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 PDF

Info

Publication number
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
Authority
KR
South Korea
Prior art keywords
fusion protein
coli
ure
urease
histidine
Prior art date
Application number
KR1019960020905A
Other languages
Korean (ko)
Other versions
KR0177313B1 (en
Inventor
장호진
김천형
황상연
유영준
Original Assignee
성재갑
주식회사 Lg 화학
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 성재갑, 주식회사 Lg 화학 filed Critical 성재갑
Priority to KR1019960020905A priority Critical patent/KR0177313B1/en
Publication of KR980002258A publication Critical patent/KR980002258A/en
Application granted granted Critical
Publication of KR0177313B1 publication Critical patent/KR0177313B1/en

Links

Classifications

    • 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)
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
    • C—CHEMISTRY; METALLURGY
    • 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)

Landscapes

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

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

재조합 대장균으로부터 헬리코박터 필로리의 우레아제 B 제조방법Method for preparing urease B of Helicobacter pylori from recombinant E. coli

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)

헬리코박터 필로리(Helicobacter pylori : H. pylori)의 우레아제 B 유전자에 히스티딘과 유비퀴틴 유전자가 연결된 융합유전자.A fusion gene whose histidine and ubiquitin genes are linked to the urease B gene of Helicobacter pylori (H. pylori). 제1항의 융합유전자를 포함하는 히스티딘-유비퀴틴-우레아제 B(His-Ub-Ure B) 융합단백질 발현벡터.A histidine-ubiquitin-urease B (His-Ub-Ure B) fusion protein expression vector comprising the fusion gene of claim 1. 제2항에 있어서, 발현벡터 pETHUB-UREB.The expression vector pETHUB-UREB of claim 2. 제2항의 발현벡터로 형질전환된 대장균.E. coli transformed with the expression vector of claim 2. 제4항에 있어서, 대장균 BL21(DE3) pLYS.S(pET HUB UREB).The E. coli BL21 (DE3) pLYS.S (pET HUB UREB) according to claim 4. 제4항의 대장균을 융합단백질의 발현에 적합한 조건에서 배양하고, 융합단백질이 발현된 재조합 대장균 세포를 구아니딘 염이 포함된 완충용액에서 분쇄하고, 원심분리한 상등액을 히스티딘 결합수지 친화성 크로마토그래피하는 단계를 포함하는 His-Ub-Ure B 융합단백질의 제조방법.E. coli is cultured under conditions suitable for the expression of the fusion protein, the recombinant E. coli cells expressing the fusion protein is pulverized in a buffer solution containing guanidine salt, and the centrifuged supernatant is subjected to histidine binding resin affinity chromatography Method for producing a His-Ub-Ure B fusion protein comprising a. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.※ Note: The disclosure is based on the initial application.
KR1019960020905A 1996-06-12 1996-06-12 Method for producing urease B of Helicobacter pylori from recombinant E. coli KR0177313B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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
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
KR980002258A true KR980002258A (en) 1998-03-30
KR0177313B1 KR0177313B1 (en) 1999-04-01

Family

ID=19461515

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1019960020905A KR0177313B1 (en) 1996-06-12 1996-06-12 Method for producing urease B of Helicobacter pylori from recombinant E. coli

Country Status (1)

Country Link
KR (1) KR0177313B1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016018465A1 (en) * 2014-07-30 2016-02-04 Medtronic, Inc. Urease purification from jack beans or other organisms

Also Published As

Publication number Publication date
KR0177313B1 (en) 1999-04-01

Similar Documents

Publication Publication Date Title
Smith et al. Expression and purification of glutathione‐S‐transferase fusion proteins
ES2070172T3 (en) PROCEDURE FOR PRODUCING PROTEINS BY RECOMBINANT DNA.
DK520187D0 (en) PEPTIDES FOR DIAGNOSIS OF HTLV-III ANTIBODIES, METHOD OF PREPARING THEM AND THEIR USE
DK38189A (en) HIS UNKNOWN FUSION PROTEINS CONTAINING GLUTATION-S-TRANSFERASE
DK0613944T3 (en) Methods for regulating the immune response using CTLA4-binding molecules and IL4-binding molecules
DE3587524D1 (en) PRODUCTION OF CHIMERIC ANTIBODY.
CN103728447B (en) The antibody labeling method of controllable quantum dots locus specificity bridge joint coupling and application thereof
KR980002258A (en) Method for preparing urease B of Helicobacter pylori from recombinant E. coli
Piao et al. Trigger factor assisted soluble expression of recombinant spike protein of porcine epidemic diarrhea virus in Escherichia coli
KR980002257A (en) Method for preparing urease A of Helicobacter pylori from recombinant E. coli
BR9714133A (en) isolated nucleic acid, molecule thereof, probe, recombinant expression vector, cell, polypeptide, fusion protein vaccine formulation and processes for producing a h. pylori, to detect the presence of a helicobacter nucleic acid in a sample, to treat or reduce a risk of infection by h. pylori in an individual and to produce a vaccine formulation
McCluskey et al. A rapid and universal tandem‐purification strategy for recombinant proteins
KR870010192A (en) Method for preparing fusion proteins and antibodies
KR980002259A (en) Method for producing species-specific protein antigen of Helicobacter pylori from recombinant E. coli
ES2075068T3 (en) PROCEDURE FOR THE PREPARATION OF A PROTEIN BY MEANS OF YEAST USING AN INDUCIBLE SYSTEM, VECTORS AND CORRESPONDING TRANSFORMED STRAINS.
NZ511623A (en) Methods of identifying antigen gene sequences from Mycoplasma
KR950018454A (en) How to produce protein C
Carden et al. Chemical cross-linking analyses of ox neurofilaments
GB1058828A (en) Process for the manufacture of artificial antigens
KR860008282A (en) Method for producing recombinant human lenin
Yang et al. Combinational biosynthesis of a fluorescent cyanobacterial holo-α-allophycocyanin in Escherichia coli
SE8800378L (en) PREPARATION OF FUSED PROTEINS OR POLYPEPTIDES
Massagué et al. [14] Affinity cross-linking of receptors for insulin and the insulin-like growth factors I and II
KR970059276A (en) Method for producing the vacuolar cytotoxin protein of Helicobacter pylori from recombinant E. coli
Reddi et al. The calcium‐binding protein of Entamoeba histolytica as a fusion partner for expression of peptides in Escherichia coli

Legal Events

Date Code Title Description
A201 Request for examination
E701 Decision to grant or registration of patent right
GRNT Written decision to grant
FPAY Annual fee payment

Payment date: 20060915

Year of fee payment: 9

LAPS Lapse due to unpaid annual fee