KR950704501A - 포유동물 발현용 완전히 손상된 공통 코작 서열(Fully Impaired Consensus Kozac Sequences for Mammalian Expression) - Google Patents

포유동물 발현용 완전히 손상된 공통 코작 서열(Fully Impaired Consensus Kozac Sequences for Mammalian Expression)

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KR950704501A
KR950704501A KR1019950701929A KR19950701929A KR950704501A KR 950704501 A KR950704501 A KR 950704501A KR 1019950701929 A KR1019950701929 A KR 1019950701929A KR 19950701929 A KR19950701929 A KR 19950701929A KR 950704501 A KR950704501 A KR 950704501A
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미첼 이. 레프
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케네쓰 제이. 울코트
아이덱 파마슈티칼즈 코포레이션
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Abstract

본 명세서에서는 가장 대표적으로는, 발현 벡터의 일부인 전사 카셋트의 우성 선변 마커와 함께 사용되는 완전히 손상된 공통 코작 서열을 개시한다, 이들 벡터들은 가장 대표적으로는 포유동물 발현 시스템 중에서 단백질의 발현에 이용된다. 본 명세서에서 정의되고 개시되고 청구된 "완전히 손상된 공통 코작"은 상기 서열(I)을 포함한다. 서열(I)에서, "X"는 아데닌(A), 구아닌(G), 사이토신(C) 또는 티민(T)/우라실(U)로 이루어진 군 중에서 선택된 뉴클레오티드이고,"Py"는 피리미딘 뉴클레오티드, 즉 C 또는 T/U이고, "ATG"는 아미노산 메티오닌을 코팅하는 코돈, 소위 "개시"코돈이고, -3 및 +1은 ATG를 기준으로 한 방향 기준 점 즉, -3은 ATG로부터 3개의 뉴클레오티드 상류를 나타내는 것이고, +1은 ATG로부터 1개의 뉴클레오티드 하류를 나타내는 것이다. 우성 선별 마커는 추가로 개시된 인공 인트론 영역을 추가로 포함한다.

Description

포유동물 발현용 완전히 손상된 공통 코작 서열(Fully Impaired Consensus Kozac Sequences for Mammalian Expression)
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1도는 공통 코작 및 몇개의 특히 바람직한 완전히 손상된 공통 코작 서열의 관련부를 나타내고,
제2도는 생쥐/인체 키메릭 면역글로불린을 발현하도록 디자인된 벡터 TCAE 5.2 및 ANEX 1 (TCAE 12)을 나타내는 도면으로, 여기서 면역글로불린 유전자들은 우성 선별 마커로서 네오마이신 포스포트랜스퍼라제를 사용하여 세로로 나란히 배치되고,
제3도는 벡터 TCAE 5.2 및 ANEX 1을 이용한 단백질 발현 정도를 비교하는 히스토그램이다.

Claims (30)

1개 이상의 우성 선별 마커를 포함하고, 이 마커의 번역 개시 시작부가 하기 서열을 포함하는, 재조합 데옥시리보핵산 기술에 의해 목적하는 단백질을 발현시키기 위한 발현 벡터.
상기에서 "Py"는 피리미딘 뉴클레오티드이고, "X"는 뉴클레오티드이고, 수치는 코돈 "ATC"에 대한 상대값이다.
제1항에 있어서, 상기 목적하는 단백질을 코딩하는 핵산 서열이 상기 우성 선별 마커와 상호연결된 발현 벡터.
제1항에 있어서, 상기 우성 선별 마커가 헤르페스 단순 바이러스 티미딘 키나제, 아데노신 데아미나제, 아스파라긴 합성효소, 살모넬라 히스 D 유전자, 크산틴구아닌 포스포리보실 트랜스퍼라제, 하이그로마이신 B 포스포트랜스퍼라제 빛 네오마이신 포스포트랜스퍼라제로 이루어진 군 중에서 선택된 발현 벡터.
제1항에 있어서, 상기 번역 개시 시작부 서열이 TxxATGCxx, CxxATGCxx, CxxATGTxx 및 TxxATGTxx(여기서, "x"는 뉴클레오티드이지만 단, ATG 코돈의 하류의 코돈 "Txx"는 중지 코돈을 코딩하지 않아야 함)로 이루어진 군으로부터 선택된 발현 벡터.
제1항에 있어서, 상기 번역 개시 시작부 서열이 TxxATGCxx(여기서, "x"는 뉴클레오티드임)인 발현 벡터.
제1항에 있어서, 상기 번역 개시 시작부 서열이 TCCATGCTT인 발현 벡터.
제1항에 있어서, 상기 번역 개시 시작부 서열이 2차 구조 내에 위치하는 발현 벡터.
제1항에 있어서, 상기 번역 개시 시작부 서열이 상기 시작부의 ATG개시 코돈으로부터 약 1000개의 뉴클레오티드 이내에 1개 이상의 프레임을 벗어난 개시 코돈을 추가로 포함하지만 상기 1000개의 뉴클레오티드 내에 프레임에 맞는 중지 코돈은 위치하지 않는 발현 벡터.
제1항에 있어서, 상기 번역 개시 시작부 서열이 상기 시작부의 ATG 개시 코돈으로부터 약 350개의 뉴클레오티드 이내에 1개 이상의 프레임을 벗어난 개시 코돈을 추가로 포함하지만 상기 350개의 뉴클레오티드 내에 프레임에 맞는 중지 코돈은 위치하지 않는 발현 벡터.
제1항에 있어서, 상기 번역 개시 시작부 서열이 상기 시작부의 ATG 개시 코돈으로부터 약 50개의 뉴클레오티드 이내에 1개 이상의 프레임을 벗어난 개시 코돈을 추가로 포함하지만 상기 50개의 뉴클레오티드 내에 프레임에 맞는 중지 코돈은 위치하지 않는 발현 벡터.
제8, 9 또는 10항에 있어서, 상기 프레임을 벗어난 개시 코돈이 공통 코작 서열의 일부인 발현 벡터.
제10항에 있어서, 상기 프레임을 벗어난 개시 코돈 및 상기 번역 개시 시작부 서열이 모두 2차 구조의 일부로서 포함되는 발현 벡터.
제8, 9 또는 10항에 있어서, 상기 번역 개시 시작부 서열이 2차 구조의 일부이고, 상기 프레임을 벗어난 개시 코돈이 상기 2차 구조의 일부가 아닌 발현 벡터.
번역 개시 시작부가 TxxATGCxx, CxxATGCxx, CxxATGTxx 및 TxxATGTxx(여기서, "x"는 뉴클레오티드이지만 단, ATG 코돈 하류의 코돈"Txx"는 중지 코돈을 코딩하지 않아야 함)로 이루어진 군으로부터 선택된, 핵산 서열에 의해 코딩되는 우성 선별 마커.
제14항에 있어서, 헤르페스 단순 바이러스 티미딘 키나제, 아데노신 데아미나제, 아스파라긴 합성효소, 살모넬라 히스 D 유전자, 크산틴구아닌 포스포리보실 트랜스퍼라제, 하이드로마이신 B 포스포트랜스퍼라제 및 네오마이신 포스포트랜스퍼라제로 이루어진 군 중에서 선택된 우성 선별 마커.
제14항에 있어서, 상기 번역 개시 시작부 서열이 TxxATGCxx(여기서, "x"는 뉴클레오티드임)인 우성 선별 마커.
제14항에 있어서, 상기 번역 개시 시작부 서열이 TCCATGCTT인 우성 선별 마커.
제14항에 있어서, 상기 번역 개시 시작부 서열이 2차 구조 내에 위치하는 우성 선별 마커.
제14항에 있어서, 상기 번역 개시 시작부 서열이 상기 시작부의 ATG개시 코돈으로부터 약 1000개의 뉴클레오티드 이내에 1개 이상의 프레임을 벗어난 개시 코돈을 추가로 포함하지만 상기 1000개의 뉴클레오티드 내에 프레임에 맞는 중지 코돈은 위치하지 않는 우성 선별 마커.
제14항에 있어서, 상기 번역 개시 시작부 서열이 상기 시작부의 ATG 개시, 코돈으로부터 약 350개의 뉴클레오티드 이내에 1개 이상의 프레임을 벗어난 개시 코돈을 추가로 포함하지만 상기 350개의 뉴클레오티드 내에 프레임에 맞는 중지 코돈은 위치하지 않는 우성 선별 마커.
제14항에 있어서, 상기 번역 개시 시작부 서열이 상기 시작부의 ATG 개시 코돈으로부터 약 50개의 뉴클레오티드 이내애 1개 이상의 프레임을 벗어난 개시 코돈을 추가로 포함하지만 상기 50개의 뉴클레오티드 내에 프레임에 맞는 중지 코돈은 위치하지 않는 우성 선별 마커.
제19,20 또는 21항에 있어서, 상기 프레임을 벗어난 개시 코돈이 공통 코작 서열의 일부인 우성 선별 마커.
제21항에 있어서, 상기 프레임을 벗어난 개시 코돈 및 상기 번역 개시 시작부 서열이 모두 2차 구조의 일부로서 포함되는 우성 선별 마커.
제19, 20 또는 21항에 있어서, 상기 번역 개시 시작부 서열이 2차 구조의 일부이고, 상기 프레임을 벗어난 개시 코돈이 상기 2차 구조의 일부가 아닌 우성 선별 마커.
ANEX 1(아메리칸 타입 컬쳐 콜렉션 기탁 번호 69120 내에 포함됨) 및 ANEX 2(아메리칸 타입 컬쳐 콜렉션 기탁 번호 69118 내에 포함됨)로 이루어진 군으로부터 선택된 발현 벡터.
목적하는 단백질을 코딩하는 핵산 서열이 상기 우성 선별 마커와 상호연결된 제1항 기재의 발현 벡터를 포함하는 플라스미드.
제26항에 있어서, 포유동물 숙주 세포의 세포 데옥시리브핵산 내에 통합된 플라스미드.
제27항에 있어서, 상기 숙주 세포가 DG44, DXB11, CV1, COS, R1610, SP2/0, P3x633-Ag8.653, BFA-1c1BPT< RAJI 및 293으로 이루어진 군으로부터 선택된 플라스미드.
제1항에 있어서, 상기 우성 선별 마커 내에 인공 인트론 삽입 영역을 추가로 포함하고, 목적하는 단백질을 코딩하는 서열이 상기 삽입 영역 내에 위치하는 발현 벡터.
제14항에 있어서, 인공 인트론 삽입 영역을 추가로 포함하는 우성 선별 마커.
※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019950701929A 1992-11-13 1993-11-12 포유동물발현용완전히손상된공통코작서열 KR100369418B1 (ko)

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