MXPA98000686A - New human human protein mp - Google Patents

Abstract

The present invention provides proteins called human HMW MP52s which have some amino acid sequence of SEQ ID NO: 1 which is produced in CHO cells. The present invention also provides a process for the production of human HMW MP52s and a pharmaceutical composition containing as an active ingredient human HMW MP52s. Human HMW MP52s can be used to treat or prevent bone diseases, etc. due to its property of promoting the morphogenesis of hue

Description

MOVE PROTEIN HUMAN HUMAN MP52a Detailed description of the invention (1) Field of the Invention This invention relates to the new HWP prstina HWW MP52s and to an inter-aJia medical composition for promoting cartilage and bone morphogenesis comprising the human HMW MP52s. In particular the medical composition is useful for treating bone diseases caused by abnormal bone metabolism such as osteoporosis, to treat fracture of bone, and for the purpose of orthopedic reconstruction, bone transplantation, cosmetic surgery and dental therapeutics. In addition, it is useful to treat cartilage disorders. (2) Description of the Prior Art Pharmaceutical compositions including vitamin D3, calcitonin, estrogen and bisphosphonate derivatives have been used in clinical practice to treat bone diseases. Their therapeutic results, however, are not entirely satisfactory, and a better pharmaceutical composition is greatly desired. The TGF-li family of growth factors such as BMP, TGF, and related inhibitory proteins have reported REF ,: 26670 be useful to heal the wound and repair the tissue. The bone morphogenetic activity of some of these proteins has also been shown. PCT Patent Application WO 93/16099 and WO 95/04819 disclose human DNA sequences encoding TGF-β type proteins, and as a preferred human protein MP52. E.E. Storm et al. reports in Nature, 1994, vol. 368, p.639-642 three mouse growth / differentiation factors, p. ex. GDF5, GDF6 and GDF7 were identified as new members of the TGF-β superfamily and that mutations in the GDF5 gene caused bradypodism in mice. The GDFS de ratin has the same amino acid sequence of the predicted / mature form as the human one of MP52 except one amino acid. However, there is no indication in this publication for using those proteins for the treatment of bone diseases. This was therefore the aim of the present invention to provide an additional growth factor which is useful as an agent for the stimulation of bone or cartilage formation. (3) Detailed Description of the Invention. Mature MP52 is considered as a protein that has 120 amino acids. This amino acid sequence is from 355 to 474 of SEQ ID NO.rl of the Listed Sequence (WO 95/04819). In fact, it has surprisingly been found by the inventors that there are cell lines which, when expressing a suitable DNA sequence, form proteins having amino acid sequence length variations, ie mature MP52 and human HMW MP52s (high molecular weight) . For the first time the invention was able to prove of human HMW MP52s having bone morphogenetic activity and are useful for preventing and / or treating bone diseases. The present invention relates to human HMW MP52s which falls within one of the following definitions: (1) a peptide containing dimeric protein having amino acid sequence from 1 to 474 of SEQ ID NO .-- 1. I « (2) a dimeric protein containing peptides having amino acid sequence from 121 to 474 of SEQ ID NO.:l. (3) a dimeric protein containing peptides having amino acid sequence 122 to 474 of SEQ ID NO.:l. (4) a dimeric protein containing peptides having amino acid sequence from 121 to 474 of SEQ ID NO: 1 and a peptide having amino acid sequence from 122 to 474 of SEQ ID NO.:l. (5) a dimeric protein containing peptides having amino acid sequence from 1 to 474 of SEQ ID NO: 1 and a peptide having amino acid sequence of 121 and / or 122 to 474 of SEQ ID NO. :1. (6) a dimeric protein containing peptides having amino acid sequence from 1 to 474 of SEQ ID NO: 1 and a peptide having amino acid sequence from 355 to 474 of SEQ ID NO. :1. (7) a dimeric protein containing peptides having an amino acid sequence of 121 and / or 122 to 474 of SEQ ID NO: 1 and a peptide having an amino acid sequence of 355 to 474 of SEQ ID NO: 1. The proteins usually exist as dimers through -S-S- bonds formed by cysteines. The peptide in this specification means a monomer. Human HMW MP52s induces cartilage formation of undifferentiated mesenchymal cells and stimulates the differentiation and maturation of osteoblasts. Because, the HMW *. Human MP52s are effective in preventing and / or treating bone diseases caused by abnormal bone metabolism such as osteoporosis. They also accelerate the healing process of bone fractures. In addition, they are useful for orthopedic reconstruction, bone transplants and dental therapeutics due to their bone morphogenetic activity. In addition, human HMW MP52s are effective in preventing and / or treating cartilage disorders caused by abnormal cartilage metabolism. A further object of the present invention is to provide a process for the production of human HMW MP52s, wherein the DNA sequence encoding human HMW MP52s as shown in SEQ ID NO.:1 is introduced into a suitable host cell and cultured under conditions that favor expression of the DNA sequence and protein formation, followed by isolation of said protein from other proteins recovered from said host cell. With the structure of the present invention, the DNA sequence depicted in SEQ ID NO.:l could be used to produce human HMW MP52s, however, also smaller portions thereof, still provide encoded MP52s human HMW and it is possible to expression of the DNA sequence in a suitable vector / host cell system. Suitable expression systems are known to one skilled in the art and can be easily determined by routine experimentation which are the minimum requirements for the length of the DNA sequence of SEQ ID NO: 1. Subsequent to the formation of the protein, the proteins are recovered from the host cell by methods known per se and finally human HMW MP52s are isolated from them. In particular the isolation of human HMW MP52s can be carried out using very precise differentiation separation methods which are known to one skilled in the art. For example, reverse phase HPLC could be considered for this purpose. A further objective of the present invention is to provide a pharmaceutical composition containing human HMW MP52s. Optionally, this composition could also include substances of usual vehicles, 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. It is possible to administer one of the human HMW MP52s separately or in a human HMW blend form MP52s. For the treatment of bone diseases caused by abnormal bone metabolism, human HMW MP52s are *; they are administered systemically by injection such as intravenous injection, intramuscular injection and intraperitoneal injection, oral administration, parenteral administration such as suppository, and any other conventional method. For the treatment of bone fracture, they are administered systemically and locally by injection, oral and parenteral administration. Also matrices containing human HMW MP52s are preferably implanted in the closed area of the fractured bone. Suitable matrices are natural polymers such as collagen and fibrin glue, and artificial polymers degradable in the living body such as poly-linked glycolic acid. In the case of orthopedic reconstruction, cosmetic surgery, bone transplantation and dental implant, human MP52s HMW for example can coat the surface of the bone and tooth to be implanted by means of collagen paste, fibrin glue and other adherent materials. This can also be applied to the tissue, bone or alveolar bone around which the bone and tooth are transplanted. In case of bone transplant this can be used for natural and artificial bone. As the bone and artificial tooth material, conventional materials such as metals, ceramics, glass and other natural or artificial inorganic substances are used. Hydroxyapatite is *, a preferable artificial substance. The artificial bone can be constructed by dense material on the inside and porous material on the other side. For example, solid steel and porous steel can be cited. Porous hydroxyapatite is one of the materials to produce artificial bone. When such porous material is used, human HMW MP52s can penetrate it. The surface of the artificial bone can be rough to retain human HMW MP52s on the surface. This also benefits the human HMW application MP52s in which part of the cancerous tissue was removed from the bone to accelerate bone reconstruction. The dose of human HMW MP52s administered is decided depending on the purpose and method of application. In general, when administered systemically, the dose is from 1 μg to 100 μg / kg. When implantation is used, the preferred dose is 30μg to 30mg per site. These purified human MP52s HMW can be formulated in any conventional manner such as injection liquid, pills, capsules and suppository. For local administration of human HMW MP52s can be included in a matrix such as collagen, fibrin glue and polylic acid glycolic acid. For the use of the implant and transplant was applied to the surface or in the porous part of the bone and tooth. This invention is illustrated by the examples. t Example 1 Production of human HMW MP52s (1) Construction of the expression vector for human HMW MP52s The vector pSK52s containing the human MP52 gene, which was provided by Dr. Hótten of Biopharm GmbH, was digested with Hind III and the fragment DNA containing the human MP52 gene was isolated by extracting 0.8% low melting point agarose gels and bound to the Hind III site of the pABstop vector, which is supplied by Dr. Gerd Zettlmeissl of Behringwerke AG. The structure of the human HMW expression vector MP52s, pMSS99 (5.0 kb) as shown in Fig. 1, was confirmed by the DNA sequence and the mapping with the restriction enzyme. The human HMW DNA sequence MP52s in pMSS99 were the nucleotides from 576 to 2279 shown in SEQ ID NO: 1 of the Sequence Listing. (2) Establishment of human MP52-producing CHO clones CH0-DUKX-B11 cells, mutants of CHO cells, which were supplied by Dr. Zettlmeissl of Behringwerke AG, were co-transfected with pMSS99 and pSVOAdhfr which were also provided by Dr. Zettlmeissl, by the method of DNA transfer by means of calcium phosphate. Afterwards, the highly productive human HMW clones MPS2s were established by the gene extension protocol using * -. methotrexate (MTX). Ten μg of pMSS99 and 2 μg of pSVOAdhfr were dissolved in 1 ml of 25 M HEPES-140 mM NaCl-0.75 mM Na2HP04 (pH 7.05), then mixed with 50 μl of 22.5 M CaCl. The resulting precipitates were coated for cells CHO-DUKX-B11 in a 10 cm dish and was stopped at room temperature for 30 min. Then 8 ml of MEM ALPHA with ribo- and deoxyribo-nucleosides (MEM +) containing 10% bovine fetus serum (FBS) was added to the cell layer to incubate in a CO 2 incubator for 4-6 h. After treatment with 10% glycerol in MEMa + containing 10% FBS at room temperature for 3 min, the cells were cultured in MEM + containing 10% FBS for 2 days. Then the cells were rearranged in MEM ALPHA without ribo- and deoxyribonucleosides (MEMcr) containing 10% dialyzed FBS to select the transformed ones. The transformed clones were isolated and tested for the expression of human HMW MP52s by Western blot analysis as described in the next session. The human HMW MP52s which produces clones was further selected by increasing the stepwise concentrations of methotrexate (MTX) to amplify the MP52 gene according to the pSVOAdhfr gene. Several clones were obtained to produce 1-3 μg of human HMW MPS2s / 106 cells / 24 hr at 400 nM MTX. * (3) Detection of human HMW MP52S in the culture supernatants The clones were examined for expression of human MP52 HMW by Western blot analysis as follows: culture supernatants (1-15 μl) were applied on SDS- PAGE (15-25% gradient polyacrylamide gel, Daiichi Puré Chemicals) under seductive 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, washed with Tris saline buffer (TBS), then treated with 10 μg / ml of chicken antibodies against human HMW MP52s diluted 10-fold in Block Ace all night. After washing the membrane with 0.1% Tween 20 in TBS (TTBS), the membrane was treated with rabbit antibodies against chicken IgG conjugated with ALP (Siqma A 9171) for 1 hr. in Block Ace diluted 10 times The membrane was washed with TTBS, then reacted with Alkaline Phosphatase from the Conjugate Substrate Kit (BIO-RAD) to visualize the bands corresponding to MP52. (4) The human HMW cell culture MP52s that produces the CHO cell line The CHO cell line with the highest productivity of human HMW MPS2s, MC-2 (Deposit No. FERM BP-5142), grew in rotary bottles containing MEM -complemented with 10% *.

FBS, MTX 400 nM, Penicillin 100 U / ml, Streptomycin 100 μg / ml. The MC-2 cells were then confluent, washed with serum free of MEMa- and then cultured in serum free of DME / F12 supplemented with 10 mM HEPES (pH 7. 3), Aprotinine 10 KIU, 1 mM sodium butyrate, 6 μg / ml sodium selenate, 5 μg / ml transferrin, 18 μg / ml amine ethanol, 9 μg / ml insulin, 100 U / ml penicillin, 100 μg streptomycin / ml. The conditioned medium was collected every day for a week. (5) Purification of human HMW MP52s The supernatant of the CHO culture and 0.1 vol. of 0.2 M sodium phosphate buffer, pH 6.0, were mixed and applied to the POROS HS column (10 ml, PerSeptive Biosystems) previously equilibrated with 50 mM NaCl, 20 M sodium phosphate buffer, pH 6.0. Proteins were eluted by linear gradient of NaCl from 0.05 to 2 M and collected with 20 fractions of 10 ml. Eluate MP52s was observed as three types of monomers and their apparent molecular weights of approximately 52, 40 and 14 kD were determined by SDS-PAGE analysis under reduced condition. These monomers form three types of homodimers (104 kD, 80 kD and 28 kD) and three types of heterodimers (92 kD: 40 kD-52 kD, 66 kD: 14 kD-52 kD, and 54 kD: 14 kD-40 kD ) and all dimers were named HMW (high *, molecular weight) human MP52s except the 28 kD homodimer that appeared to be known as a human MP52 homodimer (WO 95/04819). In this manner, the 104 kD homodimer and the 80 kD homodimer were isolated from the above fractions to examine the N-terminal amino acid sequence and biological activities. Fractions from 5S to 9 ^ were combined and concentrated approximately 10 times. The concentrate was loaded onto Superdex 200 pg (1.6 ID x 60 cm, Pharmacia) previously equilibrated with 20 mM sodium phosphate buffer, pH 7.1, containing 1 M NaCl. The elution was carried out at a flow rate of 0.5 ml / min. The fractions containing the 104 kD homodimer and the fractions containing the 80 kD homodimer were combined separately. Each was applied to reverse phase HPLC column (RESOURCE RPC, 3 ml, Pharmacia) and these were eluted with 35-40% acetonitrile. The concentrations of human HMW isolated MPS2s were determined by densitometry of the SDS-PAGE gel protein bands. Analysis of the N-terminal amino acid sequence was performed using a liquid pulse in a gas phase sequencer (Applied Biosystems model 476) for the 80 kD homodimer and the 104 kD homodimer, respectively. The results are shown in Table 1.

Table 1 HMW MP52s amino acid N-terminal 80 kD Lys Wing Arg Glu Pro Gly Pro Pro Arg Glu Pro Wing Arg Glu Pro Gly Pro Pro Arg Glu Pro Lys 104 kD Wing Pro Asp Leu Gly Gln Arg Pro Gln Gly Thr The amino acid sequences of 80 kD were from Lys 121 or Ala 122 to Arg 474 and that of 104 kD was from Ala 1 to Arg 474 in SEQ ID NO: 1 of the Sequences List. It was again found that CHO cells produced 3 types of homodimers, 104 kD, 80 kD and 28 kD, and 3 types of heterodimers, called 92 kD, 66 kD and 54 kD dimers.

Example 2 Biological activity ROB-C26 osteoprogenitor cells (Calcif. Tissue Int., Vol 49, pp. 221-225, 1991) cloned from newborn rat calvarial cells were plated in 40-well multi-well plates (Coaster) at a density of 1.5 x 104 cells / well and pre-incubated for 3 days in MEMa "containing 10% FBS." After the removal of the culture medium, fresh MEMa containing 10% FBS and serially diluted the human HMW MP52s of 80 kD or 104 kD in 10 mM HCl (2 μl / ml) was added to the cultures and incubated for 6 days changing the medium and the additives on day 3. The cell layers were washed with phosphate buffer, and extracted with 0.2% of Nonidet containing MgCl2 • 1 mM. Alkaline phosphatase activities were determined according to the procedure of Takuwa et al. (Am. J. Physiol., Vol 257, pp. E797-E803, 1989). As shown in Table 2, the treatment of ROB-C26 cells with human HMW of 80 kD or 104 kD MP52s increased the total activities of ALP per well depending on the concentration.

Table 2. Influence of human HMW of 80 kD and 104 kD MP52s on the ALP activity of the ROB-C26 cell line.

Compound Concentration ALP activity (ng / ml) (nmol / min / well) Vehicle (HCl mM 5.47 ± 0.81 1 -control treated human HMW MP52s 8.6 5.42 ± 1.09 of 80 kD 29 7.00 ± 0.89 86 14.30 ± 0.24 * 290 16.28 ± 0.19 * 860 18.41 ± 1.95 * Human HMW MP52s 11 6.51 ± 0.90 of 104 kD 38 7.54 ± 0.29 110 8.32 ± 0.12 * 380 12.07 ± 0.53 * 1100 16.98 ± 0.47 * The values represent means ± S.D. of 3 or 4 crops. * p < 0.01 compared treated control vehicle (Dunnett's test).

"List of Sequences" SEQ ID NO: 1 TYPE OF SEQUENCE: nucleotide with the corresponding protein LENGTH OF THE SEQUENCE: 2703 HEBRA: double TOPOLOGY: linear TYPE OF MOLECULE: cDNA for mRNA ORIGINAL SOURCE ORGANISM: homo sapiens CHARACTERISTICS: Peptide signal from 640 to 720 bp mature peptide from 1783 to 2142 bp OWNERS: human embryo DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: CCATGGCCTC GAAAGGGCAG CGGTGATTTT TTTCACAT ?? ATATATCGCA CTTAAATCAG 60 TTTAGACAGC ATGACATCAG AGAGTAATTA AATTGGTTTG GGTTGGAATT CCGTTTCCAA 120 TTCCTGAGTT CAGGTTTGTA AAAGATTTTT CTGAGCACCT GCAGGCCTGT GAGTGTGTGT 180 GTGTGTGTGT GTGTGTGTGT GTGTGTGTGA AGTATTTTCA CTGGAAAGGA TTCAAAACTA 240 GGGGGAAAAA AAAACTGGAG CACACAGGCA GCATTACGCC ATTCTTCCTT CTTGGAAAAA 300 TCCCTCAGCC TTATACAAGC CTCCTTCAAG CCCTCAGTCA GTTGTGCACG AGAAAGGGSß 360 CGGTTGGCTT TCTCCTTTCA AGAACGAGTT ATTTTCAGCT GCTGACTGGA GACGGTGCAC 420 GTCTGGATAC GAGAGCATTT CCACTATGGG ACTGGATACA AACACACACC CSGCAGACTT 480 CAAGAGTCTC AGACTGAGGA GAAAGCCTTT CCTTCTGCTG CTACTGCTGC TGCCCCTGCT -540 TTTGAAAGTC CACTCCTTTC ATGGTTTTTC CTGCCAAACC AGAGGCACCT TTGCTGCTGC 600 CGCTGTTCTC TTTGGTGTCA TTCAGCCGCT ATG AGA CTC CCC GGCCAGAGG AAA 654 Leu Arg Pro Lys Mat -25 CTC CTC ACT TTC TTG CTT TGG TAC CTG GCT TGG CTG GAC CTG GAA TTC 702 Leu Leu T r Phß L «u Lsu Trp Tyr Leu Wing Trp au Asp Leu Glu Phß -20 -15 -10 ATC TGC ACT GTG TTG GGT GCC CCT GAC TTG GGC CAG AGA CCC CAG GGG 750 II * Cys Thr Val su Gly Ais Pro Asp Leu Gly Gln Ara Pro Gln Gly -5 1 5 10 ACC AGG CCA GGA TTG GCC AAA GC? GAG GCC AAG GAG AGG CCC CCC CTG 798 Thr Ring Pro Gly Leu A s Lys A s Glu Wing Lys Glu Arg Pro Pro Lou 15 20 25 GCC CGG AAC GTC TTC AGG CCA GGG GGT CAC AGC TAT GGT GGG GGG GCC 846 Wing Arg Asn Val Phß Arg Pro Cly Gly His Ser Tyr Gly Gly Gly Wing 30 35 40 ACC AAT GC? AAT GCC AGG GCA AAG GGA GGC ACC GGG CAG ACA GGC 894 Thr Asn Wing Asn Wing Arg Wing Lys Gly Gly Thr Gly Gln Thr Gly Gly 45 50 55 CTG ACE CAG CCC AAG AAG GAT GAA CCC AAA AAG CTG CCC CCC AGA CCG 942 Leu Thr G n Pro Lys Lys Asp Glu Pro Lys Lys Lau Pro Pro Arg Pro 60 65 70 GGC GGC CCT GAA CCC AAG CCA GGA CAC CCT CCC CAÁ ACÁ AGG CAG GCT 990 Gly Gly Pro Glu Pro Lys Pro Gly His Pro Pro Gln Thr Arg Gln Wing 75 80 85 90 ACÁ GCC CGG ACT GTG ACC CCA AAA GGA CAG CTT CCC GGA GGC AAG GCA 1038 Thr Wing Arg Thr Val Thr Pro Lys Gly G n Leu Pro Gly Gly Lys Wing 95 100 105 CCC CCA AAA GCA GGA TCT GTC CCC AGC TCC TTC CTG AAG AAG GCC 1086 Pro Pro Lys Wing Gly Ser Val Pro Ser Ser Phe Leu Leu Lys Lys Wing 110 115 120 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 125 130 135 CCC ATC ACÁ CCC CAC GAG TAC ATG CTC TCG CTG TAC AGG ACG CTG TCC 1182 Pro lie Thr Pro His Glu Tyr Mee Leu Ser Leu Tyr Arg Thr Leu Ser 140 145 150 GAT GAC GAC AGA AAG GGA GGC AAC AGC AGC GTG AAG TTG GAG GCT GGC 1230 Asp Wing Asp Arg Lys Gly Gly Asn Ser Ser Val Lys Leu Glu Wing Gly 155 160 165 170 CTG GCC AAC ACC ATC ACC AGC TTT ATT GAC AAA GGG CAA GAT GAC CGA 1278 Leu Wing Asn Thr lie Thr Ser Phe lie Asp Lys Gly Gln Asp Asp Arg 175 180 185 GGT CCC GTG GTC AGG AAG CAG AGG TAC GTG TTT GAC ATT AGT GCC CTG 1326 Gly Pro Val Val Arg Lys Gln Arg Tyr Val Phe Asp lie Be Ala Leu 190 195 200 GAG AAG GAT GGG CTG CTG GGG GCC GAG CTG CGG ATC TTG CGG AAG AAG 1374 Glu Lys Asp Gly Leu Leu Gly Wing Glu Leu Arg lie Leu Arg Lys Lys 205 210 215 ccc tcs GAC? CG GCC AAG CCA GCG GCC CCC GGA GGC GGG CGG GCT scc 1422 Pro Ser Aso Thr Wing Lys Pro Wing Wing Pro Gly Gly Gly Arg Wing Wing 220 225 230 CAG CTG AAG CTG TCC AGC TGC CCC AGC GGC CGG CAG CCG GCC TCC TTG 1470 Gln Leu Lys Leu Ser Ser Cys Pro Ser Gly Arg Gln Pro Ala Ser Leu 235 240 245 250 CTG GAT GTG CGC TCC GTG CCA GGC CTG GAC GGA TCG GGC TGG GAG GTG 1518 Leu Asp Val Arg Ser Val Pro Gly Leu Asp Gly Ser Gly Trp Glu Val 255 260 265 TTC GAC ATC TGG AAG CTC TTC CGA AAC TTT AAG AAC TCG GCC CAG CTG 1566 Phe Asp lie Trp Lys Leu Phe Arg Asn Phe Lys Aan Ser Wing Gln Leu 270 275 280 TGC CTG GAG CTG GAG GCC TGG GAA CGG GGC AGG GCC GTG GAC CTC CGT 1614 Cys Leu Glu Leu Glu Wing Trp Glu Arg Gly Arg Wing Val Asp Leu Arg 285 290 295 GGC CTG GGC TTC GAC CGC GCC GCC CGG CAG GTC CAC GAG AAG GCC CTG 1662 Gly Leu Gly Phe Asp Arg Wing Wing Arg Gln Val His Glu Lys Wing Leu 300 305 310 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 Phß Phe Asn Glu 315 320 325 330 ATT AAG GCC CGC TCT GGC CAG GAC GAT AAG ACC GTG TAT GAG TAC CTG 1758 lia Lys Wing Arg Ser Gly Gln Asp Asp Lys Thr Val Tyr Glu Tyr Leu 335 340 345 TTC AGC CAG CGG CGA AAA CGG CGG GCC CCA CTG GCC ACT CGC CAG GGC 1806 Phe Ser Gln Arg Arg Lys Arg Arg Ala Pro Leu A the Thr Arg Gln Gly 350 355 360 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 Wing Arg Cya Ser Arg Lya Wing Leu 365 370 375 CAT GTC AAC TTC AAG GAC ATG GGC TGG GAC GAC TGG ATC ATC GCA CCC 1902 His Val Asn Phß Lys Asp Mee Gly Trp Asp Asp Trp Lie Lie Ala Pro 380 385 390 CTT GAG TAC GAG GCT TTC CAC TGC GAG GGG CTG TGC GAG TTC CCA TTG 1950 Leu Glu Tyr Glu Wing Ph * His Cys Glu Gly Leu Cys Glu Phß Pro Leu 395 400 405 410 CGC TCC CAC CTG GAG CCC ACG AAT CAT GCA GTC ATC CAO ACC CTG ATG 1998 Arg Ser His Leu Glu Pro Thr Asn His Wing Val lie Gln Thr Leu Met 415 420 425 AAC TCC ATG GAC CCC GAG TCC ACÁ CC? CCC ACC TGC GTT CCC ACG 2046 Asn Ser Met Asp Pro Glu Be Thr Pro Pro Thr Cys Cys Val Pro Thr 430 435 440 CGG CTG AGT CCC ATC AGC ATC CTC TTC ATT GAC TCT GCC AAC AAC GTG 2094 Arg Leu Ser Pro lie Ser lie Leu Phe He Asp Ser Ala? sn Asn Val 445 450 455 GTG TAT AAG CAG TAT GAG GAC ATG GTC GTG GAG TCG TGT GGC TGC AGG 2142 Val Tyr Lyß Gln Tyr Glu Asp Mee Val Val Glu Ser Cys Gly Cys Arg 460 465 470 TAG CAGCACTGGC CCTCTGTCTT CCTGGGTGGC ACATCCCAAG AGCCCCTTCC 2195 * • * 475 TGCACTCCTG GAATCACAGA GGGGTCAGGA AGCTGTGGC? GGAGCATCTA CACAGCTTGG 2255 GTGAAAGGGG ATTCCAATAA GCTTGCTCGC TCTCTGAGTG TGACTTGGGC TAAAGGCCCC 2315 CTTTTATCCA CAAGTTCCCC TGGCTGAGGA TTGCTGCCCG TCTGCTGATG TGACCAGTGG 2375 CAGGCACAGG TCCAGGGAGA CAGACTCTGA ATGGGACTGA GTCCCAGGAA ACAGTGCTTT 2435 CCGATGAGAC TCAGCCCACC ATTTCTCCTC ACCTGGGCCT TCTCAGCCTC TGGACTCTCC 2495 TAAGCACCTC TCAGGAGAGC CACAGGTGCC ACTGCCTCCT CAAATCACAT TTGTGCCTGG 2555 TGACTTCCTG TCCCTGGGAC AGTTGAGAAG CTGACTGGGC AAGAGTGGGA GAGAAGAGGA 2615 GAGGGCTTGG ATAGAGTTGA GGAGTGTGAG GCTGTTAGAC TGTTAGATTT AAATGTATAT 2675 TGATGAGATA AAAAGCAAAA CTGTGCCT 2703 4. Brief Description of the Drawings Fig. 1 shows a plasmid map of an MP52s human HMW expression vector, pMSS99 (5.0 kb). The sequence of Human HMW DNA MP52s in pMSS99 are the nucleotides of 576 to 2279 shown in SEQ ID NO: 1 of the Sequence Listing.

Claims (6)

Claims

1. - A protein called human HMW MP52, characterized in that it is selected from the group consisting of: (1) a dimeric protein containing peptides having amino acid sequence from 1 to 474 of SEQ ID NO: 1. (2) a dimeric protein containing peptides having amino acid sequence from 121 to 474 of SEQ ID NO: 1. i (3) a dimeric protein containing peptides having amino acid sequence from 122 to 474 of SEQ ID NO: 1. (4) a dimeric protein containing a peptide having amino acid sequence from 121 to 474 of SEQ ID NO: 1 and a peptide having amino acid sequence from 122 to 474 of SEQ ID NO: 1. (5) a dimeric protein containing a peptide having amino acid sequence from 1 to 474 of SEQ ID NO: 1 and a peptide having amino acid sequence 121 and / or 122 to 474 of SEQ ID NO. :1. (6) a dimeric protein containing a peptide having amino acid sequence from 1 to 474 of SEQ ID NO: 1 and a peptide having amino acid sequence from 355 to 474 of SEQ ID NO. :1. (7) a dimeric protein containing a peptide having an amino acid sequence of 121 and / or 122 to 474 of SEQ ID NO: 1 and a peptide having an amino acid sequence of 355 to 474 of SEQ ID NO: 1. and mix it.

2. - A process for the production of human HMW MP52s defined in claim 1, characterized in that it comprises *. introducing DNA containing the DNA sequence of at least 721 to 2145 as shown in SEQ ID NO: 1 into a suitable host cell and culturing the host cell under conditions that allow expression of the DNA sequence and formation of the protein.

3. A pharmaceutical composition, characterized in that it contains as active ingredient at least one of human HMW MP52s defined in claim 1.

4. The pharmaceutical composition as claimed in claim 3 for the use of orthopedic reconstruction, bone transplantation, cosmetic surgery or dental implant.

5. Use of the pharmaceutical composition as claimed in claim 3, characterized in that it promotes bone morphogenesis, treatment or prevention of damage to bone, cartilage, connective tissue, skin, mucous membranes, epithelium or teeth, for application in dental implants and for application in wound healing and tissue regeneration processes.

6. Use of the pharmaceutical composition as claimed in claim 3, characterized in that it treats osteoporosis or bone fracture. *.