MXPA96000765A - Protein and methods to produce prote - Google Patents

Abstract

A protein derived from human fibroblast, with N-terminal amino acid sequence represented in Sequences Number 1 and 2, with a molecular weight of ca. 15 kD under conditions of reduction and not reduction by (electrophoresis of polyacrylamide gel - sodium dodecyl sulfate), and with an activity to stimulate the growth of osteoblasts. The protein containing the amino acid sequence represented in Sequence Number 9 and having osteoblast growth activity. A method for preparing the protein by culturing human fibroblast and treating the conditioned medium for purification. The protein is produced by genetic engineering procedure. The protein is used for the treatment of conditions with mass reduction such as osteoporosis, or it is used as an antigen for the immunological diagnosis of the disease

Description

SPECIFICATION PROTEIN AND METHODS FOR PRODUCING PROTEIN Field of the Invention This invention relates to a new protein, basic osteoblast growth factor II (bOFG-II), which stimulates the growth of osteoblasts, and methods to produce the protein.

Previous technology • "-O Human bones are always being reconstructed through the repeated process of resorption and reconstitution.In this process, osteoblasts and osteoclasts are considered the main cells responsible for bone formation and bone resorption, respectively. typical of a The condition caused by an abnormal bone metabolism caused by bone cells is osteoporosis. It is known that this condition is caused by a condition where bone resorption, performed by osteoclasts, exceeds bone formation, the task of osteoblasts, although it has not yet been completely clarifies the mechanism of osteoporosis. Osteoporosis causes pain in the bones and weakens them, which leads to fractures. Since osteoporosis increases the number of elderly people prostrate in cane, it has become an aspect of social interest with the increase in the number of people of the third age. Therefore, it is expected that effective drugs will be developed for its treatment. It is believed that the reduction of bone mass caused by abnormal bone metabolism can be treated by inhibiting bone resorption, improving bone formation, or improving balanced metabolism. It is expected to promote bone formation by stimulating the growth, differentiation or activation of osteoblasts. Recently, cytokines, which stimulate the growth or differentiation of osteoblasts, have attracted public attention and have been studied intensively. It has been found that many cytokines stimulate the growth of osteoblasts, ie the fibroblast growth factor (FGF) (Rodan SB, et al., Endocrinology vol 121, pl917, 1987) insulin-like growth factor I (IGF -I) (Hock JM, et al., Endocrinology vol 122, p254, 1988), insulin-like growth factor II (IGF-II) (McCarthy T., et al., Endocrinology vol 124, p301, 1989), bone orfogenetic protein (BMP) (Sampath TK, et al., J. Biol. Chem., Vol 267, p20532, 1992, Knutsen R. et al., Bichem, Biophys. Res. Common, Vol. .194, p.1352, 1993, and Akira Yamaguchi, et al., Zikken Igaku, vol.10, p2003, 1992). It has also been shown that many cytokines stimulate the differentiation of osteoblasts, for example, transforming growth factor-β (TGF-β) (Centrella M., et al., J. Biol. Chem. Vol.262, p2869, 1987), insulin-like growth factor (IGF), and bone morphogenetic protein (Takuwa Y., et. Al., . r Biochem. Biophys. Res. Commun. Vol.174, p96, 1991, and Knutsen R. et al., Biochem. Biophys. Res. Commun. Vol.194, pl352, 1993). It is expected that these cytokines are effective drugs to improve bone mass by stimulating bone formation; now some of the cytokines, such as bone morphogenetic proteins in chemical tests, are being investigated in order to know their effects in the care of patients suffering from bone diseases. '.0 Examples of drugs currently used clinically for the treatment of bone diseases and to shorten the period of treatment are vitamin D3 dehydroxyl, calcitonin and its derivatives, hormones such as estradiol, ipriflavon and calcium preparations. However, these drugs do not offer satisfactory therapeutic effects, and the development of new substances for medicines is expected. As mentioned, metabolism is controlled by the balance between bone resorption and bone formation. Therefore, it is expected that cytokines will be developed that stimulate osteoblast growth and osteogenesis as drugs for the treatment of bone diseases such as osteoporosis.

Disclosure of the invention The inventors have intensively searched for osteoblast growth factors, and found a novel osteoblast growth factor. The inventors have also established methods for accumulating the protein in a high concentration and for purifying it efficiently. A cDNA clone was isolated with this protein by using partial amino acid sequences of the native bOFG-II protein. In addition, bOGF-II was produced by genetic engineering techniques with this cDNA. The aim of the invention is to provide a new osteoblast growth factor (protein) and methods to efficiently produce the protein. The inventors investigated the conditioned media for the culture of animal cells, and found the osteoblast growth factor in a conditioned medium of human fibroblast IMR-90 (ATCC-CCL186). The inventors examined the culture conditions of the IMR-90 cells, and have established the method for culturing the cells in alumina ceramic fragments to accumulate the osteoblast growth factor in a high concentration in the culture medium. The inventors found the method to efficiently purify bOGF-II by a combination of ion exchange column and / or heparin column. In addition, the inventors determined the amino acid sequences of the bOGF-II protein, designed the preparations based on these amino acid sequences and obtained cDNA fragments from bOGF-II from a cDNA library of IMR-90 cells. A cDNA clone encoding the entire protein of the present invention was isolated from a cDNA library of IMR-90 cells by hybridization, using the cDNA fragments as a probe. In addition, the inventors established a method to produce recombinant bOGF-II in the culture medium of the cells, which was transformed into the expression vector containing the cDNA. This invention relates to a protein characterized by the following features: (1) derived from human fibroblast cells, (2) molecular weight from ca. 15 kD in electrophoresis of polyacrylamide gel - sodium dodecyl sulfate (SDS-PAGE) under conditions of reduction and non-reduction, (3) a high affinity for cation exchanger and heparin, (4) reduction in the growth activity of osteoblasts by heating at 70 ° C for 10 minutes, and (5) deactivation by heating at 90 ° C for 10 minutes. The bOGF-II of the present invention is, in appearance, different from the growth factors of osteoblasts in N-terminal amino acid sequence. The N-terminal amino acid sequences of bOGF-II are shown in Sequences Number 1 and 2. The invention also relates to a method for producing bOGF-II, which includes: (1) the culture of fibroblasts - "human", (2) treatment of the culture medium by a heparin column, (3) levigation of an adsorbent fraction, (4) treatment of the eluate by an anion exchange column to obtain a non-adsorbent fraction, (5) application of the fraction to the cation exchange column, and (6) purification of the target protein by a heparin column. The purification procedure according to the invention includes all procedures having the same effect as that obtained by the method for mixing a culture medium with heparin Sepharose, etc. in an operation throughout the lot and for treatment through a column, as well as a simple method to flow the culture medium through a column of heparin-Sepharose, etc. In accordance with the invention, bOGF-II can be effectively isolated from a culture medium of human fibroblasts at a high yield. The isolation of bOGF-II is based on general means to purify biomaterial proteins, using physical and chemical properties of the target bOGF-II protein. For example, the concentration procedure includes general biochemical techniques, such as ultrafiltration, lyophilization and dialysis. The purification procedure includes combinations of various techniques for the protein purification, such as gel filtration chromatography, hydrophobic chromatography, chromatography < -'- 'reverse phase, and preparative gel electrophoresis. The human fibroblast preferably is IMR-90. The culture medium for the human fibroblast cell IMR-90 is obtained by absorption of IMR-90 human fibroblast cell in ceramic, and cultivating in DMEM medium supplemented with a 5% fetal calf serum in a cylindrical bottle in steady state for about a week. For purification, 0.1% CHAPS (3- [3-colamidopro-'* "*) pyl) -dimethylane] -1-sulfonate propane) is preferably added to a regulator as a surfactant.The protein of the invention is purified by applying the culture medium to a column of heparin (heparin-Sepharose CL6B, manufactured by Pharmacia), levigado with a regulator lOmM Tris-HCl with 2 M NaCl, pH 7.5 by applying the diluted fraction to anion exchange column Q (Hiload-Q / FF, manufactured by Pharmacia), collecting a non-adsorbent fraction and applying the fraction obtained to the cation exchange column S (Hiload-S / FF, manufactured by Pharmacia) for split three peaks, bOGF-I (0.15 M NaCl), bOGF-II (0.35 NaCl) and bOGF-III (0.55 M NaCl) in the order of levigating the activity at lower concentrations of NaCl. BOGF-II can also be isolated by the following repeated heparin column chromatography (heparin-5PW, manufactured by Toso Co. ) and can be identified by the properties previously described. It is likely that the bOGF-II protein is a factor "- of fibroblast growth from the results where it is deactivated when heating at 70 aC for 10 minutes, it is levigated by ca 1.8 M NaCl of 5 P heparin column, and is deactivated by an anti-background fibroblasts growth factor antibody. In addition, a method for producing recombinant bOGF-II was established. The method includes the following three steps; first, bOGF-II amino acid sequences are used to "0 to prepare preparation oligonucleotides, second, a fragment of bOGF-II cDNA is obtained by PCR amplification using the preparation Finally, the cDNA clone encoding all bOGF-II is isolated from a cDNA library of IMR cells -90 by hybridization, using the fragment of cDNA as a probe. In addition, bOGF-II is recovered and purified from the culture medium or cells by culturing selected eukaryotic host cells such as mammalian cells (eg Chinese hamster ovarian cells) or prokaryotes such as bacteria (e.g. -coli), which are transfered by vector with an expression promoter, and the complete bOGF-II encoded in the cDNA. The invention relates to proteins that have an activity to stimulate the growth of osteoblasts, which contain, as a part, the amino acid sequence described, or having a homology with the amino acid sequence described in more than 80%, and cDNA of the protein.

"The activity of osteoblast growth factor can be evaluated by using osteoblast cell lines of normal osteoblasts as target cells, measuring an increased incorporation of 3H-thymidine into the cells." The target cell is preferably cell mouse osteoblastic line MC3T3-E1 (J. Oral, Biol., 23, 899, 1981 and J. Cell, Bio, 96, 191, 1983) It has been shown that the cell responds to vitamin D3 and parathyroid hormone and that It grows until it is calcified in vitro in a manner similar to calcification in vivo.The activity of osteoblast growth factor is preferably measured with a serum-free medium, and can be evaluated very accurately at a high sensitivity. measuring the incorporation of 3H-thymidine 15 bOGF-II is useful as a pharmaceutical composition to treat or improve reduced bone mass in conditions such as osteoporosis and others with an abnormal bone metabolism, or as an antigen an immunological diagnosis of diseases. The bOGF-II is formulated to be a preparation Pharmaceutical, and can be administered orally or parenterally. The preparation includes bOGF-II as an effective ingredient, and can be administered safely to humans. Examples of the pharmaceutical preparation include compositions for intravenous injection or drip, suppositories, preparations nasals, sublingual preparations and tapes for percutaneous absorption. The preparation for injection is a mixture of bOGF-II in a pharmacologically effective amount and a pharmaceutically acceptable agent. The agent is a vehicle / activator that is generally added to the compositions for injection, for example, amino acids, saccharides, cellulose derivatives and other organic / inorganic compounds. When bOGF-II is mixed with the vehicle / activator to make injections, pH adjuster, regulator, stabilizer, solubilizing agent, etc. can be added. , as necessary.

Brief description of the illustrations Figure 1 shows a levigation pattern of bOGF fractions in a cation exchange column (HiLoad-S / HP ™, 2.6 x 10 cm., Manufactured by Pharmacia Co.). In Figure 1, peak 1 indicates bOGF-I, while peak 2 indicates bOGF-II, and peak 3 indicates bOGF-III. Figure 2 shows the bOGF-II pattern in an affinity column (Heparin 5P ™, 0.8 x 7.5 cm., Manufactured 20 by Toso Co.). Figure 3 shows the bOGF-II pattern in polyacrylamide gel-sodium dodecyl sulfate electrophoresis (SDS-PAGE) under non-reducing condition. In Figure 3, column 1 indicates the 25 molecular weight marker, number 2 indicates fraction A, 3 indicates fraction B, 4 indicates fraction C, 5 indicates fraction D and. - * - 6 indicates fraction E. See Illustration 2 for fractions A-E. Figure 4 shows the levigation pattern of the peptides of reduced bOGF-II PE that was digested with Asp-N endoprokinase (manufactured by Bayringer Co.) on a reverse phase column (OD-300, C18, 2.1 x 200 mm, manufactured by Applied Biosystems Co.). Figure 5 shows the bOGF-II activity tested. Figure 6 shows the structure of pQE30-OGF-II plasmid. In this drawing, 6His represents a grumo de e histidina; Phage T5 promoter represents phagopromotor T5; bla represents a gene resistant to ampicillin; Ori represents 15 replication origin in E. coli; and bOGF-II represents OGF-II cDNA, respectively. Figure 7 shows the His6-bOGF-II activity tested with MC3T3-E1. In the drawing, column 1 shows a control and column 2 shows the aggregate solution activity of 10% His-bOGF-II respectively.

Preferred Modes for Carrying Out the Invention [Examples] A detailed description of the invention is provided by the following examples. However, it should be noted that the examples are merely for illustration and the invention is not limited to them. Example 1 Natural-type production of bOGF-II 5 (1) preparation of a conditioned human fibroblast medium IMR-90. IMR-90 human fetal lung fibroblast was cultured (ATCC-CCL186) in ceramic alumina fragments (80 g) (alumina: 99.5%, manufactured by Toshiba Ceramic KK) in one or a half DMEM (manufactured by Gibco Co.) supplemented with 5% FCS and lOmM regulator HEPES (cylindrical bottle / 500ml) in steady state at 37 SC in the presence of 5% C02 for 7 to 10 days using cylindrical bottles 60 (190 cm2, lOOx 127 mm., Manufactured by Coning Co.). 5 The conditioned medium was harvested and a fresh medium was added to obtain 30L of conditioned medium IMR-90 in a culture lot. The conditioned medium obtained was designated as sample 1. 0 (2) A method for testing the activity of osteoblast growth The activity of osteoblast growth factor was evaluated by incorporating DNA into mouse osteoblasts MC3T3-E1 (donated by Dr. Masayoshi Kumegawa, 5 Professor of the Department of Dentistry of the Meikai University). In a precise way, a sample solution (50 μl) that had been diluted with an aMEM medium (manufactured by Gibco Co.) without nucleic acid content and supplemented with 0.2% BSA, was transferred to a 96-well microplate. Next, the MC3T3-E1 cells in the a-MEM medium were inoculated at 372C in air with 5% C02 for 15 to 20 hours. After the culture, 10 μl of 3H-thymidine (TKR686, manufactured by A asham Co.) diluted with phosphate buffered saline adjusted to O.lmCi / ml was added to each of the cavities. After three hours, the radioactivity of the H-thymidine incorporated in the cells was measured by a Matrix β counter (manufactured by Packard Co.). (3) Purification of bOGF-II (i) purification in heparin Sepharose CL-6B Conditioned medium IMR-90 (ca.90L) (sample 1) was filtered with a membrane filter of 0.22 μm (Milidisk hidrofi-lico, 2000 cm3, manufactured by Milipore Co.), and divided into three fractions. Each fraction was applied to heparin Sepharose CL-6B (5 x 4.1 cm) (80 ml) equilibrated with lOmM Tris-HCl 0.3M NaCl pH 7.5. After washing with 10 mM Tris HCl, pH 7.5 at a flow rate of 500 ml / hr. , a heparin adsorbent protein fraction Sepharose CL-6B is levigated with 10 mM Tris HCl, 2M NaCl, pH 7.5. This fraction was designated as sample 2. ii) Purification in HiLoad-Q / FF - "- The absorbent fraction of heparin Sepharose (sample 2) was dialyzed against 10 mM Tris HCl, pH 7.5, supplemented with CHAPS at the final concentration of 0.1%, incubated at 42C for 5 hours. overnight and divided into two fractions, each fraction was then applied to an anion exchange column (HiLoad-Q / FF 2.6 x 10 cm, manufactured by Pharmacia Co.) equilibrated with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5 to obtain a non-adsorbent fraction (1000 ml).

"" -O designated as sample 3. iii) Purification with HiLoad-S / HP The non-adsorbent fraction of HiLoad-Q (sample 3) was applied to a cation exchange column (HiLoad-S / HP, 2.6 x 10 cm , manufactured by Pharmacia Co.) balanced with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5. After washing with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein was levigated with 0-1M Nací in a linear gradient for more than 100 minutes at a flow rate of 8 ml / min. The eluate was fractionated to 12 ml / fraction. Factor activity was evaluated of osteoblast growth of each fraction (10 μl), in accordance with the method described in example (2). The activity of osteoblast growth factor was found in three peaks (peak 1: bOGF-I, peak 2: bOGF-II, peak 3: bOGF-III). The result is shown in Figure 1. 25 It was assumed that peak 3 was bOGF, from the fact that the activity of osteoblast growth factor in - ^ peak 3 was deactivated by heating at 70 aC for 10 minutes, levigated with ca. 1.8M Heparin column NaCl and neutralized by anti-human bOGF antibodies. Iv) affinity column purification (heparin-5P) The fraction of peak 2 (bOGF-II) was diluted with 240 ml of 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5, and applied to an affinity column (heparin-5P, 0.8 x 7.5 cm, manufactured by Toso Co.) equilibrated with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5. Z '^ .- O After washing with 50mM Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein was levigated with 0-2M NaCl in a linear gradient for more than 60 minutes at a flow rate of 0.5 ml / min. The eluate was fractionated at 0.5 ml / fraction. Each fraction (2 μl) was evaluated to know its factor activity of osteoblast growth. It was found that a fraction (5 mi) levigated with ca. 1-0-1.2M NaCl had osteoblast growth factor activity and was designated as sample 4. Sample 4 (5 ml) was diluted with 10 ml of 50 mM Tris-20 HCl, 0.1% CHAPS, pH 7.5, and was applied to an affinity column (heparin-5P, 0.8 x 7.5 cm, manufactured by Toso Co.) equilibrated with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5. After washing with 50mM Tris-HCl, 0.1% CHAPS, pH 7.5, the absorbed protein was levigated with 0-2M NaCl in a linear gradient at a rate of flow of 0.5 ml / min. The eluate was fractionated at 0.5 ml / fraction.

The activity of osteoblast growth factor of each fraction (2 μl) was evaluated. It was found that a fraction (5 ml) levigated with ca. 1-0-1.2M NaCl had osteoblast growth factor activity and was designated as sample 5. 5 Sample 5 (5 ml) was diluted with 10 ml of 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5, and was applied to an affinity column (heparin-5PW, 0.5 x 7.5 cm, manufactured by Toso Co.) balanced. with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5. After washing with 50mM Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein "-0 was levigated with 0.2-0.8M NaCl in a linear gradient over 60 minutes at a flow rate of 0.5 ml / min.The eluate was fractionated at 0.5 ml / fraction Each fraction (4 μl) was evaluated for its osteoblast growth factor activity, the result is shown in Figure 2. 15 (4) Molecular weight of bOGF-II The obtained OGF fraction was divided into five fractions to 2 ml / fraction (Figure 2, AE fractions) Each fraction (100 μl) was subjected to polyacrylamide gel electrophoresis - SDS under no-reduction condition. of the AE fractions (100 μl) were dialysed against water, lyophilized, dissolved in 1.5 μl of a mixture of lOmM Tris-HCl, pH8, lmM EDTA, 2.5% SDS, 0.01% bromophenol blue and incubated at 379C during all night. The 1 μl sample was analyzed by polyacrylamide gel electrophoresis - SDS with a gradient gel of 8-25% acrylamide (manufactured by Pharmacia Co.) and an electrophoresis device (Fast - «- System, manufactured by Pharmacia Co.). The following molecular weight markers were used: phospholipase b (94kD), serum albumin (67kD), ovalbum (43kD), anhydrase carbonic (30 kD), tryptin inhibitor (20.1kD), and an α-lactalbumin (14.4kD). After electrophoresis, the protein bands were visualized by silver dyeing, according to a Pharmacia Co. protocol. The result is shown in Figure 3. * "- 0 The protein band detected in the molecular mass of 15kD was proportional to the activity of osteoblast growth factor. Fractions D and E contained only the protein band at 15kD. When this protein was analyzed by polyacrylamide gel electrophoresis - low SDS In the reduction condition, a single band of proteins was detected at almost the same molecular weight as that obtained under the no-reduction condition. (5) Determination of N-terminal amino acid sequence The obtained fraction D (500 μl) was applied to a reverse column phase (BU-300, C4, 2.1 x 220 mm, manufactured by Applied Biosystems Co.) equilibrated with a mixture of 0.1% trifluoroacetic acid (TFA), 10% acetonitrile, and eluate in a linear gradient of 10-60% acetonitrile for more than 50 minutes at a flow rate of 0.2 ml / min., to obtain the desalted sample and concentrated This mixture was analyzed by a protein sequencer (477A-120A, manufactured by Applied Biosystems Co.) for N-terminal amino acid sequence. The amino acid sequence of the peptide obtained is shown in Sequence Number 1. In the sequence, "Xaa" indicates an amino acid not yet identified. (6) Determination of amino acid sequence of protein Fraction C (2000 μl) was concentrated, dissolved with 300 μl of 0.5M Tris-HCl, pH 8.5 containing lOmM EDTA, 7M guanidine hydrochloride and dithiothreitol (1 mg), incubated at room temperature for four hours, it was further supplemented with 2 μl of 4-vinylpyridine and left in the dark at room temperature overnight to obtain pyridylethylation (PE). 3 μl of 25% TFA was added to the sample. The mixture was applied to a reverse base column (BU-300, C4, 4.6 x 30 mm, manufactured by Applied Biosystems Co.) equilibrated with 10% acetonil in a linear gradient for more than 50 minutes at a flow rate of 1. ml / min. to obtain reduced bOGF-II PE. A quarter of the reduced bOGF-II PE was analyzed by a protein sequencer (477A-120A, manufactured by Applied Biosystems Co.) for N-terminal amino acid sequence. The amino acid sequence obtained from the peptide is shown in Sequence Number 2. The remaining three quarters of the reduced bOGF-II PE were digested with 0.5 μl of the Asp-N offenoprotein (manufactured - by Boehringer Mannheim Co.) in a mixture (50 μl) of 50mM phosphate buffer, pH 8.5 containing ÍM urea at 37 aC for 15 hours, was applied to a reverse phase column (OD-5 300, C18, 2.1 x 220 mm, manufactured by Applied Biosystems Co.) equilibrated with 0.1% TFA, and levigated in a linear gradient of 0-40% acetonil for more than 80 minutes at a flow rate of 0.1 ml / min. The pattern of levigation is shown in the Figure 4. The five detected peaks were analyzed "" -Or through a protein sequencer (477A-120A, manufactured by Applied Biosystems Co. for amino acid sequence N-ter inal. Each amino acid sequence of the five peptides obtained is shown in Sequences Numbers 3 to 7. (7) Efficacy of bOGF-II in the proliferation of MC3T3-El cells. The protein concentration in fraction D was evaluated, according to Lowry's method, using BSA (bovine serum albumin) as standard. This sample was diluted to lOOng / ml and their activity of osteoblast growth factor was evaluated, with duplicate intervals between continuous doses, according to the method described in paragraph (2). The result is shown in Illustration 5.

Example 2. Preparation of bOGF-II by the application of genetic engineering (1) Cloning of the bOGF-II gene 1) Cloning of the bOGF-II cDNA gene by PCR A set of preparatory elements was elaborated to amplify the cDNA in the determined amino acid sequence base of OGF-II. That is, from Glu-Tre-Glu-Tir-Gli-Pro-Cis, an N-terminal amino acid sequence, the DNA sequence encoding it was deduced and the preparative elements were synthesized mixed with the sequence of 5'-GA - (A / G) ACNGA (A / G) TA (T / C) GGNCCNTG-3 '. Here, A / G means A or G; T / C means T or C; and N represents A, G, C, or T. Asp-Lis-Lis-Gli-Fen-Tir-Lis, an internal amino acid sequence, a DNA sequence was deduced that encoded it, and the mixed preparatory elements were synthesized with the complementary sequence chain of 51 -TT (A / G) TA (A / G) AANCC (T / 0) TT (-A / G) TC-3 '. For the synthesis of the preparatory elements, we used the DNA synthesizer 394 from Perkin Elmer Co. The two types of elements (200 pmol respectively) and the single chain cDNA, derived from human fetal lung fibroblast IMR-90 polyA RNA (1 μg), like the model DNA, were used for the polymerase chain reaction (PCR). The enzyme used was EX Taq (manufactured by Takara Shuzo Co., Ltd.) The reaction solution contained 5 μl of 10 x ExTaq buffer, 4 μl of 2.5 mM dNTP, 1 μl of the cDNA solution, 0.25 μl of Ex Taq. , 29.75 μl of distilled water, and each 5 μl of the preparatory element (40 μM) in a final volume of 50 μl. The reaction condition is as follows. After maintaining the reaction mixture at 95 ° C for 3 minutes, 30 cycles of 5 incubation steps consisting of 95aC for 3 minutes, 50SC for 30 seconds, and 709C for 2 minutes were performed. After these reactions, the reaction mixture was maintained at 70aC for 5 minutes. After the reaction was finished, 8 μl of the reaction solution was subjected to electrophoresis of 4% agarose gel; several bands were detected, including approximately 12 bp fragment. The reaction solution was mixed in a volume of 4.5 μl, pCRII (original TA cloning kit, manufactured by Invitrogen Co.), a collation vector at a volume of 0.5 μl, and a ligation kit DNA (version 2) liquid 1 (manufactured by Takara Shuzo Co. Ltd.) in a volume of 5 μl, and kept at 16aC overnight. Using 5 μl of the ligation reaction solution, transformation of E.coli DH5a (prepared by BRL Co.) was allowed. The length of inserted fragments in the host plasmid inserted into the resulting ampicillin resistance bacterium was measured by PCR, and 4 chains were isolated with approximately 120 bp inserted fragments. Two types of the preparatory elements mentioned above were used in the reaction. Of the After four isolated chains, the plasmid DNA was purified, and the DNA sequences of the inserted fragments were determined. When the deduced amino acid sequence of the nucleotide sequence was analyzed, all the clones had a reading frame that matched the amino acid sequence determined from the OGF-II protein. One clone (clone # 1) was subjected to the next experiment. 2) Cloning of complete OGF-II cDNA Clone plasmid # 1 was purified, digested by an EcoRI restriction enzyme (prepared by Takara Shuzo Co. Ltd.), and subjected to agarose gel electrophoresis; OGF-II cDNA of approximately 12 bp was isolated. This fragment was labeled with 32P using a Megapri e kit (manufactured by Amersham Co.) and a32P-dCTP and served as the probe in the following experiment. Human fetal lung fibroblast double chain cDNA was synthesized IMR-90 polyA * RNA (5 μg), according to the manual of the synthesis team of cDNA Great Lengths (manufactured by Clontech Co.). PolyA + RNA was made by Fast Track (manufactured by Stratagene Co.). The method for synthesizing the double-stranded cDNA is briefly described below. The preparative elements polyA * RNA (5 μg) and Oligo (dT) 25 (dN) were mixed and converted into a final volume of 12.5 μl with the addition of distilled water, the solution was maintained at 702C for 3 minutes and left Cool on ice for 2 minutes. In this solution, 3.2 μl of distilled water, 5 μl of 5 X first chain regulator, 0.5 μl of DTT (dithiitritol), 1.3 μl of dNTP (20 mM each) and 2.5 μl (500 units = of MMLV (RNaseH ") were added. ), and the mixture was maintained at 422 C. In addition, 123.5 μl of distilled water, 40 μl of 5 X second-chain regulator, 1.5 μl of dNTP (20 mM each) and 10 μl of second-chain enzyme cocktail were added, and the mixture was maintained at 162 ° C for 2 hours.In this reaction solution 15 units of T4 poly erase were added; the resultant was maintained at 16SC for an additional 30 minutes and the reaction was stopped by adding 10 μl of 0.2M EDTA followed by chloroform and isoamyl alcohol treatments and ethanol precipitation. For the terminal of this double stranded cDNA, EcoRI-SalI-Notl (prepared by Clontech Co.) was linked. Then, the resultant was inserted into DNA from a ZAP Express phage (prepared by Stratgene Co.) which had previously been cut with an EcoRI restriction enzyme and treated with CIAP (bovine alkaline phosphatase). The obtained recombinant DNA was packed and infected with E.coli XL1-Blue MRF '(prepared by Stratagene Co.): the plates were formed on NZY agar medium (0.5% NaCl, 0.2% MgSO4 «7H20, 0.5% yeast extract, 1% NZ amine, pH 7.5, 1.5% agar). For packing, Gigapack II Gold packaging extract (made by Stratgene Co.) was used. The phage formed on the agar medium was transferred to a nylon membrane, Hybond-N (manufactured by Amersham Co.) and the phage DNA was fixed. The resulting membrane was immersed in a hybridization buffer containing 100 μg / mL of salmon sperm DNA (manufactured by Amersham Co.) and treated for 4 hours at 642C, submerged in the mentioned regulator containing the labeled DNA probe. with ^ p. heat denatured (2.5 x 105 cpm / ml), mentioned above, at 652C overnight to allow hybridization. After the membrane was washed, the 3 clones having OGF-II cDNA of about one million phages could be selected. The 3 clones were purified by two more screens. Then, XL1 cells were infected. Blue-MRF, an E. coli strand with purified phages, infected cells were coinfected with phage Assistants Exassist (prepared by Stratagene Co.). The supernatant culture was used to infect E. coli XLOLR (prepared by Stratagene Co.), and E. coli cells were obtained which became kanamycin resistant. The plasmid DNA structure was analyzed in one of these E. coli clones and the nucleotide sequence of the inserted fragment was determined. The sequence is shown in Sequence Number 8. The amino acid sequence deduced from Sequence Number 8 of OGF-II is shown as Sequence Number 9. When comparing this amino acid sequence with the amino acid sequence of wild type OGF-II , it was found that the first one has an amino acid Lis more than the C-terminal one (85B amino acid). Accordingly, the C-terminal Usage of bOGF-II purified from the conditioned medium of IMR-90 cells may have been divided by (a) carboxypeptidase (s). This means that the 3 clones contained the DNA encoding an open reading frame, consisting of 85 amino acids of OGF-II N-terminal Glu-Tre-Glu-Tir. The open reading frame was determined from the position of the first termination code and the amino acid sequence from the OGF-II protein. One of the plasmids obtained is called pBK-CMV OGF-II (3). (2) Expression of the OGF-II cDNA in E. coli The total length of the OGF-II cDNA was amplified by PCR and isolated. The preparatory elements used were Q30F 5 '-GGGGATCCGAGACAGACAATATGGTC-3' and Q30R 51 -CCAAGCTTCTACTTGCTCTGCATACT-3 '. These elements were designed in such a way that the amplified products could be digested with restriction enzymes BamHI and HindIII. Using the 20 ng model of pBK-CMV OGF-II (3) and the preparatory elements (Q30F and Q30R), the PCR was carried out. The reaction solution contained 10 μl of 10 x ExTaq buffer, 8 μl of 2.5 mM dNTP, 0.5 μl of Ex Taq, 9.5 μl of the DNA solution, 70 μl of distilled water and 1 μl of the preparatory element (100 μM each) ) in a final volume of 100 μl. After maintaining the reaction mixture at 95 ° C for 3 minutes, 25 three-step incubation cycles, consisting of 95aC for 3 minutes, 50aC for 30 seconds, and 70aC for 2 minutes, were carried out. After these reactions, the reaction mixture was maintained at 70aC for 5 minutes. After amplification, the reaction mixture was maintained at 70aC for 5 minutes. After amplification, the preparative elements were removed with Microcon 100 (Amicon Co.) and the PCR product was digested with restriction enzymes Ba HI and HindIII; thereafter, the product was mixed with 20 ng of pQE30 (manufactured by QIAGEN, Co.) which previously ") cut with BamHI and HindIII and was used for ligation, using the ligation solution, the E. coli XL2-Blue (made by Stratagene Co.) was transformed from the obtained ampicillin-resistant colonies, a clone was separated with the inserted target fragment through an analysis of the fragments of DNA digested with restriction enzymes and performing the sequence. The plasmid in this clone is called pQE30-OGF-II. E. coli XL2-Blue (pQE30-OGF-II) with this plasmid has been deposited in NIBH, Agency of Industrial Science and Technology with the number "FERM BP-5139". The structure of the plasmid The content in the deposited E. coli is shown in Figure 6. This chain was cultured by shaking in super medium (2.5% bactotripton, 1.5% bacto-yeast extract, 0.5% NaCl, 50 μg / ml ampicillin); when OD600 nm was converted to 0.8, isopropyl β-D-thio-galatopylanoside (IPTG) was added to a final concentration of 0.5 mM, and culture by shaking was continued for another 20 hours to produce OGF-II with E. coli. After finishing the culture, the chain was collected; The main band of an approximate molecular weight of 15 KD was confirmed with polyacrylamide gel electrophoresis - SDS. This molecular weight coincided perfectly with that of OGF-II that was obtained from the cultivated supernatant of IMR-90. (3) Purification of His6-bOGF-II To test the osteoblast growth activity of the protein obtained by transferring the bOGF-II gene, expression of the gene was performed using QIAex-press equipment (manufactured by QIAGEN Co.) . In this system, a protein containing marked hexamer histidine is produced, and the purification is carried out using a nickel-chelate nitrilotriacetic acid resin column. This system is usually used to effectively purify the expressed protein. By using this system, bOGF-II is produced in the form with clumps of six histidines at the N-terminus (His6-bOGF-II). Added, for 0.35 g of E. coli treated with IPTG, 1.75 ml of 10 mIMR-90 Tris-HCl, pH 8.0 containing 8 M urea and 0.1 M sodium phosphate; it was stirred for one hour at room temperature. After stirring, the sample was subjected to centrifugation at 12,000 rpm for 15 minutes at room temperature and the supernatant was collected. The supernatant was added to 8 ml of 50% resin suspension of nickel-chelate nitrile-triacetic acid (manufactured by QIAGEN Co.) which was balanced with 10 mM Tris-HCl, 8 M urea, 0.1 M sodium phosphate , pH 8.0, the mixture was stirred for 45 minutes at room temperature. This resin was transferred in a 1.6 cm column. of internal diameter and washed with 20 ml of 10 mM Tris-HCl, 8 M urea, 0.1 M sodium phosphate, pH 8.0, at a flow rate of approximately 0.5 ml / min. Next, this column was further washed with 10 mM Tris-HCl, 8 M urea, 0.1 M sodium phosphate, pH 6.3, at a flow rate of about 0.5 ml / min. Finally, the His6-bOGF-II target was levigated with 10 mM Tris-HCl, 250 mM imidazole, 8 M urea, 0.1 sodium phosphate, pH 6.3 at a flow rate of approximately 0.5 ml / min. The purified fraction of His6-bOGF-II was dialyzed to phosphate-regulated saline. (4) Osteoblast growth activity of His6-bOGF-II The osteoblast growth activity of His6-bOGF-II was tested as described in Example 1 (2), method for testing osteoblast growth activity. That is, the purified His6-bOGF-II fraction described above was added to the assay medium and 3 H-thymidine incorporation was tested in mouse osteoblast cells MC3T3-EA. The results are shown in Figure 7. From these results, it has been confirmed that the protein produced by the transfer of the bOGF-II gene has the osteoblast growth activity as well as the wild-type bOGF-II.

Possibility for use in the industry According to the invention, a new protein with osteoblast growth activity is provided, as well as the methods for producing the protein. Since the protein of the present invention has osteoblast growth activity, it is useful as a medicament for the treatment of bone mass reduction, such as osteoporosis and as an antigen for the immunological diagnosis of diseases.

Reference to deposited microorganisms Organization and domicile where the deposited Organizations: National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology Ministry of Commerce and Industry International 1-3, Higashi 1-chome, Tsukuba City, Ibaragi Prefecture Deposit date in the deposit organization: June 19, 1995 Number Deposit deposited by the organization where deposited: FERM BP-5139 This deposit was transferred from the national deposit (Accession number; FERM p-14942 of May 25, 1995) on June 19, 1995.

Sequence number: 1 Sequence length: 25 Sequence type: amino acid Chain: simple Topology: linear J-0 Molecular type: peptide Sequence Glu Tre Glu Tir Gli Pro Xaa Arg Arg Glu Met Glu Asp Tre Leu 1 5 10 15 Asn His Leu Lis Fel Leu Asn Val Leu Ser 15 20 25 Sequence number: 2 ~ Sequence length: 40 Sequence type: amino acid 20 Chain: simple Topology: linear Molecular type: peptide Sequence Glu Tre Glu Tir Gli Pro Cis Arg Arg Glu Met Glu Asp Tre Leu 25 1 5 10 15 Asn His Leu Lis Fel Leu Asn Val Leu Ser Pro Arg Gli Val Xaa 20 25 30 lie Pro Asn Cis Xaa Lis Lis Gli Fel Tir 35 40 Sequence number: 3 Sequence length: 9 Sequence type: amino acid Chain: simple Topology: linear Molecular type: peptide Sequence Asp Val His Cis Tir Ser Met Gln Ser 1 5 Sequence number: 4 Sequence length: 12 Sequence type: amino acid Chain: simple Topology: linear Molecular type: peptide Sequence Glu Tre Glu Tir Gli Pro Cis Arg Arg Glu Met Glu 1 5 10 Sequence number: 5 Sequence length: 16 Type sequence: amino acid Chain: simple Topology: linear Molecular type: peptide Sequence Asp Lis Tir Gli Gln Pro Leu Pro Gli Tir Tre Tre Lis Gli Lis 1 5 10 15 Glu Sequence number: 6 Sequence length: 25 Sequence type: amino acids Chain: simple Topology: linear Molecular type: peptide Sequence Asp Lis Lis Gli Fel Tir Lis Lis Lis Gln Cis Arg Pro Ser Lis 1 5 10 15 Gli Arg Lis Arg Gli Fel Cis Trp Cis Val 20 25 Sequence number: 7 Sequence length: 22 Sequence type: amino acid Chain: simple Topology: linear Molecular type: peptide Sequence Asp Tre Leu Asn His Leu Lis Fel Leu Asn Val Leu Ser Pro Arg 1 5 10 15 Gli Val His lie Pro Asn Cis 20 Sequence number: 8 Sequence length: 258 Sequence type: nucleic acid Chain: double Topology: linear Molecular type: cDNA to mRNA Original source: human Trait: Key trait: Presence position: 1.258 Method to determine trait: E Sequence GAG ACA 'GAA TAT GGT CCC TGC CGT AGA GAA ATG GAA GAC ATA 42 CTG AAT CAC CTG AAG TTC CTC AAT GTG CTG AGT CCC AGG GGT 84 GTA CAC ATT CCC AAC TGT GAC AAG AAG GGA TTT TAT AAG AAA 126 AAG CAG TGT CGC CCT TCC AAA GGC AGG AAG CGG GGC TTC TGC 168 GAT AAG TAT GGG CAG CCT CTC CCA GGC TAC ACC 210 ACC AAG GGG AAG GAG GAC GTG CAC TGC TAC AGC ATG CAG AGC 252 AAG TAG_258_Sequence number: 8 Sequence length: 85 Sequence type: amino acid Chain: simple Topology: linear Molecular type: protein Sequence Glu Tre Glu Tir Gli Pro Cis Arg Arg Glu Met Glu Asp Tre Leu 1 5 10 15 Asn His Leu Lis Fel Leu Asn Val Leu Ser Pro Arg Gli Val His 20 25 30 lie Pro Asn Cis Asp Lis Lis Gli Fel Tir Lis Lis Lis Gln Cis 35 40 45 Arg Pro Ser Lis Gli Arg Lis Arg Gli Phe Cis Trp Cis Val Asp 50 55 60 Lis Tir Gli Gln Pro Leu Pro Gli Tir Tre Tre Lis Gli Lis Glu 65 70 75 Asp Val His Cis Tir Being Met Gln Ser Lis *** 80 85

Claims (1)

1. Having described the foregoing invention, the content of the following CLAIMS is claimed as property: (1) A protein with the following features: 5 (a) molecular weight of approximately 15kD under conditions of reduction and not reduction by electrophoresis of polyacrylamide-sulfate gel sodium dodecyl (SDS-PAGE), (b) a high affinity with a cation exchanger and heparin, ": L0 (C) an activity to stimulate osteoblast growth, and (d) the activity is reduced by heating to 702C during 10 minutes and deactivated by heating at 90 aC for 10 minutes (2) The protein mentioned in claim 1, 15 derived from human fibroblast cells (3) The protein according to claim 1 or 2, characterized by the amino acid sequence. N-terminal represented in Sequence Number 1. (4) The protein according to claim 1 or 2, characterized by the N-terminal amino acid sequence represented in FIG. a Sequence Number 2. (5) A cDNA that encodes the amino acid sequence determined by Sequence Number 9. (6) A protein with activity to stimulate the growth of osteoblast cells, characterized by the entire amino acid sequence represented in Sequence Number 9. (7) A protein with an activity to stimulate the growth of osteoblast cells, characterized in that the protein contains the amino acid sequence represented in Sequence Number 9, as a part. (8) A protein with an activity to stimulate the growth of osteoblast cells, characterized in that the protein has a homology of a minimum of 80% with the amino acid sequence represented in Sequence Number 9. (9) Sequence of amino acids cDNA that encodes the protein with activity to stimulate the growth of osteoblast cells, characterized in that the protein has a homology of a minimum of 80% with the sequence determined by Sequence Number 9. (10) A method to produce protein that includes: culture of human fibroblast; treatment of the conditioned medium with a heparin column; levigation of the adsorbed fraction; treatment of the eluate with an anion exchange column to obtain the non-adsorbent fraction; and treating the fraction with a cation exchange column and then purifying it with a heparin column to produce the protein having the following characteristics: (a) a molecular weight of approximately 15 kD under conditions of reduction and not reduction by electrofere-sis of polyacrylamide gel - sodium dodecyl sulfate (SDS-PAGE), (b) a high affinity to a cation exchanger and heparin, (c) an activity to stimulate the growth of osteoblasts, and (d) the activity of osteoblast growth that is reduced by heating to 70 aC for 10 minutes and deactivated by heating at 90 aC for 10 minutes. (11) The method for producing the protein according to claim 10, characterized in that the protein obtained includes the amino acid sequence determined in Sequence Number 9, as a part and has an activity to stimulate the growth of osteoblast cells. (12) A host cell that is transformed by an expression vector containing cDNA encoding the amino acid sequence of Sequence Number 9 or an amino acid sequence with a minimum homology of 80% with the amino acid sequence determined by the Sequence Number 9. (13) The host cell according to claim 12, which is a cell derived from mammals, bacteria, yeast or insects. (14) The cell according to claim 13, derived from Escherichia coli. (15) The host cell according to claim 14, which is Escherichia coli pQE30-OGFII (FERM BP-5139). (16) A process for producing the protein of claims 1, 3, 4, 6, 7 or 8 which includes; culture of the host cell that is transformed by an expression vector containing cDNA encoding the amino acid sequence of Sequence Number 9 or the amino acid sequence having a homology of at least 80% with the amino acid sequence determined by the Sequence Number 9, and get the culture protein. SUMMARY OF L? INVENTION A protein derived from human fibroblast, with N-terminal amino acid sequence represented in Sequences Number 1 and 2, with a molecular weight of ca. 15 kD under conditions of reduction and not reduction by (polyacrylamide gel electrophoresis - sodium dodecyl sulfate), and with an activity to stimulate the growth of osteoblasts. The protein containing the amino acid sequence represented in Sequence Number 9 and having osteoblast growth activity. A method for preparing the protein by culturing human fibroblast and treating the conditioned medium for purification. The protein is produced by genetic engineering procedures. The protein is used for the treatment of conditions with reduction of bone mass such as osteoporosis, or it is used as an antigen for the immunological diagnosis of diseases. In compliance with article 47 of the Industrial Property / I declare in the name of the applicant / and under protest to tell the truth / that the method described here is the best known by her to carry out the invention contained herein. IN FAITH WHAT / I sign this in Mexico City D.F. , today at the twenty-seventh day of February raes of one thousand nine hundred and ninety-six.