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WO1986007063A1 - Separation de deux proteines - Google Patents

Separation de deux proteines Download PDF

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Publication number
WO1986007063A1
WO1986007063A1 PCT/JP1985/000274 JP8500274W WO8607063A1 WO 1986007063 A1 WO1986007063 A1 WO 1986007063A1 JP 8500274 W JP8500274 W JP 8500274W WO 8607063 A1 WO8607063 A1 WO 8607063A1
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WIPO (PCT)
Prior art keywords
protein
met
buffer
amino acid
amino
Prior art date
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PCT/JP1985/000274
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English (en)
Japanese (ja)
Inventor
Koichi Kato
Takao Yamada
Kenji Kawahara
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Takeda Chemical Industries, Ltd.
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Filing date
Publication date
Application filed by Takeda Chemical Industries, Ltd. filed Critical Takeda Chemical Industries, Ltd.
Priority to PCT/JP1985/000274 priority Critical patent/WO1986007063A1/fr
Priority to IL76360A priority patent/IL76360A0/xx
Priority to US06/774,811 priority patent/US5256769A/en
Priority to AT85306559T priority patent/ATE73819T1/de
Priority to EP19850306559 priority patent/EP0176299B1/fr
Priority to DE8585306559A priority patent/DE3585657D1/de
Priority to AU47496/85A priority patent/AU599575B2/en
Priority to JP60205873A priority patent/JPH0694476B2/ja
Priority to DK431485A priority patent/DK431485A/da
Priority to NZ213615A priority patent/NZ213615A/xx
Priority to KR1019850007049A priority patent/KR860002526A/ko
Priority to CN198585107875A priority patent/CN85107875A/zh
Publication of WO1986007063A1 publication Critical patent/WO1986007063A1/fr
Priority to JP6105210A priority patent/JPH07165792A/ja
Priority to JP8186491A priority patent/JPH09100296A/ja

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    • 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
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—Electrophoresis
    • C07K1/28—Isoelectric focusing
    • 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]
    • C07K14/55—IL-2
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha

Definitions

  • the present invention relates to a method for mutually separating proteins.
  • bioactive proteins such as cytokine peptide hormones has been elucidated, and recent advances in genetic engineering techniques are opening the way for mass production of these bioactive proteins and their application to clinical practice.
  • Interleukin-12 is one of the lymphokines produced by T cells activated by mitogens and antigens, and is an essential factor in the growth and differentiation of cytotoxic T cells and natural killer cells. Thus, they play an important role in these cells-mediated immune response systems.
  • interferon-a is one of the lymphokines produced by leukocytes activated by viruses and nucleic acids, and has the biological activity of acting on cells to put them into an antiviral state. And plays an important role in the tumor immune system.
  • Interleukin-12 and interferon-a are expected to be effectively used as remedies for various immunodeficiencies, infectious diseases, malignant tumors, etc. due to their biological activities.
  • the natural form of the mouse which has been isolated from the culture supernatant of human peripheral blood lymphocytes and human T cell leukemia (JURKAT strain) has several molecular weight differences. Although they are composed of molecular species, they are very similar to each other with respect to their polypeptides, and it is known that the amino terminal begins without exception with an alanine residue [Japanese Patent Application No. 59-149] No. 248 (filed July 19, 1979): Procedural of National Academy of Sciences (Pro. Natl. Acad. Sci.
  • the present inventors have succeeded in producing non-glycosylated human interleukin-12 by expressing the interleukin-12 gene of a human lymphocyte in Escherichia coli using recombinant DNA technology [ Japanese Patent Application No. 58-225079 (refer to the specification filed on January 28, 1983)].
  • the interlinkin 12 contains a polypeptide (I) consisting of the amino acid sequence shown in FIG. 1 (in the figure, X represents a hydrogen atom or a methionine residue).
  • the amino terminal amino acid As the amino terminal amino acid, it has a molecular species that starts with an alanine residue as in the case of the human natural form, and a molecular species that starts with a methionyl-alanine residue with a methionine residue added to the amino terminus.
  • interferon- ⁇ A expressed in E. coli using recombinant DNA technology contains a polypeptide consisting of the amino acid sequence shown in FIG. As the amino terminal amino acid, it has a molecular species starting from a cystine residue as in the natural human form, and a molecular species starting from a methionyl-cystine residue having a methionine residue added to the amino terminal.
  • interleukin-12 and interferon- ⁇ expressed in Escherichia coli are mixtures of the above two molecular species indicates that eukaryotes and
  • the higher order structure of the protein may be different from the molecular species in which a methionine residue is added to the amino terminus and the non-amino acid type. There may be differences in activity and biological stability. It is also possible that the addition of a methionine residue to the amino terminus could result in an increase or decrease in antigenicity. Therefore, from the viewpoint of industrial use, it is extremely significant to separate the molecular species having a methionine residue at the amino terminal from the molecular species having no methionine residue and to extract both in substantially pure form.
  • the rate of addition of methionine residues to the amino terminus may be influenced by the culture conditions and protein expression levels [Journal 'Ob' Interferon. Research (J. Interferon Res.), J_, 381 (1991 :)] has not been reported so far in which the addition rate of methionine residues could be controlled. Furthermore, during the protein purification process, a methionine residue was added to the amino terminal.
  • the methionine residue is an electrically neutral amino acid residue having a molecular weight of about 131, having moderate hydrophobicity, and having no dissociating group. Therefore, a macromolecule having a large number of dissociating groups, hydrophobic groups, and hydrophilic groups per se such as a protein [for example, the molecular weight of a polypeptide (I; X is a hydrogen atom) consisting of 133 amino acid residues]
  • a protein for example, the molecular weight of a polypeptide (I; X is a hydrogen atom) consisting of 133 amino acid residues
  • the addition of one methionine residue to the amino terminus is not expected to significantly affect the physicochemical properties of the entire protein. It is considered to be extremely difficult to separate molecular species with and without methionine residues from each other.
  • interleukin-12 and interleukin-12 and interferon-A which also have a methionine residue at the amino terminus
  • interferon- ⁇ A which further has a methionine residue at the amino terminus.
  • methods using the difference in solubility such as the solvent precipitation method using salt Xin ⁇ , mainly the difference in molecular weight, such as the Xin Xin method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis method.
  • the present inventors have conducted intensive studies for the purpose of separating interleukin-12 and interleukin-12 further having a methionine residue at the amino terminus thereof, and as a result, they have surprisingly been found. The fact that they have different isoelectric points was discovered. Since methionine is an electrically neutral amino acid, even if it is added to the amino terminal of a protein, it has no effect on the overall charge of the protein.
  • interleukin-12 and inta-leukin-12 which additionally has a methionine residue at its amino terminus, have different isoelectric points.
  • the present inventors have further studied and completed the present invention.
  • the present invention is characterized in that a mixture of a protein and a protein having a methionine residue at the amino terminus thereof is subjected to a separation means based on a difference in isoelectric point, and a protein having a methionine residue at the amino terminus. It provides a method for mutual separation from proteins.
  • a mixture of the above protein and a protein having a methionine residue at the amino terminal thereof can be usually produced by a gene recombination technique, and can be usually produced by expression using Escherichia coli, Bacillus subtilis, yeast, and animal cells.
  • proteins include various bioactive proteins, such as interferon (IFN; eg, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , etc.), interleukin (interleukin-11, interleukin-1). 2), cytokins such as cell growth factor (BGF), B cell differentiation factor (BDF), macrophage activation factor (MAF), lymphotoxin (LT), tumor necrosis factor (TNF); Transforming growth factor-1 (TGF-); peptide proteins such as erythropoietin, epidermal growth factor, insulin, and human growth hormone; hepatitis B virus antigen, influenza antigen, and foot-and-mouth disease virus antigen Peptidase (eg, tissue plasminogen activator, perokinase. Seratiopep) Daze etc.) enzymes such as Yari Zochimu; human serum albumin (H S A) blood protein components and the like.
  • IFN interferon
  • BDF B cell differentiation factor
  • MAF macrophage activation
  • the method for mutual separation of proteins according to the present invention for those having a molecular weight of 3,000 to 50,000, especially 5,000 to 30,000, and those having an amino acid number of 30 to 500, particularly 50 to 300, is preferred. It can be applied advantageously.
  • proteins having an isoelectric point of 4:11, especially 5-8 can be advantageously separated from each other, and can be used to separate proteins from proteins having a methionine residue at the amino terminus. It is preferable that the difference between the isoelectric points is about 0.01 to 0.2. ⁇
  • the mutual separation method of the present invention can be advantageously applied to interleukin-12-interferon- ⁇ produced by a genetic recombination technique. .
  • the interleukin-12 has the same biological or immunological activity as natural human interleukin-12, for example, an interleukin-12 receptor ⁇ binding ability to an anti-interleukin-12 antibody.
  • a polypeptide (I: X is a hydrogen atom) having the amino acid sequence shown in FIG. 1 or a portion required for its biological or immunological activity.
  • a fragment comprising an amino acid sequence may be used.
  • a fragment lacking one amino acid EPC Publication No. 915 39
  • these interleukin-12 may be abbreviated as IL-2, and the interleukin-12 further having a methionine residue at the amino terminal thereof may be abbreviated as Met-IL-2. .
  • Lysis Any substance having the same biological or immunological activity, for example, the ability to bind to the interferon ⁇ receptor and the anti-interferon ⁇ antibody may be used.
  • a polypeptide having the amino acid sequence shown in FIG. 2 may be used.
  • interferon-a ⁇ is preferable.
  • these polypeptides are non-glycosylated polypeptides.
  • these interferon-A are abbreviated as IF- ⁇
  • IFN- ⁇ having a methionine residue at the amino terminal of IFN- ⁇ is referred to as Met-IF-aA. May be abbreviated.
  • a mixed protein having a purity of 99% or more may be used. It may be a purified sample.
  • the mixture can be separated from each other by subjecting the mixture to separation means based on a difference in isoelectric point.
  • the isoelectric points of IL-12 and Met-IL-12 were calculated to be 7.7 and 7.5, respectively.
  • the isoelectric points of IFN- ⁇ A and Met-IFN- ⁇ A were calculated to be 6.2 and 6.3, respectively.
  • any method can be used as long as it is a method for separating proteins having a difference in isoelectric point of about 0.01 to 0.2.
  • ampholine is from LKB
  • gel used for gel isoelectric focusing is from Pharmacia as Sephadex IEF
  • LKB is as a PAG (polyacrylamide gel) plate.
  • the buffer solution was used as a polybuffer-exchanger PBE94, PBE118, polybuffer174, polybuffer96 from Pharmacia Inc.
  • the buffer solution is from Pharmacia
  • the DEAE-ion exchanger is DE AE-Toyopearl from Toyo Soda Kogyo Co., Ltd.
  • the CM-ion exchanger is CM-Toyopearl from Toyo Soda Kogyo Co., Ltd.
  • the body is available from Toyo Soda Kogyo Co., Ltd. as SP-5 PW or from Pharmacia as SP-Sephadex.
  • a plate for PH3.5-9.5 is used as a PAG plate.
  • 5.5—8.5 Plate, etc. 1 M phosphoric acid, 0.4 M HEPES, etc. as anolyte, 1 M sodium hydroxide, 0.1 M sodium hydroxide, etc. as catholyte use.
  • 1 ⁇ 100 ⁇ 100g of protein is loaded per plate, power is 1 ⁇ 200W, replacement
  • the electrophoresis is carried out preferably at -10 to 50 W and at a temperature of 0 to 20 ° C, preferably 2 to 5 ° C.
  • the electrophoresis time is 0.5 to 50 hours, usually 1.5 to 5 hours.
  • an equilibration buffer such as 0.025 M diethanolamine-hydrochloric acid buffer (pH 9.5) is used. ), 0.075 M Tris-acetate buffer (PH 9.3), etc., and 1% (vZv) Pharmamalite (8–10.5) -5.2% (v / v) Polycarbonate soft 96-hydrochloric acid buffer (PH 7.0 to 8.0), 10% (v / v) polypa, soft 96-hydrochloric acid buffer (pH 6.0) ⁇ 7.0) and 10% (vZv) buffer 96-acetic acid buffer (PH 6.0 to 7.0).
  • FPLC FPLC is performed at a flow rate of 1 to 50 mlZh, preferably 10 to 30 mlZli.
  • chromatofocusing method commercially available gels such as PBE 118 and PBE 94 (manufactured by Pharmacia) are used.
  • Column temperature is 0 to 30. C, preferably 2-5.
  • a protein and a protein having an additional k-methionine residue at its amino terminus are subjected to electrophoresis in an electric field or a carrier in a column with a PH gradient according to the difference in their isoelectric points. From each other and eluted or separated from each other.
  • separation can be achieved by applying the isocratic elution method using an ion exchanger without applying a pH gradient or a salt concentration gradient.
  • commonly used methods commonly used for protein purification such as salt folding, hydrophobic chromatography, gel filtration, ion exchange chromatography, and high performance liquid chromatography, are known. Can be used.
  • Met-IL-2 and Met-IFN- ⁇ were separated as proteins, and the present invention was the first to purify highly purified Met-IL-12 protein and Met-IFN- ⁇ . It also provides proteins.
  • the protein produced by the present invention and the protein having a methionine residue at the amino terminal thereof have the same biological or immunological activity as the corresponding natural protein, and have been purified to a high degree of purity. Since it is extremely low in proteins and heat-generating substances, it can be safely used as an injection drug substance.
  • Both IL-2 and Met-IL-2 obtained according to the present invention have an activity to grow normal T cells or natural killer cells while maintaining their functions. Therefore, IL-12 and Met-IL-2 obtained according to the present invention can be used for long-term propagation, subculture and cloning of T cells and natural killer cells in vitro. By utilizing this property, the activity of human IL-12 can be measured.
  • IL-2 and Met-IL-2 obtained by the present invention are:
  • in vitro natural killer cells that have the ability to recognize and destroy tumor antigens and have the ability to kill tumors regardless of experience of antigen sensitization, regardless of whether they have experienced antigen sensitization.
  • these killer T cells are transferred to a living body, their antitumor effect is greatly increased by simultaneously inoculating IL-12 or Met-IL-12 obtained according to the present invention.
  • IL-12 and Met-IL-2 obtained according to the present invention have no toxicity due to contaminating proteins and have low toxicity.
  • IL-12 or Met-IL-2 obtained according to the present invention as a prophylactic or therapeutic agent for tumors
  • the substance is mixed and diluted with a carrier known per se, for example, parenterally as an injection or capsule. It can be administered orally or orally. Furthermore, it can be used together with killer T cells or natural killer cells grown in vitro as described above or alone.
  • the IL-12 and Met-IL-2 of the present invention have substantially the same biological activity as that of a known natural isolated human IL-12, and can be used in the same manner. (2) Since the dissociation constant with the receptor is extremely small, administration of an extremely small amount is sufficient.
  • the IL-12 or Met-IL-2 of the present invention should be used in an amount of about 0.01 to 1 unit Zml, preferably about 0.1 to 0.1 ⁇ l. It can be used by adding it to the medium at a concentration of 5 units Zml.
  • Both IFN-A and Met-IFN-A obtained according to the present invention have the activity of acting on cells to bring them into an antiviral state. By utilizing this property, the activity of human IFN- ⁇ can be measured.
  • IFN- ⁇ A and Met-IFN- ⁇ obtained by the present invention have not only an antiviral effect, but also a cell growth inhibitory effect, an antibody production inhibitory effect, a strong effect on natural killer activity, and the like. .
  • IFN-a; A and Met-IFN-aA obtained according to the present invention have no antigenicity due to contaminating proteins and have low toxicity.
  • the substance is mixed and diluted with a carrier known per se, for example, parenterally as an injection or capsule. It can be administered orally or orally.
  • a carrier known per se, for example, parenterally as an injection or capsule. It can be administered orally or orally.
  • G lu / G 1 . ⁇ glutamate and gluta
  • FIG. 1 shows the non-dalcosylated human interleukin-12 protein obtained in Reference Example 1
  • FIG. 2 shows the non-glycosylated human interleukin obtained in Reference Example 3.
  • ⁇ Exchange 1 shows the amino acid sequence of perfluoro-a A protein (representing a hydrogen atom or a methionine residue in the figure).
  • Fig. 3 shows the results of FPLC in Example 1
  • Fig. 4 shows the results of isoelectric focusing
  • Fig. 5 shows the results of tryptic digested peptide and // bing.
  • Fig. 6 shows the results of mouth matochawcing in Example 2
  • Fig. 7 shows the results of SP-5 PW ion exchange mouth chromatography in Example 5
  • Fig. 8 shows the results of FPLC in Example 6. Not shown.
  • FIG. 9 and FIG. 10 show the construction diagrams of plasmids pTFl and PTB285 disclosed in Reference Example 3, respectively.
  • the transformant Escherichia coli ⁇ 4830 / ⁇ 285, was deposited as IFO-144-37 at IF0 and as FER ⁇ - ⁇ -189 at FRI from April 30, 1987. Have been.
  • interleukin-12 The biological activity of interleukin-12 was measured using interleukin-12-dependent cells as biochemical / biophysical * research ⁇ Communiques (B.iochem. Biophys. Res. Cotnmun.) This was performed in accordance with the method described in 10i, 36 3 (1992).
  • peak 1 was eluted at ⁇ 80 and peak 2 was eluted at PH7.9. Therefore, after fractionation, high-performance liquid chromatography using trifluoroacetic acid monoacetonitrile as an elution solvent was performed to remove the polybuffer used in FPLC.
  • PTH-amino acid phenylthiohydantoinamino acid
  • Table 1 shows the PTH-amino acids detected at each step.
  • Carboxyl terminal amino acid was analyzed as follows. That is, P1 and P2 were placed in a hydrazine decomposition glass tube, anhydrous hydrazine was added, the tube was sealed under reduced pressure, and then heated at 100 ° C. for 6 hours. After treating the obtained hydrazine hydrolyzate with benzaldehyde, the amount of free amino acid was measured using a Hitachi 8335 type amino acid analyzer. As a result, only threonine was detected in P1 and P2, and the recovery was 34.8% and 34.8%, respectively.
  • the amino acid composition Xin was added with constant boiling hydrochloric acid containing 4% thioglycolic acid and sealed under reduced pressure.Then, it was hydrolyzed with 110, 24, 48, and 72 hours, and Hitachi 83 The measurement was performed using a 5-type amino acid analyzer. Cystine and cystine were subjected to formic acid oxidation, then hydrolyzed in a constant boiling point hydrochloric acid under reduced pressure for 2 hours, and quantified as cystinic acid by an amino acid content meter. The amino acid content was determined by averaging the values obtained after 24, 48 and 72 hours of hydrolysis.
  • Fig. 3 shows the results of measuring the isoelectric points of P1 and 25P2 using an Ampholine PAG plate (manufactured by LKB). IL used as raw material
  • Non-glycosylated human interleukin a mixture of 2 and Met I L-2
  • Replacement Kin-1 2 shows two bands in isoelectric focusing, whereas P1 and P2 obtained in this example migrated as one band with different migration distances from each other.
  • the isoelectric point of P1 (IL-2) was 7.7, and that of P2 (Met IL-12) was 7.5.
  • the obtained P2 was digested with trypsin as follows to obtain a peptide map.
  • Peak 1 was collected in the first half and peak 2 in the second half so that the peaks did not overlap.
  • the yield was 0.82 mg (8.0%) for peak 1 and 1.98 mg (19.2%) for peak 2.
  • peak 1 contained 90% or more of IL-2 and peak 2 contained 95% or more of Met-IL-2.
  • non-glycosylated interleukin-12 was eluted as two peaks (peak A and peak B).
  • peak A shows Met-IL-12
  • peak B shows IL-12 with a purity of 99.5% or more. It was confirmed that it did.
  • Example 6 Separation of IFN- ⁇ and Met-IFN- ⁇ A by FPLC Glycosylated human interferon ⁇ which is a mixture of ⁇ FN-A and Met-IFN-A obtained by the method described in Reference Example 3 0.1% sodium chloride! 3.025M ammonium acetate buffer (pH 5.0) (Protein concentration, 2.96mg / ml) 1.0ml (2.96ing) in 0.025M imidazole-hydrochloric acid Desalting was carried out on a PD-10 force ram (1.5 ⁇ 5 cm, manufactured by Pharmacia) equilibrated with a buffer solution (PH6.7). The eluate (protein concentration, 1.5811 ⁇ 2 / 1111) 1.51111 (2.37!
  • PTH-amino acid The phenylthiohydantoinamino acid (PTH-amino acid) was identified by high performance liquid chromatography using a Micropak SP—C ⁇ 8 column (manufactured by Varian). Table 3 shows the PTH-amino acids detected at each step.
  • the isoelectric point of PI and P ⁇ was measured using an Ampholine PAG plate (manufactured by LKB).
  • the isoelectric point of PI (Met-IF- ⁇ A) was 6.3
  • P ⁇ The isoelectric point of IF-a A was calculated to be 6.2.
  • CM Topar (Toyo Soda Industry Co., Ltd.) column in which the Met-IL-2 containing solution obtained in Example 1 was equilibrated with 0.02-5 M ammonium acetate buffer (pH 5.0). And elute with the above buffer containing 0.15 M NaCl. The eluate is diluted with 0.15 M NaCl as appropriate, diluted with HSA to a concentration of 0.5%, filtered through a membrane filter (pore size: 0.22 m), and filtered. Aseptically dispense 1 ml each into a vial and freeze-dry to prepare Met-IL-2 for injection. This injection product is dissolved in 1 ml of distilled water for injection before use.
  • Replacement M9 medium containing 2 ml Transfer to a 51-volume jar armor containing 51, add 3 7 for 4 hours, then add 3-3 indolacrylic acid (25 gZml), and culture with aeration and agitation for another 4 hours. We obtained 2.5 £ of culture solution. The culture was centrifuged, the cells were collected, frozen at 180 ° C and stored.
  • the supernatant obtained above was permeabilized with 0.1 M Tris ⁇ HC1 buffer (pH 8.5), centrifuged at 19,000 xg for 10 minutes, and centrifuged for 10 minutes. ml was obtained. The supernatant was applied to a DE 52 (DEAE-cellulose, manufactured by ⁇ ⁇ Toman, UK) column (50 ml) equilibrated with 0.01 MTris-HC1 buffer (pH 8.5). After adsorbing the protein through the column, a linear gradient of NaC1 concentration (0 to 0.15 M NaCl, 11) was prepared to elute IL-12 to obtain 53 ml of an active fraction.
  • the active fraction 5 3011 of Kami was concentrated to 4.8 ml using ⁇ ! ⁇ -5 membrane (Amicon Co., Ltd., Ameri force), and 0.1 MT ris, HC 1 ( ⁇ 8.0 )-Gel filtration was performed using a Cefacryl S-200 (Pharmacia, Sweden) column (500 ml volume) equilibrated with 1 M NaCl buffer.
  • the active fraction (28 ml) was concentrated to 2.5 ml with a YM-5 membrane.
  • the obtained concentrate was adsorbed on an Ultrapore RPSC (America) column, and high-performance liquid chromatography was performed using trifluoroxyacetic acid-acetonitrile as an elution solvent.
  • the specific activity of the lyophilized product (white powder) of the above solution was 26,000 U / mg.
  • the plasmid PILOT 135-8 having the IL-2 gene (see Japanese Patent Application No. 58-225079 (filed on November 28, 1983), Example 1 (v'ii)) was modified with the restriction enzyme HgiAI. Cut.
  • the obtained 1294 bp DM fragment was blunt-ended with T4DNA polymerase, and EcoRI linker dTGCCATGAATTC ATGGC was ligated using T4DNA ligase.
  • Plasmid PTF1 was digested with restriction enzyme ⁇ 1 ⁇ 1 and ligated with BamHI linker. This plasmid DNA is treated with the restriction enzymes ⁇ HI and ⁇ £ RI, and then the plasmid pTB281 which has a ⁇ PL promoter at the EcoRI-BamHI site.
  • Escherichia coli 830 was processed according to the method of Kohen et al. [Procedures of National Academic Science ⁇ Prob Natl. Acad. Sci. USA USA, Vol. 69. , P. 2110 (1972)] to obtain a transformant containing the above plasmid, Escherichia coli N4830 / PTB285.
  • Transformant E. Coli N4830 / pTB285 was added to a 250 ml flask of Bacto Tripton (Difco Laboratories, America) 1% Nectoist Yeast Extract (Difco Laboratories. Ame) Jamaica) was inoculated into 50 l of a liquid medium (pH 7.0) containing 0.5%, 0.5% salt and 50 g / ml of ampicillin, and cultured at 37 ° C overnight with shaking.
  • the suspension was uniformly suspended in 100 mU, stirred at 4 ° C for 1 hour, and then centrifuged with 28, OOOXg for 20 minutes to obtain a supernatant.
  • the resulting supernatant is folded through a 0.01 M Tris-HC1 buffer (pH 8.5), and then centrifuged at 19,00 OXg for 10 minutes.
  • the supernatant obtained is 0.01 M Tris-HC1 buffer (pH 8.5) (DEAE-cellulose. ⁇ 'Sotman, England's) column equilibrated with
  • the active fraction obtained above was concentrated to 5 ml using YM-5 membrane (Amicon Co., Ltd.) and 0.1 M Tris-HCl (pH 8.0)-1 M NaCl buffer. Gel filtration was performed using Sephacryl S-200 (Pharmacia, Sweden) force column (500 ml) equilibrated in step.
  • the active fraction (40 nU) was concentrated to 3 ml with a YM-5 membrane.
  • the obtained concentrated solution was also adsorbed on an Ultrapore RPSC (Ametrix Co., Ltd.) column, and subjected to high-performance liquid chromatography using trifluoroacetic acid-acetonitrile as an elution solvent.
  • Escherichia coli 294 (ATCC 31446) / pLeI FAtrp25 having an expression plasmid incorporating the human IFN-A gene encoding the amino acid sequence shown in FIG. 2 [see Example I of EPC Publication No.
  • the culture solution 2 ⁇ obtained in (i) was centrifuged to collect the cells, and the cells were collected. Suspend in 50 mM Tris-HCl (pH 7.6) containing phenylmethylsulfonyl fluoride (PMSF), 0.2 tng / ml lysozyme, stir at 4 ° C for 1 hour, and keep at 37 ° C for 5 minutes This was further treated at 40 ° C. for 40 seconds by an ultrasonic crusher (manufactured by Altec, USA). The lysate was eccentrically separated with ll, 3Q0xg for 1 hour, and 95 ml of supernatant was collected.
  • PMSF phenylmethylsulfonyl fluoride
  • IF- ⁇ was eluted with Q.2MS acid containing 0.1% Tween 20 '(manufactured by Wako Pure Chemical Industries, Ltd.), and the active fraction was collected and adjusted to pH 4.5. After adjustment, the mixture was adsorbed on a CM cellulose column, washed thoroughly, and eluted with 0.025 MS acid ammonium buffer (PH 5.0) containing 0.15 M NaCl. The active fraction was collected again and freeze-dried to obtain 320 mg of human leukocyte IF- ⁇ A powder.
  • the proteins obtained by the separation method are each useful as pharmaceuticals and the like.

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Abstract

Procédé de séparation d'une protéine d'une autre protéine ayant un résidu de méthionine à sa terminaison aminée. Ledit procédé consiste à soumettre un mélange des protéines ci-décrites à un processus de séparation basé sur un écart de points isoélectriques entre les deux protéines.
PCT/JP1985/000274 1984-09-26 1985-05-21 Separation de deux proteines WO1986007063A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/JP1985/000274 WO1986007063A1 (fr) 1985-05-21 1985-05-21 Separation de deux proteines
IL76360A IL76360A0 (en) 1984-09-26 1985-09-10 Mutual separation of proteins
US06/774,811 US5256769A (en) 1984-09-26 1985-09-11 Mutual separation of proteins
AT85306559T ATE73819T1 (de) 1984-09-26 1985-09-16 Gegenseitige trennung von proteinen.
EP19850306559 EP0176299B1 (fr) 1984-09-26 1985-09-16 Séparation mutuelle de protéines
DE8585306559A DE3585657D1 (de) 1984-09-26 1985-09-16 Gegenseitige trennung von proteinen.
AU47496/85A AU599575B2 (en) 1984-09-26 1985-09-16 Mutual separation of proteins
JP60205873A JPH0694476B2 (ja) 1984-09-26 1985-09-17 蛋白質の相互分離方法
DK431485A DK431485A (da) 1984-09-26 1985-09-24 Gensidig adskillelse af proteiner
NZ213615A NZ213615A (en) 1984-09-26 1985-09-25 Separation of protein from its n-terminal methionine analogue
KR1019850007049A KR860002526A (ko) 1984-09-26 1985-09-25 단백질의 상호 분리 방법
CN198585107875A CN85107875A (zh) 1985-05-21 1985-09-26 蛋白质相互分离
JP6105210A JPH07165792A (ja) 1984-09-26 1994-05-19 精製された蛋白質
JP8186491A JPH09100296A (ja) 1984-09-26 1996-07-17 蛋白質の相互分離方法

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PCT/JP1985/000274 WO1986007063A1 (fr) 1985-05-21 1985-05-21 Separation de deux proteines

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WO1986007063A1 true WO1986007063A1 (fr) 1986-12-04

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WO (1) WO1986007063A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN100541185C (zh) * 2007-03-15 2009-09-16 云南神宇新能源有限公司 麻疯树种子胚乳蛋白质高分辨率的分离方法
EP2475678B1 (fr) * 2009-09-10 2014-10-08 Lonza Biologics Plc. Procédé et système de purification de polypeptides

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No relevant documents have been disclosed. *

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