CN85107875A - Protein is separated from each other - Google Patents
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- CN85107875A CN85107875A CN198585107875A CN85107875A CN85107875A CN 85107875 A CN85107875 A CN 85107875A CN 198585107875 A CN198585107875 A CN 198585107875A CN 85107875 A CN85107875 A CN 85107875A CN 85107875 A CN85107875 A CN 85107875A
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/24—Extraction; Separation; Purification by electrochemical means
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- C07K1/28—Isoelectric focusing
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
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Abstract
With valuable protein or Met-protein (methionine(Met)-protein), for example valuable proteinic N-Met (N-methionine(Met)) analogue is separated from the mixture of forming them effectively, utilizes the iso-electric point difference between protein and the Met-protein that mixture is separated.
Description
This patent relates to the method that protein is separated from each other, and is about to different proteins and is separated from each other.
As everyone knows, had the protein of various physiologically actives, such as cytokinin and peptide hormone.The progress of current genetic engineering technique has been opened up road for the mass production and the clinical application of this class physiologically active protein matter.
The production of the physiologically active protein matter that above-mentioned technology has been used to provide of a great variety comprises cytokinin, such as Interferon, rabbit, interior white corpuscle (sterilization) element, Bcell growth factor, the B cytometaplasia factor, macrophage activating factor (MAF), lymphotoxin, tumour necrosis factor etc.; Peptide hormone, such as erythropoietin, epithelial cell growth factor, Regular Insulin, human growth hormone, transforming growth factor etc.; The antigen protein that development prevention pathogenic microorganism vaccine is used, such as hepatitis b virus antigen, influenza antigens, foot-and-mouth disease virus antigen, malarial parasite antigen etc.; Enzyme is such as peptase (for example, tissue plasminogen activator, urokinase, Serratia peptase etc.), N,O-Diacetylmuramidase etc.; Reduced hematin, such as human serum protein etc., and other protein with practicality physiological property.
But this proteinoid that every employing recombinant chou method is made has all been run into from other and has normally been isolated required finished product problem the mixture.The nerve-racking problem of people is caused by following situation, i.e. " performance " by valuable protein gene password obtains " performance product " and is generally and contains the valuable protein and the second proteinic mixture, and this second protein contains valuable protein and connecting methionine(Met) (Met) base on its amino end group.This additional methionine(Met) base is to be subjected to due to the ATG initiation codon influences, and this password is that desirable gene begins the signal of " performance ", and " representation system " that also be stroma cell is owing to additional methionine(Met) base breaks away from the signal that " performance product " produces fault.In prokaryote matrix and eukaryote matrix, all this problem can occur, but appear at more in " performance " of prokaryote gene usually.Particularly more outstanding in utilizing intestinal bacteria conducts " representation system " of " performance matrix ".
Because carrying out protein synthesis in eukaryote and prokaryote begins under messenger RNA(mRNA) (RNA) password AUG effect, this password passes to amino acid with methionine(Met), thereby can think, proteinic " performance " produced the molecular species mixture, be to contain additional methionine(Met) analogue (the amino analogue of N-egg) on valuable protein and the N-end group thereof, when " performance matrix " when being intestinal bacteria, all the more so.
In fact, as everyone knows, IF-3 is the intestinal bacteria initiation factors, it comprises two quasi-molecule kinds, be that the molecule kind has the methionine(Met) base on amino end group, another kind of molecular species does not then have methionine(Met) and (sees: Hoppe Seyler's Z.Physiol.Chem., 354 as the end residue, 1415(1973)), and the amino end group of Escherichia coli protein be generally methionine(Met) and (see: Conn ﹠amp; Stumpf(1976), Outlines of Biochemistry, the 4th edition, John Wiley ﹠amp; Sons) generate the methionine(Met) analogue to recombinant chou DNA(thymus nucleic acid) the required protein of engineering generation, the bigger problem of ratio be quite generally (to see: Nature, 293,408(1981)), wherein speak of the generation of human growth hormone, bigger owing to the methionine(Met) analogue to the required proteinic ratio of reality.
When protein that every generation is required and N-methionine(Met) analogue thereof, all be difficult to they are separated from one another, this be because two quasi-molecule kinds with regard to its physical and chemical performance no matter differ atomic in that respect.
Methionine residue is that molecular weight is about 131, and has medium hydrophobic amino-acid residue, simultaneously, presents electric neutrality owing to lacking deliquescent functional group.In addition, protein is a kind of polymer with solvability gene, hydrophobic group and hydrophilic radical.For example, the molecular weight of plain-2 polypeptide of interior white corpuscle (sterilization) as shown in Figure 1, its molecular weight is about 15,420, is made up of 133 amino-acid residues (wherein, X represents hydrogen atom).Therefore can think that a methionine residue is added on the protein amino end group, in general the physical and chemical performance of protein own be there is no considerable influence.Like this, just be difficult to have the amino end group of methionine residue with not separated from one another with the molecular species of methionine residue.
Difficulty in the above-mentioned separation is to the numerous protein ubiquity, particularly to attempting from N-methionine(Met) analogue to separate in the recombinant chou rather difficulty of white corpuscle (sterilization) plain-2 and recombinant chou Interferon, rabbit, and particularly difficult when recombinant protein matter is utilized intestinal bacteria as " performance matrix ".
Interior white corpuscle (sterilization) element the-the 2nd, a kind of lymphokine, the latter is by being produced through the T of mitogen or antigen activates cell.Interior white corpuscle (sterilization) plain-2 comes down to cause the variation and the somatomedin of cytotoxin T cell and natural killer cell, and it plays an important role in the immune response system that causes by this class cell.
Can think that interior white corpuscle (sterilization) plain-2 and interferon-' alpha ' all can be effectively used to treatment and lack immunity and cause a disease, infect and cause a disease and treatment malignant tumor etc., because they have physiologically active.Blood lymphocyte is alive plain slightly by the human body end, or the interior white corpuscle of separating in the supernatant liquor by human T cell, leukemia cell (Jurkat line) substratum (bacteriocidin)-2, comprises a few quasi-molecule kinds, has different molecular weight.But, as everyone knows, all these analogues are all formed relevant with polypeptide chain to a great extent, for example, the amino end group of polypeptide is alanine residue without exception (to be seen: Japan's special permission communique specification sheets, No.149248/1984(applying date 1984.7.19), corresponding to open (laid open) No.032359 of European patent; Pro.Natl.Acad.Sci.USA, 81,2543(1984)).
Natural interferon-the α that is separated by the supernatant liquor of human leukocytes substratum comprises the subtype more than ten kinds or ten kinds; But they have many resemblances each other, and these are all relevant with the polypeptide chain structure, and for example, their amino end group is cysteine residues (seeing: Biochem.Biophys., 221,1, (1983)) bar none.
This patent the inventor successfully find, the generation of non-glycosylated human body words spoken by an actor from offstage cell (sterilization) plain-2 is (see: Japan special permission communique specification sheets No.225079/1983(files an application the date: 1983.11.28), corresponding to the EP Publication(Laid open) No.0145 390 that showed by plain-2 genes of white corpuscle in the human lymphocyte (sterilization) in Bacillus coli cells by the recombinant DNA engineering).White corpuscle (sterilization) plain-2 contains polypeptide (I) in above-mentioned; and (1) has the amino acid segment; (X represents hydrogen atom or methionine residue among the figure) as shown in Figure 1; and comprise two quasi-molecule kinds; the amino end group that is the molecule kind is that white corpuscle (sterilization) element-2(is that X is a hydrogen atom in the natural human body) alanine residue; and the amino end group of another kind of molecular species is methionyl L-Ala (being a methionyl L-Ala) residue, and contains methionine residue (being that X is a methionine residue) on amino end group.
Once had report (see: J.Interferon Res., 1,381(1981); J.Biol.Chem.; 256; 9750(1981)); for example; contain polypeptide by the recombinant DNA engineering by the interferon-' alpha ' A that intestinal bacteria showed; the latter has the amino acid segment; as shown in Figure 2; it comprises two kinds of molecular species; the amino end group amino acid that is the molecule kind is the cysteine residues as natural human interferon-alpha (being that X is a hydrogen atom); and the amino end group amino acid of another kind of molecular species is the methionyl cysteine residues, and contains methionine residue on amino end group (being that X is a methionine residue).
Might in the middle of two quasi-molecule kinds, there be the difference on the protein high-sequential configuration, and contains methionine residue and do not have methionine(Met) as the end residue at amino end group; Therefore, also just may exist in the middle of the two quasi-molecule kinds biological activity or biologically stable to show as " external " (invivo) and the difference on (in vitro).In addition, methionine residue is connected on the amino end group and can descend or increase and cause by antigenicity.Therefore, from physiology and industrial application viewpoint,, post with very high expectation for by molecular species high-purity on the border one thing of producing separated from one another in the molecular species that has and do not have methionine residue to participate on the amino end group.
Methionine residue is connected in the influence (see: Interferon Res., 1,381(1981)) that ratio on the ammonia end is subjected to culture medium condition or protein " performance level "; But still there is not the successfully ratio of control linkage methionine residue of example report.Equally, still do not have report so far: by the protein process for purification that two quasi-molecule kinds are separated from each other, said here molecular species is meant the molecular species that has and do not have methionine residue to participate on amino end group.
Although adopted many method this patent inventors both to fail to separate (a): the interior white corpuscle (sterilization) plain-2.5 that has methionine residue on amino end group does not have the proteinic separated from one another of methionine(Met) end residue, can not separate (b): separated from one another at amino end group interferon-' alpha ' A with methionine residue and the interferon-' alpha ' A that does not have methionine residue, though once attempted the method that adopted following method to be separated from each other as different proteins, for example, utilize the desalination and the solvent precipitation of different solubility, dialysis, the infrared-filtered method, gel filtration method, utilize the different SDS-polyacrylamide gel electrophoresis of molecular weight, utilize the affinity chromatograph partition method of the ratio affinity of protein antibody, but and utilize the different anti-phase HPLC (high performance liquid chromatography) partition method of hydrophobicity.
This patent inventor finds, protein and N-methionine(Met) analogue thereof have different iso-electric point and can they are separated from one another according to these characteristics surprisingly.Those skilled in the art may consider that as methionine(Met) being connected on the proteinic amino end group, because methionine(Met) is a kind of electroneutral amino acid that is, then whole proteinic electric charge can not be affected.Therefore, this is unexpected a discovery, promptly in white corpuscle (sterilization) plain-2 and interior white corpuscle (sterilization) plain-2 with the methionine(Met) that is connected on the amino end group different iso-electric points is arranged.
The object of the present invention is to provide the both valuable protein of a kind of separation, or the method for its N-methionine(Met) analogue, can from the mixture of forming by protein and N-methionine(Met) analogue thereof, separate.This N-methionine(Met) analogue is made up of the amino acid segment, this segment and valuable protein segment are closely similar, has the physiologically active that has by protein, and N-methionine(Met) analogue to connect the situation of methionine residue on its ammonia end similar to protein, the different mixtures to constitutive protein matter of the iso-electric point between available protein and the N-methionine(Met) analogue separate.
As the main polymeric material of forming by amino acid of " protein " speech system expression that uses in this specification sheets, and comprise the polypeptide class, their pass through or do not carry out chemistry or structurally-modified through glycosylation or alternate manner, for example, adopt chemistry or the enzyme reaction adopted in the known technology to reach the modification purpose.
System is illustrated in the protein that connects the methionine(Met) base on the amino end group as " methionine(Met)-protein " that uses in this specification sheets (Met-protein) speech.The present invention stresses aspect methionine(Met)-protein, and it has and the same or analogous physiologically active of valuable protein.Therefore, according to the present invention and priority declaration's methionine(Met)-protein is the class that those are made up of an amino acid segment, they and valuable proteinic amino acid segment are quite similar, the physiologically active that can provide valuable protein to have.The present invention be directed to from methionine(Met)-protein (valuable proteinic N-methionine(Met) analogue) method of separating protein and associated products.The best scheme of the present invention is about plain-2 protein of white corpuscle (sterilization) in separating from N-methionine(Met) analogue and isolates interferon-alpha proteins matter from N-methionine(Met) analogue.
Have tangible activity or effect as " physiologically active " speech system expression biology and/or the biomaterial that use in the specification sheets of the present invention, here said biomaterial comprises cell, cellular constituent, the product of cell or other biologically active, and other biomaterial, whether these biomaterials no matter live, and is in " in the vivo(body) " and still is in " vitro(is external " state, comprise have biology and immunocompetence and effect both.
Above-mentionedly reach that " mixture formed of methionine(Met)-protein adopts the mode of gene recombination engineering to produce usually, is about to intestinal bacteria, Bacillus subtilus, yeast or zooblast and transfers in " protein performance " by " protein ".
The protein of " protein " said and different physiologically actives is as follows: cytokinin, and such as Interferon, rabbit (IFN
S; IFN-α, IFN-β, IFN-γ etc.), interior white corpuscle (sterilization) plain (interior white corpuscle (sterilization) element-1, interior white corpuscle (sterilization) plain-2 etc.), Bcell growth factor (BGF), the B cytometaplasia factor (BDF), the macrophage activity factor (MAF), lymphotoxin (LT), knurl lethal gene (TNF), transforming growth factor (TGF-α); Peptide hormone such as erythropoietin, ends skin cell growth factor, Regular Insulin, human growth hormone; The pathogenic microorganism antigen protein is such as hepatitis b virus antigen, influenza antigens, foot-and-mouth disease antigen and malarial parasite antigen; Enzyme is such as peptase (for example, tissue plasminogen activator, urokinase, husky Lei Shi peptase etc.) and N,O-Diacetylmuramidase; Hemoprotein is such as human serum albumin (HSA).
The protein process for mutual separation that adopts the present invention to propose is approximately 3 for those molecular weight, 000-50,000 protein particularly advantageous, preferably molecular weight is about 5,000-30,000, or contain about 30-500 amino acid, be good to contain about 50-300 amino acid especially.
The protein method separated from one another that adopts the present invention to propose can be used to separate the protein that iso-electric point is about 4-11 effectively, and iso-electric point is good with about 5-8 especially.Preferably, the difference of iso-electric point is at least about 0.01, to be 0.1 for good at least, it would be desirable that the difference in protein and the iso-electric point between the protein that has methionine residue on the amino end group is 0.01-0.2 especially.
The separation method that adopts the present invention to propose is specially adapted to separate interferon-' alpha ' and interior white corpuscle (sterilization) element-α that the mode according to the gene recombination engineering generates.
In this case, promptly for having the biology similar and the interior white corpuscle (sterilization) plain-2 of immunologic competence to white corpuscle (sterilization) plain-2 in the natural human body, or its microorganism, or through modification, such as suppressing active modification body, all can be used as interior white corpuscle (sterilization) plain-2 and be used with interior white corpuscle (sterilization) plain-2 acceptor and plain-2 antibody of anti-interior white corpuscle (sterilization).In order to specify, has (the polypeptide I of polypeptide as shown in Figure 1 of amino acid segment; X is a hydrogen atom), and contain partial amino-acid segmental peptide fragment (fragments) and can be used what biology or immunologic competence can not lack, for example, lose an amino acid whose fragment (European patent specification of exhibition No.91539), lose four amino acid whose fragments (sees: Japan's special permission communique specification sheets: No.235638/1983 at the amino end group place of polypeptide (I), (filing an application the date: 1983.12.13)), this patent application is disclosed in announcement (Laid Open) in 1985 as Japan's special permission, publication number: 126088, and on the carbonyl end group, lack and severally contain amino acid whose fragment and all be fine.Lose the fragment of the aforementioned polypeptides (1) that or several amino acid forms or by other amino acid institute metathetical fragment, fragment such as the 125th cysteine residues is replaced (see: Japan speciallys permit prospectus No.93093/1984), its correspondence: European prospectus No.109748 by serine residue.Preferably, this peptide species is nonglycosylated polypeptide, is good with the interior white corpuscle (sterilization) plain-2 with amino acid segment especially, as shown in Figure 1.
In the following description, element-2s is abbreviated as IL-2 with interior white corpuscle (sterilization), and the interior white corpuscle (sterilization) that contains methionine residue at amino end group plain-2 is abbreviated as Met-IL-2.
In the present invention, interferon-' alpha ' kind with the biology similar or immunologic competence to natural human interferon-α as interferon-' alpha ', as in conjunction with interferon-' alpha ' acceptor or anti-interferon antibody activity.For example, can utilize the polypeptide that contains the amino acid segment, as shown in Figure 2 (polypeptide-II: wherein X is a hydrogen atom).In addition, allow to use interferon-' alpha ' biology or the requisite partial amino-acid segment of immunologic competence are formed fragment, for example lack the fragment of several amino acid and can be used at the fragment that the carbonyl end lacks several amino acid at the amino end group of interferon-' alpha ' A polypeptide (II), that is to say that permission is made up of aforementioned polypeptides (II) one or several amino acids fragments are lost, and are perhaps replaced by other amino acid.Interferon-' alpha ' A preferably.Advantage point is that this peptide species is non-glycosylated polypeptides.
In the following description, interferon-' alpha ' A is abbreviated as IFN-α A, the interferon-' alpha ' A at amino end group tool methionine residue is abbreviated as Met-IFN-α A.
Proteinic purity at least approximately will be 50% in the used said mixture of being made up of protein and methionine(Met)-protein, preferably is approximately higher than 80%, is good with about 99% especially.
The present invention proposes to can be used for separating the method for this proteinoid, is about to said mixture and separates through the separable programming of setting up according to the iso-electric point difference.
According to embodiment 1 described method, the iso-electric point of IL-2 and Met-IL-2 is respectively 7.7 and 7.5.
According to embodiment 6 described methods, the iso-electric point of IFN-α A and Met-IFN-α A is respectively 6.2 and 6.3.
In the present invention, be that to be used for the difference of iso-electric point be 0.01 to 0.2 protein technology separated from one another according to the different separable programmings of setting up of iso-electric point.For example, can adopt known mature technology and combination process, such as the technology that makes protein through the electromagnetic field migration, comprise electrophoresis such as the electrophoresis such as density gradient that adopt amphotericeledrolyte to carry out, gel and isotachophoresis etc., and with the technology of protein attachment at (for example in the elution post) on the charged carrier, and carry out elution according to the electric charge difference and separate, this electric charge is not both that concentration gradient because of salt in iso-electric point difference and pH gradient or the carrier is different to be caused.Also have certain methods to adopt, comprise chromatofocusing method (Chromato focusing), FPLC method (protein liquid chromatography fast), DEAN(diethyllaminoethyl)-CM(carbonyl methyl) or the SP(sulfopropyl) ion exchange chromatography.Employed whole reagent and instrument all have commercial product supply in the separation method that the present invention proposes.For example, the both sexes dielectric medium can LKB2r(Sweden) bought; Electric separatory gel such as gel can be available from Pharmacia 2r(Sweden), merchant's name Sephadex LEF, and available from LKB2r(Sweden) merchant's PAG(polyacrylamide gel by name) plate; Carrier that the chromatofocusing method is used and dilution buffer agent can be available from Pharmacia 2r(Sweden), merchant Polybuffer exchangers PBE94/PBE118 by name, Polybuffer 74, and Polybuffer 96; Adopt the used packed column of FPLC method dilution buffer agent can adopt Mono-P columns, Mono-Q columns(Sweden Pharmacia r); DEAE ion exchange resin can be available from Japan Cao Da company (Japan), merchant DEAE-Toycpearl by name; QAE ion exchange resin can be available from Japan Cao Da company (Japan), merchant's name CM-Toy-opearl; SP ion exchange resin can be available from Japan Cao Da company (Japan), a merchant name SP-5PW also can be available from Pharmacia 2r(Sweden), merchant's name SP-Sephadex (sees: Methods in Enzymology 5,3-27(1962), related content all has disclosed therein).
When adopting electric electrophoretic separation technique such as gel, as the available PAG pole plate of commodity (245 * 110 * 1 millimeter, the LKBr goods), for example, can be with it as pH3.5-9.5, or the pole plate of usefulness such as pH5.5-8.5; Also have the crowd to know material, such as the 1M(volumetric molar concentration) or 0.4M HEPES buffer reagent etc. can be used as negative electrode liquid, and 1M sodium hydroxide, or the aqueous sodium hydroxide solution of 0.1M etc. then can be used as anode liquid.Used suitable operational condition is:
Protein mass: 10-1,000 microgram/pole plate
Power supply: 1-200, preferably 10-50 watt
Temperature: 0-20 ℃, preferably 2-5 ℃
Transition time: 0.5-50 hour, 1.5-5 hour usually
When adopting the FPLC method, the available crowd knows that material is Mono-P colum(0.5 * 20 centimetre, Pharmacia 2r), used equilibrium buffer has 0.025M diethanolamine-hydrochloride buffer agent (pH9.5) and 0.075M three second buffer reagents (pH9.3) etc., used dilution buffer agent then has following suitable buffering system, for example: the 1%(volume ratio) Pharmalyte(pH3-10.5)-the 5.2%(volume ratio) Polybuffer 96-hydrochloride buffer agent (pH7.0-8.0) system, the 10%(volume ratio) Polybuffer 96-hydrochloride buffer agent (pH6.0-7.0) system, the 10%(volume ratio) Polybuffer 96-acetic acid buffer (pH6.0-7.0) system etc.The condition that the FPLC method is suitable for is:
Post bed ability: every gram protein 0.01-10 liter,
100-1 preferably, 100 milliliters
Flow velocity: the SV(constant speed)=0.01-10,
SV=0.1-1.0 preferably
Column temperature: 0-30 ℃, preferably 2-5 ℃
Separation method according to the present invention's proposition, can protein and Met-protein is separated from each other according to the iso-electric point difference, be about to test portion input electric field and carry out electrophoretic migration, or it is added the carrier packed column, with pH value gradient column spinner the elution from carrier of isolating product is come out then.Promptly pass through isocratic elution
*Reach the separation purpose, thereby neither need special pH value gradient
* isocratic elution, i.e. isocratic elution.-annotation of translation.Handle, also do not need the concentration gradient of salt to handle.
If necessary, adopt tradition or currently known methods also can further make with extra care according to the inventive method isolated protein from Met-protein (for example its N-methionine(Met) analogue), thereby obtain pure especially protein.Equally, adopt tradition or currently known methods also can further make with extra care according to the inventive method isolated Met-protein from protein, thereby obtain pure especially Met-protein.
For this purposes, can adopt known mature technology, such as desalination process, the hydrophobicity chromatography, gel filtration method, ion exchange chromatography, and high-effect liquid chromatography, these methods all can be used for proteinic refining usually, preferably it are used in combination.
The present invention has at first proposed a kind of possibility, is about to come by its corresponding Met-protein separation of protein that the recombinant DNA engineering produces, and causes this protein to be gone up substantially and does not contain its corresponding Met-protein, does not for example contain N-methionine(Met) analogue.Corresponding M et-protein content should approximately be no more than 3%(weight in the protein of making according to the inventive method), preferably be no more than 2%.Especially to be no more than 1% for good.
Equally, the present invention has at first proposed a kind of possibility, is about to come by its corresponding protein separation of Met-protein that the recombinant DNA engineering produces, and causes this Met-protein to be gone up substantially and does not contain corresponding proteins matter.Corresponding proteins matter content should approximately be no more than 3%(weight in the Met-protein of making according to the inventive method), preferably be no more than 2%, especially to be no more than 1% for good.
Still no-trump Met-IL-2 (methionine(Met)-Nei white corpuscle (sterilization) plain-2) or Met-IFN-α A (methionine(Met)-interferon-' alpha ' A) report as the successfully isolating example of protein so far.The present invention provides separating high-purity Met-IL-2 protein and Met-IFN-α A method of protein originally.
The protein of making according to the inventive method has and corresponding similar biology or the immunologic competence of natural protein with Met-protein, and have quite high purity, cause and seldom contained impurity or protein and pyrogen (Pyrogens) free from foreign meter.Therefore, they can be used to injection of making injection etc. safely.
The IL-2 and the Met-IL-2 that make according to the inventive method have the activity that promotes normal T cell or natural killer cell growth, keep its inherent function simultaneously.Therefore, the purpose that IL-2 and Met-IL-2 can be used as the subculture base of T cell or natural killer cell and be used to promote grow makes this class cell be in " external " (in vitro) state for a long time, or is used for vegetative propagation.In addition, above-mentioned this performance also can be used for human body IL-2 active testing aspect.
Secondly, the growth that IL-2 that obtains according to the inventive method and Met-IL-2 can optionally promote special antigen T type killer cell, tumor antigen can be discerned and eliminate to this cell, or optionally promote the growth of natural killer cell, with whether " in the body " exists the antigen sensibilized irrelevant, this natural killer cell can kill and wound oncocyte.When being expelled in the biological organism, when injecting above-mentioned killer cell simultaneously, then can improve the anti-knurl effect of T type killer cell according to IL-2 and the Met-IL2 immunoprophylaxis that the inventive method obtains.Therefore, can be used for prevention and treatment knurl, or be used for the treatment of the pathogenic disease of immunodeficiency of warm-blooded animal (for example, vole, mouse, rabbit, dog, cat, pig, horse, sheep, ox and people etc.).
The IL-2 and the Met-IL-2 that obtain according to the inventive method are high purity products, and be little for human antigen's property, and low toxicity.
The IL-2 or the Met-IL-2 that obtain according to the inventive method can be used as the medicament of preventing and treating knurl in organism, for example adopt the carrier know suitably to be mixed or dilute and make injection or capsule is used for oral or parenteral treatment.They both can singly be used, also can with the composite use of T type killer cell, or with the composite use of natural killer cell.
IL-2 and Met-IL-2 that the best mode that proposes according to the present invention makes have the basic identical biological activity with natural human IL-2.Therefore can adopt the mode identical to be used with natural product.The dissociation constant related with the IL-2 acceptor of corresponding cell is very little, therefore only needs very little dosage as a rule.
In order to be used to promote the T cell (vitro) to increase this purpose under the attitude " external ", IL-2 that the inventive method can be made or Met-IL-2 put in the medium that a kind of concentration is about 0.01-1 units per ml (being good with about 0.1-0.5 units per ml especially).
The bioactive mensuration of IL-2 can be carried out (see: Biochem, Biophys.Res.Commun., 109,363(1982)) with reference to the method that IL-2 adopted.
To be used to the promoting T cell to increase this purpose under " external " attitude is example, IL-2 that the inventive method is made or Met-IL-2 annotate concentration and are about in the medium that the 0.1-0.5 units per ml contains the cell suspension thing, for example, this cell suspension thing is a kind of T cell by the hapten sensitization, and its method for making is: with human body end serum deutero-T cell (1 * 16 slightly
6Cells/ml) lymphocyte culture fluid and B transformant (1 * 10
6Cells/ml) form through 3 days combination treatment, and the B transformant is to form through x-ray irradiation (1,500 rad) in RPMT 1640 media that contain 20% foetal calf serum.Incubation period about one month continuously, about around here medium that renews week about a time.
The IFN-α A and the Met-IFN-α A that make according to the inventive method have a kind of activity that cell changes antiviral attitude over to that influences.Utilize this characteristic, can measure the activity of human body IFN-α A.
In addition, the IFN-α A and the Met-IFA-α A that make according to the inventive method not only have antivirus action, but also the following effect of tool such as the effect that suppresses cell enlargement, suppresses to produce the effect of antibody, and the effect that promotes natural killer cell activity.
The antigenicity that IFN-α A that makes according to the inventive method and Met-IFN-α A do not cause because of protein impurities, thus hypotoxic.
Adopt IFN-α A that the inventive method makes or Met-IFN-α A as the medicine for treatment product in order to make, above-mentioned substance can be made into injection, capsule product etc. and is used for oral or the parenteral treatment, for example, and with traditional carrier dilution with mix and can use afterwards.Every day, dosage range was from 1 * 16
6To 1 * 10
8Unit is especially with 5 * 10
7To 6 * 10
7Unit is good.
In the claim and accompanying drawing of specification sheets of the present invention, amino acid is explained with shortenings sometimes.Based on IUPAC-IUB(IUPAC-International Union of Biochemistry) nomenclature mo of biochemical nomenclature commission and these shorteningss of proposing are generally used in the relevant speciality field, and example is as shown in the table.Herein, do not give and listing for being present in optical isomer in the amino acid and L-type product.
The Gly:Glycine glycine
The Ala:Alanine L-Ala
The Val:Valine Xie Ansuan
The Leu:Leucine leucine
The Ile:Isoleucine Isoleucine
The Ser:Serine Serine
The Thr:Threonine Threonine
The Cys:Cysteine halfcystine
1/2 Cys:Half cystine halfcystine
CmCys: Carboxymethylcys-
The teine Carbocisteine
The Met:Methionine methionine(Met)
Glu:Glutamic acid L-glutamic acid
Asp:Aspartic acid aspartic acid
Lys:Lysine Methionin
The Arg:Arginine arginine
The His:Histidine Histidine
The Phe:Phenylalanine phenylalanine
Tyr:Tyrosine tyrosine
The Trp:Tryptophan tryptophane
The Pro:Proline proline(Pro)
The Asn:Asparagine l-asparagine
The Gln:Glutamine glutamine
Asp/Asn:Aspartic acid and aspartic acid and asparagus fern
The Asparagine acid amides
Glu/Gln:Glutamic acid and L-glutamic acid and glutamy
Glutamine amine
Accompanying drawing content brief description
Amino acid segment (wherein X is hydrogen atom or methionine residue) in non-glycosylated human body words spoken by an actor from offstage cell (bacteriocidin)-2 protein that Fig. 1 represents to be obtained by reference example 1.Amino acid segment (wherein X is hydrogen atom or methionine residue) among non-glycosylated human interferon-α A that Fig. 2 represents to be obtained by reference example 3.Fig. 3, Fig. 4 and Fig. 5 represent respectively among the embodiment 1 to adopt the FPLC method, etc. the result that obtains of electrophoresis and the tryptic digest peptide assignment of genes gene mapping.Fig. 6, Fig. 7 and Fig. 8 represent respectively to adopt the result who adopts the FPLC method to obtain among SP-5PW ion exchange chromatography and the embodiment 6 among employing CF method (chromatofocusing method), the embodiment 5 among the embodiment 2.Fig. 9 and Figure 10 represent in the reference example 3 structure iron that adopts plasmid Plasmid PTFI and PTB285 to obtain respectively.
The present invention also will further be illustrated particularly by following embodiment and reference example, but it does not represent that the scope of the invention only limits to this.
By transformant, the intestinal bacteria DHI/PTF4 that reference example obtains, (Insti-tute for Fermentation, Osaka), number of registration is IFO-14299 in the big lonely fermentation research institute of Japan (IFO) in seasoning.On April 6th, 1984 was obtained number of registration FERMP-7578 in Japanese fermentation research institute (Fermentation Research Institute(FRI) (for the industrial science technology agency of Ministry of International Trade and Industry administration), and again the latter being obtained number of registration at FRI according to Budapest agreement is FERM BP-628, and forwarding CGMCC simultaneously to, to obtain number of registration be CGMCC No.0001.
Transformant (intestinal bacteria DH1/PTF4) is obtained number of registration IFO-14437 at IFO.On April 30th, 1985 was obtained number of registration FERMP-8199 at FRI, and number of registration FERM BP-852 is obtained at FRI again according to Budapest agreement in the back, obtained number of registration at CGMCC simultaneously.
Embodiment 1: adopt the FPLC method to separate IL-2 and Met-IL-2
With 5 milliliters of 0.005M ammonium acetate buffer reagents (pH5.0) by (Mono-p is a filler: 0.5 * 20 centimetre by Mcno-P post that the 0.025M diethanolamine-hydrochloride buffer agent (pH9.4) equilibrated FPLC method is used, Pharmacia " goods); used ammonium acetate buffer reagent contains the ⅳ by reference example 1() 5.9 milligrams of non-glycosylated human body white corpuscles (sterilization) element-2(protein concn of obtaining is 1.18 mg/ml), it is the mixture of IL-2 (interior white corpuscle (sterilization) plain-2) and Met-IL-2 (methionine(Met)-Nei white corpuscle (sterilization) element-2).Then, use the 1%(volume ratio) Pharmalyte(pH8-10.5)-the 5.2%(volume ratio) protein that adsorbed by the Mono-P post of Polybuffer 96-hydrochloride buffer agent (pH8.0) system elution.The operation of FPLC method (rapidly and efficiently can liquid chromatography) is at room temperature carried out, and flow velocity is 30 milliliters/hour.Result: separate the peak value 1(P1 obtain) (expression pH is the elution product under 8.0) and peak value 2(P2) (representing that pH is the elution product under 7.9) as shown in Figure 2.Collect then and separate, and remove used Polybuffer in the operation of FPLC method as diluting solvent through high-effect liquid chromatography knot with trifluoracetic acid-acetonitrile.
Post: Ultrapore RPSC filler (1.0 * 25
Centimetre, Altex, the U.S.)
Column temperature: 30 ℃
Diluting solvent A:0.1% trifluoracetic acid-99.9% water
Diluting solvent B:0.1% trifluoracetic acid-99.9% acetonitrile
Liquid stream time control: 0 minute.
(control of elution time)
(55%A+45%B)-4 minute
(55%A+45%B)-28 minute
(42%A+58%B)-38 minute
(34%A+66%B)-43 minute
(20%A+80%B)-44 minute
(55%A+45%B)
Elution speed: 3.0 ml/min
The every kind of solution that is obtained by above-mentioned chromatography becomes white powder through lyophilize.Represent peak value 1 and peak value 2 powder (FPLC method) respectively with P1 and P2.The P1 yield is 1.12 milligrams (19.0%), and the P2 yield is 3.01 milligrams (51.0%).
Secondly, P1 and P2 are separated albumen chemical analysis (Proteochemicalanalysis).Adopt the P1 and the P2 amino end group amino acid segment content that record with the automatic detection method of Edman that flies phase protein detection programtape (american biosystem company (Biosystem Inc.) 470A type goods) to be 45 μ g(3nmol).Phenyl thiohydantoin (PTH) amino acid (PTH-amino acid) is to detect with high-effect liquid chromatography, and what use in this process is Micropack SP-c18 post (Varian Associates, the U.S.).In each program that table 1 listed, all can detect PTH-amino acid.
Table 1
The PTH-amino acid (Pmol) that detects
Cycle P1 P2
1 L-Ala methionine(Met) L-Ala methionine(Met)
(2330) (21) (56) (2600)
2 proline(Pro) L-Ala proline(Pro) L-Ala
(1670) (77) (33) (2430)
3 Threonine proline(Pro) Threonine proline(Pro)
(819) (51) (45) (1770)
4 serine threonine serine threonines
(222) (40) (16) (789)
The amino acid whose analysis of carboxyl end group adopts following method to carry out.Being about to P1 and P2 joins and decomposes in the Glass tubing that hydrazine uses.Test with anhydrous hydrazine.Then with Glass tubing at the vacuum lower seal, and 100 ℃ of down heating 6 hours.After phenyl aldehyde processing hydrazine resolvent, adopt amino acidanalyser (835 types, FDAC product) to detect total free aminoacids.Detected result: P1 and P2 reverse-examination are measured Threonine, and its rate of recovery is respectively 34.8% and 34.0%.Therefore, the carboxyl end group amino acid of P1 and P2 can be represented with Threonine.For the analysis of amino acid component, add contain 4% thioglycollic acid azeotropic hydrochloric acid in Glass tubing, and at the vacuum lower seal, then under 100 ℃ through 24 hours, the hydrolysis of 48 hours and 72 hours.Adopt amino acid analysis to state (835 types, HIT's goods).Gelucystine and halfcystine process are with being detected quantitatively by amino acidanalyser as halfcystine behind the performic oxidation.The amino acid analysis value is according to the mean value calculation that obtains after 24 hours, 48 hours and the hydrolysis in 72 hours, and wherein the value of Serine and Threonine is zero to extrapolate and obtain by hydrolysis time.Its result is shown in table 2.Result according to amino end group amino acid segment and amino acid composition analysis, can prove conclusively: P1 contains IL-2 (interior white corpuscle (sterilization) plain-2) molecular species, as shown in Figure 1, when the X=hydrogen atom, and P2 contains Met-IL-2 (methionine(Met)-Nei white corpuscle (sterilization) plain-2) molecular species, as shown in Figure 1, when the X=methionine residue, and purity is higher than 98% and 99% respectively.
Table 2
Composition ratio
According to CDNA base chain
Amino acid P1 P2 section derived value
Aspartic acid and l-asparagine 11.8 11.8 12
Threonine 12.6 12.6 13
Serine 7.5 7.5 8
L-glutamic acid and glutamine 18.6 18.7 18
Proline(Pro) 5.3 5.3 5
Glycine 2.2 2.2 2
L-Ala 4.9 5.0 5
Halfcystine 2.6 2.8 3
Xie Ansuan 4.1 4.1 4
Methionine(Met) 4.1 5.1 4
Isoleucine 8.6 8.6 9
Leucine 21.8 21.9 22
Tyrosine 3.1 3.2 3
Phenylalanine 6.0 6.1 6
Methionin 11.9 11.9 11
Histidine 3.0 3.0 3
Arginine 4.2 4.2 4
Tryptophane 1.1 1.1 1
Secondly, the result of employing Ampholine PAG pole plate (LKB company product) mensuration P1 and P2 iso-electric point as shown in Figure 3.Non-glycosylated human body words spoken by an actor from offstage cell (sterilization) element-2(IL-2) is the mixture of two kinds of raw materials, i.e. IL-2 and Met-IL-2, and they demonstrate two kinds of energy bands when electric electrophoretic migration such as passing through.And aspect in addition, the P1 that obtains according to the embodiment of the invention and P2 are that different a kind of of migration distance can band each other.Through electrophoretic migration test PAG pole plate lacing film pH value, predict P1(IL-2) and iso-electric point P2(Met-IL-2) be respectively 7.7 and 7.5.
In addition, adopt laxative remedy through tryptic digestion P2, then obtain the peptide structure iron.Promptly as, with the TPCK-trypsin U.S. Worthington company goods of 1.25 μ g) join in the sodium hydrogen carbonate solution (pH8.0) of the 100 μ l0.02M that contain 50 μ g P2, and under 37 ℃, keep the reaction 28 hours.At last by in reaction soln, adding 400 μ l1%(volume ratios) trifluoracetic acid stops reaction.Adopt high-effect liquid chromatography (HPLC method) to handle the Digestive system that makes under the following conditions, its product structure figure as shown in Figure 4.
HPLC:5040 type (Varian Associates, the U.S.)
Post: Nucleosyl 5c18(Machelehner
Gel AG, West Germany)
Column temperature: 30 ℃
Diluting solvent A:0.1% trifluoracetic acid-99.9% water (volume ratio)
Diluting solvent B:0.1% trifluoracetic acid-99.9% acetonitrile (volume ratio)
Liquid stream time control: 0 minute
(85%A+15%B)-15 minute
(72%A+28%B)-16 minute
(64%A+36%B)-80 minute
(40%A+60%B)-85 minute
(15%A+85%B)
*
Elution speed: 3.0 ml/min
Detection method: carry out back spike (Post-Labelling) according to o-phthalaldehyde method and detect (Anal.Chem., 43,880(1971)).
With the ammonium acetate buffer reagent (pH5.0) of 500 milliliters of 0.05M by filling the PBE94(Pharmaciar goods by 0.025M diethanolamine-hydrochloride buffer agent (pH9.4) equilibrated) separator column, used ammonium acetate buffer reagent contains 545 milligrams by reference example 1(ⅳ) non-glycosylated human body words spoken by an actor from offstage cell (sterilization) element-2(protein concn of making is 1.09 mg/ml), it is the mixture of IL-2 and Met-IL-2.Adopt the 1%(volume ratio then) Pharmalyte(pH8-10.5)-the 5.2%(volume ratio) Polybuffer 96-hydrochloride buffer agent (pH8.0) carries out the CF method as eluant and separates.Operation is undertaken by following condition: 4 ℃ of temperature, 200 milliliters/hour of flow velocitys.Result: separate the peak value 1(P1 obtain) (the elution product under pH8.5) and peak value 2(P2) (the elution product under pH8.3) as shown in Figure 5.Adopt the method identical with embodiment 1 to remove after the Polybuffer, the protein yield is respectively 9.4 milligrams (17.4%) and 336 milligrams (61.6%) to P1 and P2.The amino acid whose analysis of amino end group is preferably adopted the HPLC method to analyse survey dansyl amino acid and is realized, and the latter produces with hydrochloric acid hydrolysis behind P1 and the red sulfonylation of P2 process; In adopting HPLC method process, to use
* sic does not have the concrete time.The translator annotates.
Micropack SP separator column.Finally guarantee: P1 contains purity greater than 99.6% IL-2, and P2 contains purity greater than 99.5% Met-IL-2.
Embodiment 3: adopt the DEAE-Toyopearl ion exchange chromatography to separate IL-2 and Met-IL-2
In order to regulate pH value to 8.5, the tri hydrochloride buffer agent (pH9.0) of 10 milliliters of 10mM is joined the ammonium acetate buffer reagent (pH5.0) of 10 milliliters of 0.00M, this ammonium acetate buffer reagent contains the ⅳ by reference example 1() 10.3 milligrams of non-glycosylated human body words spoken by an actor from offstage cells (sterilization) element-2(protein concn of making is 1.03 mg/ml), it is the mixture of IL-2 and Met-IL-2.Then, this composite solution fed with 10mM tri hydrochloride buffer agent (pH8.5) equilibrated fills DEAE-Toyopearl 650M(Japan Cao Da company) separator column, it will carry out the classification of pH value with 1 liter of 10mM tri hydrochloride buffer agent (pH8.5) and 1 liter of 10mM tri hydrochloride buffer agent (pH7.0) and dilute.Operate by following condition: 4 ℃ of temperature, 100 milliliters/hour of flow velocitys.Though the solvability of this law is lower than FPLC method and CF method, the result still obtains two kinds of peak values (P1 and P2).In order to prevent that peak value from overlapping each other, to the first half of P1 and P2 back half collect and separate, P1 that obtains and the yield of P2 are respectively 0.82 milligram (8.0%) and 1.98 milligrams (19.2%).The amino end group amino acid analysis result conclusive evidence that adopts the dansyl chloride method to obtain: P1 contains purity greater than 90% IL-2, and P2 contains purity greater than 95% Met-IL-2.
Embodiment 4: adopt the FPLC method to separate IL-2 and Met-IL-2
With plain-2 solution of the 5 milliliters of non-glycosylated human body words spoken by an actor from offstage of part purified cells (sterilization) by Mono-P separator column with the 0.025M diethanolamine-hydrochloride buffer agent (pH9.4) equilibrated FPLC method is used, white corpuscle (sterilization) plain-2 derives from reference example 1(ⅲ in the used this human body), wherein contain IL-2 and Met-IL-2.Then, protein 1%(volume ratio with Mono-P post absorption) Pharmalyte(pH8-10.5)-and the 5.2%(volume ratio) Polybuffer 96-hydrochloride buffer agent (pH8.0) carries out elution, elution speed is 25 milliliters/hour in this process, and column temperature is a room temperature.The result separates and to obtain two kinds of fractions: contain IL-2(P1) fraction and contain Met-IL-2(P2) fraction, the yield of P1 and P2 is respectively 25% and 54%.
Embodiment 5: adopt the SP-5PW post to separate IL-2 and Met-IL-2
0.5 milliliter of 0.05M ammonium acetate buffer reagent (pH5.0) is fed (0.75 * 7.5 centimetre on the SP-5PW post used with 0.025M phosphate buffer (pH7.4) equilibrated HPLC method, Japan Cao Da company), used ammonium acetate buffer reagent is 1.03 mg/ml by non-glycosylated human body words spoken by an actor from offstage cell (sterilization) element-2(protein concn that reference example 2 obtains).Adopt 0.025M phosphate buffer (pH7.4) elution protein.Column temperature is 35 ℃, and the buffer reagent flow velocity is 0.5 ml/min.Used chromatographic system is Varian 5500 type liquid chromatographs.
The result: white corpuscle (sterilization) plain-2 is come out by elution as two kinds of peak values (PA and PB) in non-glycosylated, as shown in Figure 7.Collect and separate each peak value (representing with mark in the drawings), and through the amino end group analysis.Can prove conclusively, PA and PB contain Met-IL-2 and the IL-2 greater than 99.5% respectively.
Embodiment 6: adopt the FPLC method to separate IFN-α A and Met-IFN-α A
1.0 milliliters of 0.12M sodium-chlor-0.025M ammonium acetate buffer reagent (pH5.0) feeding is carried out desalting treatment with 0.025M imidazoles-hydrochloride buffer agent (pH6.7) equilibrated PD-10 post, and used this ammonium acetate buffer system contains the non-glycosylated human interferon-α A(IFN-α A that is made by said method in the reference example 3) (protein concn is 2.96 mg/ml).Again 1.5 milliliters of eluants are fed with the 0.025M imidazoles-(Mono-P is a filler to hydrochloride buffer agent (pH6.7) equilibrated FPLC method Mono-P post: 0.5 * 20 centimetre, Pharmacia factory goods), employed buffer reagent to contain the 2.37 milligrams of non-glycosylated human interferons-α A(protein concn that is made by last method be 1.58 mg/ml).Use the 10%(volume ratio then) protein that adsorbed by the Mono-P post of Pclybuffer74-hydrochloride buffer agent (PH5.5) elution.The FPLC method is at room temperature operated: flow velocity is 30 milliliters/hour.Result: separate the eluant of P1(under PH5.6 obtain) and the eluant of P2(under PH5.4) as shown in Figure 8.In order to remove the used Polybuffer of FPLC method, separate two kinds of peak values that obtain through collection and will handle with high efficiency liquid chromatography, in this process, adopt trifluoracetic acid and acetonitrile as diluting solvent.
Post: Ultrapcre RPSC filler (1.0 * 25 centimetres,
Altex, the U.S.)
Column temperature: 30 ℃
Diluting solvent A:0.1% trifluoracetic acid-99.9% water
Diluting solvent B:0.1% trifluoracetic acid-99.9% acetonitrile
Liquid stream time control: 0 minute
(60%A+40%)-45 minute
(45%A+55%B)-46 minute
(60%A+40%B)
Flow velocity: 3.0 ml/min
The every kind of solution that adopts above-mentioned chromatography to make becomes white powder through lyophilize.Represent with P I and P II respectively by the powder that peak value I and peak value II make.P I yield is 0.723 milligram (24.4%), and P II yield is 0.945 milligram (31.9%).
Secondly, P I and P II will be through separating albumen chemical analysis (Proteochtmical analysis).When P I and P II be reduced with carboxymethylation after, the P I and the P II amino end group amino acid segment content that adopt the automatic detection method of Edman with gas phase protein detection programtape (the 470 class product that american biosystem company produces) to record are about 40 μ g(2.1nmol).Phenyl thiohydantoin (PTH) amino acid (PTH-amino acid) is to detect with high-effect liquid chromatography, uses Micropack SP-C18 post (Varian Associates, the U.S.) in this process.All can detect PTH-amino acid in each program that table 3 listed.
Adopt with embodiment 1 same procedure and analyze the amino acid whose result of carboxyl end group, have only L-glutamic acid to detect from P I and P II, the rate of recovery is respectively 12.5% and 14.3%.
Table 4 is listed the result who adopts the amino acid composition that records with embodiment 1 same procedure.Result according to amino end group amino acid segment and amino acid composition analysis, can prove conclusively: P I and P II contain molecular species shown in Figure 2 (Met-IFN-α A) (when the X=methionine residue) and molecular species (IFN-α A) (when X is hydrogen atom) shown in Figure 2 respectively, and their purity is higher than 98%.
Table 3
The PTH-amino acid (Pmol) that detects
Cycle
PⅠ PⅡ
1 Carbocisteine methionine(Met) Carbocisteine methionine(Met)
(28) (1630) (1370) (15)
2 aspartic acid Carbocisteine aspartic acid Carbocisteines
(20) (1250) (1220) (21)
3 leucine aspartic acid leucine aspartic acids
(22) (986) (1170) (11)
4 proline(Pro) leucine proline(Pro) leucines
(14) (963) (761) (8)
Table 4
Proportion of composing
Amino acid is according to cDNA base segment
P I P II derived value
Aspartic acid and l-asparagine 12.0 12.0 12
Threonine 9.7 9.7 10
Serine 12.9 12.5 14
L-glutamic acid and glutamine 26.1 26.0 26
Proline(Pro) 5.0 4.9 5
Glycine 4.9 4.9 5
L-Ala 8.1 8.1 8
1/2 halfcystine 3.8 3.9 4
Xie Ansuan 7.1 7.1 7
Methionine(Met) 6.1 5.1 5
Isoleucine 7.9 7.8 8
Leucine 20.8 20.6 2.1
Phenylalanine 9.9 9.8 10
Methionin 11.0 11.1 11
Histidine 3.5 3.4 3
Arginine 8.8 8.8 9
Tryptophane 2.2 2.1 2
Adopt the iso-electric point of Ampholine PAG plate (LKB company) test P I and P II.Iso-electric point is 6.3 to calculate P I (Met-IFN-α A) in view of the above, and P II (IFN-α A) iso-electric point is 6.2.
Embodiment 1: do the preparation of the IL-2 of injection
The solution that contains interior white corpuscle (sterilization) element-2(IL-2) that makes by embodiment 1, after under aseptic condition, adsorbing with 0.025M ammonium acetate buffer reagent (pH5.0) equilibrated CM-Toyopearl post (Japan Cao Da company goods), carry out elution with the above-mentioned buffer reagent that contains 0.15MNacl, preferably adding its concentration of 0.15M Nacl and HSA(then again is 0.5%) eluant is advanced the thing dilution.Use film filter (aperture 0.22 micromillimeter) to filter diluting soln at last.With the filtrate that makes the like this medicine bottle (1 milliliter every bottle) of under aseptic condition, packing into, make injection IL-2 through lyophilize.This injection preferably mixes use with 1 ml distilled water.
Embodiment 8: do the preparation of the IFN-α A of injection
Contain interferon-' alpha ' A(IFN-α A by what embodiment 6 made) solution, after under aseptic condition, adsorbing with 0.025M ammonium acetate buffer reagent (pH5.0) equilibrated CM-Toyopeal post (Japan Cao Da company goods), carry out elution with the above-mentioned buffer reagent that contains 0.15M Nacl, preferably adding its concentration of 0.15M Nacl and HSA(then again is 0.5%) eluant is diluted.Use film filter (aperture 0.22 micromillimeter) to filter diluting soln at last.With the filtrate that makes the like this medicine bottle (1 milliliter every bottle) of under aseptic condition, packing into, make injection IFN-α A through lyophilize.This injection injection preferably mixes use with 1 ml distilled water.
Still can not preferentially adopting at present process purified Met-IL-2 and being used under the situation of above-mentioned injection preparation, can adopt above-mentioned process purified IL-2 and IFN-α A to replace it respectively through purified Met-IFN-α A.
Reference example 1: the proteinic preparation of non-glycosylated human body IL-2 ... I
(ⅰ) cultivation of transformant
Transformant intestinal bacteria DH1/PTF4 (referring to: Japan's special permission communique specification sheets 225079/1983 year (file an application December 28 nineteen eighty-three), corresponding to: No.0145390 European prospectus EP Publication(laid open)) be injected in the 50 milliliters of liquid media (pH7.0) and (be contained in 250 milliliters of erlenmeyer flasks), this liquid medium contains 1%Bacto tryptones (U.S. Difco laboratory), 0.5%Bacto yeast extract (U.S. Difco laboratory), 0.5% sodium-chlor and 7 micrograms (μ g)/milliliter tsiklomitsin.On shaking turner,, developing medium is dumped in the jar fermenter that volume is 5 millis, 2.5 liters of M9 wherein are housed through after 37 ℃ of following overnight incubation
*Medium, and contain 0.5% casamino acids, 0.5% glucose and 7 mcg/ml tsiklomitsins at this medium gram.Stirred 4 hours down then with the nutrient solution aeration aerating, and at 37 ℃, behind adding 3-β-indyl vinylformic acid (25 mcg/ml), oxygenation and stirring are 4 hours again.Cultivating the cell that obtains in the meat meter by 2.5 liters that make by centrifugation will be-80 ℃ of following refrigerated storage.
(ⅱ) extract
The above-mentioned refrigeration cell that makes (12.1 gram) is suspended in the 100 liters of extracting solutions (pH7.0) that contain 7M guanidinesalt hydrochlorate and 0.1M tri hydrochloride, and this suspension was stirred 1 hour down at 4 ℃.Restrain filtration yield centrifuging lysate (Lysate) with 20 minutes 28,000 then and obtain 92 milliliters of supernatant liquors
(ⅲ) the proteinic part of IL-2 is refining
The supernatant liquor that makes by last method runs into 0.01M tri hydrochloride buffer agent (pH8.5)
* sic.Should be M-9, merchant's name.-annotation of translation.
And dialysis is come out, and with the centrifuging of 10 minutes 19,000 gram filtration yield, obtains the supernatant liquor that 94 milliliters of dialysis are come out.With this supernatant liquor join the DE52 post used by 0.01M tri hydrochloride buffer reagent (pH8.5) equilibrated adsorbed proteins (fill DEAE cellulose (DEAE-Cellulose), 50 milliliters of volumes, Britain Whatman " goods) in.At last protein is diluted with LINEAR N acl concentration gradient (0-0.15M NaCl, 1 liter).
(ⅳ) IL-2 is proteinic refining
Active ingredient (53 milliliters) is concentrated to 4.8 milliliters with YM-5 film (U.S. Amicon company goods), and carry out gel analysis with 0.1M tri hydrochloride (pH8.0)-1M NaCl buffer reagent equilibrated Sephacryl S-200 post (Sweden Pharmacia " goods, 500 milliliters of volumes).Again with the active ingredient (28 milliliters) that obtains with YM-5 membrane concentration to 2.5 milliliter.Enriched material is admitted in the Ultrapore RPSC post (U.S. Altex " goods) of absorption usefulness, adopts trifluoracetic acid-acetonitrile system to carry out high-effect liquid chromatography as moving phase and separates.
The condition that is adopted:
Post: Ultrapore RPSC filler (4.6 * 75 millimeters).
Column temperature: 30 ℃.
Solvent orange 2 A: 0.1% trifluoracetic acid-99.9% water.
Solvent B:0.1% trifluoracetic acid-99.9% acetonitrile.
Liquid stream time control: 0 minute
(68%A+32%B)-25 minute
(55%A+45%B)-35 minute
(45%A+55%B)-45 minute
(30%A+70%B)-48 minute
(100%B)
Flow velocity: 0.8 ml/min.
Detect wavelength: 230 micromillimeters.
In about 39 minutes residence time active ingredient is collected, (specific activity is 30,000U/mg thereby obtain containing 0.53 milligram of non-glycosylated human body IL-2 protein; By parent material, the active ingredient rate of recovery is 30.6%; Lipidated protein 99%(calculates according to the optical density(OD) that the SDS-polyacrylamide gel electrophoresis records).
With above-mentioned solution lyophilize then white powder, this powder have the IL-2 specific activity (26,000U/mg).
Reference example 2: non-glycosylated human body IL-2
Proteinic preparation ... II
(ⅰ) Plasmid(plasmid) structure
Plasmid PILOT 135-8 has human body IL-2 (interior white corpuscle (sterilization) plain-2) gene (referring to the embodiment 1(ⅶ in Japan's special permission communique specification sheets (225079/1983)) (file an application November 28 nineteen eighty-three), corresponding European prospectus: No.0145390 EP Publication(laid open)).
The Plasmid PILOT 135-8 available constraints enzyme HgiAI that mentions in the above-mentioned document decomposes.The 1294bp DNA fragment (fragment) that obtains is like this handled through T4 DNA polymerase has level and smooth tail end, and is connected with EcoRI by T4 dna ligase (ligase) and forms the dTGCCATGAATTCATGGCA segment.The DNA that obtains like this forms the DNA fragment through Eco RI digestion, and the latter has password ATG and the human body IL-2 gene that causes conversion.
This DNA(thymus nucleic acid) is inserted on Eco RI-Pst I point by (see: Nucleic Acids Research, 11,3077(1983)) among the Ptrp 781 that digests by the T4 dna ligase.The Plasmid PTF 1 that obtains like this has password that causes conversion and the human body IL-2 gene (Fig. 9) that breaks away from tryptophan promoter gene (trp promoter).
(ⅱ) preparation of transformant
Intestinal bacteria N4830 can be converted into the transformant (intestinal bacteria N4830/PTB285) that can transmit said Plasmid (see: Pro.Natl.Acad.Sci.U.S.A.69,2110(1972)) with the Plasmid PTB285 that the method that proposes according to people such as Cohen make.
(ⅲ) cultivation of transformant
Transformant intestinal bacteria N4830/PTB285 is injected into (being contained in 250 milliliters is subjected in the human relations Mei Shi flask) in the 50 milliliters of liquid media (pH7.0), and this liquid medium contains 1%Bacto tryptones (U.S. Difco laboratory), 0.5%Bacto yeast extract (U.S. Difco laboratory), 0.5% sodium-chlor and 50 mcg/ml benzyl ampicillins.Through cultivating under 35 ℃ after the liquid, it is in 5 liters the jar fermenter that developing medium is dumped volume, and the M-9 medium wherein is housed, and contains 0.5% casamino acids in this medium, 0.5% glucose and 50 mcg/ml benzyl ampicillins on oscillating rotator.Stirred 4 hours down then with the nutrient solution aeration aerating, and at 35 ℃, again in 442 ℃ of following oxygenations and stirring 3 hours.Cultivating the cell that obtains in the gravy by 2.5 liters that make by centrifugation will be-80 ℃ of refrigerated storage.
(ⅳ) extract
The above-mentioned refrigeration cell that makes (20 gram) is suspended in the 100 liters of extracting solutions (pH7.0) that contain 7M guanidinesalt hydrochlorate and 0.1M tri hydrochloride equably, and this suspension was stirred 4 hours down at 4 ℃.Carry out centrifuging and obtain supernatant liquor with 20 minutes 28,000 gram filtration yield then.
Plain-2 proteinic parts of white corpuscle (sterilization) are refining (ⅴ)
The supernatant liquor that obtains by last method runs into 0.01 tri hydrochloride buffer reagent (pH8.5) and dialysis is come out, and carries out centrifuging through 10 minutes 19,000 gram filtration yield then.This newly formed supernatant liquor is joined in the DE52 post of being used by 0.01M tri hydrochloride buffer reagent (pH8.5) equilibrated adsorbed proteins (filling DEAE-Cellul-ose, 500 milliliters of volumes, Britain Waffman r goods).Use LINEAR N acl concentration gradient (0-0.15M Nacl) dilution IL-2 (interior white corpuscle (sterilization) plain-2) at last and obtain active ingredient.
White corpuscle (sterilization) plain-2 is proteinic refining (ⅵ)
The active ingredient that the method for using is obtained is concentrated to 5 milliliters with YM-5 film (U.S. Amicon company goods), and use by 0.1M tri hydrochloride (pH8.0)-1M Nacl buffer reagent equilibrated and fill in the separator column (Sweden Pharmaciar goods, 500 milliliters of volumes) of Sephacryl S-200.The active ingredient that will newly make (48 milliliters) is carried dense to 3 milliliters with the YM-5 film again.Enriched material is sent in the adsorption column (U.S. Altex r goods) of filling Ultrapore RPSC, adopted trifluoracetic acid-acetonitrile to carry out high-effect liquid chromatography and separate as diluting solvent.
Operational condition:
Post: Ultrapore RPSC filler (4.6 * 75 millimeters)
Column temperature: 30 ℃
Diluting solvent A:0.1% trifluoracetic acid-99.9% water
Diluting solvent B:0.1% trifluoracetic acid-99.9% acetonitrile
Liquid stream time control: 0 minute
(68%A+32%B)-25 minute
(55%A+45%B)-35 minute
(45%A+55%B)-45 minute
(30%A+70%B)-48 minute
(100%B)
Flow velocity: 0.8 ml/min
Detect wavelength: 230 micromillimeters
Under these conditions, in about 39 minute residence time, active ingredient is collected, obtain the mixture of 10 milliliters of Met-IL-2 (methionine(Met)-Nei white corpuscle (sterilization) plain-2) and IL-2 (interior white corpuscle (sterilization) plain-2).
Reference example 3: preparation non-glycosylated human interferon-α A(IFN-α A)
(ⅰ) cultivation of transformant
Contain following (1) or (2) medium in 5 liters of (volume) jar fermenters: (1) adds 25 grams per liter glucose, 5 grams per liter casamino acidss (U.S. Difco laboratory products) in the M-9 medium, 70 mg/litre vitamins B hydrochlorides and 5 mg/litre tetracycline hydrochlorides and the nutrient substance medium made; (2) in the M-9 medium, add 25 grams per liter glucose, 4 grams per liter L-Sodium Glutamates, 27 mg/litre Fecl
26H
2O, 8 mg/litre CuSO
45H
2O, 8 mg/litre ZnSO
47H
2O, 70 mg/litre vitamins Bs
1Hydrochloride, 5 mg/litre tetracycline hydrochlorides, 50 mg/litre L-proline(Pro) and 50 mg/litre L-leucines and the medium (medium on the chemical sense) made, to transmit the intestinal bacteria 294(ATCC31446 of plasmid (Plasmid))/PLe IFA trp25 joins in the above-mentioned medium and cultivates, this plasmid has the structure gene (seeing: European prospectus No.43, the embodiment 1 in 980) of human leukocytes (Leukocyte) the IFN-α A coding that amino acid shown in Figure 2 uses.Culturing process will begin to carry out under 2.5 liters of/minute aeration aerating conditions, and stirring velocity is that per minute 1000 changes, and temperature is 37 ℃.In the culturing process, temperature is reduced to 33 ℃ under OD 3000 KU situations, to OD 5000KU reduce to 29 ℃ right, OD7000KU reduces to 25 ℃.Take this mode to carry out continuously cultivating in 48 hours.In culturing process, the decomposition oxygen concn is kept and is not less than 5%; When aminoglutaric acid concentration is low to moderate 1% or when lower, then add L-glutamic acid with 25 grams per liter speed.
(ⅱ)
The cell suspension that the cultivation gravy that 2 liters of employings (ⅰ) method is obtained comes out through centrifuging is at 100 milliliters of 50mM three hydrochloric acid (among the pH7.6), wherein contain 10% sucrose, 0.2MNacl, 10mM ethylene diamine four acetate (EDTA), 10mM spermidine, 2mM phenyl methyl sulfonic acid fluoride (PMSF) and 0.2mg/ml N,O-Diacetylmuramidase.This suspended substance stirred 1 hour down at 4 ℃, kept under 37 ℃ 5 minutes, and handled for 40 seconds with ultrasonoscope (U.S. Altex company goods) down at 0 ℃.With lysate with per hour 11,300 the gram filtration yield carry out centrifugal process, thereby obtain 95 milliliters of supernatant liquors.
(ⅲ) IFN-α A(interferon-' alpha ' A) proteinic refining
With the 95ml supernatant liquor with containing 1mM EDTA and 0.15M Nacl(TEN) 20mM tri hydrochloride (pH7.6) be diluted to 300ml, this diluting soln is joined in the anti-IFN-α A antibody adsorption column (20ml).
After this post of TEN thorough washing, again with the 0.2M acetic acid elution that contains 0.1% tween (nonionic surface active agent a kind of).Used tween is a Japanese Wako pure chemistry industrial product.Active fractions is mixed, and adjust pH is 4.5, join the adsorption column of the filling carboxymethyl cellulose that is used to adsorb then.This adsorption column of thorough washing, and contact elution with the 0.025M ammonium acetate buffer reagent (pH5.0) that contains 0.15M Nacl.Again active fractions is mixed, and obtain 320mg human leukocytes IFN-α A powder through lyophilize.
The SDS-polyacrylamide gel electrophoresis detects and shows that this molecular weight product is 19000 ± 1000.The proteinic antiviral activity of human leukocytes IFN that finally makes is 2 * 10
8Unit/milligram.With regard to other physical and chemical performance, it is similar mutually for the recombinant chou human leukocytes IFN that the amino acid of this product is formed and peptide structure iron and employing traditional sucrose are made.
Revisal 85107875
After the preceding revisal of the capable revisal of file name page or leaf
Wen Weiyi translates outside need translating among the Figure of description figure
Specification sheets 11 3 1-200 1-200 watts
11 6 colum co um
118~9 three, second buffer reagent three, acetic acid buffer
" annotation of translation " two words are left out in the 1-annotation of translation of 11 tails
15 6 1×16
61×10
6
20 6 100℃ 110℃
29 4 embodiment, 1 embodiment 7
30 fall 1-annotation of translation leaves out " annotation of translation " two words
Claims (35)
1, from the mixture of protein and methionine(Met)-protein (Met-Protein) composition, separates a kind of protein or methionine(Met)-method of protein, this Met-protein is made up of the amino acid segment, this segment and the first proteinic amino acid segment are closely similar, the physiologically active that can be formed by first protein can be provided, and Met-protein to connect the situation of methionine residue on its amino end group similar to first protein, the difference that can utilize iso-electric point between first protein and second protein is separated the mixture of constitutive protein matter.
2, according to the method for claim 1, it is characterized in that, separable programming comprises: (a) make mixture through the electric field migration, perhaps (b) adopts pH value gradient or salt concn gradient elution method from carrier protein and the elution of Met-protein to be come out in carrier according to their corresponding iso-electric points.
According to the method for claim 2, it is characterized in that 3, mixture will carry out the electric field migration by electrophoresis such as electrophoresis such as density gradient, gel or isotachophoresis (isotachophoresis).
4, according to the method for claim 2, it is characterized in that, mixture will adopt chromatofocusing method (CF method), quick protein liquid chromatography (FPLC method), diethyllaminoethyl-ion exchange chromatography, carboxymethyl-ion exchange chromatography, or the elution from carrier of sulfopropyl-ion exchange chromatography is come out.
According to the method for claim 1, it is characterized in that 5, Met-protein is made up of the amino acid segment that connects the egg amino residue on first kind of proteinic amino end group.
According to the method for claim 1, it is characterized in that 6, Met-protein mainly is made up of the first kind of proteinic amino acid segment that connects methionine residue on the amino end group.
According to the method for claim 1, it is characterized in that 7, protein and Met-protein form by the gene recombination engineering.
According to the method for claim 1, it is characterized in that 8, protein comprises cytokinin, peptide hormone, pathogenic microorganism antigen protein, enzyme or reduced hematin.
According to the method for claim 1, it is characterized in that 9, it is about 3 that protein has, 000-50,000 molecular weight.
According to the method for claim 9, it is characterized in that 10, molecular weight is 5,000-30,000.
According to the method for claim 1, it is characterized in that 11, protein system is made up of 30-500 amino acid.
According to the method for claim 11, it is characterized in that 12, protein system is made up of 50-300 amino acid.
According to the method for claim 1, it is characterized in that 13, protein and Met-protein have about 4 to about 11 iso-electric point.
According to the method for claim 1, it is characterized in that 14, protein and Met-protein have about 5 to about 8 iso-electric point.
According to the method for claim 1, it is characterized in that 15, the difference of the iso-electric point between protein and the Met-protein is approximately 0.01 at least.
According to the method for claim 1, it is characterized in that 16, the difference of the iso-electric point between protein and the Met-protein is approximately 0.1 at least.
According to the method for claim 1, it is characterized in that 17, the difference of the iso-electric point between protein and the Met-protein is at least between 0.01 and 0.2.
According to the method for claim 1, it is characterized in that 18, protein and Met-protein white matter all are nonglycosylated.
According to the method for claim 1, it is characterized in that 19, protein is a kind of cytokinin.
According to the method for claim 19, it is characterized in that 20, cytokinin is a kind of interior white corpuscle (sterilization) plain-2.
21, according to the method for claim 20, it is characterized in that interior white corpuscle (sterilization) element the-the 2nd, a kind of non-glycosylated protein matter, (X is a hydrogen atom) as shown in Figure 1 with amino acid segment.
According to the method for claim 19, it is characterized in that 22, cytokinin is a kind of Interferon, rabbit.
According to the method for claim 22, it is characterized in that 23, Interferon, rabbit is meant interferon-' alpha '.
24, according to the method for claim 23, it is characterized in that the non-glycosylated interferon-' alpha ' A that interferon-' alpha ' is made up of the amino acid segment, (X is a hydrogen atom) as shown in Figure 2.
According to the method for claim 1, it is characterized in that 25, mixture means that purity is not less than about 50% protein and the proteinic mixture of Met-.
According to the method for claim 1, it is characterized in that 26, mixture means that purity is not less than about 99% protein and the proteinic mixture of Met-.
27, according to the method for claim 1, it is characterized in that, in non-glycosylated white corpuscle (sterilization) plain-2 and have the amino acid segment of interior white corpuscle (sterilization) element-2 and on amino end group, be connected the Met-protein of methionine residue can be separated from each other by the following method, the mixture that is about to be made up of interior white corpuscle (sterilization) plain-2 and Met-protein is by the chromatofocusing method, the FPLC method, diethyllaminoethyl-ion exchange chromatography or sulfopropyl-ion exchange chromatography separate.
28, according to the method for claim 27, it is characterized in that, non-glycosylated in white corpuscle (sterilization) plain-2 formed (X is a hydrogen atom) as shown in Figure 1 by the amino acid segment.
29, according to the method for claim 1, it is characterized in that, non-glycosylated interferon-' alpha ' A and connect the protein of methionine residue at the amino end group of said interferon-' alpha ' A can be separated from each other by the following method, soon separate by the FPLC method with the mixture that protein is formed by interferon-' alpha ' A.
According to the method for claim 29, it is characterized in that 30, the non-glycosylated interferon-' alpha ' A that is made up of the amino acid segment is (X is a hydrogen atom) as shown in Figure 2.
31, the protein that is generated by the recombinant DNA method is gone up substantially and is not contained its corresponding M et-protein.
32, plain-2 protein of interior white corpuscle (sterilization) are gone up substantially and are not contained its N-methionine(Met) (N-Met) analogue.
33, interferon-alpha proteins matter is substantially free of its N-methionine(Met) analogue.
34, the protein in the claim 31, described protein are to contain to be not more than 3%(weight) its corresponding methionine(Met)-protein (Met-protein).
35, the protein in the claim 34, wherein Met-protein is meant proteinic N-Met analogue (N-methionine(Met) analogue).
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/JP1985/000274 WO1986007063A1 (en) | 1985-05-21 | 1985-05-21 | Mutual separation of proteins |
JPPCT/JP85/00274 | 1985-05-21 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN85107875A true CN85107875A (en) | 1987-04-08 |
Family
ID=13846459
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN198585107875A Pending CN85107875A (en) | 1985-05-21 | 1985-09-26 | Protein is separated from each other |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN85107875A (en) |
WO (1) | WO1986007063A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100541185C (en) * | 2007-03-15 | 2009-09-16 | 云南神宇新能源有限公司 | The high-resolution separation method of jatropha curcas seed endosperm protein |
CN104774242A (en) * | 2009-09-10 | 2015-07-15 | 朗萨生物制剂有限公司 | Method and system for polypeptide purification |
-
1985
- 1985-05-21 WO PCT/JP1985/000274 patent/WO1986007063A1/en unknown
- 1985-09-26 CN CN198585107875A patent/CN85107875A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100541185C (en) * | 2007-03-15 | 2009-09-16 | 云南神宇新能源有限公司 | The high-resolution separation method of jatropha curcas seed endosperm protein |
CN104774242A (en) * | 2009-09-10 | 2015-07-15 | 朗萨生物制剂有限公司 | Method and system for polypeptide purification |
US10221209B2 (en) | 2009-09-10 | 2019-03-05 | Lonza Biologics Plc | Method and system for polypeptide purification |
Also Published As
Publication number | Publication date |
---|---|
WO1986007063A1 (en) | 1986-12-04 |
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