HUT64593A - Method for producing human fgf muteine - Google Patents

Description

The present invention relates to a method for the production of a mutein of the human basic fibroblast growth factor (hereinafter only referred to as hbFGF), which replaces at least one amino acid of hbFGF with another amino acid and serves to promote the healing of mutein wounds.

Basic Fibroblast Growth Factor (bFGF) is a basic polypeptide with a molecular weight of about 17,000 and is mainly excreted by the pituitary gland. BFGF was first isolated as a factor with potent growth promoting effects on fibroblasts, such as BALB / C3T3 cells [D. Gospodarowicz et al., Natura, 249, 123 (1974)]. It is known that FGF has growth promoting effects on almost all mesoblast-derived cells [D. Gospodarowicz et al., National Cancer Institute Monograph, 48, 109 (1978)]. In particular, the angiogenic factor of bFGF, together with its cell growth stimulating effect, suggests that it may be used in drugs for the treatment of trauma, and may be a preventive and curative agent for thrombosis, arteriosclerosis, and the like. In the case.

The genes encoding hbFGF were cloned and expressed in animal cells by Abraham et al. (1986) The EMBO Journal 5: 2523-2528 and Kurokawa et al. (FEBS Letters 213: 189-194 (1987)). 213: 189-194 (1987)], in yeast (The Journal of Biological Chemistry, 263: 16471-16478 (1988)), and in Escherichia coli (The Journal of Biological Chemistry, 263: 16279-16302 (1988)). However, their production levels are not always sufficient and the resulting samples are very unstable to be used as a medicine. Different,

3 studies of instability have shown that substances in which the cysteine moieties of hbFGF molecules are replaced by serine moieties are more stable and have the same biological activity (Biochemical and Biophysical Research Communications, 1988, 151, 701-708). .

In addition, a method has been developed for the production of muteins in which at least one amino acid of hbFGF has been replaced by genetic engineering methods (EP 281 822).

When recombinant proteins are produced with Escherichia coli, often water-insoluble inclusion bodies, called inclusion bodies, are formed, in which the proteins accumulate. When the desired substances are isolated from such inclusions, the inclusions are generally resolved by addition of protein denaturing agents. However, as far as hbFGF muteins are concerned, the proteins thus solubilized are inactive and no technique has yet been developed for their reactivation.

HbFGF muteins can be used as medicaments if the muteins accumulate in large amounts in an active state as a result of genetic engineering techniques and are further isolated and purified in very good yields. Therefore, it is very important to develop methods that produce high frequency muteins.

Generally, when large quantities of gene products are produced by genetic engineering techniques, the choice of host-vector systems and promoters is very important and depends on the individual genes. Examination of various expression systems for efficient expression of hbFGF muteins ···· revealed that an Escherichia coli expression system using the T7 promoter [F. W. Studier et al., Journal of Molecular Biology, 189: 113-130 (1986)], is an excellent expression system for the expression of hbFGF muteins. The combination of the T7 promoter and hbFGF muteins is novel. This T7 promoter is known to be a strong promoter. However, in some cases, such as interleukin-2, prolactin, inclusions occur and accumulate in most inactive states. The authors of the present invention have described the case of interleukin-2, while the case of prolactin has been described by Paris, N., et al., 1990, Biotechnology and Applied Biochemistry, 12, 436-449.

The present inventors have conducted intensive studies to accumulate large amounts of hbFGF mutein in the active state and have found that administration of small amounts of isopropylthio-galactopyranoside is effective. The present inventors have further developed a method for efficiently isolating and purifying the hbFGF mutein, thereby completing the present invention.

The present invention thus relates to:

(1) a vector comprising a nucleotide sequence encoding a mutein in which at least one amino acid of the mature human basic fibroblast growth factor (hbFGF) is replaced by another amino acid and is preceded by a T7 promoter;

(2) a transformant transformed with the vector described in (1);

(3) the transformant described in (2), characterized in that a T7 RNA polymerase is present in the host Escherichia coli. · · ·

- 5 genes inserted after a lac promoter;

(4) a method for producing a mutein in which at least one amino acid of the mature hbFGF is replaced by another amino acid, wherein the transformant described in (2) is cultured in culture medium;

(5) the method described in (4), characterized in that about 3-500 μπιοΐ / l isopropylthio-galactopyranoside (hereinafter briefly referred to as IPTG) is added to the culture of the transformant in the logarithmic growth phase described in (3), and (6) the method described in (5), characterized in that the resulting mutein-containing solution is purified by chromatography on a cross-linked polysaccharide sulfate (a synthetic polymer containing sulfonic acid as ion-exchange group) and a synthetic polymer using a support as a support. .

According to the present invention, substitution-type muteins of mature hbFGFs can be efficiently produced. Thus, the present invention is advantageously applicable to the production of muteins on an industrial scale.

The figures are briefly described below.

Figure 1 is a CS23 rhbFGF mutein used in Example 1

The nucleotide sequence of the DNA as well as the amino acid sequence of the protein encoded by the nucleotide sequence are shown.

Figure 2 is a schematic diagram of the construction of plasmid pTB960 used in Example 1.

Figure 3 is a purified sample prepared in Example 5

An SDS-PAGE pattern and a marker can be seen.

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·erateerate

- 6 Α 4-6. Figures 1 to 5 show the results of HPLC purification of the purified sample prepared in Example 5.

7-9. Figures 1 to 8 show a schematic diagram of the construction of plasmids pHP901, pME901 and pCM901 produced in Example 8.

The mature hbFGF of the present invention is a peptide containing 146 amino acids, with Pro as the first amino acid behind the N-terminal Met and 146 as the amino acid Ser at the C-terminus.

The hbFGF muteins of the present invention include muteins in which at least one amino acid of the mature hbFGF is replaced by another amino acid (EP 281,922; Biochemical and Biophysical Research Communications, 151: 701-708 (1988)].

The number of hbFGF-forming amino acids in the mutein in which at least one of the hbFGF-forming amino acids is replaced is immaterial, provided that the characteristics of FGF, i.e., angiogenesis, cell growth stimulating and cell differentiation stimulating effects should not be lost.

The amino acids to be replaced include, for example, cysteine as well as any non-cysteine amino acid. However, cysteine is preferred. Non-cysteine amino acids include, for example, aspartic acid, arginine, glycine and valine.

When cysteine is substituted, neutral amino acids are preferred as the replacement amino acid. Examples of neutral amino acids include glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, histidine, tryptophan, serine, threonine and methionine. But especially serine and threonine are preferred.

If the amino acid to be substituted is not a cysteine, the amino acid to be substituted is selected in a different manner, for example, a hydrophilic, hydrophobic, or electrically charged amino acid other than the amino acid to be replaced. Specifically, if the amino acid to be substituted is aspartic acid, the amino acid to be substituted is asparagine, threonine, valine, phenylalanine and arginine. Especially asparagine and arginine are preferred.

When the amino acid to be substituted is arginine, the amino acid substitutent may be glutamine, threonine, leucine, phenylalanine and aspartic acid. Glutamine is particularly preferred.

When the amino acid to be substituted is glycine, the replacement amino acid may be threonine, leucine, phenylalanine, serine, glutamic acid and arginine. Threonine is particularly preferred.

When the amino acid to be substituted is serine, the replacement amino acid may be methionine, alanine, leucine, cysteine, glutamine, arginine and aspartic acid. Especially methionine is preferred.

When the amino acid to be substituted is valine, the replacement amino acid may be serine, leucine, proline, glycine, lysine and aspartic acid. Serine is particularly preferred.

Preferred amino acids to be substituted are aspartic acid, arginine, glycine, a and valine.

Preferred replacement amino acids are asparagine, glutamine, arginine, threonine, methionine, serine and leucine.

Most preferred are substituted muteins in which the cysteine amino acid is replaced by serine.

• · «• · · · · · · · · · · · · · · · · · · · ·

In the above substitution, at least two amino acids may be substituted simultaneously. It is particularly preferred that two or more amino acids are substituted

Preferred hbFGF muteins of the present invention include those in which at least a cysteine moiety of the mature hbFGF mutein is replaced by a serine moiety.

Of the muteins, particularly the CS23 recombinant hbFGF mutein (hereinafter only briefly referred to as GS23 rhbFGF mutein) is preferred, wherein the cysteine residues at positions 69 and 87 of the mature hbFGF mutein are replaced by serine residues. The amino acids of the aforementioned hbFGF are numbered, each number being assigned to the Pro following the N-terminal Met according to the sequence shown in Figure 1.

Site-specific mutagenesis is performed to produce muteins. This technique is well known, e.g., R.F. Lather and J.P. Lecoq (Genetic Engineering, 31-50, Academic Press (1983)). Oligonucleotide mutagenesis is described by M. Smith and S. Gillam (Genetic Engineering, Principles and Methods, 3, 1-32, Plenum Press, 1981).

For example, the construction of a mutein-encoding structural gene is carried out as follows:

(a) hybridizing the single-stranded DNA comprising one strand of the structural gene of hbFGF with a mutagenic oligonucleotide primer (said primer complementary to the region encoding the cysteine to be replaced or containing an antisense triplet which in some cases forms a pair with this codon, provided that this does not cause problems parity with other amino acid codon, or a few ·· "♦ • · · · · · • ··« _ν · «·;

* ······ « _# • · · · · · · · · · · ·

(In 9 cases the antisense triplet), (b) extending the primer molecule by DNA polymerase to obtain a mutant heteroduplex, and (c) replicating this mutant heteroduplex.

Phage DNA is then isolated and inserted into a plasmid to transfer the mutagenized gene.

As for the T7 promoter used in the present invention, any of the 17 types of promoters discovered on T7 DNA can be used [J.L. Oakley, Proceedings of the National Academy of Sciences, USA, 74, 4266-4270 (1977); D. Rosa, Cell, 16, 815-825 (1979); Panayotatos N. et al. (1979) 280: 35; J.J. Dunn et al., Journal of Molecular Biology, 166, 477-535 (1983), but also a phage FI10 promoter (Rosenberg et al., Gene 56, 125-135 (1987)) is preferred.

Any terminator may be used as a transcriptional terminator in the present invention, provided it is functional in Escherichia coli systems but is a T1 terminator [F.W. Studier et al., 1986, Journal of Molecular Biology 189: 113-130].

T7 RNA polymerase genes used in the practice of the present invention include the T7 gene [F.W. Studier et al., 1986, Journal of Molecular Biology, 189, 113-130.

The vectors used in the practice of the present invention are pBR322, pUC8, pUC9, pMB9, pKC7, pACYC177 and pKN410.

For use in the present invention, the vectors are prepared by incorporating the T7 promoter and T7 terminator into the above vectors. Examples of such vectors are pET-1, pET-2, pET-3, pET-4 and pET-5 [Rosenberg et al., Gene, 56, 125-135.

10 (1987)], but pET-3C may also be used (Rosenberg et al., Gene 56: 125-135 (1987)).

In the practice of the present invention, any Escherichia coli strain incorporating the T7 RNA polymerase gene (T7 gene 1) can be used as a host for the production of transformants [F.W. Studier et al., Journal of Molecular Biology, 189, 113-130 (1986)] such as MM294, DH-1, C600 and BL21. MM294 and BL21 strains, in which a lambda phage, with which a T7 1 gene is lysogenized, may be used in advance. The T7 RNA polymerase gene can also be readily recovered because the plasmid has an origin of replication other than its expression vector. In this case, for the T7 gene 1, the lac promoter is used, the expression of which is induced by isopropylthiogalactoside (IPTG).

The transformants used in the present invention are prepared by transforming strains of Escherichia coli into which said T7 1 gene (RNA polymerase gene) is incorporated, together with plasmids containing a gene transcription terminator for expression of the T7 promoter. , by methods known to those skilled in the art, such as those described in Proceedings of the National Academy of Sciences, USA, 69, 2110 (1972); and Gene, 17, 107 (1982). In this case, the host to be used is pre-transformed with plasmids containing the T7 lysozyme gene, so that the resulting transformants contain simultaneously two different plasmids.

If the transformants are cultivated, then the cultivation is · · «

Liquid media are particularly suitable for n. The medium contains a carbon source, a nitrogen source, inorganic compounds and other substances essential for the growth of transformants. Examples of carbon sources include glucose, dextrin, soluble starch and sucrose. The nitrogen source may be a solution of an inorganic or organic material, such as ammonium salts, nitrates, corn jam, peptone, casein, caseinic acid, meat extract, soybean meal and potato extract. The inorganic compound may be calcium chloride, sodium dihydrogen phosphate and magnesium chloride. In addition, yeast extract, vitamins, growth-promoting factors and the like can be added to the medium.

The pH of the medium is preferably between 6 and 8.

The medium for culturing Escherichia coli transformants is preferably M9 medium (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring).

Harbor Laboratory, New York (1972)], supplemented with glucose and casino acids. Even iron ions can be added to this medium. Sources of iron ions are substances that dissociate into iron ions in solution, or substances that can be used in iron form. Such materials are, for example, iron salts, preferably inorganic ferro or ferric salts, such as iron (II) chloride, iron (III) chloride, iron (II) sulfate, iron (III) sulfate, iron (III) phosphate and the like. iron (III) nitrate. The iron ion source is used at a concentration of 10 µm to 10 µm 4 mol / l, preferably at a concentration of about 5 x ^{10 -6 to} 5 x 10 ~ ⁵ mol / l. The culturing is generally carried out at 15-43 ° C for about 3-72 hours, preferably about 12-48 hours, if necessary with aeration or stirring.

It is advantageous to add IPTG to the system for expression of the gene. Thus, the T7 gene (RNA polymerase gene) ligated behind the lac promoter is expressed and the resulting T7 RNA polymerase specifically recognizes the T7 promoter. In the literature, IPTG is added at a concentration of 0.1 to 20 mM, preferably 1 to 2 mM, to express the lac promoter (up to 1 mM IPTG, Lobner-Olesen et al. Cell, 57: 881-889 (1989); 2 mM IPTG: Ernst H. et al., Gene.68, 345-355 (1988); 20 mM IPTG: Luck, D.N. et al., DNA, 5, 21-28 (1986)]. However, it has been discovered that proteins accumulated during expression induced by the addition of this amount of IPTG form inclusions and often result in the accumulation of inactive proteins. Based on the results of various studies on the accumulation in the dissolved state, it has been discovered that the goal can be achieved by first starting with 3-500 μιηοΐ / ΐ, preferably about 3-300 μιηοΐ / lt, more preferably 6-200 μιηοΐ / lt, and most preferably 6-80 μπιοί / ΐ- IPTG is added to the system during culture. When cultured in a high volume, such as a tank, it is preferable to add the IPTG to the system in an amount of 10-500 mol / l, more preferably 10-20θ | μιηο1 / 1, most preferably 10-80 μιηοΐ / ΐ. When cultivating in small amounts, for example in flasks, the IPTG is preferably used in an amount of 3-100 μπιο1 / 1, more preferably 6-80 μιιοΐ / ΐ. IPTG is first described in Figures 1-24. hours, preferably 3 to 12 hours after commencement of culture. hours, and is preferably used in the logarithmic growth phase. The IPTG can be administered intermittently or continuously as needed

According to. Media containing IPTG are incubated at 20-42 ° C, preferably 20-30 ° C. In some cases, for example, propylthio-galactopyranoside, methylthiogalactopyranoside, butylthio-galactopyranoside and cyclohexylthio-galactopyranoside may be used instead of isopropylthio-galactopyranoside.

The hbFGF muteins of the present invention can be isolated and purified from the cultures prepared as described above, for example, by the following methods.

When the hbFGF muteins of the present invention are extracted from cultured cells, the cells are cultured after culture by methods known to those skilled in the art, such as centrifugation. The recovered cells can then be recovered by glass beads, French-press, lysozyme treatment and / or freeze-thawing. More particularly, digestion with glass beads is advantageous.

Various methods for purifying the muteins of the present invention from the supernatant have been studied. As a result, high purity samples were obtained in large quantities by combining at least once with affinity chromatography using crosslinked polysaccharide sulfate as carrier, ion exchange chromatography in which the carrier is a synthetic polymer containing a sulfonic acid moiety, and the carrier being a synthetic polymer.

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 consisting of glucose molecules linked by β-1,4 bonds, preferably having a molecular weight of between 50,000 and 2,000,000. The individual materials may be, for example, Avicel (crystalline cellulose, Asahi Chemical Industry, Japan) or Cellulofine (Chisso Corporation, Japan).

The aforementioned agarose is a polysaccharide which is a major component of agar and in which D-galactosyl- (β 1-4) -3,6-anhydro-L-galactosyl- (al-3). Preferably, the molecular weight is between about 10,000 and 5,000,000. The individual material may be Sepharose 2B, Sepharose 6B (Pharmacia, Sweden).

The dextran mentioned above is a D-glucose polymer which contains predominantly the bonds (1-6) and is formed from glucose by microorganisms such as Leuconostoc mesenteroides. Preferably, its molecular weight ranges from 1,000 to 40,000,000.

The crosslinked polysaccharide sulfates used in the present invention are prepared from crosslinked polysaccharides, such as dextran, agarose and cellulose, as described above, by treatment with known crosslinking agents, such as 2,3-dibromopropanol, known to those skilled in the art.

Cross-linked polysaccharides are commercially available from Pharmacia (Sweden), Sephadex G10, Sephadex G-15, Sephadex G-25, Sephadex G-50 and Sephadex G-100 (Cross-linked dextran), and Sepharose CL-2B, Sepharose Under the trade names CL-4B and Sepharose CL-6B (Cross-linked Agarose). Cross-linked cellulose is also available from Chisso Corporation (Japan) under the trade name Cellulofine (cross-linked cellulose). Desired cross-linked

Polysaccharide sulfates can be prepared by reacting known sulfating agents such as chlorosulfonic acid and sulfuric anhydride esters with these crosslinked polysaccharides.

Cross-linked cellulose sulfate can be, for example, sold by Seikagaku Kogyo (Japan) under the trade name Sulfated Cellulofine (cross-linked cellulose sulfate).

The cross-linked dextran sulfate may be, for example, sulfated Sephadex.

The crosslinked agarose sulfate may be, for example, sulfated Sepharose.

The crosslinked polysaccharide sulfates used in the present invention may be in the form of the corresponding salts. Salts include, for example, sodium, potassium, ammonium and trimethylammonium salts. More specifically, the sodium salts are preferred.

The crosslinked polysaccharide sulfates used in the present invention are insoluble in water and are therefore preferably applied in a gel form after hydration.

Methods utilizing cross-linked polysaccharide sulfates for purifying hbFGF muteins in the present invention include, for example, affinity chromatography as described below.

Aqueous media containing hbFGF mutein are solutions containing hbFGF muteins. Aqueous media include, for example, water, and aqueous media, and are preferably adjusted to pH 3 to 10 with buffer solutions such as phosphate buffer, citrate buffer and TRIS-HCl buffer to prevent inactivation of hbFGF muteins.

• · · · · · · · · · · · · · · ·

The pH of the solutions containing hbFGF muteins is then adjusted to 5.09.0 and diluted with distilled water as required to have an electrical conductivity of about 15 milliohm or less. The thus prepared solutions containing hbFGF mutein are contacted with a crosslinked polysaccharide sulfate gel. Both flask and column methods can be used for this purpose. However, the columnar method is more appropriate because it is simpler. In the column method, the crosslinked polysaccharide sulfate gel is loaded into a column and washed thoroughly with an appropriate buffer, such as 50 mM citrate buffer, pH 7.0, 0.4 M to equilibrate the column. Contains NaCl. The amount of gel used will depend on the nature of the solution containing the charged hbFGF mutein, but 1-50 ml per mg hbFGF mutein is preferred.

The solutions containing the hbFGF mutein described above are then applied to the column applied. The application rate varies between about 0.15.0 vol. After loading, the column is washed extensively and the ionic strength of the buffer solution is increased by conventional means to elute and recover the hbFGF muteins. In order to increase the ionic strength, salts such as NaCl or high concentration buffer solutions are added in an amount such that the electrical conductivity increases to at least about 15 million, preferably at least about 30 million. Both batch and concentration gradient elution methods can be used for elution. When concentration gradient elution methods are used, for example, the NaCl concentration is increased from 0 mol / l to 2.0 mol / l to elute and recover. In this way highly purified hbFGF muteins can be produced.

Sulfonic acid-containing synthetic polymers used in the present invention include those in which the sulfonic acid groups are incorporated directly or indirectly into hydrophilic vinyl polymers, styrene-divinylbenzene polymers, acrylamide polymers and the like. More specifically, SP (sulfopropyl) -Toyopearl (Tosoh, Japan), wherein the sulfonic acid groups are introduced into a hydrophilic vinyl polymer, is advantageously used for recovery and operations.

During chromatography, partially purified solutions of hbFGF muteins are adjusted to a salt concentration of about 50 mmol / l or less, for example in phosphate buffer, and then allowed to adsorb to the above resin at a pH of about 5-7. Both batch and column systems can be used for adsorption, but the column system may be more advantageous for operation. Elution from the resins is accomplished by increasing the salt concentration. Both batch and concentration gradient methods can be used for elution. If the batch method is used, a buffer solution is prepared by adding NaCl to the above phosphate buffer in a concentration range of about 500 mM to 1 M. Citrate buffer may be used instead of phosphate buffer. Elution is carried out at about 1 to about 25 ° C, preferably at about 1 to about 10 ° C, more preferably at about 4 ° C.

In the present invention, if the synthetic polymers used for gel filtration can be vinyl polymers and acrylamide.

- 18 polymers. More specifically, Toyopearl HW (Tosoh, Japan), a hydrophilic vinyl polymer, is advantageously used for gel durability and operation.

When solutions containing partially purified hbFGF muteins are treated, for example, on a Toyopearl HW-50F column, buffer solutions such as phosphate buffer and citrate buffer can be used as development solutions. However, it is preferable to use 50 mM citrate buffer (pH 7.0). The treatment is carried out at about 1 to about 25 ° C, preferably at about 1 to about 10 ° C, more preferably about 4 ° C.

In addition to the methods described above, other methods may be used in the purification process. Such methods include, for example, natural products such as cellulose, agarose and dextran, as well as inorganic materials such as glass beads as carriers for ion exchange chromatography.

Similarly, gels comprising mainly natural materials such as cellulose, agarose and dextran gels based on inorganic materials such as glass beads may be used as carriers for gel filtration.

The samples thus obtained can be dialyzed and lyophilized to give dried powders. Further, it is convenient to add serum albumin to the samples as a carrier for storage to prevent the samples from adsorbing to the side of the container.

If trace amounts of reducing agents are present during purification or storage, it is useful to prevent oxidation of the samples. The reducing agent can be β-mercaptoethanol, dithiothreitol and glutathione.

• · · ··· ··· ·;

• · * ··· · · · · · · · · · · · · · · ·

According to the present invention, essentially pure hbFGF muteins can be prepared, essentially without pyrogens and endotoxins. The substantially pure hbFGF muteins of the present invention may be products containing 95% or more of the hbFGF muteins of the present invention, more preferably 98% or more.

The hbFGF muteins of the present invention obtained as described above have fibroblast growth promoting activity, vascular endothelial cell growth promoting activity and angiogenic activity, and have low toxicity. Therefore, they can be used as substances having a curative effect on burns, wounds, post-operative tissues and the like, or as therapeutic agents based on their angiogenic activity against thrombosis, arteriosclerosis and the like. They can also be used as growth promoters for cell cultures. More specifically, mutein is preferred in which at least one of the cysteine residues in the original protein is replaced by a serine moiety because of its greater stability.

When used as pharmaceutical compositions, the hbFGF muteins of the present invention can be safely administered parenterally or orally to warm-blooded animals (such as humans, mice, rats, hamsters, rabbits, dogs and cats) in powder form or as pharmaceutical formulations (e.g. , solutions and ointments) with pharmaceutically acceptable carriers, excipients and diluents.

♦ β>

- 20 ~

Injections are prepared by conventional techniques such as physiological saline or aqueous solutions of glucose or other excipients. Other pharmaceutical preparations, such as tablets and capsules, may also be prepared using conventional techniques.

When used in said pharmaceutical compositions, the hbFGF muteins of the present invention may be administered to said warm-blooded animals in an amount ranging from about 1 ng / kg body weight to 100 ug / kg body weight per day, depending on the route of administration, symptoms, etc.

Further, when the hbFGF muteins of the present invention are used as reagents to accelerate cell proliferation, they are preferably added to the culture media in an amount of about 0.01-10 pg / l of medium, more preferably about 0.1-10 pg / l of medium.

Abbreviations used to indicate bases, amino acids, and other such molecules in the specification and the drawings are those generally accepted by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used by one of ordinary skill in the art.

For example, the following abbreviations are used. If the optical isomer also occurs at amino acids, the abbreviation denotes the L-form unless otherwise indicated.

DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid

A: adenin • 9 «· V • * • · · · · · · · 9 ·· ·

Τ: thymine G: guanine C: cytosine RNA: ribonucleic acid dATP: deoxyadenosine triphosphate TTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate dCTP: deoxycytidine triphosphate ATP: adenosine triphosphate TDR: thymidine EDTA: EDTA SDS: sodium dodecyl sulfate Gly: glycine below: alanine With: valine Leu: leucine Ile: isoleucine Ser: serine Thr: threonine Cys: cysteine Met: methionine Glu: glutamic acid asp: asparagine Lys: lysine Arg: arginine His: histidine Phe: phenylalanine Tyr: tyrosine

Trp:

tryptophan

Pro:

proline

Asn:

asparagine

Gin:

glutamine.

w ··

1.

The amino acids that make up hbFGF are numbered at the N-terminal methionine of the amino acid sequence, and are numbered 1. However, in this specification, the post-Met · Pro is assigned a single serial number.

The Escherichia coli MM294 / pTB762 transformant carrying the plasmid pTB762 described in Example 1, the Escherichia coli MM294 (DE3) / pTB960 transformer produced in Example 1, the Escherichia coli MM294 (DE3) / pCM90i produced in Example 8. and the Escherichia coli DH1 / pTB1004 transformant used in Example 1, carrying the plasmid pTB1004, was deposited with the Institute for Fermentation, Osaka (IFO), Japan, and the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (FRI), Japan. Their deposit numbers and deposit dates are shown in Table 1. Escherichia coli MM294 / pTB762 was first deposited at FRI using FERM P numbers. Said deposit was then converted under the Budapest Convention and the transformants were stored at FRI with FERM BP accession numbers.

transformant Table 1 IFO FRI E.coli MM294 / IFO 14613 FERM P-9409 pTB762 (May 27, 1987) (June 11, 1987) FERM BP-1645 E.coli MM294 IFO 14979 FERM BP-2690 (DE3) / pTB960 (December 12, 1989) (December 16, 1989 E.coli MM294 IFO 15104 FERM BP-3168 (DE3) / pCM901 (October 26, 1990) (November 17, 1990) E.coli DH1 / IFO 14827 FERM BP-2283 ΡΤΒ1004 (February 2, 1989) (February 13, 1989)

The present invention will now be described in more detail by way of examples. This is not intended to limit the scope of the invention.

1, Example

Production of recombinant mutein producing rhbFGF CS23

The gene in plasmid pTB762, which codes for the mutein rhbFGF CS23, has been substituted for serine residues 69 and 87 of the four cysteines present in hbFGF [Senoo et al., Biochemical and Biophysical Research Communications, 151, 701 -708 (1988); European Patent Publication No. 281,822] is digested with restriction endonucleases EcoRI and PstI, thereby deleting the DNA fragment encoding the mutein rhbFGF CS23. Plasmid pTB1004 (EP 333.383) was then completely digested with EcoRI and PstI and ligated to the largest fragment generated by T4 DNA ligase to give plasmid pTB92i. Plasmid pTB921 is then digested with Eco RI, treated with mung bean (yaenari) nuclease to form blunt ends and digested with BglII to obtain the fragment encoding the CS23 rhbFGF mutein. Next, the pET3C vector carrying the F10 promoter of phage T7 [F.W. Studier et al., Journal of Molecular Biology, 189: 113-130 (1986)], digested with Nde I and treated with nascent bean nuclease to give a blunt end product and digested with Bam HI. A DNA fragment encoding the CS23 rhbFGF mutein was ligated to the resulting DNA fragment with T4 ligase to give expression plasmid pTB960 (Figure 2).

Escherichia coli MM294 was lysogenized with the phage DE3 lambda into which the RNA polymerase gene of phage T7 was incorporated [F.W. Studier et al., 1986, Journal of Molecular Biology, 189, 113-130], to prepare Escherichia coli MM294 (DE3). This Escherichia coli strain is then transformed with the expression vector pTB960 to give the Escherichia coli MM294 (DE3) / pTB960 recombinant (IFO 14979, FERM BP-2960), which contains the gene encoding the mRBF for mRBF3 (Figure 1).

Example 2 30 ml of LB medium (10 g / l Bacto Tryptone, 5 g / l Bacto Yeast Extrase and 5 g / l NaCl) containing ampicillin mg / ml was inoculated with a loop of recombinant Escherichia coli MM294 (DE3) / pTB960 strain (IFO). 14979, FERM BP-2690) and shaken overnight at 37 ° C. 1.5 ml of this culture was transferred to 30 ml of M9 medium (16.8 g / l Na2HPO4X12 H2O, 3 g / l KH2PO4, 1 g / l NH4Cl, 0.5 g / l NaCl and 0.246 g / l MgSO4). ₄ x 7H ₂ O) supplemented with 15 g / l glucose, 15 g / l casaminosa, 1 mg / l thiamine hydrochloride and 50 mg / ml ampicillin sodium and cultured by shaking at 37 ° C. When the turbidity of the culture reaches 100-120 Klett units, IPTG at various concentrations is added and the culture is continued for an additional 4 hours. Cells were harvested by centrifugation and stored at -20 ° C.

Two identical samples were prepared. To one sample was added 7M guanidine hydrochloride, and the cells were shaken sufficiently and allowed to stand for 1 hour. The supernatant is then obtained by centrifugation (total bFGF mutein is present). To the other sample was added 50 µg / ml lysozyme solution (10% sucrose, 10 mM EDTA, 100 mM NaCl, 1 mM)

- 26 APMSF and 10 mM TRIS-HCl pH 7.6) and allowed to stand at 4 ° C for 1 hour. The cells were then lysed by sonication (Kubota Insonater 200 M) and centrifuged to obtain a supernatant (soluble hbFGF mutein is found here). Both extracted solutions were diluted and the amount of CS23 rhbFGF mutein was determined by ELISA. The results are shown in Table 2.

Table 2

The added A has accumulated

Amount (μΜ) of IPTG rhbFGF mutein (mg / l)

Ratio of soluble CS23 rhbFGF mutein (%)

400 35 13 100 41 12 75 62 25 50 73 28 25 81 32 6.3 96 60 3.2 15 80 1.6 <5 -

As can be seen from the above, when the culture is carried out in small volumes, i.e. flasks, the effect of the present invention can be achieved by adding a small amount of IPTG. Namely, the addition of about 1 mM IPTG can induce the lac promoter. Addition of IPTG at 100 μτηοΐ / l or higher results in low productivity and accumulation of the desired product in insoluble form. In contrast, according to the present invention, adding IPTG in the amount of 3-100 μιηοΐ / ΐ, especially 6-80 gmol / l, results in a completely unexpected result that there is a significant improvement in productivity and in the proportion of protein accumulated in soluble form.

Example 3

2.5 liters of M9 medium (pH 6.8) containing 15 g / l glucose, 15 g / l casaminic acid and 5 mg / l thiamine hydrochloride were placed in a 5 l glass fermenter and cysterilized. The medium is then inoculated with 125 ml of the inoculum prepared as described in Example 2, the pH is adjusted to 6.8, and then incubated at 37 ° C with stirring (1000 rpm) and aeration (2.5 l / min). ). When the turbidity of the culture reaches 120 Klett units (3 hours after start of culture), IPTG is added at a final concentration of 10 μηοΐ / ΐ. At 8 hours after the start of culture, glucose and casamino acid were added to a final concentration of 10 g / L. The culture was continued for another 15 hours. As a result, 440 mg / l rhbFGF mutein accumulates in soluble form. In contrast, when 400 μπιοί / ΐ IPTG is added to the culture under the same conditions, the amount of CS rhbFGF mutein is only 17.5 mg / L.

Example 4

The inoculum prepared as described in Example 2 was transferred to a 5 L glass fermenter containing the same medium as in Example 3 and cultured at 30 ° C at pH 6.8 at 2.5 L / min. at aeration rate of 1000 rpm. If the turbidity reaches about 700 Klett units by the 7th hour after the start of culture, 42 μπιο1 / 1 IPTG is added.

At 8.5 hours after the start of the culture, 20 g / L glucose and g / L casinoic acid were added at pH 6.8. The culture was continued for another 36 hours. In culture medium, 860 mg / l rhbFGF accumulates in soluble form.

Example 5

2.5 liters of M-9 medium containing 15 g / L glucose, 15 g / L casaminic acid and 5 mg / L thiamine hydrochloride were placed in a 5 L glass fermenter and sterilized. Iron ions are then added in the amounts shown in Table 3 (sterilized by filtration). The resulting medium was inoculated with 125 ml of the culture prepared as described in Example 2 and the culture was continued with the following parameters: pH 6.8, 30 ° C, aeration.

2.5 rpm, stirring 1000 rpm. When the turbidity of the culture reaches about 300 Klett units (about 5.5-6 hours after the start of culture), IPTG is added to a final concentration of 42 mM and then the culture temperature is reduced to 25 ° C at the same time. and culturing was continued for an additional 23.5 hours. 7-7.5 hours after the start of culture, glucose and casamino acid were added at 15 g / l and the pH was maintained at 6.8 during culture. The results are shown in Table 3.

Table 3

The amount of iron ion added

Productivity of the produced CS23 rhbFGF mutein • ·

0 1.00 2x1010 mol / l 1.42 9x10 ”® mol / l 2.6 3.6x10® mol / l 2.06 lx10 ⁴ mol / l 1.23

(Note: the amount of iron ion added is the amount of iron ions added to the medium afterwards. Productivity shows the weight ratio of the amount of iron ion added to 0.)

6 ♦ Example One liter of LB medium containing 50 mg / ml of ampicillin sodium salt was inoculated with 1 ml of Escherichia coli MM294 (DE3) / pTB960 (IFO 14979, FERM BP-2690) and cultured for 8 hours at 30 ° C. shaking. The total amount obtained is transferred to 20 L of LB medium containing 50 mg / L ampicillin sodium salt in a 50 L fermenter and cultured at 30 ° C for 7 hours at an internal pressure of 1.0 kg / cm ² G with aeration. its value is 10 l / min and the stirring rate is 300 rpm. 18 liters of the resulting culture were then transferred to 360 liters of fermentation medium (M-9 medium supplemented with 15 g / l glucose, 15 g / l casaminosa, 5 mg / l Βχ vitamin and 5 mg / l FeSO4x77O) and culture at 30 ° C at an internal pressure of 1.0 kg / cm ² G, aeration rate of 240 rpm and agitation rate of 250 rpm. When the turbidity of the culture reaches about 1000 Klett units, IPTG is added at a final concentration of 100 μιηοΐ / ΐ and the culture temperature is reduced to 25 ° C. At the same time, 50 liters of sterile solution containing 18 kg glucose and 5.4 kg casinoic acid were added to the culture at about

After 2.5 hours, the culture was continued for another 36 hours. As a result, 1.2 g / L of CS23 rhbFGF mutein accumulates in a soluble state. From the resulting culture, wet cells were harvested by Sharpless centrifugation and stored at -80 ° C.

Example 7

Purification of the CS23 rhbFGF mutein in kg of Escherichia coli MM294 (DE3) / pTB960 frozen -80 ° C prepared as described in Example 6 was suspended in 4 L of buffer [composition: 25 mM phosphate buffer pH 6.0, 0 , 1 mmol / l p-amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF) and 2 mmol / l dithiothreitol (DTT) JA cells were lysed in a 5 L Dynomill Model KD-5 (Willybachfen, Switzerland) for 5 cycles using 4 L glass beads. The glass beads are washed with about 5 liters of buffer and the washings are collected together with the extract. About 10 liters of the resulting solution were centrifuged at 10,000 rpm for 60 minutes (Model 21, Beckman, USA) to obtain a supernatant. The supernatant thus obtained was applied to a column (25.2 cm x 50 cm) containing Sulfated Cellulofine (Seikagaku Kogyo, Japan). After adsorption, the column was washed with 25 L of 25 mM phosphate buffer, pH 7.0 containing 0.5 M NaCl. Elution was then performed with 30 L of 25 mM phosphate buffer, pH 7.0 containing 1 M NaCl. The eluate was concentrated by ultrafiltration (Pellicon Casset System, Millipore, USA) until the absorbance at 280 nmendonuclease reached 3-4. The resulting concentrated solution was dialyzed against about 60 L of 25 mM phosphate buffer, pH 6.0 overnight. The dialysate was centrifuged (4200 / min, 30 min, Beckman USA) to give a supernatant. The supernatant was applied to a column (17.0 cm x 33.0 cm) containing SP-Toyopearl 650M (Tosoh, Japan). The column was washed with 7 L of 25 mM phosphate buffer (pH 6.0) containing 200 mM NaCl and eluted with 10 L of 25 mM phosphate buffer (pH 6). , 0) containing 500 mM NaCl. The eluate was applied to a column (15.0 cm x 50 cm) containing Sulfated Cellulofine (Seikagaku Kogyo) bound to a high performance liquid chromatograph (Gilson, France). Elution is then performed with a linear gradient of 20 liters of 50 mM citrate buffer, pH 7.0, and 30 liters of 50 mM citrate buffer, pH 7.0, 2 mol / l. Contains 1 NaCl. The major fractions were collected and diluted with 10 mM citrate buffer (pH 6.0) until they had a conductivity of 20 mohm or less. The diluted solution was applied to a column (14.0 x 39.0 cm) packed with SP-Toyopearl 650M (Tosoh, Japan). The column was washed with 10 L of 25 mM citrate buffer (pH 6.0) and eluted with 10 L of 25 mM citrate buffer containing 0.5 M NaCl. The eluate was applied to a column (14.0 cm x 90.0 cm), packed with Toyopearl HW-50F (Tosoh, Japan), then loaded with 50 mmol / l ♦ ·· ·

- 33 citrates were developed with buffer pH 7.0. The major fractions were collected and sterilized by filtration through a Millipak 20 filter (Millipore, USA) to give a purified solution of the CS23 rhbFGF mutein. Yield 17.0 g (70% yield). The results of the N-terminal amino acid sequence analysis, the C-terminal amino acid sequence analysis and the amino acid composition determination are shown in Figures 4-6. It is presented in tables.

Table 4

N-terminal amino acid sequence analysis

Cycle Amino acid group (pmol) Nucleotide sequence sequence number CS23 rhbFGF mutein (750 pmol) Next amino acid

1 Pro (530) Pro 2 below (553) below 3 Leu (488) Leu 4 Pro (433) Pro 5 Glu (228) Glu 6 asp (269) asp 7 Gly (370) Gly 8 Gly (470) Gly 9 Ser (8) Ser 10 Gly (328) Gly 11 below (283) below

Table 4 (cont'd)

N-terminal amino acid sequence analysis

Cycle

Amino acid group (pmol)

From the nucleotide sequence

- 34 is the next amino acid for CS23 rhbFGF mutein (750 pmol)

12 Phe (341) Phe 13 i Pro (ISI) Pro 14 Pro (338) λ Pro 15 Gly (279) . ·. Gly 16 His (69) His 17 Phe (146) Phe 18 Lys (264) Lys 19 asp (104) asp 20 Pro (125) Pro

Model 477A Protein Sequencer (Applied Biosystems).

Table 5

C-terminal amino acid analysis

C-terminal amino acid Production%

Ser 41.0 * Hydrazinolysis method;

* Model 6330 amino acid analyzer (Beckman);

Table 6

Amino acid composition analysis

amino acid CS23 rhbFGF mutein Theoretical value Asp / Asn 11.9 12 Thr 4.8 5 Ser 10.7 12 Glu / Gln 12.3 12 Pro 9.0 9 Gly 14.9 15 below 9.0 9 Cys - 2 With 6.4 7 Met 2.1 2

Table 6 (cont'd)

Amino acid composition analysis

amino acid

CS23 rhbFGF mutein

Theoretical Value • 9 ·

He 3.8 4 Leu 13.3 13 Tyr 5.9 7 Phe 8.0 8 His 3.1 3 Lys 14.2 14 Arg 10.6 11 Trp ^{* 1} 1.1 1

- not tested;

* hydrolysis method with hydrochloric acid;

** Model 6330 amino acid analyzer (Beckman);

¹ Edelhoch method.

Furthermore, Figure 3 shows the results of SDS-PAGE under reduced conditions (100 mM DTT, 50 ° C, 15 minutes). Figure 3 (1) and (2), respectively, represent the marker and CS23 rhbFGF mutein results.

Figure 4 shows the results of a high performance liquid chromatography using a column packed with heparin-5PW (Tosoh, Japan) (7.5mm x 7.5cm) under the following conditions:

Eluents: Solution A [50 mM phosphate buffer (pH 7)]

Solution B [50 mM phosphate buffer, pH 7, 2 M NaCl].

Flow rate: 0.8 ml / min.

Detection wavelength: 280 nm.

w <·

Figure 5 shows the results of a high performance liquid chromatography using a column (4.6 mm x 150 cm) containing ODP-50 (Asahi Chemical Industry, Japan) as a carrier under the following conditions:

Eluents: Solution A (0.1% trifluoroacetic acid);

Solution B (80% acetonitrile containing 0.1% trifluoroacetic acid).

Flow rate: 1.0 ml / min.

Detection wavelength: 280 nm.

Figure 6 shows the results of a high performance liquid chromatography using a column (7.5 mm x 7.5 cm) containing G2000SW (Tosoh, Japan) as the support under the following conditions:

Developing solution: 0.1 M phosphate buffer (pH 7) + 0.1 M sodium sulfate.

Flow rate: 0.5 ml / min.

Detection wavelength: 280 nm.

Biological activity is measured by the method used to measure the growth activity of fetal calf endothelial cells to measure biological activity. As a result, activity equivalent to that of the sample can be detected.

Example 8

Preparation of recombinant CS23 rhbFGF mutein producing a tetracycline resistance marker Plasmid pTB762 containing the CS23 rhbFGF mutein containing hbFGF) was replaced by serine residues at positions 69 and 87 of the four cysteine a groups. [Senoo et al., 1988, Biochemical and Biophysical Research Communications 151: 701-708; No. 281,822] is completely digested with the restriction enzymes Aval and PstI to give a DNA fragment of about 0.45 kb containing most of the rh23FGF mutein CS23. A synthetic DNA fragment (GATCTGC) was ligated to the resulting fragment ACGTCTAGACG) with T4 DNA ligase and the resulting DNA molecule was digested with the restriction enzymes Aval and PstI to give fragment A in which the PstI restriction site was modified to a BglII site. Plasmid pTB955, to which the C128 rhbFGF mutein coding gene was deleted at the C-terminus of hbFGF (Senoo et al., 1988, Biochemical and Biophysical Research Communications, 151: 701-708), was digested with restriction enzymes SalI and BamHI. fragment of about 4.1 kb. PTB955 is completely digested with the restriction enzyme Aval and then with the restriction enzyme SalI to give fragment C of about 390 bp.

The three fragments (A, B and C) were ligated with T4 DNA ligase to give the expression plasmid pHP901, which contains the ampicillin resistance marker.

Plasmid pHP901 is completely digested with restriction enzymes EcoRI and BglII to give a fragment of about 1.1 kb containing the gene encoding the CS23 rhbFGF mutein, the T7 promoter and the T7 terminator. The fragment was ligated into pUC18 digested with EcoRI and BamHI * S * * * * *???

- 39 T4 DNA ligase to give plasmid pME901 (Figure 8).

Plasmid pME901 was completely digested with restriction enzymes EcoRV and Hindin to give a fragment of about 0.77 kb containing the gene encoding the CS23 rhbFGF mutein, the T7 promoter and the T7 terminator. The fragment was incubated with T4 DNA polymerase to fill the ends. The fragment was ligated into the Sci restriction enzyme digested plasmid pBR322 to obtain the expression plasmid pCM901, which contains the tetracycline resistance marker (Figure 9).

Escherichia coli strain MM294 was lysogenized with phage DE3 into which the RNA polymerase gene of phage T7 was incorporated [F.W. Studier et al., 1986, Journal of Molecular Biology, 189, 113-130], to prepare Escherichia coli MM294 (DE3). This Escherichia coli strain is then transformed with the expression vector pCM901 to give the Escherichia coli MM294 (DE3) / pCM901 recombinant (IFO 15104, FERM BP-3168), which contains the gene encoding the CS3 rhbFGF mutein.

EXAMPLE 9 1 L of LB medium containing 5 mg / L tetracycline hydrochloride was inoculated with 1 ml inoculum of the recombinant strain Escherichia coli MM294 (DE3) / pCM901 (IFO 15104, FERM BP-3168) and cultured for 8 hours. By shaking at 0 ° C. The entire volume is then transferred to 20 L of LB medium (5 mg / ml tetracycline hydrochloride) in a 50 L fermenter and cultured at 30 ° C with an internal pressure of 1 kg / cm ² G with an aeration rate of 10 L / min. mixing at 300 rpm until turbidity of the culture reaches 700 Klett units. The total volume of the resulting culture was then transferred to 360 liters of fermentation medium (M-9 medium containing 15 g / l glucose, 15 g / l casamino acid, 16.8 g / l Na ₂ HPO ₄ x 12 H ₂ O, 3 g / l KH). Contains ₂ PO ₄ , 1 g / l NH ₄ Cl, 0.5 g / l thiamine hydrochloride, 2.5 mg / l FeSO ₄ x 7 H ₂ O and 0.2 g / l antifoam) and culturing was started at 28 ° C with an internal pressure of 1 kg / cm @ g, aeration rate of 240 rpm and agitation at 250 rpm. If the culture was grown to about 450 Klett turbidities, 10 mg / L (42 μιηοΐ / ΐ) IPTG was added. Since the amount of sugar remaining is about 1%, a solution containing 60 g / l glucose and 15 g / l casein bridge lysate is added and the culture is continued for an additional 40 hours. As a result, 1.6 g / l of CS23 rhbFGF mutein accumulates in soluble form. The resulting culture was centrifuged in a Sharpless centrifuge to obtain wet cells and the cells were frozen at -80 ° C.

1 kg of the resulting cells was treated as described in Example 7 to give a stock solution of purified CS23 rhbFGF mutein (25.0 g). The quality of the solution is the same as that obtained in Example 7, based on both biological activity and chemical assays.

Claims (13)

PATENT CLAIMS A vector comprising a nucleotide sequence encoding a mutein in which at least one of the amino acids in the mutein constituting the mature human basic fibroblast growth factor (hbFGF) is replaced by another amino acid and is preceded by a T7 promoter. The vector of claim 1, wherein at least one of the constituent amino acids is a cysteine residue and the other is a serine residue. 3. The vector of claim 1, wherein the mutein is a mutein in which the cysteine residues 69 and 87 of the mature hbFGF are replaced by a serine residue. The transformant transformed with the vector of claim 1. The transformant of claim 1, wherein the host is Escherichia coli, which contains a T7 RNA polymerase gene after a lac promoter. Transformant according to claim 5, characterized in that it is Escherichia coli MM294 (DE3) / pTB960 (IFO 14979, FERM BP-2690). Transformant according to claim 5, characterized in that it is Escherichia coli MM294 (DE3) / pCM901 (IFO 15104, FERM BP-3168). A method for producing a mutein wherein at least one amino acid in a mature hbFGF is replaced by another amino acid, wherein the transformant is cultured in the medium of claim 4. 9. The method of claim 8, wherein about 3-500 μπιοί / ΐ isopropylthio-galactoside (IPTG) is added to the transformant of claim 5 in a logarithmic phase and cultured. The method of claim 9, wherein the IPTG is 3-300 μιηοΐ / ΐ. 11. The process of claim 10, wherein the IPTG is 6-200 gmol / L. 12. The method of claim 11, wherein the IPTG is 6-80 μιηοΐ / l. 13. The process of claim 8, wherein the resulting mutein-containing solution is purified by chromatography using a cross-linked polysaccharide sulfate as a support, a synthetic polymer having a sulfonic acid moiety as exchanger, and / or a synthetic polymer for gel filtration.