KR900018371A - 펩타이드의 개선된 제조방법 - Google Patents

펩타이드의 개선된 제조방법 Download PDF

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KR900018371A
KR900018371A KR1019890005946A KR890005946A KR900018371A KR 900018371 A KR900018371 A KR 900018371A KR 1019890005946 A KR1019890005946 A KR 1019890005946A KR 890005946 A KR890005946 A KR 890005946A KR 900018371 A KR900018371 A KR 900018371A
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yeast
promoter
heterologous protein
hybrid vector
dna sequence
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KR970005584B1 (ko
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트라이클러 한스죄르그
타까바야시 켄지
하인리히 볼프 디이터
하임 쥬타
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시바-가이기 에이지
유시피 겐-파마 에이지
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger

Abstract

내용 없음

Description

펩타이드의 개선된 제조방법
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1도는 삭카로마이세스 세레비지애의 형질 전환된 KEX 1 및 kex1 균주로부터 수확된 데설파토 히루딘의 크로마토그래피 분석도이다. 제2도는 바람직한 효모 코돈을 갖는 PHO5시그날 서열을 함유하는 히루딘 HV1유전자의 시험관내 합성을 나타내는 모형도이다. 사용된 21개의 올리고 뉴클레오티드는 번호매김된 선과 점선으로 각각 표시하였다. 제3도는 플라스미드 pDP33의 도식적인 작제도이다.

Claims (19)

  1. 이종 단백질의 암호화 DNA서열에 작동적으로 연결된 효모 프로모터를 함유한 하이브리드 벡터로 형질전환되고 카복시펩티다제 yscα활성이 결여된 효모균주.
  2. 제1항에 있어서, 하이브리드 벡터가 이종 단백질을 암호화한 제2DNA서열에 적절한 판독프레임으로 연결된 시그날 펩타이드를 암호화한 제1DNA서열에 작동적으로 연결된 효모 프로모터를 및 효모 전사 종결 시그날을 갖는 DNA서열을 함유하는 효모 균주.
  3. 제2항에 있어서, 이종 단백질이 데설파토히루딘인 효모 균주.
  4. 제1항에 있어서, yscA, yscB, yscY 및 yscS활성으로 이루어진 그룹중에서 선택된 펩티다제 활성이 추가로 결여된 효모 균주.
  5. 제1항에 있어서, 완전한 REP1, REP2 및 FLP유전자 뿐만아니라, 완전한 ORI, STB, IR1 및 IR2부위가 포함된 완전한 2-미크론 DNA를 함유한 하이브리드 벡터로 형질 전환되고 내인성 2-미크론 DNA가 없는 효모 군주.
  6. 제5항에 있어서, yscA, yscB, yscY 및 yscS활성으로 이루어진 그룹중에서 선택된 펩디다제 활성이 추가로 결여된 효모 균주.
  7. 제1항에 있어서, 하이브리드 벡터가 MFα1 프로모터, GAL1 프로모터, 당분해 효소를 암호화한 유전자의 프로모터, ADHI프로모터, TRPI프로모터 및 상향 활성화 부위를 임의로 제거시킨 PHO5프로모터로 이루어진 그룹중에서 선택된 효모 프로모터를 갖는 효모 균주.
  8. 제2항에 있어서, 하이브리드 벡터가 하루딘 시그날 서열, 효모 인버타제(invertase)의 시그날 및 프리프로(prepro)서열, α-인자, 페로몬 펩티다제(KEX1), "킬러독소(killer toxin)" 및 억제성 산포스파타제(PHO5)유전자로 이루어진 그룹중에서 선택된 제1DNA서열 및 아스퍼질러스 아와모리(Aspergillus awamori)의 글로코아밀라제 시그날 서열을 함유하는 효모 균주.
  9. 제2항에 있어서, 하이브리드 벡터가 데설파토히루딘 변이체 HV1, HV1(변형 a, b), HV2, HV2(변형 a, b, c), PA, PA의 변이체 및 데스(val2)-데설파토히루딘으로 이루어진 그룹중에서 선택된 데설파토히루딘 화합물을 암호화하는 제2DNA서열을 함유하는 효모 균주.
  10. 제1항에 따른 삭카로마이세스 세레비지애 (Saccharomyces cerevisiae)균주.
  11. 카복시펩디다제 yscα활성이 결여된 효모 균주를 하이브리드 벡터로 형질전환시킴을 특징으로 하여, 제1항에 따른 형질전환된 효모 균주를 생성하는 방법.
  12. 이종 단백질의 암호화 DNA서열에 작동적으로 연결된 효모 프로모터를 함유하는 하이브리드 벡터로 형질 전환되고 카복시펩티다제 yscα활성이 결여된 효모 균주를 배양하고, 이종 단백질을 분리시킴을 특징으로 하여, 효모에 대한 이종 단백질을 생성하는 방법.
  13. 제12항에 있어서, 하이브리드 벡터가 이종 단백질을 암호화한 제2DNA서열에 적절한 판독프레임으로 연결된 시그날 펩타이드를 암호화한 제1 DNA서열에 작동적으로 연결된 효모 프로모터 및 효모 전사 종결 시그날을 함유한 DNA서열로 이루어짐을 특징으로 하는 효모에 대한 이종 단백질을 생성하는 방법.
  14. 제12항에 있어서, 이종 단백질이 카복시펩티다제 yscα에 의해 해독후 C-말단 분해되기 쉬운 방법.
  15. 제12항에 있어서, 이종 단백질의 2개의 C-말단 아미노산이 Lys, Arg, Tyr, Ala, Leu, Gln, Glu, Asp, Asn 및 Ser로 이루어진 그룹중에서 선택되는 방법.
  16. 제13항에 있어서, 이종 단백질이 데설파토히루딘인 효모에 대한 이종 단백질을 생성하는 방법.
  17. 제16항에 있어서, 데설파토히루딘 변이체 HV1, HV1(변형 a, b), HV1, HV2(변형 a, b, c) PA, PA의 변이체 및 데스(val2)-데설파토히루딘으로 이루어진 그룹중에서 선택된 데설파토히루딘 화합물의 제조를 위한 방법.
  18. 제16항에 있어서, 데설파토히루딘 변이체 HV1의 제조를 위한 방법.
  19. 일반식(Ⅳ)의 데설파토 히루딘.
    상기식에서, X1은 디펩타이트 잔기 Val-Val이고, X2, X3및 X4는 Lgs이며, X9는 His이고, X6은 Glu-Tyr-Lys-Arg, Ser-Phe Arg-Tyr 및 Trp-Glu-Leu Arg로 이루어진 그룹중에서 선택된다.
    ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019890005946A 1988-05-04 1989-05-03 폴리 펩타이드의 개선된 제조방법 KR970005584B1 (ko)

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
GB8810524.2 1988-05-04
CH?8810524.1? 1988-05-04
GB888810524A GB8810524D0 (en) 1988-05-04 1988-05-04 Improvements in production of polypeptides
GB8812627.1 1988-05-27
GB888812627A GB8812627D0 (en) 1988-05-27 1988-05-27 Improvements in production of polypeptides
CH?8812627.1? 1988-05-27
GB898907110A GB8907110D0 (en) 1988-05-04 1989-03-29 Improvements in the production of polypeptides
CH?8907110.4? 1989-03-29
GB8907110.4 1989-03-29

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KR900018371A true KR900018371A (ko) 1990-12-21
KR970005584B1 KR970005584B1 (ko) 1997-04-18

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KR1019890005946A KR970005584B1 (ko) 1988-05-04 1989-05-03 폴리 펩타이드의 개선된 제조방법

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US (1) US6103515A (ko)
KR (1) KR970005584B1 (ko)
GB (1) GB8907110D0 (ko)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ513755A (en) * 2001-08-24 2001-09-28 Ann Rachel Holmes Protein expression system in yeast comprising a vector encoding a heterologous membrane protein and its application in screening for drugs
EP2504355B1 (en) * 2009-11-25 2015-07-22 Novo Nordisk A/S Method for making polypeptides
JP7234932B2 (ja) 2017-10-10 2023-03-08 味の素株式会社 タンパク質の製造法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4546082A (en) * 1982-06-17 1985-10-08 Regents Of The Univ. Of California E. coli/Saccharomyces cerevisiae plasmid cloning vector containing the alpha-factor gene for secretion and processing of hybrid proteins
GB8521496D0 (en) * 1985-08-29 1985-10-02 Ciba Geigy Ag Repressible yeast promoters
MY101203A (en) * 1985-12-12 1991-08-17 Ucp Gen Pharma Ag Production of thrombin iinhibitors.
US4929553A (en) * 1987-05-29 1990-05-29 Canadian Patents & Development Ltd. Protease for specific processing of secreted proteins
FR2628429B1 (fr) * 1988-03-08 1990-12-28 Transgene Sa Variants de l'hirudine, leurs utilisations et les procedes pour les obtenir
GB8809860D0 (en) * 1988-04-26 1988-06-02 Ciba Geigy Ag Process for production of polypeptides
FR2645175B1 (fr) * 1989-03-31 1994-02-18 Transgene Sa Souche de saccharomyces cerevisiaeproductrice d'une proteine heterologue et procede de preparation de ladite proteine heterologue par fermentation de ladite souche

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