JPH0328198B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a method for producing a novel physiologically active proteinaceous substance having cell proliferation-promoting effects and DNA synthesis-promoting effects. The substance produced by the present invention (hereinafter referred to as the substance of the present invention) is expected to be used as an immunostimulant and an anticancer agent. Prior Art Conventionally, the following examples are known as substances having DNA synthesis-promoting effects and cell proliferation-promoting effects. (1) PDGF (Platelet-derived growth facor) Proc.Natl.Acad.Sci., 76 , 1809 (1979) (2) TGF (Transforming growth factor) J.Biol.Chem. 257 , 5220 (1982) (3) EGF (Epidermal growth factor) Anal.Biochem. 111 , 195 (1981) (4) FGF (Fibroblast growth factor) Ann.Rev.Biochem. 45 , 531 (1976) (5) FSLA (Fibroblast Somatomedin like
85 , 267 (1980) ( 7) IGF- (Insulin like growth factor) Diabetes 31 , 2823 (1982) ) (8) MSA (Multiplication stimulating activity)
Nature 272 (23), 776 (1978) Among these, the primary structures of substances No. 1 to 4, 6 and 7 have been clarified, but they clearly differ from the substance of the present invention in their N-terminal structure. Other substances are difficult to separate from medium components and have no practical value. JP-A-59-181293 discloses a protein substance with DNA synthesis promoting activity produced by myeloid leukemia cells, which is clearly different from the substance of the present invention in terms of molecular weight and other properties. Problems to be Solved by the Invention and Means for Solving the Problems In order to obtain useful immunostimulants and anticancer agents, the present inventors searched for substances that promote DNA synthesis and cell proliferation in animal cells. As a result, they found that when myeloid leukemia cells were cultured in a protein-free medium, a new substance that significantly promoted the proliferation of Burkitt's lymphoma-derived lymphoblastoid cells accumulated in the culture supernatant. The substance is a protein substance that has the effect of promoting DNA synthesis and cell growth in animal cells,
Based on its remarkable activity, it is expected to be used as an immunostimulant and an anticancer agent. Components of the Invention The present invention provides a method for producing a novel proteinaceous substance that has the effect of promoting DNA synthesis and cell proliferation in animal cells. The substance of the present invention has the following physical and chemical properties. (1) Molecular weight: 20000±2000 Molecular weight is SDS (sodium dodecyl sulfate)
-Polyacrylamide gel electrophoresis [SDS-
PAGE method, New Experimental Chemistry Course (20) Biochemistry () p118, edited by the Chemical Society of Japan, published by Maruzensha Publishing]. Concentrating gel 3% and separating gel 16%
Analysis of acrylamide concentration showed a single band with a flow sample volume of 20μ. For staining, silver staining reagent "Daiichi" manufactured by Daiichi Chemical Co., Ltd. was used. Molecular weight markers include lysozyme (MW14400), soybean trypsin inhibitor (MW21500), carbonic anhydrase (MW31000), ovalbumin (MW45000), and bovine serum albumin (MW66200).
and phosphorylase B (MW92500) were used. All molecular weight markers are BIO-RAD
Manufactured by Lab. (2) N-terminal protein primary structure Met-Gln-Ile-Phe-Val-Lys-Thr-
Leu−Thr−Gly−Lys−Thr−Ile−Thr−Leu
−Glu−Val−Glu−Pro−X−Asp−X−ILe
-X-Asn-Val-X-Ala-X-Ile- (X indicates an unidentified amino acid.) The amino acid sequence was obtained using an Applied Biosystems 470A sequencer and Spectra Physics. Determination was made in combination with high-performance liquid chromatography (manufactured by Seiko Co., Ltd.). (3) Thermal stability: Stable at PH7.3, 50℃, 30 minutes. The substance of the present invention (lyophilized powder) was added to the culture supernatant for 20 minutes.
PBS (-) at PH7.3 (NaCl 8g/, KCl 0.2g/,
After dissolving in Na ₂ HPO ₄ 1.15 g/, KH ₂ PO ₄ 0.2 g/) and heating in water solution at 50°C for 30 minutes, cell proliferation promoting activity was measured using lymphoblastoid Namalva strain using the following method. . (4) PH stability: Stable for 24 hours at 4℃ and PH2-3. The culture supernatant of K562-T1 strain was reduced to a molecular weight of 3000.
Concentrate 200 times with a holoph eye bar and reduce this to 0.1
% acetic acid at 4° C. for 24 hours, and the cell proliferation promoting activity of the dialysate was measured using the lymphoblastoid Namalva strain by the following method. (5) Isoelectric point: 6.5±0.5 Isoelectric focusing method uses 110ml LKB8100
(LKB, Sweden) column is used, the concentration of ampholyte ampholyte is 1.9-0.625%, and a pH gradient of 3-10 is used. Cool to 4℃,
Perform electrophoresis for 48 hours at 900V and 10mA, and after completion,
Fractionate with a fraction collector and measure the PH and activity of each fraction. (6) DNA synthesis promoting activity and cell proliferation promoting activity
DNA synthesis promotion activity measurement method Test cells 1 to 5 x ¹⁰⁵ cells/ml are RPMI-
Glutamine (4 m
M), streptomycin (25 μg/ml), penicillin (25 U/ml), Hepes (10 mM), baking soda (0.01%), and calf serum (10%) were cultured in 25 ml of medium for 2 days at 37°C. culture
Centrifuge at 800 xg for 5 minutes to collect cells and collect ⁵ x 105
Cells/ml were suspended in the medium obtained by removing calf serum from the above medium, dispensed into a 24-well multi-dish plate, cultured at 37°C for 24 hours, and then replaced with fresh medium. The cells were incubated at ℃ for 16 hours. Again, replace with fresh medium, sample 0.1ml,
0.9 ml of medium was added. After culturing at 37℃ for 6 hours,
Add tritium thymidine to 0.5μCi/well and use a liquid scintillation counter to
Its uptake activity was measured. The measured values are the average values of two or more experiments. Cell proliferation promoting activity Test cell line 5-10×10 ⁵ cells/ml with RPMI-
Glutamine (4 m
M), streptomycin (25 μg/ml), penicillin (25 U/ml), Hepes (10 mM), baking soda (0.01%), and calf serum (10%) were cultured in 75 ml of medium at 37° C. for 48 hours. 800 cultures
×g, centrifuge for 5 minutes to collect cells, wash with the above medium minus serum, dispense into a 24-well multi-dish plate, add 0.9 ml of serum-free medium and 0.1 ml of sample solution. was added. 37
After culturing in a CO ₂ incubator at ℃ for 3 days, the number of cells was measured using a hemocytometer. In the case of adhesion-dependent cells, immediately before measurement, 0.1% trypsin was added to suspend the cells, followed by measurement. The activity was expressed as the value obtained by dividing the number of cells with the addition of the sample by the number of cells without the addition of the sample, with the value without the addition of the sample being 100. The DNA synthesis promoting activity and cell proliferation promoting activity for various cells of the substance of the present invention obtained by the above measurement method were as shown in Table 1. All cells in Table 1 except those marked with * are human-derived cells. [Table] Indicates no activity.
As mentioned above, the substance of the present invention has DNA synthesis promoting activity and cell growth promoting activity against various animal cells, and is expected to have the following uses. (1) It can be used as an immunostimulant because it promotes the proliferation of B-cell cells that have antibody-producing ability. (2) It promotes the proliferation of Erythro leukemia cells (K562), so it can be applied to anemia. (3) Since it promotes the proliferation of T-cell cells, it can improve cellular immune function and can be used as an anticancer agent. (4) It can be used as an anticancer agent because it promotes the proliferation of granulocytes (K562) and microphage cells (monocytes), and as a therapeutic agent for wounds because it increases DNA synthesis in epithelial cells. (5) Since it promotes the proliferation of B-cell and Myeloma cells, it can be applied to in vitro antibody production. Considering the various properties of the substance of the present invention, ubiquitin fractionated from calf thymus can be cited as a substance similar to the substance of the present invention. The 30th amino acid sequence from the N-terminus of the substance of the present invention shows almost the same sequence as ubiquitin. However, since ubiquitin has a molecular weight of approximately 8,500 and does not exhibit a proliferation-promoting effect on Namalva cells, it is clear that the substance of the present invention is a substance different from ubiquitin. Substances in which ubiquitin is attached to histone proteins, and precursors in which ubiquitin molecules are linked are known [Nature 225 , 423, 1975; Journal of
Biological Chemistry (JBC), 250 ,
7182, 1975: Nature 31 2, 663,
1984]. These substances also differ in molecular weight etc.
Furthermore, the biological activities of these substances have not been clarified. Recently, there has been a report that a gene for a ubiquitin precursor has been discovered [JBC 260 , 7609, 1985].
There is no mention of producing proteins using this.
Furthermore, since the amino acid sequence encoded by this gene contains many basic amino acids, it is considered that it is degraded by proteolytic enzymes in vivo and does not exist as a protein. The substance of the present invention can be produced as follows. The substance of the present invention can be produced by culturing human myeloid leukemia cells in a medium, allowing them to accumulate in the culture, and collecting them from the culture. As the human myeloid leukemia cells used in the present invention, any cells that can produce the substance of the present invention can be used. As a suitable example, myeloid leukemia cell line K562-T1 can be used. The K562-T1 strain is a protein-free medium-acclimated cell obtained by sequentially lowering the serum concentration of the K562 strain from Ham's F10 medium containing 10% fetal calf serum and undergoing an acclimatization period of about 1 year. The K562 stock is owned by the Proceedings of the National Academy of Sciences.
of Science (Proc.Natl.Acad.Sci.),
USA., 76 , 1293 (1979), Blood,
45, 321 (1975), GANN, 73 , 97 (1982)
This is a commonly available cell line among the known cell lines described in, etc. Media include Ham's F10 medium, Ham's F12 medium (manufactured by Flow Lab), Dulbecco's MEM medium,
Protein-free media such as MEM medium and RPMI-1641 medium (all manufactured by Nissui Pharmaceutical Co., Ltd.) and mixed media thereof are used. Glutamine may be added to the medium if necessary.
0.5-5mM, antibiotics [penicillin (25U/
ml), streptomycin (25 μg/ml), etc.
You may also add an appropriate amount of baking soda (0.01%). For culturing, various culture bottles, shears, roller bottles, spinner flasks, jar fermenters, etc. can be used. The culture medium usually has a seed cell density of 5 x 10 ⁴ to 1 x 10 ⁶ cells/ml, with a seed cell density of 30 to 40
℃ for 2 to 4 days, the substance of the present invention is mainly produced in the culture solution depending on each cell density. For example, 1
At a seed cell density of ×10 ⁵ cells/ml and incubation for 2 days at 37° C., 300 units/ml of active substance are produced in the culture medium. The substance of the present invention is collected from the culture as follows. That is, the obtained culture supernatant is concentrated using freeze-drying, ultrafiltration, strongly acidic ion exchange resin, or the like. For elution, use a weakly basic buffer or a low concentration alkaline solution. Since the concentrated solution contains many low-molecular salts and impurities, it is dialyzed with 1% acetic acid. This removes much of the impure protein as a precipitate. Acetic acid is removed by freeze-drying, and the product is further concentrated to obtain a crude product. The crude product is passed through a cation exchange resin. The active substance has only a weak interaction with the cation exchange resin and is not adsorbed but eluted. The active fractions are collected, concentrated by freeze-drying, etc., and then low-molecular substances derived from the medium are removed by gel filtration. Gel filtration agents include Cephadex (Pharmacia,
(manufactured by Huain Chemical Co., Ltd.) biogels (manufactured by Biorad Co., Ltd.), control pore glass (manufactured by Corning Glass Works Co., Ltd.), Toyo Pearl (manufactured by Toyo Soda Co., Ltd.), etc. are used. When Bio-Gel P-60 or the like is used, the active fraction is located between the void volume (Vo) and low molecular weight substances such as salts. The concentrate obtained by the above operation can be purified as a single component by further performing high performance liquid chromatography. Example 1 8×10 ⁵ myeloid leukemia cell line K562-T1
The cells/ml were placed in 20 large spinner flasks containing 8 of the following medium and cultured at 37°C for 3 days. The culture medium was Ham-F10 medium (manufactured by Flow Lab) and MEM of Darbetsuko et al.
A medium (manufactured by Nissui Pharmaceutical Co., Ltd.) was mixed at a ratio of 3:1, containing pyruvic acid (5mM), selenite (1.25×10 ^-7 M), glasstose (1mg/ml), glutamine (4mM), A protein-free medium supplemented with penicillin (25 U/ml), streptomycin (25 μg/ml) and baking soda (0.01%) was used. 120% of the culture supernatant was filtered with hollow fibers (molecular weight cut-off).
3000, manufactured by Amicon) to about 100 ml.
Approximately 100 ml of the concentrate was dialyzed against 1% acetic acid at 4°C for 24 hours. Centrifuge the resulting precipitate (12000 rpm,
10 minutes), and the supernatant was lyophilized to remove acetic acid to obtain a powder. This was dissolved in 120 ml of distilled water. The solution obtained above was passed through a 160 ml QAE-Sephadex-A50 (manufactured by Pharmacia Huain Chemical Co.) column in two portions of 60 ml each.
Elute with 150 ml of PBS(-), then add 0.1% acetic acid 190
ml and active fraction eluted with PBS(-) 300
Got ml. The activity recovery rate was approximately 8.3%, and the activity increased 1.9 times. 300 ml of this active fraction was freeze-dried, dissolved in 20 ml of distilled water, and passed through a column containing 400 ml of Bio-Gel P-60 in two portions at SV5.0. PBS(−)
The eluate was fractionated into 6 ml portions to obtain 60 ml of the 41st to 45th fractions. The recovery rate of activity was 1.7%;
Specific activity increased 13.3 times. This active fraction was analyzed using FPLC (Fast) manufactured by Pharmacia Huain Chemical Co., Ltd.
protein liquid chromatography) Mono S 5
It was passed through a column containing ml. Washed with PBS(-) and eluted with 0-0.5M saline. 1ml of eluate
The activity was found in the 53rd fraction. The recovery rate of activity was 0.2%, and the specific activity increased 3600 times. This active substance is called LGF-1. The results of the above purification process are shown in Table 2. [Table] Example 2 Purification step Bio-Gel obtained in Example 1
Using the P-60 active fraction, Namalva cells (B-cell
), K562−T1 (Erythroleukemia, non T
The proliferation promoting activity of CCRF-CEM (T-cell type) for each cell was measured as described above.
The results are shown in Table 3. [Table] Example 3 Purification step Bio-Gel P obtained in Example 1
-60 active fraction was used to test KB cells and HeLa.
Cellular DNA synthesis promoting activity was measured as described above. Table 4 shows the ³ H-thymidine incorporation rate when the active fraction was added, with the amount of ³ H-thymidine uptake taken as 1 when no active fraction was added. [Table] Effects of the invention
Provided is a method for producing a novel proteinaceous substance that has DNA synthesis-promoting effects and cell proliferation-promoting effects.

Claims (1)

[Scope of Claims] 1 Myeloid leukemia cells having the ability to produce a novel proteinaceous substance having the following physicochemical and biological properties are cultured in a medium, the proteinaceous substance is accumulated in the culture, and the proteinaceous substance is accumulated in the culture. A method for producing a novel proteinaceous substance, which comprises collecting the proteinaceous substance from a culture. (1) Molecular weight: 20000±2000 (SDS-PAGE method) (2) N-terminal protein primary structure Met-Gln-Ile-Phe-Val-Lys-Thr-
Leu−Thr−Gly−Lys−Thr−Ile−Thr−Leu
-Glu-Val-Glu-Pro-X-Asp-X-Ile-
X-Asn-Val-X-Ala-X-Ile- (X represents an unidentified amino acid.) (3) Thermal stability: Stable when treated at PH7.3, 50°C for 30 minutes. (4) PH stability: Stable to treatment at 4°C, PH 2-3 for 24 hours (5) Isoelectric point: 6.5±0.5 (6) Has a growth-promoting effect on the following cells. Human B lymphocyte cell Human T lymphocyte cell Human Myeloma cell Human Monocyte cell Human non-T non-B cell (7) It has a DNA synthesis promoting effect on the following cells. Human B lymphoid cells Human T lymphoid cells Human Myeloma cells Human Monocyte cells Human non-T non-B cells Human epithelial cells