KR950025098A - Purification of Human Erythropoietin Factor Expressed in Recombinant Insect Cells - Google Patents

Purification of Human Erythropoietin Factor Expressed in Recombinant Insect Cells Download PDF

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KR950025098A
KR950025098A KR1019940002513A KR19940002513A KR950025098A KR 950025098 A KR950025098 A KR 950025098A KR 1019940002513 A KR1019940002513 A KR 1019940002513A KR 19940002513 A KR19940002513 A KR 19940002513A KR 950025098 A KR950025098 A KR 950025098A
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epo
chromatography
insect cells
resin
cation exchange
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KR100321446B1 (en
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조중명
유영준
소홍섭
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성재갑
엘지화학 주식회사
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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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
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    • C—CHEMISTRY; METALLURGY
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    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • 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
    • 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
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/505—Erythropoietin [EPO]

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Abstract

본 발명은 인간 적혈구 성장인자(EP)를 함유하는 미생물로 형질전환된 곤충 세포에서 발현시킨 재조합 EPO를 정제하는 방법에 관한 것으로, EPO 유전자를 함유하는 미생물로 형질전환된 곤충 세포를 배양하고, 얻어진 배양물의 상청액을 농축하고, 농축된 배양액을 양이온 교환수지 크로마토그래피하여 EPO를 함유하는 분획을 수집하고, 수집된 EPO 함유 분획을 렉틴 어피니티 크로마토그래피하여 EPO를 함유하는 당단백질을 분리하고, 분리된 EPO 함유 당단백질을 젤 여과 크로마토그래피함으로써 곤충 세포에서 발현시킨 EPO의 생리적 활성도를 보존하면서 신속하고 효율적으로 정제하는 방법을 제공한다.The present invention relates to a method for purifying recombinant EPO expressed in insect cells transformed with microorganisms containing human erythrocyte growth factor (EP), which is obtained by culturing insect cells transformed with microorganisms containing EPO gene. The supernatant of the culture was concentrated, and the concentrated culture was subjected to cation exchange resin chromatography to collect fractions containing EPO, and the collected EPO-containing fractions were collected by lectin affinity chromatography to separate the glycoproteins containing EPO and separated. Gel filtration chromatography of EPO-containing glycoproteins provides a method for rapid and efficient purification while preserving the physiological activity of EPO expressed in insect cells.

Description

유전자재조합 곤충세포에서 발현시킨 인간 적혈구성장인자의 정제방법Purification of Human Erythropoietin Factor Expressed in Recombinant Insect Cells

본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음Since this is an open matter, no full text was included.

제1도는 유전자 재조합 곤충 세포로부터 인간 적혈구 성장인자의 정제과정을 개략적으로 도시한 것이고,Figure 1 schematically illustrates the purification of human erythrocyte growth factor from recombinant insect cells,

제2도는 유전자 재조합 곤충 세포에서 발현시킨 인간 적혈구 성장인자를 양이온 교환수지 컬럼으로 분리한 크로마토그램과 인간 적혈구 성장인자를 함유하고 있는 분획을 항원항체반응으로 검출한 결과를 나타낸 것이고,FIG. 2 shows the result of detecting the chromatogram of human erythrocyte growth factor expressed in recombinant insect cells by cation exchange resin column and the fraction containing human erythrocyte growth factor by antigen antibody reaction.

제7도는 본 발명의 방법에 따라 정제된 인간 적혈구 성장인자의 생리적 활성도를 측정한 결과이다.7 is a result of measuring the physiological activity of human erythrocyte growth factor purified according to the method of the present invention.

Claims (8)

인간 적혈구 성장인자(EPO)를 함유하는 미생물로 형질전환된 곤충 세포에서 발현시킨 재조합 EPO를 정제하는 방법에 관한 것으로, EPO 유전자를 함유하는 미생물로 형질전환된 곤충 세포를 배양하고, 얻어진 배양물의 상청액을 농축하고, 농축된 배양액을 양이온 교환수지 크로마토그래피하여 EPO를 함유하는 분획을 수집하고, 수집된 EPO 함유 분획을 렉틴 어피니티 크로마토그래피하여 EPO를 함유하는 당단백질을 분리하고, 분리된 EPO 함유 당단백질을 젤 여과 크로마토그래피하는 것을 특징으로 하는 방법.A method for purifying recombinant EPO expressed in insect cells transformed with microorganisms containing human red blood cell growth factor (EPO), the method comprising culturing insect cells transformed with microorganisms containing EPO gene, and the supernatant of the obtained culture And the concentrated culture solution was subjected to cation exchange resin chromatography to collect fractions containing EPO, and the collected EPO-containing fractions were collected by lectin affinity chromatography to separate the glycoproteins containing EPO, and the separated EPO-containing sugars. Gel filtration chromatography of the protein. 제1항에 있어서, 상기 인간 적혈구 성장인자(EPO)을 함유하는 베큘로바이러스로 sf-21 세포를 감염시킴을 특징으로 하는 방법.The method of claim 1, characterized by infecting sf-21 cells with baculovirus containing the human red blood cell growth factor (EPO). 제1항에 있어서, EPO를 포함한 배양액을 여과, 투석 및 원심분리하여 농축함을 특징으로 하는 방법.The method of claim 1, wherein the culture solution containing EPO is concentrated by filtration, dialysis and centrifugation. 제1항에 있어서, pH6.0 내지 pH6.5 범위의 완충용액으로 양이온 교환 크로마토그래피함을 특징으로 하는 방법.The method of claim 1, characterized in that the cation exchange chromatography with a buffer solution ranging from pH6.0 to pH6.5. 제1항에 있어서, 상기 양이온 교환 크로마토그래피가 CM-세파로즈, S-페파로즈, CM-세룰로즈, S-세룰로즈 또는 모노-S 수지를 이용함을 특징으로 하는 방법.The method of claim 1, wherein the cation exchange chromatography uses CM-Sepharose, S-Perose, CM-Cerulose, S-Cerulose, or mono-S resin. 제1항에 있어서, 상기 렉틴-어피니티 컬럼이 ConA 세파로즈 또는 린틸 렉틴 세파로즈 수지를 이용함을 특징으로 하는 방법.The method of claim 1, wherein the lectin-affinity column uses a ConA Sepharose or Lintyl Lectin Sepharose resin. 제1항에 있어서, 젤 여과 컬럼이 수퍼로즈 12, 수퍼덱스 75, 세파덱스 75 또는 세파크릴 S-100등의 수지를 이용함을 특징으로 하는 방법.The method according to claim 1, wherein the gel filtration column uses a resin such as Superrose 12, Superdex 75, Sephadex 75, or Sephacryl S-100. 제1항 내지 제7항중 어느 한 항에 있어서, 상기 각 크로마토그래피한후 항원항체반응을 이용하연 신속하게 EPO를 함유하는 분획을 검출함을 특징으로 하는 방법.8. The method according to any one of claims 1 to 7, characterized in that a fraction containing EPO is detected quickly after each chromatography followed by an antigen antibody reaction. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.※ Note: The disclosure is based on the initial application.
KR1019940002513A 1994-02-14 1994-02-14 Purification method of recombinant human erythropoietin(epo) expressed from recombinant insect cells KR100321446B1 (en)

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