EP0842274A1

EP0842274A1 - Human mp52 arg protein

Info

Publication number: EP0842274A1
Application number: EP96928406A
Authority: EP
Inventors: Michio Kimura; Tomoaki Matsumoto; Mikiko Takahashi; Shinji Kawai; Yukio Fujino
Original assignee: Biopharm Gesellschaft zur Biotechnologischen Entwicklung von Pharmaka mbH
Current assignee: Biopharm Gesellschaft zur Biotechnologischen Entwicklung von Pharmaka mbH
Priority date: 1995-08-03
Filing date: 1996-08-02
Publication date: 1998-05-20
Also published as: HUP9900362A2; CN1192237A; MX9800801A; AR003974A1; KR19990036081A; AU699708B2; WO1997006254A1; AU6789196A; IL122817A0; NO980375L; PL324822A1; BR9609983A; NO980375D0; JP2000511155A; ZA966489B; CA2227204A1

Abstract

This invention relates to a human MP52 Arg and a pharmaceutical medical composition inter alia for promoting cartilage and bone morphogenation comprising human MP52 Arg. In particular, the medical composition is useful for treating bone diseases caused by abnormal bone metabolism such as osteoporosis, for treating bone fracture and for the purpose of orthopedic reconstruction, bone transplantation, cosmetic surgery and dental therapeutics. Further, it is useful for treating cartilage disorders.

Description

HUMAN MP52 ARG PROTEIN

This invention relates to a new compound human MP52 Arg and a pharmaceutical medical composition inter alia for promoting cartilage and bone orphogenation comprising human MP52 Arg. In particular, the medical composition is useful for treating bone diseases caused by abnormal bone metabolism such as osteoporosis, for treating bone fracture and for the purpose of orthopedic reconstruction, bone transplantation, cosmetic surgery and dental therapeutics. Further, it is useful for treating cartilage disorders.

Pharmaceutical compositions including vitamin D3, calcitonin, estrogen and bisphosphonate derivatives have been used in clinical practice for treating bone diseases. Their therapeutic results, however, are not entirely satisfactory, and a better pharmaceutical composition is highly desired.

The TGF-β family of growth factors comprising BMP, TGF, and inhibin related proteins have been reported to be useful for wound healing and tissue repair. The bone morphogenetic activity of some of those proteins has been also known. PCT patent application WO 93/16099 and WO 25/04819 disclose DNA sequences encoding human TGF-β-like proteins, and as a preferred protein human MP52.

E. E. Storm et al. reported in Nature, 1994, vol.368, p.639-643 that mutations of mouse growth/differentiation factor 5 (GDF5) gene, a new member of the TGF-β superfamily, cause mice brachypodism. Mouse GDF5 has the same amino acid sequence of the predicted mature form as that of human MP52 except one amino acid. However, there is no suggestion in this publication to use those proteins for the treatment of bone diseases.

It was therefore the object of the present invention to provide a further growth factor that is useful as an agent for the stimulation of bone or cartilage formation. One embodiment of the present invention is therefore protein human MP52 Arg comprising amino acids 1 . to 121 of SΞQ ID NO: 1.

In fact, it has surprisingly been found by the invention that there are cell lines which, when expressing a suitable DNA sequence, form human MP52 Arg or a mixture of human MP52 and human MP52 Arg. For the first time the invention succeeded in providing human MP52 Arg which has bone morphogenetic activity and is useful for preventing and/or treating bone diseases.

It has also been confirmed that human MP52 Arg induces formation of cartilage from undifferentiated esenchymal cell and stimulates the differentiation and maturation of osteoblasts. Therefore, human MP52 Arg is effective for preventing and/or treating bone diseases caused by abnormal bone metabolism such as osteoporosis. It also accelerates the healing process of bone fractures. Moreover, it is useful for orthopedic reconstructions, bone transplantations and dental therapeutics because of its bone morphogenetic activity. Furthermore, MP52 Arg is effective for preventing and/or treating cartilage disorders caused by abnormal cartilage metabolism.

A further object of the present invention is a process for the production of human MP52 Arg, wherein at least a part of a DNA sequence as shown in Sequence ID N0:1 is introduced into a suitable host cell under conditions favouring expression of the DNA sequence and protein formation, followed by isolation of said protein from other proteins produced by said host cell.

Within the framework of the present invention the DNA sequence depicted in SEQ ID NO:l may be used for producing human MP52 Arg, however also shorter portions thereof, provided they still encode human MP52 Arg and expression of the DNA sequence in a suitable vector/host cell system is possible. Suitable expression systems are known to a person skilled in the art and it can easily be determined by routine experimentation what the minimum requirements for the length of the DNA sequence of SEQ ID NO:l are.

Subsequent to protein formation the proteins are recovered from the host cell by methods known per se and finally human MP52 Arg is isolated therefrom. In particular the isolation of human MP52 and MP52 Arg, which differ from one another only in respect of one amino acid, can be carried out using very precisely differentiating separation methods which are known to a person skilled in the art. One example is the electrophoretic separation of MP52 Arg performed according to the method of Davis (Ann. NY Acad. Sci., 121, 404-427, 1964) with small modifications like addition of 0.1 % Nonidet P-40 and 6 M urea. After electrophoresis the separated MP52 Arg is electroeluted from the gel pieces in the same buffer.

A further object of the present invention is a pharmaceutical composition containing human MP52 Arg. Optionally, this composition may also include usual carrier substances, auxiliary substances, diluents and/or fillers. The pharmaceutical composition according to the invention is useful for promoting bone morphogenation, treatment or prevention of damage to bone, cartilage, connective tissues, skin, mucous membranes, epithelium or teeth, for application in dental implants and for application in wound-healing and tissue regeneration processes.

For the treatment of bone diseases caused by abnormal bone metabolism, human MP52 Arg is administered in systemic by injection such as intravenous injection, muscle injection and intraperitoneal injection, oral administration, non-oral administration such as suppository, and other any conventional methods.

For the treatment of bone fracture, it is administered in systemic and locally by injection, oral and non-oral administration. Also matrix containing human MP52 Arg is preferably implanted in the area close to the fractured bone. Suitable matrixes are natural polymers such as collagen and fibrin clot, and artificial polymers degradable in living body such as polylactated glycolic acid.

In case of orthopedic reconstruction, cosmetic surgery, bone transplantation and dental implantation, human MP52 Arg for example can be coated on the surface of bone and tooth to be implanted by means of collagen paste, fibrin glue and other adhering materials. It can also be applied to the tissue, the bone or alveolar bone around which the bone and tooth is transplanted. In case of bone transplantation it can be used for both natural and artificial bone. As the material of artificial bone and tooth, conventional materials such as metals, ceramics, glass and other natural or artificial inorganic substance are used. Hydroxyapatite is a preferred artificial substance. Artificial bone can be constructed by dense material in the inner part and porous material in the other part. For example, dense steel covered porous steal can be cited. Porous hyroxyapatite is one of the materials to produce artificial bone. When such porous material is used, human MP52 Arg can be penetrated into it. The surface of artificial bone can also be roughened to keep human MP52 Arg on the surface.

It is also beneficial to apply human MP52 Arg to the part from which cancerous bone tissue is removed in order to accelerate the reconstruction of bone.

The dose of human MP52 Arg to be obtained is decided depending upon the purpose and the method of application. In general, when it is administered in systemic, the dose is from 1 μg to 100 μg/kg. When it is used for implantation, the preferred dose is from 30 μg to 30 g per site.

This purified human MP52 Arg can be formulated in any conventional form such as injection liquid, pills, capsules and suppository. For local administration human MP52 Arg can be included in a matrix such as collagen, fibrin glue and poly lactated glycolic acid. For the use of implantation and transplantation it is applied to the surface or in the porous part of bone and tooth.

This invention is illustrated by the examples.

Example 1

Construction of expression vector for MP52 Arg

The pSK52s plas id (WO 95/04819) was digested with Hind III and the DNA fragment containing the cDNA comprising the complete coding region for MP52 was isolated by extraction from 0.8 % low ^■melting agarose gels and ligated into the Hind III site of pABstop vector, which is supplied by Dr. Gerd Zettlmeiβl of Behringwerke AG. The structure of the resulting MP52 expression vector, pMSS99 (5.0 kb) , was confirmed by the DNA sequencing and the restriction enzyme mapping. The genetic elements of pMSS99 are schematically shown in Fig.l. The MP52 sequence in pMSS99 comprises the nucleotides 576-2279 in SEQ ID NO:l of the Sequence Listing.

Example 2

Establishment of CHO clones producing MP52 Arg

Dhfr-deficient CHO cells CHO-DUKX-B11 (Urlaub, G. and Chasin, L.A. (1980) Proc.Natl.Acad.Sci, USA, 77, pp.4216-4220) were cotransfected with the expression plasmid for MP52 (pMSS99) and pSVOAdhfr (Zettlmeiβl, G. et al., (1987) Bio/Technology 5 , 720- 725) by calcium phosphate-mediated DNA transfer method. Then high producer clones of MP52 Arg were established by a gene amplification protocol using methotrexate (MTX) .

Briefly, 10 μg of pMSS99 and 2 μg of pSVOAdhfr were dissolved in 1 ml of 25 M HEPES-140 M NaCl-0.75 mM Na₂HP0₄ (pH 7.05), then mixed with 50 μl of 2.5 M CaCl₂. The resultant precipitates were overlaid to CH0-DUKX-B11 cells and incubated at room temperature for 30 in. Then MEM ALPHA medium with ribo- and deoxyribo- nucleosides (MEMα*) containing 10 % fetal bovine serum (FBS) was added to the cell layer to incubate in C0₂ incubator for 4-6 h. After a treatment with 10 % glycerol in MEMoJ containing 10 % FBS at room temperature for 3 in, the cells were cultured in MEMα^1* containing 10 % FBS for 2 days. Then the cells were placed in MEM ALPHA medium without ribo- and deoxyribo-nucleosides (MEMαT) containing 10 % dialyzed FBS to select the transformants. The transformant clones were isolated and assayed for the expression of MP52 Arg by Western blot analysis as described in the next section.

Then high producer clones of MP52 Arg were established by a gene amplification protocol using methotrexate (MTX) . The MP52 Arg producing clones were further selected stepwisely in increasing concentrations of methotrexate (MTX) to amplify the MP52 gene in accordance with the pSVOAdhfr gene. Several clones were obtained which produced 1-3 μg of mature MP52 Arg/10⁶ cells/24 h at 400 nM MTX.

Example 3

Detection of MP52 Arg in the culture supernatants

Clones were examined for the expression of MP52 Arg by Western blot analysis as follows: The culture supernatants (1-15 μl) were applied on SDS-PAGE (15-25 % polyacryla ide gradient gel, Daiichi Pure Chemicals) under reducing conditions, then the proteins were transferred to a PVDF membrane (Clear Blot Membrane-P, ATTO) . The membrane was blocked with Block Ace (Dai-Nihon Seiyaku) for 1 h, rinsed with Tris-buffered saline (TBS) , then treated with 10 μg/ml of chicken antibodies to MP52 Arg in 10-times diluted Block Ace overnight. After washing the membrane with 0,1 % Tween 20 in TBS (TTBS) , the membrane was treated with rabbit anti-chicken IgG-ALP conjugate (Sigma A 9171) in 10-times diluted Block Ace for 1 h. The membrane was washed with TTBS and then reacted with Alkaline phosphatase Conjugate Substrate Kit (BIO-RAD) to visualize the bands corresponding to MP52 Arg.

Example 4

Cell culture of the MP52 Arg-producing CHO cell line

The CHO cell line with the highest productivity of MP52 Arg and MP52, MC-2 (deposited under No.FERM BP-5142 on June 21, 1995 with the National Institute of Bioscience and Human Technology, Japan) was grown with roller bottles containing MEMα^* supplemented with 10 % FBS, 400 nM MTX, 100 U/ml Penicillin and 100 μg/ml Streptomycin. After the MC-2 cells had grown to confluency, they were washed with serum-free MEMα-n and then cultured in serum-free DME/F12 medium supplemented with 10 mM HEPES (pH 7.3) , 10 KIE/ml Aprotinin, 1 mM sodium butyrate, 6 μg/ml sodium selenate, 5 μg/ml transferrin, 18 μg/ml ethanol amine, 9 μg/ml insulin, 100 U/ml Penicillin and 100 μg/ml Streptomycin. The conditioned medium was collected every day for a week.

Example 5

Purification

One liter of the culture supernatants were mixed with 0.1 volume of 0.2 M sodium phosphate buffer, pH 6.0, and applied to a POROS HS column (10 ml, PerSeptive Biosystems) . The elution was performed by a linear gradient of NaCl from 0.3 to 2 M. The eluate containing MP52 Arg was applied to reverse-phase column (RESOURCE RPC, Pharmacia) . The elution was performed by linear gradient of acetonitrile, containing 0.05 % TFA and MP52 Arg was eluted at about 35 % acetonitrile. The N-terminal amino acid sequence analysis for purified MP52 Arg was performed using a pulse liquid gas phase sequencer (Applied Biosystems model 476) . The result is shown in Table 1. It is indicated that MP52 Arg is processed proteolytically at Arg(380) -Arg(381) (amino acid positions -1 and +1 of Sequence ID NO:l) from its precursor. However, the precursor can also be processed at Arg(381) -Ala(382) (amino acid positions +1 and +2 of Sequence ID NO:l) .

Table 1

N-Terminal Amino Acid Sequence Analysis of MP52 Expressed in CHO-Cells

Cycle Amino Acid (pmol) Amino Acid Sequence of MP52

Arg Ala Pro Leu Ala Thr Arg Gin Gly Lys Arg Pro Ser Lys Asn Leu Lys Ala Arg Cys Ser Arg Lys Ala Leu His Val Asn Phe Lys Example 6

Biological activity

Osteoprogenitor-like ROB-C26 cells (Calcif. Tissue Int. vol. 49, p. 221-225, 1991) were plated onto 48-well multi-well plates (Coaster) at a density of 1.5 x 10* cells/well and pre-incubated for 3 days in MEMα^" containing 10% fetal bovine serum (FBS) . After the removal of culture medium, fresh MEMα" containing 10% FBS and serially diluted MP52 Arg in lOmM HCl (2μl/ml) was added to the cultures and incubated for 6 days with changing the medium and the additives on day 3. Cell layers were washed with phosphate-buffered saline, and extracted with 0.2% Nonidet containing 1 mM MgCl₂. Alkaline phosphatase (ALP) activities were determined according to the procedure of Takuwa et al. (Am. J. Physiol. vol. 257, p. E797-E803, 1989). As shown in Table 2, the treatment of ROB-C26 cells with MP52 Arg increased total ALP activities per well concentration-dependently.

Table 2

Influence of CHO cell-derived MP52 Arg on the ALP activity of the ROB-C26 cell line

Values represent means ± S.D. of 4 cultures. *p<0.01 compared to the vehicle-treated control (Dunnett's test)

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Biopharm Gesellschaf zur biotechnologischen

Entwicklung von Pharmaka mhH

(B) STREET: Czernyring 22

(C) CITY: Heidelberg

(E) COUNTRY: Germany

(F) POSTAL CODE (ZIP) : 69115

(ii) TITLE OF INVENTION: New Protein Human MP52 Arg (iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(v) CURRENT APPLICATION DATA:

APPLICATION NUMBER: EP 95112241.5

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2703 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:640..2142

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION:640..720

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 1780..2142

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CCATGGCCTC GAAAGGGCAG CGGTGATTTT TTTCACATAA ATATATCGCA CTTAAATGAG 60

TTTAGACAGC ATGACATCAG AGAGTAATTA AATTGGTTTG GGTTGGAATT CCGTTTCCAA 120

TTCCTGAGTT CAGGTTTGTA AAAGATTTTT CTGAGCACCT GCAGGCCTGT GAGTG^*TGTGT 180

GTGTGTGTGT GTGTGTGTGT GTGTGTGTGA AGTATTTTCA CTGGAAAGGA TTCAAAACTA 240 GGGGGAAAAA AAAACTGGAG CACACAGGCA GCATTACGCC ATTCTTCCTT CTTGGAAAAA 300

TCCCTCAGCC TTATACAAGC CTCCTTCAAG CCCTCAGTCA GTTGTGCAGG AGAAAGGGGG 360

CGGTTGGCTT TCTCCTTTCAAGAACGAGTT ATTTTCAGCT GCTGACTGGA GACGGTGCAC 20

GTCTGGATAC GAGAGCATTT CCACTATGGG ACTGGATACAAACACACACC CGGCAGACTT 480

CAAGAGTCTC AGACTGAGGA GAAAGCCTTT CCTTCTGCTG CTACTGCTGC TGCCGCTGCT 540

TTTGAAAGTC CACTCCTTTC ATGGTTTTTC CTGCCAAACC AGAGGCACCT TTGCTGCTGC 600

CGCTGTTCTC TTTGGTGTCA TTCAGCGGCT GGCCAGAGG ATG AGA CTC CCC AAA 654

Met Arg Leu Pro Lys -380

CTC CTC ACT TTC TTG CTT TGG TAC CTG GCT TGG CTG GAC CTG GAA TTC 702 Leu Leu Thr Phe Leu Leu Trp Tyr Leu Ala Trp Leu Asp Leu Glu Phe -375 -370 -365 -360

ATC TGC ACT GTG TTG GGT GCC CCT GAC TTG GGC CAG AGA CCC CAG GGG 750 lie Cyε Thr Val Leu Gly Ala Pro Asp Leu Gly Gin Arg Pro Gin Gly

-355 -350 -345

ACC AGG CCA GGA TTG GCC AAA GCA GAG GCC AAG GAG AGG CCC CCC CTG 798 Thr Arg Pro Gly Leu Ala Lys Ala Glu Ala Lys Glu Arg Pro Pro Leu -340 -335 -330

GCC CGG AAC GTC TTC AGG CCA GGG GGT CAC AGC TAT GGT GGG GGG GCC 846 Ala Arg Asn Val Phe Arg Pro Gly Gly His Ser Tyr Gly Gly Gly Ala -325 -320 -315

ACC AAT GCC AAT GCC AGG GCA AAG GGA GGC ACC GGG CAG ACA GGA GGC 894 Thr Asn Ala Asn Ala Arg Ala Lys Gly Gly Thr Gly Gin Thr Gly Gly -310 -305 -300

CTG ACA CAG CCC AAG AAG GAT GAA CCC AAA AAG CTG CCC CCC AGA CCG 942 Leu Thr Gin Pro Lys Lys Asp Glu Pro Lys Lys Leu Pro Pro Arg Pro -295 -290 -285 -280

GGC GGC CCT GAA CCC AAG CCA GGA CAC CCT CCC CAA ACA AGG CAG GCT 990 Gly Gly Pro Glu Pro Lys Pro Gly His Pro Pro Gin Thr Arg Gin Ala

-275 -270 -265

ACA GCC CGG ACT GTG ACC CCA AAA GGA CAG CTT CCC GGA GGC AAG GCA 1038 Thr Ala Arg Thr Val Thr Pro Lys Gly Gin Leu Pro Gly Gly Lys Ala -260 -255 -250

CCC CCA AAA GCA GGA TCT GTC CCC AGC TCC TTC CTG CTG AAG AAG GCC 1086 Pro Pro Lys Ala Gly Ser Val Pro Ser Ser Phe Leu Leu Lys Lys Ala -245 -240 -235

AGG GAG CCC GGG CCC CCA CGA GAG CCC AAG GAG CCG TTT CGC CCA CCC 1134 Arg Glu Pro Gly Pro Pro Arg Glu Pro Lys Glu Pro Phe Arg Pro Pro -230 -225 -220

CCC ATC ACA CCC CAC GAG TAC ATG CTC TCG CTG TAC AGG ACG CTG TCC 1182 Pro He Thr Pro His Glu Tyr Met Leu Ser Leu Tyr Arg Thr Leu Ser -215 -210 -205 -200 GAT GCT GAC AGA AAG GGA GGC AAC AGC AGC GTG AAG TTG GAG GCT GGC 1230 Asp Ala Asp Arg Lys Gly Gly Asn Ser Ser Val Lys Leu Glu Ala Gly

-195 -190 -185

CTG GCC AAC ACC ATC ACC AGC TTT ATT GAC AAA GGG CAA GAT GAC CGA 1278 Leu Ala Asn Thr He Thr Ser Phe He Asp Lys Gly Gin Asp Asp Arg -180 -175 -170

GGT CCC GTG GTC AGG AAG CAG AGG TAC GTG TTT GAC ATT AGT GCC CTG 1326 Gly Pro Val Val Arg Lys Gin Arg Tyr Val Phe Asp He Ser Ala Leu -165 -160 -155

GAG AAG GAT GGG CTG CTG GGG GCC GAG CTG CGG ATC TTG CGG AAG AAG 1374 Glu Lys Asp Gly Leu Leu Gly Ala Glu Leu Arg He Leu Arg Lys Lys -150 -145 -140

CCC TCG GAC ACG GCC AAG CCA GCG GCC CCC GGA GGC GGG CGG GCT GCC 1422 Pro Ser Asp Thr Ala Lys Pro Ala Ala Pro Gly Gly Gly Arg Ala Ala -135 -130 -125 -120

CAG CTG AAG CTG TCC AGC TGC CCC AGC GGC CGG CAG CCG GCC TCC TTG 1470 Gin Leu Lys Leu Ser Ser Cys Pro Ser Gly Arg Gin Pro Ala Ser Leu

-115 -110 -105

CTG GAT GTG CGC TCC GTG CCA GGC CTG GAC GGA TCT GGC TGG GAG GTG 1518 Leu Asp Val Arg Ser Val Pro Gly Leu Asp Gly Ser Gly Trp Glu Val -100 -95 -90

TTC GAC ATC TGG AAG CTC TTC CGA AAC TTT AAG AAC TCG GCC CAG CTG 1566 Phe Asp He Trp Lys Leu Phe Arg Asn Phe Lys Asn Ser Ala Gin Leu ^' -85 -80 -75

TGC CTG GAG CTG GAG GCC TGG GAA CGG GGC AGG GCC GTG GAC CTC CGT 1614 Cys Leu Glu Leu Glu Ala Trp Glu Arg Gly Arg Ala Val Asp Leu Arg -70 -65 -60

GGC CTG GGC TTC GAC CGC GCC GCC CGG CAG GTC CAC GAG AAG GCC CTG 1662 Gly Leu Gly Phe Asp Arg Ala Ala Arg Gin Val His Glu Lys Ala Leu -55 -50 -45 -40

TTC CTG GTG TTT GGC CGC ACC AAG AAA CGG GAC CTG TTC TTT AAT GAG 1710 Phe Leu Val Phe Gly Arg Thr Lys Lys Arg Asp Leu Phe Phe Asn Glu

-35 -30 -25

ATT AAG GCC CGC TCT GGC CAG GAC GAT AAG ACC GTG TAT GAG TAC CTG 1758 He Lys Ala Arg Ser Gly Gin Asp Asp Lys Thr Val Tyr Glu Tyr Leu -20 -15 -10

TTC AGC CAG CGG CGA AAA CGG CGG GCC CCA CTG GCC ACT CGC CAG GGC 1806 Phe Ser Gin Arg Arg Lys Arg Arg Ala Pro Leu Ala Thr Arg Gin Gly -5 ' 1 5

AAG CGA CCC AGC AAG AAC CTT AAG GCT CGC TGC AGT CGG AAG GCA CTG 1854 Lys Arg Pro Ser Lys Asn Leu Lys Ala Arg Cys Ser Arg Lys Ala Leu 10 15 20 25

CAT GTC AAC TTC AAG GAC ATG GGC TGG GAC GAC TGG ATC ATC GCA CCC 1902 His Val Asn Phe Lys Asp Met Gly Trp Asp Asp Trp He He Ala Pro

30 35 40 CTT GAG TAC GAG GCT TTC CAC TGC GAG GGG CTG TGC GAG TTC CCA TTG 1950 Leu Glu Tyr Glu Ala Phe His Cys Glu Gly Leu Cys Glu Phe Pro Leu 45 50 55

CGC TCC CAC CTG GAG CCC ACG AAT CAT GCA GTC ATC CAG ACC CTG ATG 1998 Arg Ser His Leu Glu Pro Thr Asn His Ala Val He Gin Thr Leu Met 60 65 70

AAC TCC ATG GAC CCC GAG TCC ACA CCA CCC ACC TGC TGT GTG CCC ACG 2046 Asn Ser Met Asp Pro Glu Ser Thr Pro Pro Thr Cys Cys Val Pro Thr 75 80 85

CGG CTG AGT CCC ATC AGC ATC CTC TTC ATT GAC TCT GCC AAC AAC GTG 2094 Arg Leu Ser Pro He Ser He Leu Phe He Asp Ser Ala Asn Asn Val 90 95 100 105

GTG TAT AAG CAG TAT GAG GAC ATG GTC GTG GAG TCG TGT GGC TGC AGG 2142 Val Tyr Lys Gin Tyr Glu Asp Met Val Val Glu Ser Cys Gly Cys Arg

110 115 120

TAGCAGCACT GGCCCTCTGT CTTCCTGGGT GGCACATCCC AAGAGCCCCT TCCTGCACTC 2202

CTGGAATCACAGAGGGGTCA GGAAGCTGTG GCAGGAGCAT CTACACAGCT TGGGTGAAAG 2262

GGGATTCCAA TAAGCTTGCT CGCTCTCTGA GTGTGACTTG GGCTAAAGGC CCCCTTTTAT 2322

CCACAAGTTC CCCTGGCTGA GGATTGCTGC CCGTCTGCTGATGTGACCAG TGGCAGGCAC 2382

AGGTCCAGGG AGACAGACTC TGAATGGGAC TGAGTCCCAG GAAACAGTGC TTTCCGATGA 2442

GACTCAGCCCACCATTTCTC CTCACCTGGG CCTTCTCAGC CTCTGGACTC TCCTAAGCAC 2502

CTCTCAGGAG AGCCACAGGT GCCACTGCCT CCTCAAATCA CATTTGTGCC TGGTGACTTC 2562

CTGTCCCTGG GACAGTTGAG AAGCTGACTG GGCAAGAGTG GGAGAGAAGA GGAGAGGGCT 2622

TGGATAGAGT TGAGGAGTGT GAGGCTGTTA GACTGTTAGA TTTAAATGTA TATTGATGAG 2682

ATAAAAAGCA AAACTGTGCC T 2703

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 501 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Arg Leu Pro Lys Leu Leu Thr Phe Leu Leu Trp Tyr Leu Ala Trp

-380 -375 -370 -365

Leu Asp Leu Glu Phe He Cys Thr Val Leu Gly Ala Pro Asp Leu Gly

-360 -355 -350

Gin Arg Pro Gin Gly Thr Arg Pro Gly Leu Ala Lys Ala Glu Ala Lys -345 -340 -335 Glu Arg Pro Pro Leu Ala Arg Asn Val Phe Arg Pro Gly Gly His Ser -330 -325 -320

Tyr Gly Gly Gly Ala Thr Asn Ala Asn Ala Arg Ala Lys Gly Gly Thr -315 -310 -305

Gly Gin Thr Gly Gly Leu Thr Gin Pro Lys Lys Asp Glu Pro Lys Lys

-300 -295 -290 -285

Leu Pro Pro Arg Pro Gly Gly Pro Glu Pro Lys Pro Gly His Pro Pro

-280 -275 -270

Gin Thr Arg Gin Ala Thr Ala Arg Thr Val Thr Pro Lys Gly Gin Leu -265 -260 -255

Pro Gly Gly Lys Ala -Pro Pro Lys Ala Gly Ser Val Pro Ser Ser Phe -250 -245 -240

Leu Leu Lys Lys Ala Arg Glu Pro Gly Pro Pro Arg Glu Pro Lys Glu -235 -230 -225

Pro Phe Arg Pro Pro Pro He Thr Pro His Glu Tyr Met Leu Ser Leu

-220 -215 -210 -205

Tyr Arg Thr Leu Ser Asp Ala Asp Arg Lys Gly Gly Asn Ser Ser Val

-200 -195 -190

Lys Leu Glu Ala Gly Leu Ala Asn Thr He Thr Ser Phe He Asp Lys -185 -180 -175

Gly Gin Asp Asp Arg Gly Pro Val Val Arg Lys Gin Arg Tyr Val Phe -170 -165 -160

Asp He Ser Ala Leu Glu Lys Asp Gly Leu Leu Gly Ala Glu Leu Arg -155 -150 -145

He Leu Arg Lys Lys Pro Ser Asp Thr Ala Lys Pro Ala Ala Pro Gly

-140 -135 -130 -125

Gly Gly Arg Ala Ala Gin Leu Lys Leu Ser Ser Cys Pro Ser Gly Arg

-120 -115 -110

Gin Pro Ala Ser Leu Leu Asp Val Arg Ser Val Pro Gly Leu Asp Gly -105 -100 -95

Ser Gly Trp Glu Val Phe Asp He Trp Lys Leu Phe Arg Asn Phe Lys -90 -85 -80

Asn Ser Ala Gin Leu Cys Leu Glu Leu Glu Ala Trp Glu Arg Gly Arg -75 -70 -65

Ala Val Asp Leu Arg Gly Leu Gly Phe Asp Arg Ala Ala Arg Gin Val -60 -55 -50 -45

His Glu Lys Ala Leu Phe Leu Val Phe Gly Arg Thr Lys Lys Arg Asp

-40 -35 -30

Leu Phe Phe Asn Glu He Lys Ala Arg Ser Gly Gin Asp Asp Lys Thr -25 -20 -15 Val Tyr Glu Tyr Leu Phe Ser Gin Arg Arg Lys Arg Arg Ala Pro Leu -10 -5 1

Ala Thr Arg Gin Gly Lys Arg Pro Ser Lys Asn Leu Lys Ala Arg Cys 5 10 15 20

Ser Arg Lys Ala Leu His Val Asn Phe Lys Asp Met Gly Trp Asp Asp

25 30 35

Trp He He Ala Pro Leu Glu Tyr Glu Ala Phe His Cys Glu Gly Leu 40 45 50

Cys Glu Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala Val 55 60 65

He Gin Thr Leu Met Asn Ser Met Asp Pro Glu Ser Thr Pro Pro Thr 70 75 80

Cys Cys Val Pro Thr Arg Leu Ser Pro He Ser He Leu Phe He Asp 85 90 95 100

Ser Ala Asn Asn Val Val Tyr Lys Gin Tyr Glu Asp Met Val Val Glu

105 110 115

Ser Cys Gly Cys Arg 120

- IS -

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Claims

1. A protein comprising amino acids 1 to 121 of SEQ ID NO.1 of the enclosed Sequence Listing.

2. Process for the production of a biologically active protein according to claim 1, wherein at least a part of a DNA- sequence as shown in SEQ ID NO:1 is introduced into a suitable host cell under conditions that allow expression of the DNA-sequence and protein formation, followed by isolation of said protein from other proteins produced by said host cell.

3. Pharmaceutical composition, containing as the active substance a protein according to claim 1, if desired together with usual carrier substances, auxiliary substances, diluents and fillers.

4. Use of a pharmaceutical composition according to claim 3 for promoting bone morphogenation, treatment or prevention of damage to bone, cartilage, connective tissues, skin, mucous membranes, epithelium or teeth, for application in dental implants and for application in wound-healing and tissue regeneration processes.

5. Use of a pharmaceutical composition as claimed in claim 3 for treating osteoporosis or bone fracture.

6. A parmaceutical composition as claimed in claim 3 for the use of orthopedic reconstruction, bone transplantation, cosmetic surgery or dental implantation.