FI121070B - Bone morphogenetic protein 6, DNA encoding it, nucleotide vector, recombinant host cell, pharmaceutical composition and osteogenic device - Google Patents

Description

FIELD OF THE INVENTION This invention relates to bone formation inducing proteins called bone morphogenetic proteins (BMPs), especially BMPs 6, particularly BMPs; nucleic acid molecules encoding said proteins; vectors containing said nucleic acid molecules; and host cells expressing said protein. The present invention also relates to the use of these bone morphogenetic proteins in the treatment of disorders such as those associated with bone and cartilage formation. The present invention further relates to osteogenic devices and pharmaceutical compositions containing said proteins.

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Background of the Invention

In 1945, Lancroix discovered a phenomenon related to bone formation when he demonstrated that acidic alcohol-bone extract induces heterotropic bone formation at 20 ectopic sites. Twenty years later, Urist and his colleagues decalcified the bone matrix, and when it was implanted into muscle, he noticed new cartilage and bone forming at the implant site. These findings led to the isolation and purification of a bone inducing agent, designated BMP, from bone matrices of various animal species and, years later, to the cloning and characterization of the cDNA of these new proteins. Biological activity of BMP has been determined by bioassay in which BMP is implanted in rat or mouse muscle mass, or in mammalian cell culture; where alkaline phosphatase (ALP) is measured.

Previous studies since 1965 have shown that BMPs promulgate the TGFβ superfamily and, like other members of this gene family, have multiple effects on cell motility, growth and differentiation, particularly during bone formation and tissue repair processes, but also, - embryogenesis and cancer. They are small, hydrophobic glycoproteins of molecular size that are soluble in chaotropic agents such as urea and guanidine hydroxide 35 but are resistant to several proteases, such as collagenases.

2 BMPs are produced as large precursor molecules which are processed into proteolytically mature peptides after translation. Like other members of the TGF-β superfamily, BMPs contain a sequence of seven cysteine residues in the mature C-terminal portion. In mature BMP monomers, between these cysteines there are three disulfide bonds and one disulfide bond which combines the two monomers to form a biologically active dimer.

The effect of BMP is mediated through specific receptors on the cell membrane of target cells. B1VIP receptors are serine-threonine kinases that are similar to TGF-? Receptors and are divided into two subgroups: type I and type II receptors. BMPs can only bind tightly to the receptor that forms the heterotetrameric complex. The formation of such a complex is a prerequisite for BMP signal transduction. Within the target cell, BMP signals are transferred to the nucleus using 15 specific signal transduction molecules called Smads, which are also capable of attenuating BMP signals.

To date, 16 different BMP molecules have been characterized, seven of which (BMPs 2-7 and 9) have been shown to be able to induce bone formation when implanted into ectopic sites. Based on the amino acid sequence of the mature portion, the BMPs are divided into two subgroups. BMPs 2 and 4 are 86% identical to each other and BMPs 5, 6 and 7 are 78% identical. The identity between the two subgroups is only about 56%. The amino acid sequence of BMP-3 is about 45% similar to that of BMP-2 and BMP-4, and BMP-9 is 50-55% identical * 25 to BMPs 2, 4, 5, 6 and 7 with. Due to the high homology and slight difference in size of molecules, separation, purification and identification of BMPs at the protein level is quite difficult, time consuming and expensive. For this reason, today most BMPs are produced using molecular biology techniques. Various recombinant protein techniques have been tried and both eu-30 karyotic and prokaryotic systems have been used.

Most of the research has been directed to recombinant human BMP, but the Ce / v / dae family is also an interesting area of study because of their efficient bone induction in horns. Horns are bone-bearing skeletal structures typical of the Ce / v / dae family and differ from the Bovidae horns in their growth. Horns grow from the tip of the horn and are dropped by males once a year. It has been argued that horns are the fastest growing structures in mammals, and these structures are known to be the only ones that are completely renewed every year. A certain type of endochondral ossification occurs during the formation of horns, where the process proceeds through the presence of abundant vascular cartilage, which eventually becomes bone upon calcification. The horns provide an interesting model for the regeneration of mineralizing tissue in a full-5-year-old animal and bone regeneration continues until the horns are dropped. Although the root cause of the astounding growth rate of horns has not been elucidated, several hormone BMPs are known to exist in horns. Deer horns have been shown to produce BMP-2 and BMP-4 (Feng et al. 1997 Biochim Biophys Acta 1350: 47-52; Feng et al. 1995 Biochim Biophys Acta 1263: 163-168). Additionally, BMP-3b is expressed in 10 reindeer horns (Kapanen et al. 2002 J Biomed Mat Res 59: 78-83). However, it is also possible that the horns have one or more unknown factor (s) causing rapid horn growth.

Because of their osteoinductive properties, both BMPs extracted from demineralized 15 bone matrix and BMPs produced using recombinant technology are very interesting and highly potential alternatives for bone graft. Various BMP proteins have been used in several experimental and clinical studies.

Bone morphogenetic protein 6 has been isolated from various sources, including some mammalian species such as mouse, rat, human and bovine. However, it has not been characterized by hir-embryos, unlike BMP-2, BMP-3b and BMP-4, all cloned / * from either deer or reindeer horns, both of which belong to the Cerv / dae family (Feng et al. 1997 Feng et al. 1995; Kapanen et al. 2002). Given that BMP-6 is an important regulator of chondrogenesis, it is likely that it will be expressed in horns as other members of the BMP gene family that have already been cloned.

To date, BMP-3b is the only BMP that has been characterized by reindeer horn tissue (Kapanen et al., 2002).

US 5,399,677 discloses DNA molecules encoding mutant forms of bone morphogenetic proteins. Mutant forms of BMP can be produced in bacteria and folded into the biologically active form either as a homodimer or as a hete-35 rhodimer of BMP. The method by which said mutant BMP is made is also disclosed. These mutant forms are useful because they are flawlessly folded when produced in a bacterial host.

WO 98/51354 discloses osteogenic devices and methods of using them to repair bone and cartilage defects. A method for growing new bone at a damaged bone site in a mammal comprises the step of implanting a calcium phosphate matrix containing at least one osteogenic protein into the lesion site. The osteogenic proteins in question comprise several morphogens, such as bone morphogenetic proteins.

US 6207 813 discloses purified human BMP-6 protein and processes for its production. The publication also discloses well-known pharmaceutical compositions, medical uses and methods employing the human BMP-6 protein. BMP-6 is localized to hypertrophic cartilage and is an important regulator of chondrocyte maturation. When CHO cells overexpressing murine BMP-6 protein were directly injected into the subcutaneous tissue of athymic nude mice, they developed tumors surrounded by a large connective tissue region containing a large amount of cartilage and bone. This suggests that BMP-6 induces endo-chondral bone formation in vivo. BMP-6 has also been shown to increase osteoblastic differentiation of pluripotent mesenchymal stem cells in vitro in cells transfected with a vector overexpressing murine BMP-6 protein. In addition, recombinant human BMP-6 has been shown to be involved in osteoblastic differentiation of 20 bone marrow stromal cells in vitro. The osteoinductive activity of recombinant human BMP-6 produced in CHO cells has been confirmed by an in vivo rat muscle test. Nevertheless, the biological activity of recombinant BMP-6 from any source produced in the E. coli system has not been published. Also, no one has published data on the biological activity of the murine BMP-6 protein cloned from the corneal tissue of any animal belonging to the genus Hans Cervidae.

EP 1131087 discloses the additional use of morphogenetic proteins such as BMP. It has been shown that contact of cancer cells with morphogens inhibits the growth of V 30 cancer cells and causes their differentiation away from the cancer cell phenotype. The use of morphogens can affect the cellular fate of cancer cells and help to reduce the symptoms of cancer. BMP-6 is one of the preferred morphogens shown.

While applications of some BMPs as known as bone and cartilage inducers, as well as in the alleviation of cancer symptoms, are known, there is a need for even better methods for isolating these proteins, and in particular for obtaining better morphogenetic proteins, such as more soluble than at 5 sem. Such proteins would have utility in improved therapeutic methods and applications.

Summary of the Invention 5

Surprisingly, the novel BMP-6 protein of the present invention, isolated from a reindeer and having high sequence homology to other already known bone morphogenetic proteins, has highly beneficial properties related to bone and cartilage formation. These properties are significantly better than those of the already known BMP proteins. The bone morphogenetic proteins of the present invention and their homologs are useful as bone and cartilage inducers in a variety of applications, such as therapeutic applications. The isolated bone morphogenetic protein may also contain a propeptide moiety of the protein. The presence of this moiety can stabilize BMP and alter its functional activity.

Furthermore, the cDNA of the BMP-6 protein of the present invention was truncated without causing any alteration in the protein sequence and when produced as a recombinant protein in the host cell system, the highest possible level of expression was obtained. The heparin si-20 binding site (HBS) was added upstream of the rdBMP-6 gene and this addition also allowed expression in Eco / TOP10 cells in which expression was inhibited by (without HBS) rdBMP-6 alone.

The present invention provides an isolated bone morphogenetic protein 6 (BMP-6) containing the consensus sequence PG / S / NR / KR / HR / KQ / NQA / S / NRN / S-77 R / A / KS / AT / S / NP and a heparin binding site at its amino terminus 77 containing the amino acid sequence AKHKQRKRGT.

Another aspect of the present invention relates to said isolated bone morphogenetic protein comprising a BMP propeptide.

Another aspect of the present invention relates to an isolated DNA molecule encoding said morphogenetic protein.

Yet another aspect of the present invention pertains to a nucleic acid vector which contains said isolated DNA molecule.

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Yet another aspect of the present invention pertains to a recombinant host cell comprising said DNA molecule or the aforementioned nucleic acid vector.

Yet another aspect of the present invention pertains to a bone morphogenetic protein 5 produced by growing said host cell so as to express said bone morphogenetic protein and recovering said bone morphogenetic protein from said host cell.

Yet another aspect of the present invention pertains to a recombinant host cell expressing said bone morphogenetic protein.

Yet another aspect of the present invention pertains to a pharmaceutical composition comprising said bone morphogenetic protein.

Yet another aspect of the present invention pertains to said isolated bone morphogenetic protein for use as a medicament.

Yet another aspect of the present invention pertains to the use of said isolated bone morphogenetic protein in the manufacture of a medicament for use in the treatment of bone and cartilage disorders where regeneration, repair or growth is desirable, or other diseases such as cancers.

Yet another aspect of the present invention pertains to an osteogenic device for treating said disorders, which device comprises a morphogenetic protein of said bone.

; Yet another aspect of the present invention pertains to a method of inducing cartilage and / or bone formation by treating said cartilage and / or bone with said isolated bone morphogenetic protein.

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Yet another aspect of the present invention pertains to a method of treating such bone and cartilage disorders in which regeneration, repair, or growth is desirable, or other diseases, such as cancer, by administering said isolated bone morphogenetic protein to a patient suffering from said disorder. .

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Brief summary of pictures

Figure 1 shows plasmids containing several different inserts in the PCR vector pGEM-T® (Promega).

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Figure 2 shows plasmids containing several different inserts in the expression vector pTrchis2A (Invitrogen).

Figure 3 shows plasmids containing several different inserts in the expression vector pET-22b (+) (Novagen).

Figure 4 shows a plasmid containing an insert in the expression vector p1-VEX2.4C (Roche) RTS500.

Figure 5 shows the amino acid and nucleotide sequences of the mature portion of the reindeer BMP-6 protein produced in pMU20 and pMU90. The mature portion of the reindeer BMP-6 protein is flanked, mutated nucleotide codons are indicated in gray boxes, and modified nucleotides are indicated in bold letters. Cysteines typical of the TGF-β gene family are also labeled in bold.

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Figure 6 shows the nucleotide sequences of the mature portion of the reindeer BMP-6 protein and the heparin binding site (HBS) produced in pTrcHBSrd6A and pTrcHBSrd6 plasmids. The heparin binding site is indicated in italics and in bold. The mature portion of the reindeer BMP-6 protein is flanked, the mutated nucleotide codons are marked in gray boxes and the modified nucleotides are shown in bold. Cysteines typical of the TGF-β gene family are also labeled in bold.

Figure 7 shows the amino acid and nucleotide sequences of the mature portion of the reindeer protein BMP-6-30 produced in pETrd6A and pETrd6. The mature portion of the reindeer BMP-6 protein is flanked, mutated nucleotide codons are indicated by gray boxes, and modified nucleotides are indicated by bold letters. Cysteines that are typical of the TGFβ gene family are indicated in bold.

Figure 8 shows the amino acid and nucleotide sequences of the mature portion of the reindeer BMP-6 protein and the heparin binding site (HBS) produced in pTrcETrd6A and pTrcETrd6. The heparin binding site is indicated in italics and in bold. The mature portion of the reindeer BMP-6 protein is flanked, mutated nucleotide codons 8 are indicated in gray boxes and modified nucleotides are indicated in bold letters. Cysteines typical of the TGF-β gene family are also labeled in bold.

Figure 9 shows the amino acid and nucleotide sequences of the mature portion of the reindeer BMP-6 recombinant protein produced in pMU200. The mature portion of the reindeer BMP-6 protein is flanked. Cysteines that are typical of the TGF-β gene family are also labeled in bold.

Figures 10A and B (continued) show part of the amino acid and nucleotide sequence of reindeer BMP-6. The mature portion is flanked and the cysteines typical of the TGF-β gene family are indicated in bold letters. Amino acids 1-118 before the mature portion represent the BMP propeptide.

Figure 11 shows a SDS-PAGE gel stained with Coomassie Blue dye with fractions collected during purification from A) rdBMP-6 and B) rdBMP-6 containing a heparin binding site. The zones represent 1) starting material; 2) the standard; 3) material passed through the column; 4) first washing; 5) a second wash; 6) Elution 1; 7) Elution 2; 8) elution 3; and 9) elution 204 (rd-BMP-6-HBS).

Figure 12 is an X-ray of the hind paw muscles. A) an implanted implant containing human BMP-6 protein (control) and B) an implanted implant containing BMP-6 protein of the present invention.

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Detailed Description of the Invention

The homology between the mature portions of previously known BMP-6 proteins is highest between mouse and rat (98%) and between rat and human (98%) (Table '30 L). Cloning and characterization of the mature part of reindeer BMP-6 showed that it has the highest homology with bovine BMP-6 (Wozney et al / 1998). at the amino acid; : only one amino acid in the cell was different when comparing the two polypeptides and the homology reached 99% (Table 1). Even at the nucleic acid level, the homology between the mature portions of the reindeer and bovine BMP-6 was 95%, which is as high as between the mouse and rat (Table 1). In general, BMP-6 also has homology with other types of BMP, such as BMP-5 and BMP-7.

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Origin of Reindeer Cattle Human Mouse Rat nt ah nt ah nt ah nt ah nt ah

Poro 100 100 95 99 84 95 84 93 84 95

Cattle__95 99 100 100 84 94 85 92 85 92

Human 84 95 84 94 100 100 88 96 88 98

Mouse__84 93 85 92 88 96 100 100 95 98

Rotta I 84 I 95 l 85 l 94 I 88 | 98 I 95 I 98 Ϊ00 j 100

Table 1 Homology of mature portions of BMP-6 from different mammals at nucleotide and amino acid levels (%) (nt = nucleotides; ah = 5 amino acids)

The alignment below shows the amino acid sequences of the mature portion of the human and reindeer BMP-6 protein (comparison made using ClustalX 1.8 (Thompson, JD, Higgins, DG and Gibson, TJ (1994) Nucleic Acids Research, 22: 4673-4680), 10 rdBMP-6 = reindeer BMP-6, hBMP-6 = human BMP-6, star indicates identical amino acids). The amino acid sequences of the mature portions differ in the amino terminal region with respect to seven amino acids. The major differences are likely to be due to the prolines of rdBMP-6, Pro3, and especially Pro16, which are likely to affect the folding or structure of the mature protein. The closest equivalent of reindeer BMP-6 is human BMP-6, which has been assayed for activity.

I rdBMP-6 SAPGRRRQQARNRSTPAQDVSRASSASDYNSSELKTACRKHELYVSFQDLGWQDWIIAPK

; hBMP-6 SASSRRRQQSRNRSTQSQDVARVSSASDYNSSELKTACRKHELYVSFQDLGWQDWIIAPK

20 ** *****. *****; ***. * ******* ******************** *** *******

rdBMP-6 GYAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDN

hBMP-6 GYAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDN

k'k-k'ic-ic-k ^ 'k'k'k-k-fi'k-kk ^ e-k'k-A - k -k-fr-kkkk ^ kk-fc-kkkkk- if-kkkkk k-k'ik'k'k'kifir 0 ': 25

: ,,,; rdBMP-6 SNVILKKYRNMVVRACGCH

hBMP-6 SNVILKKYRNMVVRACGCH

30 ************************************************ A "BMP-6 protein of the invention" or "bone morphogenetic protein of the invention" refers to a protein having bone morphogenetic activity (or morphogen, both of which can be used synonymously), such as the reindeer-derived BMP-6 protein described herein (SEQ ID NO: 1 in the sequential listing or rdBMP-6 above), and includes homologues, analogs thereof , derivatives and fragments. The definition also refers to the above modifications such as BMP containing a specific functional sequence, for example a heparin binding site, a propeptide or the like. Such homo logs or derivatives include functional derivatives of the protein in question, such as those derived from the parent BMP-6 protein or any other BMP of any other species. The derivatives may differ in length and may include amino acid additions, deletions or substitutions as well known to those skilled in the art. A characteristic feature of the bone morphogenetic protein of the present invention, for example as shown in the above alignment, are amino acids which are different from other known BMP-6 proteins, such as amino acids which are different with the human counterpart, especially Pro3 and Pro16. It is likely that the regions in which these amino acids are located are conserved in the bone morphogenetic protein of this invention. These may be amino acids 3-23 of SEQ ID NO: 1 or a homologue thereof, or more preferably amino acids 3-16. Amino acids 3 and 16 are prolines which have a strong effect on the folding of the mature protein and hence on the function of the protein. Amino acids 3 and 23 define the region where the amino acids differing from the human BMP-6 counterpart are located.

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On the other hand, additions, deletions, and substitutions far from said characteristic region are unlikely to cause a substantial change in the function, potency, or folding of the BMP protein of the invention. For example, homologues with deletions, such as deletion of a few amino acids, preferably 1-10 amino acids, more preferably 1-5 amino acids, most preferably 1-3 amino acids, either at the carboxyl or amino terminal end of the "V", wherein the polypeptide is short in size. ", Empi, are within the scope of this invention so long as such deletions do not affect the amino acids characteristic of the BMP of the invention. It is preferred that the homologues in question have the beneficial properties of the original reindeer BMP-4 associated with said characteristic amino acids Pro6 and / or Pro21 and Said homologues may have amino acid substitutions that do not substantially affect the function and potency of said protein. For example, an amino acid not located in or in the vicinity of an active site may be replaced by another having a similar structure and / or chemical properties. (for example, h hydrophilicity or hydrophilicity), this means conservative amino acid exchange as long as the substitution in question does not substantially alter the function or folding of the mature protein. Such substitutions are well known and understood in the art. Examples of these 11 amino acid properties divided into different groups are hydrophobic (Met, Ala, Val, Leu, lie), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gin, His, Lys, Arg) ), amino acids that affect chain orientation (Gly, Pro), and aromatic (Trp, Tyr, Phe) amino acids. Substitutions within the groups in question are unlikely to result in significant changes in the structure of the polypeptide chain backbone (e.g., pleated or helical conformation), charge, or hydrophobicity or side chain size of the molecule.

For example, the BMP homologs of the present invention comprise any bone morphogenetic protein containing or modified to contain at least a portion of the conserved amino acid or sequence as set forth above, or a corresponding region in a homologous BMP protein having different numbering. . Any currently unknown BMP-6 protein of any species modified as above is within the scope of this invention.

Compared to the known human BMP-6 protein, the reindeer BMP-6 protein has seven substituted amino acids P3, G4, A10, P16, A17, S21 and A23, as defined by the mature BMP-6- set forth in SEQ ID NO: 1. protein. These amino-20 acids are characteristic of the BMP of the present invention. More specifically, P3 and / or P16 are particularly characteristic of the BMP of the present invention.

In one embodiment of the present invention, BMP is any BMP, or homologue, derivative or fragment thereof, containing the consensus sequence between two proline: PG / S / NR / KR / HR / KQ / NQA / S / NRN / SR / A / KS / A- T / S / NP. This consensus sequence is determined from the alignments of several similarly related BMP-6, BMP-5, and BMP-7 proteins of different species as shown in the ClustalX alignment below. This consensus sequence corresponds to amino acids 3-16 of SEQ ID NO: 1. In one embodiment of the invention, the BMP of the invention is any BMP or homologue, derivative, or fragment thereof comprising the corresponding consensus sequence defined by the BMP-6 family: P-G / S-R-R-R-Q-Q-A / S-R-N-R / A-S-T-P. Other consensus sequences may also be defined, for example, defining the region around another proline (Pro16 in SEQ ID NO: 0 1), such as RN / SR / A / K-35 S / AT / S / NPAQDV, or a similar sequence that differs in length, for example 1-5 amino acids. Corresponding consensus sequences may be determined from the alignment below, or from the corresponding alignments made by aligning various closely related BMPs. The aligned BMP-6, BMP-5, and BMP-7-12 sequences are from reindeer, bovine, rat, mouse, human, chicken and African nail frog (Xenopus laevis).

BMP6_REINDEER SAPGRRRQQARNRSTPAQDVSRASSASDYNSSELKTACRKHELYVSFQDLGWQDWIIAPK

5 BMP6_B0VINE SAPGRRRQQARNASTPAQDVSRASSASDYNSSELKTACRKHELYVSFQDLGWQDWIIAPK

BMP6_RAT SASSRRRQQSRNRSTQSQDVSRGSSASDYNSSELKTACKKHELYVSFQDLGWQDWIIAPK

BMP6JVIOUSE SASSRRRQQSRNRSTQSQDVSRGSGSSDYNGSELKTACKKHELYVSFQDLGWQDWIIAPK

BMP6_H0MAN SASSRRRQQSRNRSTQSQDVARVSSASDYNSSELKTACRKHELYVSFQDLGWQDWIIAPK

BMP5_H0MAN -AANKRKNQNRNKSSSHQDSSRMSSVGDYNTSEQKQACKKHELYVSFRDLGWQDWIIAPE

10 BMP5_CHICKEN AANNKRKNQNRNKSSSHQESSRMPSVGDYNTSEQKQACKKHELYVSFRDLGWQDWIIAPE

BMP5_M00SE -AASKRKNQNRNKSNSHQDPSRMPSAGDYNTSEQKQACKKHELYVSFRDLGWQDWIIAPE

BMP7_XENOPUS SAGGKHRNQNRSKAPKSQEALRVSNIAENSSTDQKQACKKHELYVSFKDLGWQDWIIAPE 'k »· ·> r k k k k · kk kk · ·

15 BMP6_REINDEER GYAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDN

BMP 6_B0VINE GYAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDN

BMP6_RÄT GYAANYCDGECSFPLNAHMNÄTNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDN

BMP6_MOUSE GYAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDN

BMP6_HÖMAN GYAANYCDGECSFPLNAHMNÄTNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDN

20 BMP5_HUMAN GYAAFYCDGECSFPLNAHMNATNHAIVQTLVHLMFPDHVPKPCCAPTKLNAISVLYFDDS

BMP5_CHICKEN GYAAFYCDGECSFPLNAHMNATNHAIVQTLVHLMFPDHVPKPCCAPTKLNAISVLYFDDS

j '. , BMP5_MOUSE GYAAFYCDGECS FPLNAHMNATNHAIVQTLVHLMFPDHVPKPCCAPTKLNAISVLYFDDS

BMP7_XENOPUS GYAAFYCEGECAFPLNSYMNATNHAIVQTLVHFINPDTVPKPCCAPTQLNPISVLYFDDS

w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w a w

5. BMP6_REINDEER SNVILKKYRNMVVRÄCGC H

BMP6_BOVINE SN FLIGHT YRNMVVRACGCII

BMP6_RAT SNVILKKYRNMVVRACGCH

BMP6_MOUSE SNVILKKYRNMVVRACGCH

30 BMP6_HOMAN SNVILKKYRNMVVRACGCH

BMP5__H0MAN SNVILKKYRNMVVRSCGCH

'BMP5__CHICKEN SNVILKKYRNMVVRSCGCH

,:; BMP5J40USE SNVILKKYRNMVVRSCGCH

BMP7_XENOPUS SNVILKKYRNMVVRACGCH

35 k k k k-k k kk k k k kk k * kkk k

In one embodiment of the invention, the BMP is any BMP, homologue or fragment thereof comprising amino acids 3-16 of SEQ ID NO: 1.

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The position numbers of these amino acids are calculated from the amino terminus of the mature portion of any common BMP-6 protein, such as the protein shown in SEQ ID NO: 1 or a homologue, derivative or fragment thereof, wherein the amino-terminal sequence begins with amino acids SA, such as reindeer (cat-5 i.e. the above sequence alignment or SEQ ID NO: 1), or the like. If there are numbering additions or deletions in the amino acid sequence of the homologue in question, this should be taken into account when defining the positions of said essential amino acids, for example, by aligning the sequences as described, and then determining the positions of said amino acids. However, any of the homologues, derivatives, or fragments of the BMP-6 protein in question should substantially include the function and efficacy disclosed herein. However, since all known BMP-6 proteins are highly conserved, the position determination of these essential amino acids is unambiguous, as is the case with human BMP-6. These sites can also be readily determined from other BMPs (see alignment above). In general, their degree of homology at the amino acid level may be at least 70%, preferably 80%, more preferably 90%, and most preferably 93%.

In one embodiment of the invention, the BMP is any BMP or homologue 20, derivative or fragment thereof comprising amino acids 3-23 of SEQ ID NO: 1. In one embodiment of the invention, BMP is a BMP-6 protein. In yet another embodiment of the invention, BMP is BMP or a homologue, derivative or fragment thereof comprising the amino acid sequence of SEQ ID NO: 1. The homologues, derivatives, or fragments mentioned in these embodiments should contain at least one of the characteristic amino acids described above. The homologues, derivatives or fragments in question do not include known BMP-6 proteins as such, as hBMP-6, since they do not contain the amino acids that are characteristic of the BMP protein of the present invention. However, if the already known BMP is modified to include at least one of the characteristic amino acids in question, it can be considered as such a homologue, derivative or fragment.

In addition, the BMP described above contains a heparin binding site (HBS). Generally speaking, this is an amino acid sequence capable of binding heparin. Said heparin binding site is located at the amino terminus of said BMP, such as prior to SEQ ID NO: 1 or its functional homology. In one embodiment of the invention, the heparin binding site comprises the amino acid sequence AKHKQRKRGT (Figure 8) or QAKHKQRKRGT (Figure 6). Said heparin binding site may also be a functional homologue, derivative or fragment thereof.

In one embodiment of the invention, the BMP described above contains the propeptide shown in SEQ ID NO: 2 or a functional homologue, derivative or fragment thereof. The propeptide in question plays a role in controlling BMP and proper folding of the protein. Similar propeptides are known in the art (see Regulation of bone morphogenetic protein activity by pro domains and proprotein convertases. Constam DB, Robertson EJ. J Cell Biol. 1999 Jan 11; 10 144 (1): 139-49). The BMP propeptide or homologue thereof may be linked to the amino terminal end of the BMP protein, such as BMP-6, where the propeptide allows folding of the mature protein. Subsequently, the propeptide can be cleaved off.

One embodiment of the present invention provides a nucleic acid molecule, such as a DNA or RNA molecule encoding said BMP of this invention, with or without a heparin binding site or propeptide. Due to the degeneracy of the genetic code, there are several different nucleic acid sequences encoding the BMP of the invention, the heparin-binding site or the propeptide. All such nucleic acid variants are within the scope of the present invention. Preferably, the nucleic acid molecule is a DNA molecule. Non-limiting examples of such DNA sequences are shown in Figures 5-10.

One embodiment of the present invention provides a replicable vector containing the above-described nucleic acid molecule operably linked to a sequence that controls its expression. Such vectors may be used to produce recombinant BMPs of the invention in a suitable host system.

The nucleic acid encoding the BMP of the present invention can be inserted into the 30 replicable vectors for cloning or expression. Several vectors are publicly available. The vector may be, for example, in the form of a plasmid, cosmid, viral particle or phage. The appropriate nucleic acid sequence may be inserted into a vector using a variety of procedures. Generally, DNA is ligated to a suitable restriction endonuclease cleavage site using methods well known in the art. The vector may be, for example, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of such suitable vectors containing one or more of these components utilizes standard ligation techniques well known to those skilled in the art.

Generally, the BMP in question can be produced by gene expression by expression in any suitable host cell, such as a bacterial cell. Such methods are well known in the art and can be found in the literature. It is essential that the protein folds into the correct form during expression and that it contains all necessary post-translational modifications.

10 It is not always possible to produce and purify certain proteins properly, for example due to problems with solubility and refolding. In general, E. coli cannot make the post-translational modifications typical of mammalian cell systems. However, the inventors have produced a mature portion of reindeer BMP-6 as a recombinant protein in E. coli and, after purification and refolding, have shown that it is in a biologically active form.

One embodiment of the present invention provides a host cell comprising a nucleotide molecule or nucleotide vector described above. Useful cells include all prokaryotic and eukaryotic cells capable of expressing the protein of this invention. Such host cells are well known in the art and one of ordinary skill in the art can readily select a suitable host cell. One embodiment provides BMP produced by growing said cells to express said protein and recovering said produced protein from said host * target cell. Any useful method can be used for protein recovery and purification and such methods are well known in the art.

The BMP of the present invention may be used in the treatment of disorders affecting bone, cartilage, tendon or tooth defects or diseases, or the like where regeneration, repair, or growth is desirable, or other diseases. The protein of the present invention may also be used for wound healing, such as burns, surgical wounds and stomach ulcers and similar tissue repair, as well as for the treatment of cancer, as disclosed in EP1131087. Because the BMP protein generally lacks species specificity, the patient suffering from the defect in question may be any suitable animal, preferably a mammal such as a human, and the BMP protein used in the treatment may be derived from any suitable source. The use of similar BMP proteins in many therapeutic applications is well known in the art (see, for example, US 6245889 and WO 98/51354).

"Disorders Related to Bone, Cartilage, Tendon or Tooth Defects" as used herein generally refers to any disorder in which healing or rebuilding, i.e. regeneration, of bone, cartilage, tendon, or teeth is desirable. Non-limiting examples of treatment measures for disorders or diseases of bone, cartilage, tendon or teeth, or the like, include regeneration, repair, and growth of bone and dental tissue; bone regeneration, repair and growth in 10 mammals such as man; treatment measures to correct abnormalities in bone formation or regeneration; wound healing, ectopic bone induction, and healing of segmental bone damage in vertebrates; treatment measures to correct skeletal disorders and deformities; repair of major bone damage due to trauma, removal of tumors, or congenital malformations, reconstruction of reduced bone stores due to implanted internal prosthesis in repair operations, or delayed healing of fractures or unrelated bone fractures; repair of bone and cartilage defects such as '' critical size defects '', 'non-critical size defects', unrelated fractures, segmental unrelated fractures; acute fractures, cartilage defects, bone-cartilage defects, cartilage bone defects, local bone and cartilage formation; defects due to degenerative diseases; . . O; dental applications for repairing dental tissue, alveolar bone, dental cement, tooth root membrane, filling tooth root canal contents, developing and improving dental implant attachment, 25 More examples of these disorders can be found in Ann Rheum Dis, Volume 62, 2003, 73-78: Reddy AH: Cartilage Morphogenic Proteins: Role in Joint Development, Homeostasis and Regeneration.

Other diseases where the BMP of this invention is useful include cancer, fibromyalgia, psoriasis and other dermatological disorders, and rheumatic disorders and the like. Examples of such cancers and methods and compositions for treating them are disclosed in EP1131087.

One embodiment of the invention provides a BMP, such as BMP-6, for use in any of the embodiments described herein, in combination with one or more other mor-35 phagenetic proteins, such as another type of BMP or the like. Generally, this achieves synergy benefits, as is known in the art. Examples of other suitable BMP proteins include, but are not limited to, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, other BMP-6, BMP-7 and BMP-8. 17 other therapeutically useful agents can also be used, such as epidermal growth factor, fibroplastic growth factor and transforming growth factor (US 6,245,889). In one embodiment of the invention, the complementary morphogenetic protein in question is derived from a reindeer, like any other reindeer BMP protein. In one embodiment of the invention, the BMP is provided for use as a dimer, homodimer, or heterodimer in combination with another BMP protein as described above. In another embodiment, the BMP is provided for use in dimeric form or in combination with another agent or protein, as described above, for the manufacture of a medicament for the treatment of disorders as described in the Explanatory Note.

One embodiment of the invention provides an osteogenic device, such as an implant, containing the BMP of the present invention. The osteogenic device may include a biocompatible matrix, such as calcium phosphate, carboxymethylcellulose, collagen matrix, or combinations thereof. In one embodiment, the calcium phosphate matrix is a hydroxyapatite matrix. Such a matrix may allow slow release of the BMP protein and / or a suitable environment for release of the BMP protein. The osteogenic device may also include a metallic implant surrounded by said biocompatible matrix. One example of such 20 metals is titanium. Some examples of such osteogenic devices are disclosed in WO 98/51354.

Non-limiting examples of various supports, carriers, or supports for forming, for example, various osteogenic devices or the like containing the protein of the present invention have the following various forms: powder, sponge, tab, membrane, gel, web or solution or suspension; a semi-solid solution carrier which can be used for intramuscular, intravenous, bone marrow or intra-articular injection; isolated mesenchymal stem cells, any pharmaceutically acceptable carrier; powdered auto and allografts, any pharmaceutically acceptable matrix, material selected from the group consisting of hydroxyapatite, collagen, polymers (e.g., polylactic and polyglycolic acid polymers), synthetic polymers, hyaluronic acid, α-BSM, tricalcium phosphate, non-porous ceramic biopolymer, non-porous resorbable biopolymer, coral, de-35 mineralized bone, bio glass, any biodegradable material and combinations thereof; binding agents selected from the group consisting of mannitol, dextrans, white petrolatum, alkyl and methylcelluloses, wetting agents such as sodium salts, phobrin glue, mammalian fibrinogen and thrombin, and combinations and mixtures thereof. The osteogenic device may be, for example, a structurally stable, three-dimensional implant in the shape of a cube, cylinder or block or anatomically shaped or injectable. Examples of osteogenic tools, useful materials, and techniques are disclosed in Skeletal reconstruction and bioimplantation (T. Sam Lindholm, 1997, Springer-Verlag, Heidelberg, Germany).

One embodiment of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the BMP of this invention in a pharmaceutically acceptable carrier or carrier. The pharmaceutical compositions may be used to treat disorders associated with bone, cartilage, tendon or tooth damage or other diseases, wounds and other tissue damage, or any of the disorders described herein.

An embodiment of the invention provides a method of inducing bone, cartilage, tendon, tooth, or the like, wherein said bone, cartilage, tendon, tooth, or the like is treated with the BMP of the present invention or combinations thereof as described above, in vitro or in vitro. vivo. Yet another embodiment of the invention provides a method of treating the disorders described in the specification, comprising administering an isolated BMP of the invention to a patient suffering from such disorders. The BMP in question may be administered as a pharmaceutical composition or as an osteogenic device as described above. Complementary morphogenetic proteins or other useful substances may be administered in combination with the BMP of the present invention as described above to enhance the therapeutic effect.

The following description and examples describe how the mature portion of a reindeer recombinant BMP-6 with or without a heparin binding site (HBS), as disclosed in some embodiments of the present invention, is produced in E. coli. After purification and folding, osteoinductive activity was confirmed by bioassay in the mouse hindlimb muscle pocket. This in vivo bioassay has been a standard method for determining BMP activity since the discovery of the protein. It involves BMP implantation of mouse hind foot muscle and assessment of heterotropic new bone induction radiologically and histologically 35 10 to 21 days after implantation.

In the assay of BMP activity, image analysis is performed by transmitting an X-ray through the bone, which is seen on the film as an X-positive darkening 19 when compared to soft tissue. This allows radiographic detection and radiomorphometric quantification of newly formed bone obtained by implantation of BMP or other bone inducing agent at a heterotopic or orthotopic site.

5

Osteoinductivity was observed in all three groups studied (1 mg, 3 mg, and 5 mg recombinant reindeer BMP-6) and the response increased with dose (Table 2). When compared to the recombinant protein of reindeer BMP-6 against that of human, reindeer BMP-6 was a more potent inducer of bone formation, making it a potent agent for clinical applications.

Doses__1 mg 3 mg 5 mg

Poron BMP-6 __-__ (+) -__ k ____ (+) + __ ++ + ~

Poron BMP-6-HBS --- +++ (+) - (+) + (+) + + (+)

Human BMP-6 __ + - - (+) - (+) (+) - + __ (+)

Bovine BMP-6 ........ k - (+) + 15 Table 2. Comparison of the osteoinductive response of reindeer and human BMP-6 proteins at different doses (k = discontinued).

The results of the in vivo bioassay are shown in Figure 12. Figure 12A is a control using human BMP-6 as an implant and 12B is an implant containing the BMP-6 protein of the present invention. The bioassay was performed as described in Marshall R. Urist, J.J. Chang, A. Lietze, Y.K. Huo, A.G. Brownell and J. DeLange (1987): Preparation and Bioassay of Bone Morphogenetic Protein and Polypeptide Fragments, Methods Enzymol 146: 294-312.

Initially, expression of the mature part of the recombinant rdBMP-6 in E. coli caused great difficulty. Even mutations made to specific codons to make the codon usage of rdBMP-6 similar to that of E. coli had no healing effect on the expression level. Therefore, the researchers hypothesized that poor expression would be due to high GC content in the N-terminal region of the mature portion of rdBMP-6. Kos-30 ka heparin binding site (HBS) is at the beginning of the mature portion of reindeer BMP-2 and has a low GC content, a construct was made where HBS was placed at the beginning of the mature portion of rdBMP-6 and thus the inventors were able to improve recombinant rdBMP. Expression level of -6.

HBS, located in the N-terminal portion of rdBMP-2, contains 10 basic 5 amino acids and resembles known or disclosed heparin binding sites for other growth factors. These sites are believed to be important for the stability and stability of the proteins and may directly contribute to the biological activity of the proteins. It is also possible that, during development, the interaction of the HBS-containing protein with the extracellular matrix could play an important role in creating a morphogenetic gradient limiting the free diffusion of the protein. Therefore, it was assumed that HBS would also be able to improve the biological activity of the recombinant rdBMP-6 by extending the time before protein loss at the implantation site, which was also demonstrated by bioassay.

15 Examples

Example 1: Cloning and Sequencing Reindeer BIWIP-6 cDNAm 3 'to End A. Isolation of RNA

Three-year-old male reindeer horns were severed and frozen in liquid nitrogen immediately after slaughter. The frozen horns were cut into 0.5 cm pieces and stored at -70 ° C. Reindeer horn mRNA was isolated using the QuickPrep® Micro mRNA Purification Kit (Pharmacia Biotech). A portion of the reindeer horn slice was cut into small pieces (about 1 mm 3) and 0.1 g of this tissue was added to 0.6 mL of Extract-on buffer containing guanidine thiocyanate and N-lauroyl sarcosine. The tissue was homogenized on ice with Ultra Turras three times for 10 seconds and cooled between homogenization runs. 1.2 ml of Elution buffer was added and the suspension was homogenized once more for 10 seconds. The end result was a uniform suspension.

The reindeer horn homogenate and the Oligo (dT) cellulose were centrifuged at full speed [14,000 rpm, room temperature, Centrifuge 5415 C (Eppendorf)) for one minute. The buffer on the oligo (dT) cellulose precipitate was removed and the clear K? 35 homogenate was pipetted in its place. The tube was inverted to obtain a suspension of Oli-go (dT) cellulose. The suspension was gently stirred for five minutes and centrifuged at full speed [14,000 rpm, room temperature, Centrifuge 5415 C (Eppendorf)] for ten seconds. The supernatant was discarded.

The oligo (dT) cellulose was resuspended in High-Salt Buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 M NaCl], and the suspension was centrifuged at full speed [14,000 rpm, room temperature, Centrifuge 5415 ° C (Eppendorf)] for ten seconds. Washes with High-Salt Buffer were repeated five times, and twice with Low Salt Buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 M NaCl], Three ml Low Salt Buffer was added and the suspension was transferred to a MicroSpin column. The MicroSpin column was placed in an Eppendorf tube and centrifuged at full speed for five seconds. Column Oligo (dT) cellulose was rinsed three times with Low Salt Buffer.

Reindeer horn mRNA was eluted from the MicroSpin column into a clean Eppendorf tube 15 by adding 0.2 ml of 65 ° C Elution Buffer (QuickPrep® Micro mRNA Purification Kit, Pharmacia Biotech) to the column and centrifugation at full speed [14,000 rpm, Centrifuge 5415 ( Eppendorf)] for five seconds. The elution step was repeated twice. The isolated mRNA was precipitated by adding 5 µl of glycogen solution (5-10 mg / ml in DEPC-treated H2O), 20 1/10 volumes of K-acetate solution (2.5 M potassium acetate, pH 5.0) and 0.5 ml absolute to each eluant. ethanol (cooled to -20 ° C). Precipitation was allowed to proceed at -20 ° C for at least 30 minutes and the mRNA was centrifuged at full speed [14,000 rpm, 4 ° C, Centrifuge 5415 C (Eppendorf)] for 5 minutes. The precipitated mRNA was stored at -70 ° C until cDNA synthesis was performed.

25 B. Synthesis of cDNA

Reverse transcription of reindeer mRNA was performed by modification of the Time Saver ™ cDNA Synthesis Kit (Pharmacia Biotech). 3 µg of mRNA was warmed at 65 ° C for ten minutes and cooled on ice. 0.2 pmol DTT, 0.5 µg Oligo (dT) i2-18 primer and heat denatured mRNA were added to the First Strand ...... reaction mixture containing: FPLCpure ™ Cloned Murine Reverse

Transcriptase, RNAguard ™, RNase / DNase-Free BSA, dNTPs (dATP, dCTP, dGTP and dTTP) in an aqueous buffer (Time Saver ™ cDNA Synthesis Kit, 35 Pharmacia Biotech). The mixed solution was incubated at 37 ° C for one hour.

After incubation, the First Strand reaction mixture was added to the Second Strand reaction mixture containing E. coli RNasi H and E. coli DNA polymerase I and dNTPs in an aqueous buffer (Time Saver ™ cDNA Synthesis Kit, Phar-22 Mada Biotech ). The solution was gently mixed and incubated at room temperature for 30 minutes. The synthesized cDNA was stored at + 4 ° C.

C. Detection of Reindeer Horn cDNA

Part of the reindeer BMP-6 cDNA was amplified by PCR (Polymerase chain reaction) using primers (5'-CGG (C) ATCTACAAGGACTGTGTT (G) G (A) TGGG -3 ') and (3'-GTCCGG (A ) GCC (T) TGTGCCTGCCACTAA-5 ') (Table 3) designed based on the homology of the BMP-6 genes of various known mammalian species (human, rat and mouse). The PCR reaction mixture (50 µl) contained 100 ng of reindeer cDNA, 40 µm of each primer, 0.4 mM dNTP (PCR Core Kit, Roche), and 0.7 U / μΙ Expand High Fidelity (thermostable) Taq polymerase + proof reading polymerase, Roche) in Expand High Fidelity buffer containing MgC (Expand High Fidelity PCR System, Roche). The reaction was performed using 15 Mastercycler personal apparatus (Eppendorf) according to the following program: initial denaturation 94 ° C for 4 minutes, and the following 25 cycles: denaturation 94 ° C, 1 minute, priming 55 ° C for 1 minute, extension of DNA strands 72 ° C. 2 minutes. The final extension was performed at 72 ° C for 10 minutes.

23

Primers used in cloning _

Part of the reindeer BMP-6 cDNA _______

5 '-> 3' CGG (C) ATCTACAAGGACTGTGTT (G) G (A) TGGG 3 '-> 5' GTCCGG (A) GCC (T) TGTGCCTGCCACTAA

Mature part of reindeer BMP-6_________

pTrcHis2A 5 '-> 3' GGATCCGTCGGCCCCCGGGCGGCGCCGGCAG

3 '-> 5' AAGCTTGTGGCAGCCACAGGCCCGGACGAC PIVEX2.4 5 '- »· 3' CCGCGGCTCGGCCCCCGGGCGGCGCCGGCAG

3'— »5 'GGATCCTAGTGGCAGCCACAGGCCC pTrcHBS 5' -> 3 'GGTACCTCGGCCCCCGGGCGGCGCCGGCAG

_ 3 '-> · 5' AAGCTT GT GGCAGCCACAGGCCCGGACGAC

Mutated mature part of reindeer BMP-6__________

pTrcHis2A 5 '- * 3' GGATCCGTCGGCCCCGGGGCGCCGCCGCCAG

3 '-> 5' AAGCTT GT GGCAGCCGCAGGCGCGGACGAC pTrcHBS 5 '-> 3' GGTACCTCGGCCCCGGGGCGCCGCCGCCAG

3'- »5 'AAGCTTGTGGCAGCCGCAGGCGCGGACGAC

; · Q

Table 3. Primers used for normal and mutated reindeer BMP-6; 5 D. Cloning into the pGEM-T® vector The PCR product was purified using the Wizard® PCR Preps DNA Purification System kit (Promega) and ligated into the pGEM-T® vector (Figure 1) using T4 DNA-10 ligase (pGEM -T® Vector System I; Promega). 0.3 µg of purified PCR product and 2.3 µg / ml pGEM-T® vector were added to the ligation buffer containing 18 mM Tris-HCl (pH 7.8), 6 mM MgCl 2, 6 mM DTT, 0.3 mM ATP, 3% polyethylene glycol and 0.14 U / μΙ T4 DNA ligase for a total volume of 66 μΙ. The reaction was allowed to proceed at +16 ° C in a water bath which cooled to +4 ° C overnight. The resulting new plasmid was called pMSU1 (Figure 1).

24 E. Preparation of competent Escherichia coli TOP 10 F 'cells

Competent Escherichia coli TOP 10 F 'cells were prepared by the calcium chloride / magnesium chloride method. 2 ml of LB broth were inoculated with E. Coli TOP 5 10 F 'cells and grown overnight at 37 ° C with shaking (225 rpm). The next morning, 100 ml of fresh LB broth was added to 1 ml of overnight broth and grown with shaking (225 rpm) at 37 ° C until OD 600 = 0.5-0.6. The cultured cells were harvested in a centrifuge (2.500 x g, 5 minutes), resuspended in 10 ml of 0.1 M MgCl 2 solution and re-collected in a centrifuge (10, 500 x g, 5 minutes). After treatment with MgCl2, the cells were suspended in 10 ml of 0.1 M CaCl2, incubated in an ice bath for 30 minutes, and collected again by centrifugation (2.500 x g, 5 minutes). The CaCl 2 treatment was repeated, however, with a second run of 3.5 mL CaCl 2 and incubation of the cells for one hour. Glycerol (final concentration 14% (v / v)) was added to the suspension and the suspension was divided into 200 µl aliquots. Competent E. coli TOP 10 F 'cells were frozen in liquid nitrogen and stored at -70 ° C.

F. Transformation of competent Escherichia coli TOP 10 F 'cells and selection of clones containing reindeer BMP-6.

20

Competent Escherichia coli TOP 10 F 'cells were thawed in an ice bath for 15 minutes Γν. 20 μΙ of the ligation mixture (described above) was added to 100 µl of TCM (10 mM

Tris-HCl, 10 mM CaCl 2, 10 mM MgCl 2, pH 7.0) and mixed with 200 μΙ competent E. coli cells. The mixture was incubated on ice for 30 minutes before heat shock (43 ° C, 3 minutes). After heat treatment, 800 μΙ LB broth was added and cells were allowed to recover at 37 ° C for 30 minutes. Transformed cells were harvested by centrifugation at full speed for 2 minutes and resuspended in 30 µl culture broth. The cell suspension was plated on two LB plates containing 25 pg / ml ampicillin, 1 mmol IPTG (isopropyl-pD-thiogalactopyranoside) and 2.4 30 nmoles of X-gal (5-bromo-4-chloro-3-indolyl-4-D). galactoside) and cells were grown overnight at 37 ° C. Positive clones were found to be white in color due to α-complementation of the IacZ gene. The method is described in detail in Sambrook and Russel (2001) Molecular Cloning, Cold Spring Harbor Laboratory Press, New York.

35 25 Isolation of G. pMS U1 plasmid ids and sequencing of cDNA inserts

Plasmids were isolated using the Wizard® Plus Minipreps DNA Purification System kit (Promega) and further purified by ethanol precipitation. The identity of the cDNA was confirmed by sequencing on an ABI Prism (Perkin-Elmer Corporation). The sequencing reaction was performed using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) and a Mastercycler Personel PCR (Eppendorf). The PCR primers used for sequencing were (5'-TAATACGAGTCACTATAGGGCGA-3 ') and (3'-ATTTAGGTAACACTATAGAATAC-10 5') (Table 4) and program used: 25 cycles of denaturation at 94 ° C for 30 seconds, primer addition at 50 ° C. 15 seconds, chain extension at 60 ° C. Amplified PCR products were ethanol precipitated. To 10 µl of the reaction mixture was added 1 μΙ of 1.5 M Na-acetate-250 mM EDTA buffer and 95-100% ethanol to a final concentration of 75% ethanol. Precipitation was allowed to occur in an ice bath 7 for 15 minutes and then the mixture was centrifuged for 20 minutes. The supernatant was discarded and the precipitate was washed at room temperature with 125 µl of 70% ethanol. The mixture was centrifuged rapidly and the ethanol used for washing was removed as efficiently as possible. The precipitate was dried at 37 ° C for a few minutes until all ethanol had evaporated. The ABI Prism equipment was at the Department of Medical Biochemistry and Molecular Libiology at the University of Oulu and the actual sequencing was performed there. Part of the nucleotide sequence and corresponding amino acid sequence of the reindeer BMP-6 cDNA is shown in Figure 10.

Sequencing primers _____

pGEM-T @ plasmids 5 '-> 3' TAATACGACTCACTATAGGGCGA

3 '-> 5' ATTTAGGT G AC ACTATAGAATAC

pT rcHis2A-Plasmid it 5 '-> 3' AGAGGTATATATTAAT GTATCG

; 3 '-> 5' AT GGT CG ACGGCGCTATT CAG

7 plVEX2.4c Plasmids 5 '-> 3 TAATACGACTCACTATAGGGCGA

3 '-> 55 GCTAGTTATTGCTCAGCGG

: Λ 25 /, Table 4. Primers used for sequencing normal and mutated mature portion of reindeer BMP-6 26

Example 2: Expression of the recombinant normal and nucleotide mutated mature portion of reindeer BfVIP-6 in Escherichia coii TOP 10 F ', Origami B (DE3) and Rosetta (DE3) cells 5 A. Amplification of the mature portion of reindeer BMP-6 for expression vector

The mature portion of reindeer BMP-6 was amplified from pMSU1 by PCR using homologous primers: (5'-GGATCCGTCGGCCCCCGGGCGGCGCCGGCAG-3 ') and 10 (3'-AAGCTTGTGGCAGCCACAGGCCCGGACGACi-5? - GGATCCGTCGGCCCCGGGGCGCCGCCGCCAG-3 ') and (3'-AAGCTTGTGGCAGCCGCAGGCGCGGACGAC-5') (see Example 2, Part B). The primers are also seen in Table 3. The primer-15 primer had a recognition site for the Bam HI restriction enzyme and the other primer had a recognition site for the Hind III enzyme at its 3'-end.

The 50 μΙ: η reaction mixture contained, in addition to pMSU plasmid (0.05 pg) and both primers (40 pmol) 0.4 mM dNTPs (PCR Core Kit, Roche) and 0.7 U / μΙ Expand High Fi-20 delity enzyme mixtures (thermostable Taq polymerase + proof reading polymerase, Roche) and Expand High Fidelity buffer containing MgCl 2 (Roche). The PCR reaction was performed on a Mastercycler Personel (Eppendorf) according to the following program: initial denaturation 94 ° C for 4 minutes, and the following 25 cycles: denaturation 94 ° C, 1 minute, primer ligation 55 ° C 1 minute, extension of DNA strands 25 72 ° C 2 minutes. The final extension was performed at 72 ° C for 10 minutes. The PCR product was purified from the PCR reaction mix using the Wizard® PCR Preps DNA Purification System kit (Promega) and ligated into the pGEM-T® vector. The resulting, new plasmids containing the normal and nucleotide-modified mature portion of reindeer BMP-6 were called pMU2 and pMU8 (Figure 1).

B. Subcloning of the normal and nucleotide-modified mature portion of reindeer BMP-6 from the pGEM-T® vector into the expression vector pTrcHis 2A (Invitrogen) and transformation to the competent Escherichia coli TOP 10 F ', Origami B (DE) and Rosetta (DE). ) cells: 35

Subcloning of the normal and nucleotide mutated mature portion of reindeer BMP-6 from the pGEM®-T vector into the expression vector pTrcHis 2A (Figure 2) was accomplished by cleaving the mature portions of the pGEM®-T vector with Bam HI and Hind III restriction enzymes and ligand 2Tc - vector digested with the same enzymes. Digestion of pGEM®-T constructs and pTrcHis 2A (1 µg) with Bam HI (Roche) and Hind III (Roche) were performed in 10 µl of 10 mM Tris-HCl, 10 mM NaCl, 5 mM MgCl 2, 1 mM. 2-mercaptoethanol, pH 8.0 (SuRE / Cut B, Roche) using 1 U / μΙ ku-5 coat restriction enzyme. The reaction was allowed to proceed for 1.5 hours at 37 ° C, after which the restriction enzymes were inactivated at 65 ° C for 20 minutes and frozen at -20 ° C. Ligation (ligase concentration 0.1 U / μΙ) was performed in 2X Rapid Ligation Buffer (supplied with pGEM-T® vector (Promega)) in a +16 ° C water bath allowed to slowly cool to + 4 ° C. during the night.

10

The new construct was verified by sequencing (the method is described in Example 1, Part G) using primers: (5'-AGAGGTATATATTAATGTATCG-3 ') and (3 * -ATGGTCGACGGCGCTATTCAG-5'). The expression vector containing the cDNA of the normal mature portion of pTrcHis 2A and reindeer BMP-6 was called pMU20, and the vector containing pTrcHis 2A and the mutated mature portion of reindeer BMP-6 cDNA: n, was called pMU90 (Figure 2). Competent Escherichia coli TOP 10 F 'cells were transformed as described in Example 1, Part F and Origami B (DE3) and Rosetta (DE3) cells were transformed according to the manufacturer's (Novagen) instructions.

20 C. Nucleotide Mutations Converting Codons of the Natural Mature Part of Reindeer BMP-6 to Escherichia coli

To achieve the highest possible level of expression, the eight codons of the mature part of the reindeer BMP-6 were mutated to more commonly used E. coli codons.

Mutated codons encoded amino acids P6, R8, R10, P99, P103, R132, R137 and C139 (Figure 5). Thanks to the mutations, the codon frequency of the codons used increased dramatically (the differences are shown in Table 6). Mutations P6, R8, R10, R137 and C139 were made using PCR (see Example 2, Part A) and mutations P99, P103 and R132 were performed using the QuickChange ™ Site-Directed Mutagenesis Kit 30 (Stratagene) following the manufacturer's instructions. mature part: was subcloned into pTrcHis 2A (see Example 2, Part B). The oligos used to make the mutations are shown in Table 5.

28

Primer for Complementary Mutations (pMU80 -> pMU9Q) ___________

P99 and P103 5 '^ 3' CCTCATGAACCCGGAGTACGTCCCGAAGCCGTGCTGTGCG

3 '- »5' CGCACAGCACGGCTTCGGGACGTACTCCGGGTTCATGAGG R132 5W3 'CCTGAAAAAGTACCGCAACATGGTCGTCCGCGCC

_ 3 '- * 5' G GCG CGGACGACCATGTTGCGGTACTTTTTCAGG_

Table 5. Primers used in reindeer BMP-6 mutations.

5. ___ ^

Amino acid Original codon Mutated codon / __ / frequency Frequency_ F6__CCC / 5.1 CCG / 22.0 R8__CGG / 5.4 ___ CGC / 20.6 R10__CGG / 5.4 CGC / 20.6 P99__CCC / 5.1 CCG / 22.0 P103__CCC / 5.1__CCG / 22.0 R132__AGG / 1.7 CGC / 20.6 R137__CGG / 5.4__CGC / 20.6 C139 l TGT / 5.1 TGC / 6.2

Table 6. Changes in amino acid codon frequencies caused by site-directed mutations in the mature part of reindeer BMP-6. The amino acids are numbered based on the 10 sequences of recombinant rdBMP-6 produced in pMU90 (see Figure 5).

D. Insertion of a normal and nucleotide mutated mature portion of reindeer BMP-6 into pET22b (+) (Novagen) expression vector and transformation into competent Escherichia coii Origami B (DE3) and Rosetta (DE3) cells

Subcloning of the normal and nucleotide mutated mature portion of reindeer BMP-6 into the expression vector pET22b (+) (Novagen) (Figure 3) was performed as described above (see Example 2, Part B). The resulting new plasmid containing the ':: pET22b (+) vector and the normal mature portion of the cDNA 20 of the BMP-6 ligated reindeer was called pETrd6A and the plasmid containing the pET22b (+) vector and the reindeer BMP-6 ligated thereto. The mutated mature portion of the 29 cDNA was called pETrd6 (Figure 3). Competent Origami B (DE3) and Rosetta (DE3) cells were transformed as described above.

E, Expression of the recombinant protein of Poron BMP-6 in Escherichia coli 5 cultures and cell harvesting in E. coli cells [TOP 10, Origami B (DE3) or Rosetta (DE3)] containing either pMU20, pMU90, pETrd6A or pETrd6 plasmid, was grown overnight in 50 ml SOB culture broth containing ampicillin (50 pg / ml) and Rosetta (DE3) -10 cell culture also chloramphenicol (35 pg / ml), shaking at 225 ° C (225 ° C). rpm). The next morning, 24 ml of overnight growth medium was added to 1200 ml of SOB medium containing the above antibiotics and culturing was continued at + 37 ° C with shaking (225 rpm) until the OD 600 was 0.6, with cells in a logarithmic growth phase. around the middle. At this point, the induction of recombinant protein-15 was performed by adding IPTG to a final concentration of 1 mM. After induction, cells were grown for four to five hours, after which they were harvested by centrifugation. The amino acid sequences of the recombinant proteins derived from the nucleotide sequences are shown in Figure 5 (pMU20 / pMU90) and Figure 7 (pETrd6A / pETrd6).

20

Example 3: Purification and folding of the nucleotide mutated mature portion of reindeer BMP-6 A. Washing of inclusion bodies

The harvested cells were suspended by shaking in 50 mM Na-phosphate buffer (pH 7.0, 220 g cells / liter buffer). The suspension was centrifuged at 5500 x g for 45 minutes at + 4 ° C. Washing with Na-phosphate solution was repeated once. The cell pellet was weighed and stored at -70 ° C overnight. The frozen pellet with partially disrupted cells was thawed and suspended (25 mg / ml) in 20 mM Tris-HCl buffer, pH 8.5 containing 0.5 mM EDTA, shaking for 2 minutes. The suspension was centrifuged at 26,000 x g for 30 minutes at + 4 ° C and the Tris-HCl-EDTA wash was repeated once. The resulting cell pellet was weighed. After the final wash, the cell pellet was suspended (35 mg / ml) with shaking (200 rpm / minute, overnight, at room temperature) in lysis buffer (6 M GuHCl - 20 mM Na-35 phosphate - 0.5 M NaCl, pH 8.0), the remaining whole E. coli cells are disrupted and the inclusion bodies are solubilized. The suspension was centrifuged (26,000 x g, 45 minutes, at room temperature), the pellet discarded, and the recombinant protein remaining soluble in the supernatant. Finally, the supernatant is filtered through a 45 µm filter to ensure that all cell debris is removed.

B. Precipitation at isoelectric point (pl) 5

The nucleotide mutated reindeer BMP-6 recombinant protein expressed in pETrd6 in Escherichia coli Origami B (DE3) or Rosetta (DE3) cells was precipitated at its isoelectric point at pH 9.69. The isoelectric point was determined by computer-aided calculation of the reindeer BMP-6 recombinant amino acid sequence 10 (Figure 7). The precipitate was collected by centrifugation (12,000 x g, 30 min, at room temperature) and suspended in lysis buffer (6 M GuHCl - 20 mM Na phosphate - 0.5 M NaCl, pH 8.0).

C. immobilized metal affinity chromatography (IMAC) 15

Escherichia coli cells were lysed by shaking in 6 M GuHCl - 20 mM Na-phosphate in -0.5 M NaCl (pH 8.0) for 2 hours and filtered through a 45 µm filter. Pre-packaged HiTrap Chelating HP affinity beads (Amersham Pharmacia Biotech) were used in the IMAC method. The column matrices were charged with Co2 +, 20 Cu2 + or Ni2 + ions according to the manufacturer's instructions. The idea behind the purification is that the "His-tag" epitope at the end of the rdBMP-6 protein binds to a column charged with metal ions and that cellular debris from E. coli is filed through the column. The filtered, post-wash supernatant was applied to the column after the column was charged. Most of the impurities were removed by washing the column with lysis bag 25 (6 M GuHCl - 20 mM Na phosphate - 0.5 M NaCl, pH 8.0) with 5-10 fold column volume. A second wash, 5 to 10 times the column volume, was performed in a lysis buffer with 6 M urea instead of 6 M GuHCl. The reindeer recombinant BMP-6 was eluted from a HiTrap column using a pH gradient from pH 7.0 to pH 4.0 (6M urea - 20 mM Na - phosphate - 0.5 M NaCl). Fractions were analyzed by SDS-30 PAGE and the purest fractions containing recombinant rdBMP-6 were pooled for folding.

D. Folding of the mature portion of recombinant rdBMP-6 The BMP-6 fractions analyzed by SDS-PAGE were pooled and dialyzed against water. Protein precipitated by dialysis was collected by centrifugation and suspended for 2 hours at 25 ° C in 8 M urea - 0.1 M Tris / HCl, pH 8, additionally containing 100 mM DTT, 1 mM EDTA. The pH was lowered to pH 3-4 by the dropwise addition of 1 M HCl. The DTT was completely removed by dialysis at 2 ° C for 2 hours against 6 M urea in -10 miV HCl. Dialysis was continued at + 4 ° C overnight against 6 M urea. Folding of recombinant rdBMP-6 was performed by two-step dialysis. The first dialysis solution was 20 mM Tris-HCl - 150 mM NaCl - 3 M urea 5 (pH 7.5). The dialysis buffer was changed at least six times over two or three days. The desalted sample was centrifuged and the pellet dried by lyophilization. At this point, BMP-6 was 75% pure and 50% folded as determined by non-reducing SDS-PAGE. The amount of folded dimer of reindeer recombinant BMP-6 was quantitated by densitometry on Coomassie Bril-10 liant Blue stained gels.

Example 4; Insertion of a Heparin Binding Site in Front of the Mature Part of Reindeer BMP-6 15 A. Insertion of a Heparin Binding Site DNA Fragment into the pTrcHis 2A Vector

The two complementary primers shown in Table 7 were designed using the heparin binding site (HBS) of reindeer BMP-2. The cleavage sites of the Bam HI and Κρη I 20 restriction enzymes were added to the 5 'and 3' ends of HBS, respectively. The primers were initially denatured at +100 ° C for 5 minutes and then allowed to attach with a small water bath cooled from +100 ° C first to room temperature and then to +4 ° C (1 hour). Both the linked HBS fragment (1 pg) and the pTrcHis 2A vector (0.5 pg) were digested with Bam HI (1 U / µl) and Κρη I (2 U / µl) in MuIti-Core buffer (Promega). At 1.5 ° C for 1.5 hours and ligation was performed in a +16 ° C water bath which was allowed to cool slowly to + 4 ° C overnight. The new construct constructed was verified by sequencing (see Example 2, Part B) and the plasmid was called pTrcHBS (Figure 2).

Primer used in HBS Cloning____

5 '-> 3' CGGGATCCGCAAGCAAAACATAAACAGCGCAAACGCGGTACCCC 3 '-> 5' GGGGTACCGCGTTTCCGCTGTTTATGTTTTGCTTGCGGATCCCG

Table 7. Primers Used for Cloning the Nucleotide Mutated Mature Part of Reindeer BMP-6 30 Attaching the Normal and Nucleotide Mutated Mature Part of Reindeer BMP-6 to the pTrcHBS vector and competent Escherichia coli TOP 10 F ', Origami B (DE3 ) and transformation of Rosetta (DE3) cells To insert the normal and nucleotide mutated mature portion of reindeer BMP-6 into the pTrcHBS vector, the mature portion was amplified from the pMSU1 vector (Figure 1). The primers used had the Κρη I restriction site at the start of the 5 'end of the primer and the Hind III restriction site at the 3' end of the primer. The primers used to amplify the natural mature portion of reindeer BMP-6 were: 10 (5'-GGTACCTCGGCCCCCGGGCGGCGCCGGCAG-3 ') and (3'-AAGCTTGTGGCAGCCACAGGCCCGGACGAC-5') and the primers used for the recombinant BMP-6 part : (5'-GGTACCTCGGCCCCGGGGCGCCGCCGCCAG-3 ') and (3'-AAGCTTGTGGCAGCCACAGGCCCGGACGAC-5').

The primers are also in Table 3. PCR reactions, purification of PCR products and ligation to pGEM® were performed as described in Example 1, part C and D. The pGEM® vector containing the natural mature part of reindeer BMP-6 was called under the name pMU12 and the pGEM® vector containing the mutated mature portion of reindeer BMP-6 was called pMU11. Both constructs are shown schematically in Figure 1.

Subcloning of the mutated mature portion of reindeer BMP-6 from the pGEM® vector into the * -pTrcHBS vector was performed in the same manner as the same insert subclonam; pTrcHis 2A vector as described in Example 2, part B. The only difference was that the amρη I restriction enzyme replaced Bam HI and the buffer used was the Multi-Core buffer (Promega). Transformations were performed as described in Example 1, Part F and in Example 2, Part B. The new constructs were called pTrcHBSrd6A (original mature part) and pTrcHBSrd6 (mutated mature part) (Figure 2), and both constructs were verified by sequencing (see Example 2). part c).

:: C. Insertion of the normal and mutated mature portion of reindeer BMP-6 containing the heparin binding region into pET22b (+) vector and transformed into competent E. coli Origami B (DE3) and Rosetta (DE3) cells 35

The insertion of the normal and mutated mature portion of reindeer BMP-6 containing the heparin binding region into the pET22b (+) vector was performed in exactly the same way as the subcloning of the normal and mutated mature portion of reindeer BMP-6 from 33 pGEM® vectors into the pTrcHBS vector Part 2 B. Transformations were performed as shown in Example 1 Part F and Example 2 Part C. The new constructs were called pETHBSrd6A (the original mature part of reindeer BMP-6) and pETHBSrd6 (the mutated mature part of reindeer BMP-6) (Figure 3).

D. Expression of mature portion of reindeer's recombinant BMP-6 containing heparin binding region in Escherichia coli and cell harvesting

Expression was performed as previously described in Example 2, Part E. The amino acid sequences of the Re-10 recombinant proteins and the corresponding nucleic acid sequences are shown in Figure 6 (pTrcHBSrd6A / pTrcHBSrd6) and Figure 8 (pETHBSrd6A / pETHBSrd6).

Example 5: Purification and folding of the mutated mature portion of reindeer's recombinant BMP-Sn containing heparin binding site 15 (HBSrdBMP-6) A, Purification of HBSrdBMP-6 by IMAC 20 Escherichia coli cells were lysed by shaking for 2 hours at 6 M. GuHCl - 20 mM Na-phosphate - 0.5 M NaCl (pH 8.0) buffer and filtered through a 45 µm filter. Pre-packaged HiTrap Chelating HP affinity columns (Amersham Pharmacia Biotech) were used in the IMAC purification process. The column matrices were charged with Co2 +, Cu2 + or Ni2 + ions according to the manufacturer's instructions. The filtered lysate was loaded onto the column and washed with lysis buffer at 15 times the column matrix. A second wash was performed in 40 column volumes of column matrix using 6 M urea - 20 mM Na phosphate - 0.5 M NaCl (pH 7.5) buffer. The third wash solution was 0.05 M imidazole added to the second wash buffer and the wash volume was 15 times the column volume. Recombinant HBSrdBMP-6 eluted from the column with an imidazole gradient starting at 0.05 M imidazole concentration and ending at 0.5 M imidazole concentration in 6 M urea-20 mM Na-phosphate-0.5 M NaCl buffer (pH 7.5). Fractions were analyzed by SDS-PAGE and those fractions containing the purest HBSrdBMP-6 recombinant were pooled and dialyzed against 100 mM Na phosphate buffer (pH 7.5) over the weekend. The precipitated recombinant-35 tiprotein was collected by centrifugation and dissolved in 8 M urea in -100 mM Na-phosphate in -10 mM Tris-HCl (pH 7.5). Prior to purification on a heparin affinity column, the recombinant protein solution was filtered through a 45 µm filter.

34 B. Purification of recombinant HBSrdBMP-6 on a heparin affinity column.

The post-IMAC column filtrate was dispensed onto a finished HiTrap Heparin HP column (Amersham Pharmacia Biotech) equilibrated with 8 M urea -5 100 mM Na-phosphate-10 mM Tris-HCl (pH 7.5). The column was washed with 20X column volume of the same buffer and recombinant HBSrdBMP-6 was eluted from the heparin column with a NaCl gradient starting at 0 M and ending with 2 M NaCl in the same buffer. Fractions were analyzed by SDS-PAGE and Western blotting and the purest HBSrdBMP-6 fractions were pooled. The Wes-10 tern blot method used specific antibodies raised against His6 and BMP-6. The combined fractions were ready for folding.

C. Folding of Recombinant HBSrdBMP-6 Folding of recombinant HBSrdBMP-6 was performed in the same manner as folding of recombinant rdBMP-6 as described in Example 3, Part D.

Example 6. Testing for biological activity of a mutated mature portion of reindeer BMP-6 recombinant protein, whether heparin-binding region or not

The biological activity of the lyophilized recombinant HBSrdBMP-6 was tested by implantation into a mouse thigh pocket with less than 1 mg of recombinant HBSrdBMP-6 absorbed into Lyostrypt® collagen blanket. After 21 days, the hind legs were x-rayed and the implant sites were dissected and fixed in 10% neutral formalin solution. Fixed implants were cut into 4 µm sections and stained with hematoxylin-eosin solution. The sections were examined under a light microscope.

New bone formation was evaluated by X-ray imaging by determining area 30 and optical density. X-rays were transferred to a computer using an optical scanner (HP Scan Jet, Hewett-Packard, USA). The formation of ectopic and orthotopic new bone was assessed by the area of calcified tissue seen in X-rays (mm 2) using the Scion Image Beta 4.02 (Scion Corp., USA) software. The average optical density (mmAl) of a given region was measured with the same 35 devices. The optical density calibration was performed using an aluminum plate (Al) with 0.25 mmAl steps and a calibrated density giving a range of up to 4 mmAl. The biological activity of the mature portion of recombinant human BMP-6 produced by the same method was also tested for comparison by the bioassay described above.

Example 7: Expression of the mature part of reindeer recombinant BW1P-6 in Rapid 5 Translation System RTS 500 Construction of A RTS 500 expression vector pIVEX 2,4c (Roche)

Amplification of the original mature portion of reindeer BMP-6, purification of the PCR product, ligation into the pGEM-T® vector (Figure 1), transformation into competent Escherichia co // TOPI 0 F 'cells (Invitrogen), plasmid purification and the sequences of the inserts were otherwise performed as described in Example 1, except that the following primers were used to amplify the mature part of reindeer BMP-6: (5'-CCGCGGCTCGGCCCCCGGGCGGCGC-3 ') 15 and (3'-GGATCCTAGTGGCAGCCACAGGCCc for' 5 ') under the name pMU2 / 2 (Figure 1). The primers used to construct the construct were: (5'-TAATACGACTCACTATAGGGCGA-3 ') and (3'-GCTAGTTATTGCTCAGCGG-5') (Table 4). The primers used for amplification had a Ksp I (Sac II) restriction enzyme cleavage site at the 5 'end and a Bam HI 20 cleavage site at the 3' end of the primer, and these sites were utilized for subcloning the mature portion of reindeer BMP-6. Plasmids pMU2 / 2 and pIVEX 2.4c (0.5 µg) were digested using 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithioerythritol, pH 7.5 (SuRE Cut Buffer L, Roche) buffer and 1 U each. / μΙ enzyme, with a total volume of 10 pL. The restriction enzymes were inactivated before ligation and the ligation-25 reaction was performed as shown in Example 2, Part C. The new construct was called pMU500 (Figure 4).

B. Production of the mature portion of recombinant BMP-6 in the RTS 500 System The RTS 500 reaction was performed following the instructions of the Rapid Translation System RTS 500 E. coli Template Kit Instruction Manual. The amino acid sequence and the corresponding nucleotide sequence of the recombinant protein are shown in Figure 9.

36

Score

Cloning of Reindeer BMP-6 Partial cDNA The nucleotide sequence obtained from the ABI Prism sequencing reactions was computer-analyzed and compared to already known BMP sequences. The new cloned cDNA was found to be most homologous to bovine BMP-6 based on homology comparisons (nucleotide homology 95.0% and amino acid homology 99.1%, US 6,207,813). The nucleotide and amino acid homologies of the BMP-6 proteins between the different 10 mammals are shown in Table 1. The nucleotide sequence of the reindeer BMP-6 and the corresponding amino acid sequence of the reindeer BMP-6 partial cDNA are shown in Figure 10.

Expression of the mature part of reindeer BMP-6 15

First, the mature portion of reindeer BMP-6 was cloned into the pTrcHis2A vector, transformed into E. coli TOP 10 cells to obtain the pMU20 vector. Recombinant protein production was induced by IPTG. Production of the recombinant protein was confirmed by SDS-PAGE, but no induction was observed. This was thought to be due to many of the 20 rdBMP-6 codons rarely used in E. coli. These codons were mutated to be more suitable for the E. coli translation system and plasmid pMU90 was created. Nevertheless, no induction of recombinant protein was detected by SDS-PAGE, and it was concluded that the induction is due to the high GC-25 concentration at the beginning of the rdBMP-6 coding region. It was also found that the GC content of the heparin binding site at the beginning of the mature part of the reindeer BMP-2 was very low and when added to the beginning of the mature part of the reindeer BMP-6 it could also be used for purification. Therefore, the pTrcHBS vector was constructed. By cloning the original mature portion of rdBMP-6 and the mutated mature portion into the pTrcHBS vector, pTrcHBSrd6A and 30 pTrcHBSrd6 were obtained. Successful induction of both recombinants, HBSrdBMP ~ 6A and HBSrdBMP-6, was confirmed by SDS-PAGE but with mutated HBSrdBMP-; The expression level of 6 was significantly higher.

Since no vector was yet available for the production of the rdBMP-6 mature portion of recombinant 35 tiprotein, it was decided to experiment with a different vector system and a different E. coli cell line. pET22b (+) (Novagen) with His6 tag and pelB leader was selected as the new expression vector and Rosetta (DE3) (Novagen) and Origami B (DE3) (Novagen) as new E. coli lines. The original and mutated mature 37 portions of reindeer BMP-6, with or without the heparin binding region, were cloned into the pET22b (+) vector and four new plasmids were designated pETrd6A, pETrd6, pETHBSrd6A and pETHBSrd6 and Rosetta (DE3). ) that Origami B (DE3) cells were transformed with this vector individually. When analyzed by SDS-PAGE, ha-5 prevailed in all combinations for expression of rdBIVIP-6 or HBSrdBMP-6. However, as previously described for pTrcHBSrd6A and pTrcHBSrd6, nucleotide dimutations in the mature portion produced a dramatic increase in expression level when either reindeer BMP-6 alone or heparin binding site was expressed. Based on the expression experiments, mainly Rosetta (DE3) -10 cells containing the pETrd6 or pETHBSrd6 vector were used to produce the recombinant protein rdBMP-6 or HBSrdBMP-6.

Purification of rdBMP-6 15

Reindeer rdBMP-6 recombinant protein was produced in E. coli. After washes, isoelectric point precipitation, and solubilization of the inclusion bodies, the recombinant reindeer had a rdBMP-6 concentration of 85%.

The next purification step was immobilized metal affinity chromatography (IMAC). The sample was eluted from the column using a pH gradient and the rdBMP-6 had a purity of at least 75% as determined by SDS-PAGE. The mature portion of the isolated rdBMP-6 protein had a molecular weight of 20,300 Da as determined by electrophoretic mobility by SDS-PAGE under reducing conditions (Figure 11).

25 Purification of rdBMP ~ 6 ~ HBS

Reindeer rdBMP-6-HBS recombinant protein was produced in E. coli in inclusion kappa-30 (IBs). After washing, isoelectric point precipitation, and solubilization of the inclusion bodies, the recombinant reindeer had a rdBMP-6-HBS content of 85%.

A: The next purification step was immobilized metal affinity chromatography (IMAC).

The sample was eluted from the column using a pH gradient and the rdBMP-6-HBS protein was at least 75% pure as determined by SDS-PAGE. The mature portion of the isolated rdBMP-6-HBS protein had a molecular weight of 21,600 Da as determined by electrophoretic mobility by SDS-PAGE under reducing conditions (Figure 11).

Folding and Activity Testing of rdBMP-6 and rdBMP-6-HBS The in vitro folding of denatured rdBMP-6 and rdBMP-6-HBS was quantitated by densitometry by measuring the folded dimer on Coomassie Brilliant Blue stained gels. Protein folding was determined from non-reducing SDS-PAGE gels at 50%.

The osteoinductive activity of 10 rdBMP-6 increased with increasing dose size (Table 2). Reindeer rdBMP-6 recombinant protein proved to be a much more potent inducer of bone formation than that of human. The bone-forming activity of reindeer rdBMP-6-HBS was proportional to that of rdBMP-6, whereas bovine BMP-6 produced the effect at only the 15 highest amounts used.

The present invention has been described with emphasis on some preferred embodiments and applications. However, it will be apparent to those skilled in the art that variations of the embodiments disclosed may be made and practiced, and that the invention may be practiced within the scope of the following claims other than as specifically set forth herein.

39

SEQUENCE LISTING

<110> BBS-Bioactive Bone Substitutes Oy 5 <120> Bone Morphogenetic Proteins <130> OP10087 5 <160> 2 10 <170> Patent Version 3.1 <210> 1 <211> 139

15 <212> PRT

<213> Rangifer tarar tarar <4 00> 1 20 Ser Ala Pro Gly Arg Arg Arg Gin Gin Arg Asn Arg Ser Thr Pro 15 10 15

Ala Gin Asp Val Ser Arg Ala Ser Ser Ala Ser Asp Tyr Asn Ser Ser 20 25 30 25

Glu Leu Lys Thr Ala Cys Arg Lys His Glu Leu Tyr Val Ser Phe Gin 35 40 45

Asp Leu Gly Trp Gin Asp Trp lie lie Ala Pro Lys Gly Tyr Ala Ala 30 50 55 60

Asn Tyr Cys Asp Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn 65 70 75 80> 35 Ala Thr Asn His Ala lie Val Gin Thr Leu Val His Leu Met Asn Pro I. 85 90 95

Glu Tyr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala lie 100 105 110 40 'V Ser Val Leu Tyr Phe Asp Asp Asn Ser Asn Val lie Leu Lys Tyr • J * 115 120 125

Arg Asn Met Val Val Arg Ala Cys Gly Cys His 45 130 135

<210> 2 <211> 118 <212> PRT

50 <213> Rangifer tarand tarang <400> 2

Arg lie Tyr Lys Asp Cys Val Val Gly Ser Phe Lys Asn Gin Thr Phe 55 1 5 10 15

Leu lie Ser lie Tyr Gin Val Leu Gin Glu His Gin His Arg Asp Ser 20 25 30 1

Asp Leu Phe Leu Leu Gly Thr Arg Ala Val Trp Ala Ser Glu Ala Gly 40 35 40 45

Trp Leu Glu Phe Asp lie Thr Ala Thr Ser Asn Leu Trp Val Leu Thr 50 55 60 5

Pro Gin His Asn Met Gly Leu Gin Leu Ser Val Val Thr Arg Asp Gly 65 70 75 80

Leu Ser lie Ser Pro Gly Alla Gla Leu Val Gly Arg Asp Gly Pro 10 85 90 95

Tyr Asp Lys Gin Pro Phe Met Val Ala Phe Phe Lys Ala Ser Glu Ala 100 105 110 15 His Val Arg Ser Ala Arg 115

Claims (19)

An isolated bone morphogenetic protein 6 (BMP-6), characterized in that it contains the consensus sequence P-G / S / N-R / K-R / H-R / K-Q / N-Q-A / S / N-R-N / S-R / A / K- Bone morphogenetic protein 6 according to claim 1, characterized in that it contains amino acids 3-16 of SEQ ID NO: 1. 10 Bone morphogenetic protein 6 according to claim 1, characterized in that it contains the amino acid sequence of SEQ ID NO: 1. Bone morphogenetic protein 6 according to any one of the preceding claims, characterized in that it additionally contains the BMP propeptide of SEQ ID NO: 2. An isolated DNA molecule, characterized in that it encodes a bone morphogenetic protein according to any one of the preceding claims. 5 S / A-TYS / N-P and at its amino terminus a heparin binding site containing the amino acid sequence AKHKQRKRGT. Nucleotide vector, characterized in that it contains an isolated DNA molecule according to claim 5. A recombinant host cell, characterized in that it contains the nucleotide vector of claim 6. 25 Recombinant host cell according to claim 7, characterized in that it is an E. coliTOP10, Origami B (DE3) or Rosetta (DE3) cell. Pharmaceutical composition, characterized in that it contains a bone morphogenetic protein according to any one of claims 1-4. Pharmaceutical composition according to claim 9, characterized in that it contains said bone morphogenetic protein as a homodimer, or as a heterodimer with another bone morphogenetic protein. 35 Pharmaceutical composition according to claim 9 or 10, characterized in that it further comprises a bone morphogenetic protein, an epidermal growth factor, a fibroblastic growth factor or a transforming growth factor. Use of a bone morphogenetic protein according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of disorders associated with bone, cartilage, tendons or teeth where regeneration, repair or growth is desirable. 5 Use of a bone morphogenetic protein according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of cancer, fibromyalgia, psoriasis and other dermatological disorders, and rheumatic disorders. Osteogenic device, characterized in that it contains a bone morphogenetic protein according to any one of claims 1 to 4. An osteogenic device according to claim 14, characterized in that it contains said bone morphogenetic protein as a homodimer, or as a heterodimer with another bone morphogenetic protein. An osteogenic device according to claim 14 or 15, characterized in that it further comprises a bone morphogenetic protein, an epidermal growth factor, a fibroblastic growth factor or a transforming growth factor. An osteogenic device according to any one of claims 14 to 16, characterized in that it contains a biocompatible matrix. The osteogenic device according to claim 17, characterized in that said biocompatible matrix contains calcium phosphate, carboxymethylcellulose or collagen or combinations thereof. A method for inducing bone, cartilage, tendon or tooth bone formation in vitro, characterized in that the bone of any one of claims 1-4; the morphogenetic protein is applied to the bone, cartilage, tendon or tooth.