KR970074932A - Method for preparing human interleukin-6 using yeast - Google Patents

Method for preparing human interleukin-6 using yeast Download PDF

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KR970074932A
KR970074932A KR1019960018347A KR19960018347A KR970074932A KR 970074932 A KR970074932 A KR 970074932A KR 1019960018347 A KR1019960018347 A KR 1019960018347A KR 19960018347 A KR19960018347 A KR 19960018347A KR 970074932 A KR970074932 A KR 970074932A
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yeast
expression plasmid
gene
pyhil
protein
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KR1019960018347A
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KR100204504B1 (en
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유향숙
정경숙
변광호
최인표
강형식
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박원훈
한국과학기술연구원
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
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  • Analytical Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

본 발명은 인체에 존재하는 사이토카인의 일종으로서 인체의 면역반응등과 관련하여 매우 중요한 작용을 하는 물질인 인터루킨-6 (Interleukine-6)의 유전자를 포함하는 효모 발현 플라스미드 및 이를 이용하여 효모에서 인터루킨-6를 제조하는 방법에 관한 것이다.The present invention is a type of cytokine present in the human body, and a yeast expression plasmid containing a gene of interleukine-6, which is a substance which plays a very important role in relation to the immune response of the human body, and an interleukin in yeast using the same. It relates to a method for producing -6.

Description

효모를 이용한 사람의 인터루킨-6의 제조 방법Method for preparing human interleukin-6 using yeast

본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음As this is a public information case, the full text was not included.

제1도는 IL-6 유전자가 포함된 효모 발현 플라스미드, pYHIL-10Zd를 제조하는 과정을 나타낸 것이다, 제2도는 IL-6 유전자가 포함된 효모 발현 플라스미드, pYHIL-FZd를 제조하는 과정을 나타낸 것이다, 제3도는 본 발명의 발현 플라스미드로 형질전환된 효모 균주가 IL-6를 생산하는 정도를 SDS-폴리아크릴아미드겔 전기영동 및 이의 웨스턴 블럿 방법으로 나타낸 것이다, 제5도는 효모에서 발현되는 재조합 IL-6-lacZ 융합단백질의 IL-6 활성도를 나타낸 것이다.FIG. 1 shows a process for preparing a yeast expression plasmid containing IL-6 gene, pYHIL-10Zd, and FIG. 2 shows a process for preparing a yeast expression plasmid containing IL-6 gene, pYHIL-FZd. Figure 3 shows the degree of production of IL-6 by the yeast strain transformed with the expression plasmid of the present invention by SDS-polyacrylamide gel electrophoresis and its Western blot method, Figure 5 shows the recombinant IL- expressed in yeast IL-6 activity of 6-lacZ fusion protein is shown.

Claims (10)

IL-6 유전자의 발현 플라스미드.Expression plasmid of the IL-6 gene. 제1항에 있어서, IL-6 유전자는 IL-6-lcaZ 융합유전자인 것을 특징으로 하는 발현 플라스미드.The expression plasmid of claim 1, wherein the IL-6 gene is an IL-6-lcaZ fusion gene. 제1항에 있어서, IL-6 유전자가 PGK 프로모터로부터 발현되는 것을 특징으로 하는 발현 플라스미드 pYHIL-10Zd.The expression plasmid pYHIL-10Zd of claim 1, wherein the IL-6 gene is expressed from a PGK promoter. 제1항에 있어서, IL-6 유전자가 킬라틴 프로모터로부터 발현되는 것을 특징으로 하는 발현 플라스미드 pYHIL-FZd.The expression plasmid pYHIL-FZd of claim 1, wherein the IL-6 gene is expressed from a keratin promoter. 제1항의 IL-6 유전자의 발현 플라스미드로 효모 균주를 형질전환한 형질 전환체.A transformant transformed into a yeast strain with the expression plasmid of the IL-6 gene of claim 1. 제5항에 있어서, 효모 균주는 L3262 효모 균주이고 발현 플라스미드는 pYHIL-10Zd인 것을 특징으로 하는 형질전환체, L3262/ pYHIL-10Zd. (수탁번호 : KCTC 8741P).6. The transformant, L3262 / pYHIL-10Zd, according to claim 5, wherein the yeast strain is L3262 yeast strain and the expression plasmid is pYHIL-10Zd. (Accession No .: KCTC 8741P). 제5항에 있어서, 효모 세포는 L-3262 효모 세포이고 발현 플라스미드는 pYHIL-FZd.인 것을 특징으로 하는 형질전환체, L3262/ pYHIL-FZd. (수탁번호 : KCTC 8740P).The transformant, L3262 / pYHIL-FZd, according to claim 5, wherein the yeast cell is L-3262 yeast cell and the expression plasmid is pYHIL-FZd. (Accession No .: KCTC 8740P). 제6항의 효모 형질전환체를 배양하고 IL-6 단백질의 발현을 유도하여 생체 활성이 있는 IL-6 단백질을 생산하는 것을 특징으로 하는 IL-6 단백질의 제조 방법.A method for producing IL-6 protein, comprising culturing the yeast transformant of claim 6 and inducing the expression of IL-6 protein to produce IL-6 protein with bioactivity. 제8항에 있어서, 활성이 있는 IL-6 단백질은 IL-6-lacZ 융합단백질 형태인 것을 특징으로 하는 IL-6 단백질의 제조 방법.The method of claim 8, wherein the active IL-6 protein is in the form of an IL-6-lacZ fusion protein. 제9항에서 제조한 IL-6-lacZ 융합단백질을 베타갈락토시데이즈 항체를 사용한 컬럼을 이용하여 효모 형질전환체로부터 분리·정제하는 방법.A method for separating and purifying IL-6-lacZ fusion protein prepared in claim 9 from a yeast transformant using a column using a beta galactosidase antibody. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.※ Note: The disclosure is based on the initial application.
KR1019960018347A 1996-05-28 1996-05-28 Process for the production of human interleukin-6 using yeast KR100204504B1 (en)

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KR100204504B1 KR100204504B1 (en) 1999-06-15

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