JPH09100296A - Mutual separation of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To mutually separate proteins so as to produce cytokine, hormone, etc., by subjecting a mixture of a protein with a protein, to which methionine residue is added, having the same activity as the former protein to separation means by difference of isoelectric point. SOLUTION: A mixture of proteins is mutually separated in order to produce cytokine, peptide hormone, pathogenic germ antigen protein, enzyme, etc., by a method migrating a mixture of a protein with a protein obtained by adding a methionine residue to amino terminal, etc., of the protein and having similar physiological activity as the protein in an electric field by isoelectric focusing electrophoresis of density gradient, electrophoresis of gel isoelectric point or electrophoresis of constant velocity or by a method eliminating and eluting a mixture of proteins added to electron charge carrier of chromatofocusing method, fast protein liquid chromatography, ion exchange column chromatography in order from a carrier according to the difference of electron charge bringing out the difference of isoelectric point by preparation of pH gradient or salt concentration gradient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a protein and a method for separating a mixture of proteins having a methionine residue added to the protein and having the same physiological activity as the protein from each other.

[0002]

2. Description of the Related Art The existence of various physiologically active proteins such as cytokines and peptide hormones has been clarified, and recent advances in genetic engineering technology are opening the way to mass production of these physiologically active proteins and their clinical application. . Such techniques have already been applied to cytokines such as interferon, interleukin, B cell growth factor (BGF), B cell differentiation factor (BDF), macrophage activating factor (MAF), lymphotoxin (LT) and tumor necrosis factor (TNF). Transforming growth factor (TGF-α); Erythropoietin, epidermal growth factor, insulin, peptide protein hormones such as human growth hormone; For the development of vaccines such as hepatitis B virus antigen, influenza antigen, foot-and-mouth disease virus antigen, and malaria parasite antigen Useful pathogenic microbial antigen proteins; peptidases (eg, tissue plasminogen activator, urokinase, seratiopeptidase, etc.)
And enzymes such as lysozyme; human serum albumin (HSA)
It has been applied to the production of various bioactive proteins such as protein components in blood.

However, since these proteins are produced by genetic engineering techniques, a problem often arises in that the final required product must be separated from other substances in the mixture. Of particular concern is that the gene product produced by expression of the gene encoding the required protein is often a mixture of the required protein and a second protein with a methionine at the amino terminus. is there. The added methionine (Met) expresses the translation initiation codon ATG that directs the initiation of expression of the desired gene, and
This is due to the failure of the host cell expression system excluding the Met group added from the expression product. This problem occurs in both prokaryotic and eukaryotic hosts, but especially in the expression of genes in prokaryotic hosts. This is a particular problem in an expression system using E. coli as an expression host. Protein synthesis in both eukaryotic and prokaryotic cells is an mRNA encoding the amino acid methionine.
In particular, when the expression host is Escherichia coli, the protein required for protein expression is further added with methionine at the amino terminus (N-Me).
It cannot be predicted whether it is a mixture of molecular species with (t analog).

In fact, in Escherichia coli initiation factor IF-3, both a molecular species having a methionine residue at the amino terminus and a molecular species having no methionine residue are present [Hoppe, Zyrahs, Zeishlift, Fuer, Physiolyche].・ Hemi (Hoppe Seyler's Z.Physiol.Chem.), 354
Vol. 1415 (1973)] and E. coli,
The amino terminus of the whole protein is mainly methionine (Corn Stamp, Outlines of Biochemistry 4th Edition, John.
Willie and Sons, 1976) are known. For proteins produced using recombinant DNA technology, the rate of addition of methionine residues to the amino terminus is approximately 100% in human growth hormone [Nature, 2
93, p. 408 (1981)] is known. According to genetic engineering technology, it is known that the Met analogue is produced at a higher ratio than the required protein. Nature, Volume 293, p. 408
(1981) show that in the production of human growth hormone, its Met analogue is obtained in a large amount as compared with the protein actually required. When the required protein and its N-Met analogue are produced as a mixture, the difference in the physicochemical properties of the two molecular species is
It is extremely difficult to separate from each other, because they are very small, if any.

The methionine residue is an electrically neutral amino acid residue having a molecular weight of about 131, being moderately hydrophobic and having no dissociative group. Therefore, a macromolecule having a large number of dissociative groups, hydrophobic groups and hydrophilic groups, such as proteins, [eg interleukin-2 polypeptide (I; where X is a hydrogen atom) consisting of 133 amino acid residues] The molecular weight is about 15,420] It is expected that the addition of one methionine residue at the amino terminus will not usually have a large effect on the physicochemical properties of the whole protein. It is considered extremely difficult to separate the added molecular species and the non-added molecular species from each other. This difficult problem exists in many proteins, and in particular, proteins expressed using Escherichia coli as a host for expression, especially interleukin-2 and interferon produced by genetic engineering techniques, are
-There are some strict requirements when separating from Met analogues. Interleukin-2 is one of lymphokines produced by T cells activated by mitogens and antigens, and is an essential factor for proliferation and differentiation of cytotoxic T cells and natural killer cells, and these cells Plays an important role in the immune reaction system mediated by. In addition, interferon-α is one of lymphokines produced by leukocytes activated by viruses and nucleic acids, and has a biological activity of acting on cells to put them in an antiviral state, and thus prevents infection control systems and It plays an important role in the tumor immune system. Due to its biological activity, interleukin-2 and interferon-α are expected to be effectively used as therapeutic agents for various immunodeficiency diseases, infectious diseases, malignant tumors and the like. To date, natural interleukin-2 isolated from culture supernatants of human peripheral blood lymphocytes and human T-cell leukemia (JURKAT strain)
Are composed of several molecular species having different molecular weights, but they are very similar to each other with respect to their polypeptide chains, and it is known that the amino terminus always begins with an alanine residue [Japanese Patent Application No. 59-59].ー 149248
(Application July 19, 1984) Specification (EPC Publication No. 01
32359); Proc. Of National Academy of Science (Pro. Natl.
Acad. Sci. USA), 81, 2543 (1984)
Year)〕.

On the other hand, natural interferon-α isolated from the culture supernatant of human leukocytes to date consists of more than 10 subtypes, and their polypeptide chains are very similar to each other. It is known that the amino terminus always begins with a cystine residue [Arch. Biochem. Biophys.], 221, page 1.
(1983)]. The present inventors succeeded in producing non-glycosylated human interleukin-2 by expressing the interleukin-2 gene of human lymphocytes in Escherichia coli using recombinant DNA technology [Japanese Patent Application No. 58-58].
-225079 (filed on November 28, 1983); see specification; corresponding to EPC Publication No. 0145390]. The interleukin-2 contains a polypeptide (I) consisting of the amino acid sequence shown in FIG. 1 (wherein X represents a hydrogen atom or a methionine residue),
As with the natural human amino acid, the molecular species starting from an alanine residue (that is, X is a hydrogen atom) as the amino terminal amino acid, and the molecular species starting from a methionyl-alanine residue with a methionine residue added to the amino terminal (that is, X is methionine). Residues). Also, as already reported [Journal of Interferon Research
(J. Interferon Res.), Volume 1, p. 381 (1981)
Year); Journal of Biological Chemistry
(J. Biol. Chem.), 256, p. 9750 (198).
1 year)], for example, interferon-αA expressed in E. coli using recombinant DNA technology contains a polypeptide having the amino acid sequence shown in FIG. As the amino terminal amino acid, a molecular species starting from a cystine residue (that is, X is a hydrogen atom) as well as a human natural type, and a molecular species starting from a methionyl-cystine residue having a methionine residue added to the amino terminal (that is, X Has a methionine residue).

[0007]

Even if the same protein is used, there is a possibility that the higher order structure of the protein may be different between the molecular species having a methionine residue added to the amino terminus and the molecular species having no methionine residue. There may be differences in biological activity and biological stability in vivo and in vitro. It could also be possible that addition of a methionine residue to the amino terminus results in increased or decreased antigenicity. Therefore, from the viewpoints of physiological and industrial use, it is extremely significant to separate the molecular species with a methionine residue added to the amino terminus and the molecular species with no methionine residue, and to take out both in substantially pure form. Is. The rate of methionine residue addition to the amino terminus may depend on culture conditions and protein expression level [J. Interferon Res., Vol. 1, 38.
1 (1981)], but no example has been reported so far that could control the addition rate of methionine residues. Furthermore, no examples have so far been reported in which, in the process of purifying a protein, a molecular species having a methionine residue added to the amino terminus and a molecular species having no methionine residue are separated from each other. The present inventors have defined (a) interleukin-2, interleukin-2 having a methionine residue at its amino terminus and (b) interferon-αA, and interferon-αA having a methionine residue at its amino terminus. As the mutual separation method, a method utilizing a difference in solubility such as salting out and a solvent precipitation method, a dialysis method, an ultrafiltration method, a gel filtration method and an SDS-polyacrylamide gel electrophoresis method are mainly used, and a difference in molecular weight is mainly used. The method, the method utilizing the specific affinity with the antibody, the method utilizing the difference in hydrophobicity such as the reversed phase high performance liquid chromatography, etc. were tried, but they could not be separated from each other at all.

[0008]

[Means for Solving the Problems] The present inventors have proposed a protein and a protein having a methionine residue at the amino terminus thereof.
As a result of intensive research aimed at separating (N-Met analogue) from each other, the fact that they have different isoelectric points from each other was unexpectedly discovered. Since methionine is an electrically neutral amino acid, addition of methionine to the amino terminus of a protein is thought to have no effect on the overall charge of the protein. Therefore, a protein such as interleukin-2 and its amino terminus are considered to have no effect. It was a totally unexpected finding that the protein having a different methionine residue has a different isoelectric point. Based on this finding, a protein and a methionine residue were added to the protein, and a mixture of proteins having the same physiological activity as the protein (Met-protein) was subjected to separation means based on the difference in isoelectric point to obtain a protein. It has been found that a methionine-added protein (Met-protein) can be mutually separated from the protein.

That is, the present invention is characterized in that a protein and a methionine residue are added to the protein, and a mixture of proteins having the same physiological activity as the protein is subjected to separation means based on the difference in isoelectric point. It is intended to provide a method for mutually separating a protein and a protein in which a methionine residue is added to the protein.

In the present specification, the term "protein" includes both a protein having a sugar chain and a protein having no sugar chain, and a polypeptide chemically or structurally modified by, for example, a chemical reaction or an enzymatic reaction. It means a polymeric substance consisting of the primary structure of amino acids. The "Met-protein" means the above "protein" to which a methionine group is further added. The invention concerns, inter alia, the desired proteins and Met-proteins having the same or similar physiological activity. The preferred Met-protein in the present invention has sufficient amino acid sequence similarity to the desired protein to exert the physiological activity of the desired protein. The present invention provides a protein (N-) in which a methionine residue is further added to the amino terminus of the desired protein.
Met analogs) and the products of the method for separating proteins from Met-proteins. Above all,
The present invention provides for the isolation of interleukin-2 protein which is substantially free of its N-Met analog, and its N-Met.
It relates to the separation of interferon-α substantially free of analogues.

As used herein, "bioactive" refers to cells that may or may not be viable in vivo or in vitro, including physiological and immunological activities and effects,
By activity or effect on living and / or physiological substances, including parts of cells, cells or other physiologically based products and biological substances. The above-mentioned protein and a protein having a methionine residue added thereto (Met-protein)
The mixture of can be usually produced by gene recombination technology, and can be usually produced by expressing it using Escherichia coli, Bacillus subtilis, yeast and animal cells. Examples of the protein include various physiologically active proteins, for example, interferon (IFN;
(Eg, IFN-α, IFN-β, IFN-γ, etc.), interleukin (eg, interleukin-1, interleukin-2, etc.), B cell growth factor (BGF), B cell differentiation factor (B
DF), macrophage activating factor (MAF), lymphotoxin (LT), tumor necrosis factor (TNF) and other cytokines;
Transforming growth factor (TGF-α);
Erythropoietin, epidermal growth factor, insulin, peptide protein hormone such as human growth hormone; hepatitis B virus antigen, influenza antigen, foot-and-mouth disease virus antigen,
Pathogenic microbial antigen proteins such as malaria parasite antigens; peptidases (eg, tissue plasminogen activator, urokinase, seratiopeptidase) and enzymes such as lysozyme; blood serum protein components such as human serum albumin (HSA) . Among these proteins, the molecular weight is about 3,000 to 50,000, especially about 5,000.
30,000, and the number of amino acids is about 30-5
00, especially about 50 to 300, the method for mutually separating proteins of the present invention can be advantageously applied.

[0012] Also, when the isoelectric point of the protein is about 4 to 11, especially about 5 to 8, mutual separation can be advantageously carried out, and the isoelectricity between the protein and the protein having a methionine residue in the protein can be obtained. It is preferable that the difference between the points is about 0.01 or more, preferably about 0.1 or more, especially about 0.01 to 0.2. In particular, the interseparation method of the present invention can be advantageously applied to interleukin-2 and interferon-α produced by gene recombination technology. Interleukin-2 is the same biological or immunological activity as natural human interleukin-2, such as interleukin-2 receptor and anti-interleukin-2.
Any antibody having the ability to bind to an antibody may be used. Specifically, a polypeptide having the amino acid sequence shown in FIG. 1 (I; where X is a hydrogen atom), its biological or immunological properties May be a fragment consisting of a partial amino acid sequence necessary for biological activity, for example, polypeptide (I)
1 amino acid from the amino terminus of
No. 9) or a fragment lacking 4 amino acids (Japanese Patent Application No. 58-235638, filed on Dec. 13, 1983, see the specification, corresponding to Japanese Patent Application Laid-Open No. 60-126088) and a carboxyl terminal portion. Examples include fragments lacking several amino acids, and the above-mentioned polypeptide.
Those in which a part of the constituent amino acids of (I) has been deleted or substituted with other amino acids, for example, those in which the cysteine residue at position 125 has been replaced with a serine residue (JP-A-59-9309).
No. 3 publication). These polypeptides are preferably non-glycosylated polypeptides, with IL-2 having the amino acid sequence shown in FIG. 1 being particularly preferred. In the following specification of the present application, these interleukins-2 may be abbreviated as IL-2, and the interleukins-2 having a methionine residue at their amino terminals may be abbreviated as Met-IL-2.

The term interferon-α may be any as long as it has the same biological or immunological activity as that of natural human interferon-α, for example, the ability to bind to interferon α receptor or anti-interferon α antibody. For example, a polypeptide (II; where X is a hydrogen atom) having the amino acid sequence shown in FIG. 2 can be mentioned. Further, it may be a fragment consisting of a partial amino acid sequence required for the biological or immunological activity of interferon-α, for example, a fragment lacking a few amino acids of the amino terminal portion of polypeptide (II) or the number of the carboxyl terminal portion. Examples thereof include fragments lacking individual amino acids, and may be those in which a part of the constituent amino acids of the above polypeptide (II) is deleted or substituted with other amino acids. Interferon-αA is particularly preferable. Preferably these polypeptides are non-glycosylated polypeptides. In the following specification of the present application, these interferon-αA are referred to as IFN-αA.
And IFN-αA having a methionine residue at the amino terminus of IFN-αA may be abbreviated as Met-IFN-αA. As a mixture of the above protein and a protein further having a methionine residue at the amino terminus thereof, a mixed protein having a purity of 50% or more, preferably 80% or more, and particularly 99% or more is used.

In the present invention, the above mixture can be separated from each other by subjecting it to a separating means based on the difference in isoelectric point. According to the method described in Example 1, the isoelectric points of IL-2 and Met-IL-2 were calculated to be 7.7 and 7.5, respectively. Further, according to the method described in Example 6, IFN-αA and Met-IFN-αA.
The isoelectric points of were calculated to be 6.2 and 6.3, respectively.
As the separation means based on the difference of isoelectric points in the present invention, any method can be applied as long as it is a method of separating proteins having isoelectric points of about 0.01 to 0.2, for example, ampholine. Density gradient isoelectric focusing method, gel isoelectric focusing method, isophoretic electrophoresis method, etc., in which proteins are migrated in an electric field, chromatofocusing method, FPLC method (Fast Protein Liquid Chromatograph
y), DEAE (diethylaminoethyl)-, CM (carboxymethy
l)-or SP (sulfopropyl) -ion exchange column chromatographic method, etc., a protein added to a charge carrier such as an elution column is subjected to a pH gradient or a salt concentration gradient to generate a difference in isoelectric point. Examples thereof include methods known per se such as a method of sequentially desorbing and eluting proteins from the carrier according to the difference, a method of combining these methods, and the like. The reagents and instruments used in these separation methods are all commercially available and can be easily obtained. For example, ampholine is from LKB (Sweden), gel used for the gel isoelectric focusing method is Sephadex IEF from Pharmacia, and PAG (polyacrylamide gel) plate from LKB. The elution buffer is a polybuffer exchanger PBE94,
FPLC from Pharmacia (Sweden) as PBE 118, Polybuffer 74 and Polybuffer 96.
For example, Mono P column or Mono Q column and elution buffer used in the method are from Pharmacia, and DEAE-ion exchanger is DEAE-Toyopearl as Toyo Soda Kogyo Co., Ltd.
CM-Ion Exchanger as CM-Toyopearl from Toyo Soda Kogyo Co., Ltd., SP Ion Exchanger as SP-5PW
Available from Toyo Soda Kogyo Co., Ltd. or SP-Sephadex from Pharmacia. [Methods in Enzymology, No. 5
Volume, 3-27 (1962)].

Commercially available PAG plates (245 × 110
X1 mm, LKB) gel isoelectric focusing separation method using a PAG plate for pH 3.5.-9.5 plate, pH
5.5-8.5 plate, 1M phosphoric acid as anolyte,
A 0.4M HEPES buffer or the like is used, and 1M sodium hydroxide, 0.1M sodium hydroxide or the like is used as a catholyte.
Usually, 10 to 1000 μg of protein is put on one plate, and the power is 1 to 200 W, preferably 10 to 50 W
The temperature is 0 to 20 ° C, preferably 2 to 5 ° C. The migration time is 0.5 to 50 hours, usually 1.5 to 5 hours. In addition, commercially available mono P column (0.5 × 20 c
m, manufactured by Pharmacia) as an equilibration buffer, for example, 0.025M diethanolamine-hydrochloric acid buffer (pH 9.5), 0.075M Tris-acetate buffer (pH 9.3).
As an elution buffer, 1% (v / v) pharmalite (8-10.5) -5.2% (v / v) polybuffer 96-hydrochloric acid buffer (pH 7.0 to 8.0), 10% ( v / v) Poly buffer 9
A 6-hydrochloric acid buffer solution (pH 6.0 to 7.0) and a 10% (v / v) polybuffer 96-acetic acid buffer solution (pH 6.0 to 7.0) are used. Usually, 0.1 to 10 mg of protein is put on, 1 to 50
FPLC at a flow rate of ml / h, preferably 10-30 ml / h
I do.

In the chromatofocusing method, commercially available PBE118 and PBE94 (manufactured by Pharmacia) as gels
, Etc. as the equilibration buffer, 0.025M triethylamine-hydrochloric acid buffer (pH 11.0), 0.025M diethanolamine-hydrochloric acid buffer (pH 9.4) and 0.025M diethanolamine-acetic acid buffer (pH 9.4), As elution buffer 2.
2% (v / v) pharmalite (8-10.5) -hydrochloric acid buffer (pH)
7.0 to 8.0), 10% (v / v) polybuffer 96-hydrochloric acid buffer (pH 7.0 to 8.0) and 10% (v / v) polybuffer 96-acetic acid buffer (pH 6.0 to 7.0) To use. The bed volume of the column is 0.01 to 0.1, preferably 100 to 1000 ml per 1 g of protein, and the flow rate is SV =
Chromatofocusing is performed at 0.01-10, preferably SV = 0.1-1.0. The column temperature should be maintained at 0 to 30 ° C, preferably 2 to 5 ° C. According to the separation method of the present invention,
Proteins and proteins with methionine residues added (Met
-Proteins) are separated from each other according to the difference in their isoelectric points by migrating in an electric field or by sequentially desorbing and eluting from the carrier in a column having a pH gradient. Especially p
Separation can also be performed by applying an isocratic elution method using an ion exchanger without providing an H gradient or a salt concentration gradient. If desired, a substantially pure protein and a protein (Met-protein) further having a methionine residue added thereto can be obtained from a fraction containing only the protein separated according to the above method and a fraction containing a protein further having a methionine residue added thereto. Each can be collected. For this purpose, appropriate methods commonly used for protein purification such as salt folding method, hydrophobic chromatography method, gel filtration method, ion exchange chromatography method and high performance liquid chromatography method, which are known per se, are appropriately combined. You can use it.

The present invention makes it possible for the first time to separate a protein produced by genetic engineering technology so as to be substantially free of the corresponding Met-protein such as its N-Met analogue. The protein produced according to the present invention contains the corresponding Met-protein in an amount of 3% by weight or less, preferably 2%.
% Or less, especially 1 or less. Similarly, the present invention makes it possible for the first time to isolate a Met-protein produced by genetic engineering technology so as to be substantially free of its corresponding protein. The Met-protein produced according to the present invention contains the corresponding protein in an amount of 3% or less by weight, preferably 2% or less, particularly 1% or less by weight.

So far, Met-IL-2 and Met-IF
There is no report that N-αA was isolated as a protein, and the present invention for the first time also provides highly purified Met-IL-2 protein and Met-IFN-αA protein. The protein produced by the present invention and the protein having a methionine residue added thereto (Met-protein) have the same biological or immunological activity as the corresponding natural protein and are purified to high purity. Since it contains very little contaminating proteins and pyrogens, it can be safely used as an injectable drug substance. IL-2 and Met Obtained by the Present Invention
-IL-2 has the activity of proliferating normal T cells and natural killer cells while retaining their functions. Therefore, the IL-2 and Met-IL-2 obtained by the present invention can be used to proliferate, passage or clone T cells and natural killer cells in vitro for a long period of time, respectively. Note that this property can be used to measure the activity of human IL-2.

Furthermore, the IL-2 and Met-IL-2 obtained by the present invention, for example, recognize tumor-specific killer T cells that recognize and destroy tumor antigens and tumors regardless of whether or not they have undergone antigen sensitization. Natural killer cells, which have the ability to kill, can be selectively proliferated in vitro, and the IL-2 or Met-IL-2 obtained by the present invention can be simultaneously produced when these killer T cells are transferred into a living body. By inoculating it, its antitumor effect is increased, so that warm-blooded animals (eg, mouse, rat, rabbit, dog, cat,
It can be used for the prevention and treatment of tumors of pigs, horses, sheep, cattle, humans, etc. and for the treatment of immunocompromised diseases. IL-2 and Met-IL-2 obtained by the present invention have low toxicity without any antigenicity due to contaminant proteins. In order to use IL-2 or Met-IL-2 obtained by the present invention as a prophylactic or therapeutic agent for tumors, the substance is mixed and diluted with a carrier known per se, for example, parenterally as an injection or capsule. Or orally. In addition, it can be used with or without killer T cells or natural killer cells grown in vitro as described above. IL-2 and Met of the present invention
-IL-2 is a known human IL-2 isolated from nature
Since it has substantially the same biological activity as above, it can be used in the same manner as above, and since the dissociation constant of cells with the IL-2 receptor is extremely small, administration of an extremely small amount is sufficient. For use for the purpose of expanding T cells in vitro,
The IL-2 or Met-IL-2 of the present invention is about 0.01-.
1 unit / ml, preferably about 0.1-0.5 unit /
It can be used by adding it to the medium at a concentration of ml. The bioactivity of interleukin-2 can be measured by a method using interleukin-2 dependent cells [Biochemical and
Bio-Physical Research Communities (Bio
Chem. Biophys. Res. Commun.), 109, 363.
(1982)].

Specific examples of use for the purpose of proliferating T cells in vitro include, for example, T cells (1 × 10 ⁶ cells / ml) separated from human peripheral blood in RPMI 1640 medium containing 20% fetal bovine serum, and Alloantigen-sensitized T obtained by adding X-ray (1500 rads) -irradiated B cell transformants (1 × 10 ⁶ cells / ml) and performing mixed culture of lymphocytes at 37 ° C. in the presence of 5% CO ₂ for 3 days. IL-2 or Met-IL-of the present invention is added to a cell suspension containing cells.
2 at a concentration of 0.1 to 0.5 unit / ml, and culturing is continued for about 1 month while changing the medium about once a week. Both IFN-αA and Met-IFN-αA obtained by the present invention have an activity of acting on cells to put them in an antiviral state. Note that the activity of human IFN-αA can be measured by utilizing this property. Further, IFN-αA and Met-IFN-αA obtained by the present invention have not only antiviral action but also cell proliferation inhibitory action, antibody production inhibitory action, natural killer activity enhancing action and the like. The IFN-αA and Met-IFN-αA obtained by the present invention have low toxicity without any antigenicity due to contaminant proteins.
IFN-αA or Met-IFN obtained by the present invention
When -αA is used as a therapeutic agent, the substance can be mixed and diluted with a carrier known per se and administered parenterally or orally, for example, as an injection or capsule. The dose is 1 × 10 ⁶ to 1 × 10 ⁸ units, preferably 5 × 10 ^{7 to} 6 × 10 ⁷ units per day.

In the present specification, claims and drawings, when amino acids are represented by abbreviations, IUPAC-IU
It is based on the abbreviations by B Commission on Biochemical Nomenclature or abbreviations commonly used in this field, and examples thereof are given below. In addition, when an amino acid can have an optical isomer, the L-isomer is indicated unless otherwise specified. Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine 1 / 2Cys: Half cystine mCys: Carboxymethyl cystine Met: Methionine Gly: Glutamic acid Asp: Aspartic acid: Aspartic acid Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine Asp / Asn: Aspartic acid / Asparagine Glu / Gln: Glutamic acid / Glutamine

[0022]

The present invention will be described in more detail with reference to the following examples and reference examples, but the present invention is not limited thereto. The transformant Escherichia coli DH1 / pTF4 disclosed in Reference Example was designated as IFO-14299 by the Fermentation Research Institute (IFO), and from April 6, 1984, the Ministry of International Trade and Industry, Industrial Technology Institute. FERM P-757 to Institute of Microbial Technology (FRI)
8 was deposited, and the deposit was converted into a deposit under the Budapest Treaty, and is stored in the institute with the deposit number FERM BP-628. In addition, the transformant Escherichia coli N4830 / pTB285 was transferred to IFO.
Deposited as FO-14437 and as FERMP-8199 at FRI from April 30, 1985, and the deposit has been converted into a deposit under the Budapest Treaty and is kept at the institute with the deposit number FERM BP-852. .

Example 1 Separation of IL-2 and Met-IL-2 by FPLC: Non-glycosylated human interleukin which is a mixture of IL-2 and Met-IL-2 obtained in Reference Example 1 (iv). Xin-
0.005M ammonium acetate buffer containing 2 (pH 5.
0) (protein concentration, 1.18 mg / ml) 5 ml (5.9 mg) to 0.0
Mono P column for FPLC (0.5 × 20 cm, equilibrated with 25 M diethanolamine-hydrochloric acid buffer (pH 9.4))
(Pharmacia) and then 1% (v / v) Pharmalite (8-10.5) -5.2% (v / v) Polybuffer 9
The protein adsorbed on the Mono P column was eluted with 6-hydrochloric acid buffer (pH 8.0). The FPLC was performed at room temperature at a flow rate of 30 ml / h. As a result, as shown in FIG. 2, it was separated into a peak 1 eluted at pH 8.0 and a peak 2 eluted at pH 7.9. Therefore, in order to remove the polybuffer used in FPLC after collecting these,
High performance liquid chromatography was performed using a trifluoroacetic acid-acetonitrile system as an elution solvent. Column, Ultrapore RPSC (1.0 × 25 cm, Artex, USA); Column temperature, 30 ° C .; Elution solvent A, 0.1% trifluoroacetic acid-99.9% water; Elution solvent B, 0.1 % Trifluoroacetic acid-99.9% acetonitrile; elution program, 0 min (55% A + 45% B) -4 min (55% A + 4
5% B) -28 minutes (42% A + 58% B) -38 minutes (34%
A + 66% B) -43 minutes (20% A + 80% B) -44 minutes
(55% A + 45% B); elution rate 3.0 ml / min. The solutions obtained by this chromatography were each freeze-dried to obtain a white powder. Those obtained from peak 1 and peak 2 in FPLC were designated as P1 and P2, respectively. The yield of P1 is 1.12 mg (19.0 mg).
%), And the yield of P2 was 3.01 mg (51.0%). Next, the obtained P1 and P2 were subjected to protein chemical analysis. The amino terminal amino acid sequences of P1 and P2 were determined by an automated Endman degradation method using 45 μg (3 nmol) of each of P1 and P2 using a gas phase protein sequencer (Model 470A manufactured by Applied Biosystems). Phenylthiohydantoin amino acid (PTH
-Amino acid) was identified by high performance liquid chromatography using Micropack SP-C18 column (manufactured by Varian). The PTH-amino acids detected in each step are shown in [Table 1].

[0024]

[Table 1]

The analysis of the carboxyl terminal amino acid was performed as follows. That is, P1 and P2 were placed in a glass tube for hydrazine decomposition, anhydrous hydrazine was added, the tube was sealed under reduced pressure, and then heated at 100 ° C. for 6 hours. The resulting hydrazine decomposition product was treated with benzaldehyde, and free amino acids were measured with a Hitachi model 835 amino acid analyzer. As a result, only threonine was detected in both P1 and P2, and the recoveries were 34.8% and 34.0%, respectively.
Met. From this, the carboxyl terminal amino acids of P1 and P2 were identified as threonine. The amino acid composition analysis was carried out by adding a constant boiling hydrochloric acid containing 4% thioglycolic acid, sealing the tube under reduced pressure, hydrolyzing it at 110 ° C. for 24, 48 and 72 hours, and using a Hitachi model 835 amino acid analyzer. After cystine and cysteine are oxidized with formic acid,
It was hydrolyzed in constant boiling hydrochloric acid under reduced pressure for 24 hours and quantified as cysteic acid by an amino acid analyzer. The amino acid analysis value was obtained by averaging the values obtained by hydrolysis for 24, 48 and 72 hours. However, the values of serine and threonine were obtained by extrapolating the hydrolysis time to 0 hours. The results are shown in [Table 2]. From the results of amino terminal amino acid sequence analysis and amino acid composition analysis, P1 is a molecular species (IL-2) represented by X = hydrogen atom in [FIG. 1], and P2 is [FIG. 1].
X = molecular species represented by methionine residue (Met-IL-
2) was confirmed to be contained in a purity of 98% and 99% or higher, respectively.

[0026]

[Table 2]

Next, the ampholine PAG plate (LK
The results of measuring the isoelectric points of P1 and P2 by using (manufactured by Company B) are shown in FIG. Non-glycosylated human interleukin-2, which is a mixture of IL-2 and MetIL-2 used as a raw material, shows two bands by isoelectric focusing, whereas P1 and P2 obtained in this example were obtained. Were electrophoresed as one band with different migration distances from each other. Also, by measuring the pH of the PAG plate fragment after electrophoresis, the isoelectric point of P1 (IL-2) was 7.7,
The isoelectric point of P2 (MetIL-2) was calculated to be 7.5. Further, the obtained P2 was digested with trypsin as follows to obtain a peptide map. That is, 50μ
0.02M sodium hydrogen carbonate solution containing g of P2 (pH
8.0) 100 μl, 1.25 μg of TPCK-trypsin (Washington, USA) was added and reacted at 37 ° C. for 28 hours. 1% (v / v) trifluoroacetic acid in the reaction solution 4
The reaction was stopped by adding 00 μl. The obtained digested liquid was subjected to high performance liquid chromatography under the following conditions to obtain the map shown in FIG. High Performance Liquid Chromatograph: Varian (USA) 504
Type 0 column: Nucleosyl 5C18 (Machelena Gel, West Germany) Column temperature: 30 ° C Elution solvent: A liquid, 0.1% trifluoroacetic acid-99.9% water (v / v) B liquid, 0.1% tri Fluoroacetic acid-99.9% acetonitrile (v / v) Elution program: 0 min (85% A + 15% B) -15 min
(72% A + 28% B) -16 minutes (64% A + 36% B)-
80 minutes (40% A + 60% B) -85 minutes (15% A + 85)
% B) [Linear gradient] Elution rate: 3.0 ml / min Detection method: Orthophthalaldehyde method [Anal., Chem.,
Vol. 43, p. 880 (1971)]

Example 2 IL-2 and Met-IL-2 by chromatofocusing
Separation with: IL-2 and Met obtained in Reference Example 1 (iv)
-500 ml of 0.005 M ammonium acetate buffer (pH 5.0) (protein concentration, 1.09 mg / ml) containing non-glycosylated human interleukin-2, which is a mixture of IL-2.
45 mg) to 0.025 M diethanolamine-hydrochloric acid buffer solution
It was placed on a PBE94 (Pharmacia) column (2.7 x 87 cm) equilibrated with (pH 9.4), and then 1% (v / v) pharmalite (8-10.5) -5.2 was used as an eluent. % (V /
v) Chromatofocusing was performed using polybuffer 96-hydrochloric acid buffer (pH 8.0). This operation is 4
It was carried out at a flow rate of 200 ml / h at ℃. As a result, as shown in FIG. 5, peak 1 eluted at pH 8.5 and pH 8.3 were eluted.
It was separated into peak 2 which was eluted at. The protein yield after removal of polybaar by the same method as described in Example 1 was 94.8 mg (17.4%) in peak 1 and 3 in peak 2.
It was 36 mg (61.6%). The amino terminal amino acid analysis was carried out by a method of hydrolyzing with hydrochloric acid after the dansylation reaction and detecting the resulting dansyl amino acid by high performance liquid chromatography using a Micropack SP column.
Peak 1 is IL-2 with a purity of 99.6% or higher.
Was confirmed to contain Met-IL-2 in a purity of 99.5% or more.

Example 3 Separation of IL-2 and Met-IL-2 by DEAE-Toyopearl Ion Exchange Chromatography: Reference Example 1 (iv)
Containing non-glycosylated human interleukin-2, which is a mixture of IL-2 and Met-IL-2 obtained in 1.
005M ammonium acetate buffer (pH 5.0) (protein concentration: 1.03 mg / ml) 10 ml (10.3 mg) equivalent to 10 mM
After adjusting the pH to 8.5 by adding Tris-hydrochloric acid buffer solution (pH 9.0), 10 mM Tris-hydrochloric acid buffer solution (pH 8.5)
DEAE-Toyopearl 650M (manufactured by Toyo Soda Kogyo Co., Ltd.) column (1.0 × 64 cm) equilibrated with 10 mM Tris-HCl buffer (pH 8.5) (1 L) and 10 mM Tris-HCl buffer (pH 7. 0) Elution was performed by the pH gradient method using 1 L. This operation was performed at 4 ° C. and a flow rate of 100 ml / h. As a result, the resolution was inferior to that of FPLC and chromatofocusing, but two peaks (peak 1 and peak 2) were detected. The first half of the peak 1 and the second half of the peak 2 were fractionated so that the respective peaks did not overlap. The yield was 0.82 mg (8.0%) for peak 1 and 1.98 mg (19.2%) for peak 2. As a result of amino terminal amino acid analysis by the dansyl method, peak 1 contained 90% or more of IL-2, and peak 2 contained 9 Met-IL-2.
It was confirmed that the content was 5% or more.

Example 4 Separation of IL-2 and Met-IL-2 by FPLC: IL-2 and Met-IL-2 obtained in Reference Example 1 (iii)
A non-glycosylated human interleukin-2 partially purified standard (5 ml) containing E. coli was equilibrated with 0.025 M diethanolamine-hydrochloric acid buffer solution (pH 9.4) to prepare a mono-P column for FPLC.
Put on (0.5 × 20 cm, Pharmacia), then 1
% (V / v) pharmalite (8 to 10.5) -5.2% (v / v)
The protein adsorbed on the Mono P column was eluted with Polybuffer 96-hydrochloric acid buffer (pH 8.0). Dissolution rate:
25 ml / h, column temperature: room temperature. As a result, it was separated into a fraction containing IL-2 (peak 1) and a fraction containing Met-IL-2 (peak 2). Peak 1 activity recovery is 25
%, The activity recovery rate of peak 2 was 54%.

Example 5 Separation of IL-2 and Met-IL-2 by SP-5PW column: IL-2 and Met-IL obtained in Reference Example 2.
-0.005 M ammonium acetate buffer containing non-glycosylated human interleukin-2 (pH
5.0, protein concentration 1.03mg / ml) 0.5ml to 0.025M
SP-5 PW column (0.75 x 7.5c) for high performance liquid chromatography equilibrated with phosphate buffer (pH 7.4)
m; Toyo Soda Co., Ltd.) 0.025M phosphate buffer (pH)
The protein was eluted using 7.4). Column temperature is 35 ℃
The flow rate of the buffer solution was set to 0.5 ml / min. The chromatographic system used was a Varian 5500 liquid chromatograph. As a result, as shown in FIG. 7, non-glycosylated interleukin-2 was eluted as two peaks (peak A and peak B). As a result of fractionating each peak (indicated by a bold solid line in the figure) and analyzing the amino terminal amino acid, peak A was Met-IL-2 and peak B was IL-2 with a purity of 99.5% or more. It was confirmed to be included in.

Example 6 Separation of IFN-αA and Met-IFN-αA by FPLC: IFN-obtained by the method described in Reference Example 3.
0.12M Sodium Chloride-0.025M Ammonium Acetate Buffer (pH) containing unglycosylated human interferon αA, which is a mixture of αA and Met-IFN-αA.
5.0) (protein concentration, 2.96 mg / ml) 1.0 ml (2.96 m
g) 0.025M imidazole-hydrochloric acid buffer solution (pH 6.7)
It was desalted by placing it on a PD-10 column (1.5 × 5 cm, manufactured by Pharmacia) equilibrated with. The eluate (protein concentration, 1.58 mg / ml) 1.5 ml (2.37 mg) containing the non-glycosylated human interferon αA thus obtained was added with 0.025 M imidazole-hydrochloric acid buffer (pH 6.7). Place on the equilibrated mono-P column for FPLC (0.5 × 20 cm, manufactured by Pharmacia), and then adsorb on the mono-P column using 10% (V / V) polybuffer 74-hydrochloric acid buffer (pH 5.5). The protein was eluted. The FPLC was performed at room temperature at a flow rate of 30 ml / h. As a result, as shown in FIG. 8, it was separated as a peak I eluted at pH 5.6 and a peak II eluted at pH 5.4. Therefore, after collecting these, high-performance liquid chromatography using a trifluoroacetic acid-acetonitrile system as an elution solvent was performed in order to remove the polybuffer used in FPLC. Column, Ultrapore RPSC (1.0 × 25 cm, manufactured by Altex); Column temperature, 30 ° C .; Elution solvent A, 0.1% trifluoroacetic acid-99.9% water; Elution solvent B, 0.1% Trifluoroacetic acid-99.9% acetonitrile; elution program, 0 min (60% A + 40% B) -45 min (45% A + 5
5% B) -46 min (60% A + 40% B); elution rate 3.0
ml / min. The solutions obtained by this chromatography were each freeze-dried to obtain a white powder. FPLC
Obtained from peak I and peak II in
They were named PI and PII, respectively. The PI yield is 0.
723 mg (24.4%), PII yield 0.945 mg (31.
9%).

Next, the obtained PI and PII were subjected to protein chemical analysis. After reducing carboxymethylated PI and PII, 40 μg (2.1 nmol) of each was used, and the amino terminal amino acid sequences of PI and PII were determined by an automated Edman degradation method using a gas phase protein sequencer (Applied Biosystems type 470A). It was determined. Phenylthiohydantoin amino acid (PTH-
Amino acids) were identified by high performance liquid chromatography using a Micropack SP-C18 column (manufactured by Varian). The PTH-amino acids detected in each step are shown in [Table 3].

[0034]

[Table 3]

The carboxyl terminal amino acid was analyzed in the same manner as in Example 1. As a result, only glutamate was detected in both PI and PII, and the recoveries were 12.5% and 14.3%, respectively. The results of amino acid composition analysis performed in the same manner as in Example 1 are shown in [Table 4]. From the results of amino terminal amino acid sequence analysis and amino acid composition analysis, P
I is a molecular species represented by X = methionine residue in [Fig. 2]
It was confirmed that (Met-IFN-αA) and PII each contain a molecular species (IFN-αA) represented by X = hydrogen atom in FIG. 2 in a purity of 98% or more.

[0036]

[Table 4]

The ampholine PAG plate (L
As a result of measuring the isoelectric points of PI and PII using a product manufactured by KB Co., the isoelectric point of PI (Met-IFN-αA) was 6.3 PI.
The isoelectric point of I (IFN-αA) was calculated to be 6.2.

Example 7 IL-2 injectable preparation: The IL-2 containing solution obtained in Example 1 was treated with 0.025M ammonium acetate buffer (pH 5.
Adsorbed under aseptic conditions on a column of CM Toyopearl (Toyo Soda Kogyo Co., Ltd.) equilibrated with 0) and eluting with the above buffer containing 0.15 M NaCl. 0.15 M NaCl in the eluate
Is added appropriately to dilute, and HSA is added to 0.5% and filtered using a membrane filter (pore size: 0.22 μm), and the obtained filtrate is aseptically dispensed in 1 ml a vials. Lyophilize to prepare IL-2 for injection. The preparation for injection is dissolved in 1 ml of distilled water for injection at the time of use.

Example 8 IFN-αA injectable preparation: IFN-obtained in Example 6
αA-containing solution was added to 0.025M ammonium acetate buffer
CM Toyopearl equilibrated with (pH 5.0) (Toyo Soda Kogyo)
Co., Ltd. column was adsorbed under aseptic conditions to obtain 0.15M Na.
Elute with the above buffer containing Cl. 0.15M in eluate
Add NaCl appropriately to dilute, add HSA to 0.5% and filter through a membrane filter (pore size 0.22 μm), then aseptically dispense 1 ml of the obtained filtrate into vials. And freeze-dried to prepare IFN-αA for injection. The preparation for injection is dissolved in 1 ml of distilled water for injection at the time of use. The injectable preparation contains purified IL-2 and IFN-αA, purified Met-IL-2 and Met-I, respectively.
By replacing with FN-αA, an injectable preparation of Met-IL-2 and Met-IFN-αA can be obtained.

Reference Example 1 Production of non-glycosylated human interleukin-2 I: (i) Cultivation of transformant: transformant E. coli DH1 / p
TF4 [Japanese Patent Application No. 58-225079 (11 November 1983)
(Filed on 28th March) Refer to the specification; the application is JP-A-60-11
No. 5528 gazette] in a 250 ml Erlenmeyer flask with 1% Bact Trypton (Difco Laboratories, USA), 0.5% Bact East Extract (Difco Laboratories, USA), 0.5% salt and tetracycline. Liquid medium (pH 7.0) containing 7 μg / ml 50
The cells were inoculated in ml and cultivated at 37 ° C. overnight with rotary shaking. This culture solution was transferred to a 5 L jar fermenter containing 2.5 L of M9 medium containing 0.5% casamino acid, 0.5% glucose and 7 μg / ml tetracycline, and incubated at 37 ° C. for 4 hours, followed by 3-β indolyl. Acrylic acid (25 μg / ml) was added, and the mixture was further cultivated with aeration and stirring for 4 hours to obtain 2.5 L of culture solution. The culture was centrifuged to collect the cells, frozen at -80 ° C and stored. (ii) Extraction: 12.1 g of the cryopreserved cells obtained above was extracted with 7M guanidine hydrochloride and 0.1M Tris · HCl (pH 7.
0) Evenly suspended in 100 ml and stirred at 4 ° C. for 1 hour.
This lysate was centrifuged at 28,000 xg for 20 minutes to obtain 93 ml of supernatant.

(Iii) Partial purification of interleukin-2 protein: The supernatant obtained above was added to 0.01 M Tris.HCl buffer.
After dialysis against (pH 8.5), centrifugation was performed at 19,000 × g for 10 minutes to obtain 94 ml of dialysis supernatant. The dialyzed supernatant was passed through a DE52 (DEAE-cellulose, Whatman, UK) column (50 ml volume) equilibrated with 0.01 M Tris-HCl buffer (pH 8.5) to adsorb protein, and then NaC was added.
A linear concentration gradient (0 to 0.15 M NaCl, 1 L) was prepared to elute IL-2, and 53 ml of an active fraction was obtained. (iv) Purification of interleukin-2 protein: 53 ml of the above active fraction was concentrated to 4.8 ml using a YM-5 membrane (Amicon, USA), and 0.1 M Tris.HCl (pH) was added.
8.0) -1 M NaCl buffered Sephacryl S equilibrated
-200 (Pharmacia, Sweden) column (50
Gel filtration was performed using 0 ml volume. 28 ml of the active fraction was concentrated to 2.5 ml with a YM-5 membrane. The obtained concentrated liquid was adsorbed on an Ultrapore RPSC (manufactured by Altex, USA) column and subjected to high performance liquid chromatography using a trifluoroacetic acid-acetonitrile system as an elution solvent. Column, Ultrapore RPSC (4.6 x 75
mm); column temperature, 30 ° C; elution solvent A, 0.1% trifluoroacetic acid-99.9% water; elution solvent B, 0.1% trifluoroacetic acid-99.9% acetonitrile; elution program, 0 minutes (68% A + 32% B) -25 minutes (55% A + 4
5% B) -35 minutes (45% A + 55% B) -45 minutes (30%
A + 70% B) -48 minutes (100% B); elution rate, 0.8
ml / min; detection wavelength, 230 nm. Under these conditions, the active fractions with a retention time of about 39 minutes were collected to give 0.53 mg of non-glycosylated human interleukin-2 protein [specific activity, 30,000 U].
/ Mg, activity recovery from starting material, 30.6%; protein purity, 99% (by densitometry)]. The specific activity of the freeze-dried product (white powder) of the above solution was 26,000 U / mg.

Reference Example 2 Production of non-glycosylated human interleukin-2 II: (i) Construction of expression plasmid: plasmid pILOT 135-8 containing human IL-2 gene [Japanese Patent Application No. 58-58]
No. 22579 (filed on Nov. 28, 1983) Specification (corresponding to JP-A-60-115528) Example 1 (vii)]
Was cleaved with the restriction enzyme HgiAI. The obtained 1294 bp DN
The A fragment was blunt-ended with T4 DNA polymerase and
EcoRI linker dTGCCATGAATT with DNA ligase
CATGGCA was attached. The obtained DNA was digested with EcoRI to obtain a DNA fragment having the translation disclosure codon ATG and the human IL-2 gene. This DNA fragment is
The EcoRI-PstI site was digested and inserted into ptrp781 [Nucleic Acids Research, Vol. 11, p. 3077 (1983)] using T4 DNA ligase. The thus obtained expression plasmid pTF1 has a translationally disclosed codon and the human IL-2 gene downstream of the trp promoter [FIG. 9]. The plasmid pTF1 was cut with the restriction enzyme StuI and ligated with the Bam HI linker. This plasmid DNA was treated with restriction enzymes BamHI and EcoRI and then inserted into plasmid pTB281 having a λPL promoter at the EcoRI-BamHI site. The expression plasmid thus obtained was designated as pTB285 [Fig. 1
0]. (ii) Production of transformant: plasmid pTB2 obtained above
Escherichia coli N4830 at 85 [Procedures of National Academy of Science (Pro. Natl. Acad. Sci. USA), No. 69]
Pp. 2110 (1972)], and a transformant containing the above plasmid, Escherichia coli N4830 /
pTB285 was obtained.

(Iii) Culture of transformant: transformant E. c
oli N4830 / pTB285 in a 250 ml flask containing Bacto tryptone (Difco Laboratories, USA) 1%, Bacto yeast extract (Difco Laboratories, USA) 0.5%, salt 0.5% and ampicillin 50 μg / ml. 50 ml of a liquid medium (pH 7.0) containing the same was inoculated and cultivated at 37 ° C. with rotary shaking overnight. This broth was treated with 0.5% casamino acid, 0.5% glucose and 50% ampicillin.
The mixture was transferred to a 5 L jar fermenter containing 2.5 L of M9 medium containing μg / ml, and cultured at 35 ° C. for 6 hours and then at 42 ° C. for 3 hours with aeration and stirring to obtain 2.5 L of culture medium. The culture was centrifuged to collect the cells, frozen at -80 ° C and stored. (iv) Extraction: 20 g of frozen bacterial cells was added with 7M guanidine hydrochloride 0.1M
Suspend uniformly in 100 ml of extraction (pH 7.0) containing Tris-HCl,
After stirring at 4 ° C for 1 hour, the mixture was centrifuged at 28,000 xg for 20 minutes to obtain a supernatant.

(V) Partial purification of interleukin-2 protein: The obtained supernatant was mixed with 0.01 M Tris-HCl buffer (pH 8.5).
Against DE52 (DEAE-cellulose, manufactured by Whatman, UK), the supernatant obtained by centrifugation at 19,000 × g for 10 minutes after dialysis was equilibrated with 0.01 M Tris-HCl buffer (pH 8.5). After adsorbing the protein through a column (50 ml volume),
Create a linear gradient of NaCl concentration (0 to 0.15M NaCl, 1L),
IL-2 was eluted to obtain an active fraction. (vi) Purification of interleukin-2 protein: The active fraction obtained above was concentrated to 5 ml using YM-5 membrane (Amicon, USA) and 0.1 M Tris-HCl (pH 8.0) was added.
-Sefacryl S-200 equilibrated with 1M NaCl buffer
Gel filtration was performed using a (Pharmacia, Sweden) column (500 ml volume). The active fraction (40 ml) was concentrated to 3 ml with a YM-5 membrane. The obtained concentrated liquid was adsorbed on an Ultrapore RPSC (Altex Co., USA) column and subjected to high performance liquid chromatography using a trifluoroacetic acid-acetonitrile system as an elution solvent. column,
Ultrapore RPSC (4.6 × 75mm); Column temperature, 30 ℃:
Elution solvent A, 0.1% trifluoroacetic acid-99.9% water; Elution solvent B, 0.1% trifluoroacetic acid-99.9% acetonitrile; Elution program, 0 minutes (68% A + 32% B) -25 minutes (55
% A + 45% B) -35 minutes (45% A + 55% B) -45 minutes (30% A + 7
0% B) -48 minutes (100% B); elution rate, 0.8 ml / min; detection wavelength, 230 nm. Under these conditions, the active fraction with a retention time of approximately 39 minutes, Ala-IL-2
And 10 ml of a mixture of Met-Ala-IL-2 was collected.

Reference Example 3 Production of non-glycosylated human IFN-αA: (i) Culture of transformant: Escherichia coli carrying an expression plasmid incorporating the human IFN-αA gene encoding the amino acid sequence shown in FIG. Coli 294 (ATCC 31
446) / pLeIFAtrp25 [EPC release 439
No. 80 gazette, see Example I] in M-9 medium with glucose 25 g / L, sodium L-glutamate 4 g / L, FeCl ₃ .6H
₂ O 27 mg / L, CuSO ₄ / 5H ₂ O 8 mg / L, ZnSO ₄ 7H ₂ O 8 mg / L,
Vitamin B ₁ hydrochloride 70 mg / L, tetracycline hydrochloride 5 m
g / L, L-proline 50 mg / L and L-leucine 50 mg /
Inoculate 5 L jar fermenters containing 2.5 L of each L-added medium (synthetic medium), aeration 2.5 L / min, stirring 1
Culture was started at 000 rpm and 37 ℃, and OD300
33 ℃ at 0 KU, 29 ℃ at 5,000 KU, 7,000 KU
The temperature was lowered to 25 ° C and the culture was continued for 48 hours. The dissolved enzyme concentration was maintained at 5% or more during the culture. When the glucose concentration in the culture medium dropped to 1% or less, glucose was added at a rate of 25 g / L. (ii) Extraction: 2 L of the culture solution obtained in (i) was centrifuged to collect the bacterial cells, and this was collected in 100 ml of 10% sucrose, 0.2.
M NaCl, 10 mM ethylenediamine tetraacetate (ED
TA), 10 mM spermidine, 2 mM phenylmethylsulfonylfluoride (PMSF), 0.2 mg / ml lysozyme in 50 mM Tris-HCl (pH 7.6) and suspended at 4 ° C for 1 hour, and then at 37 ° C for 5 minutes. It was kept warm, and this was further treated with an ultrasonic disintegrator (Altec, USA) at 0 ° C. for 40 seconds. This lysate was centrifuged at 11,300 × g for 1 hour to collect 95 ml of supernatant.

(Iii) Purification of IFN-αA protein: 95 ml of this supernatant was added to 20 mM containing 1 mM EDTA and 0.15 M NaCl.
After diluting to 300 ml with Tris-HCl (pH 7.6) (TEN),
It was applied to an anti-IF-αA antibody column (20 ml). After thorough washing with TEN, IF-αA was further eluted with 0.2 M acetic acid containing 0.1% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) to collect active fractions, and the pH was adjusted to 4.5. Adsorbed on cellulose column and washed thoroughly, then 0.15M NaCl
Elution was carried out with 0.025 M ammonium acetate buffer (pH 5.0) containing The active fractions are again collected and freeze-dried 320
mg human leukocyte IF-αA powder was obtained. SD of this thing
The molecular weight by S-polyacrylamide gel electrophoresis was 19000 ± 1000. The specific activity of the final human leukocyte IF protein obtained here was 2 × 10 ⁸ U.
It was / mg. Also, in other physicochemical properties, amino acid composition, and peptide mapping, it showed exactly the same behavior as the recombinant human leukocyte IF produced in the conventional medium.

[0047]

INDUSTRIAL APPLICABILITY According to the present invention, a protein and a protein having a methionine residue added thereto can be separated from each other. The proteins obtained by the separation method are useful as pharmaceuticals and the like.

[0048]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 133-134 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence characteristics: X represents a hydrogen atom or a methionine residue. Sequence X-Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 5 10
15 His Leu Leu Leu Asp Leu Gln Met Ile
Leu Asn Gly Ile Asn Asn Tyr 20 25
30 Lys Asn Pro Lys Leu Thr Arg Met Leu
Thr Phe Lys Phe Tyr Met Pro 35 40
45 Lys Lys Ala Thr Glu Leu Lys His Leu
Gln Cys Leu Glu Glu Glu Leu 50 55
60 Lys Pro Leu Glu Glu Val Leu Asn Leu
Ala Gln Ser Lys Asn Phe His 65 70
75 80 Leu Arg Pro Arg Asp Leu Ile Ser Asn
Ile Asn Val Ile Val Leu Glu 85
90 95 Leu Lys Gly Ser Glu Thr Thr Phe Met
Cys Glu Tyr Ala Asp Glu Thr 100 105
110 Ala Thr Ile Val Glu Phe Leu Asn Arg
Trp Ile Thr Phe Cys Gln Ser 115 120
125 Ile Ile Ser Thr Leu Thr 130

[0049]

[SEQ ID NO: 2] Sequence length: 165-166 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence characteristics: X represents a hydrogen atom or a methionine residue Sequence X-Cys Asp Leu Pro Gln Thr His Ser Le
u Gly Ser Arg Arg Thr Leu 5 10
15 Met Leu Leu Ala Gln Met Arg Lys Ile
Ser Leu Phe Ser Cys Leu Lys 20 25
30 Asp Arg His Asp Phe Gly Phe Pro Gln
Glu Glu Phe Gly Asn Gln Phe 35 40
45 Gln Lys Ala Glu Thr Ile Pro Val Leu
His Glu Met Ile Gln Gln Ile 50 55
60 Phe Asn Leu Phe Ser Thr Lys Asp Ser
Ser Ala Ala Trp Asp Glu Thr 65 70
75 80 Leu Leu Asp Lys Phe Tyr Thr Glu Leu
Tyr Gln Gln Leu Asn Asp Leu 85
90 95 Glu Ala Cys Val Ile Gln Gly Val Gly
Val Thr Glu Thr Pro Leu Met 100 105
110 Lys Glu Asp Ser Ile Leu Ala Val Arg
Lys Tyr Phe Gln Arg Ile Thr 115 120
125 Leu Tyr Leu Lys Glu Lys Lys Tyr Ser
Pro Cys Ala Trp Glu Val Val 130 135
140 Arg Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu 145 150
155 160 Ser Leu Arg Ser Lys Glu 165

[Brief description of the drawings]

1 shows the amino acid sequence of the non-glucosylated human interleukin-2 protein obtained in Reference Example 1 (X in the figure represents a hydrogen atom or a methionine residue).

FIG. 2 shows the amino acid sequence of the non-glycosylated human interferon-αA protein obtained in Reference Example 3 (where X represents a hydrogen atom or a methionine residue).

FIG. 3 shows the results of FPLC in Example 1.

FIG. 4 shows the results of isoelectric focusing in Example 1.

FIG. 5 shows the results of tryptic digest peptide mapping in Example 1.

FIG. 6 shows the results of chromatofocusing in Example 2.

FIG. 7 shows the results of SP-5PW ion exchange chromatography in Example 5.

FIG. 8 shows the result of FPLC in Example 6.

FIG. 9 shows a construction diagram of plasmid pTF1 in Reference Example 3.

FIG. 10 shows a construction diagram of pTB285 in Reference Example 3.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07K 14/52 ZNA C07K 14/52 ZNA 14/575 14/575 14/745 14/745 14/76 14/76

Claims (26)

[Claims] 1. A protein and a protein characterized in that a methionine residue is added to the protein and a mixture of proteins having the same physiological activity as the protein is subjected to separation means based on the difference in isoelectric point. A method for mutual separation from a protein having a methionine residue added thereto. 2. The isoelectric point in which the separation step comprises (i) a method of migrating a mixture of proteins in an electric field, or (ii) a mixture of proteins added to a charge carrier by creating a pH gradient or a salt concentration gradient. 2. The separation method according to claim 1, which is a method of sequentially desorbing and eluting from the carrier according to the difference in charge that causes the difference. 3. The separation method according to claim 2, wherein the method of migrating the protein mixture in an electric field is density gradient isoelectric focusing, gel isoelectric focusing or isokinetic electrophoresis. 4. A method of desorbing and eluting a mixture of proteins added to a charge carrier in order from the carrier according to the difference in charge, is a chromatofocusing method, a fast protein liquid chromatography method, a diethylaminoethyl method. The separation method according to claim 2, which is an ion exchange column chromatography method, a carboxymethyl-ion exchange column chromatography method, or a sulfopropyl-ion exchange column chromatography method. 5. The method according to claim 1, wherein the protein having a methionine residue added thereto is a protein having a methionine residue added at the amino terminus. 6. The method according to claim 5, wherein the added methionine residue is only at the amino terminus of the protein. 7. The method according to claim 1, wherein the protein and the protein in which a methionine residue is added to the protein are proteins produced by genetic engineering technology. 8. The method according to claim 1, wherein the protein is a cytokine, a peptide hormone, a pathogenic microbial antigen protein, an enzyme or a blood protein component. 9. A protein having a molecular weight of about 3,000 to 50,000.
The separation method according to claim 1, which is 0. 10. The separation method according to claim 9, which has a molecular weight of 5,000 to 30,000. 11. The method according to claim 1, wherein the protein is a protein consisting of 30 to 500 amino acids. 12. The method according to claim 11, which is a protein consisting of 50 to 300 amino acids. 13. The method according to claim 1, wherein the protein and the protein obtained by adding a methionine residue to the protein have an isoelectric point of about 4 to 11. 14. The method according to claim 1, wherein the protein and the protein obtained by adding a methionine residue to the protein have an isoelectric point of about 5-8. 15. The separation method according to claim 1, wherein the difference in isoelectric point between the protein and the protein obtained by adding a methionine residue to the protein is about 0.01 or more. 16. The separation method according to claim 1, wherein the difference in isoelectric point between the protein and the protein obtained by adding a methionine residue to the protein is about 0.1 or more. 17. The method according to claim 1, wherein the difference in isoelectric point between the protein and the protein obtained by adding a methionine residue to the protein is about 0.01 to 0.2. 18. The method according to claim 1, wherein the protein and the protein having a methionine residue in the protein are non-glycosylated proteins. 19. The separation method according to claim 1, wherein the protein is a cytokine. 20. The separation method according to claim 19, wherein the cytokine is interferon. 21. Interferon is interferon-
The separation method according to claim 20, which is α. 22. The separation method according to claim 21, wherein interferon-α is a non-glycosylated protein consisting of the amino acid sequence shown in FIG. 2 (where X represents a hydrogen atom). 23. The method according to claim 1, wherein the purity of the mixture of the protein and the protein obtained by adding a methionine residue to the protein is about 50% or more. 24. The separation method according to claim 1, wherein the purity of the mixture of the protein and the protein obtained by adding a methionine residue to the protein is about 99% or more. 25. Non-glycosylated interferon-αA
And a mixture of non-glycosylated interferon-αA having an additional methionine residue at its amino terminus were subjected to Fast Protein Liquid Chromatography to show the non-glycosylated interferon-αA and its amino acid. The separation method according to claim 1, wherein non-glycosylated interferon-αA further having a methionine residue at the end is separated from each other. 26. Non-glycosylated interferon-αA
26 is the non-glycosylated interferon-αA represented by FIG. 2 (where X represents a hydrogen atom).