KR20190141229A - 자발적 이소펩타이드 결합 형성 속도가 향상된 단백질 및 펩타이드 태그 및 이의 용도 - Google Patents
자발적 이소펩타이드 결합 형성 속도가 향상된 단백질 및 펩타이드 태그 및 이의 용도 Download PDFInfo
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- KR20190141229A KR20190141229A KR1020197034615A KR20197034615A KR20190141229A KR 20190141229 A KR20190141229 A KR 20190141229A KR 1020197034615 A KR1020197034615 A KR 1020197034615A KR 20197034615 A KR20197034615 A KR 20197034615A KR 20190141229 A KR20190141229 A KR 20190141229A
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- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
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Abstract
Description
도 1은 M13 파지의 pIII에 표시된 SpyTag 변이체를 선택하기 위한 패닝(panning) 절차의 카툰을 도시한 것이다.
도 2는 (A) 야생형(WT) SpyCatcher 미끼로 선택한 후 회수된 SpyTag-파지의 양을 비반응성 SpyCatcher EQ와 비교하여 콜로니 형성 단위(cfu)로 정량하여 입증하는 막대 차트(평균 ± 1 sd, n = 3)이고; (B) N-말단 라이브러리(NLib1-3, 서열번호 15-17) 및 후속 C-말단 라이브러리(CLib1-10, 서열 18-27)의 최종 선택 라운드로부터 선택된 SpyTag 변이체 서열의 표이다. WT는 SpyTag의 서열(서열번호 6)을 나타내고, SpyTag002는 개선된 반응 속도를 가진 변이체, 서열번호 3을 나타낸다.
도 3은 SpyTag N-말단 라이브러리의 가장 반응성이 높은 변이체(NLib1-MBP)의 결실 변이체와 반응하는 SpyCatcher의 시간 경과 그래프를 도시한 것이다. PPVPT는 서열번호 15를 나타내고, PVPT는 서열번호 30을 나타내고, VPT는 서열번호 31을 나타내고, PT는 서열번호 32를 나타낸다. 데이터는 3반복으로 수행된 반응의 평균±1 s.d.을 나타낸다; 일부 오차 막대는 너무 작아 보이지 않는다.
도 4는 (A) 가속된 SpyCatcher 변이체에 대한 파지 디스플레이 선택 체계의 카툰을 도시한 것이다. 스트렙타비딘-비드로부터 TEV 프로테아제 용리 전에 M13 파지상의 SpyCatcher 돌연변이체를 비오티닐화된 AviTag-SpyTag-MBP 미끼에 대해 패닝한다; (B)는 cfu(평균 ± 1 s.d., n = 3)로 정량화된 WT SpyTag-MBP 또는 비반응성 SpyTag DA-MBP 대조군으로 선별한 후 회수된 SpyCatcher-파지의 양을 보여주는 막대 차트를 나타낸다.
도 5는 SpyCatcher 라이브러리 선택의 최종 라운드에서 선택된 변이체의 아미노산 서열의 정렬을 보여준다. * 변화 없음, : 매우 보전적 변화, . 보전적 변화, 및 갭은 먼 변화를 나타낸다. WT는 서열번호 7, L1C1은 서열번호 33, L1C4는 서열번호 34, L1C2는 서열번호 35, L2C1은 서열번호 36, L1C3은 서열번호 37, L1C6은 서열번호 38을 나타내고, L2C8은 서열번호 39를 나타내고, SC002는 서열번호 40을 나타낸다.
도 6은 파지-선택된 SpyCatcher 변이체의 반응 시간 경과 그래프를 보여준다. SpyTag-MBP는 PBS pH 7.5, 25℃에서 각 단백질을 1μM로 하여 SpyCatcher 및 선택된 변이체와 함께 배양하였다. 반응은 비등가열 후 쿠마시 염색과 SDS-PAGE에 의해 분석했다. 데이터는 반복 반응의 평균을 나타낸다. SpyCatcher는 서열번호 7, L1C1은 서열번호 33, L1C4는 서열번호 34, L1C2는 서열번호 35, L2C1은 서열번호 36, L1C3은 서열번호 37, L1C6은 서열번호 38을 나타내고, L2C8은 서열번호 39를 나타낸다.
도 7은 (A) L1C6 SpyCatcher 변이체의 자기 반응이 SpyCatcher002로 차단되었음을 나타내는 SDS-PAGE 겔을 보여준다. L1C6 및 SpyCatcher002는 단독으로 또는 SpyTag002-MBP와 반응 후에 SDS-PAGE하여 쿠마시 염색으로 분석했다. SpyTag002-MBP와 반응한 L1C6 이량체의 생성물뿐만 아니라, 공유 L1C6 이량체의 작은 분획이 표시된다. 반응 조건 : 10 μM(+) SpyCatcher 변이체, 13 μM(++) SpyTag002-MBP, PBS pH 7.5, 25 ℃에서 1 시간 동안; 및 (B) SpyTag의 아미노산 서열의 파트(서열번호 41)와 SpyCatcher L1C6의 N-말단(서열번호 42)의 정렬. L1C6 D5T의 N-말단(서열번호 43)은 자기 반응을 방지했다.
도 8은 SpyCatcher002와 중첩된 SpyCatcher의 시차 주사 열량측정을 나타내는 그래프를 도시한다. Tm 값이 삽입도로 표시된다.
도 9는 (A) SpyCatcher002와 SpyTag002 사이에 자발적 이소펩타이드 결합 형성의 특징을 나타내는 SDS-PAGE 겔을 보여준다. SpyCatcher002 및 SpyTag002-MBP는 pH 7.0의 숙시네이트-포스페이트-글리신 완충액에서 10 μM로 1 시간 동안 혼합하고 비등가열 후 쿠마시 염색과 SDS-PAGE로 분석했다. 미반응성 대조 단백질, SpyCatcher002 EQ 및 SpyTag002 DA-MBP도 나타냈다; 및 (B) pH 7.0의 숙시네이트-포스페이트-글리신 완충액에서 0.1 μM의 SpyCatcher002-sfGFP와 SpyTag002-MBP의 반응 또는 SpyCatcher-sfGFP와 SpyTag-MBP의 반응에 대한 시간 경과 그래프(3반복의 평균 ± 1 s.d.; 일부 오차 막대는 너무 작아 보이지 않음).
도 10은 pH 7.0의 숙신산-포스페이트-글리신 완충액에서 (A) 1 μM 및 (B) 10 μM인 SpyCatcher002-sfGFP와 SpyTag002-MBP의 반응 또는 SpyCatcher-sfGFP와 SpyTag-MBP의 반응에 대한 시간 경과 그래프를 보여준다(3반복의 평균 ± 1 s.d.; 일부 오차 막대는 너무 작아서 볼 수 없음)(B).
도 11은 3반복 측정(각 데이터 점이 표시됨)으로부터 SpyTag002-MBP와 반응하는 SpyCatcher002의 속도 상수를 정량화한 그래프이다. 각 단백질 0.5 μM을 pH 7.0, 25 ℃에서 숙시네이트-포스페이트-글리신 완충액에 제조했다. 추세선에 대한 방정식과 상관 계수가 표시된다.
도 12는 종결하는 SpyCatcher002/SpyTag002의 반응 시험을 나타내는 SDS-PAGE 겔을 보여준다. Spy-Catcher002는 SDS-PAGE 및 쿠마시 염색에 의한 분석 전에 25℃에서 1 시간 동안 숙시네이트-포스페이트-글리신 완충액 pH 7.0에서 SpyTag002-MBP와 함께 배양하였다. 단백질은 10 μM(+) 또는 20 μM(+++)이었다.
도 13은 (A) SDS-PAGE 및 쿠마시 염색에 의해 분석된, 숙시네이트-포스페이트-글리신 완충액에서 25℃에서 1분 또는 5분 동안 SpyCatcher002와 SpyTag002-MBP 반응의 pH 의존성을 도시한 그래프이다; (B) PBS pH 7.5에서(A)에서와 같은 반응의 온도-의존성을 나타내는 막대 차트이다; (C) PBS, PBS + 1 mM EDTA, 50 mM HEPES, 50 mM HEPES-완충 식염수(HBS) 또는 Tris 완충 식염수(TBS)를 사용한 25 ℃ 및 pH 7.5에서 (A)에서와 같은 반응의 완충액 의존성을 보여주는 막대 차트이다; (D) 계면활성제(PBS) 없이, 1% Triton X-100과 PBS, 또는 1% Tween-20과 PBS 하에 25℃, PBS pH 7.5에서 (A)에서와 같은 반응의 계면활성제 의존성을 보여주는 막대 차트이다; 및 (E) 25℃, pH 7.5 PBS에서 30분 또는 120분 동안의 SpyCatcher002와 SpyTag002-MBP 반응의 우레아 의존성을 보여주는 그래프이다. 모든 그래프는 3반복의 평균 ± 1 s.d.를 나타낸다; 일부 오차 막대는 너무 작아 볼 수 없다.
도 14는 (A) PBS pH 7.5, 25℃에서 각 단백질을 0.5 μM로 사용한 SpyTag002-MBP와 반응하는 MBPx-SpyCatcher 및 MBPx-SpyCatcher002의 시간 경과를 비등가열 후 SDS-PAGE 및 쿠마시 염색으로 분석하여 나타낸 그래프를 보여주며, 이는 단백질이 N-말단에 융합되었을 때 SpyCatcher보다 SpyCatcher002의 개선된 반응성이 유지되었음을 입증한다; (B) 각 단백질 2 μM에서 25℃, PBS pH 7.5 하에 1분 또는 5분 동안 SpyCatcher 또는 SpyCatcher002와 항온처리되고 SDS-PAGE 및 쿠마시 염색으로 분석된 AffiEGFR-SpyTag002의 반응성에 대한 막대 차트이다. 데이터는 3반복으로 수행된 반응의 평균 ± 1 s.d.를 나타낸다; 일부 오차 막대는 너무 작아 볼 수 없다. 이는 SpyTag002가 C-말단에 있을 때 SpyCatcher에 비해 SpyCatcher002의 향상된 반응성이 유지되었음을 보여준다.
도 15는 (A) 0.5 μM SpyTag002-MBP(서열번호 3-MBP) 또는 SpyTag002 T3H-MBP(서열번호 4-MBP); 및 (B) 0.5 μM SpyTag002-T3H-MBP(서열번호 4-MBP) 또는 SpyTag002 RG T3H-MBP(서열번호 5-MBP)와 반응하는 0.5 μM D5T SpyCatcher002(서열번호 40)에 대한 시간 경과를 나타내는 그래프를 보여준다. 반응은 25℃에서 인산염 완충 식염수(PBS) pH 7.5에서 수행하고 SDS-PAGE 및 쿠마시 염색으로 분석했고, 데이터는 3반복으로 수행된 반응의 평균 ± 1 s.d.로 나타냈다. 추세선에 대한 방정식 및 상관 계수가 제시된다. 추세선의 기울기로부터 반응의 2 차 속도 상수가 수득되며 단위는 μM-1 min-1이다.
도 16은 인산염 완충 식염수(PBS) pH 7.5, 25℃에서 0.5 μM AP-SpyTag002-MBP(서열번호 3-MBP)와 반응하는 0.5 μM D5A SpyCatcher002 변이체(서열번호 44-47)의 속도 분석을 나타내는 그래프를 보여준다. 모든 반응은 SDS-PAGE 및 쿠마시 염색으로 분석했으며, 데이터는 3반복으로 수행된 반응의 평균 ± 1 s.d.를 나타낸다. 추세선에 대한 방정식 및 상관 계수가 표시된다. 추세선의 기울기로부터 반응의 2 차 속도 상수가 수득되며 단위는 μM-1 min-1이다.
Claims (21)
- 펩타이드 및 폴리펩타이드를 포함하는 2-파트 링커로서,
a) 상기 펩타이드는 서열번호 1에 제시된 아미노산 서열을 포함하되,
(i) 위치 1의 X가 아르기닌이거나 아미노산이 없고;
(ii) 위치 2의 X가 글리신이거나 아미노산이 없고;
(iii) 위치 5의 X가 히스티딘 또는 트레오닌, 바람직하게는 히스티딘이고;
(iv) 위치 11의 X가 알라닌, 글리신 또는 발린, 바람직하게는 알라닌이고; 그리고
(v) 위치 14의 X가 아르기닌 또는 리신, 바람직하게는 아르기닌이고;
상기 위치 1의 X에 아미노산이 없을 때, 위치 2의 X에도 아미노산이 없고; 그리고,
b) 상기 폴리펩타이드는
i) 서열번호 2에 제시된 아미노산 서열;
ii) 서열번호 101에 제시된 아미노산 서열을 포함하는 (i)의 부분;
iii) 서열번호 2에 제시된 서열과 적어도 80% 서열 동일성을 갖고, 위치 34에 리신, 위치 80에 글루탐산 및 다음 중 하나 이상, 즉
1) 위치 5에 트레오닌;
2) 위치 16에 프롤린;
3) 위치 40에 아르기닌;
4) 위치 65에 히스티딘;
5) 위치 92에 프롤린;
6) 위치 100에 아스파르트산:
7) 위치 108에 글루탐산; 및
8) 위치 116에 트레오닌 중 하나 이상을 포함하는 아미노산 서열로서, 상기 명시된 아미노산 잔기들이 서열번호 2의 위치들과 동등한 위치들에 있는 아미노산 서열; 또는
iv) 서열번호 101에 제시된 서열과 적어도 80% 서열 동일성을 갖고, 위치 10에 리신, 위치 56에 글루탐산 및 다음 중 하나 이상, 즉
1) 위치 16에 아르기닌;
2) 위치 41에 히스티딘;
3) 위치 68에 프롤린; 및
4) 위치 76에 아스파르트산 중 하나 이상을 포함하는 아미노산 서열로서, 상기 명시된 아미노산 잔기들이 서열번호 101의 위치들과 동등한 위치들에 있는 아미노산 서열을 포함하는 (iii)의 부분을 포함하고,
상기 펩타이드 및 폴리펩타이드는 서열번호 1의 위치 10에 있는 아스파르트산 잔기와 서열번호 2의 위치 34 또는 서열번호 101의 위치 10에 있는 리신 잔기 사이에 이소펩타이드 결합을 자발적으로 형성할 수 있는, 2-파트 링커. - 제1항에 있어서, 상기 펩타이드가
1) 위치 5에 히스티딘;
2) 위치 11에 알라닌; 및
3) 위치 14에 아르기닌 중 하나 이상을 포함하고,
상기 명시된 아미노산 잔기들이 서열번호 2의 위치들과 동등한 위치들에 있는, 2-파트 링커. - 제1항 또는 제2항에 있어서, 상기 펩타이드가 서열번호 3 내지 5 중 어느 하나, 바람직하게는 서열번호 5에 제시된 아미노산 서열을 포함하는, 2-파트 링커.
- 제1항 내지 제3항 중 어느 한 항에 있어서, 상기 폴리펩타이드가 서열번호 2에 제시된 서열과 적어도 80% 서열 동일성을 가진 아미노산 서열을 포함하고, 상기 아미노산 서열은 위치 34에 리신, 위치 80에 글루탐산 및
1) 위치 5에 트레오닌;
2) 위치 16에 프롤린;
3) 위치 40에 아르기닌;
4) 위치 65에 히스티딘;
5) 위치 108에 글루탐산; 및
6) 위치 116에 트레오닌을 모두 포함하고,
상기 명시된 아미노산 잔기들이 서열번호 2의 위치들과 동등한 위치들에 있는, 2-파트 링커. - 제1항 내지 제3항 중 어느 한 항에 있어서, 상기 폴리펩타이드가 서열번호 2에 제시된 서열과 적어도 80% 서열 동일성을 가진 아미노산 서열을 포함하고, 상기 아미노산 서열은 위치 34에 리신, 위치 80에 글루탐산 및
1) 위치 5에 트레오닌;
2) 위치 16에 프롤린;
3) 위치 40에 아르기닌;
4) 위치 65에 히스티딘;
5) 위치 92에 프롤린;
6) 위치 108에 글루탐산; 및
7) 위치 116에 트레오닌을 모두 포함하고,
상기 명시된 아미노산 잔기들이 서열번호 2의 위치들과 동등한 위치들에 있는, 2-파트 링커. - 제1항 내지 제3항 중 어느 한 항에 있어서, 상기 폴리펩타이드가 서열번호 2에 제시된 서열과 적어도 80% 서열 동일성을 가진 아미노산 서열을 포함하고, 상기 아미노산 서열은 위치 34에 리신, 위치 80에 글루탐산 및
1) 위치 5에 트레오닌;
2) 위치 16에 프롤린;
3) 위치 40에 아르기닌;
4) 위치 65에 히스티딘;
5) 위치 92에 프롤린;
6) 위치 100에 아스파르트산;
7) 위치 108에 글루탐산; 및
8) 위치 116에 트레오닌을 모두 포함하고,
상기 명시된 아미노산 잔기들이 서열번호 2의 위치들과 동등한 위치들에 있는, 2-파트 링커. - 제1항 내지 제6항 중 어느 한 항에 있어서, 상기 폴리펩타이드가
1) 위치 12에 글리신; 및
2) 위치 22에 트레오닌 중 하나 이상을 포함하고,
상기 명시된 아미노산 잔기들이 서열번호 2의 위치들과 동등한 위치들에 있는, 2-파트 링커. - 제1항 내지 제7항 중 어느 한 항에 있어서, 상기 펩타이드 및/또는 상기 폴리펩타이드가 핵산 분자, 단백질, 펩타이드, 소분자 유기 화합물, 형광단(fluorophore), 금속-리간드 착물, 다당류, 나노입자, 나노튜브, 중합체, 세포, 바이러스, 바이러스 유사 입자 또는 이들의 조합에 접합되는, 2-파트 링커.
- 제1항 내지 제7항 중 어느 한 항에 있어서, 상기 펩타이드 및/또는 폴리펩타이드가 고체 기질 위에 고정되어 있는, 2-파트 링커.
- 서열번호 1에 제시된 아미노산 서열을 포함하는 펩타이드로서,
(i) 위치 1의 X는 아르기닌이거나 아미노산이 없고;
(ii) 위치 2의 X는 글리신이거나 아미노산이 없고;
(iii) 위치 5의 X는 히스티딘 또는 트레오닌, 바람직하게는 히스티딘이고;
(iv) 위치 11의 X는 알라닌, 글리신 또는 발린, 바람직하게는 알라닌이고; 그리고
(v) 위치 14의 X는 아르기닌 또는 리신, 바람직하게는 아르기닌이고,
위치 1의 X에 아미노산이 없을 때, 위치 2의 X에도 아미노산이 없고;
상기 펩타이드는 서열번호 2에 제시된 아미노산 서열을 포함하는 폴리펩타이드와 이소펩타이드 결합을 자발적으로 형성할 수 있고, 상기 이소펩타이드 결합은 서열번호 1의 위치 10에 있는 아스파르트산 잔기와 서열번호 2의 위치 34에 있는 리신 잔기 사이에 형성되는, 펩타이드. - 제10항에 있어서, 상기 펩타이드가 제2항, 제3항, 제8항 또는 제9항 중 어느 한 항에 정의된 바와 같은, 펩타이드.
- 폴리펩타이드로서,
i) 서열번호 2에 제시된 아미노산 서열; 또는
ii) 서열번호 101에 제시된 아미노산 서열을 포함하는 (i)의 부분;
iii) 서열번호 2에 제시된 서열과 적어도 80% 서열 동일성을 갖고 위치 34에 리신, 위치 80에 글루탐산 및 다음 중 하나 이상, 즉
1) 위치 5에 트레오닌;
2) 위치 16에 프롤린;
3) 위치 40에 아르기닌;
4) 위치 65에 히스티딘;
5) 위치 92에 프롤린;
6) 위치 100에 아스파르트산:
7) 위치 108에 글루탐산; 및
8) 위치 116에 트레오닌 중 하나 이상을 포함하는 아미노산 서열로서, 상기 명시된 아미노산 잔기들이 서열번호 2의 위치들과 동등한 위치들에 있는 아미노산 서열; 또는
iv) 서열번호 101에 제시된 서열과 적어도 80% 서열 동일성을 갖고 위치 10에 리신, 위치 56에 글루탐산 및 다음 중 하나 이상, 즉
1) 위치 16에 아르기닌;
2) 위치 41에 히스티딘;
3) 위치 68에 프롤린; 및
4) 위치 76에 아스파르트산 중 하나 이상을 포함하는 아미노산 서열로서, 상기 명시된 아미노산 잔기들이 서열번호 101의 위치들과 동등한 위치들에 있는 아미노산 서열을 포함하는 (iii)의 부분을 포함하고,
상기 폴리펩타이드는 서열번호 5에 제시된 아미노산 서열을 포함하는 펩타이드와 이소펩타이드 결합을 자발적으로 형성할 수 있고, 상기 이소펩타이드 결합은 서열번호 5의 위치 10에 있는 아스파르트산 잔기와 서열번호 2의 위치 34 또는 서열번호 101의 위치 10에 있는 리신 잔기 사이에 형성되는, 폴리펩타이드. - 제12항에 있어서, 상기 폴리펩타이드가 제4항 내지 제9항 중 어느 한 항에 정의된 바와 같은, 폴리펩타이드.
- 폴리펩타이드 및 제10항 또는 제11항에 정의된 바와 같은 펩타이드 및/또는 제12항 또는 제13항에 정의된 바와 같은 폴리펩타이드를 포함하는, 재조합 또는 합성 폴리펩타이드.
- 제10항 또는 제11항에 정의된 바와 같은 펩타이드, 제12항 또는 제13항에 정의된 바와 같은 폴리펩타이드 또는 제14항의 재조합 또는 합성 폴리펩타이드를 암호화하는 뉴클레오타이드 서열을 포함하는, 핵산 분자.
- 제15항의 핵산 분자를 포함하는, 벡터.
- 제15항의 핵산 또는 제16항의 벡터를 포함하는, 세포.
- 제10항 내지 제13항 중 어느 한 항의 펩타이드 및/또는 폴리펩타이드를 생산하거나 발현시키는 방법으로서,
a) 제15항에 정의된 바와 같은 펩타이드 및/또는 폴리펩타이드를 암호화하는 뉴클레오타이드 서열을 포함하는 벡터로 숙주 세포를 형질전환 또는 형질감염시키는 단계;
b) 상기 펩타이드 및/또는 폴리펩타이드를 발현시키는 조건하에 숙주 세포를 배양하는 단계; 및 선택적으로
c) 상기 펩타이드 및/또는 폴리펩타이드를 분리하는 단계를 포함하는, 방법. - 두 분자 또는 성분을 이소펩타이드 결합을 통해 접합시키기 위한 제1항 내지 제9항 중 어느 한 항에 정의된 바와 같은 2-파트 링커 펩타이드의 용도로서,
이소펩타이드 결합을 통해 접합된 상기 분자 또는 성분은
a) 제10항 또는 제11항의 펩타이드를 포함하는 제1 분자 또는 성분; 및
b) 제12항 또는 제13항의 폴리펩타이드를 포함하는 제2 분자 또는 성분을 포함하는, 용도. - 두 분자 또는 성분을 이소펩타이드 결합을 통해 접합시키는 방법으로서,
a) 제10항 또는 제11항의 펩타이드를 포함하는 제1 분자 또는 성분을 제공하는 단계;
b) 제12항 또는 제13항의 폴리펩타이드를 포함하는 제2 분자 또는 성분을 제공하는 단계;
c) 상기 제1 및 제2 분자 또는 성분을, 상기 펩타이드와 폴리펩타이드 사이에 이소펩타이드 결합을 자발적으로 형성할 수 있게 하는 조건 하에 접촉시켜 상기 제1 분자 또는 성분을 상기 제2 분자 또는 성분에 이소펩타이드 결합을 통해 접합시켜 복합체를 형성하는 단계를 포함하는, 방법. - 제19항의 용도 또는 제20항의 방법에 사용하는 것이 바람직한 키트로서,
상기 키트가
(a) 분자 또는 성분에 선택적으로 접합 또는 융합된, 제10항 또는 제11항의 펩타이드;
(b) 분자 또는 성분에 선택적으로 접합 또는 융합된, 제12항 또는 제13항의 폴리펩타이드; 및/또는
(c) 상기(a)에 정의된 바와 같은 펩타이드를 암호화하는 핵산 분자, 특히 벡터; 및
(d) 상기(b)에 정의된 바와 같은 폴리펩타이드를 암호화하는 핵산 분자, 특히 벡터를 포함하는, 키트.
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WO2016193746A1 (en) * | 2015-06-05 | 2016-12-08 | Oxford University Innovation Limited | Methods and products for fusion protein synthesis |
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DK3615556T3 (da) | 2021-08-30 |
EP3615556B1 (en) | 2021-06-09 |
WO2018197854A1 (en) | 2018-11-01 |
US11059867B2 (en) | 2021-07-13 |
CN110709412B (zh) | 2024-05-24 |
CN110709412A (zh) | 2020-01-17 |
AU2018258000B2 (en) | 2022-03-31 |
US20220119459A1 (en) | 2022-04-21 |
EP3615556A1 (en) | 2020-03-04 |
US11873323B2 (en) | 2024-01-16 |
ES2887004T3 (es) | 2021-12-21 |
KR102642896B1 (ko) | 2024-02-29 |
AU2018258000A1 (en) | 2019-11-07 |
GB201706430D0 (en) | 2017-06-07 |
CN118580325A (zh) | 2024-09-03 |
US20200131233A1 (en) | 2020-04-30 |
CA3060025A1 (en) | 2018-11-01 |
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