MXPA98008915A - Process to produce the bone morphogenetic protein by madurac - Google Patents

Abstract

A process for producing a morphogenetic protein by maturation by the action of a processing enzyme on a morphogenetic-bone protein precursor, which is characterized in that it comprises either introducing a vector expression of a morphogenetic-protein precursor and an expression of the vector of a processing enzyme in an animal cell strain, until the production of a morphogenetic-osseous protein by maturation, and separating it from the culture, or alternatively, adding a solution of a processing enzyme to a solution of a morphogenetic-protein precursor and incubating the obtained mixture solution . A suitable process consists in introducing a vector expression of a human MP52 precursor and a vector expression of a secretory furin variant in an established animal cell strain, culturing the resulting strain to produce the morphogenetic-osseous protein by maturation, and separating it from the culti

Description

PROCESS TO PRODUCE THE OSEA MORFONGENETIC PROTEIN. BY MATURATION Technical area The present invention presents a process for producing bone morphogenetic protein by maturation. More specifically, this invention presents a process for the production of a bone morphogenetic protein by maturation, which includes inducing the action of a processing enzyme on a bone morphogenetic protein precursor.

Background of the Invention The existence of a proteinaceous bone morphogenetic factor in the matrix bone was discovered by ürist et al. (Science, 150, pp. 893-899, 1965) and was termed "bone morphogenetic protein" (which will be abbreviated hereinafter as "BMP"). "). In recent years, many genes related to BMP have been cloned and each of them is known as belonging to the super family of the transforming growth factor-ß. (which will be abbreviated here as "TGF-ß"). From some of them, recombinant proteins have already been produced. They have been used for the confirmation of bone morphogenetic activity REF .: 28647 and its application is expected in the treatment of bone diseases- The proteins previously described supposedly correspond each of them to the TGF-β super family, from its genetic structure, until it is synthesized as a precursor in vivo and then subjected to several processes, from a maturation (active type) of peptide or odimer. The maturation of human TGF-ßl is known to be a dimer of the peptide residue with COOH terminal 112 (Nature, 316, 701-705, 1985). In practice, it is known that in mammalian cells, the introduction of a coding cDNA precursor, to produce BMP-2 or BMP-6 / Vgr-1 incorporates, in addition to the maturation of the dimer peptide, various peptides dimers (ho odimers). compounds of high molecular weight and heterodimeric monomers composed of high molecular weight monomers and a maturing monomer) having a higher molecular weight than the more mature ones in the culture supernatant (Growth Factors 7, pp. 139-150, 1992 : J. Biol. Chem. 126, pp. 1595-1609, 1994). These dimer peptides have a higher molecular weight than the maturation ones that are supposed to correspond to the low-processing molecules from the precursor of maturation. The dimeric peptide having a higher molecular weight than the maturing one will henceforth be called a "precursor dimer". Technically, it is very difficult to produce it selectively, among the various dimers described above, a dimer peptide of a certain molecular weight, for example a large-scale maturing dimer; or by efficiently carrying out the separation of the maturation dimer. Accordingly there is a strong demand to overcome such technical difficulty.

Exhibition of the Invention An objective of the present invention is to provide an efficient method to selectively produce, in a mammalian cell, a maturation dimer of the same molecular weight as the mixture of dimers of various precursor molecular weights of a large-scale bone morphogenetic protein.

In recent years, a group of proteases similar to Kex2, F rina, PC-2, PC-3, PACE4, PC6 and the like were identified as processing enzymes in contrast to the precursor of a protein in a higher animal (Biochem. 299, pp. 1-18, 1994). Among them, furine has a hydrophobic transmembrane region in the terminal COOH and locally exists in the Golgi membrane. The cDNA sequence encoding human furin has already been reported by van den Ouweland et al. (Nucleic Acids Res. 18, pp. 664, 1990). It has been revealed that furin recognizes Arg-X? -X2-Arg (where XI represents any amino acid and X2 represents any amino acid, but mainly Lys or Arg) as a 4 amino acid sequence upstream from its division site (J. Biol. Chem. 266, pp. 12127-12130, 1991). It has also been reported that when only the NH2 terminal region of furin without the transmembrane region is expressed in a mammalian cell, it is secreted outside the cell and the specific activity of cleaving the amino acid sequence remains the same at the site of secretion.

(J. Biol. Chem. 269, pp. 25830-25837, 1994).

The inventors hereby, for the first time, achieved in efficient large-scale production, in a mammalian cell, of only one maturation dimer without a precursor dimer by the use of the above-described furin based on recombinant DNA technology.

According to the present invention, a bone morphogenetic protein by maturation can be produced by inducing the action of a processing enzyme on a bone morphogenetic protein precursor.

According to the present invention, the processing enzyme is acting on the bone morphogenetic protein precursor by introducing both, an expression vector of the bone morphogenetic protein precursor and an expression vector of the processing enzyme in the mammalian cell strain. The cell strain is cultured and then, from the supernatant culture, the bone morphogenetic protein is obtained by maturation.

According to the present invention, the processing enzyme acts on the bone morphogenetic protein precursor by the addition of a processing enzyme containing the supernatant culture in a supernatant culture containing precursors of the bone morphogenetic proteins. Bone morphogenetic protein by maturation is produced by incubation of the resulting mixture.

Preferably, the production is carried out by introducing both a vector expression of a human MP52 precursor and an expression of the mutant secretory furin vector into a mammalian cell strain, culturing the cell strain and then, separating the supernatant culture from the culture supernatant. bone morphogenetic protein by maturation thus produced.

The term "bone morphogenetic protein by maturation" as used herein means a bone morphogenetic protein that is substantially composed of a sequence of homologous amino acids with amino acid residues COOH-terminal 112 of human active type TFG-ßl. The activity of the bone morphogenetic protein is assumed to exist in the homologous region of the amino acid sequence with amino acid residue with terminal COOH 112 of this human TGF-ßl active type as desired, the bone morphogenetic protein does not contain the other region of the amino acid sequence.

Examples of the processing enzymes usable in the present invention include a group of proteases similar to Kex2, furin, PC-2, PC-3, PACE4 and PC6. Among them, furine is particularly preferred. As the cDNA encoding furin, the DNA of the base sequence encoding the entire amino acid sequence of furin can be used, but the DNA of the base sequence encoding a portion of the amino acid sequence (which will be called from here "mutant furin type .secretory") that does not include a tansmembrane region and also maintains specific activity of division in the amino acid sequence.

In the preferred embodiment of the present invention, a DNA fragment containing a secretory mutant furin cDNA is cloned from a total RNA extracted from human HepG2 cell strains (ATCC HB8065) by the RT-PCR method using a synthesized DNA primer. The secretory mutant furin has, as its base sequence DNA, from nucleotide No. 163 to nucleotide No. 2014 placed forward in SEQ ID NO: 1 of the Sequence Listing. This DNA fragment is inserted into a vector expression by which an expression of the secretory mutant furin vector is constructed. The term "vector" used herein contains, at the lower end of the eukaryotic promoter, a poly (A) -added which indicates which is a restriction enzyme site for cloning a gene for expression. This expression vector may also contain a drug-resistant marker favorable for the selection of a transformant. Examples of such vector expression include a human cytomegalovirus promoter enhancer, polylinker, poly (A) - bovine growth hormone as signal addition and pRc / CMV (available from INVITROGEN Inc.) containing a resistant neomycin marker.

The present invention is applicable to a method of producing a bone morphogenetic protein belonging to the human TGF-β superfamily, more specifically, to the method of producing a morphogenetic bone protein by maturation composed of monomers having the same molecular weight, example, MP52, BMP-2, BMP-4, BMP-6 or BMP-7. As a bone morphogenetic protein, human MP52 which is presented in PCT Application WO93 / 16099 or O95 / 04819 and has bone morphogenetic activity which is particularly preferred. In a preferred embodiment, the cDNA encoding a human MP52 precursor is inserted into a vector expression to construct the expression of the human MP52 precursor vector, followed by introduction into a mammalian cell, whereby the cell line is prepared of mammals that produce the precursor and the MP52 dimer of maturation. Examples of mammalian cells adapted as host cells here include Chinese hamster ovary (CHO) cells, BHK cells, 293 cells and mouse L cells. Among them, CHO cells are preferred. In the line of human MP52 produced mammalian cells (MC-2: FERM declaration number BP-5142) thus obtained, expression of the secretory mutant furin vector is introduced to co-express both proteins, whereby they are only produced MP52 dimers maturation in their culture supernatant.

The conversion of the precursor protein in its maturation form can also be achieved by mixing a solution containing the processing enzyme with precursor containing solution and incubating the resulting mixture overnight at 32 to 40 ° C, preferably 37 ° C.

The bone morphogenetic protein by maturation available by the process of the present invention can be used for the treatment or prevention of damage to bones, cartilage or teeth or for roots of artificial teeth by adding it to pharmaceutically acceptable vehicles, additives, diluents and / or excipients such as be necessary.

For the treatment of bone diseases caused by malfunction of bone metabolism, bone morphogenetic protein by maturation can be administered in any conventional manner systemically and carefully, for example, injections such as intravenous injection, intramuscular injection or intraperitoneal injection, oral administration or parenteral administration such as suppository.

For the treatment of bone fractures, morphogenic protein by maturation can be administered systemically or locally by injection, oral administration or parenteral administration. Also preferred is the implantation of a matrix or template containing the morphogenetic protein by maturation in a region close to the fractured bone. Suitable examples of the matrix include natural polymers such as collagen or adhesive fibrin and synthetic polymers such as a copolymer of polylactic acid and glycolic acid.

In the case of orthopedic reconstructions, bone grafts or roots of artificial teeth, bone morphogenetic protein by maturation can be applied on the surface of the bone or tooth to be implanted, being covered with a paste of collagen, adhesive fibrin or other adhesive. In the case of bone grafts, it can be used for both natural and artificial bones.

The dose of bone morphogenetic protein by maturation is determined based on the purpose and method of administration. In general, the dose is 1 μg to 100 μg / Kg. in systemic administration, while it is preferably 30 μg to 30 mg. / place in local administrations.

Brief Explanation of the Drawings Fig. 1 is a plane of a plasmid of a vector expression, pDfurRC / CMV (7.2 kb), of a human secretory mutant furin.

Fig. 2 is a plane of a plasmid of a vector expression, pMSS99 (5.0 kb), of human MP52.

Fig. 3 is an analysis photo by Western blotting under reducing conditions, showing the serum free from the supernatant culture of a cell line that co-expresses the human MP52 maturation dimer and the human secretory mutant furin.

Fig. 4 is a photo of analysis by Western staining under reducing conditions, which shows that several precursors of human MP52 dimers are converted to the MP52 mature due to the human secretory mutant furin when acting on the dimer.

Description of preferred modalities The advantages of the present invention hereinafter will be described more specifically by examples.

Example 1 The production of the human MP52 maturation dimer by CHO cell lines that co-express a human secretory mutant furin and human MP52. (1) Cloning of the mutant furin cDNA type secretory human and construction of its vector expression.

The human furin protein has a structure composed of a peptide signal, a protease region similar to sub-lysine, a transmembrane region and a lateral region of cytoplasm in the order mentioned from the terminal NH2 region. In this invention, human secretory mutant furin cDNA encoding cDNA from the NH2-terminal protease region without the transmembrane region was cloned by the RT-PCR method and provided for expression.

The total RNA was extracted from the human HepG2 cell and with this as the standard, the primer 1 in upward sense (human furin cDNA sequences Nos. 931 to 914 located forward of SEQ ID NO: 1 of the List of Sequences) and primer 2 in contra-sense down (Nos .. 2095 to 2071) was subject to reverse transcription by rTth RNA polymerase. These products in combination with primer 3 in the sense (SEQ ID NO: 2 of the Sequence Listing) and the primer 4 in contrasense (SEQ ID NO: 3 of the Sequence Listing), and with primer 5 in the sense (SEQ ID NO: 4 of the Sequence Listing) and primer 6 in contrasense (SEQ ID NO: 5 of the Sequence Listing) were subjected to PCR reaction, respectively, whereby two cDNA fragments were obtained at the upper and lower ends. These fragments were pooled and inserted into the HindIII-SalI site of plasmid pUC119 (obtained from Takara Shuzo Co., Ltd.), whereby the cDNA of the secretory mutant human furin encoding 595 amino acids was obtained. The resulting cDNA was confirmed by restriction enzyme digestion and determination of a DNA base sequence. The cDNA of the human secretory mutant furin was found to have, as the base DNA sequence, nucleotides Nos. 163 through 2014 of SEQ ID NO: 1 of the Sequence Listing. The DNA sequence of this human secretory mutant furin is different from the DNA sequence reported by van der Ouweland et al., In which this base No. 165 is adenine so that an initiation codon that supposedly has an adverse effect on the translation and is therefore unnecessary, has been eliminated; and in this the base No. 2004 is adenine so that a stop codon was formed. Then the human secretory mutant furin cDNA was removed by digestion with HindIII-Xbal, followed by insertion in the HindlII-Xbal site of the pRc / CMV vector purchased from INVITROGEN INC., So pDfurpRC / CMB, an expression was prepared of the cDNA of the human secretory mutant furin as illustrated in Fig. 1. (2) Construction of the vector expression of human MP52.

From the plasmid pSK52s containing the human MP52 gene provided by Dr. Hoetten of Biopharm GmbH, a DNA fragment containing the human MP52 gene isolated by digestion with HindIII, followed by insertion in the HindIII site of the vector pABstop provided by Dr. Zettl eissl from Behringwerke AG, by means of pMSS99, an expression of the human MP52 vector as illustrated in FIG. 2. Its structure was confirmed by the determination of the DNA base sequence and digestion with a restriction enzyme. As a result the human MP52 of the DNA base sequence of pMSS99 was found to be nucleotides Nos. 576 to 2279 of SEQ ID NO: 6 of the Sequence Listing. (3) Establishment of MC2, that is, a Chinese Hamster Ovary cell line (CHO) that produces several precursors of human MP52 dimers.

In CHO-DÜKX-B11 cells provided by Dr.

Zettl eissl from Behringwerke AG, that is, mutant strains of CHO cells, pMSS99 and pSVOAdhfr provided by Dr. Zettlmeissl was introduced by the calcium phosphate co-precipitation method of DNA. Then, a high production of the human MP52 cell line was established by the method of gene amplification using methotrexate (MTX). pMSS99 (10 μg.) and pSVOAdhfr (2 μg.) were dissolved in 1 ml. of 25 mM HEPES, 140 mM NaCl and 0.75 mM NaaHP0 (pH 7.05) and followed. mix with 50 μl. of 2.5 M of Ca CI2. The precipitate was stored on CHO-DUKX-B11 cells in 10 cm dishes. and it was kept at room temperature for 30 mins. To the cell layer was added 8 ml. of ribo- and deoxyribonucleotide containing MEM-ALPHA medium (MEM-a +), containing 10% fetal calf serum and the resulting mixture was cultured for 4 to 6 hours in a CO2 incubator. Following the treatment with 10% glycerol at room temperature for 3 minutes, the cells were cultured in a MEM-a + medium containing 10% FBS. The cultured cells were then inoculated again in a medium of free ribo- and deoxyribonucleotide MEM-ALPHA (MEM-a-) containing 10% dialyzed FBS and the transformant was selected. The production of human MP52 was detected by Western blot analysis as described below in (5).

In the production medium of human MP52 cell strains, MTX was added. By successive increase of the MTX concentration, cell lines having the amplified MP52 gene were selected. In the 400 nM concentration of MTX, the production of the precursor cell line MC-2 of the human MP52 dimers of various molecular weights and the human MP52 maturation dimer was obtained. The resulting MC-2 cell line was deposited in "National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken) on June 21, 1995 under the Deposit number of FERM BP-5142. (4) Establishment of the coexpression of the human MP52 cell line and the human secretory mutant furin by the introduction of the expression vector of the human secretory mutant furin in the human MP52 producing CHO of the MC-2 cell line.

The co-expression of the human MP52 cell line and the human secretory mutant furin was established by introduction of the vector expression of the human secretory mutant furin in the CHO of the MC-2 cell line which produced several precursors of MP52 dimers. human and maturation dimers MP52 by the calcium phosphate co-precipitation method of the DNA and then select the transforming cell in the presence of 333 μg / ml. of G418 and 400 nM. of MTX.

In a manner similar to Example 1 (2), the calcium phosphate co-precipitate of the DNA was formed using pDfurpRC / CMV (4.8 μg.). The MC-2 cells were then stored in 10-cm dishes, and kept at room temperature for 30 minutes. To the cell layer, 8 ml was added. of a free ribo- and deoxyribonucleotide medium MEM-ALPHA (MEM-a-) containing 10% FBS and the resulting mixture was cultured for 4 to 6 hours in a CO2 incubator. After treatment with 10% glycerol at room temperature for 3 minutes, the cells were cultured on a MEM-a-medium containing 10% FBS for 2 days. The cultured cells were again inoculated in MEM-a medium containing 10% FBS, 400 nM MTX and 333 μg / ml. of G418 and the transformant strains were selected. The production of human MP52 was detected by Western blot analysis as described below in (5). The production of human secretory mutant furin was detected by the method of measurement of enzymatic activity as described below (6). Free serum from one of the supernatant cultures of the co-expression of the cell lines resulting from the human MP52 furin and the human secretory mutant furin was subjected to the daily exchange medium and the supernatants collected were subjected to SDS-polyacrylamide electrophoresis under recutting conditions and then analyzed by Western staining. The results are shown in the photo of Fig. 3. At each stage, a 0.5 μl portion of the culture supernatant was emptied. Stages 1, stage 2, and stage 3 are supernatant cultures of Day 1, Day 2, and Day 3 respectively. The MP52 monomer band of maturation is indicated by an arrow. It was found that the MP52 precursor did not have a large molecular weight detected in the supernatant and all the MP52 were peptides with a molecular weight of approximately 15 K, which corresponds to the maturing monomers MP52. as a result of an analysis under non-reducing conditions, these peptides formed the maturing MP52 dimers each having a molecular weight of approximately 28 K. The amount of production of the dimers by use of the cell co-expression line was approximately 8. μg / 24 hours, which was approximately 3 to 8 times as well as by the use of the MP52 expressing cell of the MC-2 line. Thus by the use of the human MP52 co-expression line and the human secretory mutant furin, the present inventors were successful for the first time in the production of MP52 maturation dimers only on a larger scale than conventional methods. (5) Detection of human MP52 in the culture supernatant by Western blot analysis Proteins from the culture supernatant were separated by electrophoresis through SDS-polyacrylamide gel (15-25% polyacrylamide gel gradient, Dalichi Kagaku Co., Ltd.) followed by transfer of the protein to a PVDF Membrane P membrane for clear staining). The membrane was blocked with "Block Ace" (Dainippon Pharmaceutical Co., Ltd.) for one hour, rinsed with Tris saline regulator (TBS) and then treated overnight with 10 μg / ml. of chicken antibodies in contrast to human MP52. The membrane was washed with TBS, containing 0.1% Tween 20 (TTBS) and followed by treatment with an antipollo IgG and rabbit alkaline phosphatase complex (Sigma A9171). The membrane was rinsed with TTBS and the band corresponding to MP52 was visualized by means of the alkaline phosphatase substrate kit (BIO-RAD). (6) Detection of the activity of human secretory mutant furin in the culture supernatant Twenty μl. of the supernatant culture were diluted by 30 μl of pure water and mixed with 200 μl of a phosphorescent substrate solution; incubate with 125 mM of MES / NaOH (pH 7.0) containing 100 mM of Boc-Arg-Arg-Val-Arg-MCA (purchased from Protein Technology Institute) and 1.25 mM of CaCl2 at 37 ° C for 10 minutes. The fluorescence of AMC was measured at an excitation wavelength of 380 nm and an emission wavelength of 450 nm. Thus the furina activity unit was defined as, the activity necessary for the release of 1 pCM per minute. (OR) . (7) MP52 purification of maturation produced by co-expression with human secretory mutant furin and analysis of the NH2 terminal amino acid sequence.

With serum free from the supernatant culture of the co-expression of the human MP52 cell strain and the human secretory mutant furin, 0.1 volume by volume of 0.2 M sodium phosphate buffer (pH 6) was mixed. The resulting mixture was applied to the HiTrap SF column (1 ml., Pharmacia) equilibrated by 20 M of a sodium phosphate buffer (pH 6.0) containing 50 mM NaCl, followed by rinsing with the same buffer. The protein was then eluted with 0.1M sodium phosphate buffer (pH 6.0), containing guanidine hydrochloride acid. The elution was applied on the HPLC column for reverse phase Resource RPC (3 ml., Pharmacia) and the protein was eluted with acetonitrile from 25 to 55%. The fraction containing the MP52 of maturation was subjected to the analysis of the NH2 terminal amino acid sequence, by a one-pulse sequencer in gas-liquid phase (Applied Biosystems model 476). the result is shown in Table 1.

Table 1.

Cycle: Sequence 1 Sequence 2 of amino acids (pmsl) of Amino Acids (pmol) 1 Arg 11.95 Wing 25.51 2 Wing 28.75 Pro 14.75 3 Pro 17.55 Leu 18.07 4 Leu 16.47 Wing 14.46 5 Wing 16.99 Thr 5.02 6 Thr '7.15 Arg 7.38 7 Arg 9.21 Gln 9.08 8 Gln 9.54 Gly 13.23 9 Gly 11.29 Lys 5.29 10 Lys 8.04 Arg 6.52 From Table 1, it was assumed that sequence 1 of amino acids and sequence 2 of amino acids were derived from the amino acid sequence of Arg 354 and that of Ala 355 of SEQ ID NO: 6 of the Sequence Listing, respectively and that its molecular proportion was approximately 1: 1.

Example 2 Conversion of the precursor of the human MP52 dimer in dimer of maturation by means of the human secretory mutant furin (1) Establishment of the CHO cell line that produces human secretory mutant furin In the CHO-DUKX-B11 cells provided by Dr. Zettlmeissl of Behringwerke AG, expression of the human secretory mutant furin vector, pDfurpRC / CMV, which was described in Example 1 (1) was introduced by the method of co -precipitation of calcium phosphate DNA. The transforming cells were selected in the presence of G418, whereby a high expression of the human secretory mutant furin strain was established.

In a similar way to that described in example 1 (3) the co-precipitate of pDfurpRC / CMV and calcium phosphate was prepared, stored on CH0-DUKX-B11 cells in a dish of 10 ems. and kept at room temperature for 30 minutes. Cell layer was added, 8 ml. of a ribo- and deoxyribonucleotide medium containing MEM-ALPHA (MEM a +) containing 10% FBS, followed by culture in a CO2 incubator for 4 to 6 hours. After treatment of the cells with 10% glycerol at room temperature for 3 minutes, they were again inoculated on a MEM-a + medium containing 10% FBS and 400 μg / ml. of G418, with which the transformant was selected. The production of the human secretory mutant furin cell line was detected by the enzymatic activity measurement method described above in Example 1 (6) to obtain a cell line having a furin activity of 500 to 1000 U / ml. 24 hours. (2) Conversion of the precursor of the human MP52 dimer by maturation by means of the human secretory mutant furin It was mixed with a serum free culture supernatant (containing the MP52 dimers of maturation and the precursor) of the CHO cell production of the MC2 line of the human MP-52, a serum free from the culture supernatant (1000 U / ml) of the cell line expressed by the secretory mutant furin human in various proportions, followed by overnight incubation at 37 ° C. After the reaction, Western blot analysis as described above in Example 1 (5) was carried out under reducing conditions and the conversion of the MP52 precursor to maturation was detected. The result is shown in the photo of Fig. 4. In each of the steps from 1 to 5, a portion of 1 μl of the supernatant culture of the cell line expressed by the secretory mutant furin hunana was emptied. The final concentrations of the human secretory mutant furin was 0 U / ml. for stage 1, 50 U / ml for stage 2, 100 U / ml. for stage 3, 200 U / ml. for stage 4 and 400 U / ml. for stage 5, respectively. The band of the maturing monomer MP52 is indicated by the arrows A and B, whereas that of the maturing monomer MP52 is indicated by the arrow C. as illustrated in FIG. 4, the incorporation of the culture supernatant of the cell line expressing the human secretory mutant furin so as to give an active final concentration of at least 200 U / ml. turned into MP52 of maturing completely. As a result, the amount of the MP52 dimer of maturation was increased approximately 3 times.

Industrial Application The maturation of the bone morphogenetic protein has conventionally been obtained by separating a mixture of morphogenetic bone protein dimers which are produced by mammalian cells and have several molecular weights, but it is difficult to selectively produce the morphogenetic protein of bone in large numbers. scale or to obtain the maturation of the bone morphogenetic protein efficiently because a technology for the efficient separation of the mixtures described above has not yet been developed, the process of the present invention however, makes it possible to prepare the bone morphogenetic protein by maturation from the precursor of bone morphogenetic protein, which facilitates the production of this on a large scale. Accordingly, the bone morphogenetic protein by maturation available by the process of the present invention is a highly pure substance composed of peptides of substantially the same molecular weight so that it is particularly suitable for use as pharmaceuticals.

LIST OF SEQUENCES SEQ ID NO: 1 LENGTH: 4180 TYPE: Amino Acid TYPE OF FILAMENT: DobLe TOPOLOGY: Linear TYPE OF MOLECULE: peptide TYPE OF FRAGMENT: ORIGINAL SOURCE: ORGANISM: homo sapiens TYPE OF TISSUE: human hepatoma CHARACTERISTICS: LOCA IZ ATION: OTHER INFORMATION: a furin processing enzyme DESCRIPTION SEQ ID NO: 1 GCGGGGAAGC AGCAGCGGCC AGGATGAATC CCAGGTGCTC TGGAGCTGGA TGGTGAAGGT " '60 CGGCACTCTT CACCCTCCCG AGCCCTGCCC GTCTCGGCCC CATGCCCCCA CCAGTCAGCC 120 CCGGGCCACA GGCAGTGAGC AGGCACCTGG GAGCCGAGGC CCTAAGACCA GGCCAAGGAG 180 ACGGGCGCTC CAGGGTCCCA GCCACCTGTC CCCCCC ATG GAG CTG AGG CCC TGG 234 Met Glu Leu Arg Pro Trp May 1 TTG CTA TGG GTG GTA GCA GCA ACA GGA ACC TTG GTC CTG CTA GCA GCT 282 Leu Leu Trp Val Val Ala Al a Thr Gly Thr Leu Val Leu Leu Ala Al a 10 15 20 GAT GCT CAG GGC CAG AAG GTC TTC ACC AAC ACG TGG GCT GTG CGC ATC • 330 Asp Wing Glp Gly Gln Lys Val Phe Thr Asn Thr Trp Ala? Al Arg He 25 30 35 CCT GGA GGC CCA GCG GTG GCC AAC AGT GTG GCA CGG AAG CAT GGG TTC 378 Pro Gly Gly Pro Wing Val Wing Asp Ser Val Wing Arg Lys His Gly Phe 40 45 50 CTC AAC CTG GGC CAG ATC TTC GGG GAC TAT TAC CAC TTC TGG CAT CGA 426 Leu Asn Leu Gly Gin lie Phe Gly Asp Tyr Tyr His Phe Trp His Arg 55 60 65 70 GGA GTG ACG AAG CGC TCC CTG TCG CCT CAC CGC CCG CGG CAC AGC CGG 474 Gly Val Thr Lys Arg Ser Leu Pro Pro His Arg Pro Arg His Ser Arg 75 80 85 CTG CAG AGG GAG CCT CAA GTA CAG TGG CTG GAA CAG CAG GTG GCA AAG 522 Leu Gln Arg Glu Pro Glp Val Gln Trp Leu Glu Gln Gln Val Ala Lys 90 95 100 CGA CGG ACT AAA CGG GAC GTG TAC CAG GAG CCC ACA GAC CCC AAG TTT 570 Arg Arg Thr Lys Arg Asp Val Tyr Gín Glu Pro Thr Asp Pro Lys Phe 105 110 115 CCT CAG CAG TGG TAC CTG TCT GGT GTC ACT CAG CGG GAC C TG AAT GTG 618 Pro Gln Gln Trp Tyr Leu Ser Gly Val Thr Gln Arg Asp Leu Asn Val 120 125 130 AAG GCG GCC TGG GCG CAG GGC TAC ACA GGG CAC GGC ATT GTG GTC TCC 666 Lys Wing Wing Trp Wing Gln Gly Tyr Thr Gly His Gly He Val Val Ser 135 140 145 150 ATT CTG GAC GAT GGC ATC GAG AAG AAC CAC CCG GAC TTG GCA GGC AAT 714 He Leu Asp Asp Gly He Glu Lys Asn His Pro Asp Leu "Ala Gly Asn 155 160 165 TAT GAT CCT GGG GCC AGT TTT GAT GTC AAT GAC CAG GAC CCT GAC CCC 762 Tyr Asp Pro Gly Wing Ser Phe Asp Val Asn Asp Gln Asp Pro Asp Pro 170 175 180 CAG CCT CGG TAC ACE CAG ATG AAT GAC AAC AGG CAC GGC ACÁ CGG TGT 810 Gln Pro Arg Tyr Thr Gln Met Asn Asp Asn Arg His Gly Thr Arg Cys 185 190 195 GCG GGG GAA GTG GCT GCG GTG GCC AAC AAC GGT GTC TGT GGT GTA GGT 858 Wing Gly Glu Val Wing Wing Val Wing Asn Asp Gly Val Cys Gly Val Gly .200 205 210 GTG GCC TAC AAC GCC CGC ATT, GGA GGG GTG CGC ATG CTG GAT GGC GAG 906 Val Wing Tyr Asn Wing Arg He Gly Gly Val Arg Met Leu Asp Gly Glu 215 220 225 230 GTG ACÁ GAT GCA GTG GA G GCA CGC TCG CTG GGC CTG AAC CCC AAC CAC 954 Val Thr Asp Ala Val Glu Ala Arg Ser Leu Gly Leu Asn Pro Asn His 235 240 245 ATC CAC ATC TAC AGT GCC AGC TGG GGC CCC GAG GAT GAC GGC AAG ACA 1002 He His He Tyr Ser Wing Being Trp Gly Pro Glu Asp Asp Gly Lys Thr 250 255 260 GTG GAT GGG CCA GCC CGC CTC GCC GAG GCC GCC TTC TTC CGT GGG GTT 1050 Val Asp Gly Pro Wing Arg Leu Wing Glu Glu Wing Phe Phe Arg Gly Val 265 270 275 AGC CAG GGC CGA GGG GGG CTG GGC TCC ATC TTT GTC TGG GCC TCG GGG 1098 Ser Gln Gly Arg Gly Gly Leu Gly Ser He Phe Val Trp 'Wing Ser Gly 280 285 290 AAC GGG GGC CGG GAA CAT GAC AGC TGC AAC TGC GAC GGC TAC ACC AAC 1146 Asn Gly Gly Arg Glu His Asp Ser Cys Asn Cys Asp Gly Tyr Thr Asn 295 300 305 310 AGT ATC TAC ACG CTG TCC ATC AGC AGC GCC ACG CAG TTT GGC AAC GTG 1194 Ser He Tyr Thr Leu Ser He Ser Ser Ala Thr Gln Phe Gly Asn Val 315 320 325 CCG TGG TAC AGC GAG GCC TGC TCG TCC ACÁ CTG GCC ACG ACC TAC AGC 1242 Pro Trp Tyr Ser Glu Wing Cys Ser Ser Thr Leu Wing Thr Thr Tyr Ser 330 335 340 AGT GGC AAC CAG AAT GAG AAG CAG ATC GTG ACG ACT GAC TTG CGG CAG 1290 Ser Gly Asn Gln Asn Glu Lys Gln He Val Thr Thr Asp Leu Arg Gln 345 350 355 AAG TGC ACG GAG TCT CAC ACG GGC ACC TCA GCC TCT GCC CCC TTA GCA 1338 Lys Cys Thr Glu Ser His Thr Gly Thr Ser Wing Ser Wing Pro Leu Wing 360 365 370 GCC GGC ATC ATT GCT CTC ACC CTG GAG GCC AAT AAG AAC CTC ACÁ TGG 1386 Ala Gly He He Ala Ala Leu Thr Leu Glu Ala Asn Lys Asn Leu Thr Trp 375 380 385 390 CGG GAC ATG CAAC CAC CTG GTG GTA CAG ACC TCG AAG CCA GCC CAC CTC 1434 Arg Asp Met Gln His Leu Val Val Gln Thr Ser Lys Pro Wing His Leu 395 400 405 AAT GCC AAC GAC TGG GCC ACC AAT GGT GTG GGC CGG AAA GTG AGC CAC 1482 Asn Ala Asn Asp Trp Wing Thr Asn Gly Val Gly Arg Lys'val Ser His 410 415 420 TCA TAT GGC TAC GGG CTT TTG GAC GCA GGC GCC ATG GTG GCC CTG GCC 1530 Be Tyr Gly Tyr Gly Leu Leu Asp Wing Gly Wing Met Val Wing Leu Wing 425 430 435 CAG AAT TGG ACC A GTG GCC CCC CAG CGG AAG TGW ATC ATC GAC ATC 1578 Gln Asn Trp Thr Thr Val Wing Pro Gln Arg Lys Cys He He Asp He 440 445 450 CTC ACC GAG CCC AAA GAC ATC GGG AAA CGG CTC GAG GTG CGG AAG ACC 1626 Leu Thr Glu Pro Lys Asp He Gly Lys Arg Leu Glu Val Arg Lys Thr 455 460-. 465 470 GTG ACC GCG TGC CTG GGC GAG CCC AAC CAC ATC ACT CGG CTG GAG CAC 1674 Val Thr Wing Cys Leu Gly Glu Pro Asn His He Thr Arg Leu Glu His 475 480 485 GCT CAG GCG CGG CTC ACC CTG TCC TAT AAT CGC CGT GGC GAC CTG GCC 1722 Wing Glp Wing Arg Leu Thr Leu Ser Tyr Asn Arg Arg Gly Asp Leu Wing 490 495 500 ATC CAC CTG GTC AGC CCC ATG GGC ACC CGC TCC ACC CTG CTG GCA GCC 1770 He His Leu Val Ser Pro Met Gly Thr Arg Ser Thr Leu Leu Wing Wing 505 510 515 AGG CCA CAT GAC TAC TCC GCC GAT GAT GGG TTT AAT GAC TGG GCC TTC ATG 1818 Arg Pro His Asp Tyr Ser Wing Asp Gly Phe Asn Asp Trp Wing Phe Met 520 525 530 ACÁ ACT CAT TCC TGG GAT GAG GAT CCC TCT GGC GAG TGG GTC CTA GAG 1866 Thr Thr His Ser Trp Asp Glu Asp Pro Ser Gly Glu Trp'Val Leu Glu 535 540 545 550 ATT GAA AAC ACC AGC GAA GCC AAC AAC TAT GGG ACG CTG ACC AAG TTC 1914 He Glu Asn Thr Ser Glu Wing Asn Asn Tyr Gly Thr Leu Thr Lys Phe 555 560 565 ACC CTC GTA CTC TAT GGC ACC GCC CCT GAG GGG CTG CCC GTA CCT CCA 1962 Thr Leu Val Leu Tyr Gly Thr Ala Pro Glu Gly Leu Pro Val Pro Pro 570 575 580 GAA AGC AGT GGC TGC AAG ACC CTC ACG TCC AGT CAG GCC TGA 2004 Glu Be Ser Gly Cys Lys Thr Leu Thr Ser Ser Gln Ala *** 585 -. 590 595 GTGGTGTGCG AGGAAGGCTT CTCCCTGCAC CAGAAGAGCT GTGTCCAGCA CTGCCCTCCA 2064 GGCTTCGCCC CCCAAGTCCT CGATACGCAC TATAGCACCG AGAATGACGT GGAGACCATC 2124 CGGGCCAGCG TCTGCGCCCC CTGCCACGCC TCATGTGCCA CATGCCAGGG GCCGGCCCTG 2184 ACAGACTGCC TCAGCTGCCC CAGCCACGCC TCCTTGGACC CTGTGGAGCA GACTTGCTCC 2244 CGGCAAAGCC AGAGCAGCCG AGAGTCCCCG CCACAGCAGC AGCCACCTCG GCTGCCCCCG 2304 GAGGTGGAGG CGGGGCAACG GCTGCGGGCA GGGCTGCTGC CCTCACACCT GCCTGAGGTG 2364 GTGGCCGGCC TCAGCTGCGC CTTCATCGTG CTGGTCTTCG TCACTGTCTT CCTGGTCCTG 2424 CAGCTGCGCT CTGGCTTTAG TTTTCGGGGG GTGAAGGTGT ACACCATGGA CCGTGGCCTC 2484 ATCTCCTACA AGGGGCTGCC CCCTGAAGCC TGGCAGGAGG AGTGCCCGTC TGACTCAGAA 2544 GAGGACGAGG GCCGGGGCGA GAGGACCGCC TTTATCAAAG ACCAGAGCGC CCTCTGATGA 2604 GCCCACTGCC CACCCCCTCA AGCCAATCCC CTCCTTGGGC ACTTTTTAAT TCACCAAAGT 2664 ATTTTTTTTAT CTTGGGACTG GGTTTGGACC CCAGCTGGGA GGCAAGAGGG GTGGAGACTG 2724 TTTCCCATCC TACCCTCGGG CCCACCTGGC CACCTGAGGT GGGCCCAGGA CCAGCTGGGG 2784 CGTGGGGAGG GCCGTACCCC ACCCTCAGCA CCCCTTCCAT GTGGAGAÁAG GAGTGAAACC 2844 TTTAGGGCAG CTTGCCCCGG CCCCGGCCCC AGCCAGAGTT CCTGCGGAGT GAAGAGGGGC 2904 AGCCCTTGCT TGTTGGGATT CCTGACCCAG GCCGCAGCTC TTGCCCTTCC CTGTCCCTCT 2964 AAAGCAATAA TGGTCCCATC CAGGCAGTCG GGGGCTGGCC TAGGAGATAT CTGAGGGAGG 3024 AGGCCACCTC TCCAAGGGCT TCTGCACCCT CCACCCTGTC CCCCAGCTCT GGTGAGTCTT 3084 GGCGGCAGCA GCCATCATAG GAAGGGACCA AGGCAAGGCA GGTGCCTCCA GGTGTGCACG 3144 TGGCATGTGG CCTGTGGCCT GTGTCCCATG ACCCACCCCT GTGCTCCGTG CCTCCACCAC 3204 CACTGGCCAC CAGGCTGGCG CAGCCAAGGC CGAAGCTCTG GCTGAACCCT GTGCTGGTGT 3264 CCTGACCACC CTCCCCTCTC TTGCACCCGC CTCTCCCGTC AGGGCCCAAG TCCCTGTTTT 3324 CTGAGCCCGG GCTGCCTGGG CTGTTGGCAC TCACAGACCT GGAGCCCCTG GGTGGGTGGT 3384 GGGGAGGGGC GCTGGCCCAG CCGGCCTCTC TGGCCTCCCA CCCGATGCTG CTTTCCCCTG 3444 TGGGGATCTC AGGGGCTGTT TGAGGATATA TTTTCACTTT GTGATTATTT CACTTTAGAT 3504 GCTGATGATT TGTTTTTGTA TTTTTAATGG GGGTAGCAGC TGGACTACCC ACGTTCTCAC 3564 ACCCACCGTC CGCCCTGCTC CTCCCTGGCT GCCCTGGCCC TGAGGTGTGG GGGCTGCAGC 3624 ATGTTGCTGA GGAGTGAGGA ATAGTTGAGC CCCAAGTCCT GAAGAGGCGG GCCAGCCAGG '3684 CGGGCTCAAG GAAAGGGGGT CCCAGTGGGA GGGGCAGGCT GACATCTGTG TTTCAAGTGG 3744 GGCTCGCCAT GCCGGGGGTT CATAGGTCAC TGGCTCTCCA AGTGCCAGAG GTGGGCAGGT 3804 GGTGGCACTG AGCCCCCCCA ACACTGTGCC CTGGTGGAGA AAGCACTGAC CTGTCATGCC 3864 CCCCTCAAAC CTCCTCTTCT GACGTGCCTT TTGCACCCCT CCCATTAGGA CAATCAGTCC • 3924 CCTCCCATCT GGGAGTCCCC TTTTCTTTTC TACCCTAGCC ATTCCTGGTA CCCAGCCATC 3984 TGCCCAGGGG TGCCCCCTCC TCTCCCATCC CCCTGCCCTC GTGGCCAGCC CGGCTGGTTT 4044 TGTAAGATAC TGGGTTGGTG CACAGTGATT TTTTTCTTGT AATTTAAACA GGCCCAGCAT 4104 TGCTGGTTCT ATTTAATGGA CATGAGATAA TGTTAGAGGT TTTAAAGTGA TTAAACGTGC 4164 AGACTATGCA AACCAG 4180 SEQ ID NO: 2 LENGTH: 29 TYPE: nucleic acid TYPE OF FILAMENT: one TOPOLOGY: linear TYPE OF MOLECULE: other nucleic acid ORIGINAL SOURCE: no ORGANISM: no FILAMENT: no CHARACTERISTICS: primer 3 in the furin sense DESCRIPTION FROM THE SEC id no: 2 GCGAAGCTTA AGACCAGGCC AAGGAGACG SEQ ID NO: 3 LENGTH: 26 TYPE: nucleic acid TYPE OF FILAMENT: one TOPOLOGY: linear TYPE OF MOLECULE: other nucleic acid ORIGINAL SOURCE: no ORGANISM: no CEPA: no CHARACTERISTICS: primer 4 in sense of furin DESCRIPTION OF SEQ ID NO: 3: CCTCGCCATC CAGCATGCGC ACCCT SEQ ID NO: 4 LENGTH: 26 TYPE: nucleic acid TYPE OF FILAMENT: one TOPOLOGY: linear TYPE OF MOLECULE: other nucleic acid TYPE OF SOURCE: no ORGANISM: no CEPA: no FEATURE: primer 5 in the furin sense DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 TGGAGGGTG CGCATGCTGG ATGGCG SEQ ID NO: 5 LENGTH: 30 TYPE: nucleic acid TIFO DE "FILAMENTO: one TOPOLOGY: linear TYPE OF MOLECULE: other nucleic acids ORIGINAL SOURCE: no ORGSNTSHO: no CEPA: no CHARACTERISTICS: primer 6 in furin sense DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 5 GGCGTCGACG CACACCACTC AGGCCTGACT SEQ ID NO: 6 LENGTH: 2703 TYPE: amino acid TYPE OF FILAMENT: double TOPOLOGY: LINEAR TYPE OF MOLECULE: peptides TYPE OF FRAGMENT: ORIGINAL SOURCE: ORGANISM: homo sapiens TYPE OF TISSUE: human embryo CARACTERI ST IC: LOCALTZACTON: OTHER T? TFORMACTO? T: bone morphogenetic MP52 protein DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6 CCATGGCCTC GAAAGGGCAG CGGTGATTTT TTTCACATAA ATATATCGCA CTTAAATGAG 60 TTTAGACAGC ATGACATCAG AGAGTAATTA AATTGGTTTG GGTTGGAATT CCGTTTCCAA 120 TTCCTGAGTT CAGGTTTGTA AAAGATTTTT CTGAGCACCT GCAGGCCTGT GAGTGTGTGT 180 GTGTGTGTGT GTGTGTGTGT GTGTGTGTGA AGTATTTTCA CTGGAAAGGA TTCAAAACTA 240 GGGGGAAAAA AAAACTGGAG CACACAGGCA GCATTACGCC ATTCTTCCTT CTTGGAAAAA 300 TCCCTCAGCC TTATACAAGC CTCCTTCAAG CCCTCAGTCA GTTGTGCAGG AGAAAGGGGG 360 CGGTTGGCTT TCTCCTTTCA AGAACGAGTT ATTTTCAGCT GCTGACTGGA GACGGTGCAC 420 GTCTGGATAC GAGAGCATTT CCACTATGGG ACTGGATACA AACACACACC CGGCAGACTT 480 CAAGAGTCTC AGACTGAGGA GAAAGCCTTT CCTTCTGCTG CTACTGCTGC TGCCGCTGCT 540 TTTGAAAGTC CACTCCTTTC ATGGTTTTTC CTGCCAAACC AGAGGCACCT TTGCTGCTGC 600 CGCTGTTCTC TTTGGTGTCA TTCAGCGGCT ATG AGA CTC CCC GGCCAGAGG AAA 654 Met Arg Leu Pro Lys -25 CTC CTC ACT TTC TTG CTT TGG TAC CTG GCT TGG CTG GAC CTG GAA TTC 702 Leu Leu Thr Phe Leu Leu Trp Tyr Leu Wing Trp Leu Asp Leu Glu Phe -20 -15 -10 ATC TGC ACT GTG TTG GGT GCC CCT GAC TTG GGC CAG AGA CCC CAG GGG 750 He Cys Thr Val Leu Gly Wing Pro Asp Leu Gly GIn Arg Pro Gln Gly -5 1 5 10 ACC AGG CGA GGA TTG GCC AAA GCA GAG GCC AAG GAG AGG CCC CCC CTG 798 Thr Arg Pro Gly Leu Wing Lys Wing Glu Wing Lys Glu Arg Pro Pro Leu 15 20 25 GCC CGG AAC GTC TTC AGG CCA GGG GGT CAC AGC TAT GGT GGG GGG GCC 846 Wing Arg Asp Val Phe Arg Pro Gly Gly His Ser Tyr Gly Gly Gly Wing 30 35 40 ACC AAT GCC AAT GCC AGG GCA AAG GGA GGC ACC GGG CAG ACÁ GGA GGC 894 Thr Asn Wing Asp Wing Arg Wing Lys Gly Gly Thr Gly GIn T hr Gly Gly 45 50 55 CTG ACA CAG CCC AAG AAG GAT GAA CCC AAA AAG CTG CCC CCC AGA CCG 942 Leu Thr Glp Pro Lys Lys Asp Glu Pro Lys Lys Leu Pro Pro Arg Pro 60 65 70 GGC GGC CCT GAA CCC AAG CCA GGA CAC CCT CCC CAÁ ACÁ AGG CAG GCT 990 Gly Gly Pro Glu Pro Lys Pro Gly His Pro Pro Gln Thr Arg Gln Wing 75 80 85 90 ACÁ GCC CGG ACT GTG ACC CCA AAA GGA CAG CTT CCC GGA GGC AAG GCA 1038 Thr Wing Arg Thr Val Thr Pro Lys Gly Gln Leu Pro Gly Gly Lys Wing 95 100 105 CCC CCA AAA GCA GGA TCT GTC CCC AGC TCC CTG CTG AAG AAG GCC 1086 Pro Pro Lys Wing Gly Ser Val Pro Ser Be Phe Leu Leu Lys Lys Wing 110 115 120 AGG GAG CCC GGG CCC CCA CGA GAG CCC AAG GAG CCG TTT CGC CCA CCC 1134 Arg Glu Pro Gly Pro Pro Arg Glu Pro Lys Glu Pro Phe Arg Pro Pro 125 130 135 CCC ATC ACA CCC CAC GAG TAC ATG CTC TCG CTG TAC AGG ACG CTG TCC 1182 Pro He Thr Pro His Glu Tyr Met Leu Ser Leu Tyr Arg Thr Leu Ser 140 145 150 GAT GCT GAC AGA AAG GGA GGC AAC AGC AGC GTG AAG TTG GAG GCT GGC 1230 Asp Wing Asp Arg Lys Gly Gly Asn Ser Ser Val Lys Leu Glu Wing Gly 155 160 165 170 CTG GCC AAC ACC ATC ACC AGC TTT ATT GAC AAA GGG CAA GAT GAC CGA 1278 Leu Wing Asn Thr He Thr Ser Phe He Asp Lys Gly Gln Asp Asp Arg 175 180 185 GGT CCC GTG GTC AGG AAG CAG AGG TAC GTG TTT GAC ATT AGT GCC CTG 1326 Gly Pro Val Val Arg Lys Gln Arg Tyr Val Phe Asp He Ser Ala Leu 190 195 200 GAG AAG GAT GGG CTG CTG GGG GCC GAG CTG CGG ATC TTG CGG AAG AAG 1374 Glu Lys Asp Gly Leu Leu 'Gly Wing Glu Leu Arg He Leu Arg Lys Lys 205 210 215 CCC GCC TCG GAC GCC AAG CCA GCG GCC CCC GGA GGG GGG CGG GCC 1422 Pro Ser Asp Thr Wing Lys Pro Wing Wing Pro Gly Gly Gly Arg Wing Wing 220 225 230 CAG CTG AAG CTG TCC AGC TGC CCC AGC GGC CGG CAG CCG GCC TCC TTG 1470 Gln Leu Lys Leu Be Ser Cys Pro Ser Gly Arg Gln Pro Wing Ser Leu 235 240 245 250 CTG GAT GTG CGC TCC GTG CCA GGC CTG GAC GGA TCT GGC TGG GAG GTG 1518 Leu Asp Val Arg Ser Val Pro Gly Leu Asp Gly Ser Gly Trp Glu Val 255 260 265 TTC GAC ATC TGG AAG CTC TTC CGA AAC TTT AAG AAC TCG GCC CAG CTG 1566 Phe Asp He Trp Lys Leu Phe Arg Asn Phe Lys Asn Ser Wing Gln Leu 270 275 280 TGC CTG GAG CTG GAG GCC TGG GAA CGG GGC AGG GCC GTG GAC CTC CGT 1614 Cys Leu Glu Leu Glu Wing Trp Glu Arg Gly Arg Wing Val Asp Leu Arg 285 290 295 GGC CTG GGC TTC GAC CGC GCC GCC CGG CAG GTC CAC GAG AAG GCC CTG 1662 Gly Leu Gly Phe Asp Arg Wing Wing Arg Gln Val His Glu Lys Wing Leu 300 305 310 TTC CTG GTG TTT GGC CGC ACC AAG AAA CGG GAC CTG TTC TTT AAT GAG 1710 Phe Leu Val Phe Gly Arg Thr Lys Lys Arg Asp Leu Phe Phe Asn Glu 315 320 325 330 ATT AAG GCC CGC TCT GGC CAG GAC GAT AAG ACC GTG TAT GAG TAC CTG 1758 He Lys Wing Arg Ser Gly Gln Asp Asp Lys Thr Val Tyr Glu Tyr Leu 335 340 345 TTC AGC CAG CGG CGA AAA CGG CGG GCC CCA CTG GCC ACT CGC CAG GGC 1806 Phe Ser Gln Arg Arg Lys Arg Arg Wing Pro Leu Wing Thr Arg Gln Gly 350 355 360 AAG CGA CCC AGC AAG AAC CTT AAG GCT CGC TGC AGT CGG AAG GCA CTG 1854 Lys Arg Pro Ser Lys Asn Leu Lys Wing Arg Cys Ser Arg Lys Wing Leu 365 370 375 CAT GTC AAC TTC AAG GAC ATG GGC TGG GAC GAC TGG ATC ATC GCA CCC 1902 His Val Asn Phe Lys Asp Met Gly Trp Asp Asp Trp He He Wing Pro 380 385 390 CTT GAG TAC GAG GCT TTC CAC TGC GAG GGG CTG TGC GAG TTC CCA TTG 1950 Leu Glu Tyr Glu Wing Phe His Cys Glu Gly Leu Cys Glu Phe Pro Leu 395 400 405 410 CGC TCC CAC CTG GAG CCC ACG AAT CAT GCA GTC ATC CAG ACC CTG ATG 1998 Arg Ser His Leu Glu Pro Thr Asn His Wing Val He Gln Thr Leu Met 415 420 425 AAC TCC ATG GAC CCC GAG TCC ACA CCA CCC ACC TGC TGT GTG CCC ACG 2046 Asn Ser Met Asp Pro Glu Be Thr Pro Pro Thr Cys Cys Val Pro Thr 430. 435 440 CGG CTG AGT CCC ATC AGC ATC CTC TTC ATT GAC TCT GCC AAC AAC GTG * 2094 Arg Leu Ser Pro He Be He Leu Phe He Asp Be Wing Asn Asn Val 445 450 455 GTG TAT AAG CAG TAT GAG GAC ATG GTC GTG GAG TCG TGT GGC TGC AGG 2142 Val Tyr Lys Gln Tyr Glu Asp Met Val Val Glu Ser Cys Gly Cys Arg 460 465 470 TAG CAGCACTGGC CCTCTGTCTT CCTGGGTGGC ACATCCCAAG AGCCCCTTCC 2195 *** 475 TGCACTCCTG GAATCACAGA GGGGTCAGGA AGCTGTGGCA GGAGCATCTA CACAGCTTGG 2255 GTGAAAGGGG ATTCCAATAA GCTTGCTCGC TCTCTGAGTG TGACTTGGGC TAAAGGCCCC 2315 CTTTTTATCCA CAAGTTCCCC TGGCTGAGGA TTGCTGCCCG TCTGCTGATG TGACCAGTGG 2375 CAGGCACAGG TCCAGGGAGA CAGACTCTGA ATGGGACTGA GTCCCAGGAA ACAGTGCTTT 2435 CCGATGAGAC TCAGCCCACC ATTTCTCCTC ACCTGGGCCT TCTCAGCCTC TGGACTCTCC 2495 TAAGCACCTC TCAGGAGAGC CACAGGTGCC ACTGCCTCCT CAAATCACAT TTGTGCCTGG 2555 TGACTTCCTG TCCCTGGGAC AGTTGAGAAG CTGACTGGGC AAGAGTGGGA GAGAAGAGGA 2615 GAGGGCTTGG ATAGAGTTGA GGAGTGTGAG GCTGTTAGAC TGTTAGATTT AAATGTATAT 2675 TGATGAGATA AAAAGCAAAA CTGTGCCT 2703 It is noted that in relation to this date the best method known by the applicant to carry out the said invention is that which is clear from the present description thereof.

Having described the invention as above, it is claimed as property in the following:

Claims (8)

Claims

1. A process for producing a bone morphogenetic protein by maturation, characterized in that a bone morphogenetic protein precursor is treated with a processing enzyme.

2. A process for producing a bone morphogenetic protein comprising the steps of: introducing a vector expression of a bone morphogenetic protein precursor and a transformed processing enzyme into a mammalian cell; culturing said cultured mammalian cell, whereby a bone morphogenetic protein is produced by maturation; and so, Separate a bone morphogenetic protein by purified maturation of the supernatant culture.

3. The process for producing a bone morphogenetic protein by maturation is considered in claim 1, which is characterized in that the bone morphogenetic protein by maturation is selected from the group consisting of lao MP52, BMP-2, BMP-4 *, BMP-6 and maturation BMP-7

4. The process for producing a bone morphogenetic protein by maturation is considered in at least one of claims 1 to 4, characterized in that the processing enzyme is furin.

5. The process for producing a bone morphogenetic protein by maturation is considered in at least one of claims 1 to 4, which are characterized in that the processing enzyme is furin consisting of a complete amino acid sequence.

6. The process for producing a bone morphogenetic protein by maturation is considered in at least one of claims 1 to 4, which are characterized in that the processing enzyme is a secretory mutant furin.

7. The process for producing a bone morphogenetic protein by maturation as considered in at least one of claims 1 to 5, is characterized in that it comprises the steps of: introducing an expression of the vector of an MP52 precursor and a secretory mutant furin transformed into a mammalian cell; culturing said cultured mammalian cell, whereby a bone morphogenetic protein is produced by maturation; and so, Separate a bone morphogenetic protein by purified maturation of the supernatant culture.

8. A process for producing a bone morphogenetic protein by maturation, which is characterized in that it comprises the steps of: Add a solution containing a processing enzyme to a solution containing a bone morphogenetic protein precursor, after that incubate the mixture.