EP0506963A1 - Production d'une muteine du facteur de croissance fibroblastique basique de l'homme - Google Patents

Production d'une muteine du facteur de croissance fibroblastique basique de l'homme

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Publication number
EP0506963A1
EP0506963A1 EP91900328A EP91900328A EP0506963A1 EP 0506963 A1 EP0506963 A1 EP 0506963A1 EP 91900328 A EP91900328 A EP 91900328A EP 91900328 A EP91900328 A EP 91900328A EP 0506963 A1 EP0506963 A1 EP 0506963A1
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EP
European Patent Office
Prior art keywords
ifo
european patent
mutein
indications
amino acid
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Legal status (The legal status 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 status listed.)
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Application number
EP91900328A
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German (de)
English (en)
Inventor
Kazuaki Kitano
Masato Kuriyama
Osamu Nishimura
Koichi Igarashi
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Takeda Pharmaceutical Co Ltd
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Takeda Chemical Industries Ltd
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Application filed by Takeda Chemical Industries Ltd filed Critical Takeda Chemical Industries Ltd
Publication of EP0506963A1 publication Critical patent/EP0506963A1/fr
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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
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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
    • C12N15/67—General methods for enhancing the expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a technique for producing a mutein of a mature human basic fibroblast growth factor (hereinafter also briefly referred to as hbFGF) in which at least one constituent amino acid of hbFGF is replaced by another amino acid which can be used as a healing promoter for wounds.
  • hbFGF human basic fibroblast growth factor
  • Basic fibroblast growth ' factor is a basic polypeptide hormone having a molecular weight of about 17,000 and is mainly secreted by a pituitary gland.
  • bFGF was first isolated as a factor exhibiting strong growth promoting action on fibroblasts such as BALB/c3T3 cell [D. Gospodarowicz, Nature 249, 123 (1974)]. It is now known that FGF exhibits growth promoting action on almost all mesoblast-derived cells [D. Gospodarowicz et al., National Cancer Institute Monograph 48, 109 (1978)].
  • the angiogenic action of the bFGF suggests a potential for the application thereof as a therapeutic medicine for traumas, and as a preventive and therapeutic medicine for thrombosis, arteriosclerosis, etc.
  • Genes coding for hbFGFs have been cloned by Abraham et al. [The EMBO Journal 5, 2523-2528 (1986)] and Kurokawa et al.
  • hbFGF muteins When recombinant proteins are produced using E. coli, water-insoluble inclusion bodies are frequently formed to accumulate the proteins. When the desired substances are isolated and purified from such inclusion bodies, the inclusion bodies are usually solubilized by adding protein denaturing agents. As for hbFGF muteins, however, the proteins thus solubilized are inactive and no technique for reactivating them has been established yet.
  • the hbFGF muteins can be used as medicines if the muteins can be accumulated in large amounts and in an active state by using genetic engineering techniques, and further isolated and purified in high yield. It is therefore an important object to establish a method for producing the muteins at high efficiency.
  • T7 promoter is known to be a strong promoter.
  • the accumulated proteins such as human interleukin-2, prolactin, form inclusion bodies, and most of them are accumulated in an inactive state.
  • the present inventors have found the interleukin-2 case, and Paris, N. et al. have found the prolactin case [Paris, N. et al., Biotechnology and Applied Biochemistry, 12, 436-449 (1990)].
  • the present inventors conducted intensive investigations to accumulate considerable amounts of the hbFGF mutein in an active state, and have found that addition of isopropylthiogalactopyranosyde in a low concentration is effective.
  • the present inventors have further established a method for isolating and purifying the hbFGF mutein efficiently, thus completing the present invention.
  • the present invention provides:
  • hbFGF human basic fibroblast growth factor
  • IPTG is added to the culture medium on a logarithmic growth phase of the transformant described in the above item (3), followed by cultivation, and (6) the method described in the above item (5), in which a resultant mutein-containing solution is purified by chromatography using a crosslinked polysaccharide sulfate, a
  • __ synthetic polymer having a sulfonic acid group as an exchange group and/or a synthetic polymer for gel filtration as a carrier.
  • the substitution type muteins of mature hbFGFs can be efficiently produced.
  • the present invention can therefore be advantageously used for industrial production of the muteins.
  • FIG. 1 shows a DNA nucleotide sequence of rhbFGF mutein CS23 used in Example 1 and an amino acid sequence of a protein for which the nucleotide sequence codes;
  • Fig. 2 is a schematic representation showing the construction of plasmid pTB960 obtained in Example 1;
  • Fig. 3 shows a SDS-PAGE pattern of a purified sample obtained in Example 5 and a marker
  • Figs. 4 to 6 show patterns of high performance liquid chromatography of the purified sample obtained in Example 5.
  • Figs. 7 to 9 are schematic representations showing the construction of plasmids pHP901, pME901 and pCM901 obtained in Example 8.
  • the mature hbFGF in the present invention is a peptide consisting of 146 amino acids, counting the amino acid Pro next to Met of the N-terminus as the 1st and the amino acid Ser of the C-terminus as the 146th, in Fig. 1. .
  • Examples of the hbFGF muteins as shown in the present invention include muteins in which at least one constituent amino acid of the mature hbFGF is replaced by another amino acid, as described in European Patent Publication No. 281,822 and Biochemical and Biophysical Research Communication 151, 701-708 (1988).
  • the number of hbFGF-constituent amino acids before substitution in the mutein which has at least one hbFGF-constituent amino acid substituted by another amino acid, it may be any number as long as FGF characteristics, such as the characteristics of angiogenesis, cell growth stimulating activity and cell differentiating activity, are not lost.
  • constituent amino acids before substitution examples include cysteine and amino acids other than cysteine.
  • cysteine is preferred.
  • the amino acids other than cysteine as the constituent amino acids before substitution include aspartic acid, arginine, glycine and valine.
  • the constituent amino acid before substitution is cysteine
  • neutral amino acids are preferred as the substituting amino acids.
  • the neutral amino acids include glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, histidine, tryptophan, serine, threonine and methionine. In particular, serine and threonine are preferred.
  • the constituent amino acid before substitution is an amino acid other than cysteine
  • there are selected as the other substituting amino acids for example, amino acids different in hydrophilicity, hydrophobicity or electric charge from the constituent amino acid before substitution.
  • the amino acid before substitution is aspartic acid
  • the substituting amino acids include asparagine, threonine, valine, phenylalanine and arginine, Asparagine and arginine are particularly preferred.
  • the amino acid before substitution is arginine
  • the substituting amino acids include glutamine, threonine, leucine, phenylalanine and aspartic acid.
  • Glutamine is especially preferable.
  • the substituting amino acids include threonine, leucine, phenylalanine, serine, glutamic acid and arginine. Threonine is particularly preferable.
  • the substituting amino acids include methionine, alanine, leucine, cysteine, glutamine, arginine and aspartic acid. In particular, methionine is preferred.
  • the substituting amino acids include serine, leucine, proline, glycine, lysine and aspartic acid. Serine is especially preferred.
  • aspartic acid Aspartic acid, arginine, glycine, serine and valine are preferably selected.
  • substituting amino acids asparagine, glutamine, arginine, threonine, methionine, serine and leucine are preferably selected.
  • the most preferred substituted muteins include a mutein in which cysteine, the constituent amino acid, is replaced by serine.
  • the substitution of at least two constituent amino acids may be simultaneously carried out.
  • Preferred examples of the hbFGF muteins in the present invention include a mutein in which at least one cysteine residue of the mature hbFGF mutein is replaced by a serine residue.
  • recombinant hbFGF mutein CS23 (hereinafter also briefly referred to as rhbFGF mutein CS23) is particularly preferred in which cysteine residues at the 69- and 87- positions of the mature hbFGF are replaced by serine residues, respectively.
  • the position of the amino acids of the above hbFGF are numbered, by counting the amino acid Pro next to Met of the N-terminus of the amino acid sequence as shown in Fig. 1 as the 1st.
  • T7 promoter used in the present invention there may be used any of 17 kinds of promoters discovered on T7 DNA [J. L. Oakley et al., Proc. Natl. Acad. Sci. U.S.A. 74, 4266-4270 (1977); M. D. Rosa, Cell 16, " 815-825 (1979); N. Panayotatos et al., Nature 280, 35 (1979); J. J. Dunn et al., J. Mol. Biol. 166, 477-535 (1983)], but a $Z_>10 promoter [A. H. Rosenberg et al., Gene 56, 125-135 (1987)] is preferably used.
  • any terminator may be used as long as it functions in E. coli systems, but a T ⁇ p terminater [F. W. Studier et al., J. Mol. .Biol. 189, 113-130 (1986) is preferably used.
  • T7 RNA polymerase genes used in the present invention include T7 gene 1. [F. W. Studier et al. , J. Mol. Biol. 189, 113-130 (1986)].
  • vectors from which the vectors used in the present invention are formed include pBR322, pUC8, pUC9, pMB9, pKC7, pACYC177 and pKN410.
  • the vectors used in .the present invention are constructed by incorporating the T7 promoter and the T7 terminate-" into the above vectors.
  • Such vectors include pET-1, pET-2, ⁇ ET-3, pET-4 and pET-5 [A. H. Rosenberg, Gene 56, 125-135 (1987)], but pET-3C (ibid.) is preferably used.
  • pET-1 pET-2, ⁇ ET-3, pET-4 and pET-5
  • pET-3C ibid.
  • any of E ⁇ coli strains into which the T7 RNA polymerase gene (T7 gene 1) [F. W. Studier et al. ,. J. Mol. Biol. 189, 113-130 (1986)-] is incorporated, such as MM294, DH-1, C600 and BL21, may be used.
  • the strains MM294 and BL21 are preferably used in which a ⁇ phage including T7 gene 1 is lysogenized.
  • the T7 RNA polymerase gene can also be harbored as a plasmid having different origin from that of expression vector.
  • the promoter for T7 gene 1 there is used the lac promoter whose expression is induced with isopropyl-1-thio- ⁇ -D-galactopyranoside (IPTG) .
  • IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside
  • the transformants used in the present invention are obtained by transforming the E_ ; _ coli.
  • T7 gene 1 RNA polymerase gene
  • the hosts to be used may be preliminarily transformed with plasmids having the T7 lysozy e gene so that the resulting transformants have two kinds of different plasmids simultaneously.
  • liquid media are particularly suitable as media used for culture.
  • Carbon sources, nitrogen sources, inorganic compounds and others necessary for growth of the transformants are contained therein.
  • the carbon sources include, for example, glucose, dextrin, soluble starch and sucrose.
  • the nitrogen sources include inorganic or organic materials such as ammonium salts, nitrates, corn steep liquor, peptone, casein, " casamino acids, meat extracts, soybean meal and potato extract solution.
  • the inorganic compounds include calcium chloride, sodium dihydrogenphosphate, and magnesium chloride.
  • Yeast extracts, vitamins, growth promoting factors and the like may be further added thereto.
  • the pH of the media is desirably about 6 to 8.
  • the medium used for cultivation of the E_ ⁇ coli transformants there is preferred, for example, M9 medium (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York, 1972) supplemented with glucose and casamino acids.
  • Iron ion sources may be further added to this medium.
  • the iron ion sources are materials which dissociate into iron ions in solution or materials utilized in iron ion form. Such materials include salts of iron, preferably ferrous or ferric inorganic salts such as ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, ferric phosphate and ferric nitrate.
  • the iron ion sources are added in an amount of about 10 -6 to 10-4 M, preferably in an amount of about 5X10 -6 to 5X10 ⁇ 5 M.
  • the cultivation is usually carried out at about 15 to 43°C for about 3 to 72 hours, preferably for about 12 to 48 hours, with aeration or agitation if necessary.
  • IPTG is added in an amount of 0.1 to 20 mM, preferably in an amount of 1 to 2 mM for expression of the lac promoter [up to 1 mM of IPTG: L ⁇ bner-Olesen et al., Cell, 57, 881-889 (1989); 2 mM of IPTG: Ernst H. et al. , Gene, 68, 345-355 (1988)]; 20 mM of IPTG: Luck, D. N.
  • IPTG is added in an amount of about 10 to 500 ⁇ M, preferably 10 to 200 ⁇ M, more preferably 10 to 100 ⁇ M, still more preferably 10 to 80 ⁇ M.
  • IPTG is added in an amount of about 3 to 100 ⁇ M, more preferably 6 to 80 ⁇ M.
  • IPTG is first added about 1 to 24 hours, preferably about 3 to 12 hours after the initiation of cultivation, and it is preferred that IPTG is added on a logarithmic growth phase. IPTG is hereafter added intermittently or continuously as required.
  • the media containing IPTG are cultivated at a temperature of about 20 to 42°C, preferably about 20 to 30°C.-
  • isopropylthiogalactopyranoside there are used in some cases, for example, propylthiogalactopyranoside, methylthiogalactopyranoside, butylthiogalactopyranoside and cyclohexylthiogalactopyranoside.
  • the hbFGF muteins in the present invention can be isolated and purified from the cultures obtained above, for example, by the following methods.
  • the cells are collected after cultivation by methods known in the art, such as centrifugation. Then the collected cells are subjected to disruption by glass beads, French press, ultrasonic treatment, lysozyme treatment and/or freeze-thawing. In particular, the disruption by glass beads is preferable.
  • the crosslinked polysaccharide sulfates used in the present invention include crosslinked cellulose sulfates, crosslinked agarose sulfates and crosslinked dextran sulfates.
  • the above cellulose is a polysaccharide composed of glucose linked by ⁇ -1,4 bonds, and its molecular weight is preferably about 50,000 to 2,000,000. Specific examples thereof include Avicel (crystalline cellulose, Asahi Chemical Industry, Japan) and Cellulofine (Chisso Corporation, Japan).
  • the above agarose is a polysaccharide which is the main component of agar, and has the recurring structure of D-galactosyl-( ⁇ l- ⁇ 4)-3,6-anhydro-L-garactosyl-( ⁇ l-3) .
  • Its molecular weight is preferably about 10,000 to 5,000,000. Specific examples thereof include Sepharose 2B, Sepharose 4B and Sepharose 6B (Pharmacia, Sweden).
  • the above dextran is a D-glucose polymer mainly comprising ⁇ (1 ⁇ 6) bonds formed, for example, by the action of a microorganism such as Leuconostoc mesenteroides on sucrose. Its average molecular weight is preferably about 1,000 to 40,000,000.
  • crosslinked polysaccharide sulfates used in the present invention are prepared by treating crosslinked polysaccharides such as the above dextran, agarose and cellulose with known crosslinking agents such as epichlorohydrin and 2,3- dibromopropanol according to methods known in the art.
  • crosslinked polysaccharides are commercially available and can be purchased from Pharmacia (Sweden) under the trade names of Sephadex G-10, Sephadex G-15, Sephadex G- 25, Sephadex G-50 and Sephadex G-100 (crosslinked dextran), and under the trade names of Sepharose. CL-2B, Sepharose CL- 4B and Sepharose CL-6B (crosslinked agarose). Also, crosslinked cellulose can be purchased from Chisso Corporation (Japan) under the trade name of Cellulofine (crosslinked cellulose).
  • the desired crosslinked polysaccharide sulfates can be synthesized by allowing known sulfating agents, such as chlorosulfonic acid and sulfuric anhydride esters, to react with these crosslinked polysaccharides.
  • sulfating agents such as chlorosulfonic acid and sulfuric anhydride esters
  • examples of the crosslinked cellulose sulfates include the product put on the market by Seikagaku Kogyo (Japan) under the trade name of Sulfated Cellulofine (crosslinked cellulose sulfate).
  • Examples of the crosslinked dextran sulfates include sulfated Sephadex.
  • crosslinked agarose sulfates examples include sulfated Sepharose.
  • the crosslinked polysaccharide sulfates used in the present invention may be in the form of the corresponding salts.
  • the salts include sodium, potassium, ammonium " and trimethylammonium salts.
  • the sodium salts are preferably used.
  • crosslinked polysaccharide sulfates used in the present invention are insoluble in water, and therefore it is preferred to use them in their gelatinous state by hydration.
  • hbFGF mutein-containing aqueous media are solutions containing the hbFGF muteins.
  • the aqueous media include water and media mainly composed of water, and are preferably adjusted to the pH range of about 3 to 10 with buffer solutions such as phosphate buffer, citrate buffer and
  • Tris-hydrochloric acid buffer to prevent inactivation of the hbFGF muteins.
  • the hbFGF mutein-containing solutions are next readjusted to a pH range of about 5.0 to 9.0, and then diluted with distilled water as is required, so that they have an electric conductivity of about 15 mU " or less.
  • the hbFGF mutein-containing solutions thus obtained are brought into contact with crosslinked polysaccharide sulfate gel. For this purpose, both batch and column methods may be used.
  • the column method is however more suitable due to its simple operation.
  • the crosslinked polysaccharide sulfate gel is filled, into a column, and thereafter to equilibrate the column it is thoroughly washed with a suitable buffer solution such as 50 mM citrate buffer (pH 7.0) containing 0.4 M NaCl.
  • a suitable buffer solution such as 50 mM citrate buffer (pH 7.0) containing 0.4 M NaCl.
  • the amount of the gel to be used depends on the nature of the loaded hbFGF mutein-containing solution, but the range of about 1 to 50 ml per mg of hbFGF mutein is preferable.
  • the hbFGF mutein-containing solutions described above are then loaded on the column.
  • the loading speed is selected in the space velocity (SV) range of about 0.1 to 5.0.
  • the column is thoroughly washed, and the ionic strength of the buffer solution is increased by conventional methods to elute and recover the hbFGF muteins.
  • salts such as NaCl are added or buffer solutions high in concentration are used so that the electric conductivity is increased to at least about 15 mU, preferably at least 30 mU * .
  • both batch and concentration gradient elution methods may be used.
  • the concentration gradient elution method for example, the concentration of NaCl is gradually increased from about 0 M to 2.0 M, thereby conducting elution and recovery.
  • highly purified hbFGF muteins can be obtained in high yield.
  • Examples of the synthetic polymers having sulfonic acid groups as exchange groups which are used in the present invention include polymers in which sulfonic acid groups are directly or. indirectly introduced into hydrophilic vinyl polymers, styrene-divinylbenzene polymers, acrylamide polymers and the like.
  • SP sulfopropyl
  • Toyopearl Tosoh, Japan
  • the hbFGF mutein-containing solutions partially purified are adjusted to about 50 mM or less in salt concentration, for example, in the case of phosphate buffer, and allowed to be adsorbed on the above resins within the pH range of about 5 to 7.
  • salt concentration for example, in the case of phosphate buffer
  • both batch and column systems may be used, but the column system is preferably used from the viewpoint of operation.
  • Elution from the resins is carried out by increasing the salt concentration.
  • both batch and concentration gradient methods may be used.
  • a buffer solution prepared by adding NaCl to the above phosphate buffer to a concentration of about 500 mM to 1 M can be used.
  • citrate buffer can be used.
  • the elution is conducted at a temperature of about 1 to 25 C, preferably ' about 1 to 10°C, more preferably about 4 C.
  • Examples of the synthetic polymers for gel filtration used in the present invention include hydrophilic vinyl polymers and acrylamide polymers.
  • Toyopearl HW Tosoh, Japan
  • the hydrophilic vinyl polymer is preferably used from the viewpoints of gel durability and operation.
  • buffer solutions such as phosphate buffer and citrate buffer can be used as developing solvents. It is however advantageous to use 50 mM citrate buffer (pH 7.0).
  • the treatment is conducted at a temperature of about 1 to 25 C, preferably about 1 to 10°C, more preferably 'about 4 C.
  • a method other than the above methods may be used as one of the purifying procedures.
  • natural products such as cellulose, agarose and dextran and inorganic materials such as glass beads can also be used as carriers for ion exchange chromatography.
  • gels mainly composed of natural products such as cellulose, agarose and dextran and gels based on inorganic materials such as glass beads.
  • the samples thus obtained can .also be dialyzed and lyophilized to form dried powders. Further, it is suitable to add serum albumin as a carrier to the samples to store them, because the samples can be prevented from being adsorbed on vessels.
  • the coexistence with trace amounts of reducing agents in the course of purification or storage is suitable to prevent the samples from being oxidized.
  • the reducing agents include S-mercaptoethanol, dithiothreitol and glutathione.
  • substantially pure hbFGF muteins essentially free from pyrogens and endotoxins can be obtained.
  • the substantially pure hbFGF muteins according to the present invention include products which contain the hbFGF muteins according to the present invention in an amount of 95% (w/w) or more as protein content, more preferably in an amount of 98% (w/w) or more.
  • the hbFGF muteins obtained by the above methods of the present invention have fibroblast growth promoting activity, vascular endothelial cell growth promoting activity and angiogenic activity, are high in stability and have low toxicity.
  • hbFGF muteins according to the present invention when used as pharmaceutical preparations, they can be safely administered parenterally or orally to warm ⁇ blooded animals (such as humans, mice, rats, hamsters, rabbits, dogs and cats), in a powder form as such, or as pharmaceutical compositions (such as injections, tablets, capsules, solutions and ointments) with pharmacologically acceptable carriers, excipients and diluents.
  • warm ⁇ blooded animals such as humans, mice, rats, hamsters, rabbits, dogs and cats
  • pharmaceutical compositions such as injections, tablets, capsules, solutions and ointments
  • the injections are prepared by conventional methods using, for example, physiological saline or aqueous solutions containing glucose or other auxiliary agents.
  • the pharmaceutical compositions such as tablets and capsules can also be prepared in accordance with conventional methods.
  • the hbFGF muteins according to the present invention are used as the above pharmaceutical preparations, they are administered, for example, to the above warm ⁇ blooded animals in an appropriate amount ranging from about 1 ng/kg body weight to 100 ⁇ g/kg body weight daily, taking into account the route of administration, symptoms, etc.
  • the hbFGF muteins according to the present invention are used as the reagents for accelerating cell cultivation, they are preferably added to culture media so as to be contained in an amount of about 0.01 to 10 ⁇ g per liter of medium, more preferably in an amount of about o.l to 10 ⁇ g per liter of medium.
  • RNA Ribonucleic acid
  • dATP Deoxyadenosine triphosphate
  • dTTP Deoxythymidine triphosphate
  • dGTP Deoxyguanosine triphosphate
  • dCTP Deoxycytidine triphosphate
  • Trp Tryptophan
  • coli DHl/pTBl004 which harbors plasmid pTBl004 (Example 1) have been deposited with the Institute for Fermentation, Osaka (IF0), Japan, and with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (FRI) , Japan. Their accession numbers and deposit dates are shown in Table 1.
  • the deposit of E. coli MM294/pTB762 in FRI was initially made under accession numbers denoted by FERM P numbers. Said deposit was converted to the deposit under the Budapest Treaty and the transformants have been stored at FRI under accession numbers denoted by FERM BP numbers.
  • Plasmid pTB762 containing .a gene coding for rhbFGF mutein CS23 in which the cysteine residues at positions 69 and 87 of four cysteine residues existing in hbFGF were replaced by serine residues [Senoo et al., Biochemical and - Biophysical Research Communication 151, 701- 708 (1988), European Patent Publication No. 281,822] was treated with EcoRI and PstI to cut out a DNA fragment coding for rhbFGF mutein CS23. Then, plasmid pTBl004 (European Patent Publication No.
  • plasmid pTB921 was Cleaved with restriction enzyme EcoRI, and mungbean (yaenari) nuclease was reacted with the product to change the termini thereof to flush ends, followed by cleavage with restriction enzyme Bglll to prepare a DNA fragment containing the gene coding for rhbFGF mutein CS23.
  • vector pET3C carrying a > 10 promoter for a T7 phage [F. W. Studier et al..
  • E. coli ⁇ M294 was lysogenized with ⁇ hage DE3 in which an RNA polymerase gene of the T7 phage was inserted [F. W. Studier et al.. Journal of Molecular Biology 189, 113-130 (1986)] to prepare E_ ; _ coli strain MM294(DE3).
  • This E. coli strain was transformed by using the expression plasmid pTB960, thereby obtaining recombinant
  • Example 2 E. coli MM294(DE3)/pTB960 (IFO 14979, FERM BP-2690) carrying the gene coding for rhbFGF mutein CS23 shown in Fig. 1.
  • Two identical samples were prepared. To one sample was added 7 M guanidine hydrochloride, and the cells were sufficiently loosened, followed by standing for 1 hour. Then, a supernatant was obtained by centrifugation (total amount of bFGF mutein) . To the other sample was added a 50 ⁇ g/ml lysozyme solution (10% sucrose, 10 mM EDTA, 100 mM NaCl, 1 mM APMSF and 10 mM Tris-HCl, pH 7.6), and the mixture was allowed to stand at 4°C for 1 hour.
  • a 50 ⁇ g/ml lysozyme solution (10% sucrose, 10 mM EDTA, 100 mM NaCl, 1 mM APMSF and 10 mM Tris-HCl, pH 7.6
  • the effect of the present invention can be obtained in the range of small amounts of added IPTG. Namely, about 1 mM of IPTG was added to induce the lac promoter. When IPTG was added in an amount of 100 ⁇ M or more, the productivity was low and most of the desired product was accumulated in an insoluble state. In contrast, according to the present invention in which IPTG was added within a range from 3 ⁇ M to 100 ⁇ M, especially 6 ⁇ M to 80 ⁇ M, ther were obtained the entirely unexpected results of significant improvements in productivity and also in ratio of the mutein accumulated as a soluble protein.
  • M-9 medium (pH 6.8) containing 15 g/1 of glucose, 15 g/1 of casamino acids and' 5 mg/1 of thiamine hydrochloride was placed in an amount of 2.5 liter in a 5 liter jar fermentor, and sterilized. Then, the medium was inoculated with 125 ml of a strain culture solution prepared in the manner described in Example 2, followed by cultivation adjusting the pH to 6.8, at 37°C, with aeration at a rate of 2.5 1/min and with stirring at 1,000 rp . When the turbidity reached 120 Klett units during cultivation (3 hours after the initiation of cultivation) , IPTG was added so as to be contained in an amount of 10 ⁇ M.
  • a seed culture solution prepared in the manner described in Example 2 was transferred to a 5 liter jar fermentor in which the same medium as with Example 3 was placed, and the cultivation was initiated at 30 C, at pH 6.8, with aeration at a rate of 2.5 1/min and with stirring at 1,000 rpm.
  • the turbidity reached about 700 Klett units 7 hours after the initiation of cultivation, 42 ⁇ M of IPTG was added.
  • 20 g/1 of glucose and 20 g/1 of casamino acids were added at pH 6.8.
  • the cultivation was carried out for 36 hours.
  • 860 mg/1 of rhbFGF mutein CS23 was accumulated in a soluble state.
  • Example 5 M-9 medium (pH 6.8) containing 15 g/1 of glucose, 15 g/1 of casamino acids and 5 mg/1 of thiamine hydrochloride was placed in an amount of 2.5 liter in a 5 liter jar fermentor, and sterilized. Then, iron ions were added thereto at concentrations shown in Table 3 after sterilization by filtration. The resulting medium was inoculated with 125 ml portions of a strain culture solution prepared in the manner described in Example 2, followed by cultivation adjusting the pH to 6.8, at 30°C, with aeration at a rate of 2.5 1/min and with stirring at 1,000 rpm.
  • IPTG IPTG was added so as to be contained in an amount of 42 ⁇ M, and at the same time the cultivation temperature was lowered to 25°C, followed ' by cultivation for 23.5 hours. 7 to 7.5 hours after the initiation of cultivation, each of glucose and casamino acids was added in a ratio of 15 g/1, and the pH was maintained at 6.8 during cultivation.
  • Table 3 The results are shown in Table 3.
  • the amount of added iron ions indicates the amount of iron ions further added to the medium.
  • the productivity indicates the weight ratio taking as 1 when the amount of added iron ions is 0.
  • the eluate was concentrated using an ultrafilter (Pellicon Casset System, Millipore, U.S.A.) until the absorbance at 280 nm became 3 to 4.
  • the resulting concentrated solution was dialyzed against about 60 liter of 25 mM phosphate buffer (pH 6.0) overnight.
  • the dialysate was centrifuged with a centrifuge (Beckman, U.S.A.) at 4,200 rpm for 30 minutes to give a supernatant.
  • the supernatant was poured into a column (17.0 cm ID X 33.0 cm) of SP-Toyopearl 650M (Tosoh, Japan).
  • the column was washed with 7 liter of 25 mM phosphate buffer (pH 6.0) containing 200 mM NaCl, and then elution was conducted using 10 liter of 25 mM phosphate buffer (pH 6.0) containing 500 mM NaCl.
  • the eluate was poured into a column (15.0 cm ID X 50.0 cm) of Sulfated Cellulofine (Seikagaku Kogyo) using a high performance liquid chromatography apparatus (Gilson, France). Then, elution was effected by a linear gradient between 20 liter of 50 mM citrate buffer (pH 7.0) and 30 liter of 50 mM citrate buffer (pH 7.0) containing 2 M NaCl. The main fractions were collected, and diluted with 10 mM citrate buffer (pH 6.0) to a conductivity of 20 mmho or less. The diluted solution was poured into a column
  • FIG. 3 shows the results of SDS-PAGE obtained under reducing conditions (100 mM. DTT, 50 C, 15 minutes).
  • (1) and (2) represent the results for the marker and rhbFGF mutein CS23, respectively.
  • Fig. 4 shows the result of high performance liquid chromatography using a column (7.5 mm ID X 7.5 cm) having heparin-5PW (Tosoh, Japan) as a carrier under the following conditions:
  • Fig. 5 shows the result of high performance liquid chromatography using a column (4.6 mm ID X 150 cm) having ODP-50 (Asahi Chemical Industry, Japan) as a carrier under the following conditions:
  • Fig. 6 shows the result of high performance liquid chromatography using a column (7.5 mm ID X 7.5 cm) having G2000SW (Tosoh, Japan) as a carrier under the following conditions: Developing solution: 0.1 M phosphate buffer (pH 7)
  • European Patent Publication No. 281,822 was completely digested with Aval and PstI to obtain a DNA fragment of about 0.45 kbase pairs containing most of rhbFGF mutein CS23.
  • a synthetic DNA GATCTGC ) was ligated to
  • pHP901 was completely digested with EcoRI and Bglll to obtain a fragment of about 1.1 kbases containing a gene coding for rhbFGF mutein CS23, T7 promoter and T7 terminator.
  • the fragment was ligated to pUCl ⁇ completely digested with EcoRI and BamHl by T4DNA ligase to obtain plasmid pME901 (Fig. 8).
  • pME901 was completely digested with EcoRV and Hindlll to obtain a fragment of about 0.77 kbases containing a gene coding for rhbFGF mutein CS23, T7 promoter and T7 terminator.
  • the fragment was incubated with T4DNA polymerase to change the termini thereof to flush ends.
  • the fragment was ligated to pBR322 digested with Seal by T4DNA ligase to obtain an expression plasmid pCM901 containing a tetracycline marker (Fig. 9).
  • E. coli ⁇ M294 was lysogenized with phage DE3 in which an RNA polymerase gene of the T7 phage was inserted [F. W. Studier et al.. Journal of Molecular Biology 189, 113-130 (1986)3 to prepare E. ' coli strain MM294(DE3).
  • This E. coli strain was transformed by using the expression plasmid pCM901, thereby obtaining recombinant E. coli MM294(DE3)/ ⁇ CM901 (IFO 15104, FERM BP-3168) carrying the gene coding for rhbFGF mutein CS23.

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Abstract

On décrit (1) un vecteur comprenant une séquence nucléotidique codant pour une mutéine dans laquelle au moins un constituant aminoacide du facteur mûr de croissance fibroblastique basique de l'homme (hbFGF) est remplacé par un autre aminoacide, ainsi qu'un promoteur T7 situé en amont de ladite séquence; (2) un transformant transformé par le vecteur de (1); (3) le transformant de (2), dans lequel un hôte est E. coli ayant un gène de polymérase d'ARN T7 en aval d'un promoteur de laque; (4) un procédé de production de la mutéine dans laquelle au moins un constituant aminoacide du hbFGF mûr est remplacé par un autre aminoacide, consistant à cultiver le transformant de (2) dans un milieu de culture; (5) le procédé de (4), selon lequel l'on ajoute au milieu de culture de 3 à 500 mum d'isopropylthiogalactopyranoside dans une phase exponentielle de croissance du transformant de (3), puis on effectue la culture; et (6) le procédé de (5), selon lequel l'on purifie par chromatographie une solution résultante contenant de la mutéine à l'aide d'un sulfate de polysaccharide réticulé, d'un polymère synthétique possédant un groupe d'acide sulfonique en tant que groupe échangeur et/ou d'un polymère synthétique de filtration par gel servant de porteur, la mutéine de hbFGF à activité biologique pouvant ainsi être efficacement obtenue.
EP91900328A 1989-12-19 1990-12-18 Production d'une muteine du facteur de croissance fibroblastique basique de l'homme Withdrawn EP0506963A1 (fr)

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KR (1) KR920703789A (fr)
CN (1) CN1055009A (fr)
CA (1) CA2070989A1 (fr)
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KR100374310B1 (ko) * 1995-02-14 2003-05-22 주식회사 엘지생명과학 변형된인간염기성섬유아세포성장인자및이의생산방법
US6274712B1 (en) 1997-12-23 2001-08-14 3-Dimensional Pharmaceuticals, Inc. Analogs of human basic fibroblast growth factor mutated at one or more of the positions glutamute 89, aspartate 101 or leucine 137
WO2002042336A2 (fr) * 2000-11-21 2002-05-30 The Texas A & M University System Chromatographie d'affinite avec fgf
WO2007035778A2 (fr) 2005-09-19 2007-03-29 Histogenics Corporation Matrice support de cellules dont la densite des pores et la porosite sont definies specifiquement de maniere uniforme verticalement et organisees de maniere non aleatoire, et procede de preparation de celle-ci
US8962556B2 (en) 2006-09-28 2015-02-24 Prochon Biotech Ltd. FGF-2 variants having N-terminal deletions and increased receptor selectivity and uses thereof
US10077420B2 (en) 2014-12-02 2018-09-18 Histogenics Corporation Cell and tissue culture container
DE102021211272A1 (de) 2021-10-06 2023-04-06 Ruhr-Universität Bochum, Körperschaft des öffentlichen Rechts Zusammensetzung enthaltend künstliche Sauerstoffträger zur Verhinderung von Organschäden in Transplantationsorganen

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EP0178863A1 (fr) * 1984-10-15 1986-04-23 Schering Corporation Systèmes d'expression utilisant des promoteurs de bactériophage T7 et des séquences de gènes
DE3723992A1 (de) * 1987-07-20 1989-02-02 Boehringer Mannheim Gmbh Verfahren zur herstellung von proteinen in loeslicher form
KR890701607A (ko) * 1987-11-24 1989-12-21 원본미기재 섬유아세포 성장 인자의 유사체
EP0326907A1 (fr) * 1988-01-26 1989-08-09 Takeda Chemical Industries, Ltd. Polypeptide, DNA et leur utilisation

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See references of WO9109126A1 *

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JPH05503006A (ja) 1993-05-27
JP3130313B2 (ja) 2001-01-31
CN1055009A (zh) 1991-10-02
HU9202039D0 (en) 1992-09-28
KR920703789A (ko) 1992-12-18
CA2070989A1 (fr) 1991-06-20
WO1991009126A1 (fr) 1991-06-27

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