KR920703839A - 신규의 유전자 및 폴리펩티드의 무세포 합성 및 분리 - Google Patents

신규의 유전자 및 폴리펩티드의 무세포 합성 및 분리

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KR920703839A
KR920703839A KR1019920700769A KR920700769A KR920703839A KR 920703839 A KR920703839 A KR 920703839A KR 1019920700769 A KR1019920700769 A KR 1019920700769A KR 920700769 A KR920700769 A KR 920700769A KR 920703839 A KR920703839 A KR 920703839A
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가와사끼 글렌
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Abstract

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Description

신규의 유전자 및 폴리펩티드의 무세포 합성 및 분리
[도면의 간단한 설명]
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음

Claims (47)

  1. (a) RNA 폴리머라제 결합 시퀀스, 리보솜 결합 시퀀스, 및 번역 개시 시그널을 함유한 5'미번역 영역으로 이루어진, mRNA를 생성할 수 있는 시험관내 발현 유니트를 조립하고; (b) 한가지 또는 그이상의 세미-랜덤 뉴클레오티드 시퀀스를 그 발현 유니트에 부착하고; (c) 발현 유니트 및 세미-랜덤 뉴클레오티드와 관련된 시퀀스를 전사하거나 복제하여 RNA를 생성하며; (d) 그 RNA를 번역하여 폴리솜을 유지하는데 충분한 조건하에 폴리솜을 생성하며; (e) 그 폴리솜을 관심 물질에 결합하고; (f) 그 관심 물질에 결합하는 그 폴리솜을 분리하며; (g) 그 분리된 폴리솜을 분쇄하여 mRNA를 유리하며; (h) 그 mRNA를 회수하고; (i) 회수된 mRNA로 부터 cDNA를 조립하고; j) 그 cDNA를 발현시켜 신규의 폴리펩티드을 생성하는 것으로 이루어진 신규 폴리펩티드의 재조방법.
  2. 제1항에 있어서, mRNA를 회수하고 cDNA를 조립하는 단계에 이어서 cDNA를 폴리머라제 사슬 반응에 의하여 확대시키는 것이 특징인 방법.
  3. 제1항에 있어서, 상기 세이-랜덤 뉴클레오티드 시퀀스가 데옥시리보핵산으로 이루어진 것이 특징인 방법.
  4. 제1항에 있어서, 상기 세미-랜덤 뉴클레오디드 시퀀스카 리보핵산으로 이루어진 것이 특징인 방법.
  5. 제1항에 있어서, 상기 발현 유니트가 적어도 한개의 RNA-지시 RNA 폴리머라제 인식 시퀀스를 함유하는 것이 특징인 방법.
  6. 제5항에 있어서, 상기 RNA-지시 RNA 폴리머라제가 Q-베타 레플리카제인 것이 특징인 방법.
  7. 제1항에 있어서, 회수 단계에 이어서 mRNA를 확대시키는 것이 특징인 방법.
  8. 제7항에 있어서, 확대 단계가 복제 시퀀스를 RNA-의존 RNA 폴리머리제로서 합성시키는 것으로 이루어진 것이 특징인 방법.
  9. 제8항에 있어서, RNA-의존 RNA 폴리머라제가 Q-베타 레플리카제인 것이 특징인 방법.
  10. 제1항에 있어서, 분리 단계가 연속 회석 또는 병류 세척 단계에 의하여 관심 물질에 결합하지 않는 폴리솜을 제거하는 것으로 이루어진 것이 특징인 방법.
  11. 제1항에 있어서, 상기 폴리솜을 분리하는 단계에 이어서, 폴리솜이 관심 물질로부터 유리되도록 선택된 엄격한 조건에 상기 폴리솜을 노출시키는 것이 특징인 방법.
  12. 제11항에 있어서, 상기 폴리솜을 노출시키는 단계가 온도를 상승시키고, 염 농도를 저하시키고, 또는 그 폴리솜의 금속이온 농도는 상승시키는 것으로 이루어진 것이 특징인 방법.
  13. (a) RNA 폴리머라제 결합 시퀀스, 리보솜 결합 시퀀스, 및 번역 개시 시그널을 함유한 5'미번역 영역으로 이루어진, mRNA를 생성한 수 있는 시험관내 발현 유니트를 조립하고; (b) 한가지 모는 그이상의 세미-랜덤 뉴클레오티드 시퀀스를 그 발현 유니트에 부착하고; (c) 발현 유니트와 세미-랜덤 뉴클레오티드와 관련된 시퀀스를 전사시켜 RNA를 생성하며; (d) 그 RNA를 번역하여 생물학적 활성 폴리펩티드를 생성하며; (e) 그 생물학적 활성 폴리펩티드를 코드화하는 RNA를 세분하고; (f) 관심 유전자가 분리되도록 단계(c)-(e)에 제시된 바와같이 전사하고, 번역하며, 세분하며; (g) 그 분리된 유전자로 부터 cDNA를 조립한 다음; (h) 그 cDNA를 발현시켜 신규 폴리펩티드를 생성하는 것으로 이루어진 신규 폴리펩티드의 제조방법.
  14. (a) RNA 폴리머라제 결합 시퀀스, 리보솜 결합 시퀀스, 및 번역 개시 시그널을 함유한 5'미번역 영역으로 이루어진, mRNA을 생성할 수 있는 시힘관내 발현 유니트를 조립하고; (b) 한가지 또는 그이상의 세미-랜덤 뉴클레오티드 시퀀스를 발현 유니트에 부착하며; (c) 발현 유니트 및 세미-랜덤 뉴클레오티드 시퀀스와 관련된 시퀀스를 복제하여 RNA를 생성하며; (d) 그 RNA를 번역하여 생물학적 활성 폴리펩티드를 생성하고; (e) 그 생물학적 활성 폴리펩티드를 코드화하는 RNA를 세분하고, (f) 관심 유전자가 분리되도록 단계 (d)-(e)에 제시된 바와같이 번역 및 세분하며; (g) 그 분리된 유전자로 부터 cDNA를 조립한 다음; (h) 그 cDNA를 발현시켜 신규의 폴리펩티드를 생성하는 것으로 이루어진 신규 폴리펩티드의 제조방법.
  15. 제14항에 있어서, RNA를 세분하는 단계에 이어서, 생물학적 활성 폴리펩티드와 관련한 신규의 유전자 시퀀스를 폴리머라제 사슬 반응물 또는 RNA-지시 RNA 폴리머라제로서 확대하는 것이 특징인 방법.
  16. 제1, 13 또는 14항의 방법에 의하여 생성된 폴리펩티드.
  17. 제1, 13 또는 14항에 있어서, 상기 리보솜 결합 부위가 성숙핵, 원시핵, 또는 비루스 리보솜 결합 시퀀스로 이루어진 것이 특징인 방법.
  18. 제1, 13 또는 14항에 있어서, 상기 리보솜 결합 시퀀스가 척추동물의 교감 번역 개시 시퀀스, GCCGCCACCATGG, 또는 작용 관련 시퀀스로 이루어진 것이 특징인 방법.
  19. 제1, 13 또는 14항에 있어서, 발현 유니트가 추가로 선택된 아미노-말단 ID 펩티드를 코드화하는, 개시 코돈에 위치한 시퀀스로 이루어진 것이 특징인 방법.
  20. 제1, 13 또는 14항에 있어서, 상기 발현 유니트가 추가로 신규 유전자의 확대, 클로닝, 복제, 정제, 및 분리를 증가시키는 시퀀스중에서 선택된 선택 시퀀스의 3'영역으로 이루어진 것이 특징인 방법.
  21. 제20항에 있어서, 상기 3' 영역이 리보솜 전위를 방해하는데 적합한 회문형 시퀀스를 포함한 것이 특징인 방법.
  22. 제20항에 있어서, 상기 3' 영역이 C-말단 ID 시퀀스를 포힘한 것이 특징인 방법.
  23. 제22항에 있어서, 상기 C-말단 ID 시퀀스가 반복 시퀀스로 이루어진 것이 특징인 방법.
  24. 제22항에 있어서, 상기 C-말단 ID 시퀀스가 항체에 결합할 수 있는 펩티드를 코드화하는 것이 특징인 방법.
  25. 제1, 13 또는 14항에 있어서, 상기 발현 유니트가 추가로 생체내에서 신규 유전자를 발현시키는데 적합한 제한 부위로 이루어진 것이 특징인 방법.
  26. 제25항에 있어서, 상기 제한 부위의 적어도 한가지가 번역 출발점에 위치한 시퀀스 CCATGG로 이루어진 것이 특징인 방법.
  27. 제1, 13 또는 14항에 있어서, 상기 발현 유니트가 T7, T3, 또는 SP6폴리머라제를 위한 촉진인자 시퀀스를 포함한 것이 특징인 방법.
  28. 제1, 13 또는 14항에 있어서, 자연 발생 DNA 또는 cDNA를 기계적, 화학적, 또는 효소적으로 단편화함으로서 세미-랜덤 뉴클레오티드 시퀀스를 발생하는 것이 특징인 방법.
  29. 제1, 13 또는 14항에 있어서, 뉴클레오티드를 화학적으로 합성하므로서 세미-랜덤 뉴클레오티드를 발생시켜 유전자 시퀀스를 형성하는 것이 특징인 방법.
  30. 제29항에 있어서, 상기 뉴클레오티드를 화학적으로 합성하는 단계는 (1) 제1코돈 위치에서 실제 동일 몰량의 C, A 및 G 그리고 실제 동일 몰량의 T에서 단지 반만 사용하고; (2) 제2코돈 위치에서 실제 동일 몰량의 C, T, 및 G. 그리고 실제 동일 몰량의 A에서 단지 반만 사용한 다음; (3) 제3코돈 위치에서 실제 동일 몰량의 C와 G 또는 T와 G를 사용하는 단계로 이루어진 것이 특징인 방법.
  31. 제1, 13 또는 14항에 있어서, 부착 단계가 추가로 발현 유니트의 5'미번역 영역의 3'말단위에 바로 상기 뉴클레오티드를 중합시키는 것으로 이루어진 것이 특징인 방법.
  32. 제1 또는 13항에 있어서, 전사 단계가 디구아노신 트리포스페이트 또는 그의 동족체의 존재하에 상기 시퀀스를 전사하는 것으로 이루어진 것이 특징인 방법.
  33. 제1, 13 또는 14항에 있어서, 번역 단계가 디구아노신 트리포스페이트 또는 그의 동족체 및 구아닐일트란스퍼라제 존재하에 상기 시퀀스를 번역하는 것으로 이루어진 것이 특징인 방법.
  34. 제1, 13 또는 14항에 있어서, 번역 단계가 무의미-억제 tRNA 존재하에 수행되는 것이 특징인 방법.
  35. 제34항에 있어서, 무의미-억제 tRNA가 티로신-삽입, 무의미-억제 tRAN인 것이 특징인 방법.
  36. 제1, 13 또는 14항에 있어서, 상기 관심 물질이 표면 항원, 수용체 단백질, 톡신, 유기 중합체, 메타볼라이트, 단백질 분자의 활성부위, 호르몬, 항체, 및 오염물질중에서 선택되는 것이 특징인 방법.
  37. 제1, 13 또는 14항에 있어서, 상기 관심 물질이 항체의 가변/과도 가변 영역인 것이 특징인 방법.
  38. 제1, 13 또는 14항에 있어서, 상기 관심 물질이 수용체 단백질인 것이 특징인 방법.
  39. 제38항에 있어서, 상기 수용체 단백질이 성장인지 수용체 단백질인 것이 특징인 방법.
  40. 39항에 있어서, 상기 성장인자 수용체 단백질이 인슐린 및 상피 성장인자중에서 선택되는 것이 특징인 방법.
  41. 제1, 13 또는 14항에 있어서, 상기 관심 물질이 비루스 표면항원, 비루스 수용체 단백질 및 CD4 중에서 선택되는 것이 특징인 방법.
  42. 제1, 13 또는 14항에 있어서, cDNA를 발현시키는 단계가 그 cDNA의 뉴클레오티드 시퀀스를 기재로한 아미노산 시퀀스를 화학적으로 합성하는 것으로 이루어진 것이 특징인 방법.
  43. 제1, 13 또는 14항에 있어서, cDNA를 발현시키는 단계가 유전공학 미생물에서 합성을 위한 발현 벡터로 뉴클레오티드 시퀀스를 클로닝하는 것으로 이루어진 것이 특징인 방법.
  44. 제1, 13 또는 14항에 있어서, cDNA를 발현시키는 단계가 뉴클레오티드 시퀀스의 시험관내 전사 및/ 또는 번역으로 이루어진 것이 특징인 방법.
  45. 제1, 13 또는 14항에 있어서, cDNA를 발현시키는 단계가 cDNA에 의하여 코드화되는 것과 실제로 동일한 폴리폡티드를 코드화하는 뉴클레오티드 시퀀스를 합성하는 것으로 이루어지며, 뉴클레오티드 시퀀스에 의하여 코드화된 폴리폡티드는 관심물질에 결합하는 폴리솜의 결합영역과 실제 동일한 것이 특징인 방법.
  46. 제1, 13 또는 14항에 있어서, 상기 cDNA을 시퀀스 코드화 톡신, 항체, 효소, 생물활성 펩티드, 및 항체에 결합할 수 있는 펩티드중에서 선택된 다른 선택 뉴클레오티드 시퀀스에 결합하는 것이 특징인 방법.
  47. RNA 폴리머라제 결합 시퀀스, 리보솜 결합 시퀀스, 번역 개시 시그널을 함유한 5'미번역 영역으로 이루어진 시험관내 발현 유니트, 및 한가지 또는 그이상의 세미-랜덤 뉴클레오티드 시퀀스를 전사시져 mRNA 라이브러리를 생성하여; 부착된 폴리펩티드 사슬을 가진 폴리솜을 유지하는 조건하에 mRNA 라이브러리를 번역하고; 관심 물질에 폴리솜을 접촉하며 관심물질에 특이하게 결합하는 폴리솜으로 부터 mRNA를 분리하는 것으로 이루어진, 관심 폴리펩티드를 코드화하는 뉴클레오티드 시퀀스의 분리방법.
    ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019920700769A 1989-10-05 1990-10-04 신규의 유전자 및 폴리펩티드의 무세포 합성 및 분리 KR0185192B1 (ko)

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ATE168416T1 (de) 1998-08-15
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AU6537390A (en) 1991-04-28
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CA2067194A1 (en) 1991-04-06
DE69032483D1 (de) 1998-08-20
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ES2118066T3 (es) 1998-09-16
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KR0185192B1 (ko) 1999-04-01
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