Bmp-11 compositions

<u style="single">Background of the invention</u>The present invention relates to a novel family of purified proteins named BMP-11, DNA molecules encoding them, and methods of obtaining them. We previously named the BMP-11 protein Activin WC. The BMP-11 protein may be useful in inducing bone and / or chondrogenesis and healing wounds and repairing tissues, or increasing the activity of other bone morphogenetic proteins. In addition, the BMP-11 protein may be useful in regulating the production of follicle-stimulating hormone for contraception, promoting neuronal survival, stimulating augmentation, and suppressing the development of ovarian cancer. U.S. Pat. No. 4,798,885 discloses DNA encoding preproinsulin α and β chains. U.S. Pat. No. 5,071,834 states two betas formulated in a pharmaceutically acceptable carrier._BA pharmaceutical composition of Actibin having a chain is disclosed. U.S. Pat. No. 5,102,807 discloses a purified inhibin protein that suppresses FSH production without suppressing luteinizing hormone production.<u style="single">Outline of the invention</u>The BMP-11 protein is a member of the TGF-β superfamily of proteins. The TGF-β superfamily includes a family of proteins known as bone morphogenetic proteins (BMPs) and a group of proteins called inhibin-β. Further, as discussed herein, when dimerized (homodimer) with another BMP-11, the BMP-11 protein becomes BMP-11 active, as further described herein. The activity can be measured by the analysis described in Examples herein. When dimerized as a heterodimer with inhibin-α protein or other inhibin-β protein, the inhibin-β / BMP-11 heterodimer is a follicle-stimulating hormone, as further described herein. It is expected to show an effect on the production of (FSH). In addition, in homodimer morphology or in heterodimer morphology with another member of the bone morphogenetic protein family, BMP-11 exhibits BMP activity, i.e. the ability to induce the formation of bone, cartilage and / or other connective tissue. Expected to show. Thus, depending on the environment surrounding BMP-11, dimers may be formed that exhibit either activin or inhibin activity or activity of either bone, cartilage and / or other connective tissue-inducing activity. Therefore, BMP-11 activity is the ability to regulate FSH production in the analysis described in Example 8 herein, or the ability to induce the formation of bone, cartilage and / or other connective tissue in the analysis described in Examples 5-7 herein. Is defined as. Proteins called inhibin and activin are produced in the gonads and are naturally present in the follicular fluid. These proteins suppress or stimulate (inhibin) the release of follicle-stimulating hormone (FSH) at the level of the anterior pituitary gland [for review, eg, Ying, S.- Y.) et al., Endocr. Rev., Vol. 9: pp. 267-293 (1988) or Ring, N. et al., See Vitamins and Hormones, Vol. 44, pp. 1-46 (Academic Press, 1988)]. Briefly, one inhibin α chain and one inhibin β (β)_AOr β_BThe dimeric protein consisting of a) chain is called activin and is characterized by its ability to suppress the release of follicle-stimulating hormone (FSH), while two activin β chains (β)._AOr β_BThe other dimer protein consisting of) is called activin and is characterized by its ability to stimulate the release of follicle-stimulating hormone (FSH) [eg, Ling et al., Nature, 321. Volumes: 779-782 (1986) or Vale et al., Nature, Volume 321: 776-779 (1986) or Manson et al., Nature, Volume 318: Pp. 659-663 (1985), or Forage et al., Proc. Natl. Acad. Sci. USA, Vol. 83: 3091-3095. (1986)]. FSH stimulates egg development in the mammalian ovary (Ross et al., Textbook of Endocrinology) Endocrinology (edited by Williams), p. 355 (1981)), and it is recognized that overstimulation of the ovaries with FSH may induce multiple ovulations. In addition, FSH is also important for testicular function. Thus, when heterodimers with members of the Inhibin α family, BMP-11 is useful as a contraceptive because of its ability to reduce fertility in female animals and reduce spermatogenesis in male animals. Can be. Administration of sufficient amounts of other inhibin can induce infertility in these animals. BMP-11 as a homodimer or a heterodimer with other protein subunits of the inhibin-β group is based on the ability of the activine molecule to stimulate FSH released from cells in the anterior pituitary gland. It may be useful as a fertility-inducing therapeutic agent. See, for example, US Pat. No. 4,798,885. In addition, BMP-11 may be useful in promoting fertilization in sexually immature mammals, resulting in extended reproductive life in livestock such as cattle, sheep and pigs. .. In addition, BMP-11 promotes neuronal survival [see, eg, Schubert et al., Nature, Vol. 344: 868-870 (1990)], inducing erythroblast differentiation. Regulation of hematopoietic function by [eg, Broxmeyer et al., Proc. Natl. Acad. Sci. USA, Proc. Natl. Acad. Sci. USA, Vol. 85: 9052-956. ] (1988) or Eto et al., Biochem.Biophys.Res.Comm., Vol. 142, pp. 1095-1103 (1987)], Suppression of development of gonad tumors [For example, Matzuk et al., Nature, Volume 360: See pages 313-319 (1992)], or increased activity of bone morphogenetic proteins [eg, Ogawa et al., Journal of Biological Chemistry (J. Biol. Chem.), Vol. 267: 14233. See page 14237 (1992)], which may be useful. In addition, the BMP-11 protein was described as described [eg, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature ( Nature), Vol. 321: 779-782 (1986) or Vale et al., Nature, Vol. 321: 776-779 (1986)], using rat and anterior pituitary cells It can be characterized by its ability to regulate the release of follicle-stimulating hormone (FSH) in established bioassays in vitro. When the BMP-11 protein of the present invention is constructed as a heterodimer together with a homodimer or other inhibin β chain, as described [Ling et al., Nature, Vol. 321 :. 779-782 (1986) or Vale et al., Nature, 321: 776-779 (1986)], showing the stimulating effect on the release of follicle-stimulating hormone from the anterior pituitary gland. It is thought that there will be. Furthermore, when the BMP-11 protein of the present invention is constructed as a heterodimer together with the inhibin α chain, as described [eg, Vale et al., Endocrinology, Vol. 91: 562. See page 572 (1972)], which is thought to suppress the release of follicle-stimulating hormone (FSH) from the anterior pituitary gland. Therefore, depending on the individual composition, it is expected that the BMP-11 proteins of the present invention may have contrasting and contradictory effects on the release of follicle-stimulating hormone (FSH) from the anterior pituitary gland. Actybin A (Inhibin β See pages 562-572 (1972)], it is believed that it will suppress the release of follicle-stimulating hormone (FSH) from the anterior pituitary gland. Therefore, depending on the individual composition, it is expected that the BMP-11 proteins of the present invention may have contrasting and contradictory effects on the release of follicle-stimulating hormone (FSH) from the anterior pituitary gland. Actybin A (Inhibin β_AHomo dimer composition) has been shown to have hematopoietic function stimulating activity [eg, Eto et al., Biochem.Biophys.Res.Comm., No. Vol. 142: 1095-1103 (1987) Murata et al., Proc. Natl.Acad.Sci.USA, Vol. 85: 2434-2438 See page (1988) and Yu et al., Nature, Vol. 330: pp. 765-767 (1987)]. The BMP-11 protein of the present invention is considered to have similar hematopoietic function stimulating activity. In addition, Lozzio et al., Blood, Vol. 45: pp. 321-334 (1975) and US Pat. No. 5,071, This activity of BMP-11 can be characterized by its ability to show the erythropoietin activity of the BMP-11 protein in biological analyzes performed using the human K-562 cell line, as described by No. 834. .. The structures of several proteins named BMP-1 to BMP-9 have already been investigated. The unique inducing activity associated with the presence of these proteins in bone may be important regulators of the bone repair process and may be required for the normal maintenance of bone tissue. It suggests that there is. The BMP-11 protein of the present invention is related to the above BMP protein and shares BMP activity such as ability to induce other connective tissues such as bone, cartilage and / or tendon or ligament as well as wound healing activity of BMP. Expected to be. In addition, the proteins of the invention are expected to act in collaboration with or perhaps synergistically with other related proteins and growth factors. Therefore, a further therapeutic method and composition of the present invention is a therapeutic amount of at least one therapeutic amount of the BMP of the present invention mixed with at least one of the therapeutic amounts of other BMP proteins disclosed in the following shared patent and application. -11 Consists of protein. Such a mixture consists of a separate BMP protein molecule or a heteromolecule consisting of different BMP moieties. In addition, the BMP-11 protein may be mixed with other agents useful in the treatment of bone and / or cartilage defects, wounds, or tissue in question. These agents include epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transformed growth factor (TGF-α and TGF-β), and k-fibroblast growth. Includes factors, parathyroid hormone (PTH), leukemia inhibitor (LIF / HILDA / DIA), insulin-like growth factor (IGF-I and IGF-II). Parts of these agents can also be used in the compositions of the present invention. Bovine BMP-11 DNA sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) and human BMP-11 DNA sequence (SEQ ID NO: 10) and amino acid sequence (SEQ ID NO:: 2) 11) is shown in the sequence listing of the present specification. Actibin protein has the ability to regulate the production of follicle-stimulating hormone (FSH), so BMP-11 may be useful as a contraceptive or fertilization-inducing therapeutic agent. In the form of a homodimer or a heterodimer with a protein in the inhibin-β group, the purified BMP-11 protein is expected to exhibit activin activity and can be used for FSH stimulation. In addition, purified BMP-11 protein can be used to induce bone, cartilage and / or connective tissue. A protein characterized by an amino acid sequence consisting of amino acids 1 to 109 shown in SEQ ID NO: 2 after culturing cells transformed with the DNA sequence consisting of nucleotides 375 to nucleotide 704 shown in SEQ ID NO: 1. The bovine BMP-11 can be produced by recovering and purifying a protein that is substantially free of the proteinaceous substance produced at the same time from the medium. Human BMP-11 is expected to be homologous to bovine BMP-11. Therefore, the present invention includes a DNA sequence encoding human BMP-11, a DNA sequence obtained by these methods, and a method for obtaining a human protein encoded by these DNA sequences. This method involves using the bovine BMP-11 nucleotide sequence or a portion thereof to design a probe for screening a human gene or fragment thereof using standard methods. DNA sequence encoding a part of human BMP-11 protein (SEQ ID NO: 3) and corresponding amino acid sequence (SEQ ID NO:: 3) 4) is shown in the sequence listing. In addition, these sequences may be used to design probes to obtain the complete human BMP-11 gene by standard methods. Human BMP-11 can be produced by culturing cells transformed with the DNA, and then recovering and purifying BMP-11 from the medium. The purified expressed protein is substantially free of other co-produced proteinaceous substances as well as other contaminants. The recovered and purified protein is expected to exhibit the ability to regulate FSH production. In addition, the protein of the invention can be characterized by its ability to regulate follicle-stimulating hormone (FSH) production in established in vitro bioassays using rat anterior pituitary cells. In addition, for example, in the rat bone formation analysis below, the BMP-11 protein can be characterized by its ability to induce the formation of bone, cartilage and / or other connective tissue. Another aspect of the invention provides a pharmaceutical composition containing a therapeutically effective amount of BMP-11 protein in a pharmaceutically acceptable excipient or carrier. The BMP-11 composition of the present invention may be useful for the regulation of follicle-stimulating hormone and may be useful for contraception. Further, the compositions of the present invention are, for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 disclosed in US Pat. Nos. 5,108,922, 5,013,649, 5,116,738, 5,106,748, 5,187,076 and 5,141,905. And BMP-7; BMP-8 disclosed in PCT Publication WO 93/18098; and BMP-9 disclosed in PCT Publication WO 93/00432; It may contain at least one other therapeutically useful agent, such as BMP-10 disclosed in No. 695. The BMP-11 composition can also be used in many uses, including regulation of follicle-stimulating hormone production, including contraception. According to the present invention, these methods include administering an effective amount of BMP-11 to a patient in need of such treatment. In addition to BMP-11, the compositions of the present invention include proteins of the inhibin-β group or other members of the proteins of the inhibin-α group, as well as epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming. It may consist of growth factors (TGF-α and TGF-β), as well as other therapeutically useful agents including insulin-like growth factor (IGF). The BMP-11 compositions of the present invention are also useful in the treatment of many bone and / or cartilage defects, periodontal disease and various forms of wounds. According to the present invention, these methods include administering an effective amount of BMP-11 protein to a patient in need of such bone and / or cartilage formation, wound healing or tissue repair. Furthermore, these methods include administering the protein of the invention mixed with at least one novel BMP protein disclosed in the shared patent and application. In addition, these methods involve administering the BMP-11 protein along with other growth factors, including EGF, FGF, TGF-α, TGF-β and IGF. A further aspect of the invention is a DNA sequence encoding expression of the BMP-11 protein. Such a sequence is a nucleotide sequence in the 5'to 3'direction shown in SEQ ID NO: 1, or hybridizes with the DNA sequence of SEQ ID NO: 1 under constriction conditions and encodes a protein having BMP-11 activity. Includes DNA sequences that are hybridized. Ultimately, allelic variants or other variants of the SEQ ID NO: 1 sequence are also included in the invention, whether or not nucleotide changes result in changes in the sequence of the peptide. A further aspect of the invention is a DNA sequence encoding expression of the BMP-11 protein. Such a sequence includes SEQ ID NO:: Same as the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 10 except for the nucleotide sequence in the 5'to 3'direction shown in 1 or SEQ ID NO: 10 and the degeneracy of the genetic code. It is a DNA sequence encoding the protein of SEQ ID NO: 11. Furthermore, a DNA sequence that hybridizes to the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 10 under austerity conditions and encodes a protein having BMP-11 activity is also included in the present invention. Ultimately, allelic variants or other variants of SEQ ID NO: 1 or SEQ ID NO: 10 also have BMP-11 activity in the peptide sequence, regardless of whether such nucleotide changes result in changes in the peptide sequence. In some cases, it is included in the present invention. A further aspect of the invention is a vector consisting of the above DNA sequences operably linked to expression regulatory sequences. A cell line transformed with a DNA sequence encoding a BMP-11 protein ligated to operate is cultured in a suitable medium, and the BMP-11 protein is recovered and purified from the cell line. These vectors can be used in the novel BMP-11 protein production method of the invention. Many known cells, both prokaryotic and eukaryotic, can be used in this method as host cells for the expression of the polypeptide. Furthermore, the present invention includes the application of the DNA sequences and vectors of the present invention to gene therapy. In such use, the vector can be transfected into the patient's cells in vitro and then the cells can be reintroduced into the patient. Alternatively, the vector may be introduced into the patient in vivo by targeted transfection. Other aspects and advantages of the present invention will become apparent in light of the following detailed description and preferred embodiments thereof. It is a DNA sequence encoding 11 proteins. Furthermore, a DNA sequence that hybridizes to the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 10 under austerity conditions and encodes a protein having BMP-11 activity is also included in the present invention. Ultimately, allelic variants or other variants of SEQ ID NO: 1 or SEQ ID NO: 10 also have BMP-11 activity in the peptide sequence, regardless of whether such nucleotide changes result in changes in the peptide sequence. In some cases, it is included in the present invention. A further aspect of the invention is a vector consisting of the above DNA sequences operably linked to expression regulatory sequences. A cell line transformed with a DNA sequence encoding a BMP-11 protein ligated to operate is cultured in a suitable medium, and the BMP-11 protein is recovered and purified from the cell line. These vectors can be used in the novel BMP-11 protein production method of the invention. Many known cells, both prokaryotic and eukaryotic, can be used in this method as host cells for the expression of the polypeptide. Furthermore, the present invention includes the application of the DNA sequences and vectors of the present invention to gene therapy. In such use, the vector can be transfected into the patient's cells in vitro and then the cells can be reintroduced into the patient. Alternatively, the vector may be introduced into the patient in vivo by targeted transfection. Other aspects and advantages of the present invention will become apparent in light of the following detailed description and preferred embodiments thereof. It is a DNA sequence encoding 11 proteins. Furthermore, a DNA sequence that hybridizes to the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 10 under austerity conditions and encodes a protein having BMP-11 activity is also included in the present invention. Ultimately, allelic variants or other variants of SEQ ID NO: 1 or SEQ ID NO: 10 also have BMP-11 activity in the peptide sequence, regardless of whether such nucleotide changes result in changes in the peptide sequence. In some cases, it is included in the present invention. A further aspect of the invention is a vector consisting of the above DNA sequences operably linked to expression regulatory sequences. A cell line transformed with a DNA sequence encoding a BMP-11 protein ligated to operate is cultured in a suitable medium, and the BMP-11 protein is recovered and purified from the cell line. These vectors can be used in the novel BMP-11 protein production method of the invention. Many known cells, both prokaryotic and eukaryotic, can be used in this method as host cells for the expression of the polypeptide. Furthermore, the present invention includes the application of the DNA sequences and vectors of the present invention to gene therapy. In such use, the vector can be transfected into the patient's cells in vitro and then the cells can be reintroduced into the patient. Alternatively, the vector may be introduced into the patient in vivo by targeted transfection. Other aspects and advantages of the present invention will become apparent in light of the following detailed description and preferred embodiments thereof. Ten allelic variants or other variants are also included in the invention if the peptide sequence still has BMP-11 activity, regardless of whether such nucleotide changes result in changes in the peptide sequence. .. A further aspect of the invention is a vector consisting of the above DNA sequences operably linked to expression regulatory sequences. A cell line transformed with a DNA sequence encoding a BMP-11 protein ligated to operate is cultured in a suitable medium, and the BMP-11 protein is recovered and purified from the cell line. These vectors can be used in the novel BMP-11 protein production method of the invention. Many known cells, both prokaryotic and eukaryotic, can be used in this method as host cells for the expression of the polypeptide. Furthermore, the present invention includes the application of the DNA sequences and vectors of the present invention to gene therapy. In such use, the vector can be transfected into the patient's cells in vitro and then the cells can be reintroduced into the patient. Alternatively, the vector may be introduced into the patient in vivo by targeted transfection. Other aspects and advantages of the present invention will become apparent in light of the following detailed description and preferred embodiments thereof. Ten allelic variants or other variants are also included in the invention if the peptide sequence still has BMP-11 activity, regardless of whether such nucleotide changes result in changes in the peptide sequence. .. A further aspect of the invention is a vector consisting of the above DNA sequences operably linked to expression regulatory sequences. A cell line transformed with a DNA sequence encoding a BMP-11 protein ligated to operate is cultured in a suitable medium, and the BMP-11 protein is recovered and purified from the cell line. These vectors can be used in the novel BMP-11 protein production method of the invention. Many known cells, both prokaryotic and eukaryotic, can be used in this method as host cells for the expression of the polypeptide. Furthermore, the present invention includes the application of the DNA sequences and vectors of the present invention to gene therapy. In such use, the vector can be transfected into the patient's cells in vitro and then the cells can be reintroduced into the patient. Alternatively, the vector may be introduced into the patient in vivo by targeted transfection. Other aspects and advantages of the present invention will become apparent in light of the following detailed description and preferred embodiments thereof.<u style="single">Array description</u>SEQ ID NO: 1 is a partial nucleotide sequence of bovine BMP-11 encoding a mature bovine BMP-11 polypeptide. SEQ ID NO: 2 is the amino acid sequence of the partial propeptide encoded by SEQ ID NO: 1 and the fully mature bovine BMP-11 polypeptide. SEQ ID NO: 3 is a partial nucleotide sequence of human BMP-11. SEQ ID NO: 4 is the partial amino acid sequence of the human BMP-11 polypeptide encoded by SEQ ID NO: 3. SEQ ID NOs: 5 and 6 are bovine BMP-11 primers used to isolate human BMP-11 or other BMP-11 proteins. SEQ ID NO: 7 is pMT2 to add an XhoI recognition site near the SV40 replication origin. A DNA sequence inserted into CXM. SEQ ID NO: 8 is a DNA sequence inserted into pMT21 to add an XhoI recognition site upstream of the DHFR gene. SEQ ID NO: 9 is a DNA sequence consisting of a part of the EMC virus reader sequence. SEQ ID NO: 10 is the DNA sequence encoding the partial propeptide and the fully mature human BMP-11 protein. SEQ ID NO: 11 is the amino acid sequence of the partial propeptide encoded by SEQ ID NO: 10 and the fully mature human BMP-11 protein.<u style="single">Detailed description of the invention</u><u style="single">BMP-11</u>The bovine BMP-11 nucleotide sequence (SEQ ID NO: 1) and the encoded amino acid sequence (SEQ ID NO: 2), and the human BMP-11 nucleotide sequence (SEQ ID NO: 10) and the encoded amino acid sequence (SEQ ID NO:: 2). 11) is shown in the sequence listing of the present specification. A host cell transformed with a DNA sequence consisting of a DNA coding sequence of nucleotides 375 to 704 of SEQ ID NO: 1 or a DNA coding sequence of nucleotides 760 to 1086 of SEQ ID NO: 10 is cultured, followed by the amino acid of SEQ ID NO: 2. The purified bovine BMP-11 protein of the present invention can be obtained by recovering and purifying a protein having the sequence represented by amino acids 1 to 109 of SEQ ID NO: 1 to 109 or SEQ ID NO: 11 or a sequence substantially homologous thereto from the medium. To manufacture. For the production of the BMP-11 protein in mammalian cells, the DNA sequence further consists of a suitable propeptide linked to the reading frame of the DNA code sequence for BMP-11. The propeptide may be a raw BMP-11 propeptide, or it may be a propeptide from another member of the TGF-β superfamily. The human BMP-11 sequence of the present invention can be obtained using the whole or fragment of the bovine BMP-11 DNA sequence or the partial human BMP-11 sequence of SEQ ID NO: 3 as a probe. Therefore, the human BMP-11 DNA sequence consists of the DNA sequences of nucleotides 28 to 185 of SEQ ID NO: 3. The human BMP-11 protein consists of the amino acid sequences of amino acids 1 to 52 of SEQ ID NO: 4. BMP-11 expressed by mammalian cells such as CHO cells is thought to exist as a heterogeneous cell population of active species of BMP-11 protein with various N-terminus. It is believed that the active species will consist of at least the cysteine residue of amino acid 6 of SEQ ID NO: 1 or SEQ ID NO: 10 or an amino acid sequence starting in the amino-terminal direction. Therefore, the DNA sequence encoding the active BMP-11 protein is SEQ ID NO:: It may consist of 1 nucleotide 375 or 390 to 701, or a nucleotide of SEQ ID NO: 10: 760 or 775 to 1086, and may consist of an additional nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 10 in the 5'direction. Let's go. By expressing in E. coli, the N-terminus of human BMP-11 was experimentally determined as follows: [M] NLGLDXDEHSSE, where X is an amino acid for which no clear signal is obtained. It is a residue and is consistent with the position of cysteine at that position. Thus, this species of BMP-11 has an N-terminus at amino acid 1 of SEQ ID NO: 1 or SEQ ID NO: 10, and the DNA sequence encoding this species is nucleotides 375 to 701 of SEQ ID NO: 1 (bovine). Alternatively, it would consist of nucleotides 760 to 1086 of SEQ ID NO: 10 (human). The apparent molecular weight of the human BMP-11 monomer was determined by SDS-PAGE to be about 12 kd. Human BMP-11 protein exists as a colorless and transparent solution in 0.1% trifluoroacetic acid. The BMP-11 protein recovered from the medium is purified from the medium by isolation from other co-produced proteinaceous substances and other contaminants present. The BMP-11 protein can be characterized by its ability to regulate FSH production. In addition, the BMP-11 protein was described as described [eg, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature ( Nature), Vol. 321: 779-782 (1986) or Vale et al., Nature, Vol. 321: 776-779 (1986)], using rat and anterior pituitary cells It can be characterized by its ability to regulate the release of follicle-stimulating hormone (FSH) in established bioassays in vitro. Furthermore It may consist of 10 nucleotides from 760 or 775 to 1086, and may consist of additional nucleotide sequences in the 5'direction of SEQ ID NO: 1 or SEQ ID NO: 10. By expressing in E. coli, the N-terminus of human BMP-11 was experimentally determined as follows: [M] NLGLDXDEHSSE, where X is an amino acid for which no clear signal is obtained. It is a residue and is consistent with the position of cysteine at that position. Thus, this species of BMP-11 has an N-terminus at amino acid 1 of SEQ ID NO: 1 or SEQ ID NO: 10, and the DNA sequence encoding this species is nucleotides 375 to 701 of SEQ ID NO: 1 (bovine). Alternatively, it would consist of nucleotides 760 to 1086 of SEQ ID NO: 10 (human). The apparent molecular weight of the human BMP-11 monomer was determined by SDS-PAGE to be about 12 kd. Human BMP-11 protein exists as a colorless and transparent solution in 0.1% trifluoroacetic acid. The BMP-11 protein recovered from the medium is purified from the medium by isolation from other co-produced proteinaceous substances and other contaminants present. The BMP-11 protein can be characterized by its ability to regulate FSH production. In addition, the BMP-11 protein was described as described [eg, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature ( Nature), Vol. 321: 779-782 (1986) or Vale et al., Nature, Vol. 321: 776-779 (1986)], using rat and anterior pituitary cells It can be characterized by its ability to regulate the release of follicle-stimulating hormone (FSH) in established bioassays in vitro. Furthermore It may consist of 10 nucleotides from 760 or 775 to 1086, and may consist of additional nucleotide sequences in the 5'direction of SEQ ID NO: 1 or SEQ ID NO: 10. By expressing in E. coli, the N-terminus of human BMP-11 was experimentally determined as follows: [M] NLGLDXDEHSSE, where X is an amino acid for which no clear signal is obtained. It is a residue and is consistent with the position of cysteine at that position. Thus, this species of BMP-11 has an N-terminus at amino acid 1 of SEQ ID NO: 1 or SEQ ID NO: 10, and the DNA sequence encoding this species is nucleotides 375 to 701 of SEQ ID NO: 1 (bovine). Alternatively, it would consist of nucleotides 760 to 1086 of SEQ ID NO: 10 (human). The apparent molecular weight of the human BMP-11 monomer was determined by SDS-PAGE to be about 12 kd. Human BMP-11 protein exists as a colorless and transparent solution in 0.1% trifluoroacetic acid. The BMP-11 protein recovered from the medium is purified from the medium by isolation from other co-produced proteinaceous substances and other contaminants present. The BMP-11 protein can be characterized by its ability to regulate FSH production. In addition, the BMP-11 protein was described as described [eg, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature ( Nature), Vol. 321: 779-782 (1986) or Vale et al., Nature, Vol. 321: 776-779 (1986)], using rat and anterior pituitary cells It can be characterized by its ability to regulate the release of follicle-stimulating hormone (FSH) in established bioassays in vitro. Furthermore It may consist of additional nucleotide sequences in the direction. By expressing in E. coli, the N-terminus of human BMP-11 was experimentally determined as follows: [M] NLGLDXDEHSSE, where X is an amino acid for which no clear signal is obtained. It is a residue and is consistent with the position of cysteine at that position. Thus, this species of BMP-11 has an N-terminus at amino acid 1 of SEQ ID NO: 1 or SEQ ID NO: 10, and the DNA sequence encoding this species is nucleotides 375 to 701 of SEQ ID NO: 1 (bovine). Alternatively, it would consist of nucleotides 760 to 1086 of SEQ ID NO: 10 (human). The apparent molecular weight of the human BMP-11 monomer was determined by SDS-PAGE to be about 12 kd. Human BMP-11 protein exists as a colorless and transparent solution in 0.1% trifluoroacetic acid. The BMP-11 protein recovered from the medium is purified from the medium by isolation from other co-produced proteinaceous substances and other contaminants present. The BMP-11 protein can be characterized by its ability to regulate FSH production. In addition, the BMP-11 protein was described as described [eg, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature ( Nature), Vol. 321: 779-782 (1986) or Vale et al., Nature, Vol. 321: 776-779 (1986)], using rat and anterior pituitary cells It can be characterized by its ability to regulate the release of follicle-stimulating hormone (FSH) in established bioassays in vitro. Furthermore It may consist of additional nucleotide sequences in the direction. By expressing in E. coli, the N-terminus of human BMP-11 was experimentally determined as follows: [M] NLGLDXDEHSSE, where X is an amino acid for which no clear signal is obtained. It is a residue and is consistent with the position of cysteine at that position. Thus, this species of BMP-11 has an N-terminus at amino acid 1 of SEQ ID NO: 1 or SEQ ID NO: 10, and the DNA sequence encoding this species is nucleotides 375 to 701 of SEQ ID NO: 1 (bovine). Alternatively, it would consist of nucleotides 760 to 1086 of SEQ ID NO: 10 (human). The apparent molecular weight of the human BMP-11 monomer was determined by SDS-PAGE to be about 12 kd. Human BMP-11 protein exists as a colorless and transparent solution in 0.1% trifluoroacetic acid. The BMP-11 protein recovered from the medium is purified from the medium by isolation from other co-produced proteinaceous substances and other contaminants present. The BMP-11 protein can be characterized by its ability to regulate FSH production. In addition, the BMP-11 protein was described as described [eg, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature ( Nature), Vol. 321: 779-782 (1986) or Vale et al., Nature, Vol. 321: 776-779 (1986)], using rat and anterior pituitary cells It can be characterized by its ability to regulate the release of follicle-stimulating hormone (FSH) in established bioassays in vitro. Furthermore [M] NLGLDXDEHSSE, where X is an amino acid residue for which no clear signal is obtained and is consistent with the position of cysteine at that position. Thus, this species of BMP-11 has an N-terminus at amino acid 1 of SEQ ID NO: 1 or SEQ ID NO: 10, and the DNA sequence encoding this species is nucleotides 375 to 701 of SEQ ID NO: 1 (bovine). Alternatively, it would consist of nucleotides 760 to 1086 of SEQ ID NO: 10 (human). The apparent molecular weight of the human BMP-11 monomer was determined by SDS-PAGE to be about 12 kd. Human BMP-11 protein exists as a colorless and transparent solution in 0.1% trifluoroacetic acid. The BMP-11 protein recovered from the medium is purified from the medium by isolation from other co-produced proteinaceous substances and other contaminants present. The BMP-11 protein can be characterized by its ability to regulate FSH production. In addition, the BMP-11 protein was described as described [eg, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature ( Nature), Vol. 321: 779-782 (1986) or Vale et al., Nature, Vol. 321: 776-779 (1986)], using rat and anterior pituitary cells It can be characterized by its ability to regulate the release of follicle-stimulating hormone (FSH) in established bioassays in vitro. Furthermore [M] NLGLDXDEHSSE, where X is an amino acid residue for which no clear signal is obtained and is consistent with the position of cysteine at that position. Thus, this species of BMP-11 has an N-terminus at amino acid 1 of SEQ ID NO: 1 or SEQ ID NO: 10, and the DNA sequence encoding this species is nucleotides 375 to 701 of SEQ ID NO: 1 (bovine). Alternatively, it would consist of nucleotides 760 to 1086 of SEQ ID NO: 10 (human). The apparent molecular weight of the human BMP-11 monomer was determined by SDS-PAGE to be about 12 kd. Human BMP-11 protein exists as a colorless and transparent solution in 0.1% trifluoroacetic acid. The BMP-11 protein recovered from the medium is purified from the medium by isolation from other co-produced proteinaceous substances and other contaminants present. The BMP-11 protein can be characterized by its ability to regulate FSH production. In addition, the BMP-11 protein was described as described [eg, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature ( Nature), Vol. 321: 779-782 (1986) or Vale et al., Nature, Vol. 321: 776-779 (1986)], using rat and anterior pituitary cells It can be characterized by its ability to regulate the release of follicle-stimulating hormone (FSH) in established bioassays in vitro. Furthermore It is believed that it will consist of 10 (human) nucleotides 760 to 1086. The apparent molecular weight of the human BMP-11 monomer was determined by SDS-PAGE to be about 12 kd. Human BMP-11 protein exists as a colorless and transparent solution in 0.1% trifluoroacetic acid. The BMP-11 protein recovered from the medium is purified from the medium by isolation from other co-produced proteinaceous substances and other contaminants present. The BMP-11 protein can be characterized by its ability to regulate FSH production. In addition, the BMP-11 protein was described as described [eg, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature ( Nature), Vol. 321: 779-782 (1986) or Vale et al., Nature, Vol. 321: 776-779 (1986)], using rat and anterior pituitary cells It can be characterized by its ability to regulate the release of follicle-stimulating hormone (FSH) in established bioassays in vitro. Furthermore It is believed that it will consist of 10 (human) nucleotides 760 to 1086. The apparent molecular weight of the human BMP-11 monomer was determined by SDS-PAGE to be about 12 kd. Human BMP-11 protein exists as a colorless and transparent solution in 0.1% trifluoroacetic acid. The BMP-11 protein recovered from the medium is purified from the medium by isolation from other co-produced proteinaceous substances and other contaminants present. The BMP-11 protein can be characterized by its ability to regulate FSH production. In addition, the BMP-11 protein was described as described [eg, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature ( Nature), Vol. 321: 779-782 (1986) or Vale et al., Nature, Vol. 321: 776-779 (1986)], using rat and anterior pituitary cells It can be characterized by its ability to regulate the release of follicle-stimulating hormone (FSH) in established bioassays in vitro. Furthermore See pages 776-779 (1986)], which can be characterized by the ability to regulate follicle-stimulating hormone (FSH) release in an established bioassay in vitro using rat anterior pituitary cells. Furthermore See pages 776-779 (1986)], which can be characterized by the ability to regulate follicle-stimulating hormone (FSH) release in an established bioassay in vitro using rat anterior pituitary cells. FurthermoreIn addition, the BMP-11 protein can also be characterized by its ability to induce the formation of bone, cartilage and / or other connective tissue. In addition, such tissue-inducing activity of BMP-11 can be characterized by its ability to induce the formation of bone, cartilage and / or other connective tissue in the analyzes described in the Examples below. The BMP-11 protein provided by the present invention also includes a factor encoded by a sequence similar to SEQ ID NO: 1 or SEQ ID NO: 10, but is the factor naturally modified (eg,)? , Allelic mutations in nucleotide sequences that can result in amino acid changes in the polypeptide) or are artificially induced. For example, the synthetic polypeptide may be a contiguous sequence in which the amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 11 are fully or partially replicated. These sequences have primary, secondary or tertiary structure and conformational features SEQ ID NO: 2 or SEQ ID NO:: By sharing with 11 inhibin-β polypeptides, they can have common BMP-11 activity. Thus, they can be used in therapeutic processes as biologically active alternatives to native BMP-11 polypeptides. Other special mutations to the BMP-11 protein sequences described herein include modification of the glycosylation site. These modifications include O-linked or N-linked glycosylation sites. For example, the absence or partial glycosylation of glycosylation results from amino acid substitutions or deletions at the asparagine-bound glycosylation recognition site. The asparagine-linked glycosylation recognition site consists of a tripeptide sequence that is specifically recognized by the appropriate glycosylation enzyme of the cell. These tripeptide sequences are either asparagine-X-threonine or asparagine-X-serine (usually X can be any amino acid). Various amino acid substitutions or deletions (and / or amino acid deletions at the second position) at one or both of the first or third amino acid positions at the glycosylation recognition site are in the modified tripeptide sequence. Does not cause glycosylation. In addition, expression of the BMP-11 protein in bacterial cells yields an unglycosylated protein without altering the glycosylation recognition site. Furthermore, the present invention includes novel DNA sequences encoding expression of the BMP-11 protein that are not bound to DNA sequences encoding other proteinaceous substances. These DNA sequences are sequenced from SEQ ID NO: 1 or SEQ ID NO: 10 in the 5'to 3'direction, and under austerity conditions, for example, 0.1XSSC at 65 ° C, 0.1% SDS [Maniatis ( Maniatis) et al., Changes in the 10 DNA sequences are also included in the present invention. Another aspect of the present invention provides a method for producing a novel BMP-11 protein. The method of the invention comprises culturing a suitable cell line transformed with a DNA sequence encoding the BMP-11 protein of the invention placed under the control of a known regulatory sequence. Transformed host cells are cultured and BMP-11 protein is recovered from the medium and purified. Purified proteins are substantially free of other co-produced proteins as well as other contaminants. Suitable cells or cell lines may be mammalian cells such as Chinese hamster ovary cells (CHO). Methods for selecting and transforming suitable mammalian host cells, culturing, amplifying, screening, producing and purifying products are known in the art. For example, Gething and Sambrook, Nature, Vol. 293: 620-625 (1981), or Kaufman et al., Molecular and Cellular Biology (Mol. Cell. Biol.), Vol. 5 (7): pp. 17501759 (1985) or Howley et al., U.S. Pat. No. 4,419,446. Another suitable mammalian cell line described in the appendix Examples is the monkey COS-1 cell line. Mammalian cell CV-1 may also be suitable. Bacterial cells are also suitable hosts. For example, various e-coli strains (eg, HB101, MC1061) are well known as host cells in the field of biotechnology. Various strains such as B. subtilis, Pseudomonas, and other Bacillus subtilis can also be used in this method. Many yeast cells known to those skilled in the art can also be used as host cells for expressing the polypeptide of the present invention. Furthermore, if desired, insect cells can also be used as host cells in the methods of the invention. For example, Miller et al., See Genetic Engineering, Vol. 8, pp. 277-298 (Plenim Press, 1986) and the references cited therein. Another aspect of the invention provides a vector for use in the expression method of these novel BMP-11 polypeptides. Preferably, the vector comprises the entire novel DNA sequence above that encodes the novel factor of the invention. In addition, the vector contains suitable expression regulatory sequences that allow expression of the BMP-11 protein sequence. A vector containing the above-mentioned modified sequence is also a specific example of the present invention. In addition, SEQ ID NO: 1 or SEQ ID NO: 10 or other sequences encoding the BMP-11 protein are manipulated to delete the propeptide sequences encoding the BMP-11 and make them the other BMP protein. It could also be made to express mature BMP-11 by substituting a sequence encoding the complete propeptide of the Actibin protein or another member of the TGF-β superfamily. The vector can be used in a method of transforming a cell line, and the vector can direct the replication and expression of the DNA sequence of the invention in a selected host cell, a selection linked to the sequence of the invention to operate. It has a regulated sequence. Regulatory sequences for such vectors are known to those of skill in the art and can be selected depending on the host cell. Such selection is usually made and does not form part of the present invention. For expression in mammalian host cells, the vector is from a sequence encoding a propeptide suitable for protein secretion by the host cell and linked to the correct reading frame of the coded sequence of the mature BMP-11 protein. It may be. A sequence encoding a suitable propeptide may be obtained from a protein in the TGF-β superfamily of proteins, eg, DNA encoding a protein such as BMP-2 or BMP-9. For example, U.S. Pat. No. 5,168, See No. 050. The disclosure is described herein by reference (in the disclosure, the DNA encoding the precursor portion of a mammalian protein other than BMP-2 is fused to the DNA encoding the mature BMP-2 protein). Incorporate into. Thus, the present invention is a DNA sequence encoding a propeptide from a member of the TGF-β superfamily of proteins, a DNA linked to the correct reading frame of the DNA sequence encoding the BMP-11 polypeptide. Includes a chimeric DNA molecule consisting of a sequence. The term "chimera" is used to mean that the propeptide is derived from a different polypeptide other than BMP-11. The protein of the present invention that regulates the production of FSH can be applied to improve fertility when expressed as a homodimer or a heterodimer with other proteins of the inhibin-β family. .. Furthermore, the proteins of the invention may be useful for contraception in the case of heterodimer construction with proteins of the inhibin-α family. The proteins of the invention that induce cartilage and / or bone formation in an environment where bone is not normally formed are applied to fractures and cartilage damage in humans and other mammals. Such preparations using the BMP-11 protein can also be used prophylactically to reduce closed and open fractures and improve artificial joint fixation. Denovo bone formation induced by osteogenic agents contributes to congenital trauma or oncological resection-induced cerebral facial skeleton defects and is also useful in cosmetic surgery. BMP-11 protein may be used in the treatment of periodontal disease and other dental treatment steps. Such agents can provide an environment for attracting osteogenic cells, stimulate the proliferation of osteogenic cells, or induce the differentiation of progenitor cells of osteogenic cells. The BMP-11 polypeptide of the present invention may also be useful in the treatment of osteoporosis. Various osteogenic, cartilage-fusing and bone-inducing factors have been described. For that discussion, see, for example, European Patent Applications Nos. 148,155 and 169, See No. 016. The proteins of the invention can also be used for wound healing and related tissue repair. Wound types include, but are not limited to, burns, cuts, and crushes (see, eg, PCT Publication WO 84/01106, for discussions on wound healing and treatment of related tissues). A further aspect of the invention is a method and composition for the treatment of bone fractures and the repair of cartilage and / or the treatment of bone defects or other symptoms associated with periodontal disease. Furthermore, the present invention comprises methods and compositions for wound healing and tissue repair. Such compositions consist of at least one therapeutically effective amount of the BMP-11 protein of the invention mixed with a pharmaceutically acceptable excipient, carrier or matrix. In addition, such preparations using the BMP-11 protein can increase nerve viability and may therefore be useful in transplantation and in symptoms showing reduced nerve viability. The BMP-11 protein of the present invention is expected to act in concert with or perhaps synergistically with other related proteins and growth factors. Therefore, a further therapeutic method and composition of the present invention comprises at least one therapeutic amount of BMP protein or growth factor of at least one therapeutic amount disclosed in the above shared patent and application. It consists of 11 proteins. Such a mixture may consist of individual molecules or heteromolecules consisting of different moieties. For example, the methods and compositions of the invention are disulfide-bonded dimers consisting of BMP-11 protein subunits and subunits derived from inhibin-α protein, inhibin-β protein or BMP proteins such as BMP-1 to BMP-10. It may consist of. Drugs that are useful with BMP-11 are epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF-α and TGF-β), and insulin-like growth factor (IGF). ) Can include various growth factors. Further therapeutic methods and compositions of the present invention are low therapeutic amounts with at least one therapeutic amount of BMP protein disclosed in the above shared patent and application. It consists of at least one BMP-11 protein of the present invention. Such a mixture may consist of individual molecules of the BMP protein or heteromolecules consisting of different BMP moieties. For example, the method and composition of the present invention may consist of a disulfide-bonded dimer consisting of a BMP-11 protein subunit and a subunit derived from one of the above "BMPs". Thus, in the present invention, one subunit consists of an amino acid sequence from amino acid 1 to amino acid 109 of at least SEQ ID NO: 2 or SEQ ID NO: 11, and the other subunit is BMP-1, BMP-2, BMP-. Purified BMP-, a heterodimer consisting of amino acid sequences for bone morphogenesis proteins selected from the group consisting of 3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP-9. Includes 11 polypeptides. A further embodiment may consist of a heterodimer of the BMP-11 moiety. In addition, the BMP-11 protein may be mixed with other agents useful in the treatment of bone and / or cartilage defects, wounds, or tissue in question. These agents include epithelial growth factor (EGF), fibroblast growth factor (EGF), platelet-derived growth factor (PDGF), transformed growth factor (TGF-α and TGF-β), and k-fibroblast growth. Includes factors, parathyroid hormone (PTH), leukemia inhibitor (LIF / HILDA / DIA), insulin-like growth factor (IGF-I and IGF-II). Parts of these agents can also be used in the compositions of the present invention. The BMP-11 protein of the present invention may be used in a composition mixed with a bone morphogenetic protein. For example, Ogawa et al., WO 92/14481 (1992); Ogawa et al., Journal of Biological Chemistry (J.Biol.Chem.), Vol. 267: pp. 14233-14237 (1992). )reference. Bone morphogenetic proteins used in such compositions include, for example, U.S. Pat. Nos. 5,108,922, 5,013,649, 5,116,738, 5,106, Includes BMP-10 disclosed in No. 695. The preparation and formulation of such physiologically acceptable protein compositions with respect to pH, isotonicity, stability, etc. are within the scope of the art. Furthermore, the therapeutic compositions of the present invention are also of value in veterinary medicine due to the lack of species specificity of BMP and TGF proteins. In particular, non-human livestock and thoroughbred horses are desirable patients for treatment with the BMP-11 of the present invention. The therapeutic methods of the present invention include administering the composition topically, systemically, or partially, such as by transplantation or device. Of course, the therapeutic composition used in the present invention is in a physiologically acceptable form without pyrogen when administered. Further, preferably, the composition is encapsulated or injected in a viscous form for delivery to the damaged site of bone, cartilage or tissue. Topical administration may be appropriate for wound healing and tissue repair. A therapeutically useful agent other than the BMP-11 protein, which may be optionally contained in the composition, is added to the BMP-11 composition simultaneously or sequentially with the BMP-11 composition in the method of the present invention. It may be administered in a modified or additional manner. Preferably, for the formation of bone, cartilage or other connective tissue, the composition delivers BMP-11 or other BMP protein to the site of tissue that is damaged and requires repair, bone and cartilage. It provides a structure for the development of cartilage and optimally contains a matrix that can be reabsorbed into the body. The matrix may provide the correct structure and suitable environment for delayed release of BMP-11 and / or other bone-inducing proteins, as well as cell wetting. Such a matrix is made from materials used in other transplanted medical devices. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. Each application of the BMP-11 composition will determine the appropriate formulation. The useful matrix for the composition is biodegradable, chemically known calcium sulphate, trilime phosphate, hydroxyapatite. , Polylactic acid and polyanhydride. Other useful materials are biodegradable and are biologically well known, such as bone, tendon or cutaneous collagen. The additional matrix consists of pure protein or extracellular matrix components. Other useful matrices are non-biodegradable and are chemically known, such as sintered hydroxyapatite, bioglass, aluminic acid, or other ceramics. The matrix may consist of a mixture of any of the above types of materials, such as polylactic acid and hydroxyapatite, or collagen and trilime phosphate. For example, the bioceramics may be changed in a composition such as calcium-aluminic acid-phosphoric acid, and the pore size, particle morphology and biodegradability thereof may be changed. Progression can be monitored by regularly assessing bone growth and / or repair. Progress can be monitored, for example, by x-ray, histomorphological measurements, and tetracycline labeling. By considering various factors that alter the action of the BMP-11 protein, such as the patient's age, gender and diet, the extent of infection, if any, the duration of administration, and other clinical factors, the dosing regimen It will be decided. The dose may be varied depending on the type of BMP protein or growth factor in the composition. The dose may vary depending on the type of matrix used. The following examples show the recovery and characterization of bovine BMP-11 protein, the use of bovine BMP-11 protein to recover human and other BMP-11 proteins, and the acquisition and recombination of human protein. The practice of the present invention in the expression of the protein by the method will be described. Example 1 It is chemically known, such as glass, aluminate, or other ceramics. The matrix may consist of a mixture of any of the above types of materials, such as polylactic acid and hydroxyapatite, or collagen and trilime phosphate. For example, the bioceramics may be changed in a composition such as calcium-aluminic acid-phosphoric acid, and the pore size, particle morphology and biodegradability thereof may be changed. Progression can be monitored by regularly assessing bone growth and / or repair. Progress can be monitored, for example, by x-ray, histomorphological measurements, and tetracycline labeling. By considering various factors that alter the action of the BMP-11 protein, such as the patient's age, gender and diet, the extent of infection, if any, the duration of administration, and other clinical factors, the dosing regimen It will be decided. The dose may be varied depending on the type of BMP protein or growth factor in the composition. The dose may vary depending on the type of matrix used. The following examples show the recovery and characterization of bovine BMP-11 protein, the use of bovine BMP-11 protein to recover human and other BMP-11 proteins, and the acquisition and recombination of human protein. The practice of the present invention in the expression of the protein by the method will be described. Example 1 It is chemically known, such as glass, aluminate, or other ceramics. The matrix may consist of a mixture of any of the above types of materials, such as polylactic acid and hydroxyapatite, or collagen and trilime phosphate. For example, the bioceramics may be changed in a composition such as calcium-aluminic acid-phosphoric acid, and the pore size, particle morphology and biodegradability thereof may be changed. Progression can be monitored by regularly assessing bone growth and / or repair. Progress can be monitored, for example, by x-ray, histomorphological measurements, and tetracycline labeling. By considering various factors that alter the action of the BMP-11 protein, such as the patient's age, gender and diet, the extent of infection, if any, the duration of administration, and other clinical factors, the dosing regimen It will be decided. The dose may be varied depending on the type of BMP protein or growth factor in the composition. The dose may vary depending on the type of matrix used. The following examples show the recovery and characterization of bovine BMP-11 protein, the use of bovine BMP-11 protein to recover human and other BMP-11 proteins, and the acquisition and recombination of human protein. The practice of the present invention in the expression of the protein by the method will be described. Example 1 it can. By considering various factors that alter the action of the BMP-11 protein, such as the patient's age, gender and diet, the extent of infection, if any, the duration of administration, and other clinical factors, the dosing regimen It will be decided. The dose may be varied depending on the type of BMP protein or growth factor in the composition. The dose may vary depending on the type of matrix used. The following examples show the recovery and characterization of bovine BMP-11 protein, the use of bovine BMP-11 protein to recover human and other BMP-11 proteins, and the acquisition and recombination of human protein. The practice of the present invention in the expression of the protein by the method will be described. Example 1 it can. By considering various factors that alter the action of the BMP-11 protein, such as the patient's age, gender and diet, the extent of infection, if any, the duration of administration, and other clinical factors, the dosing regimen It will be decided. The dose may be varied depending on the type of BMP protein or growth factor in the composition. The dose may vary depending on the type of matrix used. The following examples show the recovery and characterization of bovine BMP-11 protein, the use of bovine BMP-11 protein to recover human and other BMP-11 proteins, and the acquisition and recombination of human protein. The practice of the present invention in the expression of the protein by the method will be described. Example 1<u style="single">Cow BMP-11</u>800,000 recombinants of the bovine genomic library constructed in the vector λEMBL3 are sprinkled on 100 plates at a density of 8000 recombinant bacteriophage plaques per plate. Recombinant bacteriophage plaques are replicated and amplified with nitrocellulose from these plates. Fragments of human BMP-7 corresponding to nucleotides 1081 to 1403 (Fig. 4 of US Pat. No. 5,141,905), Feiberg et al. [Anal. Biochem., Vol. 132: 6 to 13 pages (1983)] by the random priming method³²P-labeled and hybridized to a set of filters in standard hybridization buffer (SHB) (5xSSC, 0.170SDS, 5x Den Hearts, 100 μg / ml salmon / sperm DNA) at 60 ° C for 2-3 days. .. Clean the filter under reduced tightness (4xSSC, 0.1% SDS, 60 ° C). Many positive hybridizing recombinants are found. 52 positive hybridizing recombinant bacteriophage plaques are selected for secondary screening. Recombinant plaques are replicated and amplified with nitrocellulose from these 52 plates. A set of nitrocellulose filters are hybridized to the above human BMP-7 DNA probe and washed under the same conditions with reduced tightness. Another set of filters was hybridized to a mixture of BMP-5, BMP-6 and BMP-7 probes at 65 ° C in SHB and 0.1xSSC, 0.1% at 65 ° C. Wash with SDS (austerity hybridization and wash conditions). The mixed probe consists of relatively equal amounts of human BMP-5 sequence nucleotides 1452 to 2060 (US Pat. No. 5,106,748, Figure 4).³²P-labeled DNA fragment consisting of nucleotides 1395 to 1698 of human BMP-6 sequence (Fig. 4 of US Pat. No. 5,187,076)³²Consists of a P-labeled DNA fragment and nucleotides 1081 to 1403 of the human BMP-17 sequence (US Pat. No. 5,141,905, Figure 4).³²Consists of P-labeled DNA fragments. BMP-5, BMP-6 and BMP-7 DNA fragments by random priming method³²Each probe labeled P and having the same count per minute (cpms) is mixed and added to the SHB containing the other set of nitrocellulose filters on 52 secondary plates. It hybridizes positively to human BMP-7 probes under reduced tonicity conditions and weakly or does not hybridize to mixed BMP-5 / 6/7 probes under high tonicity conditions. 14 recombinants are selected for further analysis. All 14 recombinants exhibiting these hybridization properties are plaque-purified and bacteriophage DNA is prepared from each. One of the 14 hybridization-positive regions exhibiting the above hybridization properties is named λ7r-30 and placed in a 0.5 kb SacI restriction fragment. This fragment is subcloned into a plasmid vector (pGEM-3) for DNA sequence analysis. The partial DNA sequence of clone λ7r-30 (SEQ ID NO: 1) and the derived amino acid sequence (SEQ ID NO: 2) are shown in the sequence listing. The bacteriophage λ7r-30 was deposited with the ATCC on April 7, 1993, and the accession number ATCC75439 was obtained. This deposit meets the requirements of the Budapest Treaty on International Approval of the Deposit of Microorganisms in Patent Procedures. This λ7r-30 clone encodes at least part of the bovine BMP-11 protein of the invention. The nucleotide sequence of clone λ7r-30 contains a reading frame of 456 nucleotides of nucleotides 246-701 of SEQ ID NO: 1. SEQ ID NO: The nucleotide sequence from 1 to 324 to 701 shows a reading frame of 378 nucleotides and is determined by side-by-side comparison with other BMP proteins and other proteins in the TGF-β family, bovine BMP-11. It encodes at least 126 amino acids in the C-terminal portion of the protein. The nucleotide sequences from 246 to 323 show a reading frame adjacent to the sequence encoding the estimated 126 amino acids BMP-11 peptide, while the DNA sequences (246 to 323) in this region are other. Consensus sequences that are less identical to BMP proteins and other proteins in the TGF-β family and are susceptible to multiple splices make it difficult to determine the 5'end of this exon in the bovine BMP-11 gene. I have to. The presence of an in-frame stop codon at nucleotide positions 243 to 245 indicates that the nucleotide sequence of clone λ7r-30 contains at least one exon / intron boundary of the bovine BMP-11 gene. Shown. According to knowledge of other proteins in the TGF-β family, the BMP-11 precursor polypeptide is a multibasic sequence ARG-SER-ARG-ARG consistent with the recognized proteolytic processing sequence ARG-XX-ARG. Expected to be cleaved in. Cleavage of the BMP-11 precursor polypeptide is thought to yield a mature peptide of 109 amino acids starting with the amino acid ASN at position 1. Processing of the mature form of BMP-11 is the processing of the related protein TGF-β [Gentry et al., Moll. &amp; Cell.Biol., Vol. 8, p. 4162 ( 1988); Derynck et al., Nature, Vol. 316: It is thought to include dimerization and removal of the N-terminal region in a manner similar to [p. 701 (1985)]. Therefore, the mature active species of BMP-11 consists of homodimers of two polypeptide subunits, each subunit consisting of amino acids 1-109, with an expected molecular weight of approximately 12,000 daltons. Further active species consist of amino acids 6-109, which is believed to contain the first conserved cysteine residue. Like the other members of the protein TGF-β, the carboxy-terminal region of the BMP-11 protein exhibits a more conserved sequence than the amino-terminal region. The amino acid identity of the cysteine-rich C-terminal domain (amino acids 6-109) of the BMP-11 protein with respect to the corresponding region of other proteins in the TGF-β family is as follows: BMP-2,39%; BMP-3, 37%; BMP-4,37%; BMP-5,42%; BMP-6,45%; BMP-7,42%; BMP-8,39%; BMP-9,40%; Vg1,39%; GDF-1,34%; TGF-β1,36%; TGF-β2,38%; TGF-β3,38%; Inhibin β (B), 41%; Inhibin β (A), 39%. Example 2 GDF-1,34%; TGF-β1,36%; TGF-β2,38%; TGF-β3,38%; Inhibin β (B), 41%; Inhibin β (A), 39%. Example 2 GDF-1,34%; TGF-β1,36%; TGF-β2,38%; TGF-β3,38%; Inhibin β (B), 41%; Inhibin β (A), 39%. Example 2<u style="single">Human BMP-11</u>The bovine and human BMP-11 genes are considered to be significantly homologous and therefore the bovine code sequence or part thereof is used as a probe for screening the human genomic library or similar. Used as a probe to identify human cell lines or tissues that synthesize human proteins. A human genome library, such as Stratagene Catalog No. 944201, can be screened with such probes, presumed positives can be isolated, and DNA sequences can be obtained. Evidence that this recombinant encodes part of human BMP-11 is due to homology of bovine / human protein and gene structure. Once a recombinant bacteriophage containing DNA encoding a portion of a human BMP-11 molecule is obtained, a human cell line or tissue synthesizing BMP-11 mRNA can be used as a probe in the human cryptographic sequence. Can be identified. Alternatively, the bovine BMP-11 cryptographic sequence can be used as a probe to identify such human cell lines or tissues. Briefly, RNA is extracted from selected cells or tissues and electrophoresed on a formaldehyde agarose gel to transfer to nitrocellulose, or reacted with formaldehyde to spot directly on nitrocellulose. Nitrocellulose is then hybridized with a probe derived from the bovine or human BMP-11 code sequence. Alternatively, an oligonucleotide that uses a bovine BMP-11 cipher sequence to specifically amplify part of the BMP-11 cipher sequence located in the region located between the primers used to perform the specific amplification reaction. Design a primer. Bovine and human BMP-11 sequences will allow the sequence, cDNA or genomic DNA template encoding the corresponding human BMP-11 from mRNA to be specifically amplified. If one of the above methods identifies a possible source, select the mRNA by oligo (dT) cellulose chromatography The cDNA is then synthesized and cloned into λgt10 or another λ bacteriophage vector known to those of skill in the art (ie, λZAP) by established methods (Toole et al., Supra). The above oligonucleotide primers, which are oriented towards amplification reactions, can also be used directly against genomic libraries cloned into already established human cDNA or λ bacteriophage vectors. In such cases, a library that produces a specifically amplified DNA product that encodes a portion of the human BMP-11 protein is directly used as a probe using the amplified DNA fragment that encodes BMP-11. It could be screened. Oligonucleotide primers designed based on the DNA sequence of bovine BMP-11 genomic clone λ7r-30 are believed to allow specific amplification of the sequence encoding human BMP-11. The following nucleotide primers are designed based on nucleotides 501 to 521 of the DNA sequence shown in SEQ ID NO: 1, and synthesized by an automatic DNA synthesizer. Clone into λgt10 or other λbacteriophage vector known to those of skill in the art (ie, λZAP). The above oligonucleotide primers, which are oriented towards amplification reactions, can also be used directly against genomic libraries cloned into already established human cDNA or λ bacteriophage vectors. In such cases, a library that produces a specifically amplified DNA product that encodes a portion of the human BMP-11 protein is directly used as a probe using the amplified DNA fragment that encodes BMP-11. It could be screened. Oligonucleotide primers designed based on the DNA sequence of bovine BMP-11 genomic clone λ7r-30 are believed to allow specific amplification of the sequence encoding human BMP-11. The following nucleotide primers are designed based on nucleotides 501 to 521 of the DNA sequence shown in SEQ ID NO: 1, and synthesized by an automatic DNA synthesizer. Clone into λgt10 or other λbacteriophage vector known to those of skill in the art (ie, λZAP). The above oligonucleotide primers, which are oriented towards amplification reactions, can also be used directly against genomic libraries cloned into already established human cDNA or λ bacteriophage vectors. In such cases, a library that produces a specifically amplified DNA product that encodes a portion of the human BMP-11 protein is directly used as a probe using the amplified DNA fragment that encodes BMP-11. It could be screened. Oligonucleotide primers designed based on the DNA sequence of bovine BMP-11 genomic clone λ7r-30 are believed to allow specific amplification of the sequence encoding human BMP-11. The following nucleotide primers are designed based on nucleotides 501 to 521 of the DNA sequence shown in SEQ ID NO: 1, and synthesized by an automatic DNA synthesizer.<img file="JP3681069B2_D0001.tif" />The first 9 nucleotides (underlined) of Primer C are the recognition sequences of the restriction endonuclease XbaI used to facilitate the operation of specifically amplifying the DNA sequence encoding the BMP-11 protein of the invention (underlined). Therefore, this sequence is not derived from the DNA sequence shown in SEQ ID NO: 1). The following nucleotide primers are designed based on nucleotides 701 to 678 of the DNA sequence shown in SEQ ID NO: 1, and synthesized by an automatic DNA synthesizer.<img file="JP3681069B2_D0002.tif" />The first 9 nucleotides (underlined) of Primer D are the recognition sequences of the restriction endonuclease BamHI (underlined) used to facilitate the operation of specifically amplifying the DNA sequence encoding the BMP-11 protein of the invention. Therefore, this sequence is not derived from the DNA sequence shown in SEQ ID NO: 1). The standard nucleotide symbols in the above primers are: A, adenosine; C, cytosine; G, guanine; and T, thymine. The above primers C and D are used as primers for amplifying a specific nucleotide derived from human genomic DNA. Human genomic DNA (source: peripheral blood lymphocytes) is denatured at 100 ° C for 5 minutes, then 200 μM of each deoxynucleotide triphosphate (dATP, dGTP, dCTP and dTTP), 10 mM Tris-HCl pH 8.3, 50mM KCl, 1.5mM MgCl₂Cool with ice before adding the reaction mixture containing 0.001% gelatin, 1.25 units of Taq DNA polymerase, 100 pM oligonucleotide primer C and 100 pM oligonucleotide primer D. The reaction mixture is then subjected to a temperature cycle of the following form: one cycle of 94 ° C for 3 minutes, 50 ° C for 1 minute, 72 ° C for 1 minute, then 94 ° C for 1 minute, 39 cycles of 1 minute at 50 ° C and 1 minute at 72 ° C. DNA specifically amplified by this reaction is separated from the excess oligonucleotide primers C and D used to initiate amplification by using a DNA purified resin according to the protocol under the conditions specified by the manufacturer. .. The resulting DNA product is digested with restriction endonucleases XbaI and BamHI, phenol-extracted, and then chloroform-extracted. Removal of small fragments of DNA resulting from buffer exchange and SbaI / BamHI restricted digestion, dilute the digested DNA product with 10 mM Tris-HCl pH 8.0, 1 mM EDTA, then centricon.^TM30 microconcentrator (WR Grace &amp; Company, Beverly, Massachusetts) This is done by centrifuging according to Co.), product number 4209). The resulting XbaI / BamHI digested and amplified DNA product is subcloned between the XbaI and BamHI restriction sites in the polylinker region of the plasmid vector (pBluescript). DNA sequence analysis of the obtained subclone shows that the specifically amplified DNA sequence encodes a part of the human BMP-11 protein of the present invention. The DNA sequence (SEQ ID NO: 3) and the derived amino acid sequence (SEQ ID NO: 4) of this specifically amplified DNA fragment are shown in the sequence listing. Nucleotides 1 to 27 of this sequence consist of a part of oligonucleotide primer C, and nucleotides 186 to 213 consist of a part of oligonucleotide primer D used to carry out a specific amplification reaction. Due to the function of oligonucleotide primers C and D (designed based on the bovine BMP-11 sequence) at the initiation of the amplification reaction, they are exactly consistent with the actual sequence encoding human BMP-11. It does not have to be, and therefore is not translated into a derivative of the above amino acid sequence. Standard hybridization / screening known to those of skill in the art, using DNA sequences from nucleotides 28 to 185 of SEQ ID NO: 3, specifically amplified from the human genomic DNA template, or a portion thereof, as a probe. The method allows additional human BMP-11s to be identified from the human genome or human cDNA libraries. 1.2 million recombinants of human / fetal brain cDNA library (Stratagene, Catalog No. 936206) constructed in vector λZAPII, 50 with a density of 24,000 recombinant bacteriophage plaques per plate Sprinkle on the plate. Replicas of recombinant bacteriophage plaques with nitrocellulose are made from these plates. SEQ ID NO: Oligonucleotide probes designed based on oligonucleotides 53 to 82 of 3 are synthesized by an automated DNA synthesizer. This oligonucleotide probe is γ³²Radiolabeled with P-ATP and hybridized to both sets of nitrocellulose replicas in SHB at 65 ° C. Nine positive hybridizing recombinants can be seen. One of the positive hybridizing recombinants is named λFB30.5 and plaque purified. Prepare a bacteriophage plate stock of purified λFB 30.5 cDNA clones and isolate the bacteriophage DNA. A bacterial plasmid named FB30.5, obtained by the in vivo cleavage protocol described by the supplier (Stratagene) and carried as an insert fragment of the entire λFB30.5 bacteriophage cDNA clone, is available in Maryland, USA at the request of the Budapest Treaty. Deposited with ATCC on Parklone Drive 12301, Rockville, Maryland, ATCC It is named 69619. A part of the DNA sequence of clone FB30.5 is shown in SEQ ID NO: 10. Sprinkle 1 million recombinants of the constructed human genomic library (Stratagene, Catalog No. 944201) in vector λFIX onto 50 plates at a density of 20000 recombinant bacteriophage plaques per plate. .. Replicas of recombinant bacteriophage plaques with nitrocellulose are made from these plates. Oligonucleotide probes designed based on nucleotides 57-86 of SEQ ID NO: 10 are synthesized by a DNA synthesizer, except that the GCG of nucleotides 59-61 of SEQ ID NO: 10 is accidentally replaced by CAC. This oligonucleotide probe is γ³²Radiolabeled with P-ATP and hybridized to both sets of nitrocellulose replicas in SHB at 65 ° C. Five positive hybridizing recombinants are seen. One of the positive hybridizing recombinants is named 30GEN.4 and plaque-purified. Prepare a bacteriophage plaque stock of purified 30 GEN.4 genomic clones and isolate the bacteriophage DNA. The bacteriophage stock of this genomic clone was deposited with the ATCC at Park Clone Drive 12301, Rockville, Maryland, USA, at the request of the Budapest Treaty. It is named 75775. A part of the DNA sequence of clone 30 GEN.4 is shown in SEQ ID NO: 10. It was determined that a part of the DNA sequence of the genome clone 30GEN.4 is the same as a part of the DNA sequence of the cDNA clone FB30.5. The extended portion of this overlapping sequence (nucleotides 1-198) of SEQ ID NO: 10 was used as the basis for editing the partially cryptographic sequence of the BMP-10 protein sequence. Genome clone 30GEN.4 has an additional 5'side cryptographic sequence of the human BMP-11 protein, which is believed to encode the rest of the BMP-11 precursor polypeptide, and further has initiating methionine. It is thought that it is. The partial sequence of human BMP-11 is shown in SEQ ID NO: 10, and nucleotides 1-198 are 30GEN.4 genomic clones and FB30.5. It should be noted that it is present in both cDNA clones, while nucleotides 199-1270 are all derived from the cDNA clone FB30.5. SEQ ID NO: 10 suggests a human BMP-11 precursor protein with at least 362 amino acids. Based on knowledge of other BMPs and other proteins of the TGF-β family, the precursor polypeptide is a multibasic sequence ARG- that is consistent with the proposed and accepted proteolytic processing sequence ARG-XX-ARG. It is predicted that it will be cleaved at SER-ARG-ARG (amino acid -4 to -1 of SEQ ID NO: 11). Cleavage of the human BMP-11 precursor polypeptide at this position would result in a mature peptide of 109 amino acids starting with the amino acid ASN at position 1 of SEQ ID NO: 11. Processing to the mature form of human BMP-11 involves removal of the N-terminal region in a manner similar to dimerization and processing of the related protein TGF-β (LE Gentry et al., Molecular and Cellular Biology (Molec. &amp; Cell.Biol.) Vol. 8: 4162 (1988); R. Derynck et al., Nature Vol. 316: 701 (1985)). The mature active form of human BMP-11 consists of homodimers of two polypeptide subunits, each subunit consisting of amino acids 1-108 of SEQ ID NO: 11 and is thought to have an estimated molecular weight of 12,000 daltons. Further active species consist of amino acids 7-108 of SEQ ID NO: 11, which is believed to have the first conserved cysteine residue. A heterodimer consisting of one subunit of BMP-11 and another subunit of another member of the BMP / TGF-β superfamily is also conceivable. Example 3<u style="single">Expression of BMP-11</u>To produce bovine, human or other mammalian BMP-11, the DNA encoding the protein is transferred into a suitable expression vector and mammalian cells or other preferred by conventional genetic engineering methods. Introduce into eukaryotic or prokaryotic host. The preferred expression system for biologically active recombinant human BMP-11 is believed to be stably transformed mammalian cells. Those skilled in the art may use the sequence of SEQ ID NO: 1 or SEQ ID NO: 10 or other DNA sequences encoding the BMP-11 protein, or other modified sequences and pCD [Okayama et al., Molecular and Cellular Biology (Mol. Cell Biol.), Volume 2: pp. 161-170 (1982)], pJL3, pJL4 [Gough et al., EMBO J., Volume 4: pp. 645-653 (1982)] 1985)] and known vectors such as pMT2 CXM can be used to construct mammalian expression vectors. Mammalian expression vector pMT2 CXM is a derivative of p91023 (b) (Wong et al., Science, Vol. 228: 810-815, 1985), but has an ampicillin resistance gene instead of a tetracycline resistance gene, and further. It differs from p91023 (b) in that it has an XhoI site for cDNA clone insertion. pMT2 The mechanical elements of CXM are described (Kaufman, RJ, 1985, Proc. Natl. Acad. Sci. USA). Vol. 82: pp. 689-693), adenovirus / VA gene, SV40 / replication initiation site containing 72 bp enhancer, 5'splice site and adenovirus / large adenovirus / trisection leader sequence present on late mRNA It contains adenovirus-late promoter, 3'splice receptor site, DHFA insertion fragment, SV40-early polyadenylation site (SV40), and pBR322 sequence required for growth in e-coli. plasmid pMT2 by EcoRI digestion of pMT2-VWF Get CXM. The pMT2-VWF has been deposited with the ATCC at Park Loan Drive 12301, Rockville, Maryland, USA, under accession number ATCC67122. EcoRI digestion excises the cDNA inserts present in pMT2-VWF and ligates them into a linear pMT2 that is used to transform E. coli HB101 or DH-5 into ampicillin resistance. The plasmid pMT2 DNA can be prepared by conventional methods. Next, pMT2 CXM is constructed using loop-out / in mutagenesis [Morinaga et al., Biotechnology, Vol. 84: 636 (1984)]. As a result, the bases 1075 to 1145 corresponding to the SV40 / replication origin of pMT2 and the HindIII site near the enhancer sequence are removed. In addition, the following sequence at the position of nucleotide 1145:<img file="JP3681069B2_D0003.tif" />To insert. This sequence has a recognition site for the restriction endonuclease XhoI. A derivative of pMT2 CXM, named pMT23, has recognition sites for the restriction endonucleases PstI, EcoRI, SalI and XhoI. The plasmids pMT2 CXM and pMT23 DNA can be prepared by conventional methods. pEMC2β1 derived from pMT21 is also suitable for practicing the present invention. pMT21 is derived from pMT2 derived from pMT2-VWF. As described above, EcoRI digestion cuts out the cDNA inserts present in pMT-VWF and ligates them into a linear pMT2 used to transform E. coli HB101 or DH-5 into ampicillin resistance. Is obtained. The plasmid pMT2 DNA can be prepared by conventional methods. pMT21 is derived from pMT2 through the following two modifications. First, the 76 bp 5'untranslated region of the DHFR cDNA, which contains 19 G residues extended from the G / C tailing for cDNA cloning, is deleted. In this step, the XhoI site is inserted and the following sequence is arranged just upstream of DHFR:<img file="JP3681069B2_D0004.tif" />To get. A single ClaI site is then introduced by digestion with EcoRV and XbaI, treatment with the Klenow fragment of DNA polymerase I, and ligation to the ClaI linker (CATCGATG). This removes the 250 bp fragment from the adenovirus-related RNA (VAI) region, but does not inhibit the expression or function of the VAI RNA gene. pMT21 is digested with EcoRI and XhoI and used to obtain the vector pEMC2B1. Digestion with EcoRI and PstI resulted in some EMCV readers pMT2-ECAT1 [SKJung et al., Journal of Willology (J. Virol) Vol. 63, pp. 165-1660 (1989)) ], Which is a 2752 bp fragment. This fragment is digested with TaqI to give a 508 bp EcoRI-TaqI fragment, which is purified by electrophoresis on a low melting point agarose gel. 5'TaqI overhangs and 3'XhoI overhangs with the following sequences:<img file="JP3681069B2_D0005.tif" />Is used to synthesize a 68 bp adapter and its complementary strand. This sequence matches the EMC virus leader sequence from nucleotides 763 to 827. In addition, this sequence has the ATG at position 10 within the EMC virus reader changed to ATT, followed by the XhoI site. The EcoRI-XhoI fragment of pMT21, the EcoRI-TaqI fragment of EMC virus, and the 68 bp oligonucleotide adapter are ligated to obtain the vector pEMC2β1. This vector contains SV40 / replication origin and enhancer, adenovirus / large late promoter, most cDNA copy of adenovirus / trisection leader sequence, small hybrid intervening sequence, SV40 / polyadenylation signal and adenovirus / VA. It contains the I gene, DHFR and β-lactamase markers and EMC sequences and is adequately associated with directing the expression of high levels of desired cDNA in mammalian cells. Vector construction involves modification of the BMP-11 DNA sequence. For example, the DNA of BMP-11 may be modified by removing unencrypted nucleotides on the 5'and 3'ends of the cryptographic region. The deleted non-encrypted nucleotide may or may not be replaced with another sequence known to be beneficial for expression. These vectors are transformed into host cells suitable for expression of the BMP-11 protein. In addition, delete the propeptide sequence encoding BMP-11 and replace it with a sequence encoding the complete propeptide of another BMP protein, activin protein or other member of the TGF-β superfamily. It would be possible to manipulate the sequence of SEQ ID NO: 1 or SEQ ID NO: 10 or any other sequence encoding the BMP-11 protein to express the mature BMP-11 protein. Those skilled in the art may remove or replace the mammalian regulatory sequence flanking the code sequence with a bacterial sequence to create a bacterial vector for intracellular or extracellular expression by bacterial cells. You can manipulate an array of 1 or SEQ ID NO: 10. For example, the cryptographic sequence can be further manipulated (eg, linked to another known linker, or its non-cryptographic sequence is deleted, or its nucleotide is altered by other known methods). Then, T. Taniguchi et al., Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), Vol. 77: A modified BMP-11 cipher sequence could be inserted into a known bacterial vector using a method as described on pages 5230-5233 (1980). This typical bacterial vector is then transformed into a bacterial host cell, thereby expressing the BMP-11 protein. See European Patent Publication EPA 177,343 for methods for extracellular expression of BMP-11 protein in bacterial cells. For expression in insect cells, manipulations can be performed to construct an insect vector [see, eg, the method described in published European Patent Application No. 155,476]. Yeast vectors can also be constructed using yeast regulatory sequences for intracellular or extracellular expression of the factors of the invention by yeast cells [eg, published PCT application WO86 / 00639 and European patent application Publication EPA123, Refer to the method described in No. 289]. Methods of producing high levels of the BMP-11 protein of the invention in mammalian cells include the construction of cells with multiple copies of the heterologous BMP-11 gene. Heterologous genes are ligated to amplifyable markers such as the dihydrofolate reductase (DHFR) gene, Kaufman and Sharp, Journal of Molecular Biology (J. Mol. Biol.), No. 159. By the method of Volumes: pp. 601-629 (1982), cells with increased gene copy by increasing methotrexate (MXT) levels can be selected for the marker. This approach can be used for a wide variety of different cell types. BMP-11 of the invention functionally linked to sequences of other plasmids that allow expression of BMP-11 by a variety of methods, including, for example, calcium phosphate co-precipitation and transfection, electroporation or protoplast fusion. DHFR expression plasmid pAdA26SV (A) 3 [Kaufman and Sharp, Molecular and Cellular Biology (Mol.Cell.biol.), Vol. 2, p. 1304 (1982)] can be simultaneously introduced into DHFR-deficient cells DUKX-BII. DHFR-expressing transformants were selected for growth in alpha medium containing dialyzed fetal bovine serum, followed by Kaufman et al., Molecular and Cellular Biology (Mol. Cell. Biol.), As described in Volume 5, page 1750 (1983), selection is made for amplification by proliferation when the MTX concentration is increased. Transformants are grown and expression of biologically active BMP-11 is monitored by analysis of one or more BMP-11 activity described in Examples 5-8 below. BMP-11 expression should increase with increasing levels of MTX resistance. [³⁵S] Characterize the BMP-11 polypeptide using standard methods known in the art, such as pulse labeling with methionine or cysteine and polyacrylamide gel electrophoresis. Other related BMP-11 proteins can be produced in a similar manner. Example 4<u style="single">Biological activity of expressed BMP-11</u>In order to measure the biological activity of the expressed BMP-11 protein obtained in Example 3 above, the protein was recovered from the cell culture and from other proteinaceous substances and other contaminants produced at the same time. Purify by isolating the BMP-11 protein. The purified protein can be analyzed by the analysis of BMP-11 activity described in Examples 5 to 8 below. Example 5<u style="single">W-20 bioassay</u>A.<u style="single">Description of W-20 cells</u>The use of W-20 bone marrow matrix cells as an indicator cell line is based on the conversion of these cells to osteoblasts after treatment with BMP protein [Thies et al., Journal of Bone and Minerals]. -Research (Journal of Bone and Minaral Research), Vol. 5, p. 305 (1990); and Thies et al., Endocrinology, Vol. 130, p. 1318 (1992)]. Specifically, W-20 cells are Children's Hospital in Boston, Massachusetts. A cloned bone marrow matrix cell line taken from adult mice by researchers in Dr. D. Nathan's laboratory at Hospital). Treatment of W-20 cells with certain BMP proteins causes (1) increased production of alkaline phosphatase, (2) induction of PTH-stimulated cAMP, and (3) induction of cell-induced osteocalcin synthesis. .. Although (1) and (2) show characteristics related to the phenotype of osteoblasts, osteocalcin synthesis ability is a characteristic exhibited only by mature osteoblasts. Moreover, to date, we have observed the osteoblast-like conversion of W-20 substrate cells that occurs only when treated with BMP. In this mode, the in vitro activity exhibited by BMP-treated W-20 cells correlates with the in vivo bone-forming activity known for BMP. Two in vitro analyzes useful in comparing the BMP activity of novel bone-inducing molecules are described below. B.<u style="single">W-20 Alkaline Phosphatase Analysis Protocol</u>10000 W-20 cells in 96-well tissue culture plate in 200 μl medium per well (DME containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine and 100 units / ml penicillin + 100 μg / ml streptomycin) Sprinkle at the rate of. 95% air, 5% CO₂Cells are adhered overnight at 37 ° C in an incubator. 200 μl of medium was removed from each well with a multi-channel pipettor and replaced with the same volume of test sample in DME containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine and 1% penicillin-citreptomycin. Analyze the test substance in 3 systems. Incubate test samples and standards with W-20 indicator cells for 24 hours. After 24 hours, the plate is removed from the 37 ° C incubator and the test medium cells are removed. Wash the W-20 cell layer 3 times with 200 μl calcium / magnesium-free phosphate buffered saline per well and discard these rinses. 50 μl of water distilled in a glass device is added to each well and the analysis plate is then placed in a dry ice / ethanol bath for rapid freezing. Once frozen, remove the analysis plate from the dry ice / ethanol bath and thaw at 37 ° C. This step is repeated two or more times to perform a total of three freeze-thaw steps. After completion of the step, the membrane-bound alkaline phosphatase is subjected to measurement. 50 μl analytical mixture (50 mM glycine, 0.05% Triton X-100, 4 mM MgCl₂, 5 mM p-nitrophenol phosphate, pH = 10.3) is added to each analysis well, then the analysis plate is incubated at 37 ° C for 30 minutes with shaking 60 times per minute. At the end of the 30 minute incubation, 100 μl of 0.2N NaOH is added to each well and the reaction is stopped by placing the analysis plate on ice. Read the spectroscopic absorbance of each well at a wavelength of 405 nanometers. Next, the alkaline phosphatase activity of each sample is evaluated using these values as known standards. For example, a known amount of p-nitrophenol phosphate can be used to obtain an absorbance value. This is shown in Table I.<img file="JP3681069B2_D0006.tif" />Absorbance values for known amounts of BMP can be determined and converted to μmoles of cleaved p-nitrophenol phosphate per unit time as shown in Table II.<img file="JP3681069B2_D0007.tif" />These values are then used to compare the activity of known amounts of BMP-11 with the activity of BMP-2. C.<u style="single">Osteocalcin RIA protocol</u>10 W-20 cells in 2 ml DME (containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine) in each well of a 24-well multi-well tissue culture dish.⁶Sprinkle so that it becomes an individual. 95% air, 5% CO₂Inoculate cells overnight at medium 37 ° C. The next day, the medium is replaced with DME containing 10% fetal bovine serum, 2 mM glutamine and test material in a total volume of 2 ml. Place each test substance in the well of system 3. Incubate the test material with W-20 cells for a total of 96 hours (medium exchange in the same medium at 48 hours). At the end of the 96-hour incubation, 50 μl of test medium is taken from each well and osteocalcin production is analyzed using radioimuno analysis for mouse osteocalcin. Details of the analysis can be found at Biomedical Technologies, Inc., Page Street 378, Stoughton, Massachusetts 02072. The kit manufactured by Inc.) is described. Reagents for analysis are product numbers BT-431 (mouse osteocalcin standard), BT-432 (goat anti-mouse osteocalcin), BT-431R (iodinated mouse osteocalcin), BT-415 (normal). Found as goat serum) and BT-414 (donkey anti-goat IgG). RIA for osteocalcin synthesized by W-20 cells in response to BMP treatment is performed as described in the protocol provided by the manufacturer. The values obtained for the test samples are compared to those for known standards of mouse osteocalcin and compared to the amount of osteocalcin produced by W-20 cells in response to treatment with known amounts of BMP-2. Table III shows the amount of osteocalcin synthesized by W-20 induced by BMP-2.<img file="JP3681069B2_D0008.tif" />Example 6<u style="single">Rosen-Modified Sampath-Reddi Assay</u>Sumpath and Lady, Proceedings of National Academy of Sciences USA, Vol. 80: 6591-6595 (1983), using modified versions of rat bone formation analysis, bone, cartilage and / Alternatively, evaluate the inducible activity of BMP-11 protein on other connective tissues. This modified analysis is referred to herein as the Sumpath-ready analysis modified by Rosen. Replace the Sumpath-Lady ethanol precipitation step with dialysis (if the composition is a solution) or limit filtration (if the composition is a suspension) to the water of the fraction to be analyzed. The solution or suspension is then equilibrated with respect to 0.1% TFA. The resulting solution is added to a 20 mg rat matrix. A simulated rat matrix sample that has not been treated with protein serves as a control. The material is lyophilized and the resulting powder is encapsulated in # 5 gelatin capsules. Capsules, 21-49 day old male Long Evans Rat) is transplanted subcutaneously in the abdominal chest. Half of each implant is used for alkaline phosphatase analysis [see Lady et al. Proceedings of National Academy of Sciences USA, Vol. 69: 1061 (1972)]. The other half of each implant is fixed and processed for histological analysis. 1 μm glycol methacrylate sections are stained with Von Kossa and acidic fuchsin to score the induced bone and cartilage formation found in each implant. A score of +1 to +5 indicates the area of each histological section of the implant occupied by new bone and / or chondrocytes and matrix. A score of +5 indicates that more than 50% of the implant is new bone and / or cartilage produced as a direct result of the protein in the implant. Scores +4, +3, +2 and +1 indicate that the implant contains more than 40%, 30%, 20% and 10% new cartilage and / or bone, respectively. The BMP-11 protein of the present invention can be evaluated for activity in this analysis. Example 7<u style="single">Biological activity of expressed BMP-11</u>To measure the biological activity of the expressed BMP-11 protein obtained in Example 3 above, the protein was recovered from cell culture and from other co-produced proteinaceous substances as well as other contaminants. Purification is performed by isolating the BMP-11 protein. The purified protein can be analyzed by the rat bone formation analysis described in Example 6. Purification is performed using standard methods known to those skilled in the art. SDS-PAGE acrylamides stained with silver stains [Oakley et al., Analytical Biochem. Vol. 105: 361 (1980)] [Laemmli, Nature No. Vol. 227: 680 (1970)] and Immunoblot [Towbin et al., Proc. Natl. Acad. Sci. USA, Vol. 76: 4350. A standard method such as page (1979)] is used to analyze proteins. The above description details a currently preferable specific example of the present invention. In its implementation, it is expected that those skilled in the art will make many modifications and modifications in consideration of these statements. We are confident that those modifications and modifications will be included in the appended claims. Example 8<u style="single">Tests to measure activin activity of BMP-11</u>Purification is performed using standard methods known to those skilled in the art. Like other proteins in the TGF-β superfamily, heparin sepharose could be used for purification. SDS-PAGE acrylamides stained with silver stains [Oakley et al., Analytical Biochem. Vol. 105: 361 (1980)] [Laemmli, Nature No. Vol. 227: 680 (1970)] and Immunoblot [Towbin et al., Proc. Natl. Acad. Sci. USA, Vol. 76: 4350. A standard method such as page (1979)] is used to analyze proteins. For example, Vale et al., Endocrinology, Vol. 91: 562-572 (1972); Ling et al., Nature, Vol. 321: 779-782 (1986). ) Or Vale et al., Nature, Vol. 321: 776-779 (1986), established follicle-stimulating hormone (FSH) release regulation in an in vitro bioassay using rat anterior pituitary cells. The ability can further characterize the BMP-11 protein. These disclosures are incorporated herein by reference. Alternatively, in human K-562 cells, as described by Lozzio et al., Blood, Vol. 45: pp. 321-334 (1975) and US Pat. No. 5,071,834, column 15. BMP-11 may be characterized by its ability to stimulate erythropoietin activity. These disclosures are incorporated herein by reference. In addition, Schubert, Nature Vol. 344: BMP-11 may be characterized by its activity in cell survival analysis, as described on pages 868-870 (1990). The above description details a currently preferable specific example of the present invention. In its implementation, it is expected that those skilled in the art will make many modifications and modifications in consideration of these statements. We are confident that those modifications and modifications will be included in the appended claims. Sequence Listing (1) General Information (i) Applicant: (A) Name: GENETICS INSTITUTE, INC. (B) Street Name: Cambridge Park Drive 87 (C) City Name : Cambridge (D) State: Massachusetts (E) Country: United States (F) Postal Code (ZIP): 02140 (G) Phone Number 617 876-1170 (H) Telefax: 617-876-5851 (ii) Invention Name: BMP-11 Composition (iii) Number of Sequences: 11 (iv) Computer Readable Form: (A) Medium Type: Flop Disk (B) Computer: IBM PC Compatible (C) Operating System: PC- DOS / MS-DOS (D) Software: Patent In Release # 1.0, Version # 1.25 (EPO) (2) Information about SEQ ID NO: 1: (i) Sequence Features: (A) Sequence Length: 789 Base pair (B) Sequence type: Nucleic acid (C) Number of strands: Double strand (D) Topology: Linear (ii) Sequence type: DNA (genome) (vi) Origin: ( A) Organism name: Bos Taurus (B) Strain name: Bovine Activin WC (ix) Sequence characteristics: (A) Characteristic symbols: CDS (B) Location: 324..704 ( ix) Sequence features: (A) Feature symbols: misc_feature (B) Location: 322..323 (D) Other information: / Note = "Estimated intron 3'end" (ix) Sequence features : (A) Characteristic symbol:<img file="JP3681069B2_D0009.tif" /><img file="JP3681069B2_D0010.tif" />(2) Information about SEQ ID NO: 2: (i) Sequence characteristics: (A) Sequence length: 126 amino acids (B) Sequence type: Amino acids (D) Topology: Linear (ii) Sequence type: Description of protein (xi) sequence: SEQ ID NO: 2:<img file="JP3681069B2_D0011.tif" />(2) Information about SEQ ID NO: 3: (i) Sequence characteristics: (A) Sequence length: 213 base pairs (B) Sequence type: Nucleic acid (C) Number of strands: Double-stranded (D) topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism name: Homo Sapiens (B) Strain name: Human activin WC (ix) Sequence characteristics : (A) Characteristic symbol: CDS (B) Existence position: 28 ..183 (ix) Array feature: (A) Feature symbol: misc_feature (B) Existence position: 184..185 (D) etc. Information: / Note = "Two-thirds of the codon at the end of the partial clone" (xi) Sequence description: SEQ ID NO: 3:<img file="JP3681069B2_D0012.tif" />(2) Information about SEQ ID NO: 4: (i) Sequence characteristics: (A) Sequence length: 52 amino acids (B) Sequence type: amino acids (D) Topology: Linear (ii) Sequence type: Description of protein (xi) sequence: SEQ ID NO: 4:<img file="JP3681069B2_D0013.tif" />(2) Information about SEQ ID NO: 5: (i) Sequence characteristics: (A) Sequence length: 30 base pairs (B) Sequence type: Nucleic acid (C) Number of chains: 1 Single-stranded (D) topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism name: Primer for bovine activin WC D (ix) Sequence characteristics: (A) Characteristic symbols: misc_feature (B) Location: 1..9 (D) Other information: / Note = "XbaI restriction site" (xi) Sequence description: SEQ ID NO: 5:<img file="JP3681069B2_D0014.tif" />(2) Information on SEQ ID NO: 6: (i) Sequence features: (A) Sequence length: 30 base pairs (B) Sequence type: Nucleic acid (C) Number of chains: 1 Single-stranded (D) topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism name: Primer for bovine activin WC C (ix) Sequence characteristics: (A) Characteristic symbols: misc_feature (B) Location: 1..9 (D) Other information: / Note = "BamHI restriction site" (xi) Sequence description: SEQ ID NO: 6:<img file="JP3681069B2_D0015.tif" />(2) Information about SEQ ID NO: 7: (i) Sequence features: (A) Sequence length: 15 base pairs (B) Sequence type: Nucleic acid (C) Number of chains: 1 Single-stranded (D) topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism name: pMT2 Description of DNA (xi) sequence inserted in CXM: SEQ ID NO: 7:<img file="JP3681069B2_D0016.tif" />(2) Information on SEQ ID NO: 8: (i) Sequence features: (A) Sequence length: 34 base pairs (B) Sequence type: Nucleic acid (C) Number of chains: 1 Single-stranded (D) topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism name: DNA (ix) sequence inserted in pMT21 Features: (A) Features symbol: misc_feature ( B) Location: 1..6 (D) Other information: / Note = "Pst restriction site" (ix) Sequence features: (A) Feature symbols: misc_feature (B) Location: 15..26 (D) Other information: / Note = "EcoRI and XhoI restriction sites" (xi) Sequence description: SEQ ID NO: 8:<img file="JP3681069B2_D0017.tif" />(2) Information about SEQ ID NO: 9: (i) Sequence characteristics: (A) Sequence length: 68 base pairs (B) Sequence type: Nucleic acid (C) Number of chains: 1 Single-stranded (D) topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism name: Part of EMC virus leader sequence (x) Published information: (A) Authors: Jung, S.M. Kay (Jung, SK) (C) Journal of Willology (J.Virol) (D) Volume: Volume 63 (F) pp.: 1651 ~ 1660 (G) Date: 1989 (xi) Sequence description : Sequence number: 9:<img file="JP3681069B2_D0018.tif" />(2) Information about SEQ ID NO: 10: (i) Sequence characteristics: (A) Sequence length: 1270 base pairs (B) Sequence type: Nucleic acid (C) Number of chains: 1 Single-stranded (D) topology : Linear (ii) Sequence type: DNA (genome) (vi) Origin: (A) Organism name: Human BMP-11 (vii) Direct origin (B) Clone: FB30.5 (ix) sequence Feature: (A) Feature symbol: CDS (B) Location: 1..1086 (ix) Sequence feature: (A) Feature symbol: mat_peptide (B) Location: 760..1086 (xi) Sequence description: SEQ ID NO: 10:<img file="JP3681069B2_D0019.tif" /><img file="JP3681069B2_D0020.tif" />(2) Information about SEQ ID NO: 11: (i) Sequence characteristics: (A) Sequence length: 362 Amino acids (B) Sequence type: Amino acids (D) Topology: Linear (ii) Sequence type: Description of protein (xi) sequence: SEQ ID NO: 11:<img file="JP3681069B2_D0021.tif" /><img file="JP3681069B2_D0022.tif" />