ES2367035T3 - BMP-12, BMP-13 AND COMPOSITIONS OF THE SAME TO INDUCE TENDONS. - Google Patents

Abstract

Polypeptide having the ability to induce the formation of tendon-like or ligament-like tissue, wherein said polypeptide is encoded by a DNA molecule comprising a nucleotide sequence selected from the group consisting of: (a) nucleotides no. 196, No. 199, No. 208, No. 217, No. 361, No. 388, No. 493, No. 496, No. 571 or No. 577 to No. 879 or No. 882 of SEQ ID NO: 1; (b) nucleotides encoding amino acids # -25, # 1 or # 3 to # 103 or # 104 of SEQ ID NO: 2; (c) nucleotides No. 845, No. 893 or No. 899 to No. 1201 or No. 1204 of SEQ ID NO: 3; (d) nucleotides encoding amino acids # 1 or # 19 to # 119 or # 120 of SEQ ID NO: 4; (e) nucleotides No. 410, No. 458, No. 602, No. 605 or No. 659 to No. 961 or No. 964 of SEQ ID NO: 25; (f) nucleotides encoding amino acids # 1 or # 19 to # 119 or # 120 of SEQ ID NO: 26; or (g) natural human allelic sequences and equivalent degenerate codon sequences of any one of (a) to (f), or (h) sequences that hybridize with any of (a) to (f) above under stringent hybridization conditions, for use in a procedure to induce tendon / ligament-like tissue formation in a patient in need.

Description

Field of the Invention

The present invention relates to a new family of purified proteins, and compositions containing said proteins, said compositions being useful for the induction of tendon / ligament-like tissue formation, for wound healing and for ligament repair and other tissues These proteins can also be used in compositions to increase the activity of bone morphogenetic proteins.

Background of the invention

The search for the molecule or molecules responsible for the formation of bone, cartilage, tendon and other tissues present in bone and other tissue extracts has led to the discovery of a new series of molecules called Bone Morphogenetic Proteins (BMP). Previously, the structures of several proteins, called BMP-1 to BMP-11, have been clarified. The exceptional induction activities of these proteins, together with their presence in the bone, suggest that they are important regulators of bone repair processes, and may be involved in the normal maintenance of bone tissue. There is a need to identify additional proteins that play a role in the formation of other vital tissues. The present invention relates to the identification of a family of proteins that have tendon / ligament-like tissue inducing activity, and which are useful in compositions for inducing the formation and repair of tendon / ligament-like tissue.

Summary of the invention

In a first aspect, the present invention relates to a polypeptide having the ability to induce the formation of a tendon-like or ligament-like tissue, wherein said polypeptide is encoded by a DNA molecule comprising a nucleotide sequence. selected from the group consisting of

(to)

nucleotides No. 196, No. 199, No. 208, No. 217, No. 361, No. 388, No. 493, No. 496, No. 571 or No. 577 to No. 879 or No. 882 of SEQ ID NO: 1;

(b)

nucleotides encoding amino acids # -25, # 1 or # 3 to # 103 or # 104 of SEQ ID NO: 2;

(C)

nucleotides No. 845, No. 893 or No. 899 to No. 1201 or No. 1204 of SEQ ID NO: 3;

(d)

nucleotides encoding amino acids # 1 or # 19 to # 119 or # 120 of SEQ ID NO: 4;

(and)

nucleotides No. 410, No. 458, No. 602, No. 605 or No. 659 to No. 961 or No. 964 of SEQ ID NO: 25;

(F)

nucleotides encoding amino acids # 1 or # 19 to # 119 or # 120 of SEQ ID NO: 26; or

(g)

natural human allelic sequences and equivalent degenerate codon sequences of any one of (a) to (f), or

(h)

sequences that hybridize with any of (a) to (f) above under stringent hybridization conditions.

And where said polypeptide is for use in a procedure to induce tendon / ligament-like tissue formation in a patient in need.

In another aspect, the present invention provides a pharmaceutical composition comprising a polypeptide that has the ability to induce the formation of a tendon-like or ligament-like tissue, wherein said polypeptide is encoded by a DNA molecule comprising a sequence. of nucleotides selected from the group consisting of

(to)

nucleotides No. 196, No. 199, No. 208, No. 217, No. 361, No. 388, No. 493, No. 496, No. 571 or No. 577 to No. 879 or No. 882 of SEQ ID NO: 1;

(b)

nucleotides encoding amino acids # -25, # 1 or # 3 to # 103 or # 104 of SEQ ID NO: 2;

(C)

nucleotides No. 845, No. 893 or No. 899 to No. 1201 or No. 1204 of SEQ ID NO: 3;

(d)

nucleotides encoding amino acids # 1 or # 19 to # 119 or # 120 of SEQ ID NO: 4;

(and)

nucleotides No. 410, No. 458, No. 602, No. 605 or No. 659 to No. 961 or No. 964 of SEQ ID NO: 25;

(F)

nucleotides encoding amino acids # 1 or # 19 to # 119 or # 120 of SEQ ID NO: 26; or

(g)

natural human allelic sequences or equivalent degenerate codon sequences of any one of (a) to (f), or

(h)

sequences that hybridize with any of (a) to (f) above under stringent hybridization conditions

in which the composition is to induce the formation of tendon / ligament-like tissue in a patient in need.

Thus, the present invention relates to DNA molecules encoding a tendon / ligament-like tissue-inducing protein that the inventors have designated V1-1. This new protein is now called BMP

12. The present invention also includes DNA molecules encoding BMP-12 related proteins.

BMP-12-related proteins are a subset of the BMP / TGF-β / Vg-1 family of proteins, which include BMP-12 and VL-1, which are defined as tendon / ligament-like tissue-inducing proteins encoded by DNA sequences that are cloned and identified, for example, using PCR, using specific BMP12 primers, such as primers # 6 and # 7 described below, with reduced stringency conditions. It is preferred that DNA sequences encoding BMP-12-related proteins share at least about 80% homology at the amino acid level with amino acids # 3 through # 103 of SEQ ID NO: 1.

The DNA molecules preferably have a DNA sequence encoding the BMP-12 protein, said sequence being provided in SEQ ID NO: 1, or a BMP-12 related protein as further described herein. Both BMP-12 protein and BMP-12-related proteins are characterized by the ability to induce the formation of a tendon / ligament-like tissue in the assay described in the examples.

The DNA molecules used by the invention preferably comprise a DNA sequence, as described in SEQUENCE ID NO: 1, more preferably nucleotides no. 496 to no. 882, no. 571 to no. 882 or no. 577 to no. 882 of the SEC. ID No.: 1; or DNA sequences that hybridize with the foregoing under stringent hybridization conditions and encode a protein that has the ability to form tendon / ligament-like tissue. The use of DNA molecules by the invention may also comprise a DNA sequence as described in SEQ ID NO: 25; more preferably nucleotides No. 604 or No. 658 to No. 964 of SEQ ID NO: 25.

The DNA molecules used by the invention also include DNA molecules that comprise a DNA sequence encoding a BMP-12 related protein with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 26, as well as natural allelic sequences and equivalent degenerative codon sequences of SEQ ID NO: 2 or SEQ ID NO: 26. Preferably, the DNA sequence used by the present invention encodes amino acids No. -25 to No. 104, No. 1 to No. 104 or No. 3 to No. 103 of SEQ ID NO: 2; or amino acids No. 1 to No. 120 or No. 19 to No. 120 of SEQ ID NO: 26. The DNA sequence may comprise, in a 5 'to 3' direction, nucleotides encoding a propeptide and nucleotides encoding amino acids No. -25 to No. 104, No. 1 to No. 104 or No. 3 to No. 103 of SEQ ID NO: 2; or amino acids # 1 to # 120 or # 19 to # 120 of SEQ ID NO: 26. The propeptide useful in the above embodiment is preferably selected from the group consisting of the native BMP-12 propeptide and a protein propeptide of a different member of the TGF-B superfamily or the BMP family. The invention further relates to DNA sequences that hybridize with the above DNA sequences under stringent hybridization conditions and encode a BMP-12-related protein that has the ability to induce tendon / ligament-like tissue formation.

The present invention further relates to a purified BMP-12 related protein characterized by the ability to induce tendon / ligament-like tissue formation. The BMP-12 related polypeptides preferably comprise an amino acid sequence as shown in SEQ ID NO: 2. More preferably, the polypeptide comprises amino acids # -25, # 1 or # 3 to # 103 or # 104 as set forth. in SEQ ID NO: 2; or amino acids # 1 or # 19 to # 120 as set forth in SEQ ID NO: 26. In a preferred embodiment, the purified polypeptide may be in the form of a dimer composed of two subunits, each with the amino acid sequence of SEQ ID NO: 2.

In another embodiment, the present invention comprises compositions comprising an effective amount of the BMP-12 related proteins described above. In the compositions, the protein can be mixed with a pharmaceutically acceptable carrier.

Other embodiments include chimeric DNA molecules that comprise a DNA sequence encoding a propeptide of a member of the TGP-β protein superfamily, linked in the correct reading frame to a DNA sequence encoding a BMP-12 related polypeptide . A suitable propeptide is the BMP-2 propeptide. The invention also includes heterodimeric protein molecules comprising a monomer having the amino acid sequence shown in SEQ ID NO: 2, and a monomer having the amino acid sequence of another TGF-β subfamily protein.

Description of the sequences

SEQ ID NO: 1 is the nucleotide sequence encoding human BMP-12.

SEQ ID NO: 2 is the amino acid sequence comprising the mature human BMP-12 polypeptide.

SEQ ID NO: 3 is the nucleotide sequence encoding the MP52 protein.

SEQ ID NO: 4 is the amino acid sequence comprising the mature MP52 polypeptide.

SEQ ID NO: 5 is the nucleotide sequence of a specifically amplified part of the sequence encoding human BMP-12.

SEQ ID NO: 6 is the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5.

SEQ ID NO: 7 is the nucleotide sequence of a specifically amplified part of the sequence encoding human VL-1.

SEQ ID NO: 8 is the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 7.

SEQ ID NO: 9 is the nucleotide sequence of plasmid pALV1-781, used for the expression of BMP-12 in E. coli.

SEQ ID NO: 10 is the nucleotide sequence of a fragment of the murine clone, mV1.

SEQ ID NO: 11 is the amino acid sequence of a murine protein fragment encoded by mV1.

SEQ ID NO: 12 is the nucleotide sequence of a fragment of the murine clone, mV2.

SEQ ID NO: 13 is the amino acid sequence of a murine protein fragment encoded by mV2.

SEQ ID NO: 14 is the nucleotide sequence of a fragment of the murine clone, mV9.

SEQ ID NO: 15 is the amino acid sequence of a murine protein fragment encoded by mV9.

SEQ ID NO: 16 is the amino acid sequence of a consensus sequence of the BMP / TGF-β / Vg-1 protein. He

first Xaa represents Gln or Asn; the second Xaa represents Val or Ile. SEQ ID NO: 17 is the nucleotide sequence of oligonucleotide # 1. SEQ ID NO: 18 is the amino acid sequence of a BMP / TGF-β / Vg-1 protein consensus sequence. Xaa

represents Val or Leu. SEQ ID NO: 19 is the nucleotide sequence of oligonucleotide # 2. SEQ ID NO: 20 is the nucleotide sequence of oligonucleotide # 3. SEQ ID NO: 21 is the nucleotide sequence of oligonucleotide # 4. SEQ ID NO: 22 is the nucleotide sequence of oligonucleotide # 5. SEQ ID NO: 23 is the nucleotide sequence of oligonucleotide # 6. SEQ ID NO: 24 is the nucleotide sequence of oligonucleotide # 7. SEQ ID NO: 25 is the nucleotide sequence of the human VL-1 coding sequence (BMP-13). SEQ ID NO: 26 is the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 25. SEQ ID NO: 27 is the nucleotide sequence encoding a fusion of the BMP-2 propeptide and the sequence

mature coding of BMP-12. SEQ ID NO: 28 is the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 27. SEQ ID NO: 29 is the nucleotide sequence encoding the murine mV1 protein. X01 is Val, Ala, Glu or Gly;

X02 is Ser, Pro Thr or Ala; X03 is Ser or Arg; X04 is Leu, Pro, Gln or Arg; X05 is Cys or Trp; X06 is Val, Ala, Asp or

Gly; X07 is Val, Ala, Glu or Gly; X08 is Gln, Lys or Glu. SEQ ID NO: 30 is the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 29. X01 to X08 are the same as in SEQ ID NO: 29.

SEQ ID NO: 31 is the nucleotide sequence encoding the murine mV2 protein. X01 is Pro or Thr; X02 is Val.

SEQ ID NO: 32 is the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 31. X01 and X02 are the same as in SEQ ID NO: 31. SEQ ID NO: 33 is the nucleotide sequence encoding the human BMP-12 protein. SEQ ID NO: 34 is the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 33. SEQ ID NO: 35 is the nucleotide sequence of oligonucleotide # 8.

Brief description of the figures

Figure 1 is a comparison of the sequences of human BMP-12 and a human MP52.

Detailed description of the invention

The DNA sequences of the present invention are useful for producing proteins that induce tendon / ligament-like tissue formation, as described further below. The DNA sequences of the present invention are further useful for isolating and cloning additional DNA sequences encoding BMP-12 related proteins with similar activity. These BMP-12 related proteins can be homologous to other species, or they can be related proteins within the same species.

In addition, another aspect of the invention are DNA sequences that encode the expression of a tendon / ligament-like tissue-inducing protein. These sequences include the nucleotide sequence in the 5 'to 3' direction illustrated in SEQ ID NO: 1 or in SEQ ID NO: 25, DNA sequences that, except for the degeneracy of the genetic code, are identical to the sequences DNA of SEQ ID NO: 1 or 25, and encode the protein of SEQ ID NO: 2 or

26. Also included in the present invention are DNA sequences that hybridize under stringent conditions with the DNA sequence of SEQ ID NO: 1 or 25 and encode a protein that has the ability to induce the formation of a tendon or ligament. Preferred DNA sequences include those that hybridize under stringent conditions as described in Maniatis et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to 389. Finally, they are also included in the present invention. allelic variations or other variations of the sequences of SEQ ID NO: 1 or 25, whether or not such nucleotide changes result in changes in the peptide sequence but in which the peptide sequence still has similar tissue inducing activity to tendon / ligament.

The DNA sequence of human BMP-12 (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) are set forth in the Sequence Listing. Another protein that is useful for the compositions and methods of the present invention is VL-1. VL-1 is a BMP-12 related protein that was cloned using BMP-12 sequences. The inventors have now named VL-1 under the name BMP-13. A partial DNA sequence of VL-1 (SEQ ID NO: 7) and the encoded amino acid sequence (SEQ ID NO: 8); as well as a DNA sequence encoding mature VL1 (SEQ ID NO: 25) and the encoded amino acid sequence (SEQ ID NO: 26) are set forth in the Sequence Listing. Although additional descriptions are made with reference to the BMP-12 sequence of SEQ ID NOS: 1 and 2, it will be recognized that the invention includes similar modifications and improvements that can be made in other BMP-12 related sequences, such as the sequence. of VL-1 shown in SEQ ID NO: 25 and 26.

The BMP-12 sequence shown in SEQ ID NO: 1 includes the entire mature sequence and approximately 190 amino acids of the propeptide. The coding sequence of the mature human BMP-12 protein appears to begin at nucleotide No. 496 or No. 571 and continues to nucleotide No. 882 of SEQ ID NO: 1. The first cysteine in the seven cysteine structure characteristic of TGF proteins -β begins at nucleotide No. 577. The last cysteine ends at No. 879. Thus, it is expected that DNA sequences encoding active BMP-12 species will comprise nucleotides No. 577 to No. 879 of SEQ ID. Nº: 1.

It is expected that BMP-12, expressed by mammalian cells such as CHO cells, exists as a heterogeneous population of active species of BMP-12 protein with variable N ends. It is expected that all active species contain the amino acid sequence that begins with the rest of cysteine in amino acid # 3 of SEQ ID NO: 2 and continues until at least the rest of cysteine in amino acid 103 or up to the codon of termination after amino acid 104. Other active species contain another amino acid sequence in the N-terminal direction. As further described herein, it is expected that the N-terminus of the active species produced by mammalian cells begins after the appearance of a consensus cleavage site, which encodes an Arg-X-X-Arg peptide sequence. Thus, it is expected that the DNA sequences encoding active BMP-12 proteins have a nucleotide sequence comprising the nucleotide sequence that begins at any of nucleotides # 106, 199, 208, 217, 361, 388, 493, 496 or 571 to nucleotide No. 879 or 882 of SEQ ID NO: 1.

It has been experimentally determined by expression in E. coli that the N-terminus of an active species of human BMP-12 is as follows: [M] SRXSRKPLHVDF, in which X designates an amino acid residue with no obvious signal, which is consistent with a rest of cysteine at that location. Thus, it seems that the N-terminus of this BMP-12 species is at amino acid # 1 of SEQ ID NO: 1, and a DNA sequence encoding said BMP-12 species would begin at nucleotide # 571 of SEQ ID NO: 1. The apparent molecular weight of this species of human BMP-12 dimer, as determined by SDS-PAGE, is approximately 20-22 kd in a 16% Novex tricine gel. Human BMP-12 protein exists as a clear colorless solution in 0.1% trifluoroacetic acid.

As described above, BMP-12-related proteins are a subset of the BMP / TGF-β / Vg-1 family of proteins, including BMP-12 and VL-1, which can be defined as tissue-inducing proteins similar to tendon / ligament encoded by DNA sequences that can be cloned and identified, for example, using PCR, using specific BMP-12 primers, such as primers # 6 and # 7 described below, with reduced stringency conditions. It is preferred that the DNA sequences of the present invention share at least about 80% amino acid level homology with the DNA encoding amino acids No. 3 through No. 103 of SEQ ID NO: 1. For the purposes of the present invention, the expression BMP-12 related proteins does not include the human MP52 protein. Using the sequence information of SEQ ID NO: 1 and SEQ ID NO: 3, and the comparison provided in Figure 1, it is within the skill in the art to design primers for the BMP-12 sequence that allow cloning of genes encoding BMP-12 related proteins.

An example of the BMP-12 related proteins of the present invention is VL-1, currently referred to as BMP-13. The complete mature BMP-13 protein sequence and at least a portion of the BMP-13 propeptide are provided in SEQ ID NO: 25. Like BMP-12, it is expected that BMP-13, expressed by cells of Mammalian such as CHO cells, exists as a heterogeneous population of active species of BMP-13 protein with variable N ends. It is expected that all active species contain the amino acid sequence that begins with the rest of cysteine in amino acid # 19 of SEQ ID NO: 26 and continues to at least the rest of cysteine in amino acid 119 or up to the codon of termination after amino acid 120. Other active species contain an additional amino acid sequence in the N-terminal direction. As further described herein, it is expected that the N-terminus of the active species produced by mammalian cells begins after the appearance of a consensus cleavage site, which encodes an Arg-X-X-Arg peptide sequence. Thus, it is expected that the DNA sequences encoding active BMP-13 proteins have a nucleotide sequence comprising the nucleotide sequence that begins at any of nucleotides # 410, 458, 602, 605 or 659, until nucleotide No. 961 or 964 of SEQ ID NO: 25.

To produce the purified tendon / ligament-like tissue inducing proteins useful for the present invention, a method comprising culturing a host cell transformed with a DNA sequence comprising a suitable coding sequence, particularly the DNA coding sequence from the DNA, is employed. nucleotide No. 496, No. 571 or No. 577 to No. 879 or No. 882 of SEQ ID NO: 1; and recovering and purifying from the culture medium a protein containing the amino acid sequence or a substantially homologous sequence represented by amino acids No. -25, No. 1 or No. 3 to No. 103 or No. 104 of SEQ ID NO: 2. In another embodiment, the method employed comprises culturing a host cell transformed with a DNA sequence comprising a suitable coding sequence, particularly the DNA coding sequence from nucleotide No. 605 or No. 659 to No. 961 or No. 964 of SEQ ID NO. : 25; and recovering and purifying from the culture medium a protein containing the amino acid sequence or a sequence substantially homologous to that represented by amino acids No. 1 or No. 19 to No. 119 or No. 120 of SEQ ID NO: 26

Human MP52 DNA is described in WO93 / 16099. However, this document does not disclose the ability of the protein to form tendon / ligament-like tissue, or its use in compositions for the induction of tendon / ligament-like tissue. Human MP52 was originally isolated using human embryonic tissue RNA. The nucleotide sequence of human MP52 (SEQ ID NO: 3) and the encoded amino acid sequences (SEQ ID NO: 4) are set forth in the Sequence Listing of this document. The MP52 protein appears to start at nucleotide No. 845 of SEQ ID NO: 3 and continues to nucleotide No. 1204 of SEQ No. 3. 3. The first cysteine of the seven cysteine structure characteristic of TGF-β proteins begins at nucleotide No. 899. The last cysteine ends at No. 1201. Other active species of MP52 protein may have additional nucleotides in the N-terminal direction from nucleotide No. 845 of SEQ ID NO: 3.

Purified human MP52 proteins of the present invention can be produced by culturing a host cell transformed with a DNA sequence comprising the DNA coding sequence of SEQ ID NO: 3 from nucleotide No. 845 to No. 1204, and recovering and purifying from culture medium a protein that contains the amino acid sequence or a sequence substantially homologous to that represented by amino acids No. 1 to No. 120 of SEQ ID NO: 4. It is also expected that the amino acid sequence from amino acids No. 17 or No. 19 to No. 119 or No. 120 of SEQ ID NO: 4 retain activity. Thus, it is expected that the DNA sequence of nucleotides No. 845, No. 893 or No. 899 to No. 1201 or No. 1204 encodes active proteins.

For the expression of the protein in mammalian host cells, the host cell is transformed with a coding sequence that encodes a suitable propeptide for protein secretion by the host cell, attached in a reading frame appropriate to the protein coding sequence. mature For example, see US Patent 5,168,050, in which a DNA encoding a precursor part of a mammalian protein other than BMP-2 is fused to DNA encoding a mature BMP-2 protein. Thus, the present invention includes chimeric DNA molecules that comprise a DNA sequence encoding a propeptide of a member of the TGF-β protein superfamily, which is linked in the correct reading frame to a DNA sequence encoding a tendon / ligament-like tissue induction polypeptide. The term "chimeric" is used to refer to the propeptide coming from a different polypeptide than the mature encoded polypeptide. Of course, the host cell can be transformed with a coding DNA sequence encoding the native propeptide linked in the correct reading frame to a coding sequence encoding the mature protein shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 26. The complete sequence of the native propeptide can be determined by methods known in the art using the sequences disclosed in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 25 to design a suitable probe to identify and isolate the entire clone.

The present invention also includes the new DNA sequences, without association with DNA sequences encoding other protein materials, and encoding the expression of tendon / ligament-like tissue-inducing proteins. These DNA sequences include those depicted in SEQ ID NO: 1 in a 5 'to 3' direction and the sequences that hybridize therewith under stringent hybridization conditions [eg, 0.1X SSC, 19% SDS at 65 ºC; see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to 389] and encode a protein that has tendon / ligament-like tissue-inducing activity.

Similarly, DNA sequences encoding proteins encoded by the sequences of SEQ ID NO: 1

or SEQ ID NO: 25, or proteins that comprise the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 26, but which differ in the codon sequence due to degeneracy of the genetic code or allelic variations (changes in natural bases in the population of species that may or may not result in a change in amino acids) also encode the tendon / ligament-like tissue-inducing proteins described herein. Also included in the invention are variations in the DNA sequences of SEQ ID NO: 1 or SEQ ID NO: 25 that are produced by point mutations or induced modifications (including insertion, deletion and substitution) to increase activity, half-life or production of encoded polypeptides.

Another aspect of the present invention provides a new method for producing tissue-like tissue / ligament-like tissue inducing proteins. The process of the present invention involves cultivating a suitable cell line, which has been transformed with a DNA sequence encoding a protein of the invention, under the control of known regulatory sequences. The transformed host cells are cultured and the proteins are recovered and purified from the culture medium. The purified proteins substantially lack other proteins with which they are co-produced as well as other contaminants.

Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary (CHO) cells. As described above, the expression of proteins in mammalian cells requires an appropriate propeptide to ensure protein secretion. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, exploration, product production and purification are known in the art. See, for example, Gething and Sambrook, Nature, 293: 620-625 (1981) or, alternatively, Kaufman et al, Mol. Cell Biol., 5 (7): 1750-1759 (1985) or Howley et al, US Pat.

4,419,446. Another suitable mammalian cell line, which is described in the accompanying examples, is the monkey COS-1 cell line. The mammalian CV-1 cell may also be suitable.

Suitable bacterial cells may also be suitable hosts. For example, the various strains of E. coli (for example, HB101, MC1061) are well known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like can also be used in this procedure. For the expression of the protein in bacterial cells, DNA encoding a propeptide is not necessary.

It is known that expression in bacteria of mammalian proteins, including members of the TGF-β family, produces the proteins in a non-glycosylated form, and in the form of insoluble granules, known as inclusion bodies. Techniques in this field have been described to solubilize these inclusion bodies, denature the protein using a chaotropic agent and replicate the protein in a sufficiently correct manner to allow its production in a soluble form. For example, see EP 0433225.

Alternatively, methods have been devised that prevent the formation of inclusion bodies, such as the expression of gene fusion proteins, in which the desired protein is expressed as a fusion protein with a fusion partner. The fusion protein is subsequently excised to produce the desired protein. An example of such a gene fusion expression system for E. coli is based on the use of the E. coli thioredoxin gene as a fusion partner, La Vallie et al., Bio / Technology, 11: 187-193 (1993 ).

Many strains of yeast cells known to those skilled in the art may also be available as host cells for the expression of the polypeptides of the present invention. In addition, when desired, insect cells can be used as host cells in the process of the present invention. See, for example, Miller et al., Genetic Engineering, 8: 277-298 (Plenum Press 1986) and references cited therein.

Another aspect of the present invention provides vectors for use in the method of expression of these tendon / ligament-like tissue inducing proteins. Preferably, the vectors contain the complete new DNA sequences described above that encode the new factors of the invention. In addition, the vectors contain appropriate expression control sequences that allow the expression of the protein sequences. Alternatively, embodiments of the present invention are vectors that incorporate modified sequences as described above. In addition, the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 25 could be manipulated to express a mature protein by deleting propeptide sequences and replacing them with sequences encoding the complete propeptides of BMP proteins or members of the TGF-β superfamily. Thus, the present invention includes chimeric DNA molecules that encode a propeptide of a member of the TGF-β superfamily attached in the correct reading frame to a DNA sequence encoding a protein having the amino acid sequence of the SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 26. The vectors may be used in the cell line transformation process and contain selected regulatory sequences in the operative association with the DNA coding sequences of the invention. which are capable of directing their replication and expression in selected host cells. Those skilled in the art know regulatory sequences for said vectors and can be selected depending on the host cells. Such selection is routine and not part of the present invention.

A protein of the present invention that induces tendon / ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon defects or ligaments in humans and other animals. Said preparation employing a tendon / ligament-like tissue-inducing protein may have prophylactic use in preventing injuries to tendon or ligament tissue, as well as use in improving tendon or ligament fixation to bone or other tissues, and in the repair of tendon tissue or ligament defects. Tendon-like tissue formation / de novo-induced ligation induced by a composition of the present invention contributes to the repair of congenital defects, induced by trauma or other tendon or ligament defects of another origin, and is also useful in cosmetic plastic surgery. for the union or repair of tendons or ligaments. The compositions of the invention may also be useful in the treatment of tendonitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions of the present invention can also be used in other indications in which it is desirable to cure or regenerate tendon tissue and / or ligament. These indications include, without limitation, the regeneration or repair of lesions in the periodontal ligament, such as those that occur in tendonitis, and the regeneration or repair of the tendon attachment to the bone. The compositions of the present invention may provide a means to bind tendon or ligament forming cells, stimulate the growth of tendon or ligament forming cells or induce differentiation of tendon or ligament forming cell progenitors.

BMP-12 related proteins can be recovered from the culture medium and purified by isolation from other protein materials with which they are co-produced and from other contaminants present. The proteins of the present invention can induce the formation of tendon / ligament-like tissue. These proteins can be further characterized by the ability to demonstrate tendon / ligament-like tissue formation activity in the rat ectopic implant assay described below. It is contemplated that these proteins may also have the ability to induce the formation of other types of tissue, such as ligaments.

Tendon / ligament-like tissue inducing proteins provided herein also include factors encoded by sequences similar to those of SEQ ID NO: 1 or SEQ ID NO: 25, but in which modifications are naturally provided (eg. , allelic variations in the nucleotide sequence that can result in amino acid changes in the polypeptide) or deliberately modified by genetic engineering. For example, synthetic polypeptides can totally or partially duplicate continuous sequences of amino acid residues of SEQ ID NO: 2. These sequences, because they share conformational characteristics and of primary, secondary or tertiary structure with growth factor polypeptides of Tendon / ligament-like tissue of SEQ ID NO: 2, may possess biological properties of tendon / ligament-like tissue growth factor or other tissue in common therewith. In this way, they can be used as biologically active substitutes for natural tendon / ligament-like tissue-inducing polypeptides in therapeutic compositions and procedures.

Other specific mutations of the tendon / ligament-like tissue inducing protein sequences described herein involve modifications of glycosylation sites. These modifications may involve glycosylation sites linked to O or linked to N. For example, the absence of glycosylation or only partial glycosylation results from the substitution or deletion of amino acids at glycosylation recognition sites linked to asparagine. Asparagine-linked glycosylation recognition sites comprise tripeptide sequences that are specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences can be asparagine-X-threonine, asparagine-X-serine or asparagine-X-cysteine, where X is normally any amino acid except proline. A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and / or amino acid deletion in the second position) results in an absence of glycosylation in the sequence modified tripeptide. In addition, bacterial protein expression will also result in the production of a non-glycosylated protein, although glycosylation sites are left unmodified.

The compositions of the present invention comprise a purified BMP-12 related protein that can be produced by culturing a cell transformed with the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 25 and recovery and purification of the protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 26 from the culture medium. The purified expressed protein lacks substantially other protein materials with which it is produced together, as well as other contaminants. It is contemplated that the recovered purified protein exhibits tendon / ligament-like tissue formation activity and other tissue growth activity, such as ligament regeneration. The proteins of the invention can be further characterized by the ability to demonstrate tendon / ligament-like tissue formation activity in the rat assay described below.

Compositions for inducing tendon / ligament-like tissue formation of the present invention may comprise an effective amount of a tendon / ligament-like tissue-inducing protein, wherein said protein comprises the amino acid sequence of SEQ ID NO: 2 , preferably amino acids No. -25, No. 1 or No. 3 to No. 103 or No. 104 of SEQ ID NO: 2; or amino acids No. 1 or No. 19 to No. 120 of SEQ ID NO: 26; as well as mutants and / or variants of SEQ ID NO: 2 or SEQ ID NO: 26, which has the ability to form tendon-like tissue and / or ligament.

The compositions of the present invention may further comprise additional proteins, such as additional members of the TGF-β protein superfamily, such as activins. Another aspect of the invention provides pharmaceutical compositions containing a therapeutically effective amount of a tendon / ligament inducing protein, such as BMP-12 or VL-1, in a pharmaceutically acceptable carrier or excipient. These compositions can be used to induce the formation of tendon / ligament-like tissue or other tissue. It is contemplated that such compositions can also be used for tendon and ligament repair, wound healing and repair of other tissues, such as skin repair. It is also contemplated that the proteins of the invention can increase neuronal survival and, therefore, be useful in transplantation and treatment of conditions that exhibit a reduction in neuronal survival. The compositions of the invention may further include at least one other therapeutically useful agent, such as BMP BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7 proteins, disclosed, for example, in US Patents 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT publication WO91 / 18098; BMP-9, disclosed in PCT publication WO93 / 00432; and BMP-10 or BMP-11, disclosed in the pending patent applications together with this one with the serial number 08 / 061.695 and 08 / 061.464, filed on May 12, 1993.

The compositions of the invention may comprise, in addition to tendon / ligament inducing protein such as BMP-12 or VL-1 (BMP-13), other therapeutically useful agents including MP52, epidermal growth factor (EGF), growth factor of fibroblasts (FGF), platelet-derived growth factor (PDGF), transformation growth factors (TGF-α and TGF-β) and fibroblast-4 growth factor (FGF-4), parathyroid hormone (PTH), leukemia inhibitor factor (LIF / HILDA / DIA), insulin-like growth factors (IGF-1 and IGF-II). Parts of these agents can also be used in the compositions of the present invention. For example, a composition comprising both BMP-2 and BMP-12 implanted together results in bone-like and tendon / ligament-like tissue. Such a composition may be useful for treating embryonic joint defects where tendon, ligaments and bone are formed simultaneously in contiguous anatomical locations, and may be useful for regenerating tissue at the site of tendon attachment to bone. It is contemplated that the compositions of the invention can also be used in wound healing, such as in skin healing and related tissue repair. Types of wounds include, but are not limited to, burns, incisions and ulcers. (See, for example, in PCT Publication WO84 / 0110, the analysis of wound healing and related tissue repair).

It is expected that the proteins of the invention can act in conjunction with, or perhaps synergistically, with other proteins and related growth factors. Therefore, other therapeutic methods and compositions of the invention comprise a therapeutic amount of at least one protein of the invention with a therapeutic amount of at least one of the BMP proteins described above. Said compositions may comprise molecules separated from BMP proteins or heteromolecules composed of different BMP residues. For example, a method and composition of the invention may comprise a dimer linked by disulfide bonds comprising a BMP-12 related protein subunit and a subunit of one of the "BMP" proteins described above. Thus, the present invention includes compositions comprising a purified BMP-12 related polypeptide is a heterodimer in which a subunit comprises the amino acid sequence from amino acid No. 1 to amino acid No. 104 of SEQ ID NO: 2, and Another subunit comprises an amino acid sequence for a bone morphogenetic protein selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, 2. BMP-9, BMP-10 and BMP-11. Another embodiment may comprise a heterodimer of tendon / ligament-like tissue-inducing moieties linked by disulfide bonds such as BMP-12, VL-1 (BMP-13) or MP52. For example, the heterodimer may comprise a subunit comprising an amino acid sequence from No. 1 to No. 104 of SEQ ID NO: 2 and the other subunit may comprise an amino acid sequence from No. 1 to No. 120 of SEQ ID. No.: 4 or from No. 1 to No. 120 of SEQ ID NO: 26. In addition, compositions of the present invention may be combined with other agents beneficial for the treatment of the defect, wound or tissue in question.

The preparation and formulation of said physiologically acceptable protein compositions, duly considering pH, isotonicity, stability and the like, is within the skill of the art. The therapeutic compositions are also currently valuable for veterinary applications due to the absence of species specificity in TGF-β proteins. Particularly, domestic animals and thoroughbred horses in addition to humans are desired patients for such treatment with the compositions of the present invention.

The therapeutic procedure includes administering the composition topically, systemically or locally as an implant or device. When administered, the therapeutic composition for use in the present invention, of course, is in a physiologically acceptable form without pyrogens. In addition, the composition may be desirably encapsulated or injected into a viscous form to be delivered at the site of tissue injury. Topical administration may be suitable for wound healing and tissue repair. Certain therapeutically useful agents other than proteins that may also be optionally included in the composition as described above, may be administered, alternatively or additionally, simultaneously or sequentially with the composition in the methods of the invention.

The compositions may also include an appropriate matrix and / or sequestering agent as an excipient. For example, the matrix can support the composition or provide a surface for tendon / ligament-like tissue formation and / or other tissue formation. The matrix can provide the slow release of the protein and / or the appropriate environment for its presentation. The sequestering agent may be a substance that helps facilitate administration by injection or other means, or may slow down the migration of the protein from the application site.

The choice of an excipient material is based on the properties of biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions can be biodegradable and chemically defined. Other matrices are made up of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined. Preferred matrices include collagen-based materials such as Helistat sponge (Integra LifeSciences, Plainsboro, N.J.), or collagen in an injectable form, as well as sequestering agents, which can also be biodegradable and may include alkylcellulose materials.

Another preferred class of vehicles are porous polymeric matrices in the form of particles, which include poly (lactic acid), poly (glycolic acid) polymers and copolymers of lactic acid and glycolic acid. These matrices can also include a sequestering agent. Suitable polymer matrices are described, for example, in WO 93/00050.

A preferred family of sequestering agents is that of cellulosic materials such as alkyl celluloses (including hydroxyalkyl celluloses), including methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose and carboxymethyl cellulose, the most preferred cationic salts of carboxymethyl cellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly (ethylene glycol), poly (oxyethylene oxide), carboxyvinyl polymer and polyvinyl alcohol. The amount of sequestering agent useful herein is 0.5-20% by weight, preferably 1-10% by weight with respect to the total weight of the formulation, which represents the amount necessary to prevent protein desorption. of the polymer matrix and allow for proper manipulation of the composition, but not so large as to prevent the progenitor cells from infiltrating the matrix, thus providing the protein with the opportunity to help the activity of the progenitor cells.

Other optional components useful in the practice of the present application include, for example, cryogenic protectors such as mannitol, sucrose, lactose, glucose or glycine (to protect the protein from degradation during lyophilization), antimicrobial preservatives such as methyl and propyl parabens and benzyl alcohol; antioxidants such as EDTA, citrate and BHT (butylated hydroxytoluene); and surfactants such as poly (sorbates) and poly (oxyethylenes), etc.

As described above, the compositions of the invention can be employed in methods for treating various tendon defects, such as for regeneration of tendon / ligament-like tissue in areas of tendon or ligament injury, to help repair tissue tears. of tendon, ligaments and other various types of defects or wounds in tissues. These methods, according to the invention, involve administering to a patient in need of said tendon / ligament or other tissue-like tissue repair, of a composition comprising an effective amount of a tendon / ligament-like tissue-inducing protein, such as that described in SEQ ID NO: 2, SEQ ID NO: 4 and / or SEQ ID NO: 26. These procedures may also involve the administration of a tendon / ligament-like tissue-inducing protein along with at least one of BMP proteins described above.

In another embodiment, the methods may involve the administration of a heterodimeric protein in which one of the monomers is a tendon / ligament-like tissue induction polypeptide, such as BMP-12, VL-1 (BMP-13) or MP52 , and the second monomer is a member of the TGF-β growth factor superfamily. In addition, these procedures may also include the administration of a tendon / ligament-like tissue-inducing protein with other growth factors that include EGF, FGF, TGF-α, TGF-β and IGF.

5

fifteen

25

35

Four. Five

Thus, another aspect of the invention is a therapeutic method and composition for repairing tendon / ligament-like tissue, for repairing tendons or ligaments, as well as for treating tendonitis and other conditions related to tendon or ligament defects. Such compositions comprise a therapeutically effective amount of one or more tendon / ligament-like tissue inducing proteins, such as BMP-12, a BMP-12-related protein, or MP52, mixed with a pharmaceutically acceptable carrier, excipient or matrix.

The dosage regimen will be determined by the doctor in charge of the case considering various factors that modify the action of the composition, for example, the amount of tendon or ligament tissue that is desired to form, the site of the tendon or ligament injury, the condition of the damaged tendon or ligament, the size of the wound, the type of damaged tissue, the age, sex and diet of the patient, the severity of any infection, the timing of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and the types of additional proteins in the composition. The addition of other known growth factors, such as IGF-I (insulin-like growth factor I), to the final composition may also affect the dosage.

Progress can be monitored by periodic evaluation of tendon / ligament-like tissue formation, or tendon or ligament growth and / or repair. Progress can be monitored by procedures known in the art, for example, X-rays, arthroscopy, histomorphometric determinations and tetracycline labeling.

The following examples illustrate the practice of the present invention in the recovery and characterization of human tendon / ligament-like tissue-inducing proteins and their use to recover the other tendon / ligament-like tissue-inducing proteins, obtain human proteins, express the proteins by recombinant techniques and demonstrate the ability of the compositions of the present invention to form tendon / ligament-like tissue in an in vivo model. Although the examples demonstrate the invention with respect to BMP12, with minor modifications within the skill in the art, it is believed that the same results can be obtained with MP52 and VL-1

Example 1

DNA isolation

DNA sequences encoding BMP-12 and BMP-12 related proteins can be isolated by various techniques known to those skilled in the art. As described below, oligonucleotide primers can be designed based on amino acid sequences present in other BMP proteins, Vg-1-related proteins and other TGF-β superfamily proteins. Regions containing amino acid sequences that are highly conserved within the BMP family of proteins and within other members of the TGF-β 'superfamily can be identified and consensus amino acid sequences can be constructed from these highly conserved regions based on the similarity of the corresponding regions of individual BMP / TGFβ / Vg-1 proteins. An example of said consensus amino acid sequence is indicated below.

Consensus amino acid sequence (1):

Trp-Gln / Asn-Asp-Trp-Ile-Val / Ile-Ala (SEQ ID NO: 16)

In which X / Y indicates that any amino acid residue may appear in that position.

The following oligonucleotide is designed based on the consensus amino acid sequence identified above (1):

No. 1: CGGATCCTGGVANGAYTGGATHRTNGC (SEQ ID NO: 17)

This oligonucleotide sequence is synthesized in an automatic DNA synthesizer. Conventional nucleotide symbols in the oligonucleotide primer identified above are as follows: A, adenosine; C, cytosine; G, guanine; T, thymine; N, adenosine or cytosine or guanine or thymine; R, adenosine or cytosine; And, cytosine or thymine; H, adenosine or cytosine or thymine; V, adenosine or cytosine or guanine; D, adenosine or guanine or thymine.

The first seven nucleotides of oligonucleotide # 1 (underlined) contain the recognition sequence for the BamHI restriction endonuclease to facilitate the manipulation of a specifically amplified DNA sequence encoding the BMP-12 protein and, therefore, do not originate from the consensus amino acid sequence (1) presented above.

A second consensus amino acid sequence comes from another highly conserved region of BMP / TGF-β / Vg-1 proteins as described below:

His-Ala-Ile-Val / Leu-Gln-Thr (SEQ ID NO: 18)

The following oligonucleotide is designed based on the consensus amino acid sequence identified above (2): # 2: TTTCTAGAARNGTYTGNACDATNGCRTG (SEQ ID NO: 19)

This oligonucleotide sequence is synthesized in an automatic DNA synthesizer. The same nucleotide symbols as described above are used.

The first seven nucleotides of oligonucleotide # 1 (underlined) contain the recognition sequence for the Xbal restriction endonuclease to facilitate the manipulation of a specifically amplified DNA sequence encoding the BMP-12 protein and, therefore, do not originate from the consensus amino acid sequence (2) presented above.

It is contemplated that the BMP-12 protein of the invention and other BMP / TGF-β / Vg-1 related proteins may contain amino acid sequences similar to the consensus amino acid sequences described above and that the location of this sequence within a protein BMP-12 or other new related proteins would correspond to the relative locations in the proteins from which they were obtained. It is also contemplated that this positional information derived from the structure of other BMP / TGF-β / Vg-1 proteins and oligonucleotide sequences # 1 and # 2 that have been obtained from the consensus amino acid sequences

(1) and (2) respectively, could be used to specifically amplify DNA sequences encoding the corresponding amino acids of a BMP-12 protein or other BMP / TGF-β / Vg-1 related proteins.

Based on the knowledge of BMP / TGF-β / Vg-1 protein gene structures, it is further contemplated that human genomic DNA can be used as a template to perform specific amplification reactions that would result in the identification of BMP-12 coding sequences. HMB / TGF-β / Vg-1 (BMP-12 related protein). Such specific amplification reactions of a human genomic DNA template could be initiated with the use of oligonucleotide primers # 1 and # 2 described above. Oligonucleotides # 1 and # 2 identified above are used as primers to allow specific amplification of a specific nucleotide sequence of human genomic DNA. The amplification reaction is performed as follows:

Human genomic DNA is cut (source: peripheral blood lymphocytes), provided by Ken Jacobs of the Genetics Institute, by repeated passage through a 25 gauge needle, denatured at 100 ° C for 5 minutes and then cooled on ice before if added to a reaction mixture containing a 200 µM concentration of each deoxynucleotide triphosphate (dATP, dGTP, dCTP and dTTP), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, gelatin at 0.001%, 1.25 units of Taq DNA polymerase, a 100 pM concentration of oligonucleotide # 1 and a 100 pM concentration of oligonucleotide # 2. This reaction mixture is incubated at 94 ° C for two minutes and then subjected to thermal cycles of as follows: 1 minute at 94 ° C, 1 minute at 40 ° C, 1 minute at 72 ° C for three cycles; then 1 minute at 94 ° C, 1 minute at 55 ° C, 1 minute at 72 ° C for thirty-seven cycles, followed by a 10 minute incubation at 72 ° C.

The DNA that is specifically amplified by this reaction is precipitated with ethanol, digested with the restriction endonucleases BamHI and XbaI and subjected to agarose gel electrophoresis. A region of the gel is cleaved, corresponding to the expected size of the BMP-12 or other DNA fragment encoding BMP / TGF-β / Vg-1, and the specifically contained amplified DNA fragments are electroeluted and subcloned into the vector plasmid pGEM-3 between the XbaI and BamHI sites of the polylinker. Analysis of the DNA sequence of one of the resulting BMP-12 related subclones indicates that the amplified DNA sequence specifically of the contained product encodes a portion of the BMP-12 protein of the invention.

The DNA sequence (SEQ ID NO: 5) and the amino acid sequence derived (SEQ ID NO: 6) of this specifically amplified DNA fragment from BMP-12 are shown in the SEQUENCE List.

Nucleotides No. 1-No. 26 of SEQ ID NO: 5 comprise a part of oligonucleotide No. 1 and nucleotides No. 103

- No. 128 comprise a part of the reverse complement of oligonucleotide No. 2 used to perform the specific amplification reaction. Due to the function of oligonucleotides # 1 and # 2 to initiate the amplification reaction, they may not correspond exactly to the actual sequence encoding a BMP-12 protein and, therefore, do not translate into amino acid derivation. corresponding (SEQ ID NO: 6).

Analysis of the DNA sequence of another subclone indicates that the amplified DNA product specifically contained therein encodes a part of another BMP / TGF-β / Vg1 protein (related to BMP-12) of the invention called VL-1.

The DNA sequence (SEQ ID NO: 7) and the amino acid sequence derived (SEQ ID NO: 8) of this specifically amplified DNA fragment are shown in the Sequence Listing.

Nucleotides No. 1 - No. 26 of SEQ ID NO: 7 comprise a part of oligonucleotide No. 1 and nucleotides No. 103 - No. 128 comprise a part of the reverse complement of oligonucleotide No. 2 used to perform the specific amplification reaction. Due to the role of oligonucleotides # 1 and # 2 in the initiation of the amplification reaction, they may not correspond exactly to the actual sequence encoding a VL-1 protein of the invention and, therefore, do not translate in the corresponding amino acid derivation (SEQ ID NO: 8).

The following oligonucleotide probe is designed based on the amplified BMP-12 human DNA sequence specifically set forth above (SEQ ID NO: 5) and synthesized in an automatic DNA synthesizer:

No. 3: CCACTGCGAGGGCCTTTGCGACTTCCCTTTGCGTTCGCAC (SEQ ID NO: 20)

This oligonucleotide probe is radiolabelled with 32P and is used to explore a human genomic library constructed in the λFIX vector (Stratagene, catalog no. 944201). 500,000 recombinants from the human genomic library are grown at a density of approximately 10,000 recombinants per plate in 50 plates. Duplicate nitrocellulose replicates of recombinant bacteriophage plates and hybridized with oligonucleotide probe No. 3 in conventional hybridization buffer (SHB = 5X SSC, 0.1% SDS, Denhardt 5X, 100 µg / ml salmon sperm DNA) they are left at 65 ° C overnight. The next day, the hybridization solution containing radioactively labeled oligonucleotide is removed and the filters are washed with 0.2X SSC, 0.1% SDS at 65 ° C. A single positive hybridization recombinant is identified and plaque purified. This plate purified recombinant bacteriophage clone that hybridizes with the BMP-12 oligonucleotide probe No. 3 is called λHuG-48. A stock solution is prepared in bacteriophage plate and bacteriophage DNA is isolated from the human genomic λHuG-48. The bacteriophage λHuG-48 has been deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD “ATCC” with accession number 75625 on December 7, 1993. This deposit meets the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and Regulations set forth therein. The oligonucleotide hybridization region of this recombinant, λHuG-48, is located in a 3.2 kb BamHI fragment. This fragment is subcloned into a plasmid vector (pGEM-3) and DNA sequence analysis is performed. This plasmid subclone is called PCR1-1 # 2 and has been deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD "ATCC" with accession number 69517 on December 7, 1993. This deposit meets the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of the Patent Procedure and the Regulations set forth therein. The partial DNA sequence (SEQ ID NO: 1) and the amino acid sequence derived (SEQ ID NO: 2) from the 3.2 kb DNA insert of the PCR1-1 # 2 plasmid subclone, derived from the λHuG-48 clone, are shown in the Sequence List.

It should be noted that nucleotides No. 639 - No. 714 of SEQ ID NO: 1 correspond to nucleotides No. 27 - No. 102 of the DNA fragment encoding amplified BMP-12 specifically set forth in SEQ ID NO: 5, thereby confirming that the human genomic bacteriophage clone λHuG-48 and the subclone derived PCR1-1 # 2 encode at least a portion of the BMP-12 protein of the invention. The nucleotide sequence of a part of the 3.2 kb BamHI insert of plasmid PCR1-1 # 2 contains an open reading frame of at least 882 base pairs, as defined by nucleotides No. 1-No. 882 of the SEC ID NO: 1. This open reading frame encodes at least 294 amino acids of the human BMP-12 protein of the invention. The 294 amino acid human BMP-12 protein encoded includes the complete mature human BMP-12 protein (amino acids No. 1-No. 104 of SEQ ID NO: 2) as well as the C-terminal part of the propeptide region of the translation product primary (amino acid No. -190 year -1 of ECS ID No.: 2).

Another DNA sequence of the 3.2 kb BamHI insert of plasmid PCR1-1 # 2 set forth in SEQ ID NO: 33 demonstrates the presence of an open reading frame of 1164 bp, as defined by nucleotides No. 138 to No. 1301 of SEQ ID No. 33. [NOTE that the entire sequence disclosed in SEQ ID NO: 1 is contained within SEQ ID NO: 33]. Since this sequence comes from a genomic clone, it is difficult to determine the boundary between the 5 ’end of the coding sequence and the 3’ border of the intermediate sequence (intron / non-coding sequence).

Based on the knowledge of other BMP proteins and other proteins within the TGF-β family, it is anticipated that the precursor polypeptide would be cleaved into the mutibasic sequence Arg-Arg-Gly-Arg according to a proposed consensus proteolytic processing sequence of Arg-XX-Arg. It is expected that cleavage of the BMP-12 precursor polypeptide will generate a mature 104 amino acid peptide that begins with the amino acid Ser at position # 1 of SEQ ID NO: 2. Processing of BMP-12 in the mature form involves dimerization and elimination of the N-terminal region in a manner analogous to the processing of the TGF-β related protein [Gentry et al., Molec & Cell. Biol., 8: 4162 (1988); Derynck et al. Nature, 316: 701 (1985)].

Therefore, it is contemplated that the mature active species of BMP-12 comprises a homodimer of two polypeptide subunits, each subunit comprising amino acids No. 1 to No. 104 of SEQ ID NO: 2 with an expected molecular weight of approximately 12,000 daltons. Other active species comprising at least amino acids # 3 to # 103 of SEQ ID NO: 2 are contemplated, thus including the first and last conserved cysteine residue. As with other members of the TGF-β / BMP family of proteins, the carboxy-terminal part of the BMP-12 protein has a greater sequence conservation than the part closest to the amino terminus. The percentage of amino acid identity of human BMP-12 protein in the C-terminal domain rich in cysteine (amino acids # 3 - # 104) with the corresponding region of human BMP proteins and other proteins within the TGF-β family It is as follows: BMP-2, 55%; BMP-3, 43%; BMP-4, 53%; BMP-5, 49%; BMP-6, 49%; BMP-7, 50%; BMP-8, 57%; BMP-9, 48%; BMP-10, 57%; WC activin (BMP-11), 38%; Vgl, 46%; GDF-1, 47%; TGF-β1, 36%; TGF-β2, 36%; TGF-β3, 39%; β (B) inhibin, 36%; β (A) inhibin, 41%.

The human BMP-12 DNA sequence (SEQ ID NO: 1), or a part thereof, can be used as a probe to identify a human cell line or tissue that synthesizes BMP-12 mRNA. In summary, RNA is extracted from a selected cellular or tissue source and electrophoresed on an agarose-formaldehyde gel and transferred to nitrocellulose, or reacted with formaldehyde and applied promptly on nitrocellulose directly. The nitrocellulose is then hybridized with a probe derived from the human BMP-12 coding sequence.

Alternatively, the human BMP-12 sequence is used to design oligonucleotide primers that will specifically amplify a part of the BMP-12 coding sequence located in the region between the primers used to perform the specific amplification reaction. It is contemplated that these primers derived from human BMP-12 would specifically amplify corresponding BMP-12 coding sequences from mRNA, cDNA or genomic DNA templates. Once a positive source has been identified by one of the procedures described above, mRNA is selected by oligo (dT) cellulose chromatography and cDNA is synthesized and cloned into λgt10 or other λ bacteriophage vectors known to those skilled in the art , for example, λZAP by established techniques (Toole et al., mentioned above). It is also possible to perform the amplification reaction directed to oligonucleotide primer, described above, directly in a pre-established human cDNA or genomic library that has been cloned into a bacteriophage vector λ. In these cases, a library that produces a specifically amplified DNA product that encodes a portion of the human BMP-12 protein could be directly screened, using the DNA fragment encoding amplified BMP-12 as a probe.

It is predicted that oligonucleotide primers designed based on the DNA sequence of the human BMP-12 genomic clone λHuG-48 allow specific amplification of DNA sequences encoding human BMP-12 from pre-established human cDNA libraries that are available in the market (i.e. Stratagene, La Jolla, CA or Clontech Laboratories, Inc., Palo Alto, CA). The following oligonucleotide primer is designed based on nucleotides No. 571 to No. 590 of the DNA sequence set forth in SEQ ID NO: 1 and is synthesized in an automatic DNA synthesizer:

nº 4: TGCGGATCCAGCCGCTGCAGCCGCAAGCC (SEQ ID NO: 21)

The first nine nucleotides of primer # 4 (underlined) comprise the recognition sequence for the BamHI restriction endonuclease that can be used to facilitate the manipulation of a specifically amplified DNA sequence encoding the human BMP-12 protein of the invention and, by therefore, they are not derived from the DNA sequence presented in SEQ ID NO: 1. The following oligonucleotide primer is designed based on nucleotides # 866 - # 885 of the DNA sequence set forth in SEQ ID N: 1 and is synthesized in an automatic DNA synthesizer.

No. 5 GACTCTAGACTACCTGCAGCCGCAGGCCT (SEQ ID NO: 22)

The first nine nucleotides of primer # 5 (underlined) comprise the recognition sequence for restriction endonuclease XbaI that can be used to facilitate the manipulation of a specifically amplified DNA sequence encoding the human BMP-12 protein of the invention and, by therefore, they do not come from the DNA sequence presented in SEQ ID NO: 1.

Conventional nucleotide symbols in the primers identified above are the following: A, adenine; C, cytosine; G, guanine; T, thymine.

Primers # 4 and # 5 identified above are used as primers to allow amplification of a nucleotide sequence encoding specific BMP-12 from pre-established cDNA libraries that may include the following: human fetal brain cDNA / λZAPII (Stratagene , catalog no. 936206), human liver / λUNI-ZAP XR (Stratagene, catalog no. 937200), human lung / λUNI-ZAP XR (Stratagene, catalog no. 937206) and human fetal spleen / UNI-ZAP XR (Stratagene, Catalog No. 937205).

Approximately 1 x 108 pfu (plaque forming units) of λ bacteriophage libraries containing human cDNA inserts such as those detailed above are denatured at 95 ° C for five minutes before adding to a reaction mixture containing 200 µM of each deoxynucleotide triphosphate (dATP, dGTP, dCTP and dTTP), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 1.25 units of Taq DNA polymerase, a concentration 100 pM of oligonucleotide primer No. 4 and a 100 pM concentration of oligonucleotide primer No. 5. The reaction mixture is then subjected to term cycles as follows: 1 minute at 94 ° C, 1 minute at 50 ° C, 1 minute at 72 ° C for thirty-nine cycles, followed by 10 minutes at 72 ° C.

It would be expected that the DNA that is specifically amplified by this reaction will generate a BMP-12 coding product of approximately 333 base pairs, whose internal 315 bp correspond to nucleotides No. 571 to No. 885 of SEQ ID NO: 1 and that they also include 9 bp at each end of the specific BMP-12 fragment corresponding to the restriction sites defined by nucleotides # 1 - # 9 of primers # 4 and # 5. The resulting 333 bp DNA product is digested with Restriction endonucleases BamHI and Xbal, extracted with phenol, extracted with chloroform and precipitated with ethanol.

As an alternative to ethanol precipitation, buffer exchange and extraction of small DNA fragments resulting from restriction digestion with BamHI / XbaI is performed by dilution of the digested DNA product in 10 mM Tris-HCl, pH 8.0, EDTA 1 mM followed by centrifugation through a Centricon ™ 30 microconcentrator (WR Grace & Co., Beverly, MA; product no. 4209). The resulting amplified DNA product digested with BamHI / Xbal is subcloned into a plasmid vector (ie, pBluescript, pGEM-3 etc.) between the BamHI and XbaI sites of the polylinker region. Analysis of the DNA sequence of the resulting subclones would be required to confirm the integrity of the BMP-12 coding insert. Once a positive cDNA source has been identified in this way, the corresponding cDNA library from which a specific BMP-12 sequence of 333 bp with the 333 bp insert or other specific probes could be directly scanned BMP-12 to identify and isolate cDNA clones encoding the full length BMP-12 protein of the invention.

Other methods known to those skilled in the art can be used to isolate other full length cDNAs encoding human BMP-12 related proteins, or full length cDNA clones encoding BMP-12 related proteins of the invention from species other than humans, particularly other mammalian species.

The following examples demonstrate the use of the human BMP-12 sequence to isolate BMP-12-related protein homologs in a murine genomic DNA library.

It is predicted that the DNA sequence encoding the human BMP-12 protein of the invention is significantly homologous to BMP-12 and BMP-12 related sequences of species other than humans, such that it could be used to specifically amplify sequences of DNA from those other species that encode the corresponding BMP-12 related proteins. Specifically, the following oligonucleotides are designed based on the human BMP-12 sequence (SEQ ID NO: 1) and synthesized in an automatic DNA synthesizer:

No. 6: GCGGATCCAAGGAGCTCGGCTGGGACGA (SEQ ID NO: 23)

No. 7: GGAATTCCCCACCACCATGTCCTCGTAT (SEQ ID NO: 24)

The first eight nucleotides of oligonucleotide primers # 6 and # 7 (underlined) comprise the recognition sequence for restriction endonucleases BamHI and EcoRI, respectively. These sequences are used to facilitate the manipulation of an amplified DNA sequence specifically encoding BMP-12 or a BMP-12 related protein from a species other than human and, therefore, not derived from the DNA sequence presented in SEQ ID NO: 1. Oligonucleotide primer No. 6 is designed based on nucleotides No. 607-No. 626 of SEQ ID NO: 1. Oligonucleotide primer No. 7 is designed based on the reverse complement of nucleotides No. 846-no. 865 of the DNA sequence set forth in SEQ ID NO: 1.

Oligonucleotide primers # 6 and # 7 identified above are used as primers to allow amplification of specific BMP-12 related sequences from genomic DNA derived from species other than humans. The amplification reaction is performed as follows:

Genomic DNA is cut (source: Balb c strain) by repeated passage through a 25 gauge needle, denatured at 100 ° C for five minutes and then cooled on ice before adding to a reaction mixture containing a concentration 200 µM of each deoxynucleotide triphosphate (dATP, dGTP, dCTP and dTTP), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 1.25 units of Taq DNA polymerase , a 100 pM concentration of oligonucleotide primer No. 6 and a 100 pM concentration of oligonucleotide primer No. 7. Then, the reaction mixture is subjected to thermal cycles as follows: 1 minute at 95 ° C, 1 minute at 55 ° C, 1 minute at 72 ° C for forty cycles, followed by 10 minutes at 72 ° C.

The DNA that is specifically amplified by this reaction is precipitated with ethanol, digested with the restriction endonucleases BamHI and EcoRI and subjected to agarose gel electrophoresis. A region of the gel, which corresponds to the expected size of the DNA fragment encoding murine BMP-12 or a BMP-12 related protein, is cleaved and the amplified DNA fragments specifically contained in the gel are extracted (by electroelution or by others procedures known to those skilled in the art) and is subcloned into a plasmid vector, such as pGEM-3 or pBluescript between the BamHI and EcoRI sites of the polylinker. Analysis of the DNA sequence of one of the resulting subclones, called mV1, indicates that the amplified DNA sequence specifically contained therein encodes a part of a protein that appears to be the murine homologue of the BMP-12 or VL sequence. -1 of the invention. The DNA sequence (SEQ ID NO: 10) and the amino acid sequence derived (SEQ ID NO: 11) of this specifically amplified murine DNA fragment are shown in the sequence listing.

Nucleotides No. 1-No. 26 of SEQ ID NO: 10 comprise an oligonucleotide part. Nucleotide No. 6 and nucleotides No. 246-No. 272 comprise a part of the reverse complement of oligonucleotide No. 7 used to perform the specific amplification reaction. Nucleotide No. 27 of SEQ ID NO: 10 appears to be the last nucleotide of a codon triplet, and nucleotides No. 244-No. 245 of SEQ ID NO: 10 appear to be the first two nucleotides of a codon triplet. Therefore, nucleotides No. 28 to No. 243 of SEQ ID NO: 10 correspond to a partial coding sequence of mV1. Due to the role of oligonucleotides # 6 and # 7 in the initiation of the amplification reaction, they may not correspond exactly to the actual sequence encoding the murine homologue to the human BMP-12 or VIL-1 protein of the invention and, therefore, they may not translate into the corresponding amino acid sequence derivation (SEQ ID NO: 11).

Those skilled in the art may use oligonucleotide probes designed based on the amplified murine BMP-12 or VL-1 DNA sequence specifically set forth in SEQ ID NO: 10 to identify murine BMP-12 or VL-1 encoding clones in length. complete (cDNA or genomic).

Analysis of the DNA sequence of another of the resulting subclones, called mV2, indicates that the amplified DNA sequence specifically contained therein encodes a part of a murine * BMP-12 related sequence of the invention. The DNA sequence (SEQ ID NO: 12) and the amino acid sequence derived (SEQ ID NO: 13) of this specifically amplified murine DNA fragment are shown in the sequence listing.

Nucleotides No. 1-No. 26 of SEQ ID NO: 12 comprise a portion of oligonucleotide # 6 and nucleotides No. 246-No. 272 comprise a portion of the reverse complement of oligonucleotide No. 7 used to perform the specific amplification reaction. Nucleotide 27 of SEQ ID NO: 12 appears to be the last nucleotide of a codon triplet, and nucleotides No. 244-no. 245 of SEQ ID NO: 12 appear to be the first two nucleotides of a codon triplet. Therefore, nucleotides No. 28 to No. 243 of SEQ ID NO: 12 correspond to a partial coding sequence of mV2. Due to the function of oligonucleotides # 6 and # 7 in the initiation of the amplification reaction, they may not correspond exactly to the actual sequence encoding the murine BMP-12 related protein of the invention and, therefore, does not translate into the corresponding amino acid sequence derivation (SEQ ID NO: 13).

Oligonucleotide probes designed based on the amplified murine BMP-12 related DNA sequence specifically set forth in SEQ ID NO: 12 can be used by those skilled in the art to identify clones encoding murine BMP-12 related proteins of full length ( CDNA or genomic).

Analysis of the DNA sequence of another of the resulting subclones called mV9 indicates that the amplified DNA sequence specifically contained therein encodes a part of a murine BMP-12 related sequence of the invention. This sequence appears to be the murine homologue of the BMP52 DNA sequence described in SEQ ID NO: 3. The DNA sequence (SEQ ID NO. 14) and the derived amino acid sequence (SEQ ID NO: 15) of this fragment Specifically amplified murine DNA are shown in the sequence listing.

Nucleotides No. 1-No. 26 of SEQ ID NO: 14 comprise a part of oligonucleotide No. 6 and nucleotides No. 246-No. 272 comprise a part of the reverse complement of oligonucleotide No. 7 used to perform the specific amplification reaction. Nucleotide No. 27 of SEQ ID NO: 14 appears to be the last nucleotide of a codon triplet, and nucleotides No. 244-No. 245 of SEQ ID NO. 14 appear to be the first two nucleotides of a codon triplet. Therefore, nucleotides No. 28 to No. 243 of SEQ ID NO: 14 correspond to a partial coding sequence of mV9. Due to the role of oligonucleotides # 6 and # 7 in the initiation of the amplification reaction, they may not correspond exactly to the actual sequence encoding the murine BMP-12 related protein of the invention and, therefore, not translate in the corresponding amino acid sequence derivation (SEQ ID NO: 15).

Those skilled in the art may utilize oligonucleotide probes designed based on the amplified murine BMP-12 related DNA sequence specifically set forth in SEQ ID NO: 14 to identify clones encoding murine full length BMP-12 related protein (cDNA or genomic).

Alternatively, oligonucleotide primers # 6 and # 7 identified above as primers are used to allow specific amplification of a 275 base pair DNA probe, of which 259 internal bp correspond to nucleotides No. 607 to No. 865 of the SEC ID No. 1 of the subclone of the BMP-12 PCR1-1 # 2 coding plasmid. This 275 bp DNA probe was radiolabelled with 32P and was used to explore a murine genomic library constructed in the X FI XX vector (Stratagene, catalog no. 946306). 1 million recombinants from the murine genomic library are grown at a density of approximately 20,000 recombinants per plate in 50 plates. Duplicate nitrocellulose replicates of recombinant bacteriophage plates are hybridized under conditions of reduced stringency, with 333 bp probe specifically amplified in conventional hybridization buffer (SHB = SSC 5X, 0.1% SDS, Denhardt 5X, 100 µg / ml of salmon sperm DNA) at 60 ° C overnight. The next day the hybridization solution containing radioactively labeled oligonucleotide is removed and the filters are washed, under conditions of reduced stringency, with 2X SSC, 0.1% SDS at 60 ° C. Multiple positive hybridization recombinants are identified and plaque purified. Fragments of murine genomic recombinant positive hybridization clones are subcloned into conventional plasmid vectors (i.e., pGEM-3) and subjected to DNA sequence analysis.

Analysis of the DNA sequence of one of these subclones called MvR3 indicates that it encodes a portion of the mouse gene corresponding to the mV1 PCR product (murine homologue of the human BMP-12 sequence set forth in SEQ ID NO: 1) described previously. The partial DNA sequence of this subclone and the corresponding amino acid translation are set forth in SEQ ID NO: 29 and SEQ ID NO: 30, respectively.

Analysis of the DNA sequence of another of these subclones called MVR32 indicates that it encodes a portion of the mouse gene corresponding to the mV2 PCR product (murine homologue of the human VL-1 sequence set forth in SEQ ID NO: 7) described previously. The partial DNA sequence of this subclone and the corresponding amino acid translation are set forth in SEQ ID NO: 31 and SEQ ID NO: 32 respectively.

Analysis of the DNA sequence of another of these subclones called MVR23 indicates that it encodes a portion of the mouse gene corresponding to the mV9 PCR product (murine homologue of the MP-52 sequence set forth in SEQ ID NO: 3) described above. .

In a manner similar to what has been described above to identify and isolate human genomic clones encoding the BMP-12 protein of the invention, oligonucleotide probes corresponding to the VL-1 coding sequence set forth in SEQ ID NO: 7 can be designed. and used to identify human genomic or cDNA sequences encoding the VL-1 protein (BMP-13). These oligonucleotides would be designed for specific regions for VL-1 coding sequences and, therefore, would probably come from regions of the lowest degree of nucleotide sequence identity between the sequence encoding specifically amplified VL-1 (SEQ ID NO: 7) and the BMP-12 coding sequence specifically amplified (SEQ ID NO: 5).

Alternatively, oligonucleotide primers # 4 and # 5 identified above are used as primers to allow specific amplification of a 333 base pair DNA probe, of which 315 bp internal correspond to nucleotides No. 571 to # 885 of the SEQ ID NO: 1, from the BMP-12 PCRI-1 # 2 coding plasmid subclone. This 333 bp DNA probe was radiolabelled with 32P and was used to explore a human genomic library constructed in the XDASH II vector (Stratagene, catalog no. 945203). 1 million recombinants from the human genomic library are grown at a density of approximately 20,000 recombinants per plate in 50 plates. Duplicate nitrocellulose replicates of recombinant bacteriophage plates are hybridized under conditions of reduced stringency, with the 333 bp probe specifically amplified in conventional hybridization buffer (SHB = SSC 5X, 0.1% SDS, Denhardt 5X, 100 µg / ml of salmon sperm DNA) at 60 ° C overnight. The next day, the hybridization solution containing radiolabeled oligonucleotide is removed and the filters are washed, under conditions of reduced stringency, with 2X SSC, 0.1% SDS at 60 ° C. Multiple (approximately 15) positive hybridization recombinants are identified and plaque purified.

To distinguish positive hybridization recombinants encoding the VL-1 protein of the invention from BMP-12 and other recombinants encoding BMP-12 related proteins that, as predicted, would positively hybridize with the 333 bp DNA probe generated from of the BMP-12 coding plasmid PCRI-1 # 2 used this scanning procedure, the following oligonucleotide probe is designed based on the VL-1 sequence set forth in SEQ ID NO: 7, and is synthesized in an automatic DNA synthesizer :

8: TGTATGCGACTTCCCGC [SEQUENCE ID NO: 35]

An oligonucleotide corresponding to nucleotides No. 60 to No. 76 of SEQ ID NO: 7 containing 5 nucleotide differences with the corresponding region of the BMP-12 coding sequence set forth in SEQ ID NO: 1 (nucleotides No. 672 to No. 689). One of the recombinant bacteriophage clones that hybridizes with the VL-1 oligonucleotide probe No. 8 is called λJLDc31. This recombinant bacteriophage clone is plaque purified, a bacteriophage plate stock is made and bacteriophage DNA is isolated from the human genomic clone λJLDc31. The bacteriophage λJLDc31 has been deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD “ATCC” with accession number 75922 on October 20, 1994. This deposit satisfies the requirements of the Budapest Treaty on Recognition International Deposit of Microorganisms for the Purposes of Patent Procedure and Regulations set forth therein. The oligonucleotide hybridization region of this recombinant, λJLDc31, is located in a 2.5 kb Eco RI fragment. This fragment is subcloned into a plasmid vector (pGEM-3) and DNA sequence analysis is performed. This plasmid subclone is called pGEMJLDc31 / 2.5 and has been deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD "ATCC" with accession number 69710 on October 20, 1994. This deposit meets the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and Regulations set forth therein.

The partial DNA sequence (SEQ ID NO: 25) and the amino acid sequence derived (SEQ ID NO: 26) from a part of the 2.5 kb DNA insert of the plasmid subclone pGEMJLDc31 / 2.5, derived from the λJLDc31 clone, is show in the Sequence Listing.

The DNA sequence of a 2.5 kb EcoRI insert part of plasmid pGEMJLDc31 / 2.5 is set forth in SEQ ID NO: 25.

It contains an open reading frame of 912 bp, as defined by nucleotides No. 52 to No. 963 of SEQ ID NO:

25. Since this sequence derives from a genomic clone, it is difficult to determine the boundary between the 5 ′ end of the coding sequence and the 3 ’limit of the intermediate sequence (intron / non-coding sequence). The entire open reading phase (nucleotides No. 52 to No. 963 of SEQ ID NO: 25) encodes a portion of the VL-1 protein of the invention of up to 304 amino acids.

Based on the knowledge of other BMP proteins and other proteins within the TGF-β family, it is predicted that the precursor polypeptide would be cleaved into the Arg-Arg-Arg-Arg multibasic sequence according to an Arg-consensus consensus proteolytic processing sequence. XX-Arg. It is expected that cleavage of the VL-1 precursor polypeptide will generate a mature 120 amino acid peptide that begins with the amino acid Thr at position # 1 of SEQ ID NO: 26. It is expected that processing of VL-1 in the mature form involves the dimerization and elimination of the N-terminal region in a manner analogous to the processing of the TGF-β-related protein [Gentry et al., Molec & Cell. Biol., 8: 4162 (1988); Derynck et al. Nature, 316: 701 (1985)].

Therefore, it is contemplated that the mature active species of VL-1 comprises a homodimer of two polypeptide subunits, each subunit comprising amino acids No. 1 to No. 120 of SEQ ID NO: 26 with an expected molecular weight of approximately 12,000 daltons. Other active species comprising at least amino acids # 19 to # 119 or # 120 of SEQ ID NO: 26 are contemplated, thus including the first and last conserved cysteine residues.

Using said procedure, a clone encoding mature human VL-1 (BMP-13) was obtained. The nucleotide sequence and corresponding amino acid sequence encoded by this clone are presented in the Sequence Listing in SEQ ID NOS: 25 and 26, respectively.

Example 2

BMP-12 expression

To produce human BMP-12 proteins, the DNA encoding them is transferred to an appropriate expression vector and introduced into mammalian cells or other eukaryotic or prokaryotic hosts preferred by conventional genetic engineering techniques.

To produce the human BMP-12 protein in bacterial cells, the following procedure is used.

BMP-12 expression in E. coli

An expression plasmid pALV1-781 was constructed for the production of BMP-12 in E. coli, which contains the following main characteristics. Nucleotides 1-2060 contain DNA sequences from plasmid pUC-18 [Norrander et al., Gene 26: 101-106 (1983)] including sequences containing the gene for β-lactamase that confers resistance to the antibiotic ampicillin in E strains coli host, and an origin of replication derived from colel. Nucleotides 2061-2221 contain DNA sequences for the main left (pL) promoter of bacteriophage λ [Sanger et al., J. Mol. Biol. 162: 729-773 (1982)], including, three operating sequences OL1, OL2 and OL3. The operators are the binding sites for the λcI repressor protein, whose intracellular levels control the amount of transcription initiation from pL. Nucleotides 2222-2723 contain a strong ribosome binding sequence included in a sequence from nucleotides 35566 to 35472 and 38137 to 38361 of the bacteriophage lambda as described in Sanger et al., J. Mol. Biol. 162: 729-773 (1982). Nucleotides 2724-3041 contain a DNA sequence that encodes the mature BMP-12 protein with the entire 3 ′ untranslated sequence removed. The BMP-12 DNA sequences introduced into the expression vector pALV1-781 were modified at the 5 ′ end to raise the A + T content without altering the coding capacity. These changes were made to increase the efficiency of translation initiated in the BMP-12 mRNA in E. coli. Nucleotides 3042-3058 provide a "linker" DNA sequence that contains restriction endonuclease sites. Nucleotides 3059-3127 provide a transcription termination sequence based on that of the E. coli asp A gene [Takagi et al., Nucl. Acids Res. 13: 2063-2074 (1985)]. Nucleotides 3128-3532 are DNA sequences derived from pUC-18.

Plasmid pALV1-781 was used to transform the E. coli G1724 host strain (F, lacIq, lacpL8, ampC :: λcI +) by the procedure of Dagert and Ehrlich, Gene 6:23 (1979). GI724 (ATCC, accession no. 55151) contains a copy of the wild-type λcI repressor gene stably integrated into the chromosome in the ampC locus, where it has been placed under transcriptional control of Salmonella trp promoter / operator sequences typhimurium In GI724, the λCI protein is obtained only during growth in medium without tryptophan, such as minimum medium

or a minimum medium supplemented with casamino acids such as BMI, described above. The addition of tryptophan to a GI724 culture will repress the trp promoter and inactivate the synthesis of λcI, gradually causing the induction of transcription from pL promoters if they are present in the cell.

Transformers were selected on 1.5% w / v agar plates containing IMC medium, which is composed of M9 medium [Miller, "Experiments in Molecular Genetics", Cold Spring Harbor Laboratory, New York (1972)] containing MgSO4 1 mM and supplemented with 0.5% w / v glucose, 0.2% w / v casamino acids and 100 µg / ml ampicillin. GI724 transformed with pALV1-781 at 37 ° C was grown at an A550 of 0.5 in BMI medium containing 100 µg / ml ampicillin. Tryptophan was then added to a final concentration of 100 µg / ml and the culture was incubated for a further 4 hours. During this time, the BMP-12 protein accumulates within the "inclusion bodies" fraction.

Preparation of protein monomer

18 g of frozen cells were weighed and resuspended in 60 ml of 100 mM Tris, 10 mM EDTA, phenylmethylsulfonyl fluoride [PMSF], 1 mM, pH 8.3. The cells were lysed by 3 passes through a Microfluidizer ™ [model No. MCF 100 T]. The inclusion body sediment was obtained by centrifugation at 15,000 g at 4 ° C for 20 minutes. The supernatant was decanted and the pellet was washed with 100 ml of 100 mM Tris, 1.0 M NaCl, 10 mM EDTA, 1 mM PMSF, pH 8.3. The suspension was centrifuged again at 15,000 g at 4 ° C for 10 minutes and the supernatant was decanted. Then, the pellet was washed with 100 ml of 100 mM Tris, 10 mM EDTA, 1% Triton X-100, 1 mM PMSF, pH 8.3. The suspension was centrifuged again at 15,000 g at 4 ° C for 10 minutes and the supernatant was decanted. The pellet was resuspended with 50 ml of 20 mM Tris, 1 mM EDTA, 1 mM PMSF, pH 8.3, containing 1% DTT in a glass tissue homogenizer. Then, the monomeric BMP-12 was solubilized by acidification to pH 2.5 with glacial acetic acid. The soluble fraction was isolated by centrifugation at 15,000 g for 20 minutes at 4 ° C.

The supernatant from this centrifugation was collected and chromatographed on a Sephacryl S-100 ™ molecular exclusion column (83 cm x 2.6 cm; bed ≈440 ml) in 20 ml increments. The Sephacryl S-100 ™ column was processed with a mobile phase of 1% acetic acid at a flow rate of 1.4 ml / min. The fractions corresponding to the BMP-12 monomer were detected by absorbance at 280 nm and using an computer an extinction coefficient of 18200 M-1 m-1 and a molecular weight (11667 daltons) was calculated. The material collected in the molecular exclusion column was used as the starting material for refolding reactions.

As an alternative to the above, 1.0 g of cells stored at -80 ° C are measured. Solution (3.4 ml of 100 mM TRIS, 10 mM EDTA, pH 8.5) is added. The solution is subjected to vortexing until the cells are well suspended. 40 µl of 100 mM PMSF in isopropanol are added. The cells are lysed at 6.89 MPa (1000 psi) in a French pressure cell. The inclusion bodies are centrifuged at 4 ° C for 20 minutes in an Eppendorf microcentrifuge to form sediments. The supernatants are decanted. 1.0 ml of 8.0 M degassed guanidine hydrochloride, 0.5 M TRIS, 5 mM EDTA, pH 8.5, containing 250 mM DTT are added to a sediment (4 in total). The sediment dissolves and argon is blown over the liquid for 30 seconds. Then, the solution is incubated at 37 ° C for one hour. The insoluble material is sedimented for 2-3 minutes in an Eppendorf microcentrifuge at 23 ° C. 0.5-1.0 ml of supernatant are injected into a Supelco 2 cm auxiliary cartridge (LC-304) and eluted with a gradient of acetonitrile in 0.1% TFA of 1-70% for 35 minutes. The BMP-12 elutes between 29 and 31 minutes. The fractions are pooled and the protein concentration is determined by absorbance at 280 nanometers against 0.1% TFA, using the theoretical extinction coefficient based on the amino acid content.

As a second alternative procedure to the above, frozen cell sediments obtained from E. coli transformants are thawed as described above in 30 ml of TE8.3 buffer (100: 10) (100 mM Tris-HCl pH 8, 3, 10 mM Na2EDTA, 1 mM PMSF). The cells are lysed by three passes through a Microfluidizer ™ [model No. MCF 100 T]. The sediment of initial inclusion body material is dissolved in 8 M HClguanidine, TE8.5 buffer (100: 10) (100 mM Tris-HCl, pH 8.5, 10 mM Na2EDTA containing 100 mM DTT) and incubated at 37 ° C for 1 hour. This material is centrifuged at 12,000 x g for 15 minutes at room temperature.

BMP-12 protein refolding using the CHAPS system

A sufficient volume of BMP-12 reserve is lyophilized to give 10 µg of protein. 5 µl of distilled water in glass still is added to redissolve the residue, and then 100 µl of refolding mixture is added

- (50 mM Tris, 1.0 M NaCl, 2% 3- (3-chloramidopropyl) dimethylammonium sulfonate (CHAPS) sulfonate, 5 mM EDTA, 2 mM glutathione (reduced), 1 mM glutathione (oxidized) ; at pH of about 8.5). The solution is mixed gently and stored at 23 ° C for 1-4 days. The formation of dimers is evaluated by processing an aliquot on a 16% Novex tricine gel at 125 volts for 2.5 hours, followed by staining with Coomassie Blue and allocation.

The BMP-12 dimer was purified using a C4 column of analytical RP-HPLC (reverse phase high performance liquid chromatography) (Vydac 214TP54) that was equilibrated to 1% Buffer B (diluted in buffer A) and processed for 35 minutes, during which time the protein elutes, using the following gradient (buffer A = 0.1% trifluoroacetic acid, buffer B = 95% acetonitrile, 0.1% trifluoroacetic acid [TFA] with a flow rate of 1 ml / min:

1-5 minutes of 20% buffer B 5-10 minutes of 20-30% buffer B 10-30 minutes of 30-50% buffer B 30-35 minutes of 50-100% buffer B

Protein was monitored by absorbance at 280 nm. Peak fractions of BMP-12 (eluting between 29 and 31 minutes) were collected. Purity was evaluated by SDS-PAGE. The concentration was determined by absorbance at 280 nm, and using the computer-calculated extinction coefficient and molecular weight indicated above.

BMP-12 expression in mammalian cells

Another preferred expression system contemplated for biologically active recombinant human BMP-12 is stably transformed mammalian cells.

One skilled in the art can construct mammalian expression vectors using the sequence of SEQ ID No. 1 or other DNA sequences encoding BMP-12 proteins or other modified sequences and known vectors, such as pCD [Okayama et al., Mol . Cell Biol., 2: 161-170 (1982)], pJL3, pJL4 [Gough et al., EMBO J.,

4: 645-653 (1985)] and pMT2 CXM.

The mammalian expression vector pMT2 CXM is a derivative of p91023 (b) (Wong et al., Science 228: 810-815, 1985) which differs from the latter in that it contains the ampicillin resistance gene rather than the gene of Tetracycline resistance and also contains an XhoI site for cDNA clone insertion. The functional elements of pMT2 CXM (Kaufman, RJ, 1985, Proc. Natl. Acad. Sci. USA 82: 689-693) have been described and include the adenovirus VA genes, the SV40 origin of replication that includes the enhancer of SV40 72 bp, the main late adenovirus promoter that includes a 5 'splice site and most of the tripartite leader sequence of adenovirus present in late adenovirus mRNA, a 3' splice acceptor site, an insert of DHFR, the SV40 early polyadenylation site (SV40) and pBR322 sequences necessary for propagation in E. coli.

Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF, which has been deposited with the American Type Culture Collection (ATCC), Rockville, MD (USA) with accession number ATCC 67122. EcoRI digestion cleaves the cDNA insert present in pMT2-VWF, producing pMT2 in a linear fashion that can be ligated and used to transform E. coli HB 101 or DH-5 into ampicillin-resistant bacteria. Plasmid pMT2 DNA can be prepared by conventional procedures. PMT2 CXM is then constructed using loopout / in mutagenesis [Morinaga, et al., Biotechnology 84: 636 (1984). This eliminates bases 1075 to 1145 with respect to the Hind III site near the SV40 origin of replication and pMT2 enhancer sequences. In addition, it inserts a sequence that contains the recognition site for restriction endonuclease Xho I. A derivative of pMT2CXM, called pMT23, contains recognition sites for restriction endonucleases PstI, Eco RI, SalI and XhoI. Plasmid pMT2 CXM and pMT23 DNA can be prepared by conventional procedures.

pEMC2β1 derived from pMT21 may also be suitable in the practice of the invention. pMT21 comes from pMT2 that comes from pMT2-VWF. As described above, EcoRI digestion cleaves the cDNA insert present in pMT-VWF, producing pMT2 in a linear manner that can be ligated and used to transform E. coli HB 101 or DH-5 into ampicillin-resistant bacteria. Plasmid pMT2 DNA can be prepared by conventional procedures.

pMT21 comes from pMT2 through the following two modifications. First, 76 bp of the 5 ’untranslated region of the DHFR cDNA are deleted including a stretch of 19 G residues of the G / C tail for cDNA cloning. In this procedure, an Xhol site is inserted to obtain the following sequence immediately upstream of DHFR. Second, a single ClaI site is introduced by digestion with EcoRV and Xbal, treatment with Klenow fragment of DNA polymerase I and binding to a ClaI linker (CATCGATG). This deletes a 250 bp segment of the adenovirus-associated RNA region (VAI) but does not interfere with the expression or function of the VAI RNA gene. pMT21 is digested with EcoRI and XhoI and used to obtain the vector pEMC2B1.

A part of the EMCV leader is obtained from pMT2-ECAT1 [S.K. Jung, et al., J. Virol 63: 1651-1660 (1989)] by digestion with Eco RI and PstI, resulting in a fragment of 2752 bp. This fragment is digested with TaqI to produce a 508 bp Eco RI-TaqI fragment that is purified by electrophoresis on a low melting agarose gel. A 68 bp adapter and its complementary chain are synthesized with a 5 'TaqI protruding end and a 3' XhoI protruding end having a sequence corresponding to the EMC virus leader sequence from nucleotide 763 to 827. ATG also changes in the position 10 within the leader of the EMC virus to ATT and is continued by an Xhol site. A three-way ligation of the Eco RI-XhoI fragment of pMT21; the EcoRI-TaqI fragment of the EMC virus and the 68 bp TaqI-XhoI oligonucleotide adapter results in the vector pEMC2β1.

This vector contains the origin of SV40 enhancer and replication, the adenovirus major late promoter, a cDNA copy of most of the adenovirus tripartite leader sequence, a small hybrid intermediate sequence, an SV40 polyadenylation signal and the gene of adenovirus VA I, markers of DHFR and β-lactamase and an EMC sequence, in appropriate relationships to direct the high level expression of the desired cDNA in mammalian cells.

The construction of vectors may involve the modification of the BMP-12 DNA sequences. For example, the BMP-12 cDNA can be modified by removing non-coding nucleotides from the 5 ′ and 3 ′ ends of the coding region. Deleted non-coding nucleotides may or may not be replaced by other sequences considered beneficial for expression. These vectors are used to transform appropriate host cells for the expression of BMP-12 proteins. In addition, the sequence of SEQ ID NO: 1 or other sequences encoding BMP-12 proteins can be manipulated to express the BMP-12 protein by isolation of the mature coding sequence of nucleotides 571 to 882 of SEQ ID NO: 1 and Addition to the 5 'end sequences encoding the complete propeptides of other BMP proteins.

For example, one skilled in the art can make a fusion protein in which the BMP-2 propeptide is operatively linked to the mature BMP-12 peptide by preparing a DNA vector in which the DNA sequence encoding the BMP-2 propeptide is attached in the appropriate reading frame to the DNA sequence encoding the mature BMP-12 peptide. The DNA sequence of said fusion protein is shown in SEQUENCE ID NO: 27.

One skilled in the art can manipulate the sequences of SEQ ID NO: 1 by removing or replacing mammalian regulatory sequences flanking the coding sequence with bacterial sequences to create bacterial vectors for intracellular or extracellular expression by bacterial cells, as described. previously. As another example, the coding sequences could be further manipulated (eg, linked to other known linkers or modified by deletion of non-coding sequences thereof or nucleotide alteration by other known techniques). The modified BMP-12 coding sequence could then be inserted into a known bacterial vector using procedures such as those described in T. Taniguchi et al., Proc. Natl Acad. Sci. USA, 77: 5230-5233 (1980). This exemplary bacterial vector could then be used to transform bacterial host cells and thus express a BMP-12 protein. As a strategy for producing extracellular expression of BMP-12 proteins in bacterial cells, see, for example, European Patent Application EPA 177,343.

Similar manipulations can be made for the construction of an insect vector [See, for example, the procedures described in the publication of European patent application 155,476] for expression in insect cells. A yeast vector could also be constructed using yeast regulatory sequences for intracellular or extracellular expression of the factors of the present invention by yeast cells [See, for example, the procedures described in the publication of PTC application WO 86/00639 and in the European patent application EPA 123.289).

A method for producing high levels of a BMP-12 protein of the invention in mammalian cells may involve the construction of cells containing multiple copies of the heterologous BMP-12 gene. The heterologous gene is linked to an amplifiable marker, for example, the dihydrofolate reductase (DHFR) gene for which cells containing more copies of genes can be selected for propagation in increasing concentrations of methotrexate (MTX) according to the procedures. from Kaufman and Sharp, J. Mol. Biol., 159: 601-629 (1982). This strategy can be used with several different cell types.

For example, a plasmid containing a DNA sequence for a BMP-12 of the invention can be co-introduced in operative association with other plasmid sequences that allow expression thereof and the DHFR expression plasmid pAdA26SV (A) 3 [Kaufman and Sharp, Mol. Cell Biol., 2: 1304 (1982)] in CHFR cells deficient in DHFR, DUKX-BII, by various procedures including co-precipitation with calcium phosphate and transfection, electroporation or fusion of protoplasts. Transformants that express DHFR are selected for growth in alpha medium with dialyzed fetal bovine serum and subsequently selected for growth amplification at increasing concentrations of MTX (eg, sequential steps in MTX 0.02, 0.2, 1, 0 and 5 µM) as described in Kaufman et al., Mol Cell Biol., 5: 1750 (1983). The transformants are cloned and the expression of biologically active BMP-12 is monitored by the Rosen-modified Sampath-Reddi rat assay described below in Example 5. The expression of BMP-12 would increase with increasing MTX resistance levels. . BMP-12 polypeptides are characterized using conventional techniques known in this field such as pulsatile labeling with [35S] methionine or cysteine and polyacrylamide gel electrophoresis. Similar procedures can be followed to produce other BMP-12 related proteins.

Example 3

BMP-2 Propeptide / BMP-12 Mature Peptide Fusion Preparation

To construct a vector encoding the BMP-2 propeptide / mature BMP-12 peptide fusion, the following cloning procedure was used to fuse the two sequences together.

First, a DNA restriction enzyme fragment comprising the propeptide of the human BMP-12 protein, which comprised nucleotides 1 to 843 of SEQ ID NO: 27, was cut from pBMP2nEMC. pBMP2nEMC is a U20S-39 lamba-derived plasmid (ATCC # 40345) comprising the entire coding sequence of the human BMP-2 protein with the 5 ’and 3’ untranslated sequences of BMP-2 deleted from the vector. The 5 'restriction enzyme used was Bgl II and cuts to pBMP2nEMC in the vector in nucleotide 979. The 3' restriction enzyme used was Mae II and cuts pBMP2n EMC in the BMP-2 propeptide in nucleotide 1925, near the carboxy end The resulting 954 base pair product was then gel isolated and subjected to Geneclean. Second, a restriction enzyme DNA fragment comprising the 5 'part of the human BMP-12 mature peptide DNA sequence was cut from pPCR1-1 # 2 V1-1 (ATCC No. 69517) . The restriction enzyme 5 ’used was Eae I and cuts pPCRI- # 12 V1-1 in position 3’ with respect to the N-terminus of the human BMP-12 mature peptide sequence. The resulting 259 base pair product was gel isolated and subjected to Geneclean. Third, two DNA oligos were designed and synthesized, so that when they hybridized they formed a small DNA fragment that comprised the fusion sequence of the 3 'end of the human BMP-2 propeptide and the 5' end of the peptide BMP-12 mature. The DNA fragment has a complementary 5 'Mae II adherent end that hybridizes with the 3 ’restriction enzyme fragment comprising the human BMP-12 propeptide. The hybridized oligo DNA fragment has a complementary Eae I 3 ’adherent end that hybridizes with the 5 ′ end of the restriction enzyme fragment comprising the mature human BMP-12 peptide. The oligo of the coding chain is called B2 / 12 and is 13 base pairs in length. Next, a DNA fragment encoding the 123 base pairs at the 3 ′ end of the BMP-12 mature peptide fragment was obtained as indicated below. First, a DNA fragment comprising the propeptide of the human BMP-2 protein, comprising nucleotides 1 to 846, was amplified by PCR from pBMP2ΔEMC. The 5 ’primer (oligo 655a) hybridizes to the 5’ position of the poliengarce. Primer 3 ’(BMPpro3) hybridizes with the BMP-2 propeptide at the 3 ′ end and introduces a Bgl II restriction enzyme site by silent sequence mutations. The resulting PCR product was cut with Salt I, which cleaves in the polylinker, and Bgl II. The 850 base pair restriction enzyme fragment (ending in the amino acid sequence REKR) was gel isolated and subjected to Geneclean. The mature BMP-12 peptide was amplified by PCR using a 5 'primer (oligo 5-1) that encoded the Bgl II restriction enzyme site by silent sequence mutations, and hybridization with the 5' end of a possible product of mature cleavage, beginning with the amino acid sequence SCRS. Primer 3 '(V1-1 3) hybridizes with the 3' end of the mature BMP-12 peptide and introduces a restriction enzyme site Xba I after the termination codon. The resulting PCR product was cut with Bgl II and Xba I. The 321 base pair restriction enzyme fragment was gel isolated and subjected to Geneclean.

The two restriction fragments were ligated in three ways in a vector previously cut with SalI and XbaI. The resulting construct was sequenced to check for PCR-induced errors and a silent mutation of C to T was observed in base pair 185 in the propeptide. This plasmid was called pREKRSRC. Then, pREKRSRC was cut with BgIII and Ngo MI, and the vector fragment that included the last 123 base pairs of the mature BMP12 sequence was isolated in this way. The three restriction fragments and the hybridized oligoengarce were ligated in four ways to produce pREKR-TAL with the BMP-2 propeptide with the mature cleavage site at the 3 'end fused to the 5' end (TAL) of the mature BMP peptide -12. The coding sequence of the resulting linked vector is shown in SEQ ID NO: 27.

Example 4

Biological activity of BMP-12 expressed

To measure the biological activity of the expressed BMP-12 proteins obtained in Example 2 above, the proteins are recovered from the cell culture and purified by isolating the BMP-12 proteins from other protein materials with which they are co-produced, as well as from other pollutants The purified protein can be tested according to the rat assay described later in Example 5.

Purification is performed using conventional techniques known to those skilled in the art.

Protein analysis is performed using conventional techniques such as SDS-PAGE acrylamide [Laemmli, Nature 227: 680 (1970)] stained with Coomassie Blue or silver [Oakley, et al. Anal: Biochem. 105: 361 (1980)] and by immunoblot [Towbin, et al. Proc. Natl Acad. Sci. USA 76: 4350 (1979)]

5

10

fifteen

twenty

25

30

35

40

Four. Five

Example 5

SAMPATH-REDDI test modified by ROSEN

A modified version of the rat ectopic implant assay described in Sampath and Reddi, Proc. Natl Acad. Sci. USA, 80: 6591-6595 (1983) to evaluate the activity of BMP-12 proteins. This modified test is referred to herein as the Sampath-Reddi test modified by Rosen. The assay has been widely used to evaluate the activity of cartilage and bone induction of BMPs. The ethanol precipitation step of the Sampath-Reddi procedure is replaced by dialysis (if the composition is a solution) or diafiltration (if the composition is a suspension) of the fraction to be tested against water. The solution or suspension is then equilibrated to 0.1% TFA. The resulting solution is added to 20 mg rat matrix. As a control, a sample of simulated rat matrix not treated with the protein is used. This material is frozen and lyophilized and the resulting powder is enclosed in gelatin capsules No. 5. The capsules are implanted subcutaneously in the abdominal thoracic area of male Long Evans rats 21-49 days old. Implants are removed after 10 days. A section of each implant is fixed and processed for histological analysis. 1 µm glycol methacrylate sections are stained with Von Kossa and acid fuchsin to assess the amount of tendon / ligament-like tissue formation present in each implant.

BMP-12 was implanted in rats at doses of 1, 5, 25 and 50 µm per implant for 10 days. BMP-2 at a dose of 5 µg was included as a positive control. For all doses of BMP-12 tested, no bone formation was observed.

or cartilage in the implants after ten days. Instead, the implants were filled with embryonic tendon-like tissue, which is easily recognized by the presence of dense bundles of fibroblasts oriented in the same plane and pressed together. [Tendon / ligament-like tissue is described, for example, in Ham and Cormack, Histology (JB Lippincott Co. (1979), pp. 367-369. These findings were reproduced in a second series of trials in which tissues were present. tendon / ligament similar in all implants containing BMP-12 On the contrary, BMP-2 implants, as expected, showed cartilage and bone formation but did not contain tendon / ligament-like tissue.

The BMP-12 proteins and related proteins of the present invention can be tested for activity in this assay.

Example 6

Using procedures according to the previous examples, with minor modifications within the skill of the art, human MP52 protein and murine homolog of BMP-13 protein were expressed and tested for the tendon-like tissue-inducing activity. ligament. All proteins showed comparable results, similar to those described above for human BMP-12.

The above descriptions detail currently preferred embodiments of the present invention. It is expected that those skilled in the art will come up with numerous modifications and variations in the practice of the present invention after consideration of these descriptions. These modifications and variations are considered to be included within the appended claims.

SEQUENCE LIST

(one)

GENERAL INFORMATION

(i)

APPLICANT: GENETICS INSTITUTE, INC. PRESIDENT AND FELLOWS OF HARVARD COLLEGE

(ii)

TITLE OF THE INVENTION: COMPOSITIONS TO INDUCE TENDONS

(iii) NUMBER OF SEQUENCES: 35

(iv)

POST ADDRESS:

(TO)

RECIPIENT: GENETICS INSTITUTE, INC.

(B)

STREET: 87 CambridgePark Drive

(C)

CITY: Cambridge

(D) STATE: Massachusetts

(D)

COUNTRY: USA

(AND)

ZIP CODE: 02140

(v)

LEGIBLE FORM BY COMPUTER: 23

(TO)

TYPE OF SUPPORT: Floppy Disk

(B)

COMPUTER: Compatible with IBM PC

(C)

OPERATING SYSTEM: PC-DOS / MS-DOS

(D)

SOFTWARE: PatentIn Release # 1.0, Version # 1.25

(saw)

DATA OF THE CURRENT APPLICATION:

(TO)

APPLICATION NUMBER:

(B)

SUBMISSION DATE: Together with this

(C)

CLASSIFICATION:

(vii) PREVIOUS APPLICATION DATA:

(TO)

APPLICATION NUMBER: US 08 / 164.103

(B)

DATE OF PRESENTATION: 07-DEC-1993

(C)

APPLICATION NUMBER: US 08 / 217,780

(D)

PRESENTATION DATE: MAR 25, 1994

(AND)

APPLICATION NUMBER: US 08 / 333,576

(F)

DATE OF PRESENTATION: 02-NOV-1994

(viii) MANDATORY / AGENT INFORMATION:

(TO)

NAME: Lazar, Steven R.

(B)

REGISTRATION NUMBER: 32,618

(C)

REFERENCE NUMBER / RECORD: 5202D-PCT

(ix)

TELECOMMUNICATIONS INFORMATION:

(TO)

PHONE: 617 498-8260

(B)

TELEFAX: 617 876-5851

(2)

INFORMATION FOR SEQ ID NO: 1

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 926 base pairs

(B)

TYPE: nucleic acid

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CHAIN TYPE: simple

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TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic)

(saw)

ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLON: v1-1

(ix) FEATURE:

(TO)

NAME / KEY: mat_peptide

(B)

LOCATION: 571..882

24

(ix)

CHARACTERISTIC:

(TO)

NAME / KEY: CDS

(B)

LOCATION: 1,882

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 1

(2)

INFORMATION FOR SEQ ID NO: 2

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CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 294 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 2

(2)

INFORMATION FOR SEQ ID NO: 3

(i)

CHARACTERISTICS OF THE SEQUENCE:

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(TO)

LENGTH: 1207 base pairs 5 (B) TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii) TYPE OF MOLECULE: DNA (genomic)

(saw)

ORIGINAL SOURCE: 10 (A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLON: MP52

(ix) FEATURE:

(TO)

NAME / KEY: CDS 15 (B) LOCATION: 845..1204

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 3

(2)

INFORMATION FOR SEQ ID NO: 4

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 120 amino acids

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TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Protein

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4

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(2) INFORMATION FOR SEQ ID NO: 5

(i)

CHARACTERISTICS OF THE SEQUENCE: 5 (A) LENGTH: 128 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic) 10 (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLON: fragments V1-1

(ix)

FEATURE: 15 (A) NAME / KEY: CDS

(B) LOCATION: 28..102

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 5

(2)

INFORMATION FOR SEQ ID NO: 6

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 25 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 6

(2)

INFORMATION FOR SEQ ID NO: 7 10 (i) CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 128 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear 15 (ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B)

CLON: VL-1 20 (ix) FEATURE:

(TO)

NAME / KEY: CDS

(B)

LOCATION: 28..102

image2

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7

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25 (2) INFORMATION FOR SEQ ID NO: 8

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 25 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 8

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(2)

INFORMATION FOR SEQ ID NO: 9 5 (i) SEQUENCE CHARACTERISTICS:

(TO)

LENGTH: 3585 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear 10 (ii) TYPE OF MOLECULE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLON: pALV1-781

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 9

(2)

INFORMATION FOR SEQ ID NO: 10

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 272 base pairs 5 (B) TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii) TYPE OF MOLECULE: DNA (genomic)

(saw)

ORIGINAL SOURCE: 10 (A) ORGANISM: mouse

(vii) IMMEDIATE SOURCE:

(B) CLON: mV1

(ix)

CHARACTERISTIC:

(TO)

NAME / KEY: CDS

(B)

LOCATION: 28..243

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 10

(2)

INFORMATION FOR SEQ ID NO: 11 5 (i) SEQUENCE CHARACTERISTICS:

(TO)

LENGTH: 72 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: protein 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11

(2)

INFORMATION FOR SEQ ID NO: 12

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 272 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic)

(saw)

ORIGINAL SOURCE:

(A) ORGANISM: mouse

(vii) IMMEDIATE SOURCE:

(B) CLON: mV2

(ix)

CHARACTERISTIC:

(TO)

NAME / KEY: CDS

(B)

LOCATION: 28..243

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 12

(2)

INFORMATION FOR SEQ ID NO: 13 10 (i) CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 72 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: protein 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13

(2)

INFORMATION FOR SEQ ID NO: 14

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 272 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic) 5 (vi) ORIGINAL SOURCE:

(A) ORGANISM: mouse

(vii) IMMEDIATE SOURCE:

(B) CLON: mV9

(ix)

FEATURE: 10 (A) NAME / KEY: CDS

(B) LOCATION: 28..243

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14

(2)

INFORMATION FOR SEQ ID NO: 15 15 (i) SEQUENCE CHARACTERISTICS:

(TO)

LENGTH: 72 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: protein 20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15

(2)

INFORMATION FOR SEQ ID NO: 16

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 7 amino acids

(B)

TYPE: amino acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Peptide

(saw)

ORIGINAL SOURCE:

(A) ORGANISM: BMP / TGF-beta consensus sequence

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 16

(2)

INFORMATION FOR SEQ ID NO: 17

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 27 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLON: oligonucleotide # 1

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 17

(2)

INFORMATION FOR SEQ ID NO: 18

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 6 amino acids

(B)

TYPE: amino acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Peptide

(vii) IMMEDIATE SOURCE:

(B) CLON: BMP / TGF-beta consensus sequence

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 18

(2)

INFORMATION FOR SEQ ID NO: 19

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 28 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLON: oligonucleotide # 2

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 19

(2)

INFORMATION FOR SEQ ID NO: 20

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 40 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLON: oligonucleotide No. 3

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 20

(2)

INFORMATION FOR SEQ ID NO: 21

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 29 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: oligonucleotide No. 4

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 21

(2)

INFORMATION FOR SEQ ID NO: 22

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 29 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLON: oligonucleotide No. 5

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 22

(2)

INFORMATION FOR SEQ ID NO: 23

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 28 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: oligonucleotide No. 6

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 23

(2)

INFORMATION FOR SEQ ID NO: 24

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 28 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLON: oligonucleotide No. 7

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 24

(2)

INFORMATION FOR SEQ ID NO: 25

(i)

CHARACTERISTICS OF THE SEQUENCE: 5 (A) LENGTH: 1171 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: DNA (genomic) 10 (vii) IMMEDIATE SOURCE:

(B) CLON: human VL-1 protein

(ix) FEATURE:

(A) NAME / KEY: CDS

(B)

LOCATION: 2,964 15 (ix) FEATURE:

(TO)

NAME / KEY: mat_peptide

(B)

LOCATION: 605.964

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 25

(2)

INFORMATION FOR SEQ ID NO: 26

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 321 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 26

(2)

INFORMATION FOR SEQ ID NO: 27

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 1233 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear 5 (ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B)

CLON: DNA encoding BMP2 propeptide / mature BMP-12 peptide

(ix)

CHARACTERISTIC:

(TO)

NAME / KEY: CDS 10 (B) LOCATION: 1..1233

(ix)

CHARACTERISTIC:

(TO)

NAME / KEY: mat_peptide

(B)

LOCATION: 847..1233

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 27

(2)

INFORMATION FOR SEQ ID NO: 28

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 411 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 28

(2)

INFORMATION FOR SEQ ID NO: 29

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 1203 base pairs 5 (B) TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

image2

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image2

image2

image2

image2

image2

image2

image2

image2

image2

image2

image2

image3

image2

image2

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image2

(ii) TYPE OF MOLECULE: DNA (genomic)

(vii) IMMEDIATE SOURCE: 10 (B) CLON: MV1 murine

(ix)

CHARACTERISTIC:

(TO)

NAME / KEY: CDS

(B)

LOCATION: 2,721

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 29

(2)

INFORMATION FOR SEQ ID NO: 30

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 240 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 30

image2

image2

image2

(2)

INFORMATION FOR SEQ ID NO: 31 10 (i) CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 1046 base pairs

(B)

TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

(ii) TYPE OF MOLECULE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:

(B) CLON: MV2 murine

(ix)

CHARACTERISTIC:

(TO)

NAME / KEY: CDS

(B)

LOCATION: 2,790

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 31

(2)

INFORMATION FOR SEQ ID NO: 32

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 263 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 32

(2)

INFORMATION FOR SEQ ID NO: 33

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 1345 base pairs 5 (B) TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

image4

image2

image2

image2

(ii) TYPE OF MOLECULE: DNA (genomic)

(iii) HYPOTHETICAL: NO 10 (iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:

(B) CLON: V1-1 HUMAN

(ix) FEATURE:

(TO)

NAME / KEY: CDS 15 (B) LOCATION: 138..1301

(ix)

CHARACTERISTIC:

(TO)

NAME / KEY: mat_peptide

(B)

LOCATION: 990..1301

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 33

(2)

INFORMATION FOR SEQ ID NO: 34

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 388 amino acids

(B)

TYPE: amino acid

(D)

TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Protein

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 34

(2)

INFORMATION FOR SEQ ID NO: 35

(i)

CHARACTERISTICS OF THE SEQUENCE:

(TO)

LENGTH: 17 base pairs 5 (B) TYPE: nucleic acid

(C)

CHAIN TYPE: simple

(D)

TOPOLOGY: linear

image2

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image2

image2

image2

(ii) TYPE OF MOLECULE: DNA (genomic)

(iii) HYPOTHETICAL: NO 10 (iv) ANTI-SENSE: NO

(vii) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATED: primer number 8

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35

image2

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Claims (16)

1. Polypeptide having the ability to induce the formation of tendon-like or ligament-like tissue, wherein said polypeptide is encoded by a DNA molecule comprising a nucleotide sequence selected from the group consisting of:

(to)

nucleotides No. 196, No. 199, No. 208, No. 217, No. 361, No. 388, No. 493, No. 496, No. 571 or No. 577 to No. 879 or No. 882 of SEQ ID NO: 1;

(b)

nucleotides encoding amino acids # -25, # 1 or # 3 to # 103 or # 104 of SEQ ID NO: 2;

(C)

nucleotides No. 845, No. 893 or No. 899 to No. 1201 or No. 1204 of SEQ ID NO: 3;

(d)

nucleotides encoding amino acids # 1 or # 19 to # 119 or # 120 of SEQ ID NO: 4;

(and)

nucleotides No. 410, No. 458, No. 602, No. 605 or No. 659 to No. 961 or No. 964 of SEQ ID NO: 25;

(F)

nucleotides encoding amino acids # 1 or # 19 to # 119 or # 120 of SEQ ID NO: 26; or

(g)

natural human allelic sequences and equivalent degenerate codon sequences of any one of (a) to (f), or

(h)

sequences that hybridize with any of (a) to (f) above under stringent hybridization conditions,

for use in a procedure to induce tendon / ligament-like tissue formation in a patient in need.

2.

Polypeptide of claim 1, wherein the polypeptide is encoded by a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 1.

3.

Polypeptide of claim 1, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 2.

Four.

Polypeptide of claim 1, wherein the polypeptide is encoded by a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 25.

5.

Polypeptide of claim 1, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 26.

6.

Polypeptide of any one of claims 1 to 5, wherein said polypeptide is for use in a method of treating tendonitis, tendon or ligament tears, deformities and other tendon defects

or ligaments.

7.

Polypeptide of any one of claims 1 to 6, wherein said polypeptide is present as a monomer within a heterodimeric protein molecule, and wherein the second monomer is a member of the TGF-β growth factor superfamily.

8.

Pharmaceutical composition comprising a polypeptide having the ability to induce the formation of tendon-like or ligament-like tissue, wherein said polypeptide is encoded by a DNA molecule comprising a nucleotide sequence selected from the group consisting of

(to)

nucleotides No. 196, No. 199, No. 208, No. 217, No. 361, No. 388, No. 493, No. 496, No. 571 or No. 577 to No. 879 or No. 882 of SEQ ID NO: 1;

(b)

nucleotides encoding amino acids # -25, # 1 or # 3 to # 103 or # 104 of SEQ ID NO: 2;

(C)

nucleotides No. 845, No. 893 or No. 899 to No. 1201 or No. 1204 of SEQ ID NO: 3;

(d)

nucleotides encoding amino acids # 1 or # 19 to # 119 or # 120 of SEQ ID NO: 4;

(and)

nucleotides No. 410, No. 458, No. 602, No. 605 or No. 659 to No. 961 or No. 964 of SEQ ID NO: 25;

(F)

nucleotides encoding amino acids # 1 or # 19 to # 119 or # 120 of SEQ ID NO: 26; or

(g)

natural human allelic sequences and equivalent degenerate codon sequences of any one of (a) to (f), or

(h)

sequences that hybridize with any of (a) to (f) above under stringent hybridization conditions,

in which the composition is to induce the formation of tendon / ligament-like tissue in a patient in need.

9.

Pharmaceutical composition of claim 8, wherein the polypeptide is encoded by a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 1.

10.

Pharmaceutical composition of claim 8, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 2.

eleven.

Pharmaceutical composition of claim 8, wherein the polypeptide is encoded by a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 25.

12.

Pharmaceutical composition of claim 8, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 26.

13.

Pharmaceutical composition of any one of claims 8 to 12, wherein the pharmaceutical composition

It is to treat tendonitis, tendon or ligament tears, deformities and other tendon defects or 5 ligaments. 14. Pharmaceutical composition of any of claims 8 to 13, wherein said polypeptide is present as a monomer within a heterodimeric protein molecule, and wherein the second monomer is a member of the TGF growth factor superfamily. -β. 15. Pharmaceutical composition of any of claims 8 to 14, wherein the pharmaceutical composition 10 further comprises at least one other therapeutically useful agent selected from BMP proteins, BMP-1 to BMP-11. 16. Pharmaceutical composition of claim 15, wherein the pharmaceutical composition further comprises BMP-2.