JPS6354398A - Colony formation-stimulating factor - Google Patents

Abstract

NEW MATERIAL:A glycoprotein recoverable from human urine and having the following properties. Mol. wt.: approximately 70,000 measured by a gel filtration method; isoelectric point: approximately 4.7pH; amino acid sequence of the N-terminal group: formula (- means to be unmeasurable); ratio of the glucide component to the protein component: glucide content of approximately 13-20%; the glycoprotein acts on bone marrow cells to stimulate the differentiation and multiplication of granulocyte stem cells. USE:Leukopenic leukemia remedy. PREPARATION:For example, human urine is partially purified to give a soln. contg. a colony formation-stimulating factor (CSF) having increased specific activity. The soln. is mixed with polyethylene glycol, cooled with ice and centrifuged to recover the resulting precipitates. The precititates are dissolved in a 10mM phosphoric acid buffer soln. and the produced soln. is mixed with ethanol to again result precipitates. The precipitates are again dissolved in the 10mM phosphoric acid buffer soln. and then purified by a method such as ion exchange chromatography or gel filtration to provide the novel CSF.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a human-derived colony formation stimulating factor (hereinafter referred to as C3F) consisting of a homogeneous glycoprotein. Human-derived C3F is a miraculously active substance that is extremely present in human urine, and acts on bone marrow III follicles to promote the differentiation and proliferation of rA granulocytes and monocytes. The drug's efficacy as an anti-inflammatory drug is expected to reach t3'.

C Prior Art] C3F in human urine was reported by Das et al.
of Biological Chemistry (J.

13io1. Chem,) 257.13679
(2982) ), Wong (14,lng) et al.C Journal of Cell Biochemistry (J, Ce
Biochem, ) 21.263 (1983
)), Ilatake et al. [Journal of Chromatography (J, fJromatog, )
344 (1985)) 4, and the purified product has a specific activity of 1 to 2 x 10'' units/n+g and a molecular weight of 1+46.
000 and was reported to be a subunit dimer with a molecular weight of 23,000.

The co-researchers of the present invention also focused on C3F in human urine, and found that it has a molecular weight of 75,000 to 90.000 (gel σ5?
We would like to develop the novel υN protein of
No. 140707, IJsP 4.230.691 Jino) 6Q, Cetus, Inc. has investigated C in human urine.
The N-terminal amino acid sequence was determined by Il'IP of 3F-1 (Science, 2QQ, 2
91 (1985c, whose N Song ami7) acid sequence is Glu Gluνal Ser Glu Tyr Cy
s 5er1:ys Met IIs Gly Ser
It is reported that Gly 1lis is a substance consisting of 224 heptamino acids and a molecule of ¥26,000 daltons.

As described above, several types of C3F are mixed in urine, and their biological activities are diverse and complex.

[Problems to be Solved by the Invention] An object of the present invention is to provide a novel C3F contained in urine.

[Means for solving problems]

The present invention provides 3 proteins recovered from human urine,
It is a colony formation stimulating factor with the following properties.

tal molecular weight Nigel, as determined by I filtration method, &-
370.000 (1) Isoelectric point pH 4.7 tel N-terminal amino acid sequence: Ser Pro Ser Gly -Gln -S
er Gin Pro Gin Thr Val lbeThr Ala Gln Gly (- in the sequence means it cannot be measured) +
dl Acts on bone marrow cells to promote the differentiation and proliferation of condyle ir: ¥ lineage stem silk follicles, tel Protein to carbohydrate ratio: Approximately 13-20% sugar-containing star
A. Starting material human urine, etc., especially human urine with a C3FO specific activity of at least about 10
As a partial purification method in which a l6 solution having human C3F activity partially purified to about 00 to 3000ji1/8280 can be used, there are known methods such as JP-A-54-140.
No. 707 (silicon-containing adsorbent, cation exchanger, anion exchanger, purification by gel d@filtration treatment), JP-A-59
-58629 (purification by concentration, heating, treatment with polyethylene glycol (PEG), anion exchanger), and the like.

B. Purification/Isolation Method Purification/isolation involves combining partially purified C5F with PEG fractionation, ethanol fractionation, ion exchange chromatography, gel filtration, hydrophobic chromatography, and HPLC (high performance liquid chromatography). I will do it. For example, the following process is performed for fat~(('I. The process is preferably performed in the order of +a+~(fl).

Adjust the pH of the falPEG fraction 11csF solution to about 3 to 5! f! 1 adjustment,
1) After adding 10 to 20% (W/V) of EC, 1 to 1
The mixture is left at 0° C. for 30 minutes to 3 hours, and the supernatant is collected.

Add PEG to the upper 4ff under the same conditions, Hiiragi, 20-40%
(W/V) and collect the precipitate. The recovery rate of C3F is 60-80%. Specific activity increases 2-5 times.

Note that PEG has a molecular weight of i, ooo to 10.
000, preferably about 4.000 to 6.000.

(b) Ethanol Fractionation The precipitate from the PEG fractionation is dissolved in a buffer strain having a pH of about 6 to 8, and the solution is diluted to a protein content of 5 to 15%. Ethanol fraction is heated at high temperature/ζ degree under conditions of -10 to -25℃.
After removing i:tr by 75% ethanol fractionation,
This is done by collecting the precipitate from the 85 to 95 silanotanol fraction. By going through this process, C
The recovery rate of 3F from partially purified C3F is 50-70%. The specific activity is 10 to 20 times higher than that of raw material C3F.

(C1 anion exchanger chromatography A weakly basic anion exchanger is preferably used, and an exchanger having DEAE as an exchange group (DEAE-cellulose, etc.) is used. The exchanger is ρ) 14 ~ A low-salt agricultural buffer solution with a concentration of about 5% is used, and C3F is contacted and adsorbed to 1!
'Afterwards, elute with a mild solution of pH 6 to 8. By going through this step, C5 from partially purified C3F
The recovery rate of F was 30-40%, and the specific activity was that of partially purified C.
It increases 30 to 60 times from SF.

(dl Gel filtration treatment By using an IP system suitable for separating substances with a molecular weight of 10,000 to 10,000, the specific activity can be reduced from a partial volume of 5IC3F to 280
~350 times increase. By going through this step, the recovery rate from partially purified C3F will be 15 to 25%. The column is equilibrated with a buffer of about p11G~8 and developed.

A preferred carrier is Sephacryl S-200. Tracking of CSF includes C5F activity and 280
This can be done by measuring the absorbance at nm.

Tel hydrophobic chromatography The carrier used in this treatment includes an immobilized carrier having a hydrophobic group. As the immobilization carrier, amino acid copolymer, cellulose, agarose, dextran, polyacrylamide, etc. are used, and as the hydrophobic group, )S
Nylalanine, an alkyl group having 2 to 8 carbon atoms, a phenyl group, etc. are used. A known method may be used for the immobilization.

Chroma 1-graphy uses 7g of inorganic salt with a pH of about 6 to 8.
? Straight line l by a; This can be done by the gradient gradient method. The recovery rate in this step is about 20-40%.

(fl Reverse-phase HPLC (high performance liquid chromatography)-l D) C purified by hydrophobic chromatography
3Fii! ii fraction is dialyzed to remove salts and then highly purified by conventional reverse phase HPLC techniques. This reverse phase HP
LC may be repeated multiple times, for example, by changing one body. For example, in the first treatment, Pro RPCHR5/10 (manufactured by 77 Lumacia) is used as a carrier, and then 1li- pore RP-304 (
Reverse-phase HP L was performed twice using [3io-Rad)].
It is preferable to perform C.

Through this treatment, C3F can be recovered at a recovery rate of about 1 to 15%.

C0 properties (a) Biological activity The biological activity of C5F obtained in the present invention was measured using mouse bone marrow cells on a soft agar medium (Semisolidafa).
The colony formation method was performed using R culture.

The following medium was used.

Fetal bovine serum 22 all McCoy's 5A medium 11 (2(?1)I
OIII (1x) 48ml 3.3% agar solution 10ml cells! Silent,・Yamotomoto
Total: 100 ml Chapter Manokoi 5A powder medium 12.1 g Eagle amino acid vitamin medium 2.6 g Sodium bicarbonate
1.5 g6g6 streptomycin
200 mg penicillin G potassium 200,000 units Sterile water 500 ml 1 bottle I This test was conducted with 1 copy/counterfeit and 10% sample added. That is, sample 10
Place the 0pa in the 3511I11 dish in advance, and
After adding 900 d of the cell culture solution described in 1 above and stirring well, the mixture was cultured for 7 days in a 37° C. incubator with 5% CO2. The formed colony was observed at 50x magnification and cells 5
Only colonies consisting of 0 or more were counted. As a result, the specific activity of C3F provided by the present invention is at least 5
It was Xl'05 units/mg.

(b) Molecular weight Sephadex G-75, G-200, Sephacryl S
When the molecular weight of the CSF of the present invention was measured by the gel i+liM method using -300 etc. under the following conditions, the molecular weight of the CSF of the present invention was:
It was about 70,000.

Conditions: 2 Column size: 24 x 900a+m Solvent: Phosphate buffer (pH 7) Development speed: 1.5 1/hour The molecular weight was determined by comparison with lit protein of known molecular weight.

(c) Isoelectric point Biogemica Hyophisica Acta (Bio-c
he＋＊, [1iophys, Aeta,), 1
94, 335 (+969), Acta-Namikaru Scandinavia (＾eta, (j+em.

5eand, ) % 20.820 (1966);
1
．． When the isoelectric point was determined by ,B)), it was found that the present invention C
3F was pll (4.7).

(d) Proof that it is a glycoprotein/color reaction The C3F of the present invention was dissolved in water, and a seven-color reaction was carried out to obtain the results shown in the table below. From this result, it was confirmed that the CSF of the present invention is a glycoprotein.

-Sugar Content The protein of the CSF of the present invention was quantified by the semi-microkale pool method, and it was found to be 77-80%, and the sugar content was determined to be approximately 13-20%.

(e) Determination of the amino acid sequence of the present invention C3F (specific activity 5XIO5 units/mg)
Regarding llB, Abride Biosystems' Ga
5-Phase Protein 5equencer
The N-terminal amino acid sequence was determined by automated Edman degradation using Model 470A. As a result,
It has been confirmed that the present invention proposes a novel C5F that has a different sequence from the conventionally known C3F.

Ser Pro Sar Gly-Gln-S
er Gin Pro Gin Thr Val f'
he- Thr Ala -Gin Gly (- in the sequence means not measurable) Example 1 fat C5 from polyethylene glycol treated human urine
F crude bulk liquid 1520 ml (absorbance at 280 nm 58300, l, @ CSF F activity 13300 x l
O' value) was adjusted to pll4.0 with concentrated hydrochloric acid on water cooling, and polyethylene glycol 4000 was added to 14% (W/V).
It was added so that I left it for 165 hours under water cooling.5
Centrifuge at 8.00 rpm for 30 minutes at 4°C, and add 30% (W) polyethylene glycol 4000 to the resulting supernatant 78.
/V). After being left for 1 hour under water cooling, it was centrifuged at 10,000 rpm and 4'C for 30 minutes to collect Precipitate B.

Both components have an absorbance of 12300 at 280nn+ (21% recovery) and a total C5F activity of 10300× 104 units (
The recovery rate was 77%).

(bl Ethanol treatment (IL+) The precipitate fraction obtained was mixed with 10+M sodium phosphate buffer containing 0.15M sodium chloride (pll 7.
4), adjusted to pH 7.0, and then adjusted to 90+al. Next, 45 m of 12M lithium chloride solution (pH 7°0)
1 was gradually added while cooling with ice to prevent the temperature from rising to -. After leaving in the freezer for 2 hours, 270o+l of ethanol (-20°C) was slowly added dropwise and left in the freezer again overnight [ethanol final concentration 67% (v/
v)], followed by 8. OOOrpm, 20 at -20℃
Centrifuge for 1 minute, and add ethanol (-20°C) to the resulting supernatant.
) was added to approximately 945 liters [ethanol final liter yield 90% (V
/V)), 8,0 after being aired in the freezer for 2 hours
The precipitate was collected by centrifugation at 0 rpm and 20°C for 20 minutes. Both had an absorbance of 2830 at 280 nm (recovery rate of 4.9%) and a Satoshi CSF activity of 8140 x 104 degrees (recovery rate of 61%).

Ic) Ion exchange chromatography (bl)
The solution dissolved in M sodium phosphate solution (pll 7.4) was dialyzed against 10 m sodium acetate solution 51, and the pH was adjusted to 4.5 by adding tetraacetic acid. This was mixed in advance with 50mM sodium acetate buffer (pH
DEAε-cellulose CDE- equilibrated with 4,5)
52.Whatman? J[) column <3- Q X 13
．． 5cm). After washing the column with equilibration buffer, add 50J sodium chloride solution (1115
, 4).

Both CSF fractions were then eluted with 0.1M sodium phosphate buffer (pH 7.0) containing 0.5M thorium chloride.The flow rate was 50(1) l/hr. Absorbance at 2BOnl11 520 (&Q recovery rate 0.9
9'o), total C5F activity 3980 xlO'l single O(
The twist recovery rate was 30%).

(di gel filtration (elution fraction of C3F filtered with C1)) in minimoji 1
．． - (cutting Asahi Kasei bimolecular 〒10,00t) or less)
LHhl was applied to 4011II using. Let's outline this: 0.15M chloride, Lium content, QmM Phosphate, 1. It was applied to a heptafacrylic S-200 (Pharmacia) column (5X 89 cm) equilibrated with a mild (PL! 7.4) solution, and gel filtering was performed at a flow rate of 701/hour. 18.2 ml of each fraction was collected, and A280 (absorbance at 280 nm) and C3F activity were measured for each fraction.

The final absorbance at 280 discharges was 34.5 (twist recovery rate 0).
．． 06%), Satoshi CS F activity 2310 x 104 units (
It was possible to obtain CSF with a yield of 17 stones.

(El Hydrophobic 11 Madography) Active C5F fraction used for general purpose 2XlO”44)
0.1M Na? IIPO4/1.5M (NII4
)2SO4 (11117,0), l@dialysis (4
℃), and using a TSK--7, y-nyl 5PW (To 7 Baku Sodasha!!) as a hydrophobic column, the solution was separated from (A) by the linear concentration distribution method of (B) f&△. I drew it.

The conditions are as follows.

Column: 21.5X 150sn Mild ffi liquid: (A) liquid 0. IM N, 1q old)
04 /1.5M (NH4)2SO4(11117,
0) (B) N & 0.1 Hi Naz
IIPO4 elution rate = 4 pumps/min Detection: 280 rra + absorbance/CSF activity This process yielded approximately 7 x 104 units of total C3F with a recovery of 339%. The Chroma I-Graphene customer pattern is shown in Figure 2.

(f-1) Reversed phase 11PLc (high performance liquid chromatography) active C3F fraction 1 activity 2.5XIO7 units)
Dialysis overnight (4°C) against 9mM t-thorium butyrate (pH 7,2).
H! ,, after lyophilization, 50mM sodium acetate
(pl+ 7.2) and then dialyzed again (4°C, 4 hours) against 50mM sodium acetate (pH 7,2).

Dialysis a? & reverse phase HP! , C (machine name: Pro RP
C11R5/10 (manufactured by Pharmacia) under the following conditions.

Size: 5i+aXlOcI++ Eluate 20, 1% TFA (ρII 2.5) 0.
1% TFA 8094 Acetonitrile elution quick melon: 0
．． 3+*I/min Pressure: IMPa Detection: 280 Na + Absorbance Both proteins were collected, dialyzed against 0.1 M sodium acetate, lyophilized, and further reactivated to assay C3F activity. 75.5 pg of CSF was obtained. Note that the C5F of the present invention is the beak 2 in FIG.

(f-2) After lyophilizing both active CSF components obtained in (f-1) [approximately 20 μg (protein)], 2% t'denle6dyl sodium (SDS) and 0.3 M Kushi. 60IlIM Tris-HCl buffer (pH 6,
8) was dissolved in reverse phase 1' [7] under the following conditions.
5 tt g of C3F was obtained.

Column: l1i-pore RP-304 (Rio-
Rad Jl) Column size - φ4.6I x 250
Separate mobile phase: (A) 0.1% trifluoroacetic acid (B) 80% 7cetonitrite 11 0°1% trifluoroacetic acid gradient: (B) from 0% to 1 (10% (80
Flow rate: la+l/min Detection: 215r++m The absorbance elution pattern is shown in FIG.
Met.

[Brief explanation of drawings]

Figure 1 shows the gel filtration elution pattern of CSF using Heptaphryl S-200, Figure 2 shows the elution pattern of hydrophobic chromatography, and Figure 3 shows the elution pattern of reverse high performance liquid chromatography (using RPC+1115/10). , Figure 4 shows reversed-phase high-speed, skin chromatography (R
11-304) is shown. Patent author Midori Juji Co., Ltd. Patent attorney Takashima −■−，】゛−)−・
:The first figure of the book is the number. C5F activity (x10+ focal position)

Claims (1)

[Claims] (1) A colony formation stimulating factor that is a glycoprotein that can be recovered from human urine and has the following properties. (a) Molecular weight: approximately 70,00 as measured by gel filtration method
0 (b) Isoelectric point: pH approximately 4.7 (c) N-terminal amino acid sequence: [There is an amino acid sequence] (- in the sequence means that it cannot be measured) (d) In bone marrow cells (e) Protein to carbohydrate ratio: Sugar content approximately 13-20%