JPH10271998A - Method for producing polypeptide - Google Patents

Abstract

(57) [Summary] [Problems] Interferon-in immunocompetent cells
An object of the present invention is to provide a method for producing an active polypeptide from a precursor of a polypeptide that induces γ production. Kind Code: A1 Abstract: Interferon-
The problem is solved by a method for producing an active polypeptide that induces the production of interferon-γ in immunocompetent cells, which comprises causing an interleukin-1β converting enzyme to act on a precursor of the polypeptide that induces γ production.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a physiologically active polypeptide, and more particularly, to a method for producing interferon-.gamma.
−γ ”. )).

[0002]

2. Description of the Related Art The present inventors have proposed that Immune cells
The isolation of a novel polypeptide that induces the production of FN-γ and the cloning of a cDNA encoding the polypeptide were successful and disclosed in JP-A-8-27189 and JP-A-8-193098. Since this polypeptide has both the property of inducing the production of IFN-γ, a useful bioactive protein, and the property of enhancing cytotoxicity by killer cells and inducing the production of killer cells, It is expected to be widely used as agents, antibacterial agents, antitumor agents, antiimmune disease agents and the like.

[0003] By the way, in human cells, it is said that a polypeptide produced by the expression of a gene is processed by an intracellular enzyme, and a part of the polypeptide is cleaved or a sugar chain is added. As a polypeptide to be incorporated into a drug, it is desirable that the polypeptide has undergone the same processing as in human cells, but as described in Japanese Patent Application No. 8-269105,
Human cells generally have a problem that the production amount of the polypeptide is small. The present inventors have conducted intensive studies on the cause, and in human cells, the polypeptide is
It turned out that it normally exists as a precursor having no biological activity and a molecular weight of about 24,000 daltons. Not only the polypeptide, but many cytokines are usually first produced as precursors having no biological activity,
Thereafter, it is known that it is processed by an intracellular enzyme to be converted into an active polypeptide.

[0004]

In view of such circumstances, an object of the present invention is to provide a method for producing an active polypeptide from a precursor of a polypeptide that induces the production of IFN-γ in immunocompetent cells. Is to do.

[0005]

Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and found that interleukin-1β
Converting enzyme (hereinafter, abbreviated as “ICE”) acts on the precursor of the polypeptide and causes IFN in immunocompetent cells.
It has been found to produce an active form of the polypeptide that induces the production of N-γ.

That is, the present invention provides an active form that induces the production of IFN-γ in immunocompetent cells, which comprises causing ICE to act on a precursor of a polypeptide that induces the production of IFN-γ in immunocompetent cells. SUMMARY OF THE INVENTION

[0007] ICE is composed of a subunit having a molecular weight of about 20,000 daltons, as reported, for example, by Nancy A. Thornbury et al., Nature, 356, 768-774 (1992). Molecular weight about 1
It is a kind of cysteine protease having a heterodimer structure consisting of a subunit of 000 daltons, and usually exhibits an activity in a state where the heterodimers are associated with each other. ICE is a known enzyme, and a peptide bond between aspartic acid at position 116 and alanine at position 117 in the precursor of interleukin-1β,
Furthermore, it is known that the peptide bond between the 27th aspartic acid and the 28th glycine is cleaved, respectively. However, ICE acts on a precursor of a polypeptide that induces the production of IFN-γ in immunocompetent cells, and the ICE acts at a specific site in the amino acid sequence, particularly, at position 36 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. Cleavage of the peptide bond between aspartic acid and tyrosine 37, I
It is not known to produce an active form of the polypeptide that induces the production of FN-γ. A method for producing the polypeptide utilizing this novel action of ICE is a pioneer of the present invention.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS As described above, the present invention provides an IC
It is based on the unique finding that E produces an active polypeptide from a precursor of a polypeptide that induces the production of IFN-γ in immunocompetent cells. The precursor referred to in the present invention usually has a molecular weight of about 24,000 in SDS-polyacrylamide gel electrophoresis in the presence of a reducing agent.
A dalton, for example, into a cell that naturally produces the polypeptide, or introducing a DNA containing a region encoding the polypeptide (eg, a DNA having the nucleotide sequence of SEQ ID NO: 5 in the sequence listing) Present in host cells derived from mammals transformed by Such a precursor usually has, at the N-terminus, part or all of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and as a whole, all or part of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. It has an amino acid sequence in which a part of the N-terminus has been deleted, and as it is, IFN-γ
Does not induce the production of

However, such precursors, when acted on by ICE in accordance with the present invention, have
The peptide bond between the aspartic acid at position 36 and the tyrosine at position 37 in the amino acid sequence shown in Table 1 is cleaved and converted into an active polypeptide having an amino acid sequence shown in SEQ ID NO: 3 at the N-terminus, This active form of the polypeptide induces IFN-γ production in immunocompetent cells, alone or in the presence of a cofactor as appropriate. The active polypeptide usually has at the N-terminus the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing and, as a whole, the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, in the presence of a reducing agent. Molecular weight of 18,000 to 19,50 by SDS-polyacrylamide gel electrophoresis
Indicates 0 Dalton.

As long as it acts on a precursor of the polypeptide and produces an active polypeptide that induces the production of IFN-γ in immunocompetent cells, ICE may be artificial even if it is natural. It may be created in any way, regardless of its structure, source or origin. Also, Emad s Arnhemli and others appeared in The Journal of
Biological Chemistry ”, Vol. 270, No. 9
No. 4,312 to 4,317 (1995), it is known that ICE derived from the same source contains several isoforms depending on the source. I have. Although these isoforms are said to have slightly different physiological effects, they can be used in the present invention as long as they act on precursors of the polypeptide to produce the active form of the polypeptide.

ICE can be generally obtained from cells that naturally produce ICE, or from a transformant that has become capable of producing ICE by applying recombinant DNA technology. Cells that naturally produce ICE include those derived from mammals including humans, such as epithelial cells, endothelial cells, stromal cells, chondrocytes, monocytes, granulocytes, lymphocytes, nerve cells, and cultures thereof. Cell lines obtained by the above method. Examples of the transformant include a transformed microorganism and a transformed cell obtained by introducing a DNA encoding ICE into an appropriate host derived from a microorganism or animal. These cells or transformants are cultured in a culture medium commonly used in the art, and the transformants grown from the resulting culture are separated once, or ultrasonic waves are applied together with the culture supernatant, or a hypotonic medium. Crushed by immersion or the like in the resulting crushed material or a mixture of the crushed material and the culture supernatant, for example, salting out, dialysis, filtration, concentration, fractional precipitation, ion exchange chromatography, gel filtration chromatography, Adsorption chromatography, isoelectric point chromatography, hydrophobic chromatography, reverse phase chromatography, affinity chromatography, gel electrophoresis, and appropriately combining conventional methods in the art for purifying enzymes such as isoelectric focusing Apply to IC
Collect E. DNA encoding ICE and I
Transformants capable of producing CE are described in, for example, Nancy A. Thornbury et al., Nature, Vol.
768-774 (1992) and Emad s Arnemuri et al., The Journal of Biological.
Chemistry, Vol. 270, No. 9, pages 4,312 to 4,317 (1995).

As an embodiment of the present invention, in which ICE acts on a precursor of the polypeptide, for example, ICE prepared by the above-mentioned method is contacted with a precursor of the polypeptide, or DNA encoding ICE is used. And a DNA encoding the precursor of the polypeptide may be introduced into a suitable host cell derived from a mammal, where both DNAs may be co-expressed. In the former case, cells capable of producing a precursor of the polypeptide or cells that have become capable of producing a precursor of the polypeptide by transformation are cultured. Either the ICE prepared by the above method is allowed to coexist in the culture, or the cells are separated from the culture, or the cells are crushed as necessary without separation. What is necessary is just to add. The amount of ICE to be coexisted or added is usually not more than equimolar to the precursor,
At a level at which the ICE can operate, usually at a temperature of about 4
The reaction may be carried out at a temperature of about 40 ° C. and a pH of about 6 to 9 until a desired amount of the active polypeptide is produced from the precursor of the raw material. Thus, a reactant containing the active polypeptide is obtained. In the case where the same cell produces a precursor of the polypeptide and ICE, respectively, as in the case of THP-1 cells and A-253 cells described later, it is, of course, not necessary to add ICE. Instead, after crushing the cells as necessary, the temperature and pH at which ICE can act on the precursor to produce the active polypeptide as described above.
May be used to incubate the culture containing cells or cell lysates. In such a case, it is desirable to add an appropriate amount of a reducing agent such as 2-mercaptoethanol, dithiothreitol, and reduced glutathione in order to obtain a more efficient production reaction.

In the latter case, the DN that codes the ICE
A and DNA encoding the precursor of the polypeptide may be introduced into appropriate mammalian host cells and transformed. In this case, the ICE generated by the expression of the DNA acts on the precursor of the polypeptide generated by the expression of the DNA in the same transformant to generate an active polypeptide. As host cells, for example,
Epithelial lines derived from human, monkey, mouse and hamster commonly used as hosts in the art, including 3T3 cells, C127 cells, CHO cells, CV-1 cells, COS cells, HeLa cells, MOP cells and mutants thereof. Cell lines, stromal cell lines, neuroblast cell lines and hematopoietic cells are used. DNA encoding ICE in such a host cell
And a DNA encoding a precursor of the polypeptide, for example, by introducing the known DEAE-dextran method,
Calcium phosphate method, electroporation method, lipofection method, microinjection method,
Retrovirus, adenovirus, herpes virus,
A virus infection method using vaccinia virus or the like may be used. At that time, if necessary, an appropriate promoter, enhancer, origin of replication, transcription termination site, splicing sequence, polyadenylation sequence and / or selection marker, for example, pCD, pcDL-SRα, pKY
4, pCDM8, pCEV4, pME18S, pSV2
A vector such as -gpt may be used. In order to select a target clone from the transformed cells, a colony hybridization method may be applied, or the transformed cells may be cultured in a culture medium, and a clone in which production of an active polypeptide has been observed may be selected. If this clone is cultured using a conventional culture medium in the art for culturing transformed cells, a culture containing the active polypeptide is obtained. Depending on cells or culture conditions, depending on the cells and culture conditions, the precursor of the polypeptide may be used, even if cells that naturally produce the precursor of the polypeptide are used, or cells that have become capable of producing the polypeptide by transformation. With the body, a degrading enzyme such as CPP32 or Mch-3 that degrades and inactivates the active polypeptide may be produced. In such a case, the activity of the degrading enzyme in the culture medium or the culture containing cells or cell lysates is inhibited, for example, acetyl-L-asparatyl-L-
When a proper amount of a substance such as glutamyl-L-valyl-L-aspart-1-al coexists, degradation of the active polypeptide is suppressed, and the yield increases. The recombinant DNA technology using mammalian host cells is described in, for example, Toshio Kuroki, Katsushi Taniguchi, Mitsuo Oshimura, "Experimental Medicine Cell Engineering Handbook", 1992, published by Yodosha, Takashi Yokota, Kenichi Arai Editing, “Experimental Medicine Separate Volume Bio Manual Series 3 Gene Cloning Experiment Method”, 1993
It is also described in detail in the year, published by Yodosha.

In the present invention, the activity of ICE is determined by Douglas K. Miller et al., The Journal of Biological Chemistry, Vol. 268, No. 24, 18,062 through 18,069. (1993
Year)) and the activity value (unit) is displayed. That is, 10% (w / v)
Sucrose, 0.1% (w / v) 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate (hereinafter abbreviated as "CHAPS") and 2 m
Take 395 μl of 25 mM Hepes buffer (pH 7.5) containing M dithiothreitol, and add N- (N
-Acetyl-tyrosinyl) -valinyl-alanyl-aspartic acid-7-amino-4-methylcoumarinamide (5 μl of a 10 mM dityl sulfoxide solution) and 100 μl of a test ICE solution are added, and the mixture is reacted at 30 ° C. for 1 hour. During the reaction, 7-amino-
The amount of 4-methylcoumarin was measured at a wavelength of 3 using a fluorometer.
It is monitored over time based on the intensity of fluorescence at a wavelength of 460 nm that is excited and emitted at 80 nm. One unit of ICE is defined as the amount of enzyme that releases 1 picomole of 7-amino-4-methylcoumarin per minute when reacted under such conditions.

The reaction product and the culture containing the active polypeptide thus obtained may be used directly as an IFN-γ inducer. After crushing the cells with a cell lytic enzyme and / or a surfactant, the polypeptide is separated from the cells or the crushed cells by filtration, centrifugation or the like, and purified. For purification, cells or cultures from which cell debris have been removed may be subjected to, for example, salting out, dialysis, filtration, concentration, fractional precipitation, ion exchange chromatography, gel filtration chromatography, adsorption chromatography, isoelectric focusing, hydrophobic Conventional methods in the art for purifying biologically active polypeptides such as affinity chromatography, reverse phase chromatography, affinity chromatography, gel electrophoresis, isoelectric focusing, etc. are used. Applied in combination. Then, depending on the final use form, the purified polypeptide may be concentrated and lyophilized to be in a liquid or solid state. Incidentally, the monoclonal antibody disclosed in Japanese Patent Application Laid-Open No. 8-231598 by the same applicant is extremely useful for purification of the polypeptide, and when immunoaffinity chromatography using the monoclonal antibody is used, the polypeptide having a high purity can be obtained. Can be obtained with minimal cost and effort.

As described above, the active polypeptide obtained by the method of the present invention has the property of inducing the production of IFN-γ, a useful bioactive protein, and enhancing the cytotoxicity of killer cells, Since it has the property of inducing the generation of killer cells, it is extremely effective in treating and preventing various diseases having sensitivity to IFN-γ and / or killer cells. Furthermore, since the active polypeptide obtained by the method of the present invention has a strong IFN-γ inducing ability, it can generally produce a desired IFN-γ in a small amount,
Due to its extremely low toxicity, it does not cause serious side effects even when administered in large amounts. Therefore, the active polypeptide obtained by the method of the present invention has an advantage that the desired IFN-γ production can be rapidly induced without strictly controlling the dose when used. The use of the polypeptide as a susceptibility disorder agent is described in detail in Japanese Patent Application No. 8-28722 by the same applicant.

Hereinafter, the present invention will be described based on embodiments.

[0018]

Example 1 <Preparation of Precursor> 10 μl of 10 × PCR buffer, 8 μl of 2.5 mM dNTP mixed solution in a 0.5 ml reaction tube,
0.5 μl of 5 units / μl Taq DNA polymerase
l and the same patent applicant is disclosed in Japanese Unexamined Patent Publication No. Hei.
Of the recombinant DNA "pHIGIF" disclosed in
5′-AAGGCCAGTGTGCTG chemically synthesized as a sense primer and an antisense primer based on the nucleotide sequence of cDNA encoding the precursor of the polypeptide shown in SEQ ID NO: 5 in the sequence listing
GGCCTGGACAGTCAGCAAGG-3 'and 5'-ACAGCCAGTGTGATGGCTAGTC
20 pmol of an oligonucleotide having a nucleotide sequence represented by TTCGTTTTGAACAG-3 ′ was added,
Make up to 100 μl with sterile distilled water. According to a conventional method, a cycle of reacting the mixed solution at 94 ° C. for 1 minute, at 60 ° C. for 1 minute, and at 72 ° C. for 1 minute was repeated 30 times to cause a PCR reaction.
As the PCR reaction reagent, Takara PC manufactured by Takara Shuzo
R amplification kit "was used.

The obtained reaction product is cleaved with a restriction enzyme Bst XI according to a conventional method, and 0.1 μg of the obtained DNA fragment of about 800 base pairs is dissolved in an appropriate amount of sterilized distilled water and restricted in advance. 10 ng of the plasmid vector “pRc / CMV” manufactured by Invitrogen which had been cut with the enzyme Bst XI, an appropriate amount of 10 × ligation buffer and T4 DNA ligase were added, and 10 mM ATP was added to a final concentration of 1 mM. For 18 hours to insert the DNA fragment into the plasmid vector pRc / CMV. The obtained recombinant DNA was subjected to E. coli JM109 by the competent cell method.
A transformant was introduced into the strain, and this was transformed with 5 ampicillin.
After inoculation into an L-broth medium (pH 7.2) containing 0 μg / ml and culturing at 37 ° C. for 18 hours, cells were collected from the culture and recombinant DNA was extracted by the alkali-SDS method. This recombinant DNA was named "pRCHuGF", and its base sequence was examined by the dideoxy method. As a result, it had the structure shown in FIG. As shown in FIG. 1, in the recombinant DNA “pRCHuGF”, a cDNA “HuIGIF” consisting of about 800 base pairs encoding a precursor of the polypeptide is ligated downstream of the cytomegalovirus promoter “PCMV”. I was

Separately, CHO-K1 cells derived from Chinese hamster ovary (ATCC CC
L61) supplemented with 10% (v / v) fetal bovine serum
Am's F12 medium (pH 7.2) was inoculated and grown. Proliferating cells are collected and the cells are buffered in phosphate-buffered saline
Abbreviated as "PBS". ), The cells were suspended in PBS so that the cell density became 1 × 10 ⁷ cells / ml, and 0.8 ml of the suspension was used for recombinant DNA “pRCHuG”.
F ”, placed in a cuvette together with 10 μg, cooled on ice for 10 minutes, attached to a Bio-Rad electroporation apparatus“ Gene Pulser ”, and applied a discharge pulse once.
The cuvette was immediately removed and cooled on ice for 10 minutes. The cell suspension was then removed from the cuvette and 10% (v /
v) Inoculation into Ham's F12 medium (pH 7.2) supplemented with fetal bovine serum, cultivation in a 5% CO ₂ incubator at 37 ° C. for 3 days, and then a final concentration of G418 in the culture medium of 400 μg / ml And cultured for another 3 days under the same conditions. Forty-eight colonies were selected from over 100 colonies thus obtained, and a part thereof was G418.
Was inoculated into a culture plate containing 400 μg / ml of Ham's F12 medium (pH 7.2) supplemented with 10% (v / v) fetal bovine serum, and cultured for 1 week in the same manner as described above. Then, 5.1 m was added to each well of the culture plate.
M magnesium chloride, 0.5% (w / v) sodium deoxycholate, 1% (w / v) Nonidet P-40,
10 μg / ml aprotinin and 0.1% (w / v) S
DS containing 10 mM Tris-HCl buffer (pH
An appropriate amount of 8.5) was added to lyse the cells.

Take 50 μl of each cell lysate and add 50 μl of glycerol and dithiothreitol to a final concentration of 2%.
(W / v), and the mixture was allowed to stand at 37 ° C for 1 hour, and then the polypeptide in the cell lysate was separated by SDS-polyacrylamide gel electrophoresis. Then, the polypeptide separated in the gel was transferred to a nitrocellulose membrane according to a conventional method, and prepared separately. The polypeptide was specific to the polypeptide disclosed in JP-A-8-231598 by the same patent applicant. For 1 hour in the culture supernatant of hybridoma H-1 strain producing a specific monoclonal antibody, and then washed with 20 mM Tris-HCl buffer (pH 7.5) containing 0.05% (v / v) Tween 20. To remove excess monoclonal antibody. Thereafter, the nitrocellulose membrane was immersed for 1 hour in PBS containing rabbit-derived anti-mouse immunoglobulin antibody labeled with horseradish-derived peroxidase, and buffered with 50 mM Tris-HCl containing 0.05% (v / v) Tween 20. Liquid (pH 7.
5), and immersed in 50 mM Tris-HCl buffer (pH 7.5) containing 0.005% (v / v) hydrogen peroxide and 0.3 mg / ml diaminobenzidine to develop color. Based on the degree of color development, a clone of a transformant having a high ability to produce a precursor of the polypeptide was selected and named "RCHuGF".

The transformant “RCHuGF” was transformed with 40
Ham's F12 medium containing 0 μg / ml G418 and supplemented with 10% (v / v) fetal calf serum (pH 7.
2) was inoculated into a dispensed square culture bottle, and cultured at 37 ° C. for 1 week in a 5% CO ₂ incubator while appropriately replacing the culture medium with fresh one. Thereafter, an appropriate amount of a trypsin agent "Trypsin-EDTA" manufactured by Gibco was added to the culture bottle, the cells adhered to the inner wall of the culture bottle were detached, washed with PBS, and further washed with 10 mM potassium chloride, 1.5 mM magnesium chloride and 0.1 mM ethylenediamine. The plate was washed with an ice-cooled 20 mM Hepes buffer (pH 7.4) containing disodium tetraacetate, and allowed to stand in a three-fold volume of the same buffer under ice-cooling for 20 minutes. Thereafter, the cells were disrupted according to a conventional method, centrifuged at 10,000 × g for 30 minutes, and the supernatant containing the polypeptide precursor was collected. This precursor has a molecular weight of about 24,000 in SDS-polyacrylamide gel electrophoresis in the presence of a reducing agent.
Dalton, and has an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing at the N-terminus.

[0023]

Example 2 <Preparation of ICE> In a 0.5 ml reaction tube, 1 μl of 10 × PCR buffer, 4 μl of 25 mM magnesium chloride,
0 mM dATP, 10 mM dGTP, 10 mM d
1 μl each of CTP and 10 mM dTTP, 1 μl of 50 μM random hexanucleotide, 20 units / μl 1 μl of ribonuclease inhibitor, 50 units / μl
1 μl of reverse transcriptase, and total RNA separately prepared from THP-1 cells (ATCC TIB202) which is a monocyte cell line derived from human acute monocytic leukemia according to a conventional method
Take 1 μg of each extract, and add 20 μl with sterile distilled water.
μl. This mixture was incubated at 42 ° C. for 30 minutes to perform a reverse transcription reaction, and then heated at 99 ° C. for 5 minutes to stop the reaction.

To this reaction was added 8 μl of 10 × PCR buffer.
1, 4 μl of 25 mM magnesium chloride, 5 units / μl
0.5 μl of Taq DNA polymerase, human I
5'- chemically synthesized based on the base sequence encoding CE
CTGCTCGAGACCATGGCCGACAAGG
TCCTG-3 'and 5'-GAGGCGGCCGCT
An oligonucleotide having a base sequence represented by TAATGTCCCTGGGAAGAG-3 ′ was added at 20 pmol each as a sense primer and an antisense primer, and the total amount was adjusted to 100 μl with sterile distilled water. A cycle of reacting at 1 ° C. for 1 minute was repeated 40 times to perform a PCR reaction. As a reagent for the reverse transcription reaction and the PCR reaction, "GeneAmp RNA PCR Kit" manufactured by Perkin-Elmer was used.

The newly obtained reaction product is digested with restriction enzymes XhoI and NotI in a conventional manner, and the resulting DNA fragment of about 1,200 base pairs containing the ICE coding region is preliminarily digested with the restriction enzyme. 1 ng together with 10 ng of the plasmid vector “pCDM8” manufactured by Invitrogen which had been cut with Xho I and Not I.
After adding appropriate amounts of 0x ligation buffer and T4 DNA ligase, 10 mM ATP was added to a final concentration of 1 mM, and the mixture was reacted at 16 ° C for 18 hours to convert the above DNA fragment into plasmid vector pCDM.
8 was inserted. E. coli MC1061 / P3 strain (ATCC47) was obtained by the competent cell method.
035) to give a transformant, which was 20 μg /
L-broth medium (pH 7.2) containing ml ampicillin and 10 μg / ml tetracycline, respectively,
The cells were cultured at 37 ° C. for 18 hours. Collect the cells from the culture,
Recombinant DNA was obtained by applying the alkali-SDS method to the cells.
"PCDHICE" was obtained. When the nucleotide sequence of the recombinant DNA “pCDHICE” was examined by the dideoxy method,
As shown in FIG. 2, a cDNA "HuICE" consisting of about 1,200 base pairs including a region encoding ICE was linked downstream of the cytomegalovirus promoter "PCMV".

Next, COS-1 cells derived from African green monkey kidney (ATCC CRL16) were prepared according to a conventional method.
50) was supplemented with DM supplemented with 10% (v / v) fetal bovine serum.
EM medium (pH 7.4) was inoculated and grown. Proliferating cells are harvested, washed with fresh identical medium, and cell density 2 × 1
0 ⁷ cells / ml suspended so, a recombinant was prepared above in conjunction with 0.25ml of the suspension in the cuvette D
After taking 10 μg of NA “pCDHICE” and allowing it to stand at room temperature for 10 minutes, the cuvette was attached to a Bio-Rad electroporation apparatus “Gene Pulser”, and a discharge pulse was applied once. After removing the cuvette and allowing to stand at room temperature for 10 minutes, the cells are removed and 10% (v / v)
DMEM medium (pH 7.4) supplemented with fetal bovine serum was inoculated and cultured at 37 ° C. for 2 days in a 5% CO ₂ incubator.

The supernatant was removed from the culture by suction, the cells attached to the bottom of the Petri dish were washed with PBS, an appropriate amount of PBS containing 5 mM disodium ethylenediaminetetraacetate was added, and the mixture was allowed to stand at room temperature for 10 minutes. The detached cells were collected. The cells were washed with PBS, and 20 mM Hepes buffer (pH 7) containing 10 mM potassium chloride, 1.5 mM magnesium chloride, 0.1 mM disodium ethylenediaminetetraacetate and 1 mM (4-amidino-phenyl) -methane-sulfonyl fluoride was used. .4) was added thereto, and the cells were disrupted using a homogenizer under ice-cooling. The cell lysate was centrifuged at 1,500 × g for 5 minutes to collect the supernatant, which was further centrifuged at 22,500 × g for 60 minutes.
After centrifugation for minutes, a supernatant containing ICE at 230 units / ml was obtained.

[0028]

Example 3 <Production of polypeptide> 100 mM Hepes buffer (p) containing 10% (v / v) glycerol, 0.1% (w / v) CHAPS and 2 mM dithiothreitol, respectively.
H7.4) was dissolved in 500 nM of the precursor of the polypeptide obtained by the method of Example 1 to obtain a substrate solution. An IC obtained by the method of Example 2 was added to this substrate solution.
E was added to 500 units / ml and incubated at 37 ° C. 0 minutes after the start of incubation, 10 minutes
Minutes, 30 minutes, 1 hour, 3 hours, 6 hours and 1
Eight hours later, a part of the reaction product was collected, and the reaction was stopped by adding iodoacetamide to a final concentration of 200 μg / ml. By applying the western blotting method using a monoclonal antibody disclosed by the same applicant in Japanese Patent Application Laid-Open No. Hei 8-231598 to the collected reaction product, the time-dependent change in which an active polypeptide is produced from a precursor of the polypeptide is obtained. Was examined. The results are shown in FIG.

At the same time, KG-1 cells (ATCC C), a kind of myeloid monocytic cell line derived from human acute myeloid leukemia.
The content of active polypeptide in the reaction product collected over time was estimated by a bioassay using CL246) as an immunocompetent cell. That is, KG-1
RPM of cells supplemented with 10% (v / v) fetal calf serum
A cell density of 1.5 × 1 in I-1640 medium (pH 7.4)
0 ⁶ suspended so that / ml, was dispensed at 0.1 ml / well in 96-well microplates. In the above, 10% (v
/ V) After appropriately diluting with RPMI-1640 medium (pH 7.4) supplemented with fetal bovine serum, this was dispensed into the microplate at a rate of 0.1 ml / well, and 5% C
The cells were cultured at 37 ° C. for 24 hours in an O ₂ incubator.
After the culture, 0.1 ml of the culture supernatant was collected from each well, and the IFN-γ content was measured by a conventional enzyme immunoassay. At the same time, a system was prepared in which the reaction product was omitted, and treated as above to serve as a control. Table 1 shows the results. Table 1
The IFN-γ content shown in Table 1 is based on the National Institutes of Health (NI
H) IFN-γ standard (Gg23-901)
-530) and converted to International Units (IU).

[0030]

[Table 1]

As can be seen from the Western blotting of FIG. 3, under the reaction conditions of this example, the band having a molecular weight of about 24,000 daltons corresponding to the precursor gradually disappeared by 3 hours from the start of the reaction, and Accompanying this, a band with a molecular weight of 18,200 daltons corresponding to the active polypeptide appeared. The IFN-γ content in Table 1 also agrees well with this result, with the appearance of a band with a molecular weight of 18,200 daltons gradually increasing the IFN-γ inducibility of the reactants. These results indicate that ICE acted on the precursor of the polypeptide to produce an active polypeptide that induced IFN-γ production in immunocompetent cells.

[0032]

Example 4 <Purification of polypeptide and physicochemical properties>

[0033]

Example 4-1 <Purification of polypeptide> A reaction product obtained by reacting according to the method of Example 3 for 18 hours was subjected to a 10 mM phosphate buffer (pH 6.0).
8) after dialysis against 10 mM phosphate buffer (pH
The column was loaded on a column of the gel “DEAE 5PW” for ion exchange chromatography manufactured by Tosoh, which had been equilibrated in 6.8), and 10 mM phosphoric acid was added to the column under a concentration gradient of sodium chloride that increased linearly from 0 M to 0.5 M. Buffer solution (p
H6.8) was passed through, and a fraction eluted at a sodium chloride concentration of about 0.2 to 0.3 M was collected.

[0034] The newly obtained fractions were combined and
While dialysis against
A gel for immunoaffinity chromatography using a monoclonal antibody was prepared according to the method described in JP-A-231598, and the gel was packed in a cylindrical shape inside a plastic cylindrical tube and washed with PBS. The column was loaded. A 100 mM glycine-hydrochloric acid buffer (pH 2.5) is passed through the column, and a fraction containing an active polypeptide that induces the production of IFN-γ in immunocompetent cells is collected from the obtained elution fraction, and sterilized by distillation. It was dialyzed against water, concentrated by membrane filtration, and freeze-dried to obtain a purified polypeptide solid. The yield is
This was about 60% of the theoretical yield based on the precursor used as the raw material.

[0035]

Example 4-2 <Molecular Weight of Polypeptide> The purified polypeptide obtained by the method of Example 4-1 was purified by U.K. Lemri in Nature, Vol. 227, pp. 680-685 (1970).
According to the method reported in 2% (w /
v) SDS-polyacrylamide gel electrophoresis in the presence of dithiothreitol revealed a molecular weight of about 18,000.
IFN-γ at a position corresponding to
A major band of inducible polypeptide was observed. This indicates that ICE acted on a precursor of the polypeptide having a molecular weight of about 24,000 daltons to produce a lower molecular weight active polypeptide. The molecular weight marker at this time was bovine serum albumin (6
7,000 daltons), ovalbumin (45,000)
Dalton), carbonic anhydrolase (30,0
00 Dalton), soybean trypsin inhibitor (20,
100 Dalton) and α-lactalbumin (14.4
00 Dalton).

[0036]

[Example 4-3] <N-terminal amino acid sequence of polypeptide> The protein sequencer "473A" manufactured by Perkin-Elmer was analyzed in accordance with a conventional method. Purification obtained by the method of Example 4-1 was performed. The polypeptide had the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing at the N-terminus. This means that ICE acts on the precursor of the polypeptide, and its N-terminal amino acid sequence is SEQ ID NO: 1 in the sequence listing.
Shows that the peptide bond between aspartic acid at position 36 and tyrosine at position 37 in the amino acid sequence shown in FIG.

[0037]

Example 5 <Production of Polypeptide> COS-1 cells derived from African green monkey kidney (ATCC CRL1
650) with D supplemented with 10% (v / v) fetal calf serum.
MEM medium (pH 7.4) was inoculated and grown. Proliferating cells are collected, washed with fresh identical medium, and grown at a cell density of 2 ×
10 ⁷ / suspended so ml, and the cuvette to the suspension recombinant DNA obtained by methods of recombinant DNA "pRCHuGF" and Example 2, obtained by the method of Example 1 with 0.25ml of "pCDHICE Was added thereto, and the mixture was allowed to stand at room temperature for 10 minutes, and then the cuvette was attached to a Bio-Rad electroporation apparatus “Gene Pulser”, and a discharge pulse was applied once. After removing the cuvette and allowing to stand at room temperature for 10 minutes, the cells were removed, inoculated into DMEM medium (pH 7.4) supplemented with 10% (v / v) fetal bovine serum, and
_The cells were cultured at 37 ° C. for 3 days in _two incubators.

The supernatant was removed from the culture by suction, the cells attached to the bottom of the Petri dish were washed with PBS, an appropriate amount of PBS containing 5 mM disodium ethylenediaminetetraacetate was added, and the mixture was allowed to stand at room temperature for 10 minutes. The detached cells were collected. After washing the cells with PBS, a 20 mM Hepes buffer (pH 7.4) containing 10 mM potassium chloride, 1.5 mM magnesium chloride and 0.1 mM disodium ethylenediaminetetraacetate as hypotonic solution.
Was added thereto, and the cells were disrupted using a homogenizer under ice-cooling. The cell lysate was centrifuged at 1,000 × g for 5 minutes, and the supernatant was collected. The enzyme immunoassay using a monoclonal antibody specific to the polypeptide was applied thereto, and the polysaccharide contained in the supernatant was collected. The amount of peptide was determined.

At the same time, the recombinant DNA "pRCHuGF"
And a system in which both or one of “pCDHICE” was omitted, and these were treated in the same manner as above to serve as controls. Table 2 shows the results.

[0040]

[Table 2]

The results in Table 2 show that in COS-1 cells
DNA encoding the precursor of the polypeptide and ICE
Shows that each of the above-mentioned DNAs was well expressed, and that the former ICE efficiently acted on the former precursor to produce an active polypeptide which induces the production of IFN-γ in immunocompetent cells. ing. In addition, as shown in Table 2, DN encoding the precursor of the polypeptide was used.
In the control system in which both or one of the DNAs encoding A and ICE were omitted, the IF
An active polypeptide that induced the production of N-γ was produced, but the amount was small. This fact, COS
-1 cells favorably express the DNA encoding the precursor of the polypeptide and the DNA encoding ICE, respectively.

Thereafter, the active polypeptide was purified by applying the purification method described in Example 4-1 to the culture supernatant of COS-1 cells into which both DNAs had been introduced, and the purified polypeptide obtained was subjected to purification. When the molecular weight was measured by applying the method of Example 4-2, a main band of a polypeptide capable of inducing IFN-γ was observed at a position corresponding to about 18,000 to 19,500 daltons. Furthermore, when the method of Example 4-3 was applied, the purified polypeptide had the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing at the N-terminus.

[0043]

Example 6 <Preparation of polypeptide> Immunity was attenuated by injecting rabbit-derived anti-thymic antiserum into a newborn hamster newborn by a conventional method to attenuate the immune response. A-2, a kind of epithelioid cell line derived from glandular epidermoid carcinoma
Approximately 1 × 10 ⁴ cells / 53 cells (ATCC HTB41) /
The animals were transplanted and bred for 32 days by an ordinary method. Then, a tumor mass (approximately 5 g / animal) formed subcutaneously was excised, cut into small pieces, and 10 mM potassium chloride and 1.5 mM as hypotonic solution.
After washing with 20 mM Hepes buffer (pH 7.4) containing magnesium chloride and 0.1 mM disodium ethylenediaminetetraacetate, the mixture was frozen at -20 ° C.

Next, the tumor mass was thawed and the wet mass of the tumor mass 1
After adding 1 ml of the same fresh hypotonic solution containing 10 mM 2-mercaptoethanol per 1 g, the cells were disrupted by polytron treatment according to a conventional method,
After centrifugation at rpm for 10 minutes, the supernatant was collected. In addition,
This supernatant contained about 500 units / ml of ICE. Thereafter, while incubating the supernatant at 4 ° C. or 37 ° C., the reaction product was sampled with time, and an enzyme immunoassay using a monoclonal antibody specific to the polypeptide was applied thereto. Amount was determined. FIG. 4 shows the results.

As can be seen from the results in FIG.
When the cell extract is incubated, the content of the polypeptide is remarkably increased. This indicates that the extract is effective for the latter in the A-253 cell extract containing ICE and the precursor of the polypeptide, respectively. To produce the active form of the polypeptide.
The incubation temperature is preferably low, and the amount of the polypeptide produced when incubated at 4 ° C. is 37.
Significantly higher than when incubated at ° C. Incidentally, the yield of the polypeptide when incubated at 4 ° C. and 37 ° C. reached 5.0 μg and 2.5 μg, respectively, per gram wet weight of the tumor mass.

The reaction product containing the active polypeptide thus obtained was purified by the method of Example 4-1 and lyophilized, and then the molecular weight was measured by applying the method of Example 4-2. A major band of a polypeptide capable of inducing IFN-γ was observed at a position corresponding to about 18,000 to 19,500 daltons. Further, when the method of Example 4-3 was applied, the purified polypeptide had an amino acid sequence represented by SEQ ID NO: 3 in the sequence listing at the N-terminus.
Furthermore, when dithiothreitol or reduced glutathione was used in place of 2-mercaptoethanol and reacted in the same manner as described above, all reducing agents including 2-mercaptoethanol significantly produced the polypeptide at a concentration of 1 mM. Promoted.

[0047]

As described above, the present invention provides an IC
It is based on the unique finding that E acts on a precursor of a polypeptide that induces the production of IFN-γ in immunocompetent cells to produce an active polypeptide. The present invention makes use of this action of ICE. According to the present invention, ICE can be produced from cells that naturally produce the polypeptide or D cells containing a region encoding the polypeptide.
Acting on a precursor of the polypeptide produced by a mammalian host cell transformed by NA, or introducing a DNA encoding ICE together with a DNA containing a region encoding the polypeptide into a mammalian host cell,
By co-expressing both DNAs in the same cell, the same processing as in a human cell is performed, and a desired amount of a polypeptide useful as a pharmaceutical can be produced.

The present invention is an invention having such remarkable functions and effects, and can be said to be a significant invention which greatly contributes to the art.

[0049]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 41 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met 15 10 15 Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn 20 25 30 Leu Glu Ser Asp Tyr Phe Gly Lys Leu 35 40

SEQ ID NO: 2 Sequence length: 193 Sequence type: amino acid Topology: linear Sequence type: polypeptide sequence Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met -35 -30 -25 Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn -20 -15 -10 -5 Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile 1 5 10 Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro 15 20 25 Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg 30 35 40 Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met 45 50 55 60 Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Xaa Leu Ser Cys 65 70 75 Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile 80 85 90 Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly 95 100 105 His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe 110 115 120 Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys 125 130 135 140 Glu Asp Glu Leu Gl y Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu 145 150 155 Asp

SEQ ID NO: 3 Sequence length: 5 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment type: N-terminal fragment sequence Tyr Phe Gly Lys Leu 15

SEQ ID NO: 4 Sequence length: 157 Sequence type: amino acid Topology: linear Sequence type: polypeptide sequence Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn 1 5 10 15 Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp 20 25 30 Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile 35 40 45 Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile 50 55 60 Ser Val Lys Cys Glu Lys Ile Ser Xaa Leu Ser Cys Glu Asn Lys Ile 65 70 75 80 Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys 85 90 95 Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys 100 105 110 Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu 115 120 125 Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu 130 135 140 Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp 145 150 155

SEQ ID NO: 5 Sequence length: 579 Sequence type: nucleic acid Topology: linear Sequence type: cDNA to mRNA sequence characteristics Characteristic symbol: leader peptide Location: 1..108 Determine characteristics Method: S Symbol indicating characteristics: mat peptide Location: 109..579 Method for determining characteristics: S sequence ATG GCT GCT GAA CCA GTA GAA GAC AAT TGC ATC AAC TTT GTG GCA ATG 48 Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met -35 -30 -25 AAA TTT ATT GAC AAT ACG CTT TAC TTT ATA GCT GAA GAT GAT GAA AAC 96 Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn -20 -15 -10 -5 CTG GAA TCA GAT TAC TTT GGC AAG CTT GAA TCT AAA TTA TCA GTC ATA 144 Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile 1 5 10 AGA AAT TTG AAT GAC CAA GTT CTC TTC ATT GAC CAA GGA AAT CGG CCT 192 Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro 15 20 25 CTA TTT GAA GAT ATG ACT GAT TCT GAC TGT AGA GAT AAT GCA CCC CGG 240 Leu Phe Glu Asp M et Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg 30 35 40 ACC ATA TTT ATT ATA AGT ATG TAT AAA GAT AGC CAG CCT AGA GGT ATG 288 Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met 45 50 55 60 GCT GTA ACT ATC TCT GTG AAG TGT GAG AAA ATT TCA AYT CTC TCC TGT 336 Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Xaa Leu Ser Cys 65 70 75 GAG AAC AAA ATT ATT TCC TTT AAG GAA ATG AAT CCT CCT GAT AAC ATC 384 Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile 80 85 90 AAG GAT ACA AAA AGT GAC ATC ATA TTC TTT CAG AGA AGT GTC CCA GGA 432 Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly 95 100 105 CAT GAT AAT AAG ATG CAA TTT GAA TCT TCA TCA TAC GAA GGA TAC TTT 480 His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe 110 115 120 CTA GCT TGT GAA AAA GAG AGA GAC CTT TTT AAA CTC ATT TTG AAA AAA 528 Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys 125 130 135 140 GAG GAT GAA TTG GGG GAT AGA TCT ATA ATG TTC ACT GTT CAA AAC GAA 576 Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu 145 150 155 GAC 579 Asp

[Brief description of the drawings]

FIG. 1. cDN encoding the precursor of the polypeptide
It is a figure which shows the structure of recombinant DNA "pRCHuGF" containing A.

FIG. 2: Recombinant DN containing cDNA encoding ICE
A is a diagram showing the structure of “pCDHICE”.

FIG. 3 is a halftone image displayed on a gel electrophoretic display showing a successive change in the production of an active form of the polypeptide from a precursor of the polypeptide, visualized by Western blotting.

FIG. 4 is a diagram showing the state of production of the polypeptide when an extract of A-253 cells is incubated at different temperatures.

[Description of the Signs] PCMV cytomegalovirus promoter HuIGIF cDNA encoding the precursor of the polypeptide cDNA encoding HuICE ICE

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91)

Claims (8)

[Claims] 1. An activity of inducing the production of interferon-γ in immunocompetent cells, which comprises causing an interleukin-1β converting enzyme to act on a precursor of a polypeptide that induces the production of interferon-γ in immunocompetent cells. A method for producing a polypeptide of a type. 2. The precursor has a part or all of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing at the N-terminus, and the interleukin-1β converting enzyme has the 36th amino acid sequence in the amino acid sequence shown in SEQ ID NO: 1. Aspartic acid and No. 37
2. Cleavage of the peptide bond between the tyrosine.
The method for producing the polypeptide according to the above. 3. The precursor comprises an amino acid sequence shown in SEQ ID NO: 2 in the sequence listing (however, the amino acid indicated by the symbol “Xaa” represents isoleucine or threonine). 3. The method for producing the polypeptide according to 1 or 2. 4. The method for producing a polypeptide according to claim 1, wherein the active polypeptide has an amino acid sequence represented by SEQ ID NO: 3 in the sequence listing at the N-terminus. 5. The amino acid sequence represented by SEQ ID NO: 4 in the sequence listing (provided that the symbol “Xa”
Amino acids indicated by "a" shall represent isoleucine or threonine. 1, 2, 3 having
Or a method for producing the polypeptide according to 4. 6. The method for producing a polypeptide according to claim 1, wherein the precursor is reacted with an interleukin-1β converting enzyme at a temperature of 4 to 40 ° C. in an equimolar amount or less. . 7. The method according to claim 1, wherein an interleukin-1β converting enzyme is allowed to act on the precursor in the presence of a reducing agent.
7. The method for producing the polypeptide according to 4, 5, or 6. 8. The active form of the polypeptide is subjected to salting out, dialysis, filtration, concentration, fractional precipitation, ion exchange chromatography,
4. A sample collected by gel filtration chromatography, adsorption chromatography, isoelectric focusing, hydrophobic chromatography, reverse phase chromatography, affinity chromatography, gel electrophoresis and / or isoelectric focusing. 8. The method for producing the polypeptide according to 4, 5, 6, or 7.