JPH07100720B2 - Method for producing colony stimulating factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention relates to a culture solution of human urine or human-derived monocyte-macrophage colony-stimulating factor-producing cells or human-derived monocyte-macrophage colony-stimulating factor gene recombinant cell. The present invention relates to a method for producing a novel colony stimulating factor which stimulates colony formation of mammalian monocyte-macrophage lineage cells.

[Conventional technology]

A colony stimulating factor (hereinafter abbreviated as CSF) is a hematopoietic factor that stimulates differentiation / proliferation of hematopoietic stem cells present in mammalian hematopoietic tissues such as bone marrow, and many CSFs are composed of glycoproteins. So far, a factor acting on monocyte-macrophage stem cells (M-CSF or CSF-1), a factor acting on granulocyte-monocyte stem cells (GM-CSF), a factor acting on granulocyte stem cells (G -CSF), and factors that act on pluripotent stem cells common to granulocytes, monocytes, erythrocytes and megakaryocytes (Multi-CSF, interleukin-3 or IL-3)
4 kinds of are known.

Except for Multi-CSF, the above-mentioned three kinds of human-derived CSF have cloned gene cDNAs encoding the respective amino acid sequences, and their protein structures have been clarified [GGWong
Et al., Science, 228, 810-815, 1985; ES Kawasaki
Et al., Science, 230, 291-296, 1985; S. Nagata et al., Na.
ture, 319, 415-418, 1986].

As CSF present in human urine, factors acting on monocyte-macrophage cell line (CSF-1 or M-CSF) [SKDa
s & ERStanly, Journal of Biological Chemistry, 25
7, 13679-13648, 1982], HGI-glycoprotein [JP-B-60-30291] and CSF that stimulate granulocyte colonies.
-HU [K. Motoyoshi et al., Blood, 52, 1012-1020, 1978 and Blood, 60, 1378-1386, 1982] have been reported.

Among them, CSF-1 acting on monocyte-macrophage cells is completely blunted, and cDNA encoding the protein has been cloned (ESKawa, supra).
saki et al.).

In the structure of blunted CSF-1, two polypeptides containing a sugar chain form a biologically active homodimer by a disulfide bond. This dimer produces two identical subunits by cleaving the disulfide bond with a reducing agent. Of this glycoprotein with biological activity,
The molecular weight measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 45,000-60,000 daltons. The molecular weight of the polypeptide subunit forming the dimer excluding the sugar chain is 14,000 to 17,000 daltons. The number of amino acids deduced from the cDNA considered to encode the amino acid sequence of this CSF-1 subunit is
It is a polypeptide with 224 molecules and a molecular weight of 26,000 daltons.

[Problems to be solved by the invention]

The object of the present invention is to have a therapeutic effect on leukopenia, immunodeficiency, bone marrow transplantation, etc. due to anticancer drug chemotherapy.
The purpose of the present invention is to provide a novel method for producing a glycoprotein CSF, which acts in the same manner as in Example 1, but which is physicochemically distinguished from CSF-1 and which has not been known so far.

That is, the present invention provides a novel method for producing CSF comprising a glycoprotein having the following physicochemical properties and having a colony formation-stimulating action on mammalian monocyte-macrophage lineage cells.

a) Molecular weight It is a homodimer that is dissociated into two identical subunits by a reducing agent like CSF-1, and has a molecular weight of 70,000 to 90,000 daltons measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. , The molecular weight of the subunit dissociated with a reducing agent and having lost its biological activity was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 35,000 to 45,000 daltons.

(B) Amino acid sequence of subunits The subunit proteins constituting the homodimer are shown below.
It has a sequence of 214 amino acids, The 122 and 140th asparagine (Asn) has a typical N-glycoside binding site represented by asparagine (Asn) -X-threonine (Thr) or serine (Ser), respectively. However, X represents an arbitrary amino acid.

c) Isoelectric point Isoelectric point (pI) measured by polyacrylamide gel isoelectric focusing method and sucrose density gradient isoelectric focusing method
Is 3.1 to 3.7.

d) Constituent monosaccharide of sugar chain After hydrolysis, analysis by high performance liquid chromatography revealed that mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid were identified as constituent monosaccharides of the sugar chain.

e) Circular dichroism spectrum The far-UV external CD spectrum measured by the circular dichroism dispersometer has a wavelength of
It has minimal peaks at m and 222 nm, respectively, and contains an α-helix structure.

f) Thermal stability No biological activity is lost when heated at 60 ± 0.5 ° C for 60 minutes.

g) Infrared absorption spectrum It has an infrared absorption spectrum shown in FIG.

The novel method for producing CSF of the present invention refers to the serum of a mammal immunized with a colony-stimulating glycoprotein having the above physicochemical properties and having a colony-stimulating action on mammalian monocyte-macrophage cells. A solution containing the above-mentioned colony stimulating glycoprotein in the antibody binding support prepared by isolating and purifying a specific antibody against the above colony stimulating glycoprotein, binding the specific antibody to an insoluble support Are contacted with each other to adsorb the colony stimulating glycoprotein contained in the solution, and the colony stimulating glycoprotein is eluted from the adsorbed antibody-bonded support, which is a method for producing a colony stimulating glycoprotein factor.

[Specific Description of the Invention]

 The method for producing CSF of the present invention is as follows.

(1) Production of CSF The production method of the present invention comprises the following three steps. This will be described sequentially below.

Specific antibody to colony-stimulating glycoprotein (hereinafter
Preparation of CSF antibody) Using the CSF obtained by the production method shown in Reference Example 1 described below or the production method of the present invention performed separately, mammals such as rabbits, goats, sheep and horses can be prepared. Immunize. That is, the CSF of the present invention is dissolved in a physiological saline solution to a concentration of 0.1 to 1.0 mg / ml, mixed with an equal amount of Freund's complete adjuvant, and subcutaneously injected into a mammal for 1-2 weeks.
Immunize by administration for 4 to 8 weeks. When the antibody titer in blood of the immunized animal rises, booster immunization by intravenous injection or subcutaneous injection is performed, and blood is collected 3 to 7 days after the booster immunization to collect CSF antiserum. The antibody titer to the CSF of the collected antiserum is measured by the CSF biotiter neutralization test described below. The CSF antibody titer in the antiserum is 5 × 10 ⁶ units or more per ml of the CSF biotiter titer. It is desirable to select a compatible antiserum. The collected antiserum is purified as an anti-CSF antibody of immunoglobulin G or M fraction by twice salting out with ammonium sulfate and once by DEAE-cellulose chromatography. If necessary, the anti-CSF antibody is passed through an antigen column that uses a contaminant protein that shows a cross-reaction with CSF or an anti-CSF antibody as a ligand, and only the anti-CSF antibody is adsorbed or the contaminant protein is adsorbed. Purify anti-CSF antibody.

Step of preparing antibody-bound support The insoluble support capable of binding the anti-CSF antibody is N of the antibody protein.
Any known insoluble support can be used as long as it is an insoluble support that can be chemically bonded to the H ₂ — group or the COOH— group. For example, cyanogen bromide activated or epoxidized polysaccharide gel, formylated polysaccharide gel, aminoethylated or hydrazide polymer and the like. The conditions for the binding reaction between the insoluble support and the anti-CSF antibody differ depending on the binding group of the selected insoluble support, so the antibody is adjusted so that the binding reaction is optimal. For example, for a cyanogen bromide active support, a pH of 8-10
The antibody is dissolved in the carbonate buffer of 1), in the case of the epoxidized support, into a solution having a pH of 10 or higher, and in the case of the formylated support, into the neutral solution. The temperature condition of the binding reaction also differs depending on the insoluble support, but in the case of the binding reaction of the present invention, it is desirable to carry out at a low temperature of 25 ° C or lower. Particularly in the case of a cyanogen bromide activated support, it is carried out at 4 ° C or lower. The amount of antibody to be bound is 10 to 50 mg, preferably 20 to 30 mg, per 1 g (wet weight) of the insoluble support, and the antibody concentration during the binding reaction is adjusted to 1 to 4% (w / v). After the binding reaction is completed, the antibody non-binding reactive group remaining on the support is inactivated by an appropriate treatment method to obtain an antibody-bound support.

Step of Purifying CSF with Antibody-Binding Support The antibody-binding support is washed with a buffer solution containing 0.5 to 1.0 M salt such as sodium chloride and having a pH of 6 to 8. The washed antibody-bound support is packed into a column or suspended in a buffer. The former is used as column chromatography and the latter is used as batch chromatography. As the solution containing CSF, for example, human urine concentrate, CSF-producing cell culture supernatant or CSF gene recombinant cell culture supernatant is used, which is adjusted to pH 6 to 8 and then the antibody-bound support The treatment solution is brought into contact with the antibody-bound support by equilibrating with the same buffer solution as that used for washing or by adding sodium chloride to a concentration of 0.5 to 1.0 M. Contact is carried out by column-type or batch-type chromatography. In the case of column-type chromatography, the CSF is bound to the antibody by passing the solution at room temperature or lower, preferably 10 ° C. or lower at a flow rate of 5 to 20 ml / cm ² · hour. Adsorb to the column of the support. The amount of adsorbed CSF is 5 per 1 g (wet weight) of the antibody-bound support.
00 to 20 million units are desirable. After adsorption, the above buffer solution is passed through to wash and remove contaminants. In the case of batch type chromatography, the treatment solution and the antibody-bound support are mixed at room temperature or 10 ° C. or lower, and the mixture is stirred for 1 to 10 hours. After stirring, the antibody-bound support is recovered by filtering with glass filter paper or the like. The antibody-bound support is washed with the above buffer to completely remove contaminants. The CSF specifically adsorbed to the antibody-bound support is, for example, after the dissociation of the anti-source antibody complex, for example, an acetate buffer of pH 2-3, 3-4M thiocyanate or 0.1-0.2M.
Elute from the antibody-bound support with a solution such as 2,4-dinitrophenol. For column chromatography,
The CSF is eluted by passing the eluent through the column or, in the case of batch chromatography, suspending the antibody-bound support in the eluent and stirring. The CSF obtained here is pure CSF from which impurities have been removed. The production method of the present invention comprises the above steps,
Further, Examples 1 to 3 which will be described later are shown and described in detail.

(2) Physicochemical properties of CSF The CSF obtained by the above manufacturing method has the following physicochemical properties. In this physicochemical property test, CSF purified by the method of Example 1 described later was used.

(A) Molecular weight In the absence of a reducing agent, Laemmli (Nature, Vol. 227, 680-685)
Page, 1970), the molecular weight was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
It was 70,000-90,000 Daltons.

Next, it was reduced with 0.2 M mercaptoethanol, and when measured by the same method, it was dissociated into subunits having a molecular weight of 35,000 to 45,000 daltons (Fig. 1).

FIG. 1 is a electrophoresis diagram of CSF obtained by the production method of the present invention by sodium dodecyl sulfate-polyacrylamide electrophoresis, where A to E are non-reducing (dimers), F and G are molecular weight marker proteins, H to L represent reductions (subunits), and the numbers on the vertical axis represent molecular weights (× 10 ³ daltons).

(B) a subunit protein of the amino acid sequence NH ₂ - terminal amino acid sequence was analyzed by a conventional method in the gas phase amino acid sequencer purified CSF. Next, the purified CSF was denatured with 6M guanidine, alkylated with monoiodoacetic acid, desalted, digested with trypsin and decomposed with cyanogen bromide. Tryptic digestion and cyanogen bromide degrading peptide Vy
Fractionated by dac C-18 reverse phase high performance liquid chromatography,
A decomposed peptide fraction was obtained, and each fraction was analyzed by a gas phase amino acid sequencer to analyze the amino acid sequence of the peptide fragment. The amino acid primary structure of the subunit protein was determined from the amino acid sequences of tryptic digested and cyanogen bromide degrading peptide fragments and the nucleotide sequence of mRNA cloned by the present inventors. The results are shown in Table 1.

NH ₂ - From glutamate, amino terminal to the 149th glutamine is identical to the known CSF-1, 0.99
The 65 amino acids from position to 214 were completely different from the known ones.

As the COO-terminal amino acid, proline was detected at the 214th position depending on the molecular weight of the subunit protein. 1
The 22nd and 140th asparagine are Asn-X-Ser / Thr
It has a typical N-glycoside bond structure of, and was presumed to bind the sugar chain at this site. Here, X represents an arbitrary amino acid.

(C) Constituent Monosaccharide of Sugar Chain The constituent monosaccharide of the sugar chain bound to the polypeptide was hydrolyzed and released, and then analyzed by high performance liquid chromatography. Aldose, sialic acid for anion exchange column,
Hexosamine was fractionated by a cation exchange column by a borate buffer gradient elution method, post-column labeled with cyanoacetamide or arginine, and then identified by a fluorescent method. Although the sugar chain contained in the CSF molecule was heterogeneous and difficult to quantify, mannose, galactose, N-acetylglycosamine, N-acetylgalactosamine, and N-acetylneuraminic acid were used as constituent monosaccharides. Was identified.

(D) Isoelectric point As a result of measuring the isoelectric point by the polyacrylamide gel isoelectric focusing method and the sucrose density gradient isoelectric focusing method, the pI was 3.1 to 3.7.

(E) Circular dichroism (CD) spectrum The CD spectrum in the far ultraviolet region was measured with a circular dichroism dispersion meter (J-600 manufactured by JASCO) (Fig. 2).

FIG. 2 shows a CD spectrum of CSF obtained by the production method of the present invention, where the horizontal axis represents wavelength (nm) and the vertical axis represents ellipticity (md
eg) is shown. Minimal peaks were observed at wavelengths of 208 nm and 222 nm, and it was presumed that the secondary structure of this CSF contains an α-helix structure.

(F) Thermostability This CSF was dissolved in a dilute buffer (pH 7.0) at a concentration of 1 μg / ml and heated at 60 ± 0.5 ° C. for 60 minutes to measure its colony stimulating activity (described later). Almost no decrease in activity was observed.

(G) Infrared absorption spectrum The infrared absorption spectrum of the freeze-dried powder of this CSF measured by a Fourier transform infrared spectroscope (5DXC manufactured by Nicolet Co.) by a transmission measurement method (KBr window) is as shown in FIG. It was In FIG. 3, the horizontal axis represents wave number (cm ⁻¹ ) and the vertical axis represents transmittance.

This CSF is strong absorption in 1650cm ^-1, 1201cm ^-1 and 1133cm ^-1, 1
It showed moderate absorption at 537 cm ^-1 , 1432 cm ^-1 and 1068 cm ^-1 .

The production method of the present invention will be described in detail by further showing experimental examples.

(Experimental Example 1: Preparation of anti-CSF antibody using rabbit) 0.4 mg of CSF of the present invention obtained by the production method of Reference Example 1 described later is applied to a rabbit (white breed) having a body weight of 2.5 to 3.0 kg.
The solution dissolved in physiological saline to a concentration of / ml was mixed with an equal volume of Freund's complete adjuvant and the
l was subcutaneously administered to the back of the rabbit at an interval of once a week for 4 weeks for immunization, and at 5 weeks, booster immunization by intravenous injection was performed. Five days after the booster immunization, blood was collected to obtain anti-CSF serum. During the immunization period, ear vein blood was collected at 1-week intervals to test the increase in CSF antibody titer.

The anti-CSF antibody titer was determined by the following CSF bioactivity neutralization test.
That is, the collected serum was serially diluted with physiological saline, and 0.5 ml thereof was added to 0.5 ml of a CSF (2,000 unit / ml, 0.02 μg / ml) solution.
And mix for 5 minutes at room temperature. After mixing, add 0.1 ml to McCoy's 5A medium containing 1 × 10 ⁵ mouse bone marrow cells, 20% fetal bovine serum and 0.3% agar, and incubate at 37 ° C. under 7.5% CO ₂ aeration.
Incubate for a day. After culturing, the colonies formed by CSF are counted. The anti-CSF antibody titer was calculated by the following formula.

Anti-CSF antibody titer (neutralizing unit / ml) = (number of colonies formed without addition of serum)-(number of colonies formed with addition of serum)
Table 1 shows the change in blood anti-CSF antibody titer of 3 rabbits immunized with x (serum dilution factor) x 10 CSF.

50 ml of No. 1 and No. 3 rabbit sera shown in Table 1 were purified by the following procedure. The purification was performed at 4 ° C. Add 50 ml of antiserum with stirring to a saturated ammonium sulfate solution to 40% (v / v),
Precipitate the antibody. The precipitate is redissolved in distilled water, and a saturated ammonium sulfate solution is added again so as to be 33% (v / v) to precipitate the antibody. The precipitate was dissolved in distilled water, dialyzed against 0.02 M Tris-hydrochloric acid buffer (pH 8.6), and then passed through a DEAE-cellulose column preliminarily equilibrated with the above Tris-hydrochloric acid buffer to remove DEAE-cellulose. The IgG fraction adsorbed was collected and purified as an anti-CSF antibody of the IgG fraction. Obtained anti-CS
F antibody was 480 mg and 460 mg, respectively.

(Experimental Example 2. Preparation of antibody-bound support) As an insoluble support, cyanogen bromide-activated Sepharose 4B was used.
(Pharmacia) and Formyl-Cellulofine (Chisso) were used to examine the optimal binding conditions with the anti-CSF antibody. Cyanogen bromide activated Sepharose 4B
When using, use anti-CSF antibody with 0.1M carbonate buffer (pH 9.0)
To be an antibody solution. Cyanogen bromide-activated Sepharose 4B was swollen in 1 mM hydrochloric acid solution, washed with 0.1 M carbonate buffer solution, suction filtered to remove the buffer solution, and 1 g of cyanogen bromide-activated Sepharose 4B gel (wet weight ) Was placed in a 50 ml Erlenmeyer flask.

Add 2 ml of each of the 4 concentrations of antibody solution shown in Table 2 at 4 ℃
The antibody was bound by shaking for time. 0.1M after coupling
After washing with a carbonate buffer, the reactive groups of the support were inactivated with 0.2 M Tris-HCl buffer (pH 7.0).

When using formyl-cellulofine,
The CSF antibody was dissolved in 0.1 M sodium phosphate buffer (pH 7.0) to give an antibody solution. Formyl-cellulofine was washed with distilled water and 0.1 M sodium phosphate, then suction filtered to remove the buffer solution, and formyl-cellulofine 1 g
(Wet weight) was taken into a 50 ml Erlenmeyer flask. 2 ml of an antibody solution having four concentrations shown in Table 3 was added, the mixture was stirred at room temperature for 2 hours, 7 mg of sodium cyanoborohydride was added, and the mixture was further stirred at room temperature for 10 hours to bind the antibody. After binding, wash with 0.2 M Tris-HCl buffer (pH 7.0), add 2 ml Tris-HCl buffer containing 5 mg sodium cyanoborohydride and stir at room temperature for 4 hours to inactivate the reactive groups. Let

Tables 2 and 3 show the antibody binding amounts of the cyanogen bromide-activated Sepharose 4B and formyl-cellulofine obtained as above.

The antibody concentration for efficiently binding a large amount of anti-CSF antibody to the insoluble support was 20 to 30 mg / ml for both cyanogen bromide-activated Sepharose 4B and formyl-cellulofine.
Was the concentration of. When the concentration was low, the binding rate was high, but the amount of antibody bound was small, and when the concentration was high, the amount of antibody bound was high, but the binding rate was low.

(Experimental Example 3 Purification of CSF by Antibody Support) As an antibody-bound support, the same as in Example 1, formyl- was used.
Antibody binding using Cellulofine as insoluble support 31 mg / g
Prepare a gel antibody-supported support and DEAE human urine as CSF.
-Specific activity 2x obtained by cellulose and gel filtration treatment
Purification of CSF was examined by batch chromatography using a solution containing 10 ⁵ units / mg (purity less than 1%) of partially purified CSF.

The antibody-bound support was washed with 0.02 M phosphate buffer (pH 7.0) containing 0.5 M sodium chloride, and suction filtered.

Then, the solution containing this CSF was preliminarily equilibrated with the above-mentioned phosphate buffer. A solution containing CSF was added to 1 g of the antibody-bound support at four addition amounts shown in Table 4, and the solution was added at 10 ° C.
After stirring for 6 hours at 0.degree. C., wash thoroughly with the above buffer solution and
CSF was eluted with 2M acetic acid-hydrochloric acid buffer (pH 2.5).

In order to purify CSF at a high recovery rate, as shown in Table 4, CSF was added in an amount of 0.6 to 1.5 per 1 g (wet weight) of the antibody-bound support.
It was appropriate to load x10 ⁷ units. Further, the collected CSF was tested for specific activity and purity by SDS-PAGE.

The results are shown in Table 5.

As shown in Table 5, the partially purified CSF was purified at least 245 times by the affinity chromatography with formyl-cellulofine, and the purity by SDS-PAGE was a uniform glycoprotein of 90% or more.

(3) Biological activity of CSF In the same manner as in the above item (2), CSF purified by the method of Example 1 was used for this colony stimulating activity and specific activity test.

(A) Colony stimulating activity and specific activity The CSF colony stimulating activity of the CSF obtained by the production method of the present invention was measured by a colony forming test method using mouse bone marrow cells on a single layer soft agar gel. Mc containing CSF sample containing 0.3% agar, 20% fetal calf serum (FCS) and 1 × 10 ⁵ mouse bone marrow cells
Mix with 1 ml of Coy's 5A medium and incubate at 37 ° C under 7.5% CO ₂ aeration.
Cultured for a day. After culturing, 50 or more cell aggregates were determined as colonies, and the number of formed colonies was counted. The colony stimulating activity was expressed in units, and one unit was defined as the amount of CSF required to form one colony. The specific activity was represented by the number of colonies (unit) formed per mg of CSF protein.
As a result, the CSF of the present invention had a specific activity of 5.2 × 10 ⁷ units / mg · protein. When the formed colonies were stained with hematoxylin-eosin and morphologically classified, 95% or more of the colonies were formed from monocytes-macrophages.

(B) in vitro and mouse bone marrow monocytes in in vivo - in macrophage stem cell promoting effect on the growth of (CFU-M) b-1 ) in vitro test C ₅₇ BL mouse bone marrow cells flat adsorption method, Non-adsorbed bone marrow cells in McCoy's 5A medium containing 20% FCS 1 × 10 ⁶ cells / m
CSF obtained by the production method of the present invention when added to a concentration of 1
0 (control), 100 units / ml, 500 units / ml, 1,000 units / ml
And 2,000 units / ml, respectively, and cultured at 37 ° C. for 24 hours under aeration of 7.5% CO ₂ . After culturing, each bone marrow cell was washed by centrifugation, diluted 5 times with the same medium, and 1,000 units of CSF, 0.3% agar and
Add 0.1 ml to 1 ml of McCoy's 5A medium containing 0% FCS,
The cells were cultured at 37 ° C for 7 days under aeration of 7.5% CO ₂ . After culturing, 50 or more cell aggregates were converted into monocyte-macrophage stem cells (CFU
-M), and the number of CFU-M formed was measured. The results are shown in Table 6.

As shown in Table 6, the number of CFU-M in mouse bone marrow cells increased depending on the concentration of CSF added.

b-2) In vivo test 0 (physiological saline) per 1 kg of body weight, 80 × 10 ⁴ units, 160 × 10 ⁴ units and 320 × 10 ⁴ units CSF for C ₅₇ BL mice (5 / group) 1 CSF It was administered intraperitoneally once a day for 3 consecutive days. On the day after the end of the administration, the femur bone marrow and spleen were removed from each mouse, and the number of monocyte-macrophage stem cells (CFU-M) in the bone marrow and spleen was adjusted to 1,000 units of CSF using the soft agar plate. It was measured by a colony formation test by the method. The results are shown in Tables 7 and 8.

As shown in Tables 7 and 8, an increase in CFU-M in the bone marrow and spleen was observed by the administration of 80 × 10 ⁴ units / kg body weight of CSF, and a remarkable increase was observed in the administration of 160 × 10 ⁴ units / kg body weight or more. An increase was observed.

The CSF obtained by the production method of the present invention having the above-mentioned physicochemical properties and biological activities is compared with known similar substances as follows.

The CSF obtained by the production method of the present invention is composed of a biologically active homodimer in which two polypeptides containing a sugar chain are disulfide-bonded like CSF-1, and has a molecular weight of 70,000. ~ 90,000 Daltons, greater than that of CSF-1. Further, the CSF-constituting subunit polypeptide of the present invention has 214 amino acids and has a molecular weight of 35,000 to 45,000 daltons, which is larger than that of CSF-1 of 14,000 to 17,000 daltons. In addition, comparing the amino acid sequence of the subunit with CSF-1, NH ₂
-The amino acid sequences from the 1st to 149th positions at the end were the same, but the amino acid sequences from 150th to 214th positions were CSF.
-1, which is different from that predicted from the cDNA of -1.
It was not encoded on one gene. Therefore, the CSF obtained by the production method of the present invention has some commonities with CSF-1, but is known genetically and structurally.
Was found to be a separate factor.

Reference Example 1 200 l of urine of a healthy person was adjusted to pH 8.5, the precipitate was removed by filtration, and the mixture was concentrated and desalted with an ultrafiltration membrane having a cut-off molecular weight of 50,000 daltons (Amicon, H10 x 50). Next, p
It was adjusted to H7.0 and sterilized by heating in a sealed container at 60 ° C for 10 hours. After sterilization, the precipitate was removed by centrifugation (5,000 xg for 30 minutes), and then mixed with DEAE-cellulose equilibrated with 0.02M phosphate buffer (pH 7.2) to adsorb. DEAE-cellulose was washed with 0.02 M phosphate buffer and 0.05 M salt-added 0.02 M phosphate buffer (pH 7.2), and then eluted with 0.25 M salt-added buffer (pH 7.2). The eluate was concentrated with an ultrafiltration membrane (H1P10 manufactured by Amicon), and a Sephacryi S-300 (Pharmacia, φ4 × 80 cm) was used to prepare a 1 M ammonium sulfate addition buffer (pH 7.
Gel filtration was performed in 2). Molecular weight range for gel filtration 70,000-1
A phenyl-Sepharose 4B column (Pharmacia Co.,
φ2 × 20 cm) and then eluted with 0.5 M ammonium sulfate addition buffer (pH 7.2). The eluate was concentrated with an ultrafiltration membrane (Asahi Kasei, NM-3) and subjected to high performance liquid chromatography on a TSKG-3,000SW column (Toyo Soda, φ4 x 600 mm x 2) to obtain a molecular weight range of 70,000 to 150,000 daltons. Fractions were obtained. This fraction was concentrated again and the Hi-Pore PR-304
0.1% on reverse phase column (Bio-Rad, φ4 × 150mm)
Acetonitrile containing trifluoroacetic acid 0-100% (p
H2.0) was subjected to high-performance liquid chromatography with a linear concentration gradient to elute CSF, and the purified specific activity was 1.4 × 10 ⁸ units / mg of protein, which was obtained from the 214 amino acid sequence shown in Table 1. A CSF consisting of a dimer of the subunit protein was obtained. The degree of purification of CSF in each step of the above production process is as shown in Table 9.

Next, examples of the present invention will be described.

Example 1 An anti-CSF antiserum was collected from 10 rabbits immunized with the CSF obtained in Reference Example 1 and having a sufficiently increased antibody titer.
About 4 g of anti-CSF antibody purified by the method of 1. was obtained. The anti-CSF antibody was dialyzed against 0.1 M phosphate buffer (pH 7.0) to adjust the concentration to 20 mg / ml. 200 ml of the antibody solution was added to 100 g of formyl-cellulofine previously washed with distilled water and 0.1 M phosphate buffer, and the mixture was stirred at room temperature for 2 hours, then 700 mg of sodium cyanoborohydride was added, and the mixture was further stirred for 16 hours. Then, formyl-cellulofine and anti-CSF antibody were bound to prepare an antibody-bound support. After binding, 0.2M Tris-
After washing with hydrochloric acid buffer, 200 ml of Tris buffer containing 500 mg of sodium cyanoborohydride was added, and the mixture was stirred at room temperature for 4 hours to inactivate unreacted groups. The antibody-bound support was then washed extensively with 0.02M phosphate buffer containing 0.5M NaCl. Antibody-bound support is 29.5 mg anti-CS / g support.
F antibody was bound. Next, 1,000 l of normal human urine was concentrated by an ultrafiltration concentrator, desalted, and then adsorbed on DEAE-cellulose to remove non-adsorbed contaminants and eluted with a 0.3 M NaCl solution to give an eluate. Sodium chloride was added to a concentration of 0.5 M to prepare a solution containing CSF. The specific activity of this CSF was 2 × 10 ⁵ units / mg. 100 g of the above antibody-bonded support
To this, add a solution containing this CSF (500 ml in total),
The mixture was stirred overnight at 10 ° C or lower and subjected to batch type chromatography. After stirring, the antibody-bound support was collected by filtration with a glass filter, and the antibody-bound support was thoroughly washed with 0.02 M phosphate buffer containing 0.5 M NaCl. After washing
500 ml of 0.2M acetate buffer (pH 2.5) was added, and the mixture was stirred at 10 ° C for 1 hour to elute CSF. After adjusting the pH of the eluate to 7.0, it was concentrated and desalted with an ultrafiltration membrane to obtain about 10 mg of purified CSF consisting of a dimer of subunit protein consisting of 214 amino acid sequences as shown in Table 1. It was The specific activity of purified CSF is 5.2 × 1
0 ⁷ units / mg, purity by SDS-PAGE method was 90% or more.

Example 2 A column (φ15 × 100 mm) was packed with 10 g of the same antibody-bound support as the antibody-bound support prepared in Example 1, and 1.0 MN
It was washed by passing 0.02M phosphate buffer (pH 7.0) containing aCl. Using this column, CSF was isolated and purified from the CSF-producing cell culture supernatant. Human pancreatic cancer cells (referred to as MIA-PaCa-2 cells) were used as CSF-producing cells. MIA-PaCa-2 cells were cultured in a Dulbecco's MEM medium containing 5% fetal bovine serum for 7 days, and the culture supernatant liquid 1 was concentrated with an ultrafiltration membrane to prepare a solution containing CSF. The specific activity of this CSF was 4 × 10 ⁵ units / mg. Sodium chloride was added to 100 ml of this solution to a concentration of 1.0 M, and then the solution was passed through the column at 10 ° C. After passing the solution, the column was washed with a phosphate buffer containing 1.0 M NaCl, and then CSF was eluted with 3.5 M sodium thiocyanate. The eluate was dialyzed against 0.02M phosphate buffer, concentrated with an ultrafiltration membrane, and concentrated as shown in Table 1.
2 subunit protein consisting of 214 amino acid sequence
About 1 mg of purified CSF consisting of monomers was obtained. This purified CSF is 7.8
× 10 ⁷ units / mg, purity by SDS-PAGE method was 90% or more.

〔The invention's effect〕

In the production method of the present invention, a person who contains CSF (CSF having physicochemical properties defined in item (2) of [Detailed Description of the Invention]: hereinafter abbreviated as the present CSF) This CSF can be purified from urine or other solutions such as the culture medium of the present CSF producing cells or the present CSF gene recombinant cells, and by using the antibody-bound support, a solution coexisting with various contaminants or biological factors can be prepared. Since the present CSF can be selectively and conveniently isolated and purified, and has an excellent action of removing heterologous proteins other than humans in the present CSF, inexpensive and highly purified present CSF can be supplied as a drug.

[Brief description of drawings]

FIG. 1 shows sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS) of CSF obtained by the production method of the present invention.
FIG. 2 and FIG. 3 show the far-UV CD spectrum and infrared absorption spectrum of CSF obtained by the production method of the present invention, respectively. In Fig. 1, A to E ... non-reduced products (dimers) F, G ... molecular weight marker proteins H to L ... reduced products (subunits)

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Muneo Yamada 752 Sakae Terrace, 752 Kuchi, Takatsu-ku, Kawasaki-shi, Kanagawa Prefecture (72) Inventor Hajime Yokota 2-5-1-406 Shirokane, Minato-ku, Tokyo (72) Inventor Kunio Ujiie 2-10-16 Nakaikenoike, Ota-ku, Tokyo Morinaga Milk Industry Co., Ltd. Ikegami Dormitory Examiner Reiko Imamura (56) References International Publication 87/6954 (WO, A)

Claims (2)

[Claims] 1. 1 ml of a mammal immunized with a colony stimulating glycoprotein having a stimulatory effect on colony formation of mammalian monocyte-macrophage cells and having the physicochemical properties of a) to g) shown below. An antiserum that neutralizes 5 × 10 ⁶ units or more of the colony-stimulating glycoprotein biological titer is collected, and a specific antibody against the above colony-stimulating glycoprotein is isolated and purified from the antiserum. At a concentration of 20 to 30 mg / ml, an insoluble support is bound to 10 to 50 mg per g to prepare an antibody-bound support, and the antibody-bound support is contacted with a solution containing the above colony-stimulating glycoprotein, and the solution is added. Adsorb the colony stimulating glycoprotein contained therein, and elute the colony stimulating glycoprotein from the adsorbed antibody-bonded support to give a purity of 90% or more and a specific activity of 5.2 × 10 ⁷ units / mg protein or more. Having the above colony stimulating sugar A method for producing a colony-stimulating glycoprotein factor, which comprises obtaining a protein: a) a molecular weight: a homodimer composed of two identical subunits,
The molecular weight measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 70,000 to 90,000 daltons, and the molecular weight measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the subunit dissociated with a reducing agent to eliminate biological activity is , 35,000 to 45,000
It is Dalton; b) Amino acid sequence of subunits The subunit proteins that make up the homodimer are shown below.
It has an amino acid sequence of 214, and the 122nd and 140th asparagine (Asn) are asparagine (Asn) -X, respectively.
-N represented by threonine (Thr) / serine (Ser)-
It has a glycosidic binding site, where X represents any amino acid; c) Isoelectric point Isoelectric point (pI) measured by polyacrylamide gel isoelectric focusing method and sucrose density gradient isoelectric focusing method
Is 3.1 to 3.7; d) Constituent monosaccharide of sugar chain The constituent monosaccharide of the sugar chain analyzed by high performance liquid chromatography after hydrolysis is mannose, galactose, N-acetylglycosamine, N-acetylgalactosamine and N-.
Acetylneuraminic acid; e) Circular dichroism spectrum The far-UV CD spectrum measured by a circular dichroism disperser has a wavelength of
It has a minimum peak at m and 222 nm, and contains an α-helix structure; f) Thermal stability No biological activity is lost by heating at 60 ± 0.5 ° C for 60 minutes; g) Infrared absorption spectrum Infrared absorption in the spectrum, 1650 cm ^-1, strong absorption at 1201cm ^-1 and 1133cm ^-1, 1537cm ^-1, 1432cm ^-1 and 1068cm
^-1 has a medium absorption. 2. A solution containing a colony stimulating glycoprotein is selected from the group consisting of human urine, a culture solution of colony stimulating glycoprotein producing cells, a culture solution of colony stimulating glycoprotein gene producing cells and concentrates thereof. The method for producing a colony-stimulating glycoprotein factor according to claim (1), characterized in that