KR980002258A - Method for preparing urease B of Helicobacter pylori from recombinant E. coli - Google Patents
Method for preparing urease B of Helicobacter pylori from recombinant E. coli Download PDFInfo
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- KR980002258A KR980002258A KR1019960020905A KR19960020905A KR980002258A KR 980002258 A KR980002258 A KR 980002258A KR 1019960020905 A KR1019960020905 A KR 1019960020905A KR 19960020905 A KR19960020905 A KR 19960020905A KR 980002258 A KR980002258 A KR 980002258A
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- fusion protein
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- urease
- histidine
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- 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
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- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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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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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01005—Urease (3.5.1.5)
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Abstract
본 발명은 대장균을 이용하여 헬리코박터 필로리(Helicobacter pylori : H. pylori)의 항원 결정부위 단백질인 우레아제 B(Urease B)를 융합단백질의 형태로 제조하는 방법에 관한 것으로, 헬리코박터 필로리의 우레아제 B 유전자에 히스티딘과 유비퀴틴 유전자가 연결된 융합유전자를 포함하는 His-Ub-Ure B 융합단백질 발현벡터로 형질전환된 대장균을 융합단백질의 발현에 적합한 조건에서 배양하고, 융합단백질이 발현된 재조합 대장균 세포를 구아니딘 염이 포함된 완충용액에서 분쇄하고, 원심분리한 상등액을 히스티딘 결합수지 친화성 크로마토그래피하는 단계를 포함하는 본 발명의 His-Ub-Ure B 융합단백질의 제조방법에 의하면 H. pylori 감염진단과 백신 개발에 유용하게 사용될 수 있는 His-Ub-Ure B 융합단백질을 높은 수율로 대량 생산할 수 있다.The present invention relates to a method for producing urease B (Urease B), an antigenic determinant protein of Helicobacter pylori (H. pylori) using E. coli, in the form of a fusion protein. Escherichia coli transformed with His-Ub-Ure B fusion protein expression vector containing a fusion gene linked to histidine and ubiquitin genes were cultured under conditions suitable for expression of the fusion protein, and the recombinant E. coli cells expressing the fusion protein were guanidine salts. According to the method for preparing the His-Ub-Ure B fusion protein of the present invention, which comprises the step of pulverizing in the buffer solution and centrifuging the supernatant, the histidine-binding resin affinity chromatography. H. pylori infection diagnosis and vaccine development It is possible to mass-produce His-Ub-Ure B fusion protein which can be usefully used in high yield.
Description
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음Since this is an open matter, no full text was included.
제1도는 우레아제 B(Urease B) 유전자를 본 발명의 대장균 발현벡터 pETHUB-UREB로 클로닝하는 과정을 도시한 것이다.Figure 1 shows the process of cloning the urease B (Urease B) gene with the E. coli expression vector pETHUB-UREB of the present invention.
제2도는 본 발명의 발현벡터 pETHUB-UREB로 형질전환된 대장균을 배양하여 얻은 세포 침전물을 변성 폴리아크릴아미드 겔 전기영동한 결과를 나타낸 것이다.Figure 2 shows the result of denatured polyacrylamide gel electrophoresis of the cell precipitate obtained by culturing E. coli transformed with the expression vector pETHUB-UREB of the present invention.
제3도는 본 발명의 발현벡터 pETHUB-UREB로 형질전환된 대장균을 배양하여 얻은 세포 침전물을 변성 폴리아크릴아미드 겔 전기영동한 후 유비퀴틴 단일 항체를 이용하여 웨스턴 블롯팅(western blotting)한 결과를 나타낸 것이다.Figure 3 shows the result of Western blotting using ubiquitin single antibody after denatured polyacrylamide gel electrophoresis of cell precipitate obtained by culturing E. coli transformed with the expression vector pETHUB-UREB of the present invention. .
Claims (6)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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KR1019960020905A KR0177313B1 (en) | 1996-06-12 | 1996-06-12 | Method for producing urease B of Helicobacter pylori from recombinant E. coli |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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KR1019960020905A KR0177313B1 (en) | 1996-06-12 | 1996-06-12 | Method for producing urease B of Helicobacter pylori from recombinant E. coli |
Publications (2)
Publication Number | Publication Date |
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KR980002258A true KR980002258A (en) | 1998-03-30 |
KR0177313B1 KR0177313B1 (en) | 1999-04-01 |
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KR1019960020905A KR0177313B1 (en) | 1996-06-12 | 1996-06-12 | Method for producing urease B of Helicobacter pylori from recombinant E. coli |
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Families Citing this family (1)
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WO2016018465A1 (en) * | 2014-07-30 | 2016-02-04 | Medtronic, Inc. | Urease purification from jack beans or other organisms |
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1996
- 1996-06-12 KR KR1019960020905A patent/KR0177313B1/en not_active IP Right Cessation
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