JP2813370B2 - Method for producing human interleukin 5 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an activated human interleukin 5 (hereinafter referred to as hIL).
More specifically, the present invention relates to a method for producing hIL-5 from animal cell culture in a high yield without using an antibody.

BACKGROUND OF THE INVENTION B cell proliferation and differentiation into immunoglobulin secreting cells is controlled by several soluble factors derived from macrophages and T cells (Kishimoto, T., & Hirano, T. et al.
(1988) Annu. Rev. Immunol. 6,485-512). Interleukin 5 (IL-5), also called a T cell replacement factor, was first reported as a factor that induces terminal differentiation of late B cells into immunoglobulin-secreting cells (Takatsu, K., T).
anaka, K., Tominaga, A., Kumahara, Y., & Hamaoka, T. (19
80) J. Immunol. 125, 2646-2653). More recently, mouse IL-5 has been purified to homogeneity and
5 cDNAs were cloned and sequenced (Taka
tsu, K., Harada, N., Hara, Y., Takahara, Y., Yamada, G., Dob
ashi, K., & Hamaoka, T. (1985) J. Immunol, 134, 382-38
9; and Kinashi, T., Harada, N., Severinson, E., Tanabe,
T., Sideras, P., Konishi, M., Azuma, C., Tominaga, A., Berg
stedt-Lindquist, S., Takahashi, M., Matsuda, F., Yaoit
a, Y., Takatsu, K., & Honjo, T. (1986) Nature324,70-7
3). Thereafter, human IL-5 cDNA was also isolated using mouse IL-5 cDNA as a probe (Azuma, C., Tanabe, T., Konis).
hi, M., Kinashi, T., Noma, T., Matuda, F., Yaoita, Y., Takat
su, K., Hammarstrom, L., Edvard-Smith, CI, Severinso
n, E., & Honjo, T. (1986) Nucleic Acid. Res. 14, 9149-
9158; and Sideras, P., Noma, R., & Honjo, T. (1988) Imm
unol. Rev. 102, 189-212).

In addition to B cell differentiation activity, human IL-5 has recently been induced to induce eosinophil differentiation (Campbell, HD, Tucker, WQJ, Hor).
t, Y., Martinson, ME, Mayo, G., Clutterbuck, EJ, Sande
rson, CJ & Young, I. (1987) Proc. Natl. Acad. Sci. US
84, 6629-6633; and Yamaguchi, Y., Suda, T., Suda, J., Egu
chi, M., Miura, Y., Harada, N., Tominaga, A. & Takatsu, K.
(1988) J. Exp. Med. 167, 43-56), stimulation of B cell proliferation (Sanderson, CJ, O'Garra, A., Warren, DJ & Klaus, G.).
GB (1986) Proc. Natl. Acad. Sci. US 83, 437-440; and
Takatsu, K., Tominaga, A., Harada, N., Mita, S., Matsumot
o, M., Takahashi, T., Kikuchi, Y., & Yamaguchi, N. (198
8) Immunol. Rev. 102, 107-135) and enhancement of cytotoxic T cell induction (Takatsu, K., Kikuchi, Y., Takahashi, T., H
onjo, T., Matsumoto, M., Harada, N., Yamaguchi, N., & Tom
inaga, A. (1987) Proc. Natl. Acad. Sci. US 84, 4234-423.
Evidence has been shown to have a wide variety of biological activities, including 8). Based on such findings, studies for utilizing the biological activity of IL-5 in clinical fields such as cancer treatment have also been started.

However, to utilize IL-5 in the clinical field, the biological activity spectrum of IL-5 should be more completely determined,
Preclinical and clinical evaluations need to be initiated, which requires large amounts of purified hIL-5. However, the amount of hIL-5 isolated by Azuma, C. et al. Or Sideras, P. et al. Described above is very small and not an amount that can be used for bioactivity spectrum studies or preclinical studies. Moreover, the conventional purification of hIL-5 requires hIL-5 for purification.
Because of the use of antibody-5, it is highly possible that the antibody will be contaminated in the final IL-5 preparation, and such an antibody will be completely lost when IL-5 is clinically applied. Antibodies have many drawbacks that hinder industrial mass production, such as the need to eliminate them, the lack of stability between production lots, and the high production cost of antibodies.

(Problem to be Solved by the Invention) An object of the present invention is to provide a method for industrially mass-producing hIL-5 without using an antibody.

Another object of the present invention is to provide a method for producing an active dimer hIL-5 in a high yield.

(Means for Solving the Problems) Animal Cell Culture Animal cells that can be used in the method of the present invention are not limited as long as they can express hIL-5. Preferably, it is a culturable animal cell into which an expression vector containing a chromosome-derived hIL-5 gene or hIL-5 cDNA has been introduced.
More preferably, Chinese hamster ovary (CHO) transfected with the cloned human IL-5 chromosomal gene, pdKCR-hIL-5gene-dhfr (kindly provided by Dr. Yu Honjo (Kyoto University)). Cells.

Depending on the type of animal cell used, various conventional methods such as a suspension culture method, a roller bottle culture method, and a microcarrier culture method can be used. In addition, a medium used for the culture can be used by modifying a conventional medium such as a MEM medium or a ham F12 medium as necessary, depending on the type of the cultured cells. Preferably, after the cells are grown in a growth medium containing serum, the growth medium is replaced with serum-free IL-5.
HIL-5 can be recovered from the production medium by replacing it with the production medium.

Purification of hIL-5 The culture supernatant was collected, and ammonium sulfate was added to the obtained supernatant for about 80 hours.
Precipitate hIL-5 in the supernatant by adding at a% saturation concentration. The ammonium sulfate precipitation is preferably carried out at a low temperature, more preferably at 4 ° C. The resulting precipitate is collected by appropriate means such as centrifugation,
After dialysis, it is used for the next purification step.

The obtained fraction containing hIL-5 is subjected to dye column chromatography in order to remove contaminating proteins. For example, it has been found that a carrier having a dye such as Matrex blue adsorbs many proteins such as albumin, but does not adsorb IL-5. By utilizing this property, hIL-5 can be efficiently separated from most other protein components in the culture solution. Preferably, matrix blue A (Amicon (Am
icon)) is used as a carrier for column chromatography.

The unbound fraction obtained in the previous step is loaded on an anion exchange column for chromatography. Elution can be performed by changing the ion concentration. In this case, hIL-5
Are eluted before contaminating proteins. The column chromatography is preferably performed at a low temperature and a pH of preferably about 6 to about neutral. Preferred anion exchange column chromatography is column chromatography using DEAE-Sepharose, and elution is performed using NaCl
Or a suitable concentration gradient of KCl 0 to 0.4M.

The active fraction obtained by the anion exchange column chromatography in the previous step is chromatographed on a column packed with a carrier having a hydrophobic functional group. The hIL-5 protein is adsorbed on the column by hydrophobic bonds, and is effective in removing non-hydrophobic contaminating proteins. A preferred example of this column chromatography is column chromatography using phenyl-sepharose.

However, the order of the three types of chromatography, that is, the dye column chromatography, the anion exchange column chromatography, and the hydrophobic column chromatography is not limited, and may be performed from any of them.

The active fractions purified by the above three kinds of chromatography are desalted and concentrated by dialysis, ultrafiltration or the like, and then further purified by molecular size by gel filtration column chromatography. Preferred gel filtration column chromatography is column chromatography using a Sephadex-based or Sephacryl-based gel, particularly preferably Sephacryl S-200 (Pharmacia).

The thus obtained hIL-5 is purified 500 times or more compared to the initial culture solution, and is substantially pure from the results of amino acid sequence analysis and amino acid composition analysis using the obtained sample. It is considered to be hIL-5 and can therefore be used directly in clinical trials and the like.

Further, for purified hIL-5 obtained by the method of the present invention, SDS-polyacrylamide electrophoresis under reducing and non-reducing conditions, Western blot analysis, amino acid sequence analysis, amino acid composition analysis, glycosidase treatment,
By performing analysis such as isoelectric focusing, active hIL-5 is bound by a disulfide bond.
In addition to elucidating the relationship between the glycerol and the biological activity and sugar chains, the physical and chemical properties of hIL-5 were elucidated.

Next, the present invention will be described in more detail with reference to examples.
In addition, each operation | movement of an Example is. The test was performed at a temperature as low as about 4 ° C. where possible.

Example 1 Purification of rhIL-5 Expressed in CHO Cells A plasmid containing the genomic sequence of hIL-5 and cDNA of dihydrofolate reductase (dhfr) (see FIG. 1) was used for CHO.
Transform cells and dihydrofolate reductase cDNA
Was used as a selectable marker to increase the copy number of the hIL-5 coding sequence. Selecting clones resistant to 1 μM methotrexate, and selecting the selected cells free of ribonucleosides and deoxyribonucleosides;
The cells were subcultured in α-minimal essential medium (MEMα ⁻ ) supplemented with 10% fetal bovine serum (PES) dialyzed in the presence of 1 μM methotrexate.

The cells obtained were grown to confluence in a roller bottle (850 cm ² , Corning 25140) containing Ham's F12 medium (abbreviated as F12) medium supplemented with 3% bovine serum (CS). I let it. The cells were then washed twice with PBS and incubated for 48 hours with 100 ml of F12 medium, during which the medium was changed twice. This washing medium was discarded, and the cells were further incubated in 500 ml of a serum-free medium supplemented with BSA (1 mg / ml) for 3 days, after which the conditioned medium (CM) (production medium) was recovered. The same procedure was repeated, adding new production medium. Cell viability after the last harvest was greater than 90% as determined by trypan blue exclusion.

Ammonium sulfate was added to the supernatant containing the obtained rhIL-5 (recombinant hIL-5) at 4 ° C. to 80% saturation to cause precipitation.
By this operation, rhIL-5 was concentrated approximately 40-fold. The precipitate was collected by centrifugation, dissolved in 20 mM Tris / HCl buffer (pH 7.4) and dialyzed extensively against a large excess of the same buffer.

The dialyzed protein concentrate is added to a 20 mM Tris-HCl buffer (pH
Matrex Blue A (Amicon (A
micon)) was loaded onto a column (5.0 x 40 cm) and eluted with the same buffer at a flow rate of approximately 2 ml / min. In this column, since rhIL-5 is obtained as an unbound fraction, rhIL-5
5 was effective in separating BSA, which is the main component of the production medium.

Next, the unbound fractions from the Matrix Blue A column were pooled (total volume about 1000 ml) and equilibrated with 20 mM Tris-HCl buffer (pH 7.4) DEAE-Sepharose CL-6B (Pharmacia). Flow rate on column (2.5 × 20cm)
Loaded at 1 ml / min. After sample loading, the column was washed with equilibration buffer and eluted with a linear gradient of sodium chloride 0-0.4M. A chromatogram as shown in FIG. 2 was obtained, and the active fraction containing rhIL-5 was eluted before most of the contaminating proteins. Here, open circles (○) indicate biological activity, and solid circles (●) indicate protein content.

Active fractions eluted from the DEAE-Sepharose column were pooled and ammonium sulfate was added to a final concentration of 0.2 g / ml. The sample was then equilibrated with 20 mM Tris-HCl buffer pH 7.4 containing 0.2 g / ml ammonium sulfate, Phenyl-Sepharose 4B.
(Pharmacia) column (1.5 × 20 cm). rhI
L-5 binds to the carrier in the column by a hydrophobic bond,
Non-hydrophobic proteins pass through the column. rhIL-5 is 0.
Elution was performed with a 20 mM Tris / HCl buffer (pH 7.4) containing 1 g / ml ammonium sulfate.

Active fractions were pooled and 20 mM Tris / 0.15 M NaCl
Dialysis with hydrochloric acid buffer (pH 7.4), ultrafiltration membrane (YM
-10 membrane: concentrated using Amicon (Amicon). Sephacryl S-equilibrated the concentrate with 20 mM Tris / HCl buffer (pH 7.4) containing 0.15 M sodium.
Load on a 200 (Pharmacia) column (2.5 x 100 cm)
Subsequently, the same buffer was passed through the column, and the active fraction flowing out was collected.

The specific activity of rhIL-5 after the final purification step is about 1.7 × 10 ³
Unit / mg and was purified approximately 500-fold with a 45% recovery of activity (Table 1).

The structure and properties of rhIL-5 obtained in Example 1 were clarified by the following experiments.

Example 2 Properties of purified rhIL-5 Molecular weight measurement by SDS-polyacrylamide gel electrophoresis FIG. 3 shows the SDS-polyacrylamide of purified rhIL-5 (1
2.5%) shows the results of gel electrophoresis analysis. Under reducing conditions (FIG. 3 (2)), rhIL-5 showed considerable heterogeneity with a major band of the protein at a molecular weight of about 22,000 and a weak band at about 20,000. These bands are broad, suggesting that each band component contains even less heterogeneity. When analyzed under non-reducing conditions (Fig. 3 (I)), about 40,000
, Two bands were detected. It is noteworthy that the ratio of these two bands differs from culture to culture.

SDS-polyacrylamide electrophoresis was performed using Laemli (Laemml
i, UK (1970) Nature 227, 680-685). When analyzing under reducing conditions, 2
-Mercaptoethanol (2-ME) (final concentration 5%) was used. The protein was observed by silver staining (Daiichi Pharmaceutical Co., Tokyo). As standard markers, phosphorylase b (94 kDa), bovine serum albumin (67 kDa), egg albumin (43 kDa), and carbonic anhydrase (30 kD)
a), soybean trypsin inhibitor (20.1 kDa), and α-
Lactalbu (14.4 kDa) was used.

Western blot analysis of rhIL-5 Purified rhIL-5 was subjected to SDS-polyacrylamide electrophoresis and analyzed by Towbin, H., Stahelin, T., & Gord.
an, J. (1979), Proc. Natl. Acad. Sci. USA 76, 4350-435.
The cells were transferred to nitrocellulose paper according to the method of 5), and subjected to Western blot analysis using rabbit polyclonal antiserum against hIL-5 fusion protein derived from E. coli. Immunospecific proteins were detected using the immunoperoxidase method recommended by the manufacturer (Bio-Rad). Since all bands detected by SDS-polyacrylamide gel electrophoresis were found to be stained by the antiserum (FIG. 4), the heterogeneity of the hIL-5 polypeptide was also confirmed by Western blot. These results suggest that rhIL-5 is secreted as a dimer regardless of its apparent molecular weight heterogeneity.

Crosslinking of rhIL-5 molecules To demonstrate the formation of dimers, crosslinking experiments were performed.
The crosslinker disuccinimide suberic acid (DSS) (Pierce, Rockford, Ill.) Was dissolved in dimethyl sulfoxide to a 25 mM solution immediately before use. 1μ of disuccinimide suberic acid solution was added to 10
RhIL dissolved in 0 mM sodium phosphate buffer (pH 8.0)
-5 (0.78 μg) in 25 μl and the reaction was allowed to proceed at 25 ° C. for 90 minutes. The reaction mixture was subjected to SDS-polyacrylamide electrophoresis under reducing conditions (FIG. 5 (2)). In addition, the purified rhIL-5 was analyzed by SDS-polyacrylamide gel under reducing conditions without cross-linking (FIG. 5 (1)).
Both were compared. When 1mMDSS is used as a crosslinking agent, 40
The presence of a kDa band was detected. These bands are DSS
It was not found in the absence, further suggesting that rhIL-5 exists as a dimer. Incidentally, the molecular weight of rhIL-5 has been calculated to be 43,000 from sedimentation equilibrium experiments.

Analysis of peptide sequence Using 200 μg of purified rhIL-5,
The amino acid sequence was analyzed using a 477A gas phase sequencer from Applied Biosystems (Foster, CA). Phenylthiohydantoin derivatives were obtained by reverse phase HPLC (Hewick, RM, Hunkapiller,
MW, Hood, LE & Dreyer, WJ (1981) J. Biol. Chem. 25
6,7990-7997) (see FIG. 6). Thr
A single amino acid sequence consisting of 20 amino acid residues except for -22 was obtained.

Amino acid analysis 0.15 ml of 6N-HCl was added to purified rhIL-5 (40 µg), hydrolyzed at 110 ° C for 24 hours in a vacuum-sealed tube, and amino acids were analyzed using Hitachi's amino acid analyzer (model 835-50). Analysis was carried out. The results are shown in Table 2. The amino acid composition of rhIL-5 obtained according to Example 1 is cDNA
From the expected hIL-5. M, which is the ninth amino acid from the C-terminus and is the only Met in the molecule
It is noteworthy that a Met residue corresponding to et-126 could be detected. That is, these results were obtained in this way.
The primary structure of the hIL-5 polypeptide is very uniform,
It further suggests that 19 amino acids are removed from the precursor of hIL-5 as a leader sequence upon secretion outside the cell.

Determination of position of disulfide bond 100 μg of purified rhIL-5 was added to PBS (pH 7.4) containing 5 M urea.
(100μ), 0.26μg of Achromobacter protease I (AP-I) was added, and the mixture was reacted at 37 ° C for 2 hours. The resulting rhIL-5 fragment by reverse phase HPLC (C _4, YMC-ShimaHisa Ltd.) were loaded and eluted with a linear gradient of 0-80% 0.1% trifluoroacetic acid presence acetonitrile. on the other hand,
The same rhIL-5 fragment was treated with 0.5M 2-mercaptoethanol and fractionated on an HPLC column in the same manner to identify and disperse a fraction (59% acetonitrile) whose elution position changed. The obtained rhIL-5 fragment was lyophilized, dissolved in 0.5 M Tris / HCl buffer (pH 7.5) containing 50 mM dithiothreitol, and fractionated under the above conditions.
Two fractions were obtained at 56% positions. As a result of determining the amino acid sequence of each fragment, they corresponded to the sequences corresponding to Asn ⁴⁰ → Lys ⁷⁰ and Cys ⁸⁶ → Ser ¹¹⁵ , respectively.

On the other hand, purified rhIL-5 (4 nmole) was converted to 50 mM containing 5 M urea.
After dissolving in a citrate buffer (pH 5.2), 0.2 mM 2-2′-dithiopyridine was added, and the mixture was reacted at room temperature for 15 minutes.
The free -SH group was quantified by the absorbance at
The presence of the group was not detected. From the above results, rhIL-5
Cys ⁴⁴ and cys ⁸⁶ present in the molecule are both involved in the formation of a -SS- bond, and two Cys ⁴⁴ -SS
-It was concluded that a Cys ⁸⁶ bond had been formed.

Effect of Glucosidase Treatment on Properties of rhIL-5 The active fraction obtained from the DEAE-Sepharose column chromatography step of Example 1 was
Sepharose 4B column chromatography was applied. The column was equilibrated with 20 mM phosphate buffer and eluted with 0.2 M α-methyl mannoside at a flow rate of 0.2 ml / min. The results are shown in FIG. 7 (biological activity is represented by a bar graph, and protein is represented by a line graph (●)). As shown in FIG.
Both IL-5 mutants bound to bean-lectin-Sepharose and eluted with 0.2 M α-methyl mannoside.
This suggests that both variants were glycosylated. To examine the potential role of glycosylation in the heterogeneity observed in purified rhIL-5,
Purified rhIL-5 was digested with endoglycosidase F.

Endoglycosidase F treatment Purified rhIL-5 was purified from Flavobacterium meningo.
With endoglycosidase F (Boehringer Mannheim, West Germany) from Septicum ( Flavobacterium meningosepticum ) at 37 ° C.
Incubated for 8 hours in 20 mM Tris / HCl buffer (pH 7.4). This enzyme cleaves high-mannose, mixed and complex oligosaccharide chains from glycoproteins, releasing carbohydrates with a complete N, N'-diacetylchitobiosyl moiety at the reducing end (Tarentino, AL, Gomez, CM & Plumme
r, TH (1985) Biochemistry 24,4665-4671). 25μg
RhIL-5 protein at 0.6 U / ml endoglycosidase F
The oligosaccharide moiety was almost completely removed within 24 hours by incubation under base treatment conditions. Even without the addition of detergent, the enzyme was active against rhIL-5 as a substrate. After incubation, the protein is removed from the enzyme preparation by TSK G300SW
The gel was separated by gel filtration chromatography.
The apparent molecular weight of hIL-5 treated with endoglycosidase F was found to be 18,000 by SDSY-polyacrylamide gel electrophoresis performed under reducing conditions (FIG. 8).

Analysis by isoelectric focusing Heterogeneity of rhIL-5 was also analyzed by isoelectric focusing. Isoelectric focusing was performed using an ampholine gel plate (pH 3.0 to 9.5) using a model 2117 multiphore (MULT
IPHOR) (LKB, Sweden). Untreated rhIL−
5 is lane 2 in FIG. 9, treated with endoglycosidase F rh
IL-5 is shown in lane 3 in FIG. 9 in the pI marker protein (soybean trypsin inhibitor; 4.55, β-lactoglobin A; 5.2
0, bovine carbonic anhydrase B; 5.88, human carbonic anhydrase B; 6.55, and myoglobin-basic band; 7.35) are shown in FIG. SDS
-Similar to polyacrylamide gel electrophoresis, rhIL-5 showed considerable heterogeneity (pI range 5.2-6.0). After treatment with endoglycosidase F, the pI value of rhIL-5 was mainly
Changed to 5.9. However, a minor band with a pI value of 6.1 was still observed. These results indicate that rhIL-
The heterogeneity observed in the charge and molecular weight of 5 suggests that it is primarily the result of glycosylation differences of the same polypeptide.

Molecular weight measurement by gel filtration before and after treatment with endoglycosidase F Next, the molecular weight of rhIL-5 before and after treatment with endoglycosidase F was determined by gel filtration chromatography,
0.15 for TSK G3000SW column (Toso, Tokyo)
The measurement was performed by elution using a 20 mM Tris / HCl buffer (pH 7.4) containing MNaCl at a flow rate of about 1 ml / min. In FIG. 10, A represents untreated rhIL-5, B represents rhIL-5 treated with endoglycosidase F, C represents alkylated rhIL-5,
1 to 5 are marker proteins, 1 is glutamate dehydrogenase (290 kD), 2 is lactate dehydrogenase (1
42 kD), 3 is enolase (67 kD), 4 is adenylate kinase (32 kD), and 5 is cytochrome c (12.4 kD). TS
The activity of untreated rhIL-5 eluted from the KG3000SW column showed a single peak, with an apparent molecular weight of 63 kD. On the other hand, rhIL-5 treated with endoglycosidase F
Apparent molecular weight corresponded to 40 kD. These results
Deglycosylated rhIL-5 as well as glycosylated rhIL-5
Are also oligomers (probably dimers).

Assay for rhIL-5 The B cell differentiation activity of IL-5 was reported by Harada et al.
N., Takahashi, T., Matsumoto, M., Kinashi, T., Ohara, J., K
ikuchi, Y., Koyama, N., Severinson, E., Yaoita, Y., Honjo,
T., Yamaguchi, N., Tominaga, A., & Takatsu, K. (1987) P
roc, were measured by the induction of BCL ₁ cells IgM secretion according to the method described in Natl.Acad.Sci.US 84,4581-4585).
Briefly, cells (2 × 10 ⁴ cells / 200 μ culture) were mixed with a dilution series of hIL-5, seeded in 96-well plates, and incubated at 37 ° C. in the presence of 5% CO _2. Cultured for 3 days. The culture solution was 1% FBS, 500 U / ml penicillin G and 50
Consists of RPMI-1640 with μg / ml streptomycin. After culturing for 3 days, the amount of IgM in the supernatant was determined by particle concentration fluorescence immunoassay (PCFIA) (Jolly, ME, Wang, C., -HJ, E
kenberg, SJ, Zuelke, MS, & Kelso, DM (1984) J. Im
mol. Meth. 67, 21-35). Specifically, 0.5 (v / v)% pre-coated with anti-mouse IgM goat serum
Polystyrene latex particles (diameter 0.8 μM; Dow Chemical) 20 μ with 96-well Epion
n) To each well of the plate (Pandex, Mandalay, Illinois) was added, followed by 50 μl of cell-free supernatant and 20 μl of purified goat anti-mouse IgM with fluorescent label affinity. The reaction plate was gently shaken, then incubated for 15 minutes at room temperature and placed in a fluorimeter (FCA, Pandex). After washing with PBS containing 1% non-immunized goat serum, the fluorescent label bound to the particles was excited at a wavelength of 485 nm and the fluorescence at 535 nm was measured. One unit of IL-5 was defined as the amount required for half the maximal response. Table 3 shows the activity of each of purified rhIL-5, rhIL-5 treated with endoglucosidase F, rhIL-5 treated with N-ethylmaleimide, and rhIL-5 treated with both 2-mercaptoethanol and N-ethylmaleimide. showed that. Treatment of purified rhIL-5 with endoglycosidase F does not destroy the biological activity of rhIL-5. Thus, the specific activities of glycosylated and glycosylated forms of rhIL-5 are considered to be the same.

Effect of disulfide bonds on the biological activity of rhIL-5 To examine the effect of disulfide bonds on the biological activity of IL-5, alkylated rhIL-5 was prepared by treating rhIL-5 with 2ME followed by NEM.

Methods for Alkylation of rhIL-5 Alkylation of rhIL-5 is essentially carried out by Brewell.
er) and the method disclosed by Riem (Bre
wer, CF, & Riehm, JP (1967) Anal. Biochem. 18, 248
−255). Simply put, rhIL-5 (15
μg) was treated with 2-ME (1 mM) at 37 ° C. for 15 hours,
Then, it was dialyzed against a 20 mM sodium acetate buffer (pH 5.5). Then, rhIL-5 was added to 1 mM N-ethylmaleimide (N
EM) (Sigma, St. Louis, Montreal) for 1 hour at room temperature and 0.15 MN
It was dialyzed against a 20 mM Tris / HCl buffer (pH 7.4) containing aCl. To test the biological activity of alkylated rhIL-5,
Residual rhIL-5 was separated by gel filtration column chromatography on TSKG3000SW.

Analysis by SDS-polyacrylamide gel electrophoresis showed that the alkylated IL-5 showed a protein band corresponding to a molecular weight of about 22,000 even under non-reducing conditions. In addition, alkylated hIL-5 has an apparent molecular weight of 2 from the G3000SW column.
Elution was performed at a position corresponding to 2,000, which means that 2ME and
After treatment with NEM, this suggests that IL-5 has dissociated into monomeric form (FIG. 10C). As shown in Table 3, alkylated hIL-5 in which the formation of disulfide bonds was prevented was
gM induction activity was greatly reduced. Treatment with NEM alone without treatment with 2ME had little effect on rhIL-5 activity. Furthermore, rhIL-5 was shown to be present in a dimer after treatment with NEM alone. These results strongly suggest that disulfide bond formation of dimers is important for the biological activity of rhIL-5.

(Effects of the Invention) The method for producing hIL-5 of the present invention is a purification method that enables industrial production without using an antibody. The yield is as high as rhIL-5 can be used directly in the clinical field and the like. Can be purified at a constant rate. Therefore, the method of the present invention can overcome the problems associated with the use of antibodies, such as contamination of the antibody protein into the final product, difficulty in mass production, denaturation of the antibody protein, and problems of antibody preparation. .

[Brief description of the drawings]

FIG. 1 shows the hIL-5 expression plasmid pdKCR-hIL-5gene-
It is a schematic diagram showing the structure of dhfr. FIG. 2 is a DEAE-Sepharose column chromatogram of rhIL-5. FIG. 3 is a photograph of SDS-polyacrylamide gel electrophoresis of rhIL-5. FIG. 4 is a photograph of rhDS-5 subjected to SDS-polyacrylamide gel electrophoresis and stained with antiserum. FIG. 5 is a photograph of SDS-polyacrylamide gel electrophoresis of rhIL-5. FIG. 6 is an amino acid sequence diagram of the N-terminal of purified rhIL-5. FIG. 7 is a chromatogram of lentil-lectin-Sepharose 4B column chromatography of rhIL-5. FIG. 8 is a photograph of SDS-polyacrylamide gel electrophoresis of rhIL-5. FIG. 9 is a photograph of isoelectric focusing of rhIL-5. FIG. 10 is a graph showing the results of gel filtration column chromatography of rhIL-5.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ identification code FI C12R 1:91) (58) Investigated field (Int.Cl. ⁶ , DB name) C12P 21/02 C12N 15/24 C07K 1 / 36 C07K 14/54

Claims (5)

(57) [Claims] 1. a) culturing animal cells into which the gene of human interleukin 5 has been introduced, and separating the culture supernatant; b) containing human interleukin 5 by adding ammonium sulfate to the culture supernatant Collecting the precipitate; c) separating the fraction containing human interleukin 5 obtained in the above step with a dye column chromatography adsorbing contaminating proteins, a column chromatography of an anion exchange resin, and a column of a carrier having a hydrophobic functional group. Subjecting it to a purification step in which chromatography is performed in any order; d) human interleukin 5 obtained in step (c) above.
Is further purified by gel filtration column chromatography; a method for producing activated human interleukin 5 comprising the steps of: 2. The method according to claim 1, wherein the order of column chromatography in the step (c) according to claim 1 is the order of dye column chromatography, anion exchange column chromatography, and hydrophobic column chromatography. . 3. The method according to claim 1, wherein the activated human interleukin 5 is a dimer linked by a disulfide bond. 4. The carrier for dye column chromatography is Matrex Blue A, the carrier for anion exchange column chromatography is DEAE-Sepharose, the carrier for hydrophobic column chromatography is phenyl-Sepharose 4B, The method according to claim 1, wherein the carrier for filtration column chromatography is Sephacryl S-200. 5. The method according to claim 1, wherein the animal cell is a CHO cell into which pdKCR-hIL-5gene-dhfr has been introduced.