CA2070989A1 - Production of human basic fgf mutein - Google Patents

Abstract

Disclosed are (1) a vector comprising a nucleotide sequence coding for a mutein in which at least one constituent amino acid of a mature human basic fibroblast growth factor (hbFGF) is replaced by another amino acid, and a T7 promoter upstream therefrom; (2) a transformant transformed by the vector of (1); (3) the transformant of (2), in which a host is E. coli having a T7 RNA
polymerase gene downstream from a lac promoter; (4) a method for producing the mutein in which at least one constituent amino acid of the mature hbFGF is replaced by another amino acid, which comprises cultivating the transformant of (2) in a culture medium; (5) the method of (4), in which about 3 to 500 µm of isopropylthiogalactopyranoside is added to the culture medium on a logarithmic growth phase of the transformant of (3), followed by cultivation, and (6) the method of (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, whereby the hbFGF mutein having biological activity can be efficiently produced.

Description

~O 91~0912~ PCr/JP90~01646 DESCRIPTION
PRODUCTION OF ~IUMAN BASIC FGF M~TEIN 8 Technical Field 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.
Backqround Art Basic fibroblast growth factor (bFGF) 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 tha~ FGF exhibits growth promoting action on almost all mesoblast-derived cells [D. Gospodarowicz et al., National Cancer Institute Monoqraph 48, 109 (1978)]. In particular, the angiogenic action of the bFGF, together with its cell growth promoting action, 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. [FEBS Letters 2 , 189-194 (198`7)~, and expressed in . . .

~'O91/~9126 PCT/JPgO/01~6 ~o~Q9 - 2 - ~

animal cells [FEBS Letters 213, 189-194 (1987)], yeast lThe Journal of Biological Chemistry 263, 16471-16478 (1988)] and Escherichia coli [The Journal of Bioloqical Chemistry 263, 16297-16302 (1988)]. ~owever, their production is not always sufficient and the resulting samples are too unstable to use as medicines. As a result of various studies to solve the unstability, it has been revealed that compounds in which cysteine residues of hbFGF molecules are replaced by serine residues have high stability and exhibit the same biological activity [Biochemical and Biophysical Research Communication 151, 701-708 (1988)].
Further, there has been developed a method for producing a mutein in which at least one constituent amino acid of hbFGF is replaced by another amino acid by genetic engineering techniques (European Patent Publication No.
281,822).
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 hb~GF 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 ~ -' ~

WO91/09t26 PCr/JP90/01646 isolated and purified in high yield. It is therefore an important object to establish a method for producing the muteins at high efficiency.
In general, when gene products are produced in large amounts by using genetic engineering techniques, the selection of host-vector systems and promoters is important and differs depending on each gene. Various studies on efficient expression systems of hbFGF muteins have revealed that an E. coli gene expression system using a T7 promoter lF. W. Studier et al., Journal of Molecular Bioloqy 189, 113-130 (1986)] is excellent for expression of the genes of the hbFGF muteins. The combination of the T7 promoter and hbFGF mutein ~enes is novel. This T7 promoter is known to be a strong promoter. In some cases, however, 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 hàve found the interleukin-2 case, and Paris, N. et al. have found the prolactin case [Paris, N. et al., BiotechnoloqY -and Applied BiochemistrY, 12, 436-449 (1990~].
Disclosure of_Invention 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. In addition, the present inventors have further established a method for isolating : . ~
" '. ' ' :

' ' ' . :~

WO91/0912fi PCT/JP90/01646 and purifying the hbFGF mutein efficiently, thus completing the present invention.
The present invention provides:
(1) a vector containing a nucleotide sequence coding for a mutein in which at least one constituent amino acid of a mature human basic fibroblast growth factor (hbFGF) is replaced by another amino acid, and a T7 promoter upstream therefrom, (2) a transformant transformed by the vector described in the above item (1), (3) the transformant described in the above item (2), in which a host is E. coli having a T7 RNA polymerase gene downstream from a lac promoter, (4) a method for producing the mutein in which at least one constituent amino acid of the mature hbFGF is replaced by another amino acid, which comprises cultivating the transformant described in the above item (2) in a culture medium, (5) the method described in the above item (4), in which about 3 to 500 ~M of isopropylthiogalactopyranoside (hereinafter also briefly referred to as 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 ~ 91/~9126 5 ~CT/JP90/01646 '~`` 2070989 synthetic polymer having a sulfonic acid group as an exchange group and/or a synthetic polymer for gel filtration as a carrier.
According to the present invention, 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.
Brief descriPtion of the Drawinqs Fig. l shows a DNA nucleotide sequence of rhbFGF mutein CS23 used in Example l 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 l;
Fig. 3 shows a SDS-PAGE pattern of a purified sample obtained in Example 5 and a marker; and 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 pHP9Ol, pME9Ol and pCM9Ol obtained in Example 8.
Detailed Description of the Inventions 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. l.
Examples of the hbFGF muteins as shown in the present invention include muteins in which at least one constituent . .
~: .

~VO9l/09l26 PCT/JP90/01~6 " ~S3 ~
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).
As to the number of hbFG~-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 cf angiogenesis, cell growth stimulating activity and cell differentiating activity, are not lost.
Examples of the constituent amino acids before substitution include cysteine and amino acids other than cysteine. In particular, cysteine is preferred. The amino acids other than cysteine as the constituent amino acids before substitution include aspartic acid, arginine, glycine and valine.
When the constituent amino acid before substitution is cysteine, neutral amino acids are preferred as the substituting amino acids. Specific examples of 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.
When 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 WO9~/09l2~ PCT/JP90/01646 ~ 7 ~ 2070989 different in hydrophilicity, hydrophobicity or electric charge from the constituent amino acid before substitution.
Specifically, when 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.
When the amino acid before substitution is arginine, the substituting amino acids include glutamine, threonine, leucine, phenylalanine and aspartic acid. Glutamine is especially preferable.
When the constituent amino acid before substitution is glycine, the substituting amino acids include threonine, leucine, phenylalanine, serine, glutamic acid and arginine.

Threonine is particularly preferable.
When the constituent amino acid before substitution is serine, the substituting amino acids include methionine, alanine, leucine, cysteine, glutamine, arginine and aspartic acid. In particular, methionine is preferred.
When the constituent amino acid before substitution is valine, the substituting amino acids include serine, leucine, proline, glycine, lysine and aspartic acid. Serine is especially preferred.
As the original constituent amino acids before substitution, aspartic acid, arginine, glycine, serine and valine are preferably selected.
As the substituting amino acids, asparagine, glutamine, arginine, threonine, methionine, serine and leucine are .: .., . - ' : - -... - ,' . . , -~:

~091/~9l26 PCT/JP90/01646 ~ 8 -preferably selected.
The most preferred substituted muteins include a mutein in which cysteine, the constituent amino acid, is replaced by serine.
In the above substitution, the substitution of at least two constituent amino acids may be simultaneously carried out. In particular, it is preferable to substitute two or three constituent amino acids.
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.
As the mutein, 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.
In order to produce the muteins, site-directed mutagenesis is employed. This technique is well known and described in R. F. Lather and J. P. Lecoq, Genetic Enqineerinq, p.31-50, Academic Press (1983). Mutagenesis directed to oligonucleotide is described in M. Smith and S.
Gillam, Genetic Enqineering: Principles and Methods, vol.3, p.l-32, Plenum Press (1981).

.
' :
~. :

WOsl/osl26 PCT/JP90/01646 ~ - 9 - 207~989 The production of a structural gene which encodes the mutein is carried out, for example, by the steps of:
(a) hybridizing a single-stranded DNA comprising a single strand of the structural gene of the hbFGF with a mutagenic oligonucleotide primer (the above primer is complementary to a region including a codon for cysteine to be replaced by this single strand or including an anti-sense triplet which forms a pair with this codon in some cases, provided this does not apply to disparity with other codons 10 for the amino acid than the above codon, or with the anti- ;
sense triplet in some cases), (b) elongating the primer with DNA polymerase to form a mutational heteroduplex, and (c) replicating this mutational heteroduplex.
Then, phage DNA for transferring the mutagenized gene is isolated and introduced into a plasmid.
As the 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 ~ 10 promoter [A. H. Rosenberg et al., Gene 56, 125-135 (1987)~ is preferably used.
As a transcription terminator used in the present invention, any terminator may be used as long as it functions in E. coli systems, but a T ~ terminater [F. W.

WO91/09126 PCT/JP90/n1646 ~9 ~
Studier et al., Jol. Biol. 189, 113-130 (1986~ is preferably used.
The T7 RNA polymerase genes used in the present invention include T7 gene l [F. W. Studier et al., J._Mol.

Biol. 189, 113-130 (1986)~.

Examples of 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 terminater into the above vectors. Such vectors include pET 1, pET-2, pET-3, pET-4 and pET-5 [A. H. Rosenberg, Gene 56, 125-135 (1987)], but pET-3C (ibid.) is preferably used.
As the host for the transformant used in the present invention, any of E. coli strains into which the T7 RNA
polymerase gene (T7 gene l) [F. W. Studier et al., J. Mol.
Biol. 189, 113-130 (1986)] is incorporated, such as MM294, DH-l, C600 and BL21, may be used. The strains MM294 and BL21 are preferably used in which a ~phage including T7 20 gene l is lysogenized. The T7 RNA polymerase gene can also -be harbored as a plasmid having different origin from that of expression vector. In this case, as the promoter for T7 gene l, there is used the lac promoter whose expression is induced with isopropyl-l-thio-~-D-galactopyranoside (IPTG).
The transformants used in the present invention are obtained by transforming the E. coli strains into which the above T7 gene l (RNA polymerase gene) have been ., ~ , . :

:.
- .

~ '0 91/09126 ~ PCI/JP90/01646 ~ t ~ 2070989 incorporated, with the plasmids having the gene-transcription terminators for T7 promoter expression, according to methods known in the art such as the methods described in Proc. Natl. Acad. Sci. U.S.A. 69, 2110 (1972) and Gene 17, 107 (1982). In this case, the hosts to be used may be preliminarily transformed with plasmids having the T7 lysozyme gene so that the resulting transformants have two kinds of different plasmids simultaneously.
When the transformants are cultivated, li~uid 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. Examples of 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.
As 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 '', ' ' ' ~ ' , ..... ., ~

~!0 91/()9126 ~o9~,9 - 12 - PCr/JP90~01646 sources may be further added to this medium. The iron ion sources are materials which dissocia~e 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 43C for about 3 to 72 hours, preferably for about 12 to 48 hours, with aera~ion or agitation if necessary.
For the gene expression, it is preferable to add IPTG
in the course of cultivation. Thereby, T7 gene 1 (RNA
polymerase gene) ligated downstream from the lac promoter is expressed, and T7 phage RNA polymerase 1 thus produced specifically recognize the T7 promoter. According to the prior art, 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: ~ ner-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. et al., DNA, 5, 21-28 (1986)]. However, it has been revealed that proteins accumulated in inducing expression by addition of IPTG of this amount form inclusion bodies, which sometimes results in accumulation of inactive proteins. As a result of various studies on methods for accumulating the .

~ 091/09126 PCT/JP90/01646 ~ 13 - 2070989 proteins in a dissolved state, it has been discovered that the object is first attained by adding about 3 to 500 ~M, preferably about 3 to 300 ~M, more preferably 6 to 200 ~M, and most preferably about 6 to 80 ~M of IPTG in the course of cultivation. When large-scale cultivation such as tank cultivation is carried out, it is preferred that IPTG is added in an amount of about lO to 500 ~M, preferably lO to 200 yM, more preferably lO to lO0 yM, still more preferably lO to 80 ~M. ~hen small-scale cultivation such as flask cultivation is carried out, it is preferred that IPTG is added in an amount of about 3 to lO0 ~M, more preferably 6 to 80 ~M. IPTG is first added about l 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 42C, preferably about 20 to 30C. In lieu of 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.
When the hbFGF muteins in the present invention are extracted from the cultivated cells, the cells are collected .. . ..

, . - :. . . .

~ pCTtJP90/01646 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.
Various studies have been made on methods for purifying the hbFGF muteins according to the present invention from the supernatants obtained above. As a result, high-purity samples have been obtained in very high yield by combining affinity chromatography using a crosslinked polysaccharide sulfate as a carrier, ion exchange chromatography whose carrier is a synthetic polymer having a sulfonic acid group as an exchange group and chromatography using a synthetic polymer for gel filtration as a carrier with one another at least once.
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 o~ agar, and has the recurring structure of .
: :
. ' .

0 91/0912~ P~T/JP90/01646 '~''`"'`'` 2070g89"~
D-galactosyl-tBl~4)-3~6-anhydro-L-garactosyl~ 3)~ Its molecular weight is preferably about lO,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 a(l~6) bonds formed, for example, by the action of a microorganism such as Leuconostoc mesenteroides on sucrose. Its average molecular weight is preferably about l,000 to 40,000,000.
The 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 15 methods known in the art.
The crosslinked polysaccharides are commercially available and can be purchased from Pharmacia (Sweden) under the-trade names of Sephadex G-lO, Sephadex G-l5, Sephadex G-25, Sephadex G-50 and Sephadex G-lO0 (crosslinked dextran), 20 and under the trade names of Sepharose CL-2B, Sepharose CL- s 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 25 polysaccharide sulfates can be synthesized by allowing known sulfating agents, such as chlorosulfonic acid and sulfuric anhydride esters, to react with these crosslinked polysaccharides.

- ~ . , . . . , - - - - . : - . - ... . .

.
- : , q~9~9 - 16 - PCT/JP90/01~6 ~``""'' ~
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.
Examples of the crosslinked agarose sulfates include sulfated Sepharose.
The crosslinked polysaccharide sulfates used in the present invention may be in the form of the corresponding salts. Examples of the salts include sodium, potassium, ammonium and trimethylammonium salts. In particular, the sodium salts are preferably used.
The 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.
The methods for purifying the hbFGF muteins using the crosslinked polysaccharide sulfates in the present invention include affinity chromatography described below.
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.

~()91/t)9126 PCT/JP90/01646 The hbFGF mutein-containinq solutions are next readjusted to a p~ 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 m ~ 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. In the case of the column method, 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. The amount of the gel to be used depends on the nature of the loaded hbFGF mutein-containing solution, but the range of about l 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 O.l to SØ After loading, 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.
In order to increase the ionic strength, 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 lS m U, preferably at least 30 m U. For elution, both batch and concentration gradient elution methods may be -.

.

~091/09126 ~,Q9~ - 18 - PCT/JP90/~1646 ~,~
~":l used. When the concentration gradient elution method is used, ~or example, the concentration of NaCl is gradually increased from about 0 M to 2.0 M, thereby conducting elution and recovery. Thus, 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. In particular, SP (sulfopropyl)-Toyopearl (Tosoh, Japan) in which sulfonic groups are introduced into the hydrophilic vinyl polymer is preferably used from the viewpoints of recovery and operation.
When chromatographed, 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. For adsorption, 20 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. For elution, both batch and concentration gradient methods may be used. When the batch method is used, for example, 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. In lieu of the ,. ~ ' '.
.. . . .

`:

~'O 91/09126 PCI/JP90/01646 9 20~ ~989 phosphate buffer, citrate buffer can be used. The elution is conducted at a temperature of about 1 to 25C, preferably about 1 to 10C, more preferably about 4 C.
Examples of the synthetic polymers for gel filtration 5 used in the present invention include hydrophilic vinyl polymers and acrylamide polymers. In particular, Toyopearl HW (Tosoh, Japan), the hydrophilic vinyl polymer, is preferably used from the viewpoints of gel durability and operation.
When the hbFGF mutein-containing solutions partially purified are treated, for example, on a column of Toyopearl HW-50F, 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 25C, preferably about 1 to 10C, more preferably about 4C.
A method other than the above methods may be used as one of the purifying procedures. In such methods, for example, 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.
As carriers for gel filtration, there can be similarly used 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 ..... . . ~... . .
~. .. - ,. . .
. ~ : . . . ' :
.: , :, `
.~ -.
.

~osl~ns~26 PC'r/JP90/01646 ~ 9S9 - 20 -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 ~-mercaptoethanol, dithiothreitol and glutathione.
According to the present invention, 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 accordinq to the present invention in an amount of 95% (w/w) or more as protein - 15 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. They can therefore be used as healing promoters for burns, wounds, postoperative tissues and the like, or as therapeutic medicines based on their angiogenic activity for thrombosis, arteriosclerosis and the like. They can also be used as reagents for promoting cell cultivation. In particular, the mutein in which at least one of constituent cysteine residues is replaced by a serine residue is preferable because of their high stability.

W() 91/09126 PCI/JP90/01646 -- 21 -- .
~;~` 207~989 When the hbFGF muteins according to the present invention are 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.
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.
When 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.
Further, when 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 abou~ 0.01 to 10 ~g per liter of medium, more preferably in an amount of about 0.1 to 10 ~g per liter of medium.
When bases, amino acids and so on are indicated by the abbreviations in this specification and the drawings, the -~091/09~26 PCT/JP90/01646 ~ ~9~0 - 22 -abbreviations adopted by I~PAC-IUB Commission on Biochemical Nomenclature or commonly used in the art are employed. For example, the following abbreviations are used. When the optical isomer is capable of existing with respect to the amino acids, the L-form is represented unless otherwise specified.
DNA : Deoxyribonucleic acid cDNA : Complementary deoxyribonucleic acid A : Adenine T : Thymine G : Guanine C : Cytosine RNA : Ribonucleic acid dATP : Deoxyadenosine triphosphate dTTP : Deoxythymidine triphosphate dGTP : Deoxyguanosine triphosphate . .
dCTP : Deoxycytidine triphosphate ATP : Adenosine triphosphate Tdr : Thymidine EDTA : Ethylenediaminetetraacetic acid SDS : Sodium dodecyl sulfate Gly : Glycine Ala : Alanine -Val : Valine 25 Leu : Leucine :
Ile : Isoleucine Ser : Serine .:

; . . ~ '~' : .

~091/09126 - 23 - PCT/JP9OtO1646 ,~ 20709-~9 Thr : Threonine Cys : Cysteine Met : Methionine Glu : Glutamic acid Asp : Arginine Lys : Lysine Arg : Arginine His : Histidine Phe : Phenylalanine Tyr : Tyrosine Trp : Tryptophan Pro : Proline Asn : Asparagine Gln : Glutamine Referring to Fig. l, with respect to the number of the ~ -hbFGF-constituent amino acids, Met of the N-terminus of the amino acid sequence is numbered as the 1st. In this specification, however, Pro next to the Met is numbered as the 1st.
Transformant E. coli MM294/pTB762 carrying plasmid pTB762 used in Example l described below, transformant E.
coli MM294(DE3)/pTB960 produced in Example l, transformant E. coli MM294(DE3)/pCM9Ol produced in Example 8 and _. coli DHl/pTBl004 which harbors plasmid pTBl004 (Example l) have been deposited with the Institute for Fermentation, Osaka (IFO), Japan, a~d with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of . .. : . - - , ~ . : :.
.': ', , 091/091'6 PCT/JP90/01646 ~g~ ~
Internationa~ rade 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 ~P numbers.

Table 1 Transformant IFO FRI

E. coli MM294/IFO 14613 FERM P-9409 pTB762 (May 27, 1987) (June 11, 1987) . . .
E. coli MM294IFO 14979 FERM BP-2690 (DE3)/pTB960(December 12,1989) (December 16,1989) 15E. coli MM294IFO 15104 FERM BP-3163 tDE3j/pCM901tOctober 26, 1990) (November 17, 1990) E. coli DHl IFO 14827 FERM BP-2283 /pTB1004(February 2, 1989) (February 13, 1989) The present invention will hereinafter be described in detail with the following Examples. It is understood of course that these are not intended to limit the scope of the invention.
Best Mode for carrYing Out the Invention Example 1 Preparation of rhbFGF Mutein CS23 Production Recombinant Plasmid pTB762 containing a gene coding for rhbFGF
mutein CS23 in which the cysteine residues at positions 69 ' , ~91/~912~ PCT/JP90/01646 ;~ ? 2 0 7 0 9 8 9 and 87 of four cysteine residues existing in hbFGF were replaced by serine residues [Senoo et al., Biochemlcal 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 pTB1004 (European Patent Publication No. 336,383) was completely digested with EcoRI and PstI, and thereafter this fragment was ligated to the resulting largest fragment by T4 DNA ligase to construct plasmid pTB921. Then, the 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 BglII to prepare a DNA fragment containing the gene coding for rhbFGF mutein CS23. Additionally, vector pET3C carrying a ~ 10 promoter for a T7 phage [F. W. Studier et al., Journal of Molecular Biolo~y 189, 113-130 (1986)] was cleaved with restriction enzyme NdeI, and the product was treated with mungbean nuelease to ehange the termini thereof to flush ends, followed by reaction with restriction enzyme BamHI. A DNA fragment containing rhbFGF mutein CS23 was ligated to the resulting DNA fragment by T4 ligase to obtain expression plasmid pTB960 tFig. 2).
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 Bioloqy 189, 113-130 (1986)] to prepare E. eoli strain MM294(DE3). This .

.
, '091/09126 PCT/JP90/01646 ~ ~9~9 26 - ~

E. coli strain was transformed by using the expression plasmid pTB960, thereby obtaining recombinant E. coli MM294(DE3)/pTB960 (IFO 14979, FERM BP-2690) carrying the gene coding for rhbFGF mutein CS23 shown in Fig. 1.
Exa~le 2 30 ml of LB medium (10 g/l of bactotryptone, 5 g/l of bactoyeast extract and 5 g/l of NaCl) containing 50 mg/l of sodium ampicillin was inoculated with one loopful of the recombinant E. coli MM294~DE3~/pTB960 (IFO 14979, FERM BP-2690), and incubated at 37 C overnight with shaking. 1.5 mlportions of this culture solution were transferred to 30 ml of M-9 medium (16.8 g/l of Na2HPO4 12H2O, 3 g/l of KH2PO4, 1 g/l of NH4Cl, 0.5 g/l of NaCl and 0.246g/1 of MgSO4 7H2O) supplemented with 15 g/l of glucose, 15 g/l of casamino - 15 acids, 1 mg/l of thiamine hydrochloride and 50 mg/l of sodium ampicillin, and the cultivation with shaking was carried out at 37C. When the culture was grown to a Klett unit of 100 to 120 in turbidity, IPTG was added to obtain various concentrations and the cultivation was further continued for 4 hours. Cells were collected by centrifugation and stored at -20C.
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

. - ~ - ' -Ogl/09126 PCT/JP90/01646 ,~; 2070989 NaCl, 1 mM APMSF and 10 mM Tris-HCl, pH 7.6), and the mixture was allowed to stand at 4C for 1 hour. Then, the cells were disrupted by ultrasonication (Kubota Insonater 200 M), followed by centrifugation to obtain a supernatant `
tamount of soluble hbFGF mutein). Both the extracted solutions were diluted, and then the amount of rhbFGF mutein CS23 was determined using an ELISA. The results are shown in Table 2.

Table 2 Amount of Amount of accumulated Formation ratio of added IPTG rhbFGF mutein CS23 soluble rhbFGF mutein (~M) (mg/l) CS23 (~) 6.3 g6 60 3.2 15 80 1.6 < 5 As is shown above, in the case of small-scale cultivation such as cultivation in flasks, 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 . - . - -.

-,~
, ~ ' ~091/~91~6 ~9 PCT/JP90/01646 ~ 9 ~ 28 -state. In contrast, according to the present invention in which IPTG was added within a ranye from 3 ~M to lO0 ~M, especially 6 ~M to 80 ~M, there were obtained the entirely unexpected results of significant improvements in productivity and also in ratio of the mutein accumulated as a soluble protein.
_ample 3 M-9 medium (pH 6.8~ containing 15 q/l of glucose, 15 g/l of casamino acids and 5 mg/l 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 ~xample 2, followed by cultivation adjusting the pH to 6.8, at 37C, with aeration at a rate of 2.5 l/min and with stirring at l,000 rpm. 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 lO ~M. 8 hours after the initiation of cultivation, glucose and casamino acids were further added thereto so as to be contained in an amount of lO g/l, repectively. The cultivation was further continued for 15 hours as a whole. As a result, 440 mg/l of rhbFGF mutein CS23 was accumulated in a soluble state. In contrast, when 400 ~M of IPTG was added under the same co~ditions, the production of rhbFGF mutein CS23 was only l7.5 mg/l.

:. ' ' " .
.

WO91/~9126 PCT/JP~0/01646 ~ 29 - 207~989 .
Example 4 -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 30C, at pH
6.8, with aeration at a rate of 2.5 l/min and with stirring at 1,000 rpm. When the turbidity reached about 700 Klett units 7 hours after the initiation of cultivation, 42 ~M of IPTG was added. 8.5 hours after the initiation of cultivation, 20 g/l of glucose and 20 g/l of casamino acids were added at pH 6.8. The cultivation was carried out for 36 hours. In the culture solution, 860 mg/l of rhbFGF
mutein CS23 was accumulated in a soluble state.
Example 5 M-9 medium (pH 6.8) containing 15 g/l of glucose, 15 g/l of casamino acids and 5 mg/l 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 30C, with aeration ~-t at a rate of 2.5 l/min and with stirring at 1,000 rpm. When the culture was grown to a Xlett unit of about 300 in turbidity (about 5.5 to 6 hours after the initiation of cultivation), IPTG was added so as to be contained in an .. . - : :

-~o sl/nsl2fi ~aaQ PCr/JP90/01646 amount of 42 ~M, and at the sarne time the cultivationtemperature was lowered to 25C, 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/l, and the pH was maintained at 6.8 during cultivation. The results are shown in Table 3.

Table 3 Amount of added Productivity of rhbFGF mutein iron ion CS23 o 1.00 2X10 M 1.42 9X10 6 M 2.60 3.6X10 5 M 2.06 lX10 4 M 1.23 (Note: 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.) ExamPle 6 1 liter of LB medium containing 50 mg/l of sodium ampicillin was inoculated with 1 ml of the master cells of recombinant E. coli MM294(DE3)/pTB960 (IFO 14979, FERM BP-2690)l and the cultivation with shaking was carried out at 30C for 8 hours. The total amount thereof was transferred to 20 liter of LB medium (containing 50 mg/l of sodium ampicillin) placed in a 50 liter culture tank, and the ... . ., , . :

- : -. ' .' ', ' .
, .

~091/0912(- PCT/JP90/01646 ; 2070989 cultivation was carried out at 30C, under an inner pressure of 1.0 kg/cm2G, with aeration at a rate of 10 l/min and with stirring at 300 rpm for 7 hours. Then, 18 liters of the resulting culture solution was transferred to 360 liter of a fermentation medium (M-9 medium containing 15 g/l of glucose, 15 g/l of casamino acids, 5 mg/l of vitamin Bl and 5 mg/l of FeSO4 7H20), and the cultivation was initiated at 30 C, under an inner pressure of 1.0 kg/cm G, with aeration at a rate of 240 l/min and with stirring at 250 rpm. When the culture was grown to a Klett unit of about 1,000, IPTG
was added in an amount corresponding to 100 ~M, and the cultivation temperature was lowered to 25C. At the same time, a 50 liter sterilized solution containing 18 kg of glucose and 5.4 kg of casamino acids was added at a rate of about 2 1/hour, and then the cultivation was continued for 36 hours. As a result, 1.2 g/l of rhbFGF mutein CS23 was accumulated in a soluble state. The cultivated product thus obtained was subjected to a Sharpless centrifuge to collect wet cells, and the cells were frozen at -80C to store them.
Example 7 Purification of rhbFGF Mutein CS23 1 kg of the cells (E. coli MM294(DE3)/pTB960) freeze-stored at -80C in Example 6 was suspended in 4 liter of a buffer containing 25 mM phosphate buffer (pH 6.0), 0.1 mM
p-amidinophenylmethanesulfonyl fluoride hydrochioride (APMSF) and 2 mM dithiothreitol (DTT). The cells were disrupted 5 cycles under ice cooling with a 5 liter Dynomill :, . -: . , , ~091/()912( 9~ 32 - PCT/~IP90/0l646 Model KD-5 (Willyb~chfen, Switzerland) using 4 liter of glass beads. The glass beads were washed with about 5 liter of the buffer, and the washings were collected together with the extract. About 10 liter o~ the resulting solution was centrifuged at 10,000 rpm (Model 21, Beckman, U.S.A.) for 60 minutes to give a supernatant. The supernatant thus obtained was poured into a column (25.2 cm ID X 50 cm) of Sulfated Cellulofine (Seikagaku Kogyo, Japan). After adsorption, the column was washed with 25 liter of 25 mM
phosphate buffer (pH 7.0) containing 0.5 M NaCl. Then, elution was effected using 30 liter of 25 mM phosphate buffer (pH 7~0) containing 1 M NaCl. 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 . ,,: , . . . .
, : -: : - .. : .

WO9l/~9l2~. P~/JP~0/01646 r~ 3 3 2 0 7 0 9 8 9 gradient between 20 liter of 50 mM citrate buffer (pil 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 (14.0 cm ID X 39.0 cm) of SP-Toyopearl 650 M (Tosoh, Japan).
The column was washed with 10 liter of 25 mM citrate buffer (pH 6.0), and then elution was conducted by using 10 liter of 25 mM citrate buffer (pH 6.0) containing 0.5 M NaCl. The eluate was poured into a column (14.0 cm ID X 90.0 cm) of Toyopearl HW-50F (Tosoh, Japan), and developed by using 50 mM citrate buffer (pH 7.0). The main fractions were coilected, and sterilized by passing through a Millipak 20 :
filter (Millipore, U.S.A.) to provide a purified stock solution of rhbFGF mutein CS23. The yield was 17.0 g tthe percent yield-was 70%). The results of N-terminal amino - acid sequence analysis, C- terminal amino acid analysis and amino acid composition analysis are shown in Tables 4 to 6, respectively.

' .~

. '' .

.

.
. ;`~: ' ,", , ', WO9l/~)sl2~. ~ P~/JP90/01646 ~ 9~ 34 - ~

Table 4 N-terminal amino acid sequence analysis Amino acid residue (pmol) Amino acid deduced rhbFGF mutein CS23 from nucleotide Cycle No. (750 pmol) sequence 1 Pro (530) Pro 2 Ala (553) Ala 3 Leu (488) Leu 4 Pro ~433) Pro Glu (228) Glu 6 Asp (269) Asp 7 Gly (370) Gly 8 . Gly (470) Gly 9 Ser ( 8) Ser Gly (328) Gly 11 Ala (283) Ala 1512 Phe (341) Phe 13 Pro (181) Pro 14 Pro (338) Pro ~ 15 Gly ~279) Gly : 16 His ( 69) His . 17 Phe (146~ Phe ~, 18 Lys (264) Lys 19 Asp (104) - Asp Pro (125) Pro . * Analyzed with a Model 477A protein sequencer (Applied . 25 :. Biosystems) .; ' '.

., .
:

'() ')1/1)912~ PCT`/~lPgO/01646 - 35 - 2070~89 Table 5 C-terminal amino acid analysis C-terminal amino acid Yield (%) Ser 41.0 * Hydrazinolysis method ** Analyzed with a Model 6330 amino acid analyzer (Beckman) Table 6 Amino acid composition analysis _ Amino acid rhbFGF mutein CS23 Theoretical value Asp/Asn 11.9 12 : Thr 4.8 5 Ser 10.7 12 Glu/Gln 12.3 12 Pro 9-0 9 `' 15 Gly 14.9 15 Ala 9-0 9 Cys Val 6.4 7 Met 2.1 2 Ile 3.8 4 Leu 13.3 13 20 Tyr 5-9 . 7 Phe 8.0 8 His 3.1 3 Lys 14.2 14 Arg 10.6 11 Trpl) 1.1 1 -; - Not assayed * Hydrolysis method with hydrochloric acid ** Analyzed with a Model 6330 amino acid analyzer ~Beckman~
1) Edelhoch method : : . . : . , s" - '-, . ~ ' ~: ~ ' :: , ::
~' :~ , -:

~V()91/~912(. PC~`/J1~0/01646 ~ - 36 ~

Further, Fig. 3 shows the results of SDS-PAGE obtained under reducing conditions (l00 mM DTT, 50 C, 15 minutes).
Referring to Fig. 3, (l) 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:
Eluents: A solution [50 mM phosphate buffer (p~ 7)]
B solution [50 mM phosphate buffer (pH 7) containing 2 M NaCl]
Flow rate: 0.8 ml/min Detection wave length: 280 nm Fig. 5 shows the result of high performance liquid 15 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:
Eluents: A solution (0.1% trifluoroacetic acid) B solution (80% acetonitrile containing 0.1%
~ trifluoroacetic acid) Flow rate: l.0 ml/min Detection wave length: 280 nm 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:
:
', . .
: .

.-: . , .. , ~ , . - :. . , :~. ~ , , : . :
: -: : . .
~ . . . .
.
. . ,~ : ., ., :
'~ . . ~, , :, : ~

~()91/0~1~6 _ 37 PCT/JPgO/016~6 ```` 2070989 Developing solution: 0.l M phosphate buffer (pH 7) -~ 0.1 M sodium sulfate Flow rate: O.S ml/min Detection wave length: 280 nm The biological activity was assayed by the method in which the growth promoting activity of fetal calf cardiac endothelial cells was given as an indication of the biological activity. As a result, an activity equivalent to that of the sample was shown.
Example 8 Preparation of rhbFGF Mutein CS23 Production - Recombinant Having a Tetracycline Resistant Marker 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 completely digested with AvaI 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 ACGTCTAGACG
the resulting fragment with T4DNA ligase, and then the resultant was digested with AvaI and PstI to obtain a fragment A in which a PstI cleavage site was modified to a BglII cleavage site. Thereafter, plasmid pTB955 into which a gene coding for rhbFGF mutein C128 being deleted ~ C-terminus of hbFGF [Seno et al., European Journal of :.

,; .
. . .

` ' .

~ . : :
-: : .:: : . :
. :: :. ~ .. ... : . : ~
.: . ., ~ : - : : : -:: :
-. . . .. :: .

~-'09l/~)')l2~ 9~ 8 - Pcrl lPso/ol646 fi~, Biochemistry, 188, 239-245 (l990)] was completely digested with SalI and BamHI to obtain a fragment B of about 4.1 kbase pairs. pTB955 was completely digested with AvaI and SalI to obtain a fragment C of about 390 base pairs.
The three fragments A, B and C were ligated with T4DNA
ligase each other to obtain an expression plasmid pHP901 containing an ampicillin resistant marker (Fig. 7).
~ pHP901 was completely digested with EcoRI and'BglII 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 pUC18 completely ; digested with EcoRI and BamHI by T4DNA ligase to obtain plasmid pME901 (Fig. 8).
pME901 was completely digested with EcoRV and HindIII
to obtain a fragment of about 0.77 kbases containing a gene coding ~or 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 ScaI 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 BioloqY 189, 113-130 ~1986)] to prepare E. coli strain MM294 (DE3) . This E. coli strain was transformed by using the expression plasmid pCM901, thereby obtainlng recombinant '' .
., .
' ' .. . . .
: - :: - ; - .

.. : ., ~ -: ~ . . .
.

WO '~ 912~ - 3 ~ - 7 ~ 9 8 9 PcrtJ~o/0l646 f'.~`?;
E. coli MM294(DE3)/pCM901 (IEO LS]04, FERM BP-3168) carr~ing the gene coding for rhbFGF mutei.n CS23.
Example 9 1 liter of LB medium containing 5 mg/1 of tetracycline hydrochloride was inoculated with 1 ml of the master cells of recombinant E. coli MM294(DE3)/pCM901 (IFO 15104, FERM
BP-3168), and the cultivation with shaking was carried out at 30C for 8 hours. The total amount thereof was transferred to 20 liter of LB medium (containing 5 mg/l of tetracycline hydrochloride) placed in a 50 liter culture tank, and the cultivation was carried out at 30C, under an inner pressure of 1.0 kg/cm2G, with aeration at~a rate of 10 : l/min and with stirring at 300 rpm up to a Klett unit of 700. Then, the total amount of the resulting culture solution was transferred to 360 liter of a fermentation medium (M-9 medium containing 15 g/l of glucose, 15 g/l of casamino acids, 16.8 g/1 of Na2HPO4 12H2O, 3 g/1 of KH2PO4, 1 g/l of NH4Cl, 0.5 g/l of sodium chloride, 0.5 g/1 of MgSO4'7H2O, 5 mg/1 of thiamin hydrochloride, 2.5 mg/1 of FeSO4- 7H20 and 0.2 g/l of antifoaming agent), and the cultivation was initiated at 28C, under an inner pressure of 1.0 kg/cm2G, with aeration at a rate of 240 l/min and with stirring at 250 rpm. When the culture was grown to a Klett unit of about 450, 10 mg/l (42 ~M) of IPTG was added thereto. Since the remaining sugar was about 1%, a solution . containing a mixture of glucose ~60 g/l) and casein hydrolysate (15 g/l) was fed and the cultlvation was : .
~, : .

.: -, ' - .' ~
.

~ 9~ T'CT/JP90/01646 ~ 9~ - 40 -continued for 40 hours. As a result, l.fi g/l of rhbFGF
mutein CS23 was accumulated in a soluble state. The cultivated product thus obtained was subjected to a Sharpless centrifuge to collect wet cells, and the cells were frozen at -80C to store them.
l kg of the.obtained cells were treated by the manner similar to Example 7 to obtain a purified rhbFGF mutein CS23 original solution (yield 25.0 g). Quality of the solution was equal to that of the sample obtained in Example 7 in both of a biological activity and physical and chemical assay.

~.

:

:

.~, .~'~' ,~., :
~ .

: -::, : . .: . ~ . .

~0 ~1/()912~ PCr/JP90/Olfi46 ., .
Incorporated by reference Nature 249, 123 (1974) National Cancer Institute Monograph 48, 109 (1978) The EMBO Journal 5, 2523-2528 (1986) FEBS Letters 213, 189-194 (1987) The Journal of Biological Chemistry 263, 16471-16478 (1988) The Journal of Biological Chemistry 263, 16297-16302 (1988) Blochemical and Biophysical Research Communication 151, 701-708 (1988) European Patent Publication No. 281,822 Journal of Molecular Biology 189, 113-130 (1986) Biotechnology and Applied Biochemistry, 12, 436-449 (1990) Biochemical and Biophysical Research Communication 151, 701-708 (1988) Genetic Engineering, p.31-50, Academic Press (1983) Genetic Engineering: Principles and Methods, vol.3, p.l-32, Plenum Press (1981) Proc. Natl. Acad. Sci. U.S.A. 74, 4266-4270 (1977) Cell 16, 815-825 (1979) Nature 280, 35 (1979) J. Mol. Biol. 189, 113-130 (1986) Gene 56, 125-135 (1987) Proc. Natl. Acad. Sci. U.S.A. 69, 2110 (1972) Gene 17, 107 (1982) Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York,-1972 Cell, 57, 881-889 (1989) Gene, 68, 345-355 (1988) DNA, 5, 21-28 (1986) European Journal of Biochemistry, 188, 239-245 (1990) :
: .

-'~ :

Claims (13)

WHAT IS CLAIMED IS:

1. A vector containing a nucleotide sequence coding for a mutein in which at least one constituent amino acid of a mature human basic fibroblast growth factor (hbFGF) is replaced by another amino acid, and a T7 promoter upstream therefrom.

2. The vector as claimed in claim 1, in which the at least one constituent amino acid is at least one cysteine residue and the another amino acid is a serine residue.

3. The vector as claimed in claim 1, in which the mutein is a mutein in which cysteine residues at the 69- and 87-positions of the mature hbFGF constituent amino acids are replaced by serine residues.

4. A transformant transformed by the vector as claimed in claim 1.

5. The transformant as claimed in claim 4, in which a host is Escherichia coli having a T7 RNA polymerase gene downstream from a lac promoter.

6. The transformant as claimed in claim 5, which has a characteristics of E. coli MM294(DE3)/pTB960.

7. The transformant as claimed in claim 5, which has a characteristics of E. coli MM294(DE3)/pCM901.

8. A method for producing a mutein in which at least one constituent amino acid of a mature hbFGF is replaced by another amino acid, which comprises cultivating the transformant as claimed in claim 4 in a culture medium.

9. The method as claimed in claim 8, in which about 3 to 500 µM of isopropylthiogalactopyranoside (IPTG) is added to the culture medium on a logarithmic growth phase of the transformant as claimed in claim 5, followed by cultivation.

10. The method as claimed in claim 9, in which 3 to 300 µM
of IPTG is employed.

11. The method as claimed in claim 10, in which 6 to 200 µM
of IPTG is employed.

12. The method as claimed in claim 11, in which 6 to 80 µM
of IPTG is employed.

13. A method as claimed in claim 8, 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.