JP2004073208A - Osteolytic composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material having sufficient soft bone and/or hard bone osteogenetic ability so as to effectively perform inducing soft bone and/or hard bone osteogensis, wound healing, tissue restoration and the like. <P>SOLUTION: The proteins exhibiting charactors of BMP-5, BMP-6 or BMP-7 are provided, a method for producing the proteins is provided and a medicinal composition containing the proteins and for inducing hard bone and/or soft bone ostogenesis, wound healing, tissue restoration and the like is provided. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a protein useful for forming bone and / or cartilage. In particular, the present invention relates to a few groups of purified proteins called BMP-5, BMP-6 and BMP-7 proteins (where BMP is a bone morphogenetic protein) and methods of obtaining them. . These proteins may exhibit cartilage and / or bone formation induction ability. They can be used for induction of bone and / or cartilage formation, wound healing and tissue repair.

Various substances and treatment methods have been proposed for treating bone and cartilage damage. However, some of these substances have insufficient cartilage and / or chondrogenesis-inducing ability, and when they are not sufficiently treated for induction of bone and / or cartilage formation, wound healing and tissue repair, etc. There were many.

Therefore, an object of the present invention is to find a substance having a sufficient ability to induce cartilage and / or bone formation, and to effectively use the substance to induce bone and / or cartilage formation, wound healing, and tissue repair. .

鑑 In view of the above problems, the present inventors have conducted intensive studies and found a protein useful for formation of hard bone and / or cartilage, and completed the present invention. In particular, the present invention provides a small group of purified proteins called BMP-5, BMP-6 and BMP-7 proteins (where BMP is a bone morphogenetic protein) and methods for obtaining them. It is.

蛋白 The protein of the present invention and a pharmaceutical composition containing the same enable effective induction of bone and / or cartilage formation, wound healing and tissue repair.

The present invention provides a group of BMP-5 proteins. Purified human BMP-5 proteins are substantially free of other proteins produced therewith and are characterized by an amino acid sequence comprising amino acids # 323 to # 454 shown in Table III.

The amino acid sequences # 323 to # 454 are encoded by a DNA sequence containing nucleotide # 1665 to nucleotide # 2060 in Table III. In addition, the BMP-5 protein may be characterized by an apparent molecular weight of 28000-30000 daltons as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under reducing conditions on SDS-PAGE, the protein electrophoreses with a molecular weight of about 14,000-20,000 daltons. It is believed that these proteins can stimulate, promote or otherwise induce cartilage and / or bone formation.

This invention further provides a bovine BMP-5 protein comprising amino acids # 9 to # 140 listed in Table I. Amino acid sequences # 9- # 140 are encoded by a DNA sequence comprising nucleotides # 32- # 427 of Table I. In addition, these proteins may be characterized by an apparent molecular weight of 28,000 to 30,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under reducing conditions on SDS-PAGE, the protein electrophoreses with a molecular weight of about 14,000-20,000 daltons. It is believed that these proteins can induce cartilage and / or bone formation.

The human BMP-5 protein of the invention cultivates cells transformed with a DNA sequence comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence shown in Table III comprising nucleotides # 699 to # 2060. It can be manufactured by the following. A BMP-5 protein containing the same or substantially the same amino acid sequence as shown in Table III from amino acid # 323 to amino acid # 454 is recovered from the culture medium, isolated and purified.

Cattle BMP-5 protein can be obtained by culturing cells transformed with a DNA sequence containing the same or substantially the same nucleotide sequence as shown in Table I containing nucleotides # 8 to # 427, and adding amino acids # 9 to A protein comprising the amino acid sequence shown in Table I or a portion thereof containing amino acid # 140 or a portion thereof can be produced by recovering and purifying from a culture medium.

This invention provides a group of BMP-6 proteins. Purified human BMP-6 proteins are substantially free of other proteins produced therewith and are characterized by an amino acid sequence comprising amino acids # 382 to # 513 shown in Table IV. The amino acid sequence containing amino acids # 382 to # 513 is encoded by the DNA sequence from nucleotide # 1303 to nucleotide # 1698 in Table IV. In addition, these proteins may be characterized by an apparent molecular weight of 28,000 to 30,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under reducing conditions on SDS-PAGE, the protein electrophoreses with a molecular weight of about 14,000-20,000 daltons. It is believed that these proteins can stimulate, promote or otherwise induce cartilage and / or bone formation.

{Circle around (2)} The present invention further provides a bovine BMP-6 protein characterized by an amino acid sequence containing amino acids # 121 to # 222 shown in Table II. The amino acid sequence from # 121 to # 222 is encoded by the DNA sequence in Table II from nucleotide # 361 to nucleotide # 666 in Table II. In addition, these proteins may be characterized by an apparent molecular weight of 28,000 to 30,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under reducing conditions in SDS-PAGE, the protein electrophoreses with a molecular weight of about 14,000-20,000 daltons. It is believed that these proteins can induce cartilage and / or bone formation.

The human BMP-6 protein of the present invention can be produced by culturing cells transformed with a DNA sequence containing nucleotides # 160 to # 1698 or a substantially similar sequence shown in Table III. The BMP-6 protein containing amino acid # 382 to amino acid # 513 or a substantially similar sequence is recovered from the culture medium, separated and purified.

Cattle BMP-6 protein is a DNA or substantially similar sequence comprising the same or substantially the same nucleotide sequence as shown in Table I, including nucleotides # 361 to # 666 shown in Table II. And the protein containing amino acids # 121 to # 222 shown in Table II is recovered and purified from the culture medium.

This invention provides a group of BMP-7 proteins. Purified human BMP-7 proteins are substantially free of other proteins produced therewith. The human BMP-7 protein is characterized by an amino acid sequence comprising amino acids # 300 to # 431 shown in Table V. These amino acid sequences # 300 to # 431 are encoded by the DNA sequence from nucleotide # 994 to nucleotide # 1389 in Table V. In addition, the BMP-7 protein may be characterized by an apparent molecular weight of 28000-30000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under reducing conditions on SDS-PAGE, the protein electrophoreses with a molecular weight of about 14,000-20,000 daltons. It is believed that these proteins can stimulate, promote or otherwise induce cartilage and / or bone formation.

The human BMP-7 protein of the invention cultivates cells transformed with a DNA sequence comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence shown in Table V comprising nucleotides # 97 to # 1389. It can be manufactured by the following. A BMP-7 protein containing the same or substantially the same amino acid sequence as shown in Table V from amino acid # 300 to amino acid # 431 is recovered from the culture medium, separated and purified.

The invention further provides a method for obtaining related human protein (s) or other mammalian cartilage and / or bone morphogenetic protein (s) by utilizing the above proteins. The above methods are well known to those skilled in the art of genetic engineering. In one method for obtaining the above proteins, a human BMP-5, BMP-6 and BMP-7 coding sequence or a portion thereof is utilized to screen a human genomic and / or cDNA library. The human genomic and / or cDNA sequences are separated by designing the sequence. In another method within the skill of the art, other mammalian BMP cartilage and / or bone morphogenetic proteins can be obtained by using the bovine and human BMP proteins of this invention.

After identifying the nucleotide sequence, the protein is produced by culturing cells transformed with the nucleotide sequence. This sequence, or a portion thereof, encodes a protein that hybridizes under stringent conditions to the nucleotide sequence of a BMP-5, BMP-6 or BMP-7 protein and exhibits cartilage and / or bone formation activity. The expressed protein is recovered and purified from the culture medium. Purified BMP proteins are substantially free of other proteinaceous materials and other contaminants produced with them.

BMP-5, BMP-6 and BMP-7 proteins may be characterized by their ability to promote, stimulate or otherwise induce cartilage and / or bone formation. It is further believed that the ability of these proteins to induce cartilage and / or bone formation can be demonstrated by the ability to demonstrate cartilage and / or bone formation activity in the rat bone formation assay described below. In addition, the proteins of this invention are expected to be active in this rat bone formation assay at a concentration of 10 μg to 500 μg per gram of bone formed. More specifically, it is believed that these proteins may be characterized by a score of at least +2 for 1 μg of protein in the rat bone formation assay described below using the prototype or modified scoring method.

Another aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a BMP-5, BMP-6 or BMP-7 protein in a pharmaceutically acceptable excipient or carrier. Yet another composition comprises at least one BMP-5, BMP-6 or BMP-7 protein. Thus, it is believed that the composition may comprise more than one BMP protein of the invention, since the BMP-5, BMP-6 and BMP-7 proteins may act in concert or possibly synergistically with each other. The composition of the present invention is used for inducing bone and / or cartilage formation. These compositions can also be used for wound healing and tissue repair.

Yet another composition of the invention comprises a BMP-5, BMP-6 or BMP-7 protein of the invention, in addition to at least one other therapeutically useful agent, such as the co-owned January 14, 1988. BMP-1 and BMP-2 disclosed in International Publication WO88 / 00205 and International Publication WO89 / 10409 published November 2, 1989 (these also include BMP-2A, BMP-2 And BMP-3 and BMP-4 (which have also been previously named BMP-2B, BMP-2 IIs). Other therapeutically useful agents include growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), tumor growth factors (TGF-α and TGF-β) and platelet-derived growth factor (PDGF ).

The compositions of the invention may also include suitable matrices, for example, for the purpose of delivering and / or supporting the composition and / or providing a bone and / or cartilage forming surface. The matrix may provide a slow release of the BMP protein and / or a suitable environment for displaying the BMP protein of the invention.

組成 The composition of the present invention can be used in a method for treating some bone and / or cartilage defects and periodontal disease. They can also be used in various types of wound treatment and tissue repair methods. According to the invention, these methods require the administration of a composition of the invention to a patient in need of bone and / or cartilage formation, wound healing or tissue repair as described above. Accordingly, the method includes administering a therapeutically effective amount of a protein of the invention. Also, these methods can include administering a protein of the invention in conjunction with at least one of the "BMP" proteins disclosed in the co-owned application. In addition, these methods can also include administering a protein of the invention with other growth factors, including EGF, FGF, TGF-α, TGF-β and PDGF.

Another aspect of the present invention is a DNA sequence encoding the expression of the protein of the present invention. The sequences include nucleotide sequences in the 5'-3 'direction shown in Table IV, or DNA sequences that hybridize under stringent conditions to the DNA sequences in Table IV, and include cartilage and / or Encodes a protein that has the ability to induce bone formation. The cartilage and / or bone formation can be demonstrated in the rat bone formation assay described below. It is believed that these proteins may be active in this assay at a concentration of 10 μg to 500 μg per gram of bone formed. More specifically, these proteins are believed to exhibit the ability to score at least +2 with 1 μg of protein in a raft bone formation assay. Finally, alleles or other variants of the sequences in Tables IV are also included in the present invention, regardless of whether the peptide sequence changes as a result of the nucleotide modifications.

別 Another aspect of the present invention provides a vector comprising the above DNA sequence operably linked to an expression control sequence. These vectors are a novel method for producing the protein of the present invention, and comprise the steps of converting a cell line transformed with a DNA sequence that directs the expression of the protein of the present invention operably linked to an expression control sequence into a suitable one. The present invention can be used in a method for culturing in a suitable culture medium and recovering and purifying the protein of the present invention therefrom. The claimed method may use some prokaryotic and eukaryotic known cells as host cells for polypeptide expression. The recovered BMP proteins are purified by separating them from other proteinaceous materials and other contaminants produced with them.

Other aspects and advantages of the invention will be apparent upon consideration of the following detailed description and preferred embodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION Purified human BMP-5 protein can be produced by culturing host cells transformed with the DNA sequences in Table III. The expressed BMP-5 protein is separated and purified from the culture medium. The purified human BMP-5 protein is expected to be characterized by an amino acid sequence comprising amino acids # 323 to # 454 shown in Table III. Accordingly, the purified BMP-5 human cartilage and / or bone protein of the present invention comprises a DNA sequence comprising nucleotides # 699 to # 2060 shown in Table III operably linked to a heterologous regulatory control sequence. Alternatively, culturing a host cell transformed with the substantially homologous sequence and culturing a protein containing the amino acid sequence from amino acid # 323 to amino acid # 454 or the substantially homologous sequence shown in Table III It is produced by collecting and purifying from a medium.

In another embodiment, the DNA sequence comprises nucleotides encoding amino acids # 323- # 454. Accordingly, the BMP-5 protein is characterized by a DNA sequence comprising nucleotides # 1665-nucleotide # 2060 shown in Table III or a substantially homologous sequence operably linked to a heterologous regulatory control sequence. It is produced by culturing the transformed host cell, and recovering and purifying from the culture medium a protein containing an amino acid sequence containing amino acids # 323 to amino acid # 454 or a substantially homologous sequence shown in Table III. You. Purified human BMP-5 proteins are substantially free of other proteinaceous materials and other contaminants produced with them.

The purified BMP-5 cartilage and / or bone protein of the present invention can be used to transform a host cell transformed with the DNA sequence from nucleotide # 8 to nucleotide # 578 or a substantially homologous sequence shown in Table I. It is produced by culturing, and recovering and purifying from the culture medium a protein containing an amino acid sequence containing amino acids # 9 to # 140 or a substantially homologous sequence shown in Table I. The purified BMP-5 bovine protein and the BMP proteins of the present invention are all substantially free of other proteinaceous and other contaminants produced therewith.

Purified human BMP-6 protein can be produced by culturing host cells transformed with the DNA sequences in Table IV. The expressed protein is separated and purified from the culture medium. The purified human BMP-6 protein of the present invention is expected to be characterized by an amino acid sequence comprising amino acids # 382 to # 513 shown in Table IV. Thus, these purified BMP-6 human cartilage / bone bone proteins of the present invention comprise a DNA sequence comprising nucleotides # 160 to # 1698 shown in Table IV operably linked to a heterologous regulatory control sequence. Alternatively, culturing a host cell transformed with the substantially homologous sequence and culturing a protein containing the amino acid sequence from amino acid # 382 to amino acid # 513 or the substantially homologous sequence shown in Table IV It is produced by collecting, separating and purifying from a medium.

Another embodiment may utilize a DNA sequence comprising nucleotides encoding amino acids # 382 to # 513. Accordingly, purified human BMP-6 protein is a DNA sequence comprising nucleotides # 1303 to # 1698 or a substantially homologous sequence shown in Table IV operably linked to a heterologous regulatory control sequence. By culturing the host cell transformed with the above, and recovering and purifying from the culture medium a protein containing an amino acid sequence containing amino acid # 382 to amino acid # 513 or a substantially homologous sequence shown in Table IV. Can be manufactured. Purified human BMP-6 proteins are substantially free of other proteinaceous materials and other contaminants produced with them.

The purified BMP-6 cartilage / bone bone protein of the present invention can be obtained by culturing host cells transformed with a DNA sequence containing nucleotides # 361 to # 666 or substantially homologous sequences shown in Table II. And a protein containing an amino acid sequence comprising amino acids # 121 to # 222 or a substantially homologous sequence shown in Table II from the culture medium. In another embodiment, the bovine protein is obtained by culturing a host cell transformed with a sequence comprising nucleotides # 289 to # 666 of Table II, and recovering and purifying the protein comprising amino acids # 97 to # 222. It is manufactured by Purified BMP-6 bovine proteins are substantially free of other proteinaceous materials and other contaminants produced with them.

Purified human BMP-7 protein can be produced by culturing host cells transformed with the DNA sequences in Table V. The expressed protein is separated and purified from the culture medium. The purified human BMP-7 protein is expected to be characterized by an amino acid sequence comprising amino acids # 300 to # 431 shown in Table V. Accordingly, these purified BMP-7 human cartilage / bone bone proteins of the present invention comprise a DNA sequence comprising nucleotides # 97 to # 1389 shown in Table V operably linked to a heterologous regulatory control sequence. Alternatively, the host cell transformed with the substantially homologous sequence is cultured, and the protein containing the amino acid sequence from amino acid # 300 to amino acid # 431 or the substantially homologous sequence shown in Table V is cultured. It is produced by collecting, separating and purifying from a medium.

Another embodiment may utilize a DNA sequence that includes nucleotides encoding amino acids # 300- # 431. Purified BMP-7 protein is transformed with a DNA sequence comprising nucleotides # 994 to # 1389 or a substantially homologous sequence shown in Table V operably linked to a heterologous regulatory control sequence. Cultivated host cells and recovering and purifying from the culture medium a protein containing an amino acid sequence containing amino acids # 300 to amino acid # 431 or a substantially homologous sequence shown in Table V. Purified human BMP-7 proteins are substantially free of other proteinaceous materials and other contaminants produced with them.

In addition, BMP-5, BMP-6 and BMP-7 proteins may be characterized by the ability to exhibit cartilage and / or bone formation activity. This activity can be demonstrated, for example, in the rat bone formation assay described in Example 3. In addition, these proteins are expected to show activity at a concentration of 10 μg to 500 μg per gram of bone formed in the assay. In addition, these proteins may be characterized by the ability to score at least +2 at 1 μg in this assay using the prototype or modified scoring methods described in more detail below.

Additionally, BMP-5, BMP-6 and BMP-7 proteins may be characterized by an apparent molecular weight of 28000-30000 daltons as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under reducing conditions on SDS-PAGE, the protein electrophoreses with a molecular weight of about 14,000-20,000 daltons.

Also, the proteins defined in this specification are similar to the sequences in Table IV, but are naturally-occurring (eg, allelic variants of a nucleotide sequence that can cause amino acid changes in the polypeptide) or intentionally. And the factor encoded by the modified sequence. Similarly, synthetic polypeptides that entirely or partially replicate the contiguous sequence of amino acid residues in Tables IV are also included in the invention. These sequences share primary and secondary or tertiary structure and conformational properties with other cartilage / bone proteins of the present invention, thereby sharing common bone and / or cartilage growth factor biological properties with them. I can do it. That is, they can be used as bioactive substitutes for natural proteins in therapeutic processes.

の 他 Other specific mutations of the protein sequences of the invention described herein include modifications of glycosylation sites. These modifications can include O-linked or N-linked glycosylation sites. For example, the absence or only partial glycosylation of glycosylation may be due to amino acid substitutions or deletions at the asparagine-binding gericosylation recognition site present in the sequence of the protein of the invention, as shown in Table IV. It is. The asparagine-linked glycosylation recognition site comprises a tripeptide sequence that is specifically recognized by a suitable cellular glycosylation enzyme. These tripeptide sequences are asparagine-X-threonine or asparagine-X-serine, where X is usually any amino acid. Various amino acid substitutions or deletions at one or both of the first or third amino acid positions of the gericosylation recognition site (and / or the amino acid deletion at the second position result in the modified tripeptide sequence being non-glycosylated. Expression of the altered nucleotide sequence produces a variant that is not glycosylated at that site.

(4) The present invention also includes a novel DNA sequence encoding the expression of the protein of the present invention without accompanying a DNA sequence encoding another proteinaceous substance. These DNA sequences include those drawn in the 5'-3 'direction in Table IV. In addition, under stringent hybridization conditions, see T. Maniatis et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982) p. Sequences that hybridize to DNA sequences and demonstrate cartilage and / or bone formation activity in rat osteogenesis assays are included.One example of one stringent hybridization condition described above is a high-temperature hybridization at 65 ° C. in 6-4 × SCC. Hybridization, followed by a 1 hour wash in 0.1 × SCC at 65 ° C. Alternatively, typical stringent hybridization conditions are 4 × SCC in 50% formamide at 42 ° C.

Similarly, it encodes a protein that resembles the protein encoded by the sequence in Tables IV, but has a synonymous or allelic variation in the genetic code (naturally occurring in a population of species that may or may not result in an amino acid change). DNA sequences that differ in codon sequence due to base modification) also encode the proteins of the invention described herein. Variants in the DNA sequence of Table IV due to point mutations or inducible modifications (including insertions, deletions and substitutions) to enhance activity, half-life or productivity of the encoded polypeptide are also included. , Included in the present invention.

In another aspect, the invention provides a method for obtaining a related human protein or other mammalian BMP-5, BMP-6 and BMP-7 proteins. One method of obtaining the above proteins is to use standard techniques for human genes or fragments thereof, for example, by utilizing the human BMP-5, BMP-6 and BMP-7 coding sequences disclosed herein. Examine human genomic libraries. In addition, by using the sequence thus identified as a probe, a human cell line or tissue that synthesizes a similar cartilage / bone bone protein can be identified. A cDNA library is synthesized and screened with probes derived from human or bovine coding sequences. The human sequence thus identified is transformed into a host cell, the host cell is cultured, and the protein is recovered, separated and purified from the culture medium. The purified protein is expected to exhibit cartilage and / or bone formation activity in the rat bone formation assay of Example 3.

Another embodiment of the present invention provides a novel method for producing the BMP-5, BMP-6 and BMP-7 proteins of the present invention. The method of the present invention comprises culturing, under the control of a known regulatory sequence, a suitable cell or cell line transformed with the above DNA sequence encoding the expression of the protein of the present invention. Regulatory sequences include promoter fragments, terminator fragments and other suitable sequences that direct the expression of the protein in a suitable host cell. Methods for culturing suitable cell lines are within the skill of the art. The transformed cells are cultured, and the BMP protein expressed thereby is recovered, separated, and purified from the culture medium using purification techniques well known to those skilled in the art. Purified BMP proteins are substantially free of other proteinaceous materials and other contaminants produced with them. The purified BMP protein of the present invention is substantially free of substances that naturally coexist with the protein of the present invention.

Suitable cells or cell lines can be mammalian cells, such as Chinese hamster ovary cells (CHO). The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, and production and purification of the product are known in the art. See, for example, Gessing and Sambrook, "Nature, 293: 620-625 (1981) or, alternatively, Kauffman et al., Mol. Cell. Biol., 5 (7): 1750-1759 (1985) or U.S. Pat. See 4419446. Other suitable mammalian cell lines include, but are not limited to, the COS-1 cell line and the CV-1 cell line.

Bacterial cells may also be suitable hosts. For example, various strains of Escherichia coli (eg, HB101, MC1061) are well known as host cells in the field of biotechnology. Also, various strains such as Bacillus subtilis, Pseudomonas, and other bacteria can be used in this method.

Many strains of yeast cells well known to those skilled in the art may also be available as host cells for expressing the polypeptides of the present invention. In addition, if desired, insect cells can be utilized as host cells in the methods of the present invention. See, e.g., Miller et al., "Genetic Engineering # 8: 277-298 (Plenum Press 1986) and references cited therein.

別 Another aspect of the present invention provides a vector used in the protein expression method of the present invention. The vector contains a novel DNA sequence encoding the BMP-5, BMP-6 and BMP-7 proteins of the present invention. In addition, the vector may also include appropriate expression control sequences to allow expression of the protein sequence. Alternatively, vectors incorporating the truncated or modified sequences described above are also an embodiment of the present invention and are useful for producing the proteins of the present invention. Said vector may be used in a method of transforming a cell line, and may contain selected regulatory sequences associated therewith so as to function the DNA coding sequence of the invention, which may direct replication and expression in a selected host cell. May be included. The regulatory sequences useful in the vector are well known to those skilled in the art and can be selected by the chosen host cell. The selections are made in conventional manner and do not form a part of the present invention. A host cell transformed with the vector and its progeny used for the production of BMP-5, BMP-6 and BMP-7 proteins and their progeny are also provided by the present invention.

れ ば If familiar with the art, the DNA sequences of this invention and known vectors, such as pCDr [Okayama et al., Mol. Cell. Biol. 2: 161-170 (1982)] and pJL3, pJL4 [Gaff et al., EMBO Journal, 4: 645-553 (1985)]. Similarly, those of skill in the art will be able to manipulate the sequences of this invention by deleting or replacing mammalian regulatory sequences in the proximity of the coding sequence and replacing the bacterial sequence with a bacterial cell for intracellular or extracellular expression by bacterial cells. Vectors can be created. For example, the coding sequence may be further manipulated (eg, by binding to other known linkers or deleting non-coding sequences therefrom, or by modifying the nucleotides present there by other known techniques). ). The modified coding sequence is then, for example, Taniguchi et al., "Insertion into Known Bacterial Vectors Using the Method Described in" Proceedings of the National Academy of Sciences of the US A.M. 77: 5230-5233 (1980) ". This illustrative bacterial vector can then be transformed into a bacterial host cell, whereby the protein of the invention is expressed, in order to effect extracellular expression of the cartilage and / or bone protein of the invention in the bacterial cell. For example, see European Patent Application EPA177343.

Similar operations can be performed for the construction of insect vectors for expression in insect cells [see, for example, the method described in published European Patent Application 155476]. In addition, yeast vectors can be constructed using yeast regulatory sequences for intracellular or extracellular expression of the factor of the invention in yeast cells. [See for example the methods described in published PCT application WO 86/00639 and European patent application EPA 123289].

Methods for producing proteins of the invention at high levels from mammalian cells require the construction of cells containing multiple copies of the heterologous gene encoding the protein of the invention. Heterologous genes can be obtained by increasing the concentration of amplifiable markers, such as cells containing multiple gene copies, according to the method of Kauffman and Sharp, Journal of Molecular Biology, 159: 601-629 (1982). May be linked to a dihydrofolate reductase (DHFH) gene that can be selected for extension in methotrexate (MTX). This method can be used with several different cell types.

For example, a plasmid containing the DNA sequence of the protein of the invention operably associated with another plasmid sequence allowing expression and the DHFR expression plasmid pAdA26SV (A) 3 "Kaufmann and Sharp, Mol. Cell. Biol. 2: 1304 (1982)] can be co-transfected into DHFR-deficient CHO cells, DUKX-BII, by calcium phosphate co-precipitation and transfection, electrical manipulation or protoplast fusion.Kaufman et al., Mol. Cell. Biol., 5 : 1750 (1983), DHFR expressing transformants were selected for growth in alpha medium containing dialyzed fetal calf serum, followed by increasing concentrations of MTX (0.02, 0.2, 1.0 and In a series of steps in 5 μM MTX) Selected for amplification by length. Protein expression should increase in accordance with increased levels of MTX resistance.

The transformant is cloned and the protein of the invention is recovered, separated and purified from the culture medium. Characterization of the expressed protein can be performed using standard techniques. For example, a characterization assay may include pulse labeling with " ³⁵ S] methionine or cysteine, or polyacrylamide gel electrophoresis, and a bioactive protein as determined by the Rosen-modified Sampath-Lady rat bone formation assay described in Example 3. Monitor expression: By following similar methods, other related proteins can be produced.

The protein of the present invention has therapeutic applicability for fracture and cartilage skipping in humans and other animals to induce cartilage and / or bone formation in an environment where bone and / or cartilage is not formed normally. Formulations using the protein of this invention may have prophylactic use in reducing closure and complex fractures and improving fixation of artificial joints. New bone formation induced by osteogenic agents contributes to the repair of cranial and facial defects by congenital, traumatic or tumor resection, and is also useful in cosmetic plastic surgery. The proteins of the present invention can be used in the treatment of periodontal disease and other dental restoration methods. The agent may provide an environment for attracting osteogenic cells, stimulating the growth of osteogenic cells, or inducing the differentiation of osteogenic cell progenitors. A variety of bone formation, cartilage induction and bone induction factors have been described. For a discussion thereof, see, for example, European Patent Applications 148155 and 169016.

蛋白 The proteins of this invention can also be used in wound healing and related tissue repair. Wound types include, but are not limited to, burns, cuts and ulcers. For a discussion of wound healing and related tissue repair, see, for example, PCT Publication WO 84/01106.

態 様 Another aspect of the invention includes methods and compositions for repairing fractures and bone and / or cartilage defects or other conditions associated with periodontal disease. In addition, the invention includes therapeutic methods and compositions for wound healing and tissue repair. The composition comprises a therapeutically effective amount of at least one of the BMP proteins, BMP-5, BMP-6 and BMP-7 of the present invention in admixture with a pharmaceutically acceptable excipient, carrier or matrix. Including.

It is anticipated that the proteins of this invention may act in concert with each other or with other related proteins and growth factors, or perhaps synergistically. Accordingly, the therapeutic methods and compositions of the present invention include one or more of the proteins of the present invention. Accordingly, yet another therapeutic method and composition of the present invention provides a therapeutically effective amount of at least one protein of the present invention, wherein the therapeutically effective amount of at least one protein of the present invention is other than those disclosed in the above-mentioned co-owned published international applications WO88 / 00205 and WO89 / 10409. "BMP" comprises at least one therapeutically effective amount of the proteins BMP-1, BMP-2, BMP-3 and BMP-4. The methods and compositions of the present invention may include a protein of the present invention or a portion thereof in combination with the "BMP mouth protein or a portion thereof described above.

The combination may include individual independent molecules or heteromolecules of the protein, eg, a heterodimer formed by a portion of each protein. For example, the methods and compositions of the invention can include a heteromolecule formed by combining a BMP protein of the invention, or a portion thereof, with a portion of another "BMP" protein.

治療 Yet another therapeutic method and composition of the invention comprises a protein of the invention or a portion thereof in combination with another agent that is beneficial in treating the bone and / or cartilage defect, wound or tissue in question. These agents include various growth factors, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet growth factor (PDGF), tumor growth factors (TGF-α and TGF-β), Includes K-fibroblast growth factor (KFGF), parathyroid hormone (PTH), leukemia inhibitory factors (LIF / HILDA, DIA) and insulin-like growth factors (IGF-I and IGF-II). Some of these agents may also be used in the compositions of the present invention.

製品 Physiologically acceptable products and preparations of protein compositions with due consideration of pH, isotonicity, stability, etc. are within the skill of the art. The therapeutic compositions are also valuable for medicinal medical applications due to the apparent lack of species specificity in cartilage and bone growth factor proteins. Livestock and purebred horses, in addition to humans, are desirable patients for such treatment with the proteins of the invention.

The method of treatment comprises administering the composition locally, systemically or locally as an implant or device. Upon administration, the therapeutic compositions used in this invention will, of course, be pyrogen-free and in a physiologically acceptable form. Further, the composition may be encapsulated or injected, desirably in a viscous form suitable for delivery to cartilage and / or still bone or the site of tissue injury. Topical administration may be suitable for wound healing and tissue repair.

Preferably, when aiming at bone and / or cartilage formation, the composition can deliver the BMP protein of the present invention to the site of bone and / or cartilage injury and provide a structure for bone and cartilage development, Optimally, it may include a matrix that can be absorbed into the body. The matrix can slowly release BMP proteins or other factors, including the composition. The matrix may be formed of materials currently used in other in-plant medical applications.

Matrix materials are selected based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interfacial properties. The particular applicability of the compositions of this invention will define an appropriate formulation. Matrices that can be used in the composition can be biodegradable, chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polybutyric acid, and polyanhydrides. Other possible materials are biodegradable and biologically well defined, for example bone or skin collagen. Still other matrices are composed of pure proteins or extracellular matrix components. Other possible matrices are non-biodegradable and chemically defined, for example, sintered hydroxyapatite, bioglass, aluminates or other ceramics. The matrix may be composed of any combination of the above types of materials, such as polybutyric acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can be modified in compositions, such as calcium-aluminate-phosphate, and processing can alter pore size, particle size, particle shape and biodegradability.

The administration system is determined by the attending physician in consideration of various factors that modify the action of the protein of the present invention. Factors that can modify the action of the protein of this invention include the weight of the bone desired to be formed, the site of the bone damage, the condition of the damaged bone, the size of the wound, the type of damaged tissue, the age, gender and diet of the patient, The extent of infection, if any, the time of administration and other clinical factors. The dosage may vary depending on the type of matrix used in the reconstitution and the type of bone and / or cartilage protein present in the composition. Also, the addition of other known growth factors to the final composition, such as EGF, PDGF, TGF-α, TGF-β, and IGF-I and IGF-II can affect the dose.

定期 Progress can be monitored by regular assessment of cartilage and / or bone growth and / or repair. For example, progress can be monitored using X-rays, histomorphometry and tetracycline labeling.

The following examples illustrate the recovery and characterization of the cartilage and / or bone proteins of the invention and the recovery of the corresponding human protein (s) by use of these proteins and the expression of said proteins by recombinant techniques. The method of practicing the invention will be described.

Example I
Isolation of bovine cartilage / osteoinductive protein R. In accordance with the method of the Proceedings of the National Academy of Sciences of USA, Vol. 70: 3511 (1973), omitting some extraction steps as described below, Prepare bone powder (20-120 mesh, Heritrex). 10 kg of this ground powder are demineralized by continuous change of 0.6N HCl at 4 ° C. for 48 hours with vigorous stirring. The resulting suspension is extracted with 4 l of 50 l of 2 M CaCl ₂ and 10 mM of ethylenediaminetetraacetic acid (EDTA) for 16 h, then in 50 l of 0.5 M EDTA for 4 h. After washing the residue three times with distilled water, 20 l of 4 M guanidine hydrochloride (GuCl), 20 mM Tris (20 mM), as described in Clinical Orthopedics and Related Research, Vol. 171: 213 (1982). pH 7.4) Resuspend in 1 mM N-ethylmaleimide, 1 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride. After 16-20 hours, remove the supernatant and transfer into an additional 10 l of GuCl buffer. The residue is extracted for a further 24 hours.

The crude GuCl extracts were combined and concentrated approximately 20-fold on a Pellicon apparatus equipped with a 10,000 molecular weight blocking membrane, then 50 mM Tris, 0.1 M NaCl, 6 M urea (pH 7.2), first buffer in the first column Dialyze with. After prolonged dialysis, the protein is loaded onto a 4 l DEAE cellulose column and the unbound fraction is collected.
The unbound fraction is concentrated and dialyzed against 50 mM NaAC, 50 mM NaCl (pH 4.6) in 6 M urea. This unbound fraction is applied to a carboxymethyl cellulose column. Proteins that did not bind to the column were removed by thorough washing with the initial buffer and the proteins having bone and / or chondrogenic activity as measured by the Rosen modified Sampath-Ready assay (described in Example III below). The material containing is desorbed from the column with 50 mM NaAc, 0.25 mM NaCl, 6 M urea (pH 4.6). The protein resulting from this step elution is concentrated 20-40 fold, and then 80 mM _KPO 4, diluted 5-fold with 6M Urea (pH 6.0). The pH of the solution is adjusted to 6.0 with 500mM K ₂ PO _4. This sample is applied to a hydroxyapatite column (LKB) equilibrated with 80 mM KPO ₄ , 6 M urea (pH 6.0), and all unbound proteins are removed by washing the column with the same buffer. Proteins with bone and / or chondrogenic activity are eluted with 100 mM KPO ₄ (pH 7.4) and 6 M urea.

The protein is concentrated approximately 10-fold and solid NaCl is added to a final concentration of 0.15M. This material, 50mM _KPO 4, 50mM NaCl, heparin equilibrated with 6M urea (pH 7.4) - applied to Sepharose column. After thoroughly washing the column with the initial buffer solution, proteins having bone and / or cartilage inducing activity are eluted with 50 mM KPO ₄ , 700 mM NaCl, 6 M urea (pH 7.4). This fraction was concentrated to a minimum volume and 0.4 ml aliquots were applied to a serial connection of Super Rose 6 and Super Rose 12 columns equilibrated with 4 M GuCl, 20 mM Tris, pH 7.2, The column is developed at a flow rate of 0.25 ml / min. A protein exhibiting bone and / or cartilage inducing activity corresponds to a protein of approximately 30,000 daltons.

上 記 Pool the above fractions from the Superrose column, dialyze against 50 mM NaAc, 6 M urea (pH 4.6) and apply to a Pharmacia Mono S HR column. The column is developed with a gradient to 1.0 M NaCl, 50 mM NaAc, 6 M urea (pH 4.6). Pool the active bone and / or chondrogenic fractions. This material was applied to a 0.46 × 25 cm Vdac C4 column in 0.1% TFA and the column was developed with a gradient to 90% acetonitrile, 0.1% TFA (1 ml / min in 60 minutes). 31.5% acetonitrile, 0.1% TFA to 49.5% acetonitrile, 0.1% TFA). Active substance elutes at approximately 40-44% acetonitrile. The fractions are assayed for cartilage and / or osteogenic activity. The active substance is further fractionated on a Mono Q column. The protein was dialyzed against 6 M urea, 25 mM diethanolamine (pH 8.6), and then applied to a 0.5 x 5 cm Mono Q column (Pharmacia) to give 6 M urea, 25 mM diethanolamine (pH 8.6) and 0.1 M Develop with a gradient of 5M NaCl, 6M urea, 25mM diethanolamine (pH 8.6). The fraction is brought to pH 3.0 with 10% trifluoroacetic acid (TFA). An aliquot of the appropriate fraction is iodinated by one of the following methods: J. McConney et al., International Archives of Allergy 29: 185-189 (1966); E. FIG. Bolton et al., Biochem @ J. 133: 529 (1973); F. Bowen-Pope, Journal of Biological Chemistry 237: 5161 (1982). The iodinated proteins present in these fractions are analyzed by SDS gel electrophoresis.

Example II
Characterization of bovine cartilage / osteoinductive factors Approximately 5 μg of protein from Example I in 6 M urea, 25 mM diethanolamine (pH 8.6), approximately 0.3 M NaCl to 0.1% with respect to SDS, 50 mM Tris / HCl 0.1% SDS (pH 7.5) ) For 16 hours. The dialyzed material is then electrophoretically concentrated on a dialysis membrane with a small amount of the ¹²⁵ I-labeled counterpart (Funkapillar et al., Methods in Enchimology 91: 227-236 (1983)). This material (approximately 100 μl in volume) is loaded on a 12% polyacrylamide gel and subjected to SDS-PAGE without reducing the sample with dithiothreitol [Lamry, U.S.A. K. Nature, Vol. 227: 680-685 (1970)]. The molecular weight is determined relative to a pre-stained molecular weight standard (Bethesda Research Labs). After autoradiography of the unfixed gel, the band at approximately 28,000-30000 daltons is cut off and the protein is electrophoretically eluted from the gel (Funkapillar et al., Supra). Based on a similar purified bone fraction described in the co-pending "BMP" application, in which bone and / or cartilage activity is found in the region of 28000-30000, this band shows that the bone and / or cartilage-derived fraction It is inferred that it contains a picture.

B. Subunit Characterization The composition of the isolated bovine bone protein subunits is also determined. The eluted protein is reduced completely, alkylated in 2% SDS using iodoacetate and standard methods, and reconcentrated by electrophoretic packing. Next, the fully reduced and alkylated sample was subjected to a further SDS-PAGE on a 12% gel, and the resulting region of approximately 14000-20,000 daltons with a doublet appearance was analyzed on the auto-fixed gel. Position is determined by radiography. Therefore, it remains in this 28000-30000 region. A weak band enters the 28000-30000 region. Therefore, the interpretation and expression can be interpreted that this 28000-30000 dalton protein produces a large area of 14,000-20,000 daltons, or that it contains two broad bands of approximately 14,000-16,000 and 16,000-20000 daltons. You can also.

Example III
Rosen Improved Sampath-Lady Assay Sampath and Lady, Proceedings of the National Academy of Sciences of USA, 80: 6591-6595 (1983). The protein of the present invention is used for evaluating bone and / or cartilage activity. This improved assay is referred to herein as the Rosen Improved Sampath-Ready Test. The ethanol precipitation step of the Sampath-Ready method is replaced by dialysis (if the composition is a solution) or diafiltration (if the composition is a suspension) of the fraction to be assayed against water. The solution or suspension is then redissolved in 0.1% TFA and the resulting solution is added to 20 mg of rat matrix. A sham rat matrix sample not treated with this protein is used as a control. The powder obtained by freezing and freeze-drying this material is enclosed in a # 5 gelatin capsule. Capsules are implanted subcutaneously at the abdominal thoracic site of 21-49 day old male Long Evans rats. Implants are removed after 7-14 days. Half of each implant was analyzed for alkaline phosphatase [A. H. Lady et al., Proceedings of the National Academy of Sciences, Vol. 69: 1601 (1972)].

固定 Fix the other half of each implant and process for histological analysis. Glycol methacrylate sections (1 μm) are stained with von Kossa and acid fustin or toluidine blue to score the amount of induced bone and cartilage formation present in each implant. The terms +1 to +5 indicate that the area of each histological section of the implant was occupied by new bone and / or chondrocytes and newly formed bone and matrix. Two types of scoring are described here. In the case of the first scoring, a score of +5 indicates that more than 50% of the implants are new bone and / or cartilage produced directly as a result of the proteins in this implant. The scores +4, +3, +2 and +1 indicate that more than 40%, 30%, 20% and 10% of the implants contain new cartilage and / or bone, respectively. The second scoring method (hereinafter referred to as the improved scoring method) is as follows: Three non-adjacent sections from each implant are evaluated and averaged. "+/-" indicates uncertain fixation of cartilage or bone, "+1" indicates that> 10% of each section is new cartilage or bone, "+2" is> 25%, "+3" is> 50%, "+4" indicates ~ 75%, and "+5" indicates> 80%. Individual implant scores are tabulated to show the variability of the assay.

The dose response characteristics of the cartilage and / or osteoinductive protein comprising the sample in the matrix sample indicate that the amount of bone and / or cartilage formed increases with the amount of cartilage / osteoinductive protein in the sample. It is possible that. It is possible that the control sample does not result in any bone and / or cartilage formation.
Like other cartilage and / or osteoinductive proteins such as the "BMP" protein described above, the formed bone and / or cartilage is expected to be physically confined to the space occupied by the matrix. The sample is further analyzed by SDS gel electrophoresis and isoelectric focusing followed by autoradiography. The activity is correlated with the pI of the protein band. To evaluate the purity of the protein in a particular fraction, an extinction coefficient of 1 OD / mg-cm is used as a protein evaluation criterion, and the protein is subjected to SDS-PAGE followed by autoradiography with silver staining or radioiodination.

Example IV
A. Bovine Protein Composition Gel slices in the approximate 14,000 to 20,000 dalton range described in Example IIB were fixed with methanol-acetic acid-water using standard methods, rinsed briefly with water, and then washed with 0.1 M ammonium bicarbonate. Sum up. After the gel slices are diced with a razor blade, proteins are digested from the gel matrix by adding 0.2 μg of TPCK-treated trypsin (Worthington) and incubating the gel at 37 ° C. for 16 hours. The resulting digest is then subjected to RPHPLC using a C4 Vydak RPHPLC column and a gradient of 0.1% TFA-water, 0.1% TFA, water-acetonitrile. The resulting peptide peak is monitored by UV absorption at 214 and 280 nm and subjected to direct amino-terminal amino acid sequence analysis using an Applied Biosystems gas phase sequenator (model 470A). When the amino acids are represented by the standard three letter symbols, one tryptic fragment having the following amino acid sequence is isolated by standard methods. In this sequence, "Xaa" is an unknown amino acid, and the amino acids in parentheses indicate uncertainty in the sequence:
Xaa-His-Glu-Leu-Tyr-Val-Ser-Phe- (Ser)
The following four oligonucleotide probes are designed based on the amino acid sequences of the fragments immobilized by trypsin immobilized above and synthesized by an automatic DNA synthesizer.
Probe # 1: GTRCTYGANATRCANTC
Probe # 2: GTRCTYGANATRCANAG
Probe # 3: GTRCTYAAYATRCANTC
Probe # 4: GTRCTYAAYATRCANAG
The standard nucleotide symbols in the probes defined above are: A, adenosine; C, cytosine; G, guanine; T, thymine; N, adenosine or cytosine or guanine or thymine; R, adenosine or guanine; And Y, cytosine or thymine.
Each of the probes consists of a collection of oligonucleotides. Since the genetic code is synonymous (one or more codons can encode the same amino acid), a mixture of oligonucleotides containing all possible nucleotide sequences encoding the trypsinized amino acid sequence is synthesized. These probes are radioactively labeled and used for screening a bovine cDNA library described below.

B. Beef BMP-5
From total DNA isolated from fetal calf bone cells by the method of Geron-Rovey et al. (A) Isolate the contained RNA. This total RNA was obtained from Dr. Marion Young (National Institute of Dental Research, National Institutes of Health). A cDNA library is prepared in Lambda gt10 (Tour et al., Supra) and plated on 50 plates at a rate of 8000 recombinants per plate. These recombinants (400,000) were probed with probes 1, 2, 3 using tetramethylammonium chloride (TMAC) hybridization method (see Uznyi et al., Science, Vol. 242: pp. 1528 to 1534 (1988)). Screen on double nitrocellulose filters using combinations of and 4. 28 positives are obtained, which are replated for the second round. Make a double nitrocellulose replica (copy) again. One set of filters screens for probes # 1 and # 2 and the other for probes # 3 and # 4. Six are obtained for the former and 21 for the latter. A phage plate stock is prepared by removing pathogens from one of the six, called HEL5, and bacteriophage DNA is isolated. This DNA is digested with EcoRI and subcloned into M13 and pSP65 (Promega Biotech, Madison, Wis.) [Melton et al., Nucleic Acids Research 12: 7035-7056 (1984)]. The DNA sequence of this fragment and the derived amino acid sequence are shown in Table I.

DNA sequence analysis of this fragment at M13 indicates that it encodes the desired tryptic peptide sequence disclosed above and that the derived amino acid sequence is a basic residue (Lys Lys) as expected by the specificity of trypsin. ) Precedes. In the sequence of Table I, the underlined amino acids # 42 to # 48 correspond to the tryptic fragments identified above, from which oligonucleotide probes are designed. The derived amino acid sequence Ser-Gly-Ser-His-Gln-Asp-Ser-Ser-Arg of amino acids # 15 to # 23 disclosed in Table I is disclosed in the aforementioned "BMP" publication WO 88/00205. And similarity to the trypsin-degraded fragment sequence Ser-Thr-Pro-Ala-Gln-Asp-Val-Ser-Arg found in the purified bone preparation of 28000-30000 daltons described in WO 89/10409. Attention. This fragment disclosed in Table I is part of the DNA sequence encoding the bovine BMP-5 protein. The DNA sequence shown in Table I encodes a partial peptide consisting of 140 amino acid residues (the first 7 nucleotides are from the adapter used in the cloning process) and is a 5 bp clone of the 420 base pair. 'Indicates open reading frames from below. The in-frame stop codon (TAA) indicates that this clone encodes the carboxy terminal portion of bovine BMP-5.

C. Beef BMP-6
The remaining positive clones isolated by the probes # 1, # 2, # 3 and # 4 (the second group containing 21 positive substances) are screened by HEL5 and hybridized under reducing hybridization conditions. Identify yet another clone [5 × SSC, 0.1% SDS, 5 × Denhardt, 100 μg / ml salmon sperm DNA standard hybridization buffer (SHB) at 65 ° C., 2 × SSC 0.1% Wash at 65 ° C. in SDS]. A phage plate stock is prepared by removing the pathogenic bacteria of this clone, and bacteriophage DNA is isolated. The DNA sequence and the amino acid sequence derived from part of this clone are shown in Table II. This sequence represents a portion of the DNA sequence encoding the bovine BMP-6 cartilage / bone protein of the present invention.
In the sequence of Table II, the first underlined portion of amino acid # 97 to amino acid # 105 corresponds to the purification of 28000 to 30000 daltons described in the above-mentioned "BMP" publications WO88 / 00205 and WO89 / 10409. Corresponds to the tryptic degradation fragment found in bovine bone preparations (and its reduced form in approximately the 18,000-20,000 dalton reduced form). The sequence of amino acids # 124- # 130, second underlined in Table II, corresponds to the tryptic fragments identified above, from which oligonucleotide probes are designed.
The DNA sequence of Table II shows an open reading frame of 666 base pairs starting from the 5 'end of the sequence of Table II encoding a partial peptide consisting of 222 amino acid residues. The stop codon in frame (TGA) indicates that this clone encodes the carboxy terminal portion of bovine BMP-6 protein. Based on knowledge of other BMP proteins and other proteins of the TGF-β family, the precursor polypeptide is cleaved at three basic residues (ArgArgArg) to residue 90 or It is expected to produce a mature peptide starting at 91.

Example V
A. Human protein composition A human cell line that synthesizes BMP-5 and / or BMP-6 mRNA is identified by the following method. RNA is isolated from various human cell lines, poly A-containing RNA is selected by chromatography on oligo dT cellulose, electrophoresed on formaldehyde-agarose gel, and transferred to nitrocellulose. A nitrocellulose replica of this gel is hybridized to a single-stranded M13 ³² P-labeled probe corresponding to the BMP-5 EcoRI-BglII fragment containing nucleotides 1 to 465 of the sequence in Table I. A strongly hybridizing band is detected in the lane corresponding to the human osteosarcoma cell line U-20SRNA. The other one nitrocellulose replica is hybridized to the single stranded ^{M13 32} P-labeled probe (corresponding to Table II nucleotides 106-261) containing PstI-SmaI fragment of bovine BMP-6. It can be seen that some RNA species in the lane corresponding to U-20S RNA hybridized with this probe.
A cDNA library is created from U-20S polyA-containing RNA into vector lambda ZAP (Stratagene) using established techniques (such as Tours). 750000 recombinants of this library are sown on a plate to create double nitrocellulose replicas. The Smal fragment of bovine BMP-6 corresponding to nucleotides 259 to 751 in Table II is labeled by nick translation and hybridized to both sets of filters in SHB at 65 ° C. One set of filters was under stringent conditions (0.2 × SSC, 0.1% SDS, 65 ° C.) and the other was under less stringent conditions (1 × SSC, 0.1% SDS, (65 ° C.). Note that many transcripts hybridize with the recombinants (approximately 162). Pick 24 and reseed for the second round. Make three nitrocellulose replicas for each plate. One hybridized to the BMP-6Smal probe, one to the nick translated BMP-6PstI-SacI fragment (nucleotides 106-378 in Table II) and the third to the nick translated BMP-5 Xbal fragment. (Nucleotides 1 to 76 in Table I). Hybridization and washing are performed under stringent conditions.

B. Human BMP-5 protein Excludes the plaques of 17 clones that hybridize more strongly to the third probe than to the second probe. DNA sequence analysis of one of these, U2-16, indicates that it encodes human BMP-5. U2-16 was deposited with the American Type Culture Collection (ATCC) (Rockville, MD) on June 22, 1989 under accession number ATCC 68109. This deposit and the other deposits described herein are made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of patent proceedings and its regulations (the Budapest Treaty). U2-16 includes an insert of approximately 2.1 Kb. The DNA sequence and derived amino acid sequence of U2-16 are shown in Table III below. This clone is expected to contain the complete nucleotide sequence required to encode the human BMP-5 protein. The cDNA sequence in Table III encodes a 1362 bp open reading frame and an in-frame stop codon (TAA) encoding a 454 amino acid protein, preceded by a 700 bp 5 'untranslated region with a stop codon in every frame. It contains the following 90 bp 3 'untranslated region.

This 454 amino acid protein has a molecular weight of approximately 52,000 daltons as predicted by its amino acid sequence and is considered to be the major translation product. Based on knowledge of other BMP proteins and other proteins within the TGF-β family, this precursor polypeptide is cleaved at the tribasic peptide Lys Arg Lys and is a 132 amino acid mature peptide beginning at amino acid # 323 “Asn” Is expected to be generated. Processing of the mature form of BMP-5 is expected to involve dimerization and elimination of the N-terminal region in a manner similar to that of the related protein TGF-β [L. E. FIG. Gentry et al., Molecular and Cellular Biology 8: 4162 (1988); Dernick et al., Nature 316: 701 (1985)].

Therefore, it is possible that the mature active species of BMP-5 contains a homodimer of two polypeptide subunits, each subunit consisting of amino acids # 323- # 454 with an expected molecular weight of approximately 15,000 daltons. Further, the active BMP-5 species may be, for example, a proprotein subunit or a proprotein dimer linked to the mature subunit. Yet another active species can include amino acids # 329- # 454, which include a homologous tryptic sequence found in purified bovine material. Also contemplated is a BMP-5 protein consisting of amino acids # 353- # 454, and thus containing the first conserved cysteine residue.

The sequence Ser-Ser-Ser-His-Gln-Asp-Ser-Ser-Arg of amino acids # 329- # 337 underlined in Table III corresponds to amino acids # 15- # 23 of Table I described in Example IV above. Shares the homology with the bovine sequence. Each of these sequences is a trypsin-degrading fragment sequence, Ser-, found in a 28,000 to 30,000 dalton purified bone preparation, as described in the aforementioned "BMP" published applications WO 88/00205 and WO 89/10409. It shares homology with Thr-Pro-Ala-Gln-Asp-Val-Ser-Arg (and its reduced form at approximately 18000-20,000 daltons).

The sequence His-Glu-Leu-Tyr-Val-Ser-Phe of amino acids # 356- # 362 underlined in Table III corresponds to the tryptic degradation fragment identified in the bovine bone preparation described above, Design more oligonucleotide probes.

The trypsin-degrading sequence His-Gul-Leu-Tyr-Val-Ser-Phe- (Ser) is contained in the bovine and human cartilage / bone protein BMP-2A sequence, for example, as described in WO 88/00205. It is noted that the sequence found is similar to His-Pro-Leu-Tyr-Val-Asp-Phe-Ser. Human BMP-5 shares homology with other BMP molecules and other members of the superfamily of TGF-β molecules. The carboxy-terminal 102 amino acid residues rich in human BMP-5 cysteine share the following homology with the BMP proteins disclosed herein and in WO 88/00205 and WO 89/10409 above: 61% match with BMP-2; 43% match with BMP-3; 59% match with BMP-4; 91% match with BMP-6; and 88% match with BMP-7. Human BMP-5 further shares the following homology: 38% match with TGF-β3; 37% match with TGF-β2; 36% match with TGF-β1; Muller inhibitor (MIS); A 25% match with testis glycoprotein that results in regression of Muller's canal during development of the male embryo; 25% match with inhibin α; 38% match with inhibin β _B ; 45% match with inhibin β _A ; 56% agreement with Xenopus factors [Weeks and Melton, Cell 51, 861-867 (1987)] that may be involved in the induction of mesoderm in early embryonic development; And a 57% agreement with Dpp, a product of Drosophila drosophila decapentapresic locus, which is involved in various other developmental processes in late development [ Jet, etc., 84 pp Nature # 325, Volume 81 (1987)].

C. Human BMP-6 Protein Six clones that hybridize more strongly to the second probe than to the second probe described in Example VA are taken and introduced into a plasmid. Restriction mapping, Southern blot analysis and DNA sequence analysis of these plasmids indicate that there are two clones. Clones U2-7 and U2-10 hybridize more strongly to the second probe than the other four clones and are based on having closer DNA homology to bovine RMP-6 in Table II. And a sequence encoding human BMP-6. The DNA sequence data derived from these clones indicates that they encode a partial polypeptide consisting of 132 amino acids including the carboxy terminus of the human BMP-6 protein. U2-7 was deposited with the American Type Culture Collection (ATCC) (Rockville, MD) under the provisions of the Budapest Treaty on June 23, 1989, under the accession number 68021.

A primer extended cDNA library is created from U-20S using the oligonucleotide GGAATCCAAGGCAGAATTGTG, whose sequence is based on the 3 'untranslated sequence of human BMP-6 from clone U2-10. The library is screened with oligonucleotides of the sequence CAGAGTCGTAATCGC derived from the sequences encoding BMP-6 of U2-7 and U2-10. Hybridization is performed in a standard hybridization buffer (SHB) at 42 degrees Celsius under washing conditions of 42 degrees Celsius, 5 × SSC, and 0.1% SDS. The clearly hybridizing clone is isolated. One of these clones, DNA insert PEH6-2, indicates that it extends further in the 5 'direction than U2-7 or U2-10. The primer-extended cDNA library assembled from the above U-20S mRNA is screened with oligonucleotides of the sequence GCCTCTCCCCCTCCGACGCCCCGTCCTCGT derived from the sequence near the 5 'end of PEH6-2. Hybridization is performed at 65 ° C. in SHB, and washing is performed at 65 ° C. in 2 × SSC, 0.1% SDS. Distinctly hybridizing recombinants are isolated and analyzed by restriction mapping and DNA sequence analysis.

The 5 'sequence of the insert of one of these distinctly hybridizing recombinants, PE5834 # 7, is used in the design of oligonucleotides of the sequence CTGCTGCTCCTCTCGCTGCCCGGAGCGC. A cDNA library using an arbitrary primer [synthesizing as a library using (dT) ₆ as a primer except using (dN) ₆ as a primer] was used to hybridize at 65 ° C. in SHB using this oligonucleotide. And screened by washing at 65 ° C. in 1 × SSC, 0.1% SDS. The clearly hybridizing clone RP10 is identified and isolated, and the DNA sequence from the 5 'end of the insert is determined. This sequence is used to design an oligonucleotide having the sequence TCGGGCTCCCTGTACCCGGCGCTCAAGACGCAGGAGAAGCGGGGAGATGCA. A human placenta cDNA library (Stratagene catalog # 936203) was hybridized with this oligonucleotide at 65 ° C. in SHB and 65 ° C. in 0.2 × SSC, 0.1% SDS. Screen by washing with. A distinctly hybridizing recombinant, designated BMP6C35, is isolated. DNA sequence analysis of the insert of this recombinant shows that it encodes the complete human BMP-6 protein.

BMP6C35 has been deposited with the American Type Culture Collection (1230: Park Lane Drive, Rockville, MD, USA) under the provisions of the Budapest Treaty on March 1, 1990, under accession number 68245. Was done. The DNA sequence of most of the BMP6C35 insert and the amino acid sequence derived therefrom are shown in Table IV. This DNA sequence contains an open reading frame of 1539 base pairs encoding a human BMP-6 protein precursor with 513 amino acids. The methionine codon, a possible initiator, is preceded by a 159 base pair 5 'untranslated sequence with a stop codon in all three open reading frames. The stop codon at nucleotides 1699-1701 is followed by a 3 'untranslated sequence of at least 1222 base pairs. Note that U2-7 has a C group at position 1221 of SMP6C35 corresponding to the T group, and U2-7 also has a G group at position BMP6C35K1253. These do not cause amino acid differences in the encoded protein, but probably indicate allelic variations.

Using the oligonucleotide TCGGGCTCTCTGTACGGGCGGCTCAAGACGCAGGAGAAGCGGAGATGCA, a 1 × 10 ⁶ recombinant nitrocellulose replica is hybridized to this oligonucleotide at 65 ° C. in SHB and washed with 0.2 × SSC, 0.1% SDS at 65 ° C. By doing so, a human genomic library (Tour et al., Supra) is screened. Purify the clearly hybridizing clones. The region to which the oligonucleotide hybridizes is located in the approximately 1.5 kb Pst fragment. DNA sequence analysis of this fragment confirms the 5 'sequence shown in Table IV.

The sequence Ser-Thr-Gln-Ser-Gln-Asp-Val-Ala-Arg of amino acids # 388 to # 396, which is initially underlined in Table IV, is the cattle disclosed in Table II above. Correspond to the analogous sequence Ser-Thr-Pro-Alg-Gln-Asp-Val-Ser-Arg in the sequence The sequence His-Glu-Leu-Tyr-Val-Ser-Phe of amino acids # 415 to # 421, which is second underlined in Table IV, corresponds to the tryptic fragment identified above, from which the oligonucleotide probe To design. The trypsin degradation sequence His-Glu-Leu-Tyr-Val-Ser-Phe- (Ser) is a sequence found in other BMP proteins, for example, the sequence His-Pro-Leu-Tyr-Val-Asp- found in cattle. It is noted that it resembles the human cartilage / bone protein BMP-2 described in Phe-Ser and Publication No. WO 88/00205. Thus, BMP-6 is a new member of the BMP subfamily of TCF-β-like molecules, which is the molecule BMP-2, BMP-3, BMP-4 described in Publications WO 88/00205 and WO 89/10409. , And BMP-5 and DMP-7 described herein.

Based on knowledge of other BMPs and other proteins and other proteins of the TCF-β family, RMP-6 is synthesized as a precursor molecule and cleaves between this precursor polypeptide and amino acids # 381 and # 382. To produce a mature polypeptide of 132 amino acids with a theoretical molecular weight of approximately 15 kd. The thermogenic form of DMP-6, like BMP-5, contains three possible N-linked glycoproteination sites per polypeptide chain.

Processing of BMP-6 to the thermophilic form is expected to involve dimerization and removal of the N-terminal region in a manner similar to that of the related protein TGF-β [L. E. FIG. Gentry et al. (1988); (1985), supra]. Active BMP-6 protein molecules are considered to be dimers. Furthermore, the mature active species of BMP-5 is a homodimer in which the protein molecule is composed of two polypeptide subunits, each subunit containing amino acids # 382 to # 513 disclosed in Table IV. It is possible that. Other active species of BMP-5 are considered to be, for example, phoprotein dimers or proprotein and mature subunits. Another active BMP-5 protein consists of amino acids # 388- # 513, and may thus include a tryptic fragment found in purified bovine material. Another BMP-5 protein according to the invention consists of amino acids # 412 to # 513 and thus contains the first conserved cysteine residue.

Comparison of the murine Vgr-1 sequence [Lyons et al., PNAS 86: 4554 (1989)] with human BMP-6 shows that the degree of amino acid sequence identity is greater than 92%. Murine Vgr-1 appears to be a rat homologue of BMP-6. Human BMP-6 shares homology with other BMP molecules and other members of the TGF-β superfamily molecule. The cysteine-rich carboxy-terminal 102 amino acid residues of human RMP-6 share the following homology with the BMP proteins disclosed herein and in WO 88/00205 and WO 89/10409. 61% match with BMP-2; 44% match with BMP-3; 60% match with BMP-4; 91% match with BMP-5; and 87% match with BMP-7. Human RMP-6 further shares the following homology: 41% match with TGF-β3; 39% match with TGF-β2; 37% match with TGF-β1; Muller inhibitor (MIS) , i.e. coincidence of glycoprotein and 26% of testicular causing regression of development in Müllerian male embryo; inhibin α and 25% match; inhibin beta _B and 43% of the match; inhibin beta _a and 49% of the match; 58% agreement with Vgl, a Xenopus factor that may be involved in the induction of mesoderm early in embryonic development [Weeks and Melton (1987) supra]; and is required for dorsoventral identification early in embryonic development and late in development 57% agreement with Dpp, a product of Drosophila drosophila decapentapresic locus, for a variety of other developmental processes [Paget et al., (1987) ),the above].

D. The remaining four clones of Example VC above, representing a second group of human BMP-7 protein clones, encode a novel polypeptide designated as BMP-7. One of these clones, U2-5, was deposited with the ATCC on June 22, 1989 under accession number ATCC 68020 under the provisions of the Budapest Treaty. It was determined that this clone did not contain the entire coding sequence of BMP-7. Oligos of the sequence GCGAGCAATGGAGGATCCAG (shown based on the 3 'non-coding sequence of U2-5) were used to generate a primer-extended cDNA library from U-2OS mRNA (Toll et al.). Oligonucleotide GATCTCGCGCTGCAT (shown based on BMP-7 coding sequence) by hybridizing 500,000 recombinants of this library with 42 ° C SHB and washing in 42 ° C 5X SSC, 0.1% SDS. Sorted by. Several hybridized clones were obtained. The DNA sequence analysis and derived amino acid sequence of one of these clones, PEH7-9, is shown in Table V. PEH7-9 was deposited with the American Type Culture Collection (ATCC) on November 17, 1989, Rockville, Maryland under the Budapest Treaty under the accession number ATCC 68182. PEH7-9 contains a 1448 base pair insertion. This clone, PEH7-9, was expected to contain all of the nucleotide sequences required to encode the BMP-7 protein. The cDNA sequence in Table V contains a 1292 base pair open reading frame encoding a 431 amino acid protein, preceded by a 96 base pair 5 'untranslated region with a stop codon for all frames, and Includes a 60 base pair 3 'untranslated region after the stop codon TAG.

This protein of $ 431 amino acids has a molecular weight of 49,000 daltons as predicted by its amino acid sequence and is predicted to represent the first translation product. Based on knowledge of other BMP proteins and other proteins in the TGF-β family, it is expected that the precursor polypeptide will cleave between amino acids # 299 and # 300 to yield a 132 amino acid mature peptide.

Processing to the mature form of BMP-7 is expected to include dimerization of the proprotein and removal of the N-terminal region in a manner similar to that of the related protein TGF-B (L. E. Gentry et al. (1988) supra and R. Delnick et al. (1985) supra). Therefore, the mature active species of BMP-7 consists of a homodimer of two polypeptide subunits consisting of amino acids # 300-431 as shown in Table V, each subunit having a calculated weight of 15,000 daltons. Is expected. Other active BMP-7 species are predicted to be, for example, protein dimers or protein subunits bound to the mature subunit. Additional active species may include amino acids # 309- # 431 of the above species in Table V and may include the tryptic sequences found in the purified ash material. It is also anticipated that the BMP-7 protein will contain amino acids # 330- # 431, thereby containing the first protected cysteine residue.

The underlined sequences of Asn-Gln-Glu-Ala-Leu-Arg of amino acids # 309- # 314 in Table V are 28,000 as described in "BMP" published WO 88/00205 and WO 89/10409 above. This is the same sequence as the amino acid of tryptic fragment # 5 found in purified bone products of ３０30,000 daltons. The underlined sequence in Table V of amino acids # 333- # 339 His-Glu-Leu-Tyr-Val-Ser-Phe is the tryptic identified in a bovine bone marrow product as described above, from which an oligonucleotide probe was assigned. Corresponds to a fragment.

Like BMP-5 and BMP-6, human BMP-7 has homology to other BMP molecules and other members of the TGF-β superfamily of molecules. The cysteine-rich carboxy-terminal 102 amino acid residues of human BMP-7 have 60% homology to BMP-2, having the following homology to the BMP proteins of WO88 / 00205 and WO89 / 10409 as described herein and above. It is 43% homologous to BMP-3, 58% homologous to BMP-4, 87% homologous to BMP-6 and 88% homologous to BMP-5. Human BMP-7 also has the following homology. 40% homologous to TGF-β3, 40% homologous to TGF-β2, 36% homologous to TGF-β1, to Mueller inhibitor (MIS), a testicular glycoprotein that causes degeneration of the Muellerian duct during the development of male embryos 29% homology, 25% homology to inhibin-α, 44% homology to inhibin-βB, 45% homology to inhibin-βA, 57% homology to Vgl, a Xenopus factor that can be involved in mesoderm induction in early embryogenesis ( Weaks and Melton, (1987) supra) and the Drosophila decapentaplasic locus required for dorsal-abdominal clarification of early embryogenesis and involved in various other developmental stages later in development. It is 58% homologous to the product Dpp (Paget et al. (1987), supra).

The present invention includes the genomic sequences of BMP-5, BMP-6 and BMP-7. To obtain these sequences, the cDNA sequences described herein are used as probes to select genomic libraries using techniques well known to those skilled in the art.

The above procedures and additional methods well known to those skilled in the art can be used to isolate other related proteins of interest by utilizing bovine or human proteins as a probe source. The other proteins mentioned above can likewise be used in wound repair, wound healing and tissue repair, among others.

Example VI
Expression of BMP protein In order to produce the bovine, human or other mammalian BMP-5, BMP-6 or BMP-7 protein of the present invention, DNA encoding the The expression vector is transfected and introduced into a mammalian cell or other preferred eukaryotic or prokaryotic host. It is contemplated that the preferred expression system for the biologically effective human recombinant proteins of the present invention will be mammalian cells that have been stably transformed. For transient expression, SV40-transformed African Green Monkey kidney COS-1 or COS-7 is selected and produced in modest amounts of the encoded protein, usually in a plasmid, for 1-4 days. In order to stably express BMP-5, BMP-6 or BMP-7 at a high level, a cell line of Chinese Hamster Ovary (CHO) is preferable. Thus, preferred mammalian cells are considered to be CHO cells.

The transformed host cells are cultured, and the BMP protein of the present invention expressed thereby is recovered, isolated and purified. The expressed protein is confirmed using standard techniques. For example, confirmation includes pulse labeling with [ ³⁵ S] methionine or cysteine and analysis by polyacrylamide electrophoresis. Recombinantly expressed BMP proteins are free of co-producing and naturally occurring co-localized proteinaceous substances and do not include other contaminants such as those found in culture.

A. Vector Construction As noted above, a number of expression vectors known in the art can be used to express the BMP proteins of the invention. The vector used in the following examples is pMT21, which is a derivative of pMT2, but other vectors are also suitable for practicing the present invention.

@ PMT2 is derived from pMT2-VWF, which has been deposited with the American Type Culture Collection (ATCC), Rockville, MD (USA) under the accession number ATCC 67122, according to the provisions of the Budapest Treaty. EcoRI digestion excise the complementary DNA insertion site present in pMT-VWF to produce linear pMT2, which can be ligated and transformed into Escherichia coli HB101 or DH-5 normally ampicillin resistant. it can. Plasmid pMT2 DNA can be prepared by a conventional method.

PMT21 is constructed using loop-out / in mutagenesis [Morinaga et al., Biotechnology 84: 636 (1984)]. Thereby, bases 1075 to 1170 (all) are removed. In addition, insert the following sequence: 5'TCGA3 '. This sequence completes a new restriction site, XhoI. This plasmid will contain three unique cloning sites, PstI, EcoRI and XhoI.

Further, pMT21 is digested with EcoRV and XhoI, the digested DNA is treated with the Klenow fragment of DNA polymerase I, and a ClaI linker (NE Bio Labs, CATCGATTG) is ligated. In this way, starting from the HindIII site near the SV40 origin of replication and the enhancer sequence of pMT2, bases 2171 to 2420 are removed and a unique ClaI site is introduced, but the adenovirus VAI gene remains intact.

B. Construction of BMP-5 vector The derivative of the BMP-5 complementary DNA sequence shown in Table III was obtained from nucleotide no. 699-No. It consists of 2070 nucleotide sequences and is specifically amplified. Oligonucleotides CGACCTGCAGCCACCATGCATCTCTACTGTA and TGCCTGCAGTTTAATATTAGTGGCAGC were used as primers and the nucleotide sequence no. 699-No. 2070 is amplified from the insertion site of clone U2-16 described in Example V. By this method, the nucleotide sequence CGACCTGGCAGCCACC is replaced by nucleotide No. 699, and the nucleotide sequence CTGCAGGCA is replaced by nucleotide no. Introduced immediately after 2070. By adding these sequences, a PstI restriction endonuclease recognition site is created at both ends of the amplified DNA fragment. The DNA product amplified by this method is digested with the restriction endonuclease PstI and subcloned into the PstI site of pMT2 derived pMT21 described above. The resulting clone is called H5 / 5 / pMT.

The insertion site of H5 / 5 / pMT is excised by digestion with PstI and subcloned at the PstI site into the plasmid vector pSP65, resulting in BMP5 / SP6. BMP5 / SP6 and U2-16 were digested with the restriction endonucleases NsiI and NdeI and the nucleotides no. 704-No. The insertion site corresponding to 1876 is excised. The resulting 1173 nucleotide NsiI-NdeI fragment of clone U2-16 ligates to the NsiI-NdeI site of BMP5 / SP6 from which the corresponding 1173 nucleotide NsiI-NdeI fragment has been removed. The resulting clone is called BMP5mix / SP64.

直接 Perform direct DNA sequence analysis of BMP5mix / SP64 to confirm that the nucleotide sequence produced by amplification is identical to that shown in Table III. Clone BMP5mix / SP64 was digested with the restriction endonuclease PstI and the nucleotides no. 699-No. The insertion site consisting of 2070 and the additional sequence containing the PstI recognition site described above is excised. As a result, a 1382 nucleotide PstI fragment is subcloned into the PstI site of pMT2 derived pMT21. This clone has the BMP5mix / pMT21No. No. 2.

C. Construction of BMP-6 vector 160-No. Derivatives of the BMP-6 complementary DNA sequence shown in Table IV, consisting of 1706 nucleotide sequences, are produced by a series of techniques known to those skilled in the art. Clone BMP6C35, described in Example V, was digested with the restriction endonucleases ApaI and TaqI and the nucleotides No. 231-No. The 1476 nucleotide portion of the insertion site consisting of 1703 is excised. Synthetic oligonucleotides with a SalI restriction endonuclease site converter are available from the No. 1476 ApaI-TaqI fragment of the BMP-6 complementary DNA sequence. 160-No. 230 and no. 1704-No. It is designed to replace the nucleotide corresponding to 1706. 5 'lost
(TCGACCCACCATGCCGGGGCTGGGGCGGAGGGCGCAGTGGCTGTG CTGGTGGT GGGGGCTGTGCTGCAGCTGCCTGCGGGCC and CGCAGCAGCTGCACAGCAGCCCCCACCACCAGCACAGCCACTGCGCC CTCCGCCCCAG CCCCGGCATGGTGGG)
And the oligonucleotide / SalI converter for replacing the 3 ′ (TCCGACTGGTTT and CGAAACCAG) sequences anneal independently of each other. The annealed 5 'and 3' converters were then ligated to the 1476 nucleotides ApaI-TaqI described above, and the Nos. A 1563 nucleotide fragment is created consisting of the nucleotide sequence 160-1706 and additional sequences designed to create SalI restriction endonuclease sites at both ends. The resulting 1563 nucleotide fragment is subcloned into the SalI site of pSP64. This clone has the BMP6 / SP64 No. No. 15.

BMP6 / SP64 No. Analysis of 15 DNA sequences confirms that the 5 'and 3' sequences replaced by the converter are identical to the sequences shown in Table IV. BMP6 / SP64 No. The 15 insertion sites are excised by digestion with the restriction endonuclease SalI. The resulting 1563 nucleotide SalI fragment was subcloned into the XhoI restriction endonuclease site of pMT2 derived pMT21 and is referred to herein as BMP6 / pMT21.

D. Construction of BMP-7 vector 97-No. Derivatives of the BMP-7 sequence shown in Table V, consisting of 1402 nucleotide sequences, are specifically amplified. The oligonucleotides CAGGTCCGACCCACCATGCACGTGCGCTCA and TCTGTCGACCCTCGGAGGGACTAGTGGC were used as primers from the insertion site of the clone PEH7-9 described above as the primers. 97-No. Used to amplify 1402. According to this method, the nucleotide sequence CAGGTCCGACCCACC is changed to nucleotide No. 97, and the nucleotide sequence GTCGACAGA at nucleotide no. It will be inserted immediately after 1402. By adding these sequences, SalI restriction endonuclease recognition sites are created at each end of the amplified DNA fragment. The amplified DNA product obtained by this method is digested with the restriction endonuclease SalI and subcloned into the SalI site of the plasmid vector pSP64, resulting in BMP-7 / SP6No. 2 is obtained.

{Clone BMP-7 / SP6 No. 2 and PEH7-9 were digested with the restriction endonucleases NcoI and StuI and the nucleotides no. 363-No. The insertion site corresponding to 1081 is excised. The resulting 719 nucleotide NcoI-StuI fragment of clone PEH7-9 is a BMP7 / SP6No. Ligation to two NcoI-StuI sites. The resulting clone is called BMP7mix / SP6.

Direct DNA sequence analysis of BMP7mix / SP6 was performed, and the 3 ' 1082-No. Confirmed to be identical to the nucleotide sequence of 1402, but the 5 'portion contains one incorrectly inserted nucleotide.

Amplification of nucleotide sequences (No. 97 to No. 1402 in Table V) using PEH7-9 as a template is repeated as described above. The amplified DNA product obtained by this method is digested with the restriction endonucleases SalI and PstI. By this digestion, the nucleotide Nos. 97-No. The 747 nucleotide fragment consisting of 833 and the additional sequence of the 5 'priming oligonucleotide that created the SalI restriction endonuclease recognition site described above are excised. This 747 SalI-PstI fragment is subcloned into a SalI-PstI digested pSP65 vector, resulting in 5'BMP7 / SP65. By DNA sequence analysis, 5'BMP7 / SP65No. 1 was inserted in nucleotide V. of Table V. 97-No. 362 is shown to consist of the same sequence.

Clones BMP7mix / SP6 and 5'BMP7 / SP65 are digested with the restriction endonucleases SalI and NcoI. Nucleotide No. of Table V obtained. 363-No. BMP7mix / SP65 3 'NcoI-SalI fragment consisting of SEQ ID NO: 1042 and nucleotide no. 97-No. The 5 'SalI-NcoI fragment of 5' BMP7 / SP65 consisting of N. 362 was ligated together at the NcoI restriction site and nucleotides no. 97-No. A 1317 nucleotide fragment consisting of 1402 and additional sequences derived from 5 'and 3' oligonucleotide primers creating SalI restriction sites at both ends of the fragment are produced. This 1317 nucleotide SalI fragment ligates to the SalI site of pMT2 derived pMT2Cla-2. This clone is called BMP7 / pMT2.

挿入 The insertion site of BMP7 / pMT2 is excised by digestion with the restriction endonuclease SalI. The resulting 1317 nucleotide SalI fragment is subcloned into the SalI restriction site of vector pSP64. This clone has the BMP7 / SP64 No. 2d. BMP7 / SP64 No. The insertion site of 2d was excised by digestion with SalI, and the nucleotide No. 97-No. The SalI fragment consisting of 1402 is subcloned into the XhoI restriction endonuclease site of pMT2 derived pMT21 described above.

Example VII
Transient COS cell expression In order to obtain transient expression of BMP-5, BMP-6 and BMP-7 proteins, each BMP5mix which is a kind of vector containing BMP-5, BMP-6 or BMP-7 cDNA is used. / PMT21 # 2, BMP6 / pMT21 # 2, or BMP7 / pMT are transduced into COS-1 cells using electroporation. Other suitable transduction methods include DEAE-dextran and lipofection. After about 48 hours, the cells were examined for both intracellular and secretory expression of BMP-5, BMP-6 or BMP-7 protein by metabolic labeling with [ ³⁵ S] methionine and polyacrylamide gel electrophoresis. Intracellular BMP was examined for cells treated with the N-linked sugar addition inhibitor, tunicamycin. In tunicamycin treated cells, the unglycosylated primary translation product migrates as a uniform band of predictable size and can often be more easily discriminated on a polyacrylamide gel than the glycosylated form of the protein. In each case, the protein in the tunicamycin treated cells is compared to a duplicate plate of transduced but untreated COS cells.

A. BMP-5 COS expression The results indicate that the intracellular forms of BMP-5 at about 52 Kd and 57 Kd are produced by COS cells. The 52Kd protein is as large as can be expected from the primary sequence of the BMP-5 cDNA clone. After cell treatment with tunicamycin, only the 52Kd form of BMP-5 was generated, indicating that the 57Kd protein is a glycosylated derivative of the 52Kd primary translation product. The 57Kd protein is secreted into the conditioned medium and apparently has not been efficiently processed by the COS cells into pro and mature peptides.

B. BMP-6 COS expression Intracellular BMP-6 is present in untreated COS-1 cells as approximately 61 Kd and 65 Kd doublets. In the presence of tunicamycin, only the 61Kd protein was observed, indicating that the 65Kd protein is a glycosylated derivative of the 61Kd primary translation product. This is close to the molecular weight expected from a BMP-6 cDNA clone. In the absence of tunicamycin, the major component protein secreted from COS-1 cells is a 65Kd glycosylated, unprocessed truncated form of BMP-6. In addition, there are also 46Kd and 20Kd peptides at less than 65Kd, which may indicate processed and mature peptides, respectively.

C. BMP-7 COS expression BMP-7 protein in tunicamycin-treated COS-1 cells appears as a doublet at 44Kd and 46Kd. In the absence of tunicamycin, 46 Kd and possibly 48 Kd of protein are produced. These probably indicate the potential of glycosylated derivatives of the BMP-7 primary translation product. The 48Kd protein is the major BMP component secreted from COS-1 cells, indicating inefficient BMP-7 cleavage at the propeptide dibasic cleavage site.

Example VIII
CHO cell expression DHFR-deficient CHO cells (DUKX Bll) were transduced by electroporation with one of the above BMP-5, BMP-6 or BMP-7 expression vectors, and expressed in a nucleotide-free medium to express DHER. Select Other methods for transduction include, but are not limited to, CaPO ₄ precipitation, protoplast fusion, microinjection, lipofection, and the like. To more conveniently obtain high expression, cells can be selected in a nucleotide-free medium supplemented with 5 nM, 20 nM or 100 nM MTX. Since the DHFR selectable marker is physically linked to the BMP cDNA as the second gene in the bicistron coding region, cells expressing DHFR should also express BMP encoded in the upstream cistron. Either a single clone or a collection of merged clones is expanded and analyzed for BMP protein expression. Cells are selected stepwise with increasing MTX concentrations (5 nM, 20 nM, 100 nM, 500 nM, 2 nM, 10 nM and 100 nM) and contain multiple copies of the expression vector DNA by gene amplification, and thus secrete large amounts of BMP protein. Get.

し Using standard techniques, screen for BMP RNA and protein expression or activity, and clone or reclon highly expressing cell lines at appropriate selection levels to obtain a more homogeneous population of cells. The BMP DNA sequence, BMP RNA and protein expression of the cell lines obtained are then examined. The appropriate cell line can then be used for producing a recombinant BMP protein.

A. CHO expression of BMP-5 The above-mentioned BMP-5 vector BMP5mix / PMT21 # 2 is morphologically introduced into CHO cells by electroporation, and cells for DHFR expression are selected. Clonal cell lines are obtained from individual colonies stepwise selected for MTX resistance and examined for BMP-5 protein secretion. In certain cases, cell lines can be kept as a population and cloned at a later stage of MTX selection.

Example V. B. As described above, BMP-5 cDNA encodes a protein of about 52 Kd. Following intracellular processing, including but not limited to propeptide cleavage, glycosylation, and dimer or multimer formation, a number of BMP-5 peptides are produced. Under reducing conditions, there are at least four peptide candidates as processing forms of the BMP-5 protein that can be discriminated by SDS PAGE: 65 Kd peptide, 35 Kd peptide and a doublet of about 22 Kd molecular weight. Other smaller amounts of BMP-5 peptides may be present. When compared to other related BMP molecules and the processing of the related protein TGF-beta, the 65Kd protein may correspond to unprocessed BMP-5, the 35Kd form may correspond to the propeptide, and the 22Kd doublet may correspond to the mature peptide. There is.
Material from the BMP-5 cell line is analyzed by a two-dimensional gel system. In the first dimension, the protein is electrophoresed under non-reducing conditions. It is then reduced and electrophoresed in a second polyacrylamide gel. The disulfide-linked dimer or multimer passes below the diagonal across the second reducing gel. Analysis of the BMP-5 protein indicates that large amounts of the mature BMP-5 peptide can produce a homodimer of about 30-35 Kd that transforms into a 22 Kd doublet observed in one-dimensional reducing gels. The fraction of the mature peptide is clearly in a disulfide bond complex with the propeptide. The amount of this complex is smaller when compared to the mature homodimer. In addition, some of the unprocessed proteins can obviously produce homodimers or homomultimers.

B. CHO expression of BMP-6 The above-mentioned BMP-6 expression vector BMP6 / pMT21 is transduced into CHO cells, and stable transformants are obtained through DHFR expression in the same manner as described in Part A for BMP-5. Select The mature active form of BMP-6 is predicted to include amino acids # 382-513 of Table IV. Secreted BMP-6 protein is predicted to be processed in a similar manner as described above for BMP-5, other related BMP molecules and the related protein TGF-β [Gentry et al .; Delnick et al., Supra. ].
C. CHO expression of BMP-7 The above-mentioned BMP-7 expression vector BMP7 / pMT21 is transduced into CHO cells, and a stable transformant is selected for BMP-5 through DHFR expression in the same manner as described above. The mature active form of BMP-7 is predicted to include amino acids # 300- # 431 of Table V. Secreted BMP-7 protein is predicted to be processed in a similar manner as described above for BMP-5, other related BMP molecules and the related protein TGF-β [Gentry et al .; Delnick et al., Supra. ].

Example IX
Biological activity of expressed BMP protein To measure the biological activity of the expressed BMP-5, BMP-6 and BMP-7 proteins obtained in Examples VII and VIII above, the BMP protein was recovered from the culture medium, BMP protein is separated and purified from co-produced proteinaceous substances and other contaminants. The protein is partially purified on a heparin Sepharose column and further purified using standard purification techniques known to those skilled in the art.

For example, the conditioned medium supernatant after transduction collected from the culture is concentrated approximately 10-fold by ultrafiltration over a YM10 membrane and then dialyzed against 20 mM Tris, 0.15 M NaCl, pH 7.5 (starting buffer). I do. This material is then applied to a heparin Sepharose column in starting buffer. The non-adsorbed protein is removed by washing with a starting buffer, and the adsorbed protein contains the protein of the present invention and is desorbed by washing with 20 mM Tris, 2.0 M NaCl, pH 7.4. The protein adsorbed by the heparin column is, for example, concentrated about 10-fold with Centricon 10 and reduced in salt, for example by diafiltration with 0.1% trifluoroacetic acid. An appropriate amount of the resulting solution is mixed with 20 mg of rat matrix and the in vivo bone and / or chondrogenic activity is determined by the Rosen modified Sampath-Ledge assay. The mock transduction supernatant fraction is used as a control.

Further purification can be performed by preparative NaDodSO ₄ / PAGE [Emuri, Nature, Vol. 227, pp. 680-685 (1970)]. For example, about 300 μg of protein can be applied to a 1.5 mm-thick 15% gel and recovered by adding L- [35S] methionine-labeled BMP protein purified on heparin-sepharose in the same manner as described above. . Proteins can be visualized by copper staining of adjacent lanes [Lie et al., Analytical Biochemistry. 166: 308-312 (1987)]. Scraped appropriate band, extracted with 0.1% NaDodSO ₄ / 20mM Tris, pH 8.0. The supernatant can be acidified to pH 3 using 10% CF ₃ COOH, and the protein desalted on a 5.0 × 0.46 cm Vydac C4 column (The Separation Group, Hesperia, Calif.), 0.1% % expand from _CF 3 COOH at 90% acetonitrile /0.1%CF ₃ COOH gradient solvent.

{Circle around (7)} After approximately 7 days, the transplanted tissue containing the rat matrix to which the specific amount of human BMP-5, BMP-6 or BMP-7 of the present invention has been added is removed, and developmental evaluation is performed. Representative sections from each transplant are stained for new bone mineral using Hong Kosa and acid fuchsin, and for matrix formation corresponding to cartilage using toluidine blue. The types of cells present in the sections and the extent to which these cells exhibited a phenotype were evaluated and indicated numerically as described in Example III.

Activity can also be tested with host cell extracts. Purification is performed in the same manner as described above, except that urea 6M is contained in all buffers.

The foregoing description details the preferred embodiments of the present invention. In fact, it is expected that many modifications and changes will occur to those skilled in the art upon reviewing these descriptions. These modifications and changes fall within the scope of the claims appended hereto.

INDUSTRIAL APPLICABILITY The protein of the present invention and a pharmaceutical composition containing the same have excellent cartilage and / or chondrogenesis-inducing ability and can be used for induction of bone and / or cartilage formation, wound healing, tissue repair, and the like. The protein of the present invention and a pharmaceutical composition containing the same are particularly useful in the field of regenerative medicine.

Claims (11)

A protein encoded by the DNA sequence described in (a) to (c) below and having BMP-5, BMP-6 or BMP-7 properties:
(a) a DNA sequence encoding BMP-5,
(i) contains nucleotide positions # 699 to # 2060 of Table III;
(ii) contains nucleotide positions # 1665- # 2060 of Table III,
(iii) contains a sequence that hybridizes to the sequence of (i) under stringent hybridization conditions and encodes a protein having the biological properties of BMP-5;
(iv) including, for any one of the sequences (i) to (iii), a sequence that is degenerate or allelic, or
(v) having a nucleotide sequence encoding the amino acid sequence of amino acids # 323 to # 454 in Table III;
(b) a DNA sequence encoding BMP-6,
(i) contains nucleotide positions # 160 to # 1698 of Table IV,
(ii) contains nucleotide positions # 1303- # 1698 of Table IV,
(iii) contains, under stringent hybridization conditions, a sequence that hybridizes to the sequence of (i) and encodes a protein having the biological properties of BMP-6;
(iv) including a sequence that is synonymous or allelic with respect to any one of the sequences (i) to (iii), or
(v) having a nucleotide sequence encoding the amino acid sequence of amino acids # 382 to # 513 in Table IV;
and
(c) a DNA sequence encoding BMP-7,
(i) contains nucleotide positions # 994 to # 1389 of Table V,
(ii) Under stringent hybridization conditions, hybridize to the sequence of (i), encode the amino acid sequence at the N-terminal position corresponding to the amino acid position encoded by the codon beginning at nucleotide position # 994, and BMP- A sequence encoding a protein having 7 biological properties,
(iii) With respect to any one of the sequences (i) to (ii), comprising a sequence that is synonymous or allelic, or
(iv) Those having a nucleotide sequence encoding the amino acid sequence of amino acids # 300 to # 431 in Table V. A protein exhibiting BMP-5, BMP-6 or BMP-7 properties, obtained by a method comprising the following steps:
(a) culturing a host cell transformed with the vector containing the DNA according to claim 1 in an appropriate culture medium, and (b) recovering the protein from the culture medium And isolate and purify. (4) The protein according to (1) or (2), which further exhibits cartilage and / or bone formation induction ability. A method for producing a BMP protein, comprising the following steps:
(a) culturing a host cell transformed with the vector containing the DNA according to claim 1 in an appropriate culture medium, and (b) recovering the protein from the culture medium And isolate and purify. A protein exhibiting the properties of BMP-7, obtained by a method comprising the following steps:
(a) culturing CHO cells transformed with a DNA sequence encoding amino acids # 1 to # 431 of Table V or a DNA sequence containing nucleotides # 97 to # 1389 of Table V in an appropriate culture medium;
(b) recovering, isolating and purifying the BMP-7 protein from the culture medium; A method for producing a BMP-7 protein, comprising the following steps:
(a) culturing CHO cells transformed with a DNA sequence encoding amino acids # 1 to # 431 of Table V or a DNA sequence containing nucleotides # 97 to # 1389 of Table V in an appropriate culture medium;
(b) recovering, isolating and purifying the BMP-7 protein from the culture; A pharmaceutical composition for treating a patient in need of cartilage and / or bone formation, which comprises the protein according to any one of claims 1 to 3 or 5 as an active ingredient. 8. The composition of claim 7, further comprising a pharmaceutically acceptable matrix. The composition according to claim 8, wherein the matrix comprises hydroxyapatite, collagen, polylactic acid and tricalcium phosphate. (4) A pharmaceutical composition for wound healing and tissue repair, comprising the protein according to any one of claims 1 to 3 or 5 as an active ingredient. DNA comprising the BMP-7 DNA sequence of ATCC Accession No. 68020.