CN1055009A - The preparation of people's basic FGF-mutein - Google Patents
The preparation of people's basic FGF-mutein Download PDFInfo
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- CN1055009A CN1055009A CN90110115A CN90110115A CN1055009A CN 1055009 A CN1055009 A CN 1055009A CN 90110115 A CN90110115 A CN 90110115A CN 90110115 A CN90110115 A CN 90110115A CN 1055009 A CN1055009 A CN 1055009A
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- mutein
- amino acid
- hbfgf
- transformant
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
- C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
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- C12N15/67—General methods for enhancing the expression
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Abstract
Disclosed in this is (1) a kind of carrier that contains a kind of mutein nucleotide sequence coding, in this mutein, become at least a component amino acid in the rh-bFGF (hbFGF) to be replaced, and the T7 promotor is in its upstream extremity by another kind of amino acid; (2) transformant of the carrier of a kind of quilt (1) conversion; (3) prepare the method for mutein, the method for (4) (3), the isopropylthio galactopyranose glycosides (IPTC) of wherein about 3~500 μ m is added in the substratum by the logarithmic phase of transformant, and the method for (5) (4) further comprises purge process.
Description
The invention relates to the technology of the mutein of a kind of one-tenth rh-bFGF of preparation (below write a Chinese character in simplified form into hbFGF), wherein at least a component amino acid of hbFGF is replaced by another kind of amino acid, and this mutein can be used as wound healing promotor.
Prostatropin (bFGF) is the hormone of a peptide species, and it has about 17,000 molecular weight, and is mainly secreted by pituitary body.BFGF has the factor of very strong growth-promoting effect and is separated (D.Gospodarowicz, Nature 249,123(1974)) inoblast (as the BALB/c3T3 cell) as a kind of.The known FGF of people has growth-promoting effect (D.Gospodarowicz et al., Nature Cancer Insitute Monograph 48,109(1978)) to nearly all mesoblastema that derives from.Especially bFGF generates the effect of blood vessel, all proposes used as the treatment medicine of wound with as the possibility of prevention and treatment of diseases medicine such as thrombosis, arteriosclerosis together with its cell growth-promoting effect.
To the gene of hbFGF coding by people such as Abraham (The EMBO Journal 5,2523~2528(1986)) and people (FEBS Letters 213 such as Kurokawa, 189-194(1987)) cloning, and in zooblast, express (The Journal of Biological Chemistry 263,16471-16478(1988)) and expression in escherichia coli (The Journal of Biological Chemistry 263,16297-16302(1988)).Yet, this material production and inadequate, to such an extent as to and final sample is too unstable can not be as medicine.Have high stability and show identical biological activity for solving this compounds that instable various result of study has been found that the cysteine residues for the hbFGF molecule is replaced by serine residue
(Biochemical and Biophsical Research Communication 151,701~708(1988)〕。
And then, studied a kind of method for preparing a kind of mutein again, wherein at least a component amino acid of hbFGF is replaced (seeing the open No.281 of European patent, 822) by gene engineering by another kind of amino acid.
When adopting intestinal bacteria to prepare recombinant protein, often form the interior inclusion of water-insoluble with cumulative protein.When desired substance is isolated the inclusion in this class and during purifying, inclusion is dissolved by the protein denaturant that is added usually in this.Yet for the hbFGF mutein, so this protein of dissolved is inactivation, does not also have to study the technology of its reactivate of sening as an envoy at present.
If the use gene engineering, active state can be accumulated and be in to this mutein in a large number, but and then high productivity separates and purify, then the hbFGF mutein can be as medicine.Thereby a key issue is exactly to set up the method for this mutein of a kind of produced in high yields.
Usually, when adopting gene engineering to prepare a large amount of gene product, it is important then selecting host's carrier system and promotor, and gene is separately depended in this different selection.The result of study of various effective expression systems for the hbFGF mutein has disclosed a kind of bacillus coli gene expression system (the F.M.Studier et al. that adopts the T7 promotor, Journal of Molecular Biology 189,113-130(1986)) be to express the fabulous system of hbFGF mutain plasmagene.The combination of T7 promotor and hbFGF mutain plasmagene is novel.This T7 promotor is considered to a kind of strong promoter.Yet in some cases, inclusion in this cumulative protein (as human body interleukin-2, prolactin antagonist) forms, and its great majority all are with inactivation attitude cumulative.The example of leukin in it has been observed by the present inventors that, Paris, people such as N. have been found prolactin antagonist example (Paris, N.et al, Biotechnology and Applied Biochemistry, 12,436-449(1990)).
The present inventor furthers investigate for accumulation hbFGF protein a large amount of, that be in active condition, and the isopropylthio galactopyranose glycosides of having found at present to add lower concentration is effective.In addition, present inventor and then also set up a kind of the separation effectively and purifying hbFGF method of protein has finished the present invention like this.
The invention provides:
(1) contains carrier to a kind of mutein nucleotide sequence coding, in this mutein, become at least a component amino acid of rh-bFGF (hbFGF) to be replaced, and the T7 promotor at its upstream end by another kind of amino acid;
(2) a kind of transformant that is converted by carrier described in above-mentioned (1);
(3) transformant described in above-mentioned (2), wherein the host has a t7 rna polymerase gene that is in lac promotor downstream end;
(4) a kind of method for preparing mutein, at least a component amino acid of ripe hbFGF is replaced by another kind of amino acid in this mutein, and this method is included in and cultivates the transformant described in above-mentioned (2) in the substratum;
(5) method described in above-mentioned (4), wherein, about 3~500 μ M(mmoles) isopropylthio galactopyranose glycosides (below be abbreviated as IPTG) joins in the substratum in the logarithmic phase of above-mentioned (3) described transformant, then cultivates; And
(6) method described in above-mentioned (5), the solution that wherein contains the mutein product uses chromatography to carry out purifying, and adopt cross-linked polysaccharides vitriol, have sulfonic acid group in return the synthetic polymer of group and/or the synthetic polymer that is used for gel-filtration as carrier.
According to the present invention, the ripe hbFGF mutein of substituted type can prepare in a large number, thereby the present invention can be used for this mutein of suitability for industrialized production.
Fig. 1 shows the dna nucleotide sequence of the rhbFGF mutein CS23 that uses among the embodiment 1 and nucleotide sequence coded proteinic aminoacid sequence;
Fig. 2 is the structural representation of the plasmid pTB960 that obtains among the embodiment 1;
Fig. 3 shows the SDS-pAGE figure and the mark of the purifying sample that obtains among the embodiment 5; And
Fig. 4 to Fig. 6 has provided the high speed liquid chromatography figure by the purifying sample that obtains among the embodiment 5;
Fig. 7 to Fig. 9 is the plasmid pHP901 that is obtained by embodiment 8, the structural representation of pME901 and pCM901.
Sophisticated hbFGF of the present invention is a kind of peptide that is made of 146 seed amino acids, in Fig. 1, with the amino proline that is next to the terminal methionine(Met) of N-as number one, and with the Serine of C-end as No. 146.
The example of the hbFGF mutein that provides in the present invention comprises this proteinoid, at least one component amino acid that is ripe hbFGF is replaced by another kind of amino acid, as European patent prospectus No.281,822 and Biochemical and Biophysical Research Communication 151,701-708(1988)) described in.
As for this kind mutein (wherein at least a amino acid of hbFGF is replaced by another kind of amino acid) amino acid whose number of the component of hbFGF before replacement, it can be an any amount, only otherwise the characteristic (as vascularization characteristic, cell growth stimulating activity and cell differentiation activity) of forfeiture FGF.
The amino acid whose example of component comprises halfcystine and the amino acid different with halfcystine replacing up till now.What particularly point out is that halfcystine is preferable.Other amino acid are amino acid whose aspartic acid, arginine, glycine and the Xie Ansuan of comprising of component replacing previous crops except that half Guang base acid.
When component amino acid was halfcystine before replacing, neutral amino acids was preferable as substituted amino acid.The object lesson of neutral amino acids comprises glycine, Xie Ansuan, L-Ala, leucine, Isoleucine, tyrosine, phenylalanine, Histidine, tryptophane, Serine, Threonine and methionine(Met).Especially Serine and Threonine are preferable.
When component amino acid is a seed amino acid except that halfcystine before replacing, is chosen in wetting ability, hydrophobicity and electric charge and is different from and replaces up till now amino acid whose other amino acid of component as substituted amino acid.Particularly, when amino acid was aspartic acid before replacing, substituted amino acid comprised l-asparagine, Threonine, Xie Ansuan, phenylalanine and arginine, and l-asparagine and arginine are preferable.
When amino acid was arginine before replacing, substituted amino acid comprised glutamine, Threonine, leucine, phenylalanine and aspartic acid.Glutamine is particularly preferred.
When component amino acid was glycine before replacing, substituted amino acid comprised Threonine, leucine, phenylalanine, Serine, L-glutamic acid and arginine.Threonine is particularly preferred.
When component amino acid was Serine before replacing, substituted amino acid comprised methionine(Met), L-Ala, leucine, halfcystine, glutamine, arginine and aspartic acid, and methionine(Met) is preferable.
When component amino acid was Xie Ansuan before replacing, substituted amino acid comprised Serine, leucine, proline(Pro), glycine, Methionin and aspartic acid, and Serine is preferred.
Concerning as primary component amino acid, aspartic acid, arginine, glycine, Serine and Xie Ansuan are preferred before replacement.
Concerning as substituted amino acid, l-asparagine, glutamine, arginine, Threonine, methionine(Met), Serine and leucine are preferred.
Best substituted mutein comprises a kind of like this mutein, and promptly component amino acid cysteine is wherein replaced by Serine.
In above-mentioned substitution reaction, can carry out at least two kinds of amino acid whose replacements of component simultaneously.Preferably replace two to three kinds of component amino acid.
The preferred embodiment of hbFGF mutein in the present invention comprises that at least a cysteine residues of sophisticated hbFGF mutein is replaced by serine residue and the mutein that obtains.
As mutein, the hbFGF mutein CS23(of reorganization hereinafter also is abbreviated as rhbFGF mutein CS23) be particularly preferred, wherein the cysteine residues that is in 69 and 87 positions of sophisticated hbFGF is replaced by serine residue respectively.The amino acid position of above-mentioned hbFGF is prefaceization, and its prefaceization is not hold the amino proline of methionine(Met) as number one, as shown in Figure 1 with being in close proximity to N-in the aminoacid sequence.
For preparing this mutein, adopted locating point mutagenesis.This technology has been widely known by the people and has been discussed at Gentic Engineering P13-50, Academic Press(1983 by R.F.Lather and J.P.Lecoq) in.Be oriented to oligonucleotide mutagenesis and discussed at Gentic Engineering:Principles and Methods by M.Smith and S.Gillam, Vol 3, P1-32, Plenum Prese(1981) in.
The structure gene of this mutein of preparation coding, for example available following step preparation:
(a) a kind of single stranded DNA that will contain hbFGF structure gene strand is hybridized with a kind of mutagenic oligonucleotide primer, (above-mentioned primer is complementary in such district, promptly this district comprises the codon that halfcystine is replaced by this strand, or comprise a kind of in some cases with this codon paired antisense triplet, verified, this situation can not be applied to be different from other amino acid whose codons that remove above-mentioned codon, or be different from some cases antisense triplet)
(b) extend this primer with archaeal dna polymerase, form a kind of mutation heteroduplex nucleic acid molecule, and
(c) duplicate this mutation heteroduplex nucleic acid molecule.
Then, will be used for shifting phage DNA through the gene of mutagenic treatment separates and is incorporated into a kind of plasmid.
The used T7 promotor of the present invention can adopt 17 kinds of promotors on T7DNA, finding any (J.L.Oakley et al., Proc.Natl.Acid.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)), and φ 10 promotors (A.H.Rosenberg et al., Gene 56,125-135(1987)) are preferred the uses.
As the used transcription termination region of the present invention, any terminator all can be adopted, and need only it and in Escherichia coli system, work, and T φ terminator (F.W.Studier et al., J.Mol.Biol.189,113-130(1986)) be preferred the use.
Be used for t7 rna polymerase of the present invention and comprise T7 gene 1 (F.W.Studier et al., J.Mol.Biol.189,113-130(1986)).
The carrier example that is used to form carrier of the present invention comprises pBR322, pUC8, pUC9, pMB9, pRC7, pACYC177 and pKN410.
The carrier that is used for the present invention is by going into T7 promotor and the fusion of T7 terminator in the above-mentioned carrier to form.This class carrier comprises pET-1, pET-2, and pET-3, pET-4 and pET5 (A.H.Roseberg, Gene 56,125~135(1987)), and pET-3C(is the same) be preferred.
As the host who is used for transformant of the present invention, any t7 rna polymerase gene (T7 gene 1) (F.W.Studier et al., J.Mol Biol.189 of having mixed, coli strain 113-130(1986)) all can use, for example MM294, DH-1, C600 and BL21.Bacterial strain MM294 and BL21 are preferred the uses, and the lambda particles phage that wherein comprises T7 gene 1 is by lysogenization.This t7 rna polymerase gene also can be used as a kind of have with expression vector not the homologous plasmid deposit.In this case, be to adopt the lac promotor to the promotor of T7 gene 1, its expression is with sec.-propyl-1-sulfo--β-D-galactopyranose glycosides (IPIG) inductive.
Being used for transformant of the present invention can be by using method as known in the art, with the plasmid that the T7 promoter expression is had the genetic transcription terminator will be fusion above-mentioned T7 gene 1(RNA pol gene) intestinal bacteria transform and obtain, these methods are as being described in Proc.Natl.Acad.Sci.U.S.A.69,2110(1972) and Gene 17, in 107(1982).In these cases, used host can transform with the plasmid with T7 N,O-Diacetylmuramidase group earlier, and the transformant that obtains like this has two kinds of different plasmids simultaneously.
When this transformant was cultivated, liquid substratum was to be specially adapted to as the cultivation medium.Carbon source, nitrogenous source, mineral compound and the essential material of other transformant growth also are contained in wherein.Carbon source comprises, for example glucose, dextrin, dissolving starch and sucrose.Nitrogenous source comprises inorganic or organic substance, and for example ammonium salt, nitrate, cereal steeping fluid, peptone, casein, casamino acids, meat soak meter, soyabeen grists and potato extracting solution.The example of mineral compound comprises calcium chloride, Sodium phosphate dibasic and magnesium chloride, yeast extract, VITAMIN, positive growth factor and analogue and then also adds wherein.
The pH value of substratum wishes it is 6 to 8.
As the substratum that is used to cultivate the E.coli transformant, even more preferably, as M9 substratum (Miller, Journal of Experiments in Molecular Genetics 431-433, Cold Spring Harbor Laboratory, New York, 1972), added glucose and casamino acids in addition.Iron ion also can join in this substratum.Iron ion be those dissociable one-tenth iron ions in solution material or be the material that can the iron ion form uses.This class material comprises molysite, is preferably divalence or ferric iron inorganic salt, for example iron protochloride, iron(ic) chloride, ferrous sulfate or ferric sulfate, tertiary iron phosphate and iron nitrate.The add-on of iron ion is about 10
-6To 10
-4Mole, preferable add-on is 5 * 10
-6To 5 * 10
-5Mole.Culturing process was carried out about 3 to 72 hours at 15 ° to 43 ℃ usually, was preferably about 12 to 48 hours, ventilated if necessary or stirred.
For genetic expression, reasonable is to add IPTG in culturing process.Thus, be connected in the T7 gene 1(RNA pol gene of lac promotor downstream end) expressed, and the T7 phage rna polymerase 1 of preparation identifies the T7 promotor thus.According to prior art, for expressing the lac promotor, the IPTG add-on is 0.1 to 20 mmole, is preferably 1 to 2 mmole (reaching people such as 1 mmole IPTG:L φ bner-Olesen, Cell, 57 881-889(1989); People such as the IPTG:Ernst H. of 2 mmoles, Gene, 68,345-355(1988); The IPTG:Luck of 20 mmoles, people DNA such as D.N., 5,21-28(1986)).Yet have been found that the IPTG cumulative protein in abduction delivering by adding such amount forms inclusion body, it obtains the protein accumulation of inactivation sometimes.Have been found that in culturing process about various results of study with the method for dissolved state cumulative protein, the IPTG that adds about 3 to 500 μ M can at first achieve the goal, preferably add about 3 to 300 μ M, 6 to 200 μ M more preferably, and best be about 6 to 80 μ M.When carrying out large scale culturing such as tank culture, the add-on of IPTG is good with about 10 to 50 μ M, is preferably 10 to 200 μ M, be more preferably 10 to 100 μ M, and the best is 10 to 80 μ M.When carrying out small-scale when cultivating such as during flask culture, the preferable add-on of IPTG is 3 to 100 μ M, more preferably 6 to 8 μ M.It is in after beginning to cultivate 1 to 24 hour, be preferably in 3 to 12 hours, and IPTG preferably to add in logarithmic phase that IPTG at first adds.After this IPTG adds with intermittent type or continous way on demand.The substratum that contains IPTG is in temperature about 20 ° to 40 ℃, is preferably about 20 to 30 ℃ and cultivates down.As the surrogate of isopropylthio galactopyranose glycosides, under some occasion, can use, as propyl dithiocarbamate galactopyranose glycosides, methyl sulfo-galactopyranose glycosides, butyl sulfo-galactopyranose glycosides and cyclohexyl thio galactopyranose glycosides.
HbFGF mutein in the present invention can separate and purifying from above-mentioned substratum, for example, can utilize following method.
When hbFGF mutein of the present invention extracted from culturing cell, these cells employing methods known in the art for example centrifuging were collected after cultivation.Then with the cell collected by glass sphere, French extruding ultrasonication, N,O-Diacetylmuramidase is handled and/or freeze-thaw to break.It is especially preferred breaking with glass sphere.
People are studied by the whole bag of tricks of the hbFGF mutein of above-mentioned supernatant liquor acquisition according to the present invention purifying.The result is that high productivity has obtained highly purified sample, the method that adopts is the combination of following several method: promptly: use the affinity chromatography of a kind of cross-linked polysaccharides vitriol as carrier, carrier is that a sulfonic acid group is arranged is the ion exchange chromatography of the synthetic polymer of cation exchange groups, use the chromatography of the synthetic polymer of gel-filtration, this method is combined each other use at least once as carrier.
Make cross-linked polysaccharides vitriol used in this invention comprise cross-linked cellulose vitriol, Sepharose vitriol and sephadex vitriol.
Above-mentioned Mierocrystalline cellulose is the polysaccharide of being made up of the chain attachment glucose of β-1,4, its molecular weight preferable about 50,000 to 2,000,000.Its specific examples comprises the Avicel(Microcrystalline Cellulose, and Asahi Chemical Industry is Japan) with Cellulofine(Chisso Corporation, Japan).
Above-mentioned agarose be a kind of be the polysaccharide of main ingredient with agar, have D-galactosyl-(β 1 → 4)-3, the repeating structure of 6-dehydration-L-galactosyl-(α 1 → 3).Its molecular weight is preferably 10,000 to 5,000,000, and its specific examples comprises agarose 28(Sepharose 2B), Sepharose 4B and Sepharose 6B(Pharmacia, Sweden).
Above-mentioned dextran is a kind of D-glucose polymkeric substance, and it mainly contains α (1 → 6) chain, for example by effect forms to sucrose with a kind of microorganism such as Leuconostoc mesenteroides.Its molecular-weight average is preferable to be about 1,000 to 40,000,000.
The preparation that is used in the cross-linked polysaccharides vitriol among the present invention is by with cross-linked polysaccharides, and with known linking agent such as Epicholorohydrin and 2, the 3-dibromo-propanol is handled according to methods known in the art and obtained as above-mentioned dextran, agarose and Mierocrystalline cellulose.
These cross-linked polysaccharides are business-like, can be from Pharmacia(Sweden) buy, commodity are called Sephadex G-10, Sephadex G-15, Sephadex G-25, Sephadex G-50 and Sephadex G-100(sephadex), and commodity Sepharose CL-2B by name, Sepharose CL-4B and Sepharose CL-6B(Sepharose).Cross-linked cellulose also can be from Chisso Corporation(Japan) buy, commodity are called the Cellulofine(cross-linked cellulose).The cross-linked polysaccharides vitriol of being wanted can react with these cross-linked polysaccharides and synthetic obtaining as chloro sulfonic acid and sulphuric anhydride ester by with following known sulfur acidizing reagent.
The example of these cross-linked cellulose vitriol comprises the Kogyo(Japan by Seikagaku) product of putting on market, commodity are called Sulfated Cellulofine(cross-linked cellulose vitriol).
The example of sephadex vitriol comprises the Sephadex of sulphating.
The example of Sepharose vitriol comprises the Sepharose of sulphating.
The form that to be used for cross-linked polysaccharides vitriol of the present invention can be corresponding salt.The example of its salt comprises sodium salt, sylvite, ammonium salt and leptodactyline.Sodium salt is particularly preferred.
Be used for cross-linked polysaccharides vitriol of the present invention and be not dissolved in water, thereby preferably use with its colloid attitude by hydration.
Adopt the cross-linked polysaccharides vitriol purification hbFGF method of protein among the present invention to comprise following affinity chromatography.
The water culture medium that contains the hbFGF mutein is the solution that contains the hbFGF mutein.This water culture medium comprises water and substratum, mainly is made up of water, preferably uses buffered soln, as phosphate buffer, nitrate buffer reagent and the agent of Tris-hydrochloride buffer, regulates the pH value scope, loses activity to prevent this hbFGF mutein.
The solution that then will contain the hbFGF mutein is readjusted in the pH value scope about 5.0 to 9.0, uses distilled water diluting then as required, so that its specific conductivity is about 15m υ or lower.The mutein solution that contains hbFGF that obtains is thus contacted with cross-linked polysaccharides vitriol gel phase.For this purpose, batch method and column chromatography all can use.Yet because column chromatography is easy and simple to handle and more suitable.In column chromatography, this cross-linked polysaccharides vitriol gel is filled in the post, uses a kind of suitable buffered soln subsequently, and the nitrate damping fluid (PH7.0) that for example contains the 50mM of 0.4M NaCl fully washs this post, so that its equilibration.The amount of the gel of required usefulness depends on the character that contains the hbFGF protein soln of filling, is preferred and every milligram hbFGF mutein is about 1 to 50 milliliter scope.
The above-mentioned solution that will contain the hbFGF mutein then is packed in the post.Filling speed is selected air speed (SV) scope about 0.1 to 5.0.After filling, with the post thorough washing, with the ionic strength of ordinary method raising buffered soln, with wash-out and recovery hbFGF mutein.For improving ionic strength, can add wherein such as the NaCl salt, perhaps use the buffered soln of high density, specific conductivity is brought up to the υ at least about 15m like this, preferably 30m υ at least.For elution process, all can use with the concentration gradient elution process in batches.When adopting the concentration gradient elution process, for example the concentration of NaCl is brought up to 2.0M by about OM gradually, carries out wash-out and recovery like this.The hbFGF mutein of the high purifying of result is made with high yield.
Can be used for of the present invention have sulfonic group in return the example of the synthetic polymer of group comprise that those sulfonic acid groups are direct or non-polymkeric substance that are introduced directly into hydrophilic vinyl polymer, vinylbenzene-divinyl benzene polymers, acrylamide polymer and analogue.From reclaiming and the operation viewpoint, sulfonic group is introduced in the SP(sulfopropyl of hydrophilic vinyl polymer)-Toyopearl(Tosoh, Japan) be preferred especially the use.
When carrying out chromatogram, the concentration that the partially purified hbFGF of containing mutein solution is adjusted to salt is about 50mM or lower, for example, in the situation with phosphate buffer, it is adsorbed on the above-mentioned resin, and the PH scope is 5 to 7.For adsorption step, all can use with the column chromatography system, but from the operation viewpoint, the column chromatography system is preferred the use in batches.Adopt the method that improves salt concn to carry out wash-out from resin.To elution step, all can use with E-test in batches.When using batch processes, for example, a kind ofly form the buffered soln that concentration is about 50mM to 1M in the above-mentioned phosphate buffered saline buffer and can be used by NaCl is joined.The nitrate damping fluid can replace phosphate buffered saline buffer and be used.Elution process is to be about 1 to 25 ℃ in temperature, preferablely is about 1 to 10 ℃, preferably carries out under about 4 ℃.
The example of the synthetic polymer that is used for gel-filtration that the present invention is used comprises hydrophilic vinyl polymer and acrylamide polymer.From colloidal weather resistance and operation viewpoint, Toyopearl HW(Tosoh, Japan), the hydrophilic ethylene polymkeric substance is preferred the use.
When the partially purified solution that contains the hbFGF mutein is for example handled, all can be used as the expansion solvent such as phosphate buffered saline buffer and this class damping fluid of nitrate damping fluid on Toyopearl HW-50F chromatographic column.Yet, more advantageously use the nitrate damping fluid (PH7.0) of 50mM.Treating processes is at about 1 to 25 ℃, is preferably at about 1 to 10 ℃, and the best is to carry out under about 4 ℃.
Additive method except that aforesaid method also can be used as one of purification step.In these class methods, for instance, natural product such as Mierocrystalline cellulose, agarose and dextran and inorganic materials such as glass sphere also can be used as the carrier in the ion exchange chromatography.
As the carrier of gel-filtration, also can use similarly mainly by natural product, the gel that (as Mierocrystalline cellulose, agarose and dextran) formed, and based on the gel of inorganic materials (as glass sphere).
The sample of Huo Deing also can be dialysed also lyophilize and be formed the dry state powder like this.And then, serum albumin can be added in the sample, store them as carrier, sample is avoided being attracted on the container thus.
Purify or storage process in, to be convenient to suppress sample oxidized with the reductive agent coexistence of tracer level.This reductive agent comprises beta-mercaptoethanol, dithiothreitol (DTT) and gsh.
According to the present invention, pyrogen-free and endotoxic pure substantially hbFGF mutein have in essence been made.The pure substantially hbFGF mutein that makes according to the present invention comprises the product that contains hbFGF mutein of the present invention, and it measures with protein content, accounts for 95%(W/W), preferably account for 98%(W/W) or more.
The hbFGF mutein that is made by aforesaid method of the present invention has fibroblast growth promoting activity, vascular endothelial cell growth promotes activity active and the generation blood vessel, and has high stability and hypotoxicity.Thereby they can be used as the accelerator for concrescence of tissue and similar wound after burn, wound, the operation, or generate vasoactive according to it, as the medicine of treatment thrombosis, arteriosclerosis and similar disease.They also can be used as the reagent that promotes cell cultures.What particularly point out is that at least a component cysteine residues is preferred by this mutein that a kind of serine residue replaced, and this is because its high stability.
When hbFGF mutein of the present invention is used as medicinal preparations, they can or be administered orally in warm-blooded animal (for example people, mouse, rat, hamster, rabbit, dog and cat) by administered parenterally, can powder type, or with being suitable for medicinal carrier, vehicle and diluent as medicinal compositions, for example with injection liquid, tablet, capsule, solution or ointment.
Injection liquid can for example use physiological saline or contain glucose or the formation soup of other assistant agents with traditional method preparation.Its medicinal compositions also can prepare according to conventional methods as tablet or capsule.
When hbFGF mutein of the present invention when the above-mentioned medicinal preparations, the optimal dose that they deliver medicine to above-mentioned warm-blooded animal be every day about 1ng/kg body weight to 100 μ g/kg body weight, consider the approach of administration and illness itself or the like.
And then, when hbFGF mutein of the present invention is used as the reagent that quickens cell cultures, preferably it being joined in the substratum, add-on is every liter of about 0.01 to 10 μ g of substratum, preferable add-on is that every liter of substratum is about 0.1 to 10 μ g.
When the alkali in this specification sheets and the accompanying drawing, amino acid were represented with abbreviation, then used abbreviation was biochemical vocabulary or the abbreviation commonly used in the art that IUPAC-IUB Commission adopts, as adopts following abbreviation.When the optically active isomer of this amino acid existed, unless otherwise indicated, then represented was left-handed type (L type).
DNA: thymus nucleic acid
CDNA: complementary DNA (cDNA)
A: VITAMIN B4
T: thymus pyrimidine
G: guanine
C: cytosine(Cyt)
RNA: Yeast Nucleic Acid
DATP: deoxyadenosine triphosphate
DTTP: deoxythymidine triphosphate
DGTP: deoxyguanosine triphosphate
DCTP: deoxycytidine triphosphate
ATP: adenosine triphosphate
Tdr: thymidine
EDTA: ethylenediamine tetraacetic acid (EDTA)
SDS: sodium laurylsulfonate
Gly: glycosides propylhomoserin
Ala: L-Ala
Val: Xie Ansuan
Leu: leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: halfcystine
Met: methionine(Met)
Glu: L-glutamic acid
Asp: arginine
Lys: Methionin
Arg: arginine
His: Histidine
Phe: phenylalanine
Tyr: tyrosine
Trp: tryptophane
Pro: proline(Pro)
Asn: l-asparagine
Gln: glutamine
Referring to Fig. 1, with regard to the amino acid whose preface of hbFGF-component was imitated, the Met of the N-end of this aminoacid sequence was arranged as No. 1.And in this manual, the Pro behind the adjacent Met is compiled to No. 1.
The transformant E.Coli.MM294/PTB762 that is loaded with plasmid pTB762 that is used for following embodiment 1, the transformant E.Coli MM294(DE3 that in embodiment 1, makes)/PTB960 and the transformant E.Coli MM294(DE3 that in embodiment 8, makes)/PCM901 entrusted the InStitute for Fermentation of Japan, Osaka(IFO), and the Fermentation Research Institute of Japan, Agency of Industrial Science and Technology, Ministry of International Trade and Industry(FRI) preservation.They go into to hide registration number and deposit the date and list in table 1.Beginning at the E.Coli of FRI preservation MM294/PTB762 is to go into to hide registration number with FERM P number mark.Said deposita is transferred to the deposita by Budapest Treaty, and this transformant is deposited with the registration number of going into to hide of FERM BP number sign at FRI.
Table 1
Transformant IFO FRI
E.Coli MM294 IFO14613 FERM P-9409
PTB762 (1987.5.27) (1987.6.11)
FERM BP-1645
E.Coli MM294 IFO14979 FERM BP-2690
(DE3)/PTB960 (1989.12.12)?(1989.12.16)
E.Coli MM294 IFO15104 FERM BP-3168
(DE3)/PCM901?(1990.10.26) (1990.11.17)
After this present invention will describe in detail with the following example.What will of course be appreciated that is that the purpose of these embodiment is not to be used for limiting the scope of the invention.
Embodiment 1
The recombinant chou of preparation rhbFGF mutein CS23
The gene plasmid PTB 762 that contains promising rhbFGF mutein CS23 coding is through handling with EcoRI and PstI, cut and be rhbFGF mutein CS23 coded DNA segment, and in this mutein four be in 69 of cysteine residues among the hbFGF and 87 locational cysteine residues and replaced (people such as Senoo by serine residue, Biochemical and Biophysical Research Communicatton 151,701-708(1988), European patent discloses 281,822).With EcoRI and PstI plasmid PTB1004(European patent is disclosed 336,383 then) complete digestion, be connected on this resulting largest fraction by the T4DNA ligase enzyme in this segment after this, then constitute plasmid PTB921.Plasmid PTB921 splits with restriction enzyme EcoRI then, make mung-bean nuclease then make its end become concordant end with this product reaction, then, then obtain containing the dna segment of rhbFGF mutein encoding gene for this reason with restriction enzyme Bg1II division.In addition, be loaded with T7 phage (people such as F.W.Studier, Journal of Molecular Biology 189 113-130(1986) a kind of φ 10 promotors are split with restriction enzyme, this product is handled with mung-bean nuclease and then its end is become concordant end then, then carries out the reaction with restriction enzyme BamHI again.The dna segment that will contain rhbFGF mutein CS23 with the T4 ligase enzyme is connected on the resulting DAN segment, and the result obtains expression plasmid PTB960(Fig. 2).
E.Coli MM294 and lambda particles phage DE3 lysogenization are with preparation E.Coli bacterial strain MM294(DE3), in this phage, inserted a kind of rna polymerase gene (people such as F.W.Studier of this phage, Journal of Molecular Biology 189,113-130(1986).By expression plasmid PTB960 this E.Coli bacterial strain is transformed, obtains to have the genetic recombinants E.Coli MM294(DE3 that is shown in Fig. 1 thus for rhbFGF mutein CS23 coding)/PTB960(IFO14979, FERM BP-2690).
Embodiment 2
Recombinant chou E.Coli MM294(DE3 with a bacterium cup amount)/PTB960(IFO14979, FERM BP-2690) is inoculated in the LB substratum (NaCl of the bacterium tryptone of 10 grams per liters, the bacterium yeast extract of 5 grams per liters and 5 grams per liters) of the sodium ampicillin that contains 50mg/l of 30ml, then 37 ℃ of following wave and culture a whole nights.This nutrient solution of 1.5ml is moved on to the (Na of 16.8 grams per liters in the M-9 substratum of 30ml
2HPO
412H
2The KH of O, 3 grams per liters
2PO
4, 1 grams per liter NH
4The MgSO of the NaCl of Cl, 0.5 grams per liter and 0.246 grams per liter
47H
2O), add the glucose of 15 grams per liters, the casamino acids of 15 grams per liters, the vitamin of 1 mg/litre and the sodium ampicillin of 50 mg/litre in this nutrient solution, then at 37 ℃ of following wave and culture.When this culture grows into the turbidity of 100-120Klett unit, add IPTG to obtain multiple concentration, then this cultivation is proceeded four hours, deposit through the centrifugation collecting cell and under-20 ℃.
Prepare two parts of identical samples.The Guanidinium hydrochloride that adds 7M in a sample makes cell fully scatter then, then leaves standstill 1 hour.After centrifugation obtains supernatant liquor (total amount of bFGF mutein).(TriS-HCl of the NaCl of 10% sucrose, the EDTA of 10mM, 100mM, the APMSF of 1mM and 10mM PH=7.6), left standstill this mixture 1 hour then under 4 ℃ to add the lysozyme soln of 50 mcg/ml in another sample.Use ultrasonic disruption method (Kubota Insonater 200M) to make these cytoclasis again, then obtain a kind of supernatant liquor (the hbFGF mutein that solubility is arranged) through centrifugation.To these two kinds solution dilutions through extracting, determine the amount of rhbFGF mutein CS23 with a kind of ELISA method, it the results are shown in table 2.
Table 2
IPTG add-on rhFGF mutein solubility rhbFGF sudden change
The semi-invariant protein C S23's of (μ m) CS23
(mg/l) rate of formation (%)
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 implied above, when in cultivating on a small scale, cultivating, can add a small amount of IPTG scope and reach effect of the present invention as retort bottle.That is to say, approximately add 1mM IPTG and then obtain
The ac promotor.When adding IPTG with 100 μ M or bigger amount, low and the most required product of productive rate is accumulated with insoluble attitude, in contrast, according to the present invention, IPTG is with 3 μ M~100 μ M, particularly add fashionablely with the amount of the scope of 6 μ M to 80 μ M, the result has just obtained the result that is all beyond one's expectations, promptly is significantly improved at productive rate and aspect soluble protein accumulation ratio.
To contain 15g/l glucose, 15g/l casamino acids, and the M-9 substratum (PH=6.8) of 5mg/l vitamin place the fermentor tank of one 5 liter capacity with 2.5 liters amount, carry out disinfection then.Strain cultured solution (preparing by mode described in the embodiment 2) with 125ml is inoculated in this substratum again, then PH is transferred to 6.8, under 37 ℃, with 2.5 liters/minute flow ventilation, to cultivate under 1000 rev/mins the stirring velocity.When turbidity reaches 120Klett unit in (beginning to cultivate back 3 hours) between this incubation period, add IPTG and make its amount reach 10 μ M.After beginning to cultivate 8 hours, add glucose and casamino acids again, cultivate and proceed long 15 hours altogether so that its content is respectively 10g/l.The rhbFGF mutein reaches 440mg/l with the soluble state semi-invariant as a result.Otherwise, under similarity condition, add 400 μ M IPTG, only produce the rhbFGF mutein CS23 of 17.5mg/l.
To transfer in the fermentor tank of one 5 liter capacity with the seed culture fluid that mode described in the embodiment 2 obtains, the substratum identical with embodiment 3 is housed in this jar, then at 30 ℃, PH=6.8, ventilation flow rate is 2.5 liters/minute, to begin cultivation under 1000 rev/mins the speed agitation condition.When cultivating beginning after 7 hours, when turbidity reaches about 700 Klett units, add the IPTG of 42 μ M.After beginning to cultivate 8.5 hours, under PH=6.8, add glucose and the 20g/l casamino acids of 20g/l.Cultivate thus and carried out 36 hours.In this nutrient solution, accumulated the rhbFGF mutain CS23 of the soluble state of 860mg/l.
Embodiment 5
To contain glucose, the 15g/l of 15g/l casamino acids, and the M-9 substratum (PH6.8,2.5 liters) of the vitamin of 5mg/l place one 5 liters fermentor tank and sterilization.Then iron ion is being added in this jar through the sterilization back with the concentration shown in the table 3.The strain cultured solution for preparing in mode described in the embodiment 2 to a 125ml of the culture medium inoculated of gained then transfers to 6.8 with PH, ventilates at 30 ℃, the flow that divides with 2.5l/, cultivates under 1000 rev/mins the rotating speed stirring condition.(greatly about beginning to cultivate the back 5.5~6 hours) adds IPTG so that its content reaches 40 μ M, and meanwhile, culture temperature reduced to 25 ℃ when culture grows to about 300 Klett turbidity units, then cultivates 23.5 hours again.After beginning to cultivate 7~7.5 hours, the ratio with 15g/l adds glucose and casamino acids separately, and in culturing process pH value is remained on 6.8.The results are shown in table 3.
Table 3
Iron ion add-on rhbFGF mutein CS23 productive rate
0 1.00
2×10
-6M 1.42
9×10
-6M 2.60
3.6×10
-5M 2.06
1×10
-4M 1.23
(annotate: what this iron ion add-on was represented is that the back adds the iron ion amount in this substratum.This productive rate is represented a kind of weight ratio, and when adding, the iron ion amount was 0, getting this ratio was 1)
Embodiment 6
To 1 liter of LB substratum of the sodium ampicillin that contains 50mg/l with 1ml recombinant chou E.Coli MM294(DE3)/PTB960(IFO14979, FERM BP-2690) chief cell inoculate, again in 30 ℃ of wave and culture 8 hours.With its all amount go to filling in 20 liters the culture tank of LB substratum (sodium ampicillin that contains 50mg/l), of one 50 liter capacity again in 30 ℃, 1.0kg/cm
2Depress in the gauge pressure, with 10
/ minute flow ventilation, to cultivate 7 hours under 300 rev/mins the stirring velocity.Then 18 liters of the culture solution of gained are moved in 360 liters the fermention medium and (contain the glucose of 15g/l, the casamino acids of 15g/l, the vitamins B of 5mg/l
1And the FeSO of 5mg/l
47H
2The M-9 substratum of O), again in 30 ℃, at 1.0kg/cm
2Depressing in the G, is to begin under 250 rev/mins of conditions to cultivate with 240 liters/minute flow ventilation, stirring velocitys.When this nutrient solution reaches about 1000 Klett units, add IPTG with the amount that is equivalent to 100 μ M, culture temperature is reduced to 25 ℃ then.Meanwhile, will contain 18kg glucose, 5.4kg casamino acids 50 liters of thimerosals with about 2 liters/time speed add wherein, continue then to cultivate 36 hours.Result's accumulation obtains the rhbFGF mutein CS23 of the soluble state of 1.2g/l.With the product cultivated like this with husky sharpie's centrifuge Sharple separated and collected to wet cell, in-80 ℃ with its freezing depositing.
Embodiment 7
Purifying rhbFGF mutein CS23
To be suspended in the damping fluid of dithiothreitol (DTT) (DTT) of 4 liters right-amidino groups phenyl methanesulfonamide acyl fluorides hydrochloride (APMSF) that contains 25mM phosphate buffered saline buffer (PH=6.0), 0.1mM and 2mM in-80 ℃ of freezing cells of depositing (E.Coli MM294(DE3)/PTB960) among the 1kg embodiment 6.With the Dynomill model KD-5 instrument of 5 liters of capacity (Willybachfen Switzerland), adopts 4 liters of granulated glass spherees, under ice-cooled with 5 cycles of this cytoclasis.Wash this granulated glass sphere with this about 5 liters damping fluid, again washing lotion is collected with extracting solution.About 10 liters of these solution of gained are carried out centrifugation with 10,000 rev/mins rotating speed, and (Model 21, Beckman.U.S.A.) reach 60 minutes, and the result obtains a kind of supernatant liquor.Pour the supernatant liquor that is obtained into Sulfated Cellulofine(Seikagaku Kogyo, and Japan) in the post (internal diameter 25.2cm * 50cm).This post is washed with the phosphate buffered saline buffer that contains 0.5M NaCl (PH7.0) of 25 liters of 25mM in the absorption back.Finish this elution process with 30 liters of phosphate buffered saline buffers (PH=7.0) that contain the 25mM of 1M NaCl then.(Pellicon Casset System, Millipore U.S.A.) concentrate this eluate and demonstrate 3~4 until the absorbancy at the 280nm place with a ultra-fine filter.The concentrated solution that obtains is carried out dialysis a whole night to the phosphate buffered saline buffer (PH6.0) of about 60 liters 25mM.Separate this dialysis thing 30 minutes with a bench centrifuge (Beckman.U.S.A.) with 4,200 rev/mins rotating speed, then obtain a kind of supernatant liquor.This supernatant liquor is poured in the post (by Tosoh, Japan makes for internal diameter 17.0cm * 33.0cm, SP-Toyopearl 650M).Wash this post with 7 liters of phosphate buffered saline buffers (PH6.0) that contain the 25mM of 200mM NaCl, use 10 liters again, the phosphoric acid buffer that contains 500mM NaCl (PH6.0) of 25mM carries out wash-out.Pour this eluate into Sulfated Cellulofine(Seikagaku Kogyo) and post (the 15.0cm internal diameter * 50.0cm), use high speed liquid chromatography instrument (Gilson, France).Finish this wash-out by the linear gradient between the citrate buffer that contains 2M NaCl (PH7.0) of the citrate buffer (PH7.0) of 20 liters of 50mM and 30 liters of 50mM.Collecting main fraction and being diluted to specific conductivity with the citrate buffer (PH6.0) of 10mM is mmho or littler.The solution that this is diluted is poured a kind of SP-Toyopeal 650M(Tosoh into, and post Japan) is (among the internal diameter 14.0cm * 39.0cm).Wash this post with 10 liters of citrate buffers (PH6.0) that contain the 25mM of 0.5M NaCl, the citrate buffer that contains 0.5M NaCl (PH=6.0) with 10 liters of 25mM carries out elution process again.Pour this eluate into Toyopearl HW-50F(Tosoh, post Japan) (in the 14.0cm internal diameter * 90.0cm), uses the citric acid (PH7.0) of 50mM to launch again.Collect main distillate fraction, and (Millipore sterilizes U.S.A.), then obtains a kind of rhbFGF mutein CS23 storage solution of purifying by Millipak 20 filters.Output is 17.0 grams (yield percentage is 7%).-terminal amino acid sequential analysis, C-terminal amino group acid assay and amino acid composition analysis are shown in respectively among the table 4-6.
The sequential analysis of table 4-terminal amino acid
Ring amino-acid residue (Pmol) is derived by nucleotide sequence
The amino acid of rhbFGF mutein CS23
(750Poml)
1 Pro(530) Pro
2 Ala(553) Ala
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 Ala(283) Ala
12 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
20 Pro(125) Pro
* use a Model 477 A protein sequence analyzers (applying biological system) to analyze.
Table 5 C-terminal amino group acid assay
C-end amino acid productive rate (%)
Ser 41.0
* hydrazinolysis method
* analyzes with one 6330 type amino acidanalyser (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
Gly 14.9 15
Ala 9.0 9
Cys - 2
Val 6.4 7
Met 2.1 2
Ile 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
-do not identify
* hydrochloric acid hydrolysis method
* is with 6330 type amino acidanalysers (Beckman)
1) Edelhoch method
And then Fig. 3 shows the result of the SDS-PAGE of gained under reductive condition (100mM DTT, 50 ℃, 15 minutes).Referring to Fig. 3, (1) and (2) represents this mark and rhbFGF mutein CS23 respectively.
Fig. 4 shows with heparin-5PW(Tosoh, Japan) be carrier post (result of the high speed liquid chromatography method analysis of internal diameter 7.5mm * 7.5cm), its operational condition is as follows:
Elutriant: A solution (50mM phosphate buffered saline buffer (PH7))
B solution (phosphate buffered saline buffer (PH7) that contains the 50mM of 2M NaCl)
Flow velocity: 0.8ml/ branch
Detect wavelength: 280nm.
Fig. 5 shows the Industry with ODP-50(Asahi Chemical, Japan) do carrier post (analytical results of the high speed liquid chromatography method of internal diameter 4.6mm * 150cm), analysis condition is as follows:
Elutriant: A solution (0.1% trifluoroacetic acid)
B solution (80% the acetonitrile that contains 0.1% trifluoroacetic acid)
Flow velocity: 1.0ml/ branch
Detect wavelength: 280nm
Fig. 6 illustrates and uses G2000SW(Tosoh, Japan) do carrier post (analytical results of the high speed liquid chromatography method of 7.5mm internal diameter * 7.5cm), analysis condition is as follows:
Developping solution; 0.1M the sodium sulfate of phosphate buffered saline buffer (PH=7)+0.1M
Flow velocity: 0.5ml/ branch
Detect wavelength: 280nm
Biological activity identifies with a kind of like this method, promptly to the measured quantity of the growth-promoting activity of tire calf heart endothelial cell as this bioactive index.The result has provided the active suitable a kind of activity of sample therewith.
Embodiment 8
Preparation has the recombinant chou of a kind of rhbFGF mutein CS23 of tetracyclin resistance mark
The gene plasmid PTB 762 that contains promising rhbFGF mutein CS23 coding is by AvaI and PstI complete digestion, the result obtains containing the dna segment of the nearly 0.45K base pair of most of rhbFGF mutein CS23, four cysteine residues that are present on 69 and 87 of hbFGF in this rhbFGF mutein CS23 are replaced (people such as Senoo by serine residue, Biochemical and Biophysical Research Communicalion 151,701-708(1988), the open NO.281 of European patent, 822).With the T4DNA ligase enzyme with synthetic DNA (
) be connected on the segment of gained, then this resultant is then obtained a segment A with AvaI and PstI digestion, therein, a PstI spilting of an egg site is modified to BglII spilting of an egg site.After this, plasmid PTB955 (has lacked (people such as Seno of a kind of gene for rhbFGF mutein C128 coding of the C-end of hbFGF, European Journal of Biochemistry, 188,239-245, (1990)) insert wherein) a kind of segment B of about 4.1K base pair obtained by SalI and BamHI complete digestion.PTB955 is by AvaI and SalI complete digestion, and the result obtains a segment C that 390 base pairs are arranged approximately.
With the T4DNA ligase enzyme these three segment A, B are connected with C and then obtain a kind of expression plasmid PHP901(Fig. 7 that contains the amicillin resistance mark).
With EcoRI and BglII the PHP901 complete digestion is just obtained the segment of an about 1.1K base pair, it contains a kind of gene, a T7 promotor and a T7 terminator to rhbFGF mutein CS23 coding.With the T4DNA ligase enzyme this segment is received and to be gone up the result with the pUC18 of EcoRI and BamHI complete digestion and obtain plasmid pME901(Fig. 8).
With EcoRI and HindIII the pME901 complete digestion is then obtained a gene that contains for rhbFGF mutein CS23 coding, the segment of about 0.77K base pair of a T7 promotor and a T7 terminator.Hatch this segment so that its end is changed into concordant end with the T4DNA polysaccharase.This segment is received the expression plasmid pCM901(Fig. 9 that on the pBR322 of ScaI digestion, then obtains to contain tetracycline marker with the T4DNA ligase enzyme).
Make E.Coli μ M294 lysogenization with preparation E.Coli bacterial strain MM294(DE3 with phage DE3), in phage DE3, inserted the rna polymerase gene (people such as F.W.Studier of this T7 phage, Journal of Molecular Biology, 189,113-130(1986)).By this E.Coli bacterial strain is transformed, obtain having the recombinant chou E.Coli MM294(DE3 of the gene of this rhbFGF mutein CS23 coding thus)/PCM901(IFO15104, FERM BP-3168).
Embodiment 9
With 1ml recombinant chou E.Coli MM294(DE3)/PCM901(IFO15104, FERM BP-3168) chief cell 1 liter the LB-substratum that contains 5mg/l tetracycline salt acidulants is inoculated, then in 30 ℃ of wave and culture 8 hours.Its whole amounts are moved to (the tetracycline salt acidulants that contains 5mg/l) in 20 liters the LB substratum of containing in the culture tank of one 50 liter capacities, then at 30 ℃, 1.0kg/cm
2Depressing in the gauge pressure and following ventilation (10 liters/minute of flows) and rotating speed is that cultivating until Klett unit under 3000 rev/mins the stirring velocity is 700.Then the nutrient solution of gained is all moved in 360 liters the fermention medium and (contain the glucose of 15g/l, the casamino acids of 15g/l, the Na of 16.8g/l
2HPO
412H
2The KH of O, 3g/l
2PO
4, 1g/l NH
4The NaCl of Cl, 0.5g/l, the MgSO of 0.5g/l
47H
2The vitamin of O, 5mg/l, the FeSO of 2.5mg/l
47H
2The M-9 substratum of the antifoams of O and 0.2g/l), then at 28 ℃, 1.0kg/cm
2Depress in the gauge pressure, begin to cultivate under the flow ventilation that divides with 240l/, 250 rev/mins the rotating speed stirring condition.When the turbidity of this nutrient solution reaches about 450Klett unit, to wherein adding 10mg/l(42 μ M) IPTG.Because surplus sugar concentration is 1%,, continue then to cultivate 40 hours so add a kind of solution that contains glucose (60g/l) and caseic hydrolysate (15g/l) mixture.The result accumulates the rhbFGF mutein CS23 of the 1.6g/l of soluble state.The cultured products that obtains is like this collected wet cell with husky sharpie's centrifuge Sharple, again with this cell freezing depositing under-80 ℃.
Handle the cell of this gained of 1kg with the mode similar, then obtain the rhbFGF mutein CS23 original solution (output 25.0g) of purifying to embodiment 7.The quality of this solution is all suitable with sample in embodiment 7 aspect biological activity and physics and chemical assay.
Claims (13)
1, a kind of carrier that contains a kind of mutein nucleotide sequence coding, the component amino acid of at least a one-tenth rh-bFGF (hbFGF) is replaced by another kind of amino acid in this protein, and the T7 promotor is positioned at its upstream extremity.
2, the carrier of claim 1, at least a therein component amino acid is the residue of at least a halfcystine, and another kind of amino acid is the residue of Serine.
3, the carrier of claim 1, this mutein is a kind ofly to be in cysteine residues on 69-and the 87-position by the displaced a kind of mutein of serine residue in sophisticated hbFGF component amino acid therein.
4, a kind of by the transformant that carrier transformed in the claim 1.
5, the transformant of claim 4, the host is the intestinal bacteria with T7 pol gene (Escherichia Coli) that are in lac promotor downstream end therein.
6, the transformant of claim 5, it has E.Coli MM294(DE3)/characteristic of PCM960.
7, the transformant of claim 5, it has E.Coli MM294(DE3)/characteristic of PCM901.
8, at least a component amino acid of the sophisticated hbFGF of preparation is by the method for the mutein that another kind of amino acid replaced, and it is included in the transformant of cultivating claim 4 in a kind of substratum.
9, method is according to Claim 8 added the isopropylthio galactopyranose glycosides (IPTG) of about 3~500 μ M by the logarithmic phase of the transformant in the claim 5 to this substratum therein, then cultivates.
10,, use the IPTG of 3~300 μ M therein according to the method for claim 9.
11,, use the IPTG of 6~200 μ M therein according to the method for claim 10.
12,, use the IPTG of 6~80 μ M therein according to the method for claim 11.
13, method according to Claim 8, the solution chromatographic purification that contains the mutein product therein, adopt a kind of cross-linked polysaccharides vitriol, a kind of have a sulfonic group in return the base synthetic polymer and/or a kind of synthetic polymer of gel-filtration that is used for as carrier.
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JP (1) | JP3130313B2 (en) |
KR (1) | KR920703789A (en) |
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CA (1) | CA2070989A1 (en) |
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US6274712B1 (en) | 1997-12-23 | 2001-08-14 | 3-Dimensional Pharmaceuticals, Inc. | Analogs of human basic fibroblast growth factor mutated at one or more of the positions glutamute 89, aspartate 101 or leucine 137 |
JP4262979B2 (en) * | 2000-11-21 | 2009-05-13 | ザ・テキサス・エイ・アンド・エム・ユニバーシテイ・システム | FGF affinity chromatography |
EP1926459B1 (en) | 2005-09-19 | 2015-01-07 | Histogenics Corporation | Cell-support matrix having narrowly defined uniformly vertically and non-randomly organized porosity and pore density and a method for preparation thereof |
EP2083846B1 (en) | 2006-09-28 | 2015-07-15 | Hepacore Ltd. | N-terminal fgf variants having increased receptor selectivity and uses thereof |
US10077420B2 (en) | 2014-12-02 | 2018-09-18 | Histogenics Corporation | Cell and tissue culture container |
DE102021211272A1 (en) | 2021-10-06 | 2023-04-06 | Ruhr-Universität Bochum, Körperschaft des öffentlichen Rechts | Composition containing artificial oxygen carriers to prevent organ damage in transplant organs |
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DE3723992A1 (en) * | 1987-07-20 | 1989-02-02 | Boehringer Mannheim Gmbh | METHOD FOR PRODUCING PROTEINS IN SOLUBLE FORM |
JPH02504468A (en) * | 1987-11-24 | 1990-12-20 | アムジエン・インコーポレーテツド | Fibroblast growth factor analog |
EP0326907A1 (en) * | 1988-01-26 | 1989-08-09 | Takeda Chemical Industries, Ltd. | Polypeptide, DNA and its use |
-
1990
- 1990-12-18 JP JP03501005A patent/JP3130313B2/en not_active Expired - Fee Related
- 1990-12-18 CA CA002070989A patent/CA2070989A1/en not_active Abandoned
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EP0506963A1 (en) | 1992-10-07 |
WO1991009126A1 (en) | 1991-06-27 |
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JPH05503006A (en) | 1993-05-27 |
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