μg/mL가 되도록 sACE2, sACE2를 포함하는 나노디스크(NDA) 각각을 50 μL만큼 각 웰에 분주하고, MCA 기질 50 μL를 각 웰에 첨가 한 후, 흡수파장 320 nm/방출파장 420 nm로 기질전환능력을 측정하였다. 그 결과, sACE2는 약 10,000 RFU에서 포화상태로 접어든 것을 확인할 수 있었으며, sACE2를 포함하는 나노디스크(NDA)는 12,000 RFU이상의 측정값을 얻을 수 있었다 (도 5).Specifically, 50 μL of each nanodisc (NDA) containing sACE2 and sACE2 is dispensed into each well so that the concentration of sACE2 is 0.125 μg/mL, and 50 μL of MCA substrate is added to each well, and then the absorption wavelength is 320 The substrate conversion ability was measured at nm/emission wavelength 420 nm. As a result, it was confirmed that sACE2 entered the saturation state at about 10,000 RFU, and the nanodisc (NDA) containing sACE2 was able to obtain a measured value of 12,000 RFU or more (FIG. 5).

이를 통해 sACE2(수용성 ACE2) 보다 sACE2가 함입된 나노디스크(NDA)의 기질전환능이 더 뛰어난 것을 확인할 수 있었는데, 이는 본 발명의 나노디스크(NDA)는 막구조화 단백질이 지질-ACE2를 안정화 시켜주므로 sACE2가 지닌 본래의 효소로써의 작용을 sACE2(수용성 ACE2) 보다 더 효과적으로 진행할 수 있었음을 의미한다.Through this, it was confirmed that the substrate conversion ability of the nanodisc (NDA) containing sACE2 was superior to that of sACE2 (water-soluble ACE2). It means that the original enzyme action of sACE2 (soluble ACE2) could proceed more effectively than that of sACE2 (soluble ACE2).

<110> MVRIX Research & Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Nanodisc with angiotensin converting enzyme 2 and its usage for disease from angiotensin converting enzyme 2 deficiency <130> YP-22-019 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 2460 <212> DNA <213> Homo sapiens <400> 1 atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60 attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120 tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180 aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240 caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300 cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360 aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420 tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480 aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540 gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600 gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660 cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720 catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780 tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840 ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900 gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960 cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020 aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080 tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140 tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200 catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260 attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320 aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380 tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440 aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500 gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560 taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620 aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680 ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740 gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800 aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860 aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920 tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980 cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040 tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100 gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160 agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220 atatggctga ttgtttttgg agttgtgatg ggagtgatag tggttggcat tgtcatcctg 2280 atcttcactg ggatcagaga tcggaagaag aaaaataaag caagaagtgg agaaaatcct 2340 tatgcctcca tcgatattag caaaggagaa aataatccag gattccaaaa cactgatgat 2400 gttcagacct cctttggggg tggaggctct caccatcacc accatcatca ccaccatcac 2460 2460 <210> 2 <211> 820 <212> PRT <213> Homo sapiens <400> 2 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735 Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val 740 745 750 Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg 755 760 765 Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile 770 775 780 Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp 785 790 795 800 Val Gln Thr Ser Phe Gly Gly Gly Gly Ser His His His His His His 805 810 815 His His His His 820 <210> 3 <211> 2265 <212> DNA <213> Artificial Sequence <220> <223> Soluble angiotensin converting enzyme 2 <400> 3 atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60 attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120 tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180 aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240 caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300 cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360 aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420 tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480 aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540 gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600 gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660 cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720 catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780 tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840 ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900 gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960 cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020 aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080 tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140 tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200 catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260 attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320 aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380 tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440 aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500 gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560 taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620 aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680 ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740 gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800 aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860 aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920 tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980 cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040 tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100 gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160 agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220 gggggtggag gctctcacca tcaccaccat catcaccacc atcac 2265 <210> 4 <211> 755 <212> PRT <213> Artificial Sequence <220> <223> Soluble angiotensin converting enzyme 2 <400> 4 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735 Pro Pro Val Ser Gly Gly Gly Gly Ser His His His His His His His 740 745 750 His His His 755 <110> MVRIX Research & Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Nanodisc with angiotensin converting enzyme 2 and its usage for disease from angiotensin converting enzyme 2 deficiency <130> YP-22-019 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 2460 <212> DNA <213> Homo sapiens <400> 1 atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60 attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120 tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180 aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240 caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300 cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360 aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420 tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480 aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540 gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600 gattatt gga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660 cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720 catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780 tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840 ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900 gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960 cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020 aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080 tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140 tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200 catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260 attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320 aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380 tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440 aagcgagaga tagttggg gt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500 gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560 taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620 aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680 ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740 gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800 aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860 aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920 tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980 cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040 tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100 gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160 agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220 atatggctga ttgtttttgg agttgtgatg ggagtgatag tggttggcat tgtcatcctg 2280 atcttcactg ggatcagaga tcg gaagaag aaaaataaag caagaagtgg agaaaatcct 2340 tatgcctcca tcgatattag caaaggagaa aataatccag gattccaaaa cactgatgat 2400 gttcagacct cctttggggg tggaggctct caccatcacc accatcatca ccaccatcac 2460 2460 <210> 2 <211> 820 <212> PRT <213> Homo sapiens <400> 2 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735 Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val 740 745 750 Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg 755 760 765 Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile 770 775 780 Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp 785 790 795 800 Val Gln Thr Ser Phe Gly Gly Gly Gly Ser His His His His His His 805 810 815 His His His His 820 <210> 3 <211> 2265 <212> DNA <213> Artificial Sequence <220> <223> Soluble angiotensin converting enzyme 2 <400> 3 atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60 attgaggaac aggccaagac atttttggac aagtttaacc acgaagcc ga agacctgttc 120 tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180 aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240 caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300 cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360 aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420 tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480 aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540 gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600 gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660 cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720 catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780 tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840 ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900 gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960cc taatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020 aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080 tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140 tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200 catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260 attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320 aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380 tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440 aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500 gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560 taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620 aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680 ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740 gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800 aattcttt tg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860 aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920 tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980 cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040 tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100 gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160 agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220 gggggtggag gctctcacca tcaccaccat catcaccacc atcac 2265 <210> 4 <211> 755 <212> PRT <213> Artificial Sequence <220> <223> Soluble angiotensin converting enzyme 2 <400> 4 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 As p Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 50 5 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735 Pro Pro Val Ser Gly Gly Gly Gly Ser His His His His His His His 740 745 750His His His 755

Claims (10)

인지질로부터 형성된 납작한 원반 형태의 이중층 구조로서, 친수성기는 외부로 배향되고, 소수성기는 내부로 배향되어 있는 지질 이중층 (lipid bilayer);
상기 지질 이중층의, '소수성기가 외부로 노출되어 있는 측면'을 소수성 결합으로 둘러싸는 막구조화 단백질 (membrane scaffold protein, MSP); 및
상기 지질 이중층 내부와 소수성 결합되어 있는 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)를 포함하는 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)의 기질 전환능이 향상된 나노디스크(nanodisc).
A lipid bilayer having a flat disc-shaped bilayer structure formed from phospholipids, in which hydrophilic groups are oriented outward and hydrophobic groups are oriented inward;
Membrane scaffold protein (MSP) surrounding the 'side where the hydrophobic group is exposed' of the lipid bilayer with a hydrophobic bond; and
Angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE2) containing angiotensin converting enzyme 2 (ACE2) hydrophobically bonded to the inside of the lipid bilayer substrate conversion ability of an improved nanodisc (nanodisc).
 삭제delete 제1항에 있어서,
상기 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)는,
트랜스멤브레인 도메인 (transmembrane domain)이 지질 이중층의 소수성 부위에 결합되어 있는 것을 특징으로 하는 나노디스크(nanodisc).
According to claim 1,
The angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE2) is,
Nanodisc characterized in that the transmembrane domain (transmembrane domain) is bound to the hydrophobic region of the lipid bilayer.
 삭제delete 제1항에 있어서,
상기 인지질은,
포스파티딜콜린(phosphatidylcholine), 포스파티딜세린(phosphatidylserine), 포스파티딜에탄올아민(phophatidylethalolamine), 포스파티딜글리세롤(phophatidylglycerol) 및 포스파티딜이노시톨(phophatidylinositol) 중 선택되는 어느 하나 이상인 것을 특징으로 하는 나노디스크(nanodisc).
According to claim 1,
The phospholipids are
Nanodisc (nanodisc), characterized in that at least one selected from phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol.
 제1항에 있어서,
상기 인지질은,
POPC(l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) 또는 DOPS(1,2-dioleoyl-sn-glycero-3-phospho-L-serine)인 것을 특징으로 하는 나노디스크(nanodisc).
According to claim 1,
The phospholipids are
Nanodisc, characterized in that POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) or DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine).
 제1항에 있어서,
상기 막구조화 단백질은,
헬릭스(helix) 구조를 갖는 양친매성 단백질인 것을 특징으로 하는 나노디스크(nanodisc).
According to claim 1,
The membrane-structured protein is
Nanodisc (nanodisc), characterized in that the amphiphilic protein having a helix (helix) structure.
 제7항에 있어서,
상기 막구조화 단백질은,
아포리포단백질(apolipoprotein) 또는 아포리포단백질의 '헬릭스 구조 및 양친매성 특성'이 유지된 아포리포단백질의 절편인 것을 특징으로 하는 나노디스크(nanodisc).
8. The method of claim 7,
The membrane-structured protein is
Nanodisc, characterized in that it is a fragment of an apolipoprotein in which the 'helix structure and amphiphilic properties' of apolipoprotein or apolipoprotein are maintained.
 제1항에 있어서,
상기 안지오텐신 전환효소 2는,
수용성 안지오텐신 전환효소 2인 것을 특징으로 하는 나노디스크(nanodisc).
According to claim 1,
The angiotensin converting enzyme 2 is,
Nanodisc characterized in that the water-soluble angiotensin converting enzyme 2 (nanodisc).
 제1항에 있어서,
상기 안지오텐신 전환효소 2의 기질 전환능은,
안지오텐신 II을 안지오텐신 1-7로 전환시키는 것을 특징으로 하는 나노디스크(nanodisc).According to claim 1,
The substrate conversion ability of the angiotensin converting enzyme 2 is,
Nanodisc, characterized in that it converts angiotensin II to angiotensin 1-7.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023158159A1 (en) * 2022-02-21 2023-08-24 엠브릭스 주식회사 Nanodisc comprising angiotensin converting enzyme 2
KR102610178B1 (en) * 2023-02-07 2023-12-06 엠브릭스 주식회사 Nanodisc containing membrane scaffold protein fused with angiotensin converting enzyme 2

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101994279B1 (en) 2017-11-03 2019-06-28 서울대학교산학협력단 Manufacturing method of nanodisc comprising an olfactory receptor protein and nanodisc comprising an olfactory receptor protein manufactured by the same
KR102181991B1 (en) * 2016-07-15 2020-11-23 성균관대학교산학협력단 Nano-perforator and pharmaceutical composition comprising the same for preventing or treating viral infection
KR20210123235A (en) 2020-04-02 2021-10-13 조선대학교산학협력단 Use of ACE2 as treatment of COVID-19

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102181991B1 (en) * 2016-07-15 2020-11-23 성균관대학교산학협력단 Nano-perforator and pharmaceutical composition comprising the same for preventing or treating viral infection
KR101994279B1 (en) 2017-11-03 2019-06-28 서울대학교산학협력단 Manufacturing method of nanodisc comprising an olfactory receptor protein and nanodisc comprising an olfactory receptor protein manufactured by the same
KR20210123235A (en) 2020-04-02 2021-10-13 조선대학교산학협력단 Use of ACE2 as treatment of COVID-19

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023158159A1 (en) * 2022-02-21 2023-08-24 엠브릭스 주식회사 Nanodisc comprising angiotensin converting enzyme 2
KR102610178B1 (en) * 2023-02-07 2023-12-06 엠브릭스 주식회사 Nanodisc containing membrane scaffold protein fused with angiotensin converting enzyme 2

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