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(19) 



3 



Europaisches Patentamt 
European Patent Office 
Office europeen des brevets 



(12) 



(n) EP 0 416 578 B1 

EUROPEAN PATENT SPECIFICATION 



(45) Date of publication and mention 
of the grant of the patent: 
31.07.1996 Bulletin 1996/31 

(21) Application number: 90117079.5 

(22) Date of filing: 05.09.1990 



(51) mtci 6: C12N 15/12, C12N 15/16, 
C07K 14/51, C12N 1/21, 
C12P 21/02, A61K 38/16, 
A61K 38/22 



(54) Protein, DNA and use thereof 

Protein, DNA und ihre Verwendung 
Proteine, ADN et leur utilisation 



s 

(O 

T— 

o 

Q. 

LU 



(84) Designated Contracting States: 


(56) References cited: 


AT BE CH DE DK ES FR GB GR IT LI LU NL SE 


WO-A-88/00205 WO-A-89/09788 


(30) Priority: 06.09.1989 JP 229250/89 


• Lyons etal., 1989, Proc.Nat.Acad.Scl.USA, 86, p. 


20.07.1990 JP 190774/90 


4554-4558 




• CELL, vo. 51, 4. Dezember 1987, Cambridge, NA 


(43) Date of publication of application: 


US pages 861-867; D.L. WEEKS etal: ° A Maternal 


13.03.1991 Bulletin 1991/11 


mRNA localized to the vegetal hemisphere in 




xenopus eggs codes for a growth factor related 


(73) Proprietors: 


to TGF-beta". 


• TAKEDA CHEMICAL INDUSTRIES, LTD. 


• EMBO JOURNAL, vol. 8, no. 4 April 1989, 


Chuo-ku, Osaka 541 (JP) 


EYNSHAM, Oxford GB pages 1057-1065; LESLIE 


• Chichibu Onoda Cement Corporation 


DALE et al: "Developmental expression of the 


Minato-ku, Tokyo (JP) 


protein product of Vgl, a localized maternal 




mRNA in the frog Xenopus laevis" 


(72) Inventors: 


• SCIENCE, vo. 242, 1988, LANCASTER, PA US 


• Murakami, Kazuo 


pages 1528-1534; J.M. WOZNEY et al: "Novel 


Tsukuba, Ibaraki 305 (JP) 


regulators of bone formation: molecular clones 


• Ueno, Naoto 


and activities" 


Tsukuba, Ibaraki 305 (JP) 




• Kato, Yukio 


Remarks: 


Toyonaka, Osaka 560 (JP) 


The file contains technical information submitted 




after the application was filed and not included in this 


(74) Representative: 


specification 


von Kreisler, Alek, Dipl. -C hem. et al 




Patentanwalte 




von Kreisler-Selting- Werner 




Postfach 10 22 41 




50462 K6ln (DE) 





Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give 
notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in 
a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 
99(1) European Patent Convention). 



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Printed by Jouve, 76001 PARIS (FR) 



EP 0 416 578 B1 



Description 

BACKGROUND OF THE INVENTION 

5 The present invention relates to a DN A containing a DNA segment coding for a Xenopus laevis bone morphogenetic 

protein analogous to a bone morphogenetic protein (hereinafter referred to as BMP), in particular a precursor protein 
(or a precursor polypeptide) and a mature protein (or a mature polypeptide) of the Xenopus laevis BMP, and a method 
for preparing the precursor protein and the mature protein. 

In this specification, the term "precursor protein" includes a protein which includes an amino acid sequence of a 

10 mature peptide Xenopus Laevis BMP and has all or a portion of an amino acid sequence coded with a Xenopus laevis 
BMP DNA segment at the N-terminus ; the C-terminus or both termini thereof. 

Recently, it has been revealed that transforming growth factor-beta (TGF-beta : TGF-P) having a bone morphoge- 
netic activity not only controls cell proliferation, but also has various biological activities such as control of cell differ- 
entiation. In particular, the bone morphogenesis-promoting activity of TGF-p has been noted, and attempts have been 

15 made to use TG F for treatment of fractures and osteoporosis, making use of the cartilage-bone induction activity thereof 
[M. Noda et al. f J. Endocrinology 124, 2991-2994 (1989); M. E. Joyce et al., J. Bone Mineral Res. 4, S-259 (1989); 
and S. M. Seyedin et al., J. Biol. Chem. 281 , 5693-5695 (1986)). More recently, however, four kinds of bone morpho- 
genetic proteins (BMPs) which are different from one another in molecular structure have been identified as a factor 
promoting morphogenesis of bones and cartilages. Of these four kinds, human BMP-1 , human BMP-2A, human BMP- 

20 2B and human BMP-3 are novel peptides, though they are very similar in structure to TGF-p, and there has been a 
report that they induce morphogenesis of bones and cartilages when subcutaneously or intramuscularly implanted in 
animals [J. M. Wozney et al., Science 242 , 1528-1534 (1988)]. 

The above peptides having bone morphogenetic activity are isolated and purified from bones in which the peptides 
are considered to be localized, or from human osteosarcoma cells (U2-OS) which are thought to produce the peptides. 

25 However, such a method has problems because the procedure is complicated and the desired peptides are obtained 
only in small amounts. 

SUMMARY OF THE INVENTION 

30 Important contributions will be made to future studies and medical treatment, if a similar peptide having the bone 

morphogenetic activity can be collected from Xenopus laevis and further prepared by recombinant technique. As a 
result, the following information was obtained, thus arriving at the present invention. 

Namely, the present inventors first succeeded in cloning five kinds of DNA coding for BMP-2A and related ONAs 
(Xenopus laevis BMPs) and subsequently three kinds of complementary DNAs, eight kinds of DNAs in total, by using 

35 a complementary DNA of a rat inhibin pA chain equally belonging to the TGF-p family as a probe. Further, the present 
inventors identified portions of the bases of the DNAs, clarified the amino acid sequences (see formulae (I), (II), (III), 
(IV) and (V) of Fig. 3 and formulae (VI), (VII) and (VIII) of Fig. 4) of the Xenopus laevis BMPs (referred to as B9, M3, 
C4, A4, A5, Xbr22, Xbr23 and Xbr41 ), and succeeded in pioneering their mass production by recombinant technique. 
In accordance with the present invention, there are provided (1) a Xenopus laevis BMP, 

40 

wherein said protein is a mature protein containing an amino acid sequence having an amino acid sequence 
represented by Nos. 15 to 1 30 of formula (I) shown in Fig. 3, an amino acid sequence represented by Nos. 14 to 
127 of formula (II) shown in Fig. 3, an amino acid sequence represented by Nos. 6 to 63 of formula (IV) shown in 
Fig. 3, an amino acid sequence represented by Nos. 6 to 65 of formula (V) shown in Fig. 3, an amino acid sequence 
45 represented by Nos. 282 to 398 or Nos. 298 to 398 of formula (VI) shown in Fig. 4, or an amino acid sequence 

represented by Nos. 328 to 426 of formula (VIII) shown in Fig. 4, and 

wherein said protein is a precursor protein containing an amino acid sequence having an amino acid sequence 
represented by formula (I), (II), (IV) or (V) shown in Fig. 3, or formula (VI) or (VIII) shown in Fig. 4; (2) a DNA 
comprising a DNA segment coding for the Xenopus laevis BMP as defined in (1) and a DNA comprising a DNA 

50 segment coding for the Xenopus laevis BMP wherein said DNA segment comprises a nucleotide sequence cor- 

responding to the nucleotide sequence represented by formula (1), (2), (3), (4), (5), (6), (7) or (8) shown in Fig. 2: 
(3) a non-human transformant bearing the DNA containing the DNA segment coding for the Xenopus laevis BMP 
as defined in (2) which is not Xenopus laevis ; (4) a method for preparing the Xenopus laevis BMP which comprises 
culturing the non-human transformant described in (3), producing and accumulating a protein in a culture and 

55 collecting the protein thus obtained. (5) a composition for therapy of fracture or osteoporosis containing the Xeno- 

pus laevis BMP defined in (1); and (6) a method for preparing the composition as defined in (5). 



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BRIEF DESCRIPTION OF THE DRAWINGS 

Fig. 1 shows simplified restriction enzyme maps of DNA sequences containing Xenopus laevis BMP precursors 

or mature peptide DNA segments; 
5 Figs. 2(1 ) to 2(8) show nucleotide sequences of the DNA segments of Xenopus laevis BMPs, B9, M3, C4, A4, A5, 

BMP-2A, BMP-2B and Vgr-1 , respectively, and the amino acid sequences deduced therefrom; 

Fig. 3 shows amino acid sequences of the Xenopus laevis BMPs deduced from the nucleotide sequences of the 

DNA segments shown in Figs. 2(1) to 2(5), comparing them with the amino acid sequences of known proteins 

having a bone morphogenetic activity; and 
io Fig. 4 shows amino acid sequences of the Xenopus laevis BMPs deduced from the nucleotide sequences of the 

cDNA segments shown in Figs. 2(6) to 2(8). 

DESCRIPTION OF THE PREFERRED EMBODIMENTS 

is The mature Xenopus laevis BMP of C4, one of the Xenopus laevis BMPs, of the present invention, which has a 

relationship to TGF-p and is a peptide consisting of 98 or 114 amino acid residues, has an amino acid sequence 
represented by Nos. 6 to 11 9 or Nos. 22 to 11 9 of formula (III) shown in Fig. 3. The molecular weight thereof is calculated 
at about 25,000, excepting sugar chains, when a dimer is formed. 

The amino acid sequence of this peptide is different from that reported by Wozney et al. in 3 or 4 amino acid 

20 residues per molecule. 

Fig. 3 shows amino acid sequences of five kinds of novel Xenopus laevis BMPs obtained in the present invention, 
comparing them with the amino acid sequences of known proteins having a bone morphogenetic activity. In these 
amino acid sequences, the same amino acid residue as with pA is represented by V, and an amino acid residue 
different from that of pA is represented by one letter symbol based on pA. CONSENSUS shown in Fig. 3 indicates 

25 amino acid residues common to all the BMPs shown in Fig. 3. The illustration of CONSENSUS results in introduction 
of gaps ■-■ in the formulae in Fig. 3. Accordingly, the number representing the precursor and mature protein portions 
is counted excluding these lacking portions. 

Fig. 4 shows amino acid sequences of three kinds of novel Xenopus laevis BMPs deduced from cDNAS, subse- 
quently discovered by the present inventors. 

30 For DNA sequences, the DNA segments coding for the Xenopus laevis BMPs of the present invention correspond 

to the nucleotide sequences of formulae (1) to (8) (corresponding to B9, M3, C4, A4, A5, Xbr22, Xbr23 and Xbr41 ; 
respectively) shown in Fig. 2 or are portions thereof. Any functional portion can be used so long as bone morphogenetic 
activity is not lost. Wozney et al. reports the amino acid sequences, but does not elucidate the nucleotide sequences. 
As used herein the term correspond permits conservative additions, deletions and substitutions. Preferably the DNA 

35 segments coding for the BMPs of the present invention have the nucleotide sequences of formulae (1 ) to (8). 

With respect to the portion relating to the mature BMPs [the amino acid sequence represented by Nos. 15 to 130 
of formula (I) shown in Fig. 3, the amino acid sequence represented by Nos. 14 to 127 of formula (II) shown in Fig. 3 : 
the amino acid sequence represented by Nos. 6 to 119 or Nos. 22 to 119 of formula (III) shown in Fig. 3, the amino 
acid sequence represented by Nos. 6 to 63 of formula (IV) shown in Fig. 3, the amino acid sequence represented by 

40 Nos. 6 to 65 of formula (V) shown in Fig. 3, the amino acid sequence represented by Nos. 282 to 398 or Nos. 298 to 
398 of formula (VI) shown in Fig. 4, the amino acid sequence represented by Nos. 288 to 401 or Nos. 304 to 401 of 
formula (VII) shown in Fig. 4, or the amino acid sequence represented by Nos. 328 to 426 of formula (VIII) shown in 
Fig. 4], the DNA sequences of the present invention differ from the DNA sequence of TGF-p, and therefore are novel. 
As the DNA sequences coding for the BMP mature peptides of the present invention, any DNA sequences may 

45 be used as long as they contain nucleotide sequences coding for the amino acid sequences of the BMP mature peptides. 
For example, DNA sequences corresponding to the nucleotide sequences represented by formulae (1 ) to (8) or portions 
thereof are preferably used. More preferably the DNA sequences contain the nucleotide sequences represented by 
formulae (1) to (8). 

The nucleotide sequences represented by formulae (1) to (8) are the Xenopus laevis BMP DNA sequences ob- 
50 tained in the present invention. Examples of the nucleotides coding for the Xenopus laevis BMP amino acid sequences 
represented by formulae (I) to (VIII) include Nos. 693 to 1040 of formula (1 ), Nos. 1 34 to 475 of formula (2), Nos. 435 
to 728 of formula (3), Nos. 183 to 356 of formula (4), Nos. 149 to 328 of formula (5), Nos. 249 to 1442 of formula (6), 
Nos. 104 to 1 306 of formula (7) and Nos. 86 to 1 363 of formula (8). 

An expression vector having the DNA sequence containing the nucleotide sequence coding for the BMP of the 
55 present invention can be prepared, for example, by the following process: 

(a) Messenger RNA (MRNA) is isolated from BMP-producing cells. 

(b) Single stranded complementary DNA (cDNA) is synthesized from the mRNA, followed by synthesis of double 



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stranded DNA. 

(c) The complementary DNA is introduced in a cloning vector such as a phage or a plasmid. 

(d) Host cells are transformed with the recombinant phage or plasmid thus obtained. 

(e) After cultivation of the transformant thus obtained, the plasmid or the phage containing the desired DNA is 
5 isolated from the transformant by an appropriate method such as hybridization with a DNA probe coding for a 

portion of the BMP or immunoassay using an anti-BMP antibody. 

(f) The desired cloned DNA sequence is cut out from the recombinant DNA. 

(g) The cloned DNA sequence or a portion thereof is ligated downstream from a promoter in the expression vector. 

JO The mRNAs coding for the BMPs can be obtained from various BMP-producing cells such as ROS cells. 

Methods for preparing the mRNAs from the BMP-producing cells include the guanidine thiocyanate method [J. M. 
Chirgwin et al., Bio-chemistry 18, 5294 (1979)]. 

Using the MRNA thus obtained as a template, cDNA is synthesized by use of reverse transcriptase, for example, 
in accordance with the method of H. Okayama et al. IMolecular and Cellular Biology 2 , 161 (1979); ibid. 3, 280 (1983)]. 
* s The cDNA thus obtained is introduced into the plasmid. 

The plasmids into which the cDNA is introduced include, for example, pBR322 [Gene 2 , 95 (1 977)], pBR325 [Gene 
.4, 121 (1978)], pUC12 IGene 19, 259 (1982)] and pUC13 (Gene 19, 259 (1982)], each derived from Escherichia coli , 
and pUBH0 derived from Bacillus subtilis IBiochemical and Biophysical Research Communication 112 , 678 (1983)]. 
However, any other plasmids can be used as long as they are replicable and growable in the host cells. Examples of 
20 the phage vectors into which the cDNA may be introduced include a.gt11 [R. Young and R. Davis, Proc. Natl. Acad. 
Sci. U.S.A. 80 , 1194 (1983)]. However, any other phage vectors can be used as long as they are growable in the host 
cells. 

Methods for introducing the cDNA in the plasmid include, for example, the method described in T Maniatis et al., 
Molecular Cloning , Cold Spring Harbor Laboratory, p. 239 (1 982). Methods for introducing the cDNA in the phage vector 
25 include, for example, the method of T V. Hyunh et al. fDNA Cloning, A Practical Approach 1, 49 (1985)]. 

The plasmid thus obtained is introduced into the appropriate host cell such as Escherichia and Bacillus . 

Examples of Escherichia described above include Escherichia coli K12DH1 [Proc. Natl. Acad. Sci. U.S.A. 60, 160 
(1968)], M103 INucleic Acids Research 9, 309 (1981)], JA221 [Journal of Molecular Biology 120 , 517 (1978)], HB101 
[Journal of Molecular Biology 41, 459 (1969)] and C600 [Genetics 39 , 440 (1954)]. 
30 Examples of Bacillus described above include Bacillus subtilis MM14 [Gene 24 , 255 (1983)] and 207-21 [Journal 

of Biochemistry 95, 87 (1984)]. 

Methods for transforming the host cell with the plasmid include, for example, the calcium chloride method or the 
calcium chloride/rubidium chloride method described in T. Maniatis et al., Molecular Cloning , Cold Spring harbor Lab- 
oratory, p.249 (1982). 

3£ When the phage vector is used, for example, the phage vector can be transduced into multiplied Escherichia coli , 

using the in vitro packaging method. 

Xenopus laevis cDNA libraries containing Xenopus laevis BMP cDNA can be obtained by numerous techniques 
well known in the art including purchasing them from the market, though obtainable by the methods described above. 
For example, the cDNA library of Xenopus laevis is available from Clontech Laboratories, Inc., U.S.A. 
40 Methods for cloning the Xenopus laevis BMP DNA from the Xenopus laevis DNA library include, for example, the 

plaque hybridization method using phage vector Xcharon 28A and rat inhibin (activin) pA cDNA as probes [T. Maniatis 
et al., Molecular Cloning, Cold Spring Harbor Laboratory, (1982)]. 

The Xenopus laevis BMP DNA thus cloned is subcloned in plasmids such as pBR322, pUC12, pUC13, pUC19, 
pUC1 1 8 and pUC1 1 9 to obtain the Xenopus laevis BMP DNA, if necessary. 
45 The nucleotide sequence of the DNA sequence thus obtained is determined, for example, by the Maxam-Gilbert 

method [A. M. Maxamand W. Gilbert. Proc. Natl. Acad. Sci. U.S.A. 74, 560 (1 977)] or the dideoxy method [J. Messing 
et al., Nucleic Acids Research 9 , 309 (1981)], and the existence of the Xenopus laevis BMP DNA is confirmed in 
comparison with the known amino acid sequence. 

As described above, the DNA sequence [Xenopus laevis BMP DNAs represented by formulae (1) to (8)] coding 
50 for the Xenopus laevis BMPs are obtained. 

Fig. 1 shows the restriction enzyme fragment maps of the DNA sequences containing the DNA segments coding 
for the Xenopus laevis BMPs obtained in Example 1 described below. Fig. 2 shows the nucleotide sequences repre- 
sented by formulae (1) to (8) of the DNA sequences as determined by the dideoxy method, and Figs. 3 and 4 show 
the amino acid sequences represented by formulae (I) to (V) and formulae (VI) to (VIII), respectively, which were 
55 ascertained form the above nucleotide sequences. 

The DNA sequence coding for the Xenopus laevis BMP cloned as described above can be used as it is, or after 
digestion with a restriction enzyme if desired, depending on the intended use. 

A region intended to be expressed is cut out from the cloned ONA and ligated downstream from the promoter in 



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EP 0 416 578 B1 



a vehicle (vector) suitable for expression, whereby the expression vector can be obtained. 

The DNA sequence has ATG as a translation initiating codon at the 5'-terminus thereof and may have TAA, TGA 
or TAG as a translation terminating codon at the 3'-terminus. The translation initiating codon and translation terminating 
codon may be added by use of an appropriate synthetic DNA adaptor The promoter is further ligated in the upstream 
s thereof for the purpose of expressing the DNA sequence. 

Examples of the vectors include the above plasmids derived from E. coli such as pBR322, pBR325, pUC12 and 
pUC13, the plasmide derived from B. subtilis such as pUB110, pTP5 and pC194, plasmids derived from yeast such 
as pSH19 and pSH15, bacteriophage such as Aphage, and animal viruses such as retroviruses and vaccinia viruses. 

As the promoters used in the present invention, any promoters are appropriate as long as they are suitable for 
io expression in the host cells selected for the gene expression. 

When the host cell used for transformation is Escherichia , it is preferable that a trp promoter, a lac promoter, a 
recA promoter, a XPL promoter, a Ipp promoter, etc. are used. When the host cell is Bacillus , it is preferable that a 
PHOS promoter, a PGK promoter, a GAP promoter, an ADH promoter, etc. are used. In particular, it is preferable that 
the host cell is Escherichia and the promoter is the trp promoter or the A.PL promoter. 
is When the host cell is an animal cell, an SV-40 derived promoter, a retrovirus promoter, a metallothionein promoter, 

a heat shock promoter, etc. are each usable. 

An enhancer, a certain DNA sequence important for promoter activity in a cell, is also effectively used for expres- 
sion. 

By using the vector containing the DNA sequence coding for the Xenopus laevis BMP mature peptide thus con- 
20 structed, the transformant is prepared. 

The host cell include, for example, Escherichia , Bacillus, yeast and animal cells. 

Specific examples of the above Escherichia and Bacillus include strains similar to those described above. 

Examples of the above yeast include Saccharomyces cerevisiae AH22, AH22R", NA87-1 1 A and DKD-5D. 

Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell (CHO), mouse L cell and human 
25 FLcell. 

The transformation of the above Escherichia is carried out, for example, according to the method described in 
Proc. Natl. Acad. Sci. U.S.A. 69 , 2110 (1972) or Gene J7, 107 (1982). 

The transformation of the above Bacillus is conducted, for example, according to the method described in Molecular 
& General Genetics 168 , 111 (1979). 
30 The transformation of the yeast is carried out, for example, according to the method described in Proc. Natl. Acad. 

Sci. U.S.A. 75, 1 929 (1 978). 

The transformation of the animal cells is carried out, for example, according to the method described in Virology 
52,456(1973). 

Thus, there is obtained the transformant transformed with the expression vector containing the DNA sequence 
35 coding for the Xenopus laevis BMP mature peptide. 

When bacterial transformants are cultured, a liquid medium is particularly suitable as a medium used for culture. 
Carbon sources, nitrogen sources, inorganic compounds and others necessary for growth of the transformant are 
contained therein. Examples of the carbon sources include glucose, dextrin, soluble starch and sucrose. Examples of 
the nitrogen sources include inorganic or organic materials such as ammonium salts, nitrates, corn steep liquor, pep- 
40 tone, casein, meat extracts, soybean meal and potato extract solution. The inorganic compounds include, for example, 
calcium chloride, sodium dihydrogenphosphate and magnesium chloride. Yeast extract, vitamins, growth promoting 
factors and so on may be further added thereto. 

The pH of the medium is preferably about 5 to 8. 

As the medium used for cultivation of Escherichia , there is preferred, for example, M9 medium containing glucose 
45 and Casamino Acids (Miller, Journal of Experiments in Molecular Genetics 431-433 , Cold Spring Harbor Laboratory, 
New York, 1972). In order to make the promoter act efficiently, a drug such as 3p-indolylacrylic acid may be added 
thereto if necessary. 

When the host cell is Escherichia , the cultivation is usually carried out at about 15 to 43°C for about 3 to 24 hours, 
with aeration or agitation if necessary. 
50 When the host cell is Bacillus , the cultivation is usually carried out at about 30 to 40°C for about 6 to 24 hours, 

with aeration or agitation if necessary. 

When yeast transformants are cultured, there is used, for example, Surkholder minimum medium [K. L. Sostian 
et al., Proc. Natl. Acad. Sci. U.S.A. 77, 4505 (1980)] as the medium. The pH of the medium is preferably adjusted to 
about 5 to 8. The cultivation is usually carried out at about 20 to 35°C for about 24 to 72 hours, with aeration or agitation 
55 if necessary. 

When animal cell transformants are cultured, examples of the media include MEM medium containing about 5 to 
20% fetal calf serum IScience 122 , 501 (1952)], DMEM medium f Virology 8. 396 (1959)], RPMI1640 medium [Journal 
of the American Medical Association 1 99 , 519 (1967)] and 199 medium [Proceeding of the Society for the Biological 



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Medicine 73, 1 (1950)]. The pH is preferably about 6 to 8. The cultivation is usually carried out at about 30 to 40°C for 
about 15 to 60 hours, with aeration or agitation if necessary. 

The above Xenopus laevis BMP mature peptide can be isolated and purified from the culture described above, for 
example, by the following method. 

s When the Xenopus laevis BMP mature peptide is to be extracted from the cultured cells, the cells are collected by 

methods known in the art after cultivation. Then, the collected cells are suspended in an appropriate buffer solution 
and disrupted by ultrasonic treatment, lysozyme and /or freeze-thawing. Thereafter, a crude extracted solution of the 
Xenopus laevis BMP mature peptide is obtained by centrifugation or filtration. The buffer solution may contain a protein 
denaturant such as urea or guanidine hydrochloride, or a surface-active agent such as Triton X-100. 

10 When the Xenopus laevis BMP precursor protein or mature peptide is secreted in the culture solution, a supernatant 

is separated from the cells by methods known in the art after the conclusion of cultivation, and then collected. 

The separation and purification of the Xenopus laevis BMP precursor protein or mature peptide contained in the 
culture supernatant or the extracted solution thus obtained can be performed by an appropriate combination of known 
separating and purifying methods. The known separating and purifying methods include methods utilizing solubility 

is such as salt precipitation and solvent precipitation, methods mainly utilizing a difference in molecular weight such as 
dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in elec- 
tric charge such as ion-exchange column chromatography, methods utilizing specific affinity such as affinity chroma- 
tography, methods utilizing a difference in hydrophobic ity such as reverse phase high performance liquid chromatog- 
raphy and methods utilizing a difference in isoelectric point such as isoelectro-focusing electrophoresis. Methods using 

20 an antibody to a fused protein expressed by fusing BMP complementary DNA or DNA with E. coli-derived DNA lacZ 
can also be used. 

Illustrative examples of the methods for expressing the BMP in the present invention include methods in which 
genes are introduced into CHO cells to produce the BMP in large amounts as described in Wang et al., Proc. Natl. 
Acad. Sci. U.S.A. 807 , 2220-2224 (1 990). 
25 The activity of the Xenopus laevis BMP precursor protein or mature peptide thus formed can be measured by an 

enzyme immunoassay using a specific antibody. If the products have a bone morphogenetic activity, this activity may 
also be measured as an index. 

The cells, such as animal cells or coH, transinfected or transformed with the DNA sequences of the present 
invention allow large amounts of the Xenopus laevis BMP mature peptides to be produced. Hence, the production of 
so these peptides can be advantageously achieved. 

It has become clear that the Xenopus laevis BMP mature peptides prepared here promote the synthesis of prote- 
oglycan which is a main component of a cartilage matrix, and the peptides can also be utilized for analysis of the 
mechanism of organism, particularly human bone-cartilage morphogenetic reaction, and as therapeutic agents for 
fracture or osteoporosis. 

35 in such instances one would administer an effective amount of the protein to a mammal. An effective amount is 

the amount of protein needed to promote the synthesis of proteoglycan in cartilage cells. Typically, this ranges from 

0.001 to 35 u.g per kg/body weight. The precise amount for a particular purpose can readily be determined empirically 

by the person of ordinayl skill in the art based upon the present disclosure. 

When one uses the protein 1or therapeutic purpose care is taken to purify it and render it substantially free of 
40 bacterica and pyrogens. This can be done by standard methods. 

When the BMPs are used as therapeutic agents for fracture or osteoporosis, they can be administered parenterally 

in the forms of solutions, injections and ointments, solely or in combination with pharmaceutical^ acceptable additional 

components, such as vehicles, binders, dispersants, plasticizers or diluents. 

The preferable administration forms include (1) administration of the agent to cutis surface near a diseased part, 
45 (2) injection of the agent into a diseased part, (3) discission of a diseased part followed by direct administration of the 

agent thereto. The preferable dose in fractue therapy for adult people is 0.1 to 2000 pg more, preferably 20 to 400 u.g 

for adult people once a day. The preferable dose in osteoporosis for adult people is 0. 1 to 200 jag once a day, for about 

one to 30 days. The concentration of the therapeutic agent is, preferably, 0.001 to 0.2% in the form of a solution, 0.001 

to 0.2% in the form of an injections, and 0.0001 to 0.2% in the form of an ointment. 
so There have been described above in detail the cloning of the DNA sequences coding for the Xenopus laevis BMPs, 

the preparation of the expression vectors for the Xenopus laevis BMP mature peptides, the production of the trans- 

formahts by using the transformants and their utility. 

When nucleotides, amino acids and so on are indicated by the abbreviations in this specification and drawings, 

the abbreviations adopted by IUPAC-IUB Commission on Biochemical Nomenclature or commonly used in the art are 
ss employed. For example, the following abbreviations are used. When the amino acids are capable of existing as optical 

isomer, the L-forms are represented unless otherwise specified. 

DNA : Deoxyribonucleic acid 



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EP 0 416 578 B1 





cDNA : 


Complementary deoxyribonucleic acid 




A • 

A . 


Adenine 




1 . 


Thymine 




Cji . 


Guanine 


c 


• 

O . 


Cytosine 




DMA • 

HNA . 


Ribonucleic acid 




mRNA : 


Messenger ribonucleic acid 




dATP : 


Deoxyadenosine triphosphate 




dTTP : 


Deoxythymidine triphosphate 


10 


dGTP : 


Deoxyguanosine triphosphate 




dCTP : 


Deoxycytidine triphosphate 




ATP : 


Adenosine triphosphate 




EDTA : 


Ethylenediaminetetraacetic acid 




SDS : 


Sodium dodecyl sulfate 


15 


Gly or G : 


Glycine 




Ala or A : 


Alanine 




Val or V : 


Valine 




Leu or L : 


Leucine 




Me or 1 : 


Isoleucine 


20 


Ser or S : 


Serine 




Thr or T : 


Threonine 




Cys or C : 


Cysteine 




Met or M : 


Methionine 




Glu or E : 


Glutamic acid 


25 


Asp or D : 


Aspartic acid 




Lys or K : 


Lysine 




Arg or R : 


Arginine 




His or H : 


Histidine 




Phe or F : 


Phenylalanine 


30 


Tyr or Y : 


Tyrosine 




Trp or W : 


Tryptophan 




Pro of P : 


Proline 




Asn or N : 


Asparagine 




Gin or Q : 


Glutamine 



35 

With respect to the Xenopus laevis BMP mature peptides of the present invention, a portion of the amino acid 
sequence may be modified, namely there may be addition, elimination or substitution with other amino acids as long 
as the bone morphogenetic activity is not lost. 

The present invention will hereinafter be described in detail with the following Examples. It is understood of course 

40 that these Examples are not intended to limit the scope of the invention. 

Transformants E coli HB101/pXar3 (coding for protein M3), E coli HB101/pXar4 (coding for protein A4), E coli 
HB101/pXar5 (coding for protein A5), E coliHB101/pXar9 (coding for protein B9) and E coli HB101/pXar14 (coding 
for protein C4) each obtained in Example 1 described below were deposited with the Institute for Fermentation, Osaka, 
Japan (IFO) under the accession numbers IFO 14928, IFO 14929, IFO 14930, IF0 14931 and IF0 14932, respectively, 

45 on August 28, 1989. These transformants were also deposited with the Fermentation Research Institute, Agency of 
Industrial Science and Technology, Ministry of International Trade and Industry, Japan (FRI) under the Budapest Treaty 
under the accession numbers FERM BP-2578, FERM BP-2579, FERM BP-2580, FERM BP-2581 and FERM BP-2582, 
respectively, on September 2, 1989. 

The transformants E coli HB101/pXbr22 (coding for Xenopus laevis BMP-2A), E. coli HB101/pXbr23 (coding for 

so Xenopus laevis BMP-2B) and E coli H B1 0 1 /pXbr41 (coding for protein Xenopus laevis Vgr- 1 ) each obtained in Example 
2 described below were deposited with the Institute for Fermentation, Osaka, Japan (IFO) under the accession numbers 
IFO 15080, IFO 15081 and IFO 15082, respectively, on August 10, 1990. These transformants were also deposited 
with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade 
and Industry, Japan (FRI) under the Budapest Treaty under the accession numbers FERM BP-3066, FERM BP-3065 

55 and FERM BP-3067, respectively, on August 16, 1990. 



7 



EP 0 416 578 B1 



Example 1 

Preparation of Xenopus laevis Liver-Derived DNA Library 

5 (1 ) Preparation of Xenopus laevis Chromosome DNA 

The liver (1 g) of Xenopus laevis was powdered in liquid nitrogen, and 10 ml of buffer (1) [100 ug/ml proteinase 
K, 0.5% Sarkosil, 0.5 M EDTA (pH 8.0)] was added thereto, followed by incubation at 50°C for 2 hours. The resulting 
DNA sample was treated with phenol, and then dialyzed against buffer (2) [10 mM EDTA, 10 mM NaCI, 50 mM Tris- 

io HCI (pH 8.0)] to remove phenol. RNase was added thereto to a final concentration of 1 00 u.g/ml, and the mixture was 
incubated at 37°C for 3 hours, followed by phenol treatment twice. The aqueous layer was dialyzed against buffer (3) 
[1 mM EDTA, 10 mM Tris-HCI (pH 8.0)]. Thus, about 1 mg of liver-derived chromosome DNA was obtained. This DNA 
(10 fig) was partially cleaved with restriction enzyme Sau3AI, and the resulting product was subjected to equilibrium 
density gradient centrifugation using CsCI. Fractions containing DNA Iragments having lengths of 10 to 20 kb were 

is selected and introduced into fragments obtained by cleaving phage charon 28 DNA with BamHI and used as a vector. 
This reaction called "ligation" was conducted at 15°C for 16 hours. The charon 28 vector into which the Xenopus laevis 
chromosome DNA was thus introduced was contained in a phage head (in vitro packaging). This procedure was carried 
out by using a commercial packaging kit (Gigapack Gold, Stratagene). This recombinant phage was amplified by in- 
fection with E. coli LE392. Specifically, the phage was mixed with excess LE392 to allow LE392 to adsorb the phage 

20 at 37°C for 10 minutes. Then, the mixture was plated on NZYM medium (containing 1 3% agar), followed by incubation 
overnight. 

(2) Screening . 

25 The total number of the phage clones was estimated to be about 1 ,000,000 from the number of the plaques pro- 

duced in a dish. As a probe (DNA used for detection of a desired gene by hybridization), there was used rat activin pA 
cDNA [Molecular Endocrinology 1, 368-396 (1987)] labeled with 32 P by a random priming method. The plaques tran- 
scribed from the dish to a nitrocellulose membrane were returned to neutrality (0.2 M Tris, 0.6 M NaCI, pH 7.4) through 
alkali treatment (immersion in 0.1 N NaOH, 0.6 M NaCI for 30 seconds). After completion of the treatment described 

30 above, the membrane was heated in a vacuum thermostat at 80°C for 1 hour. After heating, the membrane was im- 
mersed in a hybridization solution (50% formamide, 5 X Denhardt's solution, 5 X SSPE, 0. 1 % SDS, 1 00 ug/ml denatured 
salmon sperum DNA) to incubate it at 42°C for 4 hours. Then, the membrane was allowed to stand in the mixture 
solution of the above hybridization solution and the DNA probe at 60° C overnight. This procedure was carried out in 
a plastic bag. The next day, the nitrocellulose membrane was taken out of the bag, and washed with a solution of 2 X 

35 SSC and 0.1% SDS for 15 minutes and with a solution of 0.1 X SSC and 0.1% SDS for 15 minutes, increasing the 
temperature stepwise, until the cpm value of the membrane reached about 1,000 cpm. After washing, the washing 
solution was removed by filter paper, and then the membrane was subjected to autoradiography. The plaque containing 
the desired gene was identified by exposure of a Fuji X-ray film. The genes were cloned by repetition of the above 
plaque hybridization. 

40 20 X SSC contains 0.3 M sodium citrate (pH 7.0) and 3 M NaCI; 20 X SSPE contains 0.2 M sodium phosphate, 

20 m EDTA and 3 M NaCI (pH 7.4); and Denhardt's solution contains 1% Ficoll, 1% polyvinylpyrrolidone and 1% BSA 
(Pent ex Fraction V). 

(3) Determination of Nucleotide Sequence (Sequencing) 

45 

All of the five isolated clones A4, A5, B9, C4 and M3 were each subcloned into plasmid pUC19. In subcloning each 
clone into plasmid pUC19, subcloning was carried out utilizing a restriction enzyme recognition site which produced a 
fragment hybridized with the probe for each clone. However, for cloning clone A4, a commercial Bg1 II linker was used 
to ligate a Smal site. 

so The plasmids were each transformed into competent cell HB101 (E. coli) prepared by the rubidium chloride method 

to obtain five kinds of transformants E. coli HB101/pXar3 (coding for protein M3), E^ coli HBl01/pxar4 (coding for 
protein A4), E. coli HB101/pxar5 (coding for protein A5), E. coli HB101/pXar9 (coding for protein B9) and E. coli 
HB101/pXar14 (coding for protein C4), respectively. 

For determination of the nucleotide sequence, a deletion mutant of each clone was prepared, and the shortest of 
55 fragment hybridized with the probe was selected. The nucleotide sequence was determined from pUC1 9 by the direct 
Sanger method (or the dideoxy method). 

For translation of the nucleotide sequence to an amino acid sequence or for screening of homology, a software 
for genetic analysis (GENETYX, Nippon SDC) was used. 



8 



EP 0 416 578 B1 



Homology at Nucleic Acid Level 


TYX nucleotide 


Rat Act pA, % 


Rat Act pA, % 


Human TGF P2, % 


xVgl % 


M3% 


A4% 


AS 


70.3(101) 


47.5 (314) 


43.8 (169) 


48.5 (171) 


54.7 (258) 


63.7 (328) 


A4 


69.5 (0.5) 








55.4 (251) 




M3 


63.6 (332) 


53.9 (672) 


33.1 (689) 








In the above table, numerical values in parentheses indicate the length compared (bp). 


Homology at Amino Acid Level 


TYX nucleotide 


Rat Act pA, % 


Rat Act PA, % 


Human TGF P2, % 


xVgl % 


M3% 


A4% 


A5 


58.8 (34) 


44.1 (34) 


37.2 (43) 


50.0 (38) 


26.0 (77) 


67.6 (68) 


A4 


41.3(63) 


44.1 (34) 


39.5 (43) 


52.6 (38) 


30.3 (66) 




M3 


50.3(149) 


49.4(162) 


32.8 (128) 


40.6 (106) 







In the above table, numerical values in parentheses indicate the length compared (bp). 



Example 2 



Preparation of Xenopus laevis Unfertilized Egg-Derived DNA Library 

2$ 

(1 ) Preparation ol Xenopus laevis BMP -2 A Probe 

A probe was prepared by fragmentation of chromosome DNA Xarl4 coding for Xenopus laevis BMP-2A with re- 
striction enzymes Pstl and Hindlll, and three kinds of cDNAs, Xbr22, Xbr23 and Xbr41 were isolated by screening of 

50 a Xenopus laevis unfertilized egg cDNA library by a hybridization method. The comparison with the structure of the 
Xenopus laevis BMP chromosome DNA already isolated revealed that Xbr22, Xbr23 and Xbr41 coded for proteins 
having homology with Xenopus laevis BMP-2A, Xenopus laevis BMP-2B and mouse Vgr-1 reported by Lyon et al. 
IProc. Natl. Acad. Sci. U.S.A. 806 , 4554-4558 (1 989)], respectively 

The Xenopus laevis unfertilized egg cDNA library was provided by the Salk Institute (C. Kintner). This library was 

35 prepared based on XgtlO. This recombinant phage was amplified by infection with E. coli NM514. Specifically, the 
phage was mixed with excess NM514 to allow NM514 to adsorb the phage at 37°C for 10 minutes. Then ; the mixture 
was plated on N2YM medium (containing 13% agar), followed by incubation overnight. 



(2) Screening 

The total number of the phage clones was estimated to be about 1,200,000 from the number of the plaques pro- 
duced in a dish. As a probe (DNA used for detection of a desired gene by hybridization), there was used a DNA fragment 
(185 bp) obtained by cleaving Xar1 4 with restriction enzymes Pstl and Hindlll and labeled with 32 P by a random priming 
method. The plaques transcribed from the dish to a nitrocellulose membrane were returned to neutrality (0.2 M Tris, 
0.6 M NaCI, pH 7.4) through alkali treatment (immersion in 0. 1 N NaOH, 0.6 M NaCI for 30 seconds). After completion 
of the treatment described above, the membrane was heated in a vacuum thermostat at 80° C for 1 hour. After heating, 
the membrane was immersed in a hybridization solution (50% formamide, 5 X Denhardt's solution, 5 X SSPE, 0.1% 
SDS, 100 u^g/ml denatured salmon sperm DNA) to incubate it at 42°C for 4 hours. Then, the membrane was allowed 
to stand in the mixture solution of the above hybridization solution and the DNA probe aV60°C overnight. This procedure 
was carried out in a plastic bag. The next day, the nitrocellulose membrane was taken out of the bag, and washed with 
a solution of 2 X SSC and 0.1% SDS for 15 minutes, increasing the temperature stepwise, until the cpm value of the 
membrane reached about 1,000 cpm. After washing, the washing solution was removed by filter paper, and then the 
membrane was subjected to autoradiography. The plaque containing the desired gene was identified by exposure of 
a Fuji X-ray film. The genes were cloned by repetition of the above plaque hybridization. 

20 X SSC contains 0.3 M sodium citrate (pH 7.0) and 3 M NaCI; 20 X SSPE contains 0.2 M sodium phosphate, 
20 m EDTA and 3 M NaCI (pH 7.4); and Denhardt's solution contains 1% Ficoll, 1% polyvinylpyrrolidone and 1% BSA 
(Pentex Fraction V). 



9 



EP 0 416 578 B1 



(3) Determination of Nucleotide Sequence (Sequencing) 

AH of the three isolated clones Xbr22, Xbr23 and Xbr41 were each subcloned into plasmid pUC19. In subcloning 
each clone into plasmid pUC19, subcloning was carried out utilizing a restriction enzyme recognition site which pro- 
s duced a fragment hybridized with the probe for each clone. 

The plasmids were each transformed into competent cell HB1 01 (E. coli) prepared by the rubidium chloride method 
to obtain three kinds of transformants E. coli HB101/pXbr22 (coding for Xenopus laevis BMP-2A), E. coli HB101/pXbr23 
(coding for Xenopus laevis BMP-2B) and jE. coli HB101/pXbr41 (coding for protein Xenopus laevis Vgr-1 ), respectively. 
For determination of the nucleotide sequence, a deletion mutant of each clone was prepared, and the shortest 
io fragment that hybridized with the probe was selected. The nucleotide sequence was determined from pUC19 by the 
direct Sanger method (or the dideoxy method). 

For translation of the nucleotide sequence to an amino acid sequence or for screening of homology, a software 
for genetic analysis (GENETYX, Nippon SDC) was used. 

Figs. 2(6) to 2(8) show the respective nucleotide sequences, and Figs. 4(VI) to4(VIII) show the respective amino 
*5 acid sequences. 

Example 3 

In order to examine the biological activity of the Xenopus laevis BMP-related gene products, each of Xbr22, Xbr23 
20 and Xbr41 cDNAs was inserted into expression vector pCDM8 (Invitrogen, U.S.A.) for animal cells, and expressed in 
a COS cell(African green monkey kidney cell). The resulting culture supernatant was used for determination of the 
biological activity. 

Each of the Xbr22, Xbr23 and Xbr41 cDNAs to which Xhol tinkers were ligated at both ends thereof was inserted 
into the Xhol restriction enzyme-cleaving site of pCDM8 to use it for transfection (introduction of DNA). 3 X 10 6 cells 

25 were subcultured in a 1 00 mm diameter plastic dish, and the medium was removed after 24 hours, followed by washing 
once with 10 ml of TBS (Tris-buffered saline). 300 ul of a DNA solution (1 .5 u,g DNA) diluted with TBS was mixed with 
300 u,l of a 0.1% DEAE-dextran solution, and the combined solution was added dropwise to the cells. After standing 
at ordinary temperature for 1 5 minutes, the cells were washed once with 300 uJ of TBS, and then incubated in Dulbecco's 
modified Eagle's medium (DMEM, containing 10% FBS, 100 U/ml penicillin, 100 mcg/mt streptomycin and 100 uM 

30 chloroquine). After 3 hours, the cells were washed twice with TBS and incubated in DMEM (containing 10% FBS, 100 
U/ml penicillin and 1 00 mcg/ml streptomycin). After 24 hours, the cells were washed three times with TBS and incubated 
in DMEM (containing 100 U/ml penicillin and 100 mcg/ml streptomycin) for 4 days, followed by recovery of the medium. 
The recovered medium was centrifuged at 2,000 rpm for 5 minutes to obtain a culture supernatant. 

The culture supernatant thus obtained was used for determination of the biological activity as a sample containing 

35 Xenopus laevis BMP2-A, BMP-2B or protein Vgr-1 . Namely, each of the samples was added to the medium of rabbit 
chondrocytes in monolayer cultures [Y Katoetal., Exp. Cell Res. 130 , 73-81 (1980); Y. Kato et al., J. Biol. Chem. 265 , 
5903-5909 (1990)] to examine their effect on the synthesis of proteoglycan, the main component of a cartilage matrix. 
As a result, the control in which the COS cell was transfected with the expression vector alone and the medium con- 
ditioned by untreated COS cells did not affect the synthesis of proteoglycan, as shown in the following table. In contrast, 

40 the above three kinds of proteins obtained in the present invention strongly promoted the synthesis of proteoglycan 
by the cartilage cells. The maximum activity of Xenopus laevis BMP-2A, BMP- 2B and Vgr-1 was stronger than that 
of TGF-beta-1. The synthesis of proteoglycan was determined by measuring 35 S-sulfate incorporation into gly- 
cosaminoglycans [Y. Kato etal., Exp. Cell Res. 130 , 73-81 (1980); Y. Kato et al., J. Biol. Chem. 265 , 5903-5909 (1990)]. 
These results show that the BMPs of Xenopus laevis promote the differentiation of cartilages, and suggest that the 

45 BMPs of other animals have similar effects. The BMPs are therefore expected to be applied to therapeutic agents for 
healing acceleration of fractures and for various diseases of cartilages and bones (such as arthritis and osteoporosis). 

* Kind of Cell 

Rabbit costal chondrocytes maintained on 6-mm diameter plastic wells. 

* Kind of Marker 

50 35s lu.Ci/ in 100 ul medium per well 

* Kind of Medium 

A 1:1 (V/V) mixture of DMEM and Ham's F-12 medium supplemented with 0.3% fetal bovine serum. 



55 



10 



EP 0 416 578 B1 



% to 



Ma 
IN KJ < 


» rltl CI A Ui V KS 




Count 


Mean 


+ 


S.D. 


Control 


1 


Control 


5193 


4328 


4269 


4695 


+ 


351 


100 






4565 


4727 


5089 










2 


x BMP 2 A 1 5pl 


2362 


2749 


2758 


2362 


+ 


185 


56 


3 


XBMP2A 1/3 5jj1 


12198 


15502 


21891 


16530 


+ 


4023 


352 


4 


XBMP2A 1/10 5yl 


10004 


9738 


8848 


9530 


+ 


494 


203 


5 


XBMP2B 1 5m1 


3171 


2906 


3219 


3099 


+ 


138 


66 


6 


XBMP2B 1/3 5ul 


11315 


9750 


13139 


11401 


+ 


1385 


243 


7 


XBMP2B 1/10 5yl 


12426 


13457 


13324 


13069 


+ 


458 


278 


8 


xVgr-1 1 5ul 


5188 


2833 


4416 


4146 


+ 


980 


88 


9 


xVgr-1 1/3 5yl 


7486 


8834 


7202 


7841 


+ 


712 


167 


10 


xvgr-l 10 5yl 


15286 


15645 


13032 


14654 


+ 


1156 


312 


11 


pCDM8 5ul 


3604 


2694 


2927 


3075 


+ 


386 


65 


12 


PCMD8 lyl 


2637 


4219 




3428 


+ 


791 


73 


13 


DNA(-) 5pl 


3625 


4050 


4714 


4130 


+ 


448 


88 


14 


DNA(-) lyl 


5695 


4657 




5176 


+ 


519 


110 


15 


DME 5yl 


3614 


8963 


3850 


5476 


+ 


2468 


117 


16 


DME lul 


4384 


3874 


5760 


4675 


+ 


799 


100 


17 


TGF-B1 3ng/ml 


9381 


12474 


10922 














10058 


11546 


11155 


10923 


+ 


998 


233 


18 


Ins. 5 g/ml 


19431 


20476 


22746 














25066 


27835 


24965 


23420 


+ 


2876 


499 


19 


Ins. 3 g/ml 


13620 


15378 


11987 














11240 


12699 


12666 


12932 


+ 


1313 


275 



pCDM8: A culture solution of the cells into which pCDM8 



is introduced as a vector 

DNA(-): A culture solution which is in contact with the 
cells, which do not produce the BMPs 

DME: A solution which is not in contact with the cells 
Ins.: Insulin 



11 



EP 0 416 578 B1 



Experiments Procedure 

Rabbil chondrocytes were isolated from growth plates of ribs of 3- to 4- week old male New Zealand rabbits, as 
previously described (Y Kato et al. Exp. Cell Res ). Cells were seeded at a density 10 4 cells / 6-mm diameter plastic 

£ culture well in 0.1 ml of Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum and 
antibiotics. When cultures became confluent, the cells were preincubated for 24 hours in 0.1 ml of a 1:1 mixture of 
DMEM and Ham's F-12 medium supplemented with 0.3% fetal bovine serum (DF). The cells were then transferred to 
0. 1 ml of the same medium (DF) supplemented with 1 or 5 uJ of the medium that was conditioned by various COS 
cells: [The conditioned medium was diluted or not diluted with DMEM (a final concentration of 10 or 30%)]. After 3 

10 hours, 5 ul of DMEM supplemented with 1 u.Ci of 35 S0 4 2 - was also added, and incubation was continued for a further 
17 hours (Y Kato et al. Exp. Ceil Res. ). 



Claims 

15 

Claims for the following Contracting States : DE, GB, FR, IT, NL, SE, CH, LI, BE, AT, LU, GR, DK 

1. A Xenopus laevis bone morphogenetic protein; wherein said protein is a mature protein containing an amino acid 
20 sequence having an amino acid sequence represented by Nos. 1 5 to 1 30 of formula (I) shown in Fig. 3, an amino 

acid sequence represented by Nos. 1 4 to 1 27 of formula (II) shown in Fig. 3, an amino acid sequence represented 
by Nos. 6 to 63 of formula (IV) shown in Fig. 3, an amino acid sequence represented by Nos. 6 to 65 of formula 
(V) shown in Fig. 3, an amino acid sequence represented by Nos. 282 to 398 or Nos. 298 to 398 of formula (VI) 
shown in Fig. 4, or an amino acid sequence represented by Nos. 328 to 426 of formula (VIM) shown in Fig. 4. 

25 

2. A Xenopus laevis bone morphogenetic protein, wherein said protein is a precursor protein containing an amino 
acid sequence having an amino acid sequence represented by formula (I), (II), (IV) or (V) shown in Fig. 3, or 
formula (VI) or (VIM) shown in Fig. 4. 

30 3. A DNA comprising a DNA segment coding for a Xenopus laevis bone morphogenetic protein according to claim 1 
or 2. 

4. A DNA comprising a DNA segment coding for a Xenopus laevis bone morphogenetic protein, wherein said DNA 
segment comprises a nucleotide sequence corresponding to the nucleotide sequence represented by formula (1 ), 

35 (2), (3), (4), (5), (6), (7) or (8) shown in Fig. 2. 

5. A non-human transformant bearing a DNA comprising a DNA segment according to claim 3 or 4, wherein said 
transformant is not Xenopus laevis . 

40 6. A transformant in accordance with claim 5, which has the characteristics of Escherichia coli HB101/pXar3 (FERM 
BP-2578). 

7. A transformant in accordance with claim 5, which has the characteristics of Escherichia coli HB101/pXar4 (FERM 
BP-2579). 

45 

8. A transformant in accordance with claim 5, which has the characteristics of Escherichia coli HB101/pXar5 (FERM 
BP-2580). 

9. A transformant in accordance with claim 5, which has the characteristics of Escherichia coli HB101/pXar9 (FERM. 
so BP-2581). 

10. A transformant in accordance with claim 5, which has the characteristics of Escherichia coli HB101/pXar14 (FERM 
BP-2582). 

55 11. A transformant in accordance with claim 5, which has the characteristics of Escherichia coli HB101/pXbr22(F€RM 
BP-3066). 

12. A transformant in accordance with claim 5, which has the characteristics of Escherichia coli HB101/pXbr23(FERM 



12 



EP 0 416 578 B1 



BP-3065). 

13. A transformant in accordance with claim 5, which has the characteristics of Escherichia coli HB1 01 ZpXbr41 (FERM 
BP-3067). 

5 

14. A method for preparing a Xenopus laevis bone morphogenetic protein according to claim 1 or 2 which method 
comprises culturing a non-human transformant as defined in claim 5, producing and accumulating the protein in 
a culture, and collecting the protein thus obtained. 

io 1 5. A composition for therapy of fracture or osteoporosis which contains an effective amount of a Xenopus laevis bone 
morphogenetic protein according to claim 1 or 2 and pharmaceutical acceptable additional components. 

16. A method for preparing a composition for therapy of fracture or osteoporosis which comprises admixing an effective 
amount of a Xenopus laevis bone morphogenetic protein according to claim 1 or 2 with pharmaceutical^ acceptable 
' 5 additional components. 



Claims for the following Contracting State : ES 

20 1. A method for preparing a Xenopus laevis bone morphogenetic protein, wherein said protein is a mature protein 
containing an amino acid sequence having an amino acid sequence represented by Nos. 15 to 130 of formula (I) 
shown in Fig. 3, an amino acid sequence represented by Nos. 14 to 127 of formula (II) shown in Fig. 3, an amino 
acid sequence represented by Nos. 6 to 63 of formula (IV) shown in Fig. 3, an amino acid sequence represented 
by Nos. 6 to 65 of formula (V) shown in Fig. 3, an amino acid sequence represented by Nos. 282 to 398 or Nos. 

25 298 to 398 of formula (VI) shown in Fig. 4, or an amino acid sequence represented by Nos. 328 to 426 of formula 

(VIII) shown in Fig. 4, or wherein said protein is a precursor protein containing an amino acid sequence having an 
amino acid sequence represented by formula (I), (II), (IV) or (V) shown in Fig. 3, or formula (VI) or (VIII) shown in 
Fig. 4, which method comprises culturing a non-human transformant bearing a DNA comprising a DNA segment 
coding for the protein, wherein said transformant is not Xenopus laevis , producing and accumulating the protein 

30 in a culture, and collecting the protein thus obtained. 

2. A method for preparing a DNA comprising a DNA segment coding for a Xenopus laevis bone morphogenetic protein 
as defined in claim 1 , which method comprises cloning the DNA from a Xenopus laevis DNA library. 

35 3. A method for preparing a DNA comprising a DNA segment coding for a Xenopus laevis bone morphogenetic 
protein, wherein said DNA segment comprises a nucleotide sequence corresponding to the nucleotide sequence 
represented by formula (1), (2) ; (3), (4), (5), (6), (7), or (8) shown in Fig. 2, which method comprises cloning the 
DNA from a Xenopus laevis DNA library. 

40 4. A method for preparing a non-human transformant bearing a DNA comprising a DNA segment as prepared in claim 
3 or 4, wherein the prepared transformant is not Xenopus laevis , which method comprises transforming a host 
with said DNA. 

5. A method in accordance with claim 4, wherein the prepared transformant has the characteristics of Escherichia 
^5 coli HBl01/pXar3 (FERM BP-2578). 

6. A method in accordance with claim 4, wherein the prepared transformant has the characteristics of Escherichia 
coli HB101/pXar4 (FERM BP-2579). 

50 7. a method in accordance with claim 4, wherein the prepared transformant has the characteristics of Escherichia 
coli HBl01/pXar5 (FERM BP-2580). 

8. A method in accordance with claim 4, wherein the prepared transformant has the characteristics of Escherichia 
coli HBl01/pXar9 (FERM BP-2581). 

55 

9. A method in accordance with claim 4, wherein the prepared transformant has the characteristics of Escherichia 
coli HB101/pXar14 (FERM BP-2582). 



13 



EP 0 416 578 B1 



10. A method in accordance with claim 4, wherein the prepared transformant has the characteristics of Escherichia 
coli HB101/pXbr22 (FERM BP-3066). 

11. A method in accordance with claim 4, wherein the prepared transformant has the characteristics of Escherichia 
5 coli HB101/pXbr23 (FERM BP-3065). 

12. A method in accordance with claim 4, wherein the prepared transformant has the characteristics of Escherichia 
coli HBl01/pXbr41 (FERM BP-3067). 

10 13. A method for preparing a composition for therapy of fracture or osteoporosis which method comprises admixing 
an effective amount of a Xenopus laevis bone morphogenetic protein as prepared according to claim 1 with phar- 
maceutical^ acceptable additional components. 



Patentanspruche 



Patentanspruche fur folgende Vertragsstaaten : DE, GB, FR, IT, NL, SE, CH, LI, BE, AT, LU, GR, DK 

20 1. Xenopus 7aews-Knochenmorphogenese- Protein, wobei das Protein ein reifes Protein ist, das eine Aminosauren- 
sequenz mit einer Aminosaurensequenz, welche durch die Nr. 15 bis 130 der in Fig. 3 dargestellten Formel (I) 
dargestellt wird, einer Aminosaurensequenz, welche durch die Nr. 14 bis 127 der in Fig. 3 dargestellten Formel 
(II) dargestellt wird, einer Aminosaurensequenz, welche durch die Nr. 6 bis 63 der in Fig. 3 dargestellten Formel 

(IV) dargestellt wird, einer Aminosaurensequenz, welche durch die Nr. 6 bis 65 der in Fig. 3 dargestellten Formel 

(V) dargestellt wird, einer Aminosaurensequenz, welche durch die Nr. 282 bis 398 Oder Nr. 298 bis 398 der in Fig. 
4 dargestellten Formel (VI) dargestellt wird, oder einer Aminosaurensequenz, welche durch die Nr. 328 bis 426 
der in Fig. 4 dargestellten Formel (VIII) dargestellt wird, enthalt. 

2. Xenopus /aews-Knochenmorphogenese -Protein, wobei das Protein ein Vorlauferprotein ist, das eine Aminosau- 
30 rensequenz mit einer Aminosaurensequenz enthalt, welche durch die in Fig. 3 dargestellte Formel (I), (II), (IV) 

oder (V) oder in Fig. 4 dargestellte Formel (VI) oder (VIII) dargestellt wird. 

3. DNA umfassend ein DN A-Segment, das fur ein Xenopus /aeWs-Knochenmorphogenese-Protein gemaG Anspruch 

1 oder 2 kodiert. 

35 

4. DNA umfassend ein DNA-Segment, das fur ein Xenopus /aews-Knochenmorphogenese- Protein kodiert, wobei 
das DNA-Segment eine Nukleotidsequenz umfaGt, die der Nukleotidsequenz entspricht, welche durch die in Fig. 

2 dargestellte Formel (1), (2), (3), (4), (5), (6), (7) oder (8) dargestellt wird. 

40 5. Nicht-humane Transformante, die eine DNA tragt, welche ein DNA-Segment gemaG Anspruch 3 oder 4 umfaGt, 
wobei die Transformante nicht Xenopus laevis ist. 

6. Transformante gemaG Anspruch 5, welche die Eigenschaften von Escherichia co//HB101/pXar3 (FERM BP-2578) 
hat. 

45 

7. Transformante gemaG Anspruch 5, welche die Eigenschaften von Escherichia co// HB101/pXar4 (FERM BP-2579) 
hat. 

8. Transformante gemaG Anspruch 5, welche die Eigenschaften von Escherichia co//HB101/pXar5 (FERM BP-2580) 
so hat. 

9. Transformante gemaG Anspruch 5, welche die Eigenschaften von Escherichia co//HB101/pXar9 (FERM BP-2581 ) 
hat. 

55 10. Transformante gemaG Anspruch 5, welche die Eigenschaften von Escherichia -coli HB1 01 /pXarl 4 (FERM BP- 
2582) hat. 

11. Transformante gemaG Anspruch 5, welche die Eigenschaften von Escherichia coli HB101/pXbr22 (FERM BP- 



14 



EP 0 416 578 B1 



3066) hat. 

12. Transformante gemaG Anspruch 5, welche die Eigenschaften von Escherichia coli HB101/pXbr23 (FERM BP- 
3065) hat. 

5 

13. Transformante gemaG Anspruch 5, welche die Eigenschaften von Escherichia coli HB101/pXbr41 (FERM BP- 

3067) hat. 

14. Verfahren zum Herstellen eines Xenopus laevis- Knochenmorphogenese- Proteins gemaG Anspruch 1 oder 2, wo- 
io bei das Verfahren das Kultivieren einer in Anspruch 5 definierten nicht-humanen Transformanten, das Erzeugen 

und Anreichern des Proteins in einer Kultur und Isolieren des auf diese Weise erhaltenen Proteins umfaGt. 

15. Zusammensetzung zur Fraktur- oder Osteoporosetherapie, die eine wirksame Menge eines Xenopus /aews-Kno- 
chenmorphogenese-Proteins gemaG Anspruch 1 oder 2 und pharmazeutisch annehmbare Zusatzbestandteile ent- 

15 halt. 

16. Verfahren zum Herstellen einer Zusammensetzung zur Fraktur- oder Osteoporosetherapie, welches das Mischen 
einer wirksamen Menge eines Xenopus /aew's-Knochenmorphogenese-Proteins gemaG Anspruch 1 oder 2 mit 
pharmazeutisch annehmbaren Zusatzbestandteilen umfaGt. 

20 

Patentanspruche fur folgenden Vertragsstaat : ES 

1. Verfahren zum Herstellen eines Xenopi/s/aev/s-Knochenmorphogenese-Proteins, wobei das Protein ein reifes 
25 Protein ist, das eine Aminosaurensequenz mit einer Aminosaurensequenz, welche durch die Nr. 15 bis 130 der 

in Fig. 3 dargestellten Formel (I) dargestellt wird, einer Aminosaurensequenz, welche durch die Nr. 14 bis 127 der 
in Fig. 3 dargestellten Formel (II) dargestellt wird, einer Aminosaurensequenz, welche durch die Nr. 6 bis 63 der 
in Fig. 3 dargestellten Formel (IV) dargestellt wird, einer Aminosaurensequenz, welche durch die Nr. 6 bis 65 der 
in Fig. 3 dargestellten Formel (V) dargestellt wird, einer Aminosaurensequenz, welche durch die Nr. 282 bis 398 

30 oder Nr. 298 bis 398 der in Fig. 4 dargestellten Formel (VI) dargestellt wird, oder einer Aminosaurensequenz, 

welche durch die Nr. 328 bis 426 der in Fig. 4 dargestellten Formel (VIII) dargestellt wird, enthalt oder wobei das 
Protein ein Vorlauferprotein ist s das eine Aminosaurensequenz mit einer Aminosaurensequenz enthalt, welche 
durch die in Fig. 3 dargestellte Formel (I), (II), (IV) oder (V) oder in Fig. 4 dargestellte Formel (VI) oder (VIII) 
dargestellt wird, wobei das Verfahren das Kultivieren einer nicht-humanen Transformanten, die eine DNA tragt, 

35 welche ein DNA-Segment umfaGt, das fur das Protein kodiert, wobei die Transformante nicht Xenopus laevis ist, 

das Erzeugen und Anreichern des Proteins in einer Kultur und Isolieren des auf diese Weise erhaltenen Proteins 
umfaGt. 

2. Verfahren zum Herstellen einer DNA, die ein DNA-Segment umfaGt, das fur das in Anspruch 1 definierte Xenopus 
40 laevis- Knochenmorphogenese-Protein kodiert, wobei das Verfahren das Klonieren der DNA aus einer Xenopus 

/aews-DNA-Bank umfaGt. 

3. Verfahren zum Herstellen einer DNA, die ein DNA-Segment umfaGt, das fur ein Xenopus /aeWs-Knochenmorpho- 
genese-Protein kodiert, wobei das DNA-Segment eine Nukleotidsequenz umfaGt, die der Nukleotidsequenz ent- 

45 spricht, welche durch die in Fig. 2 dargestellte Formel (1), (2), (3), (4), (5), (6), (7) oder (8) dargestellt wird, wobei 

das Verfahren das Klonieren der DNA aus einer Xenopus /aews-DNA-Bank umfaBt. 

4. Verfahren zum Herstellen einer nicht-humanen Transformante, die eine DNA tragt, welche ein in Anspruch 3 oder 
4 hergestelltes DNA-Segment umfaGt, wobei die hergestellte Transfotmante nicht Xenopus laevis ist und das 

50 Verfahren das Transformieren eines Wirts mit der DNA umfaGt. 

5. Verfahren gemaG Anspruch 4, wobei die hergestellte Transformante die Eigenschaften von Escherichia coli 
HBl01/pXar3 (FERM BP-2578) hat. 

55 6. Verfahren gemaG Anspruch 4, wobei die hergestellte Transformante die Eigenschaften von Escherichia coli 
HB101/pXar4 (FERM BP-2579) hat. 

7. Verfahren gemaG Anspruch 4, wobei die hergestellte Transformante die Eigenschaften von Escherichia coli 



15 



EP 0 416 578 B1 



HB101/pXar5 (FERM BP-2580) hat. 

8. Verfahren gemaB Anspruch 4, wobei die hergestelfte Transformante die Eigenschaften von Escherichia coli 
HB101/pXar9 (FERM BP-2581) hat. 

s 

9. Verfahren gemaB Anspruch 4, wobei die hergestelfte Transformante die Eigenschaften von Escherichia coli 
HB101/pXar14 (FERM BP-2582) hat. 

10. Verfahren gemaB Anspruch 4, wobei die hergest elite Transformante die Eigenschaften von Escherichia coli 
io HB101/pXbr22 (FERM BP-3066) hat. 

11. Verfahren gemaB Anspruch 4, wobei die hergestellte Transformante die Eigenschaften von Escherichia coli 
HBl01/pXbr23 (FERM BP-3065) hat. 

*5 12. Verfahren gemaB Anspruch 4, wobei die hergestelfte Transformante die Eigenschaften von Escherichia coli 
HB101/pXbr41 (FERM BP-3067) hat. 

13. Verfahren zum Herstellen einer Zusammensetzung zur Fraktur- oder Osteoporosetherapie, welches das Mischen 
einer wirksamen Menge eines gemaB Anspruch 1 hergestelften Xenopus /aews-Knochenmorphogenese -Proteins 
20 mit pharmazeutisch annehmbaren Zusatzbestandteilen umfaBt. 



Revendications 

25 

Revendications pour les Etats contractants suivants : AT, BE, CH, DE, DK, FR, GB, GR, IT, LI, LU, NL, SE 

1. Proteine intervenant dans la morphogenese des os de Xenopus laevis , qui est une protgine mature dont la se- 
quence d'acides amines comporte la sequence des acides amines n° 15 a 130 de la formule (I) indiquGe dans la 

30 figure 3, ta sequence des acides amin6s n° 14 a 127 de la formule (II) indiquee dans la figure 3, la sequence des 

acides amines n° 6 a 63 de la formule (IV) indiquee dans la figure 3, la sequence des acides amines n° 6 a 65 de 
la formule (V) indiqu6e dans la figure 3, la sequence des acides amines n° 282 a 398 ou n° 298 a 398 de la formule 
(VI) indiquee dans la figure 4, ou la s6quence des acides amines n° 328 a 426 de la formule (VIII) indiquee dans 
la figure 4. 

35 

2. Prot6ine intervenant dans la morphogenese des os de Xenopus laevis , qui est une proteine pr6curseur dont la 
sequence d'acides amines comporte une sequence d'acides amin6s representee par I'une des formules (I), (II), 
(IV) et (V) indiqu6es dans la figure 3 ou par I'une des formules (VI) et (VIII) indiqu6es dans la figure 4. 

40 3. ADN comportant un segment d'ADN codant une p rote in e intervenant dans la morphogenese des os de Xenopus 
laevis , conforme a I'une des revendications 1 et 2. 

4. ADN comportant un segment d'ADN codant une prot6ine intervenant dans la morphogenese des os de Xenopus 
laevis , dans lequel ledit segment d'ADN comporte une sequence de nucleotides qui correspond a une sequence 

45 de nucleotides representee par I'une des formules (1), (2), (3), (4), (5), (6), (7) et (8) indiquees dans la figure 2. 

5. individu transforme non-humain qui h6berge un ADN comportant un segment d'ADN, conforme a I'une des reven- 
dications 3 et 4, mais qui n'est pas un individu Xenopus laevis . 

50 6. Individu transforme conforme a la revendication 5, presentant les caract6ristiques d 'Escherichia coli HB101/pXar3 
(FERM BP-2578). 

7. Individu transform^ conforme a la revendication 5, presentant les caract6ristiques d 'Escherichia coli HB101/pXar4 
(FERM BP-2579). 

55 

8. Individu transforme* conforme a la revendication 5, presentant les caracteristiques d 'Escherichia coli HB10l/pXar5 
(FERM BP-2580). 



16 



EP 0 416 578 B1 



9. Individu transforms conforme a la revendication 5, presentant les caracteristiques d 'Escherichia coli HB1 01/pXar9 
(FERM BP-2581). 

1 0. I ndividu transf orm6 conforme a la revendication 5, presentant les caracteristiques d 'Escherichia coli HB101 /pXarl 4 
s (FERM BP-2582). 

11.1 ndividu transf orme conforme a la revendication 5 , presentant les caracteristiques d 'Escherichia coli HB101 /pXbr22 
(FERM BP-3066). 

10 12. I ndividu transforme conforme a ia revendication 5, presentant les caracteristiques d 'Escherichia coli HB101/pXbr23 
(FERM BP-3065). 

13. I ndividu transforme conforme a la revendication 5, presentant les caracteristiques d 'Escherich ia coli HB101/pXbr41 
(FERM BP-3067). 

15 

14. Procede de preparation d'une proteine intervenant dans la morphogenese des os de Xenopus laevis , conforme a 
Tune des revendications 1 et 2, lequel precede comporte le fait de cultiver un individu transforme non-humain 
conforme a ia revendication 5, le fait de laisser la proteine produite s'accumuler dans la culture, et le fait de recueilltr 
la proteine ainsi obtenue. 

20 

15. Composition destinee au traitement des fractures ou de I'osteoporose, qui contient une quantite efficace d'une 
proteine intervenant dans la morphogenese des os de Xenopus laevis , conforme a I'une des revendications 1 et 
2, et des composants supplementaires admissibles en pharmacie. 



25 16. Procede de preparation d'une composition destinee au traitement des fractures ou de I'osteoporose, qui comporte 
le fait de meianger une quantite efficace d'une proteine intervenant dans la morphogenese des os de Xenopus 
laevis ; conforme a I'une des revendications 1 et 2, avec des composants supplementaires admissibles en phar- 
macie. 

30 

Revendications pour I'Etat contractant suivant : ES 



1. Procede de preparation d'une proteine intervenant dans la morphogenese des os de Xenopus laevis , qui est une 
proteine mature dont la sequence d'acides amines comporte la sequence des acides amines n° 15 a 130 de la 

35 formule (I) indiqu6e dans la figure 3, la sequence des acides amines n° 14 a 127 de la formule (II) indiquee dans 

la figure 3, la sequence des acides amines n° 6 a 63 de la formule (IV) indiqu6e dans la figure 3, la sequence des 
acides amines n° 6 a 65 de la formule (V) indiquee dans la figure 3, la sequence des acides amines n° 282 a 398 
ou n° 298 a 398 de la formule (VI) indiquee dans la figure 4, ou la sequence des acides amines n° 328 a 426 de 
la formule (VIII) indiqu6e dans la figure 4, ou qui est une proteine precurseur dont la sequence d'acides amines 

40 comporte une sequence d'acides amines representee par Tune des formules (I), (II), (IV) et (V) indiquees dans la 

figure 3 ou par I'une des formules (VI) et (VIII) indiqu6es dans la figure 4, lequel proc6d6 comporte le fait de cultiver 
un individu transforme non-humain qui heberge un ADN comportant un segment d'ADN codant ladite proteine, 
mais qui n'est pas un individu Xenopus laevis , le fait de laisser la proteine produite s'accumuler dans la culture, 
et le fait de recueillir la proteine ainsi obtenue. 

45 

2. Procede de preparation d'un ADN comportant un segment d'ADN codant une proteine intervenant dans la mor- 
phogenese des os de Xenopus laevis , definie dans la revendication 1, lequel procede comporte le fait de doner 
cet ADN a partir d'une bibliotheque d'ADN de Xenopus laevis. 

so 3. Procede de preparation d'un ADN comportant un segment d'ADN codant une proteine intervenant dans la mor- 
phogenese des os de Xenopus laevis , dans lequel ledit segment d'ADN comporte une sequence de nucleotides 
qui correspond a une sequence de nucleotides representee par I'une des formules (1 ), (2), (3), .(4), (5), {6), (7) et 
(8) indiquees dans la figure 2, lequel procede comporte le fait de doner cet ADN a partir d'une bibliotheque d'ADN 
de Xenopus laevis . 

55 

4. Procede de preparation d'un individu transforme non-humain qui heberge un ADN comportant un segment d'ADN, 
prepare conform6ment a I'une des revendications 2 et 3, ('individu translorme prepare n'etant pas un individu 
Xenopus laevis , lequel procede comporte le fait de transformer un h6te avec ledit ADN. 



17 



EP 0 416 578 B1 



5. Precede conforme a la revendication 4, dans lequel I'individu transforme prepare presente les caracteristiques 
d 'Escherichia coli HB101/pXar3 (FERM BP-2578). 

6. Procede conforme a la revendication 4, dans lequel I'individu transforms prepare presente les caracteristiques 
5 d 'Escherichia coli HB101/pXar4 (FERM BP-2579). 

7. Procede conforme a la revendtcation 4, dans lequel I'individu transforme prepare presente les caracteristiques 
d 'Escherichia coli HB101/pXar5 (FERM BP-2580). 

10 8. Procede conforme a la revendication 4, dans lequel I'individu transforme prepare presente les caracteristiques 
d 'Escherichia coli HB101/pXar9 (FERM BP-2581). 

9. Procede conforme a la revendication 4, dans lequel I'individu transforme prepare presente les caracteristiques 
d 'Escherichia coli HB101/pXar14 (FERM BP-2582). 

is 

10. Precede conforme a la revendication 4, dans lequel I'individu transforme prepare presente les caracteristiques 
d 'Escherichia coli HB101/pXbr22 (FERM BP-3066V 

11. Precede conforme a la revendication 4, dans lequel I'individu transforme prepare presente les caracteristiques 
20 d 'Escherichia coli HB101/pXbr23 (FERM BP-3065). 

12. Procede conforme a la revendication 4, dans lequel I'individu transforme prepare pr6sente les caracteristiques 
d'Escherichia coli HB101/pXbr41 (FERM BP-3067). 

25 13. Procede de preparation d'une composition destinee au traitement des fractures ou de rosteoporose, qui comporte 
le fait de meianger une quantite efficace d'une proteine intervenant dans la morphogenese des os de Xenopus 
]aeyjs : pr6par6e conformement a la revendication 1 , avec des composants suppiementaires admissibles en phar- 
macie. 

30 



35 



40 



45 



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55 



18 



EP 0 416 578 B1 



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X 0 


1 LJ 


H- > 




CD H 


r* > »^ 


> O co 




co > cn 


co > o 


•o H O 


> O 


o o 


T LJ 


*~ LJ 


« o 


*< H 


r o 


r > 


» H 


«< > 


c H 


CD > 


r > 


oo 




»- O co 


co > to 


»< > a 


who 


X OO 


a> 






CO 





V, J 


r\ 

v J 


LJ 




»** V. J 




~ LJ 




w O 


•> LJ 


H H 


co 3> 


"1 LJ 


u O w 


•o O 


r - > cn 


i-H J> <D 


*< [> o 




CO J> 


» H 


> > 


> O 


CO J> 


O 


3 H 


B> > 


< O 


s o 


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co H 


no O N5 


r > to 


■5 O 05 


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(D > o 


r > 


o o 


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»~ > 


co Cj 


s Cj 


r > 


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*< > 


"J O 


co a 


o H 


s o 


> > 


H- > tO 


u > tO 


» H *4 


3 0 *- 


< Cj o 


soo 


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M. > 


Cj 


co H 


> > 


o o 


*5 Cj 


»— Q 


oc] O 


o 


M > 


H 


i— 1-3 


H 


o H to 


CD H w 


CO > od 


< O to 


o 0 O 


b> H O 


i H 


H 


> > 


< Cj 




a> H 


w O 


H 


Co H 


O O 


o O 




*i H 


c > 


r* H to 


< O to 


o H CD 


o» H co 


c > o 




H > 


H > 


3" 0 


3" O 


i O 


i H 


"0 0 


SO 


*5 O 




o O 


» o 


>o 


r H 


» > co 


CD H to 


*o HO 




r > o 


> oo 


*« 


w 


cn 


•0 



C J 


LJ 




»** 


r* >> 


C J 


" t*> 




CO > 


H 


n c j 


H 


CD H 


Li 


c O 


H 


< O co 


n *3 


cp h O 


> o 


> 


o 


X o 


o 




CO 


m h 


o 


X o 


> 


> 


o 


CO H 


o 


> > t- 


G 


CO > 


O oo 


s H o 


zo 


Cj O 




»- > 




c > 


2S 


CO > 


O 


CD O 


H 


t o 


O 


r > 


a 


v: > m 


CO 


to > cn 


O CD 


H H o 


H O 


*i a 


CO 




O 


o o 


> O 


*- > 


i o 


c J> 


ft o 


Co > 


o o 


CD Q 


o 


-3 H n- 




T3 H 0i 


tOO 


sr H O 


m OO 


o H 


o H 


> O 


< O 


CO > 


» H 


•o H 


— H 


< a 


<o 


o> H 


ft H 


H- > 


O 




> > L- 


O *J 


i o »— 


ft oo 


ft > o 


*o o 


r o 


T O 


CD H 


o a 


c > 


>o 


r H 


-o 


• H 


b> > 


c O 


~> 


>o 


H H- 


ft > H- 


CD H 05 


•o On 


>0O 


H>o 




D- 


ft 





»> 




LJ 








LJ 




o 




> 




o 




O - 




H ^ - 




0 o 




> 




> 




o 




> 




o 




0 




0 




o 




H n> 




H O 




H 




O 




> 








> 




O 




H 




O 




> CO 




O o 




> 




o 




o 


0 w 


> 




o 




o 




> 




> 




> iU 


CO 


HO 




H 




O 


1 


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> 




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O 




O 




O 




> cn 




HO 




H 




> 








H 




> 




H 




H 




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> 0) 




OO 





23 



EP 0 416 578 B1 



H 


> 


H 


O 


O 


H 


H 


> 


H 


O 


H 


O H 


> 


H 


H 


<< O 




0 


a 


to 0 


> 


H 




> O 


H 


> 


> 


T O 


Ho 


O oo 


O o 


<* H <J 


H - 


> en 


> CO 


# H 03 


HO 


no 


Oo 


# > o 


H 


o 


H 


* o 


> 


a 


> 


O 


H 


> 


H 


O 


> 


a 


> 


> 


> 


o 


H 


o 


Q 


H 


H 


H 


a 


H 


H 


H 


O co 


O 00 


> oo 


>S 


H 


O o? 


Ho 


O ^ 


HO 


a o 


OO 


O O 


H 


> 


0 


O 


Q 


> 


H 


O 


H 


o 


H 


O 


> 


o 


H 


> 


O 


> 


O 


> 


O 


> 


> 


o 


O 


> 


> 


o 


> CO 


Hod 


> CO 








> >- 


> cn 


no 


H O 


>o 


OO 


> 


H 


H 


> 




H 


a 


o 


> 


H 


> 


> 


H 


> 




o 


o 


> 


a 


> 


> 


H 


> 


> 


> 


O 


> 


o 


a co 


> 00 


> 00 




H*> 


a oo 


o to 


> 0) 


>o 


>o 


>o 


>o 


H 


o 


z 


o 


> 


> 


H 


> 


> 


> 


H 


H 


H 


> 


> 


o 


a 


H 


H 


> 


a 


> 


H 


O 


H 


H 


H 


> 


O co 


Hoo 


H oo 


O -J 


> cn 


O co 


O o» 


H-3 


a o 


no 


>o 


HO 


> 


H 


> 


H 


H 


H 


> 


> 


H 


H 


> 


> 


O 


H 


H 


H 


> 


H 


> 


> 




H 




H 




H 


> 


H 




> CO 


Hco 


H-J 




OO 


H* 


O a> 




Ho 


Ho 


Oo 



H 


O 


O 


H 


> 


> 


>o 


> > 


> o 


CO > 


(0 > 


to > 


•o H 


3 O 


•o O 


O O 


~ > 


SO 


-> 


— H 


-•> 


c Oa 


ro O 03 


to H cn 


> >-3 


V O 


r H cn 


to >0 


i OO 


(8 H o 


3 H 


o O 


c > 


OO 


r > 


>> 




vc > 


to > 


c > 


CO > 


» O 


r > 


> o 


a»H 


V > 


- o 


» O 




o H 


i H 


<: o o 


O H 0> 


H > cn 


o» H oo 




sr O 0> 


~>o 


» OO 


T > O 


<o 


OH 


>> 


» H 


«< a 


CO > 


- > 


a n 


3 O 


r H 


<o 


SO 


ft H 


• H 




c > 


-O 


o) h 


r > 


T3 O 


>o 


«< > o: 


1 O03 


t-O CJi 


CD > CO 


0 O 03 


& > ^ 


> >o 


H >o 


~ > O 


co > 


srO 


-H 


* H 


i > 


« O 


HH 


OO 


<o 


v > 




» H 


n H 


c > 


*-> 


a o 


r o 


OO 


-> 


» H 


-> 


» > -0 


c O 03 




> OO 




H > od 


it >o 


o Qo 


srOO 


•o O 




i H 


S> 


>o 


rH 


o H 


"-O 


a> H 


r- O 


* O 


c O 


< o 


~> 


<o 


» H 


-H 


» H 


>- n 


o > 


-O 


< o*j 


to H 05 


> > cn 


cd H 


o Ow 


to > CO 


I- o o 


•sOo 


3 O O 


o o 


S> 


W H 


- > 


o H 


© O 


c Q 


r*0 


H 


O O 


r o 


<o 


- O 


• H 


a. H 


vj O 


c O 


-O 


OH 


HH 


>> 




<< > 05 


co > 05 


» oro 


1 H 05 


D OO 


o o o 


r O o 


H > O 




<t 


sr 




c 





24 



EP 0 416 578 B1 



o 


O 


O 


o 


H 


O 




H 


o 


> 


o 


H 


o 




r H 


r > 


> o 


to H 


H 


H 




« H 


<< > 


-o 


o 0 


H 


O 




c a 


w O 


o> H 


1 H 


H 


> 




o o 


r > 


*v O 


o o 


O 


o 






*< > 


n O 


- o 


H 


> 




c Oco 


to > ro 


o H *- 




H 


z 






no O cn 


r O co 


O O co 


O o 


H - 




v >o 


•3 D o 


a> Ho 


->o 


H O 


HO 




1 H 


o > 


c O 


3 > 


O 


> 




a o 


r O 


coH 


>o 


H 


> 




- > 


« H 


a> O 


u> > 




O 




c a 


c H 


n > 




H 


> 




> o 


S O 


H> 


> O 


a 


> 




- o 




srO 


tfi > 


o 


o 




Op > 


w H 


•» H 


o 


H 


H 




HHu 


< O to 


> > to 


r >n- 


> 


O 




*< > to 


» Ho 


"3 O O 


«< > ^ 


O 0D 


H 63 




n H O 




w oo 


» o O 




o o 




K O 


>> 


oo 


H> 


3- > 


o 




> 


tfl > 


-> 


=-o 


© o 


o 




« H 


3 H 


3 > 


T H 


> 


> 




O H 


H 


o o 


<o 


=r > 


H 




^ o 


t^H 


— o 


o. h 


a > 


H 




« H 


a> O 


v: O 


-O 


> a 


H 




oo 


r > 


r > 


HH 


to > 


> 




t- > C#3 


v: > to 


*c > to 


Vtf > H* 


3 > 


> 




C > CO 


» O *o 




t H cn 


O > CD 


O a 




OCO 


>o o 


>>o 


COO 


-Oo 


> o 




-o 


CO > 


^ o 


-> 


c O 


O 




*< o 


* H 


w O 


c > 


- O 


> 




ro 


S> 


t> o 


H H 


— o 


> 




a H 


o H 


o 


« > 


(D > 


o & 


> 0) 


c H 


r* O 


o H 


^ H 


ro 


> H 


• 


OH 


o o 


>> 


rH 




o if 




V! Q 


-o 


CO > 


a H 


» H 


o 






«< H to 


s H ro 


C > H- 


>a*- 


> 


o a 


HHOD 


r > to 


HD H 05 


HO 


o 




»-> o 


i QO 


*< > o 


3- Ho 


» Ho 


oo 




c o 


•o O 


a O 


a O 


>o 


> 




T>H 


>o 


> > 


>> 


•» H 


o 






(0 > 


(a > 


tfi > 


« H 


o 




» o 


•v H 


» H 


3 H 


O 


o 




no O 


> O 


w H 


OO 


> 


> 




n O 


u> ;> 


» o 


-> 


> 






o H 


T3 H 


*j > 


3 O 


H 


o 




r n cfi 


H H to 


r > ro 


>>►- 


Op 


> 




a H cn 


i Q co 


v: > co 


t 'O <3 


> - 


> cn 




COO 


t oo 


w >0 


woo 


oo 


HO 




>> 


~> 


>o 


>> 


> 


O 




i O 




-O 


i o 


H 


> 




<* > 


o H 


0) > 


WJ > 


H 


O 




CoH 




>> 


r > 


> 


> 




a O 


~H 




«< > 


> 


> 




n H 


• H 


00 > 


» o 


> 


> 






> O 


O H 




a 


o 








<< O to 


T O H* 


Oh 


o 






» no 


cn H ^ 


ft > 00 


O to 


H 0) 






^ o O 


W > O 


>00 


>o 


>o 






n 


a 


•1 










o 




09 









25 



EP 0 416 578 B1 



o 


O 


> 


o 


o 


> 




oo 


O O 


oo 


wH 


o 


> 




-o 


-> 


~o 


» O 


o 


o 




«< o 


c O 


* H 


n 0 


> 


o 




<o 


rH 


SO 


~> 


o 


H 




a> H 


» H 


— > 


-H 


> 


H 






c O 




o O 


> 


H 




O H 03 


O O to 


> O 


> O h- 


o 


> 




"< Oh 


G cn 


— O CD 


— O 05 


> -0 


O - 




« Ho 




&> O O 


0) O O 


Ho 


HO 




>o 


HH 


r > 


r > 


H 


O 




IA J> 


a 


<< > 


<< > 


> 


o 




•o H 


•o O 


» o 


» o 


> 


H 






> o 


r > 


>> 


o 


o 




=r H 


to > 


<< > 




H 


o 




» O 


•o H 


tO > 


* o 


O 


H 




*o O 


>Q 


coH 


>> 




O 




T O w 


» > ro 


«> O to 


i o *- 


- > 


H 




o > to 


•o O o> 


i Oo 


on >»> 


o H OD 


O to 




r no 


H Ho 


r > o 


HHO 


r H o 


Ho 




» H 


T O 


vc > 


i O 


a O 


H 




c G 


*D O 


to > 


•o O 


c O 


O 




> > 


~> 


H> 


r > 


O > 


O 




i o 


~H 


3- 0 


« > 


- > 


o 




M > 


© H 


1 > 


» > 


*< > 


> 




C/3 H 


- > 


> > 


>o 


> H 


H 




a> n 


-H 


i O 


i o 


~o 


H 




n H 


» H to 


« O to 


n > i-» 


p> O 


O 






> O <3 


OHh 


H>oi 


H H co 


O 03 






— O O 


«< o o 


=rOO 


r- > o 


>o 






• H 


» H 


1 > 


i > 


> 






O 


CO > 


H> 


O > 


O 






n O 


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-H 


> 


> 




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i 0 


i H 


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o 






r H 


r > 


r o 


O 


o 


oi r 




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»-H 


> 




c O 




c O 


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o 


to 




> O to 


r > to 


DO H 




o 




m > od 


v: > to 


1 O 05 


a o o 


o 






•o HO 


» OO 


o Oo 


t >o 


>o 






HH 


*T3 O 


H> 


o a 


> 






«< > 


T O 


sr O 


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> 






n H 


o 0 


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> 






oo 


r o 


> > 




o 






-> 


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n O 


»-o 


H 






C > 


c H 


ft O 


3 O 


H 






>o 


r O 


H> 


o 


o 






O to 


o H to 


=r O m 


O h 


H 






c O co 


c Hw 


n > «o 


>t- 


Hen 






HHO 


<0O 


>>o 


HO 


Ho 






*< > 


p H 


GO > 


H 


> 






i H 


- O 


3 H 


O 


> 






a o 


> > 


>> 


> 


O 






M-> 


to > 


0) J> 


O 


o 








8 O 


» H 


H 


> 






OH 


TJH 


OO 


O 








<< o 


=rH 


-G 


> 


> 






CD O 03 


o O ro 


Oh 


> H- 


H 






GOO 


r > * 


r > oo 


O to 


O O 






*- > o 


y > o 


*< > o 


>o 


>o 








c* 


•1 









26 



EP 0 416 578 B1 



F i & . ^ {6) 
pxbr22 (BMP2A) 

10 20 30 40 SO 60 70 80 90 100 

GAATTCTCTTCCCTCTCACCGGCCTCTCGTCTCTACTCACCTCCCGGCGACCCCGGCTGGACTGAGACACTCGCTGCCACTATGTGCGACAACTCACCGA 

110 120 130 140 150 160 170 180 190 200 

CTGGGCTCGACTGGACGCGCGGACTTGTCTCCCTCCTCTGGGGACCAGCGACTTGAACTAAAGACTCGAGTGATTGTGGAAAAAACACGCGGGGAGCAGA 

210 220 230 240 250 260 270 280 290 300 

AAACCCACATCGAGACACAAACTCGGCGACTAAATCGCTCAGGTTGACAATGGTCGCTGGGATCCACTCTCTGCTCCTGCTGCAGTTTTACCAGATCTTG 

HVAGI HS LLLLQFYQIl 

310 320 330 340 350 360 370 380 390 400 

CTGAGCGGCTGCACCGGGCTCGTCCCAGAGGAAGGCAAACGCAAGTATTCCGAAT.CCACTCGCTCGTCTCCGCAGCAGTCCCAACAAGTCCTCGACCAGT 
LSGCTGLVPEEGKRKYSES'TRSSPQOSQOVLDOF 

410 420 430 440 450 460 470 480 490 500 

TTGAGCTTCGGCTGCTCAATATGTTCGGCTTGAAGAGGAGGCCGACGCCTGGCAAAAATGTTGTGATCCCCCCCTACATGTTGGACTTGTACCACCTGCA 
E LR L LN HFGLKRRPTPGKNVVIPPYMIDLYHLH 

510 520 530 540 550 560 570 580 590 600 

CTCGGCTCAGTTGGCCGATGATCAAGGAAGTTCTGAGGTGGACTATCACATGGAGCGGGCGGCTAGCAGAGCCAACACAGTGAGGAGCTTTCACCATGAA 
S A GLADDQGSSEVDYHMERAASRANTVRSFHHE- 

610 620 630 640 650 660 670 680 690 700 

GAATCCATGGAAGAAATTCCAGAGTCTGGTGAGAAAACAATCCAACGATTCTTCTTCAACCTTTCTTCAATTCCAGATGAGGAGCTGGTCACGTCTTCTG 
ESHEEIPESGEKTIORFFFNLSSIPDEELVTSSE 

710 720 730 740 750 760 770 780 790 800 

AGCTCCGGATTTTTCGAGAGCAGGTCCAAGAGCCATTTAAGACTGACGGCAGCAAACnCATCGGATTAATATTTATGACATTGTCAAGCCAGCGGCGGC 
LRIFREQVQEPFKTDGSKLHRINI YDIVKPAAA 

810 820 830 840 850 860 870 880 890 900 

TGCCTCCCGGGGCCCTGTTGTAAGACTATTGGACACCAGACTGATCCATCATAATGAAAGCAAATGGGAAAGTTTTGAIGTGACGCCGGCAATTACACGG 
ASRGPVVRLLDTRLIHHNESKV.ESFDVTPA'ITR 

910 920 930 940 950 960 970 980 990 1000 

TGGATTGCACATAAACAGCCTAACCATGGGTTTGTTGTTGAAGTGACTCACTTGGACAATGACACAAATGTGCCCAAGAGGCATGTGAGGATTAGTAGGT 
VIAKKQPNH GFVVEVTHLDNDTNV PKRHVRISRS 

1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 
CTTTAACCCTGGATAAAGGTCACTGGCCTCGGATACGGCCATTATTGGTAACTITTAGCCATGATGGCAAAGGACATGCTCTTCACAAAAGACAAAAACG 
L'TLDKGHVPRIRPLLVTFSHDGKGHALHKRQKR 

1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 
GCAAGCTAGGCACAAACAACGTAAACGCC7TAAATCGAGCTGCAGGAGGCATCCGTTGTACGTAGATTTCAGTGACGTTGGTTGGAATGACTGGAT7GTT 
QARHKQRKRLKSSCRRHPL YVDFSOV GVNDVIV 

1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 
GCXCCACCTGGGTATCATGCCTTTTACTGCCACGGGGAAIGTCCn^ 

AP PGYHAFYCHGECPFPLADHLNSTNHAIVQTLV 

1310 1320 1330 1340 1350 1360 1370 1380 1390 1400 
TGAATTCCGTCAACACAAACATTCCCAAAGCnGC^ 
NS VNTNIPKACCVPTELSAISMLYLDENEKVVL 

1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 
AAAGAATTATCAAGACATGGTCGTGGAGGGGTGCGGGTGCCGTTAGGCGGGGACACACAAGCCAGAGACAAGAAAGCTGACACTTTAATATTTCCTTTTG 
KNYQDMVVEGCGCR* 

1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 
GAGACTATATTTATGCTTTGAAAAATGATGAAACAATTATTTTGAAAATATATTTAT<aTCTACACGGAGGCTGGGAAGCAAATATTTTAATCAGAGAAAT 

1610 1620 1630 1640 1650 1660 1670 1680 1690 1700 

ATTCCTTTTTAGTTGTACATTTTTATAAGGGTTTGTACCCAGCACATGAAGTATAATGGTCAGATTCCTATTTTGTATTTATTTACCATTATAACCACTT 

1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 

TTTAAGGAAAAAAATAGCTGTTTTGTATTTATATGTAATCAACAGAGAAAATATAGGGTTTGTAAATATGTTACTGAAAGTGTTTTTTTCTTCTTTTTTT 

1810 1820 1830 1840 1650 1860 1870 1880 1890 1900 
TAAATTATGTATACACAGCTGGTTATATGGCAAGTTTTTTATATTTTCTATAAAGCTAATTTCAAGGTCATTAGTTATAAACTTGATGATGTGTTGGTTC 

1910 1920 1930 1940 1950 1960 1970 1980 1990 
ATTGGTAAATCCTCCATATTGTGCAATTAACATGCATTTTTATAATGTACGAAGTCCAGTCCATTGTGCATTGCTTTGCAAATTTAGAATTC 



27 



EP 0 416 578 B1 



F i ff . 2 (7) 
P x b r 2 3 (BMP 2 8) 

10 20 30 40 50 60 70 80 90 100 

G6AATTCCGGCCCCACTGAGCTTTTCCACACATTTTTTGT6TCCAACATTGGCTGTCAAGAATCATGGAATGTTTTTCTATGCCTTGTTTTCTGTCAAGA 

110 120 130 140 150 160 170 180 190 200 

CATCATGATTCCTGGTAACCGAATGCTGATGGTCATTTTATTAAGCCAAGTCCTGCTCGGAGGCACTAACTATGCCAGCCTGATACCTGACACGGGCAAG 
MIPGNRHLHVILLSOVILGGTNYASLIPDTGK 

210 220 230 240 250 260 270 280 290 300 

AAGAAAGTCGCGGCCGACATTCAGGGAGGAGGTCGCAGGTCGCCTCAGAGCAATGAGCTCTTGCGGGATTTCGAGGTGACGCTGCTGCAGATGTTCGGAC 
KKVAADIQGGGRRSPOSNE LLRDFEVTLLQMFGL 

310 320 330 340 350 360 370 380 390 400 

TCCGCAAGCGGCCGCAGCCCAGTAAGGATGTGGTGGTTCCCGCTTATATGCGCGACCTGTACAGGCTTCAGTCAGCGGAGGAGGAGGATCAACTGCACGA 
RKRPQPSKDVVVPAYH RDLYR LQSAEEEDELHD 

410 420 430 440 450 460 470 480 490 500 

TATCAGCATGGAGTACCCCGAGACACCCACCAGCCGCGCCAACACCGTGAGGAGCTTCCATCACGAGGAACATTTGGAGAATCTACCAGGCACAGAAGAA 
I SMEYPEIPISRANTVRSFHHEEHLENLPGTE E 

510 520 530 540 550 560 570 580 590 600 

AATGGAAATTTCCGTTTTGTGTTCAACCTCAGCAGCATTCCAGAGAATGAGGT6ATTTCTTCAGCAGAACTGAGACTCTATAGAGAACAAATAGACCATG 
NGNFRFVFNLSSIPENEVISSAELRLYREQIDH G 

610 620 630 640 650 660 670 680 690 700 

GTCCAGCGTGGGATGAGGGTTTCCACCGGATAAATATATATGAAGTTATGAAACCCATCACAGCAAACGGACACATGATAAATAGGCTGCTGGACACGAG 
PAVDEGFHRINI YE VMKPITANGHHINRLLDTR 

710 720 730 740 750 760 770 780 790 800 

GGTAATCCACCACAATGTGACACAGTGGGAAAGTTTTGATGTAAGCCCTGCAATTATGAGGTGGACCCTGGATAAACAGATAAACCATGGGCTTGCCAIT 
VIHHNVTQVESFDVSPAIMRVTLDKQINHGLAI 

810 820 830 840 850 860 870 880 890 900 

GAGGTCATTCACCTCAACCAAACAAAAACTTATCAGGGGAAGCATGTAAGGATAAGTCGATCTTTATTACCTCAAAAGGATGCAGACTGGTCACAGATGA 
EVIHLNQTKIYQGKHVRISRSLLPQKDADVSQMR 

910 920 930 940 950 960 970 980 990 1000 

GACCACTTTTAATTACATTCAGCCATGATGGCAGGGGGCATGCACTGACTAGGAGGTCAAAAAGAAGTCCAAAACAGCAGAGACCCCGTAAAAAAAATAA 
PLLITFSHDGRGHALTRRSKRSPK QORPRKKNK 

1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 
ACACTGCCGGAGACATTCTCTTTATGTGGATTTCAGCGATGTGGGCTGGAATGATTGGATTGTGGCACCTCCTGGATACCAGGCCTTTTACTGCCATGGA 
HCRRHSLYVDFSDVGVNDVIVAPPGYQAFYCHG 

1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 
GATTGTCCATTTCCCTTGGCTGATCACCTAAACTCAACTAACCATGCTATTGTACAAACTCTGGTAAACTCTGTTAACTCAAGCATCCCAAAAGCATGCT 
DCPFPLADHINSTNHAIVQTLVNSVNSSIPKACC 

1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 
GCGTCCCCACAGAACTGAGTGCTATCTCCATGCTTTATTTGGATGAATATGACAAAGTCGTCCTTAAAAACTACCAGGAGATGGTGGTGGAAGGGTGTGG 
VPTELSAISHLYLDEYDKVVLKNYQEMVVEGCG 

1310 1320 1330 1340 1350 1360 1370 1380 1390 1400 
GTGCCGTTGAGTCTGAGATCCAAACAAAAGACTGTTAACGGCTGGACTICTTTCCACTGAACATTCACCTTGACCTTATTTATGACTTTTATGTGTAAAT 
C R * 

1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 
GTTTTTTTGACAATATGATCATATATTTTGACAAAATATATTTATAACTACGTATTAAAAGAAAAAAAAAAAATAAAATAAGTCATTATTTTAAACATAA 

1510 1520 1530 1540 1550 
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGGAATIC 



28 



EP 0 416 578 B1 



F" X . 12. 8 ) 
pxbr41 (Vjrl'i 

10 20 30 40 50 60 70 60 90 100 

GAATTCCGATATGGAATGTAAAAATACTGGTGAATTATGGGAAGTCCGACACAGACCACTAACTTC/GCATCTTATCTTTGACAAAATGAATGCTTTGAC 

H H A L T 

110 120 130 140 ISO 160 170 ISO 190 200 

AGTAAAGAGAAGATTGCCTGTGCTGCTTTTTCTTTTTCACATTTCACTGAGTTCCATCTCGTCAAATACAATATTGGAGAATGATTTCCACTCTAGTTTT 
VKRRLPVLLFLFHISLSSISSNTILENDFHSSF 

210 220 230 240 250 260 270 280 290 300 

GTCCAGAGAAGACTAAAAGGCCACGAACGCAGAGAGATTCAAAAAGAGATCTTGACTATTTTAGGXTTGCAACACAGACCAAGGCCATATTTACCGGAGA 
VGRRLKGHERREIOKEILTILGLQHRPRPYLPEK 

310 320 330 340 350 360 370 380 390 400 

AAAAGAAGTCTGCACCATTATTCATGATGGATTTATACAATGCAGTAAATATTGAAGAGATGCATGC7GAAGATGTTTCCTACAGCAATAAGCCGATCTC 
KKSAPLFMHDLYNAVNIEEHHAEDVSYSNKPIS 

410 420 430 440 450 460 470 480 490 500 

CCTAAATGAAGCTTTTTCACTGGCCACTGACCAAGAGAATGGCTTTCTTGCACATGCCGACACAGTTATGAGTTTTGCTAATTTAGTTGACAATGACAAC 
LNEAFSLATDO E N GFLAHADTVMSFANLVDNDN 

510 520 530 540 550 560 570 580 590 600 

GAATTGCATAAAAACTCCTATCGCCAAAAATTCAAGTTTGATCTAACTGATATCCCACTTGGAGATGAACTGACAGCCGCTGAATTTCGAATTTATAAAG 
EL H K NSYRQKFKFDLTDIPLGDELTAAEF R IYKD 

610 620 630 640 650 660 670 680 690 700 

ATTATGTACAAAATAACGAGACATACCAGGTCACCATCTACCAGGTGCTTAAGAAGCAAGCCGACAAA6ATCCTTATCTTTTCCAGGTAGACTCAAGAAC 
YVQNNETYQVTIYQVLKKQADKDPYLFQVDSRT 

710 720 730 740 750 760 770 780 790 800 

CATCTGGGGCACAGAAAAGGGATGGCTGACGTTTGATATTACTGCAACTGGTAATCACTGGGTGATGAACCCACATTACAACCT7GGATTGCAGTTATCA 
IVGTEKGVLTFDITATGNHVVHNPHYNLGLOLS 

810 820 830 840 850 860 870 680 890 900 

GTAGAGAGTATGGATATGCAAAATGTTAATCCCAGGCTTGTGGGCCTTGTTGGAAAGAATGGTCCTCAAGACAAACAGCCATTTATGGTGGCATTCTTTA 
VESNDNQNVKPRLVGLVGKNGPQDKQPFHVAFFK 

910 920 930 940 950 960 970 980 990 1000 

AGACCTCAGATATCCATCTCCGCAGTGTTCGATCTACTAGCAATAAGCACTGGAATCAGGAAAGAGCCAAGACCTACAAGGAGCAAGATAATT7ACCTCC 
TSDIHLRSVRSTSNKHVNQERAKTYKEQDNLPP 

1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 

AGCAAATATTACTGATGGCATCATGCCCCCTGGAAAACGTCGTTTTTTAAAGCAAGCTTGCAAGAAACATGAACTGTTTGTAAGTTTCCGCGATCTTGGT 
ANI TDGIKPPGRRRFLKQACKKH ELF VSFRDLG 

1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 
TGGCAAGACTGGATAATTGCACCTGAAGGATATGCTGCCTACTATTGTGATGGAGAATGTGCTTTCCCACTTAACTCTTTCATGAAT6CCACAAACCATG 
VQDUIIAPEGYAAYYCDGECAFPLNSFHNATNHA 

1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 
CCATTGTACAAACGTTGGTACATTTCATTAACCCAGAGACTGTCCCTAAGCCATGCTGTGCACCAACTCAGCTCAATGGTATTTCTGTTTTATACTTTGA 
I V QTLVHFINPETVPKPCCAPTQLNG ISVLYFD 

1310 1320 1330 1340 1350 1360 1370 1380 1390 1400 
TGACAGTGCCAATGTTATATTAAAGAAATACAAAAATATGGTGGTTCAAGCCTGTGGTTGCCATTGACAATAGCAGTTATTCTGTTTTTAACAGTCATTT 
DSANVILKKYKNNVVQACGCH* 

1410 1420 1430 1440 1450 1460 1470 1480 1490 2500 
TAATGGTATTGTCCTTATCGTTTATTTTAAAGTAGAGATACTTGACCATCACACTTAAAAAAATGCATTGTACACCTTAACGGATGAAAAGATTTTGTTT 

1510 

TTGCATGATTTCGGAATTC 



29 



EP 0 416 578 B1 



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• H H H ' * • ^ ' HxO50>>50©' = 
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S ' T CO 50 TJ r 

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H H H H * " * Z ' Z © * Z 70 

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30 



EP 0 416 578 B1 



F i e: . 4 - 1 
(VI) BMP2A 

W V A G I H S L L L L Q F Y 0 I L 
LSGC TGLVPEEGKRKYSESTRSS PQQSQQVLDQF 

ELRLLNMFGL KRRPTPGKNVVIPPYMLDLYHLH 
SAQL ADDQGSSE VDYHHERAASRANTVRSFHHE 
ESMEEIPESGEKTIQRFFFKLSSIPDEELVTSSE 

LRIFREOVOEPFKTDGSKLHRINIYDIVKP AAA 
ASRGPVVRLLDTRLIHHNESKVES FDVTPAITR 
VIAHKOPNHGFVVEVTHLDNDTN VPKRHVRISRS 

LTLDKGHVPRIRPLLVTFSHDGKGHA LHKROKR 
QARHK.QRKRLRSSCRRHPLYVDFSDVGUNDVIV 
APPGYHAFYCH GECPFPLADHLNSTNHAIVOTLV 
NSVNTNIPKACCVPTELSAISMLYLDEN EKVVL 
KNYQDHVVEGCGCR* 



31 



EP 0 416 578 B1 



F i er . 4 - 2 
(Vfl) BMP2B 

M IPGNRHLMVILLSOVLLGGTMYASLIPDTGK 
KKVAADIQGGGRR. SPQSNELLRDFEVTLLQHFGL 
RKRPGPSKDVVVPAYMRDLYRLQSAEEEDELHD 
ISHEYPETPTSRAN TVRSFHHEEHL.ENLPGTEE 
NGNFRFVFNLSSIPENEVISSAELRLYREQIDHG 
PAWDEGFHRINIYEVMKPITANGHMINRLLDTR 
VIHHNVTOVESFDVSPAIHRUTLOKQINHGLAI 
EVIHLNQTKTYQGKHVRISRSLLPQKDADfc? SQHR 
PLLITFSHDGRGHALIRRSKR-SPKQQRPRKKNK 
HCRRHS LYVDFSDVGWNDVIVAPP GYQAFYCHG 
DCPFPLADHLNST NHAIVOTLVKSVNSSIPKACC 
VPTELSAISHLYLDEYDKVVLKNYQEHVVEGCG 
C R * 



32 



EP 0 416 578 B1 



CVBE) (Vgrl) 

M N A L T 

VKRRLPVLLFLFHISLSSISSNTIL EN DFHSSF 
VQRRLK GHERREIQKEILTILGLQKRPRPYLPEK 
KKSAPLFHHDLYNAVN1EEMHAEDVS YSNKPIS 
LN EAFSLATDQE NGFLAHADTVMSFANLVDNDN 
ELHKNSYROKFKFDLTDIPLGDELTAAEFRIY KD 
YVQNNETYQVTIYQVLKKGADKDPYLFQVDSR7 
ItfGTEKGVLTFDITATGNHVVHNPH'YNLGLGLS 
VESHDMQNVNPRLVGLVGKNGPQDKQPFMVAFFK 
TSDI HLRSVRSTSNKHVNQERA KTYKEQDNLPP 
AKITDGIHPPGKRRFLKQACKKHELFV SFROLG 
YQDVIIAPEGYAAYYCDGECAFPLNSFMNAINHA 
IVQTLVHFINPETVPKPCCAPTQLNGISVLYFD 
DSANVILKKYRNMVVQACGCH* 



33 



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