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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) InternatiODal Patent Oassificatfon ^ : 
C12N 15/12, A61K 37/02, 9/22 



Al 



(11) International Publication Number: WO 94/01557 

(43) International Publication Date : 20 January 1 994 (20.0 1 .94) 



(21) International Application Number: PCT/JP93/00952 

(22) International Filing Date : 9 July 1 993 (09.07.93) 



(30) Priority data: 
4/206996 



13 July 1992(13.07,92) 



JP 



(71) Applicant (for all designated States except US): SUMITO- 

MO METAL INDUSTRIES.. LTD. {JP/JPJ; 5-33, Kita 
4-chome, Chuo-ku, Osaka-shi, Osaka-fu 541 (JP). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only) : KANGAWA, Kenji 
[JP/JP]; 50-D-12-104, Aoyamadai 3-chome, Suita-shi, 
Osaka-fu 565 (JP). HINO, Jun (JP/JPJ; 5-1-301, Nishi- 
midorigaoka l-chome, Toyonaka-shi, Osaka-fu 565 (JP). 
FUKUDA, Kenji [JP/JP]; 12-16, Konan 3.chome, Miya- 
zaki-shi, Miyazaki-ken 880 (JP). TAKAO, Makoto [JP/ 
JP]; 75-1-403, Kitaichi-cho, Nara-shi, Nara-ken 630 (JP). 
TAKESHITA, Norimatsu [JP/JP]; 75-1-304, Kitaichi- 
cho. Nara-shi, Nara-ken 630 (JP). KONNO. Yasuhiko 
[JP/JP]; 75-1-102. Kitaichi-cho. Nara-shi* Nara-ken 630 
(JP). 



(74) Agents: OGAWA, Nobuo et al.; Room 646. Shin-Tokyo 
BIdg., 3-1, Marunouchi 3-chome, Chiyoda-ku. Tokyo 
100 (JP). 



(81) Designated States: AU. CA, KR, US, European patent 
(AT, BE. CH. DE, DK. ES. FR, GB. GR, IE, IT. LU. 
MC, NL, PT. SE). 



Published 

With international search report. 



(54) Title: BONE FORMATION-INDUCING PROTEIN 



(57) Abstract 



Disclosed are a protein having a high activity for inducing bone formation. A DNA encoding the protein, a method for 
producing the protein and a pharmaceutical composition comprising the protein as an active ingredient. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identily States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Austria 


FR 


France 


MR 


Mauritania 


AU 


Australia 


CA 


Gabon 


MW 


Malawi 


BB 


Barbados 


GB 


United fGngdom 


NE 


Niger 


BE 


Bctg^uiD 


GN 


Guinea 


NL 


Netherlands 


BF 


Burkina Faso 


GR 


Greece 


NO 


Norway 


BC 


Bulgaria 


HU 


Hungary 


NZ 


New Zealand 


BJ 


Benin 


IE 


Ireland 


PL 


Poland 


BB 


Bratil 


IT 


Italy 


PT 


Portugal 


BY 


Belarus 


JP 


Japan 


RO 


Romania 


CA 


Canaila 


KP 


Democratic People's Republic 


RU 


Russian Federation 


CP 


Central African Republic 




of Korea 


SO 


Sudan 


CC 


Congo 


KR 


Republic of Korea 


SB 


Sweden 


CH 


Switzerland 


UZ 


Kazakhstan 


SI 


Slovenia 


CI 


C6te d'lvoire 


U 


Uecfalenttcin 


SK 


Slovak Republic 


CM 


Cameroon 


LK 


Sri Lanka 


SN 


Senegal 


CN 


China 


IV 


Luxembourg 


TO 


Chad 


cs 


Ceecho&lovakia 


LV 


Latvia 


TC 


Togo 


C2 


Ctech Republic 


MC 


Monaco 


UA 


Ukraine 


DE 


Germany 


MG 


Madagascar 


US 


United States of America 


DK 


Denmark 


ML 


Mali 


UZ 


Uzttektstan 


ES 


Spain 


MN 


Mongolia 


VN 


Viet Nam 


Fl 


Finland 











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Title of the Invention 

Bone format ion- inducing protein 

Field of the Invention 

The present invention relates to a hovel bone format ion- inducing 
protein, a DNA encoding the protein, a process for producing the 
protein and a pharmaceutical composition containing the protein as an 
active ingredient. In particular, the present invention relates to a 
novel bone formation-inducing protein which can be obtained by 
expression of a novel DNA derived from a vertebrate, a process for 
producing the protein which comprises culturing cells transformed by 
integrating the DNA thereinto, and a pharmaceutical composition for 
treating osteoporosis, bone deficiency such as partial deficiency of 
jawbone caused by alveolar pyorrhea, and bone fracture. 

Background of the Invention 

With the aging of the population, osteoporosis has become a 
social and medical problem. Hitherto, calcitonin, female hormones, 
active vitamin Ds. anabolic steroids and the like have been used for 
treating the disease. However, these agents cannot completely cure the 
disease; they can only alleviate the disease but not form bone. When 
vitamin Ds is used for treatment of the disease, adverse effect such as 
hyperka lures is become a problem. 

Therefore, there is no effective therapy for osteoporosis. This 
can also be said of alveolar pyorrhea because the teeth deciduation 
caused by it is due to a partial deficiency of bone and the disease 
tends to afflict the aged. 



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It can be seen from the above that osteoporosis and alveolar 
pyorrhea can be completely cured if a bone formation is induced at the 
bone-lost site, since these diseases are due to the deficiency of bone. 
Also in curing a fracture, the induction of bone formation produces 
several advantages, such as shortening of the time required for healing 
and for the rehabilitation subsequent to the healing, and reduction of 
medical expense. 

On the other hand. Urist et al established an experiment system 
for an ectopic bone morphogenesis using a bone matrix decalcified with 0. 
6 N hydrochloric acid (Science. ]50^ 893-899 (1965)). Subsequently, 
they experimentally demonstrated that this bone morphogenetic 
phenomenon is caused by a protein firmly attached to collagen in a 
calcified tissue and they called the protein a bone morphogenetic 
protein (BMP). Up to now. it has been reported that BMP can be 
extracted from an osteosarcoma or decalcified bone or teeth by using 4M 
guanidine hydrochloride or 6M urea as an extracting solvent and be 
purified using an ion exchange or gel filtration technique IProc. Natl. 
Acad. Sci.. USA. 85. 9484-9488 (1988): J. Bio. Chem.. 264^ 13377- 
13380 (1989): J. Bio. Chem,. 265^ 13198-13205 (1990)]. Some reports 
have been made on DNA sequences encoding BMP. For example. Japanese 
Unexamined Patent Publication for International Application 
(hereinafter referred to as "J. P. KOHYO") No. Hei 2-500241. J. P. KOHYO 
No. Hei 3-503649 and Japanese Unexamined Patent Publication (hereinafter 
referred to as 'J. P. KOKAI") No. Hei 3-195495 each describes such DNA 
sequence. 

However, the bone morphogenetic activity of BMP either purified 
in the manner as described above or encoded by the DNA sequence is 
insufficient as a therapeutic agent for the above-mentioned bone-lost 
diseases. Therefore, it is desired to develop a new protein having an 

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improved bone formation inducing-activity. 

Suniiiiary of the Invention 

The object of the present invention is to provide a novel bone 
formation-inducing protein having an improved activity. 

Another object of the present invention is to provide a DNA 
which encodes the novel bone formation-inducing protein. 

Another object of the present invention is to provide a process 
for producing the novel bone formation-inducing protein. 

Another object of the present invention is to provide a 
Pharmaceutical composition containing the novel bone format ion- inducing 
protein as an active ingredient. 

These and other objects of the present invention will be 
apparent from the following description and Examples. 

The above objects were achieved based on the discovery that a 
certain mRNA which encodes a protein having the improved bone formation 
inducing-activity exists in bone of vertebrates. 

The first aspect of the present invention relates to a protein 
comprising amino acid sequence of amino acids 1 to 110 in SEQ. ID No:l 
(hereinafter referred to as BIP as occasion demands) or analogous 
sequences thereto. The protein of amino acids 1 to 110 in SEQ. ID No:l 
is one of the maturation proteins and the protein of amino acids -368 to 
110 in SEQ. ID No:l is a precursor protein thereof. 

The second aspect of the present invention relates to a DNA 
which encodes the bone format ion- inducing protein of the present 
invention. The DNA can be obtained by isolating a mRNA from a tissue 
of a vertebrate such as human and rat. constructing a cDNA library with 
the isolated mRNA and then screening the cDNA library using a certain 
probe, which is amplified by carrying out PCR using primers designed by 

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referring to cDNA of the prior art. to obtain clones of interest. 

The third aspect of the present invention relates to a method 
for producing the protein. The method comprises (a) transforming a cell 
with the DNA and (b) culturing the transformant. 

The fourth aspect of the present invention relates to a 
pharmaceutical composition containing the bone format ion- inducing 
protein or active fragment thereof as an active ingredient. The bone 
formation-inducing protein of the present invention is useful for 
therapy of a disease involving a deficiency of bone such as osteoporosis, 
alveolar pyorrhea and the like, and bone fracture. 

Brief Description of the Drawings 

Fig.l illustrates the technique for constructing the expression 
vector for the human bone format ion- inducing protein of the present 
invention (hBIP). 

Fig. 2 shows the restriction map of the expression vector for the 
human bone formation-inducing protein of the present invention. 

Fig. 3 illustrates the technique for constructing the expression 
vector for the rat bone formation-inducing protein of the present 
invention (rBIP). 

Fig. 4 shows the restriction map of the expression vector for the 
rat bone formation-inducing protein of the present invention. 

Figs. 5 to 10 show the histological observations of the pellets 
12 days after they were implanted. 

Figs. IIA to HE show the autoradiograms of Northern blot 
analysis for poly(A)RNAs extracted from various tissues of rat. 

Fig. 12 illustrates the technique for making the original amino 
acid sequence of process site of the precursor protein of the present 
invention correspond to the consensus sequence. 

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Fig. 13 illustrates the method for constructing the stable 
expression vector for the human bone format ion- inducing protein of the 
present invention. 

Detailed Description of the invention 

The present invention includes a protein comprising amino acid 
sequence of amino acids 1 to 110 in SEQ. ID No:l or analogous sequences 
thereto. The protein of amino acids 1 to 110 in SEQ. ID No:l is one of 
the maturation proteins and the protein of amino acids -368 to 110 in 
SEQ. ID No:l is a precursor protein thereof. 

"Analogous sequences thereto' means amino acid sequences which 
are substantially homologous with the amino acid sequence of amino 
acids 1 to 110 in SEQ. ID No:l and constitute proteins having a bone 
formation-inducing activity as high as that of the protein comprising 
the amino acid sequence of amino acids 1 to 110 in SEQ. ID No:l. There 
is at least one difference between the amino acid sequence of amino 
acids 1 to 110 in SEQ. ID No:l and such analogous sequences in number or 
kind of amino acid contained in the sequences. Examples of the 
difference include those caused by a replacement, an insertion or a 
deletion of at least one amino acid. The proteins comprising these 
analogous sequence are also maturation proteins. The amino acid 
sequence of amino acids 1 to 110 in SEQ ID No:3 may be mentioned as 
example of the analogous sequence to the amino acid sequence of amino 
acids 1 to 110 in SEQ. ID No:l. There are two differences between the 
two amino acid sequences in kind of amino acid contained in the 
sequences. 

"Protein comprising amino acid sequence of amino acids 1 to 110" 
means a protein having a longer amino acid sequence than amino acids 1 
to 110 in SEQ. ID No:l and maintaining the bone formation inducing- 

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activity. For example, the following proteins are included: the 
precursor protein of amino acids -368 to 110 in SEQ. ID No:l. proteins 
analogous thereto such as a protein in which any region of amino acids 
-368 to -1 in SEQ. ID No:l is replaced with another sequence, and 
proteins cleaved from the precursor protein of amino acids -368 to 110 
at any position upstream of amino acid 1 in SEQ. ID No:l. in particular, 
at amino acid -1 or -2 in SEQ. ID No:l. 

The protein of the present invention needs no further structual 
feature insofar as it has the above-mentioned amino acid sequence. 
Therefore, the proteins of the present invention may include any 
modified proteins such as a glycosylated protain. a dimerized protain. 
a glycosylated and dimerized protain and a mixture of these. 

The present invention also includes a DNA which encodes the bone 
format ion- inducing protein of the present invention. Examples of the 
DNA include DNAs comprising base sequence of nucleotides 1191 to 1520 
in SEQ. ID No:l and analogous sequences thereto. DNAs of which the base 
sequences are only a little analogous to the DNAs comprising base 
sequence of nucleotides 1191 to 1520 in SEQ. ID No: I but are capable of 
encoding the bone format ion- inducing protein of the present invention. 
Examples of the DNA sequence of nucleotides 1191 to 1520 in SEQ. ID No:l 
include DNA sequence of nucleotides 87 to 1520 in SEQ. ID No:l. which is 
one encoding the precursor protein of amino acids -368 to 110 in SEQ. ID 
No:l mentioned above. 

"Analogous sequences thereto" means base sequences which are 
substantially homologous with the base sequence of nucleotides 1191 to 
1520 in SEQ. ID No:l. There is at least one difference between the base 
sequence of nucleotides 1191 to 1520 in SEQ. ID No:l and such analogous 
sequences in number or kind of codon contained in the sequences. 
Examples of the difference include those caused by a replacement, an 

" - 6 - 



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insertion or a deletion of at least one codon. 

"DMAs of which the base sequences are only a little analogous to 
the DNAs comprising base sequence of nucleotides 1191 to 1520 in SEQ. ID 
No:l but are capable of encoding the bone formation-inducing protein of 
the present invention" means those which have not few different codons 
from the base sequence of nucleotides 1191 to 1520 in SEQ. ID No:l but 
can consequently express the bone formation-inducing protein of the 
present invention. Of course, it is possible to synthesize such DNA by 
chaining codons which each encodes an amino acid necessary to the bone 
formation-inducing protein of the present invention. 

It is needless to say that a DNA subjected to any alteration 
only in coding region for polypeptide to be released from the pr-ecursor 
protein during processing can encode the bone formation-inducing 
protein of the present invention and therefore the DNA is included in 
the present invention. 

A detailed description will now be made on the method for 
producing the bone format ion- inducing protein and coding DNA of the 
present invention. 

For example, the coding DNA for the bone formation-inducing 
protein of the present invention can be obtained as follows: 

Initially, a mRNA which is a template of the cDNA of the present 
invention is extracted from a tissue of a vertebrate and then isolated. 

The vertebrate may be a mammal. Examples of the mammar include human, 
rat and bovine. In particular, human and rat. such as neonatal rat. 
are preferred. Examples of the usable tissue include a femur, the head 
of a femur, a calvaria and the like. Preferred are head of femur of 
human and calvaria and femur of neonatal rat. However, the tissue is 
not limited to those mentioned here. The mRNA extraction and isolation 
can be carried out using a conventional technique. After the mRNA 



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isolation, a cDNA library is constructed using the isolated mRNA. The 
construction can also be carried out using a conventional technique. 

On the other hand, a DNA probe is amplified for screening the 
target clones present in the cDNA library on the basis of 
oligonucleotide primers. The oligonucleotide primers can be designed by 
referring to the cDNA encoding the bone morphogenetic protein of the 
prior art. 

After labeling the probe thus obtained, screening of the above- 
mentioned CDNA library is carried out using the labeled probe to obtain 
hybridized clones. When this probe is used, not only clones 
corresponding to the cDNA of the present invention but also clones 
corresponding to DNAs of the prior art hybridize. However, the clones 
of the present invention hybridize with the probe more weakly than those 
of the prior art. Therefore, the clones of the present invention can 
be separated from clones of the prior art by comparing the signal 
strength of the hybridization in the Southern method. 

Once the cDNA sequence of the present invention is determined, 
the cDNA can be easily synthesized by referring to the sequence. 

The bone formation-inducing protein of the present invention can 
be obtained by constructing an expression vector by integrating the 
above-mentioned cDNA thereinto, subjecting the expression vector to a 
transformation or transfection of a host cell, culturing the host cell, 
and isolating the protein secreted within the host cell or into the 
culture medium. It is also possible to secrete the protein in milk of 
a transgenic animal, such as goat or bovine, which is constructed using 
a DNA containing the above-mentioned cDNA of the present invention under 
a control of casein promoter and the like. 

The expression vector usable for the pr-esent invention may be 
composed of one or more DNAs dirived from plasmid. virus and phage into 

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which the cDNA of the present invention can be inserted and which is can 
be introduced into the host cell. The expression vector should contain 
a transcription promoter sequence, an enhancer sequence and/or operator 
sequence which control the transcription, a suitable ribosome binding 
site sequence, and a sequence for termination of the transcription and 
the translation. According to circumstances, the expression vector may 
contain a DNA sequence necessary for replicating itself in the host 
cells or a dehydrofolate reductase (dhfr) gene enabling the introduced 
DNA to amplify in the presence of methotrexate which is an inhibitor of 
dhfr. Examples of the expression vector in the case where an animal 
cell is used as the host cell include pcDL-SR a296 (Mol. Cell. Biol. 8^ 
466. 1988). pCDM8 (B. Seed. Nature 329j_ 840. 1987). pMAM neo (F. Ue et 
al.. Nature 294^ 228. 1981). BCMG neo (Karasuyama et al.. Eur. J. 
Immuno..l8. 97. 1988) and pSV2 dhfr (F. Lee et al.. supra ). pcDL-SR a 
296 and pSV2 dhfr are preferable. Further, insect cells such as pAc373 
(G. E. Smith et al.. Proc. Natl. Acad. Sci. USA 82. 8404. 1985). 
yeasts such as pAM80 (Miyanohara et al.. Proc. Natl. Acad. Sci. USA 80. 
1. 1983) and E. coli such as pTrc99A (E. Amann et al.. Gene 69. 301. 
1988) can be used as the expression vector. These expression vectors 
may be modified if necessary. 

Examples of the suitable host cells for expression of the bone 
formation-inducing protein of the present invention include COS-1 cell. 
Verots cell. CHO cell, mouse C127 cell, human 293 cell. Syrian hamster 
BHK cell, human Naraalwa cell and monkey Vero cell. Preferable host 
cells are COS-1 cell, human 293 cell and Syrian hamster BHK cell. 
Although the medium used for transformation or transfection of the host 
cell varies depending on the host cell used. DMEM containing fetal 
bovine serum. aMEM. Haml2. RPMI-1640 and the like are usable. DMEM is 
preferable for COS-1 cell and aMEM is preferable for CHO cell. 

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Preferable combinations of expression vector and the host cell 
are. for example. pcOL-SRa296 vector /COS-1 cell in case of a transient 
expression and pSV2 dhfr vector/Syrian hamster BHK cell in case of a 
stable expression. 

After being subjected to transformation or transfection. the 
host cells are allowed to culture under the condition suitable for 
expressing the protein. If the protein is secreted in the medium, it 
may be directly purified from the medium by removing the host cells. On 
the other hand, if the protein is accumulated within the host cells, it 
is isolated from cell lysates. 

in the biological synthesis of the bone formation-inducing 
protein of the present invention, its precursor protein, which is not 
active, is formed in a host cell in the first place and subsequently 
processed to the maturation protein, which is active, at its process 
site with a protease. It is desirable for the amino acid sequence of 
this process site of the precursor protein to be efficiently cleaved 
with the protease in order to produce the maturation protein of the 
present invention to a high degree. The fact that many known precursor 
proteins have amino acid sequence of Arg-X-Arg/Lys-Arg (X representing 
an essential amino acid) as process site has been known and Nakayama et 
al showed that this sequence is a consensus sequence (Nakayama et al. J. 
Biol. Chem. 267. 16335-16345 (1992)). The precursor protein is cleaved 
at C-terminus of the last Arg in this sequence and this processing with 
the protease should be efficient. 

On the other hand, it is considered that the process site of the 
precursor protein of the human bone formation-inducing protein of the 
present invention has the amino acid sequence of Ala-Arg-Arg-Lys which 
corresponds to amino acids -4 to -1 in SEQ. ID Nd:l. However, this 
sequence is different from the above-mentioned consensus sequence. 

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Therefore, it is preferable to make the original amino acid sequence 
correspond to the above-mentioned consensus sequence in order to produce 
the human bone formation-inducing protein of the present invention 
efficiently. For this, it is possible to employ, for example, a 
technique in which an original DNA sequence encoding an amino acid 
sequence containing the process site of the precursor protein of the 
present invention (corresponding to nucleotides 1179 to 1190 in SEQ. ID 
No:l) is replaced with a DNA sequence designed to encode an amino acid 
sequence containing the consensus sequence. For example, a synthesized 
DNA containing a base sequence which encodes the process sequence of 
huDian BMP-2 type (Arg-Glu-Lys-Arg)(J.P. KOHYO No. Hei 2-500241) or human 
proactivin A type (Arg-Arg-Arg-Arg)(D. Huylebroeck et al.. Mol. 
Endocrynol. 4. 1153 (1990)) can be employed for this technique in order 
to make the process sequence of the human bone formation-inducing 
protein of the present invention correspond to the consensus sequence, 
since the process sequences of human BMP-2 type and human proactivin A 
type correspond to the consensus sequence. 

When a stably-producing cell is constructed using the thus- 
replaced DNA and a suitable host cell, it is possible to produce the 
human bone formation-inducing protein of the present invention more 
efficiently. Examples of host cells suitable for constructing the 
stably-producing mutant include human 293 cell. Syrian hamster BHK cell, 
human Namalwa cell, monkey Vero cell and the like. 

Further, the present invention includes a pharmaceutical 
composition containing the bone format ion- inducing protein or active 
fragment thereof obtained in the manner as described above as an active 
ingredient. 

The composition is useful for therapy of a disease involving 
osteoporosis, a bone deficiency such as alveolar pyorrhea and the like. 

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and bone fracture. 

The pharmaceutical composition of the present invention may be 
administered through various routes, for example, orally or by 
injection or by implantation in a bone-lost site and. for this purpose, 
it is usually prepared in the form of. for example, a suitable 
formulation for oral administration .injection or implantation. 
Particularly, the injection may be prepared in the form of a formulation 
suitable to reach to the bone-lost site and. in case of the 
implantation, the active ingredient may be implanted in the bone-lost 
site with a matrix. Examples of the suitable formulations for oral 
administration include powder, granule, tablet, capsule, solution for 
internal use, emulsion or suspension. 

The bone formation-inducing protein of the present invention may 
be administered alone or in the form of a mixture with one or more 
pharmaceutical ly-acceptable carriers suitable for the formulation 
concerned. These formulations can be prepared using conventional 
techniques for preparing a pharmaceutical composition. For example, 
they may be prepared by dissolving, emulsifying or suspending an 
ingredient or ingredients thereof in a suitable solvent. 

Examples of carriers suitable for the powder, the granule, the 
tablet, the capsule and the like include excipients such as lactose, 
glucose. D-mannitol. starch, crystalline cellulose, calcium -carbonate, 
kaolin and the like; binding agents such as starch paste solution, 
gelatin solution, hydroxypropyl cellulose, hydroxypropyl methyl 
cellulose, polyvinylpyrrolidone, ethanol and the like: disintegrators 
such as starch, gelatin powder, carboxymethyl cellulose, carboxyraethyl 
cellulose calcium salt and the like: lubricants such as magnesium 
stearate. talc and the like: and coating agents such as hydroxypropyl 
methyl cellulose, acetyl cellulose, white sugar, titanium oxide and the 

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like. Coloring agents, flavoring agents and the like can be optionally 
added to these formulations. 

Examples of carriers suitable for the solution for internal use 
include preservatives such as benzoic acid, esters of p-hydroxy benzoic 
acid, dehydroacetic acid sodium salt and the like: suspending agents 
such as gum arable, tragacanth. carboxymethyl cellulose sodium salt, 
methyl cellulose, egg yolk, surfactants and the like: edulcorants such 
as white sugar, syrup, citric acid and the like. Coloring agents, 
stabilizers and the like can be optionally added to the solution. The 
solvent used for the solution is mainly purified water. However, 
ethanol. glycerin, propylene glycol and the like are usable. 

Examples of the solvents suitable for the injection include 
distilled water, water for injection, non-aqueous solvents such as 
ethanol. glycerin, propylene glycol, macrogol and the like. Examples of 
carriers suitable for the injection include buffers such as sodium 
monohydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate 
and the like: isotonic agents such as glucose, sodium chloride and the 
like: preservatives such as phenol, thiroerosal. esters of p-hydroxy 
benzoic acid and the like: stabilizers such as sodium hydrogen sulfite 
and the like. Painkillers, solubilizers and the like can be optionally 
added to the injection. 

The matrix for the implantation can be either a biologically- 
absorbable or biological ly-unabsorbable one. Examples of biologically- 
absorbable matrices include non-biological substances such as 
hydroxyapatite. polyClactic acid), calcium sulfate and the like: and 
biological substances such as type 1 collagen, bone^nd the like. €n 
the other hand, examples of biological ly-unabsorbable matrices include 
ceramics, titanium and the like. The active ingredient can be 
implanted with one or more these matrices. 



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Generally, the injection is mainly applied to treat osteoporosis 
and. as described above, the implantation in a bone-lost site is applied 
to treat alveolar pyorrhea or fracture. However, oral administration 
and injection can also be applied to treat alveolar pyorrhea or bone 
fracture in addition to the implantation. 

The dose of the active ingredient contained in the pharmaceutical 
composition of the present invention varies widely depending on the 
administration route, type of formulation, kind of disease, age and sex 
of the patient, and the like. However, in general, it is 0.01 to 100 
mg/day for adult. When the active ingredient is implanted in the bone- 
lost site in the form of a mixture with collagen, the mixing ratio of 
the active ingredient to collagen is 4X10"* to 4 xiQ-'wtl preferably 
4 xio-« to 4 xio-^wt^. more preferably 4xio-* to 4 x\r'vit%. The 
amount of the mixture to be implanted can be suitably determined by the 
physician according to the severity of the disease. 

By the present invention, prevention and complete cure of 
osteoporosis and the teeth deciduation caused by alveolar pyorrhea 
become possible and the time required for healing a fracture can be 
shortened. 

The following examples are given to further illustrate the 
present invention but are not meant in any way to restrict the effective 
scope of the invention. 

Example 1 

Isolation of cDNA encoding the human bone formation-inducing protein 
Preparation of probe 

The following primers: 

Primer 1 5' -A€CCATCAAATCATCCTACC-3' 

Primer 2 5' -TCTGCAAGCGCAAGACTCTA-3' 

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were synthesized using an automatic DNA synthesizer and then a DNA 
fragment was amplified using 250nM of Primer 1 and 250nM of Primer 2 
from lue/m ul of human placenta chromosomal DNA by the PCR method in 
the following solution for 25 cycles: 
Solution for PCR: 

lOniM Tris-hydrochloride (pH 8.3). 50mM KCl. 1.5raM MgCh. 0.00^ 
gelatin. 200//M dATP. 200 uU dCTP. 200 uU dCTP. 200 uU dTTP. 
2.5 units AmpliTaq CPerkin Elmer Cetus) 
Condition for PCR: 

94r for 1 minute. 37 'C for 2 minutes and 72 'C for 3 minutes 
After the reactions, the DNAs amplified were electrophor^sed on 
agarose gel to recover a DNA fragment having about ISObp using the glass 
adsorption method. This fragment was ligated with EcoRI adapter 
(Pharmacia) using T4 DNA ligase in the conventional manner and then the 
resulting fragment was ligated to the recognition site for restriction 
enzyme EcoRI of plasmid BlueScriptIISK+ (Stratagene). A competent 
cells of Lcoll 109) prepared by the method of Inoue et al (Inoue 
et al.. Gene. 96. 23. 1990) were transformed with this reaction 
solution. 

The resulting transformant was cultured in LB medium and then 
the plasmid DNA was isolated from lysate of the transformant by the 
alkali-SDS method (Birnboim et al.. Nucleic Acids Res.. 7. 1513. 1979). 
Subsequently, the plasmid DNA was subjected to removal of RNA present 
therein by RNaseA treatment and deproteinized by the PEG 
(polyethyleneglycol) precipitation method and the phenol extraction 
method and then reprecipitated by adding ethanol. The sequence of the 
Plasmid DNA thus obtained was determined by the dideoxynucleotide 
method with T7 DNA polymerase (Sanger et al.. Proc. Natl. Acad. Sci. 
USA.. 74. 5463. 1977) after denaturing the DNA by the method of Hattori 

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et al (Hattori et al.. Analytical Biochemistry 15Z 232. 1986). The 
base sequence is shown in SBQ ID No: 2. 

Next, this plasmid DNA was digested by restriction enzyme EcoRI 
and the digest was electrophoresed on 1% agarose gel to recover a 
fragment having about 180bp using the glass adsorption method. This 
fragment was used as the probe. 
Constructio n of cDNA library of human bone tisR.iP 

l.Smg of total RNA was isolated from 42g of head of human femur 
by the acid guanidine tiocyanate/phenol/chloroform extraction method 
(Analytical Biochemistry 162, 156. 1987) and. from l.Snig of the RNA 
thus isolated. poly(A)RNA was purified using oligo-dT latex (Nippon 
Roche). Double stranded DNA were synthesized from 5 ui oi the poly(A) 
RNA according to the method of Gubler and Hoffman. After ligating this 
CDNA with EcoRI adapter using T4 DNA ligase. the ligated cONA was 
electrophoresed on 1% agarose gel to obtain a fraction having 2 to 5kb 
through an extraction. These extracted cDNA were ligated to the 
recognition site of restriction enzyme BcoRl of lambda phage AgtlOarm 
(Murray et al.. Mol. gen. Genet.. 15a 53. 1977)(Bethesda laboratory) 
and then an in vitro packaging (Collins et al.. Proc. Natl. Acad. Sci. 
USA.. 71. 4242. 1978) was conducted to construct the cDNA library. 
Cloning of the cDNA 

Lcoli CeoOfhl (DNA cloning. L 56. 1985) was infected with 
recombinant phage contained in the above cDNA library and then 
1.000.000 of the resulting plaques were fixed onto a nitrocellulose 
filter (Benton et al.. Science. 196, 180. I«77). The filter was 
Prehybridized for 20 hours at 37 in a hybridization solution (20% 
formamide. 6xSSPE. 0.1% SDS. 100 u%Ml salmon sperm DNA) and then was 
hybridized with the above-prepared probe, which was labeled with («- 
'''P)dCTP using a multi prime- labeling kit (Amarsham). at 42 "C in the 

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above hybridization solution. Subsequently, this filter was washed in 2 
xSSC. 0.1^ SDS solution at 50"C and 16 positive clones were obtained by 
autoradiography. The positive clones were purified in the conventional 
manner and DNAs thereof were prepared. These DMA fragments, which were 
inserted into the recombinant phage DNA. were identified to be those of 
interest by the method of Southern (J. Mol. Biol.. 98. 503. 1975) using 
hybridization with the above probe. Base sequences of these DNA were 
determined to be identical with each other and the clones were 
designated as HE24. 25 and 27. respectively. 
Determination of base sequence of the cDNA 

The recombinant phage DNA thus-obtained was digested by 
restriction enzyme NotI and then the digest was electrophoresed on 
agarose gel to isolate the DNA fragment. After purification by the 
glass adsorption method, the purified DNA fragment was inserted into 
the NotI site of plasmid BlueScriptnSK+ to subclone. The E.coli 
containing HE24 was deposited with the Fermentation Research Institute. 
Agency of Industrial Science and Technology on July 3, 1992 (Acceptance 
No. PERM P- 13045). 

The partial base sequence of this cDNA was determined by the 
dideoxynucleotide method (Sanger et al.. supra ) and shown in SEQ. ID 
No:l together with the amino acid sequence deduced therefrom. 

From these results, it can be seen that the human bone 
formation-inducing protein is contained in the amino acid sequence of 
478 amino acids encoded by the base sequence of nucleotides 87 to 1520 
in SEQ. ID No:l. That is. the protein of the 478 amino acids was 
biologically synthesized in the form of a precursor protein, which 
includes a secretion signal, and then subjected to processing at the 
process site of amino acid -2. -1 or 1 in SEQ. ID No:l to form the 
maturation proteins having 112. Ill or 110 amino acids, respectively. 

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Example 2 

Isolation of cDNA encoding the rat bone formation-inducing protein 
Constructio n of cDNA library of rat bone tissup 

5.8ing of total RNA was isolated from 2g of femur of neonatal rat 
by the acid guanidine tiocyanate/phenol /chloroform extraction method 
(Analytical Biochemistry 162, 156. 1987) and poly(A)RNA was purified 
from 2mg of the isolated RNA using oligo-dT latex (Nippon Roche). A 
double stranded DNA was synthesized from 5 of the poly(A)RNA 
according to the method of Gubler and Hoffman. After ligating this cDNA 
with EcoRI adapter using T4 ligase. the ligated cDNA was 
electrophoresed on 1% agarose gel to obtain a fraction having 1.2 to 5kb 
through an extraction. This extracted cDNA was ligated with the 
recognition site of restriction enzyme EcoRI of lambda phage A gtlOarm 
(Murray et al.. Mol. gen. Genet.. 150, 53. 1977) (fie thesda laboratory) 
and then an in vitro packaging (Collins et al.. Proc. Natl. Acad. Sci. 
USA.. 71. 4242. 1978) was conducted to construct the cDNA library. 
Cloning of the cDNA 

Leon CeoOfhl (DNA L 56. 1985) was infected with recombinant 
Phage contained in the above cDNA library and then 1..O00.000 of the 
resulting plaques were fixed onto nitrocellulose filters (Benton €t al 
Science. 196,180.1977). The filters were prehybridized at 37r in a 
hybridization solution m formamide. 6xSSPE. 5 xDenhardfs. 0.5% SDS. 
100 ug/ml salmon sperm DNA) and then were hybridized with the probe 
obtained in Example 1. which was labeled with (a-»^P)dCTP using 
multiprime-labeling kit (Amarsham). at 42 'C in the above hybridization 
solution. Subsequently, after these filters were washed with 2 xSSC. 0. 
1% SDS solution at 50'C and 24 positive clones were obtained through 
autoradiography. The positive clones were purified in the conventional 

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manner and DNAs thereof were prepared. These DNA fragments, which were 
inserted into the recombinant phage, were identified by the method of 
Southern hybridization (J. Mol. Biol.. 98. 503. 1975) using the above 
probe. Base sequences of these DNA were determined to be identical 
with each other and the clones were designated as RB12. 45 and RC27. 
respectively. 

Determination of base sequence of the cDNA 

The recombinant phage DNA thus-obtained was digested by 
restriction enzyme Not I and then the digest was electrophoresed on 
agarose gel to isolate the DNA fragment. After purification by the 
glass adsorption method, the purified DNA fragment was inserted into 
the NotI site of plasmid BlueScriptnSK+ to subclone. The E. coli 
carrying RB45 was deposited with Fermentation Research Institute, 
Agency of Industrial Science and Technology on July 3. 1992 (Acceptance 
No. PERM P-13046). 

The base sequence of this cDNA was determined by the 
dideoxynucleotide method (Sanger et al.. supra ) and shown in SEQ 10 
No: 3 together with the amino acid sequence deduced therefrom. 

From these results, it can be seen that the rat bone formation- 
inducing protein is composed of 476 amino acids encoded by the base 
sequence of nucleotides 60 to 1487 in SEQ. ID No:3. The protein of the 
476 amino acids was further subjected to processing to form the 
maturation protein having 112. Ill or 110 amino acids, which is encoded 
by the base sequence of nucleotides 1152 to 1487. 1155 to 1487 or 1158 
to 1487 in SEQ. ID No: 3. 

Example 3 

Expression of the human bone formation-inducing protein in COS-1 cell 
To demonstrate that the human bone formation-inducing protein of 

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the present invention has bone formation-inducing activity, the cDNA 
obtained in Example 1 was ligated with a transient expression vector and 
the ligated vector was introduced into COS-1 cells (Gluzman: Cell. 23. 
175. 1981) to express the protein. For the protein secreted in the 
supernatant of the medium used, the activity was measured. 
Construction of the expression vector 

PcDL-SRD obtained by modifying pcDL-SRa296 reported by Takebe 
et al (Takebe et al.. Mol. Cell. Biol.. 8(1) 466. 1988) was used as the 
transient expression vector. Namely, this vector was obtained by 
digesting vector pcDL-SR a296 by restriction enzymes Kpnl and Pstl. 
blunting with DMA polymerase I and inserting EcoRI linker thereinto. 

First, non-coding regions were removed from the cDNA coding for 
the human bone formation-inducing protein of the present invention by 
the method illustrated in Fig.l. I„ detail. HE24 was digested by 
restriction enzyme Nco! to remove the non-coding region present on the 
5- -side thereof and. after blunting of the terminus of the digest using 
DNA polymerase I. the digest was further digested by restriction enzyme 
Sail to isolate NcoA-Sall fragment which contained the initiation 
codon and was a fragment present on the 5' -side. 

On the other hand. HE24 was also digested by restriction enzymes 
Sail and Kpnl to isolate Sal-Kpn fragment. 

These two DNA fragments were ligated to the vector which was 
obtained by digesting BlueScriptIISK+ by restriction enzyme Hindlll. 
blunting the terminus thereof using DNA polymerase 1 and then digesting 
again by restriction enzyme Kpnl. The resulting plasmid was digested by 
restriction enzymes EcoRV and Kpnl to isolate EV-Kpn fragment. 
Next, the following two primers for PCR: 

C-1 5*-GATATCTCACCGGCAGGCACAGGTC-3' 
C-2 5--TCCCCGAGGTACCTGAAGGT-3' 



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were synthesized in order to amplify a cDNA region corresponding to C- 
terminus of the protein of the present invention. 

The PCR reaction was carried out on 1 uU of Primer C-1 and 1 u 
M of Primer C-2 using HE24 as a template in the following solution for 
32 cycles: 
Solution for PCR 

lOmM Tris-hydrochloride (pH 8.3). 50mM KCl. l.SmMMgCls. 0.0015K 

gelatin. 200/zM dATP. 200 uU dCTP. 200 uU dGTP. 200 dTTP. 

5 units AmpliTaq CPerkin Elmer Cetus), 500pg HE 24 plasmid 

DNA/50 u\ 
Condition for PCR 

92'C for 1 minute. 50 'C for 2 minutes and 72 "C for 3 minutes 

After the reaction, the DNAs were electrophoresed to separate 
them from the reaction solution and then the DNA fragment of interest 
was isolated and purified using the glass adsorption method. 

From the fragment thus-obtained. KpnI-EcoRV <Kpn-EV) fragment, 
which is DNA corresponding to the C-terminus side, was isolated and 
iigated to T-vector in which dTTP was attached to the 3' -terminus of 
the recognition site for restriction enzyme EcoRV of plasmid Blue 
ScriptIISK+ and then the base sequence thereof was determined in the 
same manner as in Example 1. 

The resulting plasmid was digested by restriction enzymes Kpnl 
and EcoRV to isolate the Kpn-EV fragment. This fragment was Iigated 
with the above-isolated EV-Kpn fragment and BlueScriptnSK+ which was 
digested by restriction enzyme EcoRV and then treated with alkaline 
phosphatase to dephosphorize. As a result, a cDNA was obtained, in 
which all of the coding regions remained and no non-coding region 
remained. 

This cDNA was isolated from the BlueScriptllSKVEV by digesting 

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by restriction enzyme EcoRV and then ligated to the blunted terminus of 
the EcoRI recognition site of the above-mentioned pcDL-SRD vector to 
construct an expression vector for the human bone formation-inducing 
protein (SRD-hBIP). A restriction map of the plasmid thus-obtained is 
shown in Fig. 2. 

Transfectio n with the expression vector for the human bone formation - 
inducing protein and production of the protein 

According to the method of Sambrook et al (Sambrook et al.. 
Molecular Cloning: Spring Habor Laboratory Press. 16. 41. 1989). the 
vector SRD-hBIP thus-obtained was transfected into COS-1 cells to 
produce the human bone format ion- inducing protein. 

A lOOram culture dish containing medium (10ml of DMEM containing 
10 v/\% fetal calf serum) was inoculated with 1 xio« of COS-1 cells, 
incubated at 37 "C for 18 hours with CO2 gassing and then washed with 
10ml of phosphate buffered saline (PBS(-)) twice. To this. 2u% of 
mixture of SRD-hBIP DNA and DEAE dextran was added. After incubating 
the cells at room temperature for 15 minutes. 5ml of DMEM containing 10 
fetal calf serum and 100 //M of chloroquine was added thereto. 
After culturing the cells at 37"C for 3.5 hours with ^% CO2 fassing. 
the medium was removed and then 900 /zl of 10 \/v% DMEM containing fetal 
calf serum and 10 v/v% dimethyl sulfoxide (DMSO) was added thereto. 
The cells were incubated at room temperature for 2 minutes without 55K CO2 
gassing, washed with 5ml of PBS(-) and 5ml of DMEM containing 10% fetal 
calf serum in this order and then 10ml of DMEM/105K fetal calf serum was 
added thereto. After incubating the cells at 3rc for 24 hours with 5% 
CO2 gassing, the medium was removed and then 10ml of DMEM containing 2% 
fetal calf serum was added thereto to further incubate at 37'C with 5^ 
CO2 gassing. After 5 days, the cells were removed from the medium by 
centrifugation at 10.000 rpra at 4'C for 10 minutes to obtain th€ 

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conditioned medium. 

To l,400ml of the conditioned medium, urea and Tris- 
hydrochloride (pH 7.0) was added to yield a final concentration of 6M 
urea and 50mM Tris-hydrochloride (pH 7.0). The mixture was loaded to a 
Heparin-Sepharose column (20ml, Pharmacia) preliminarily equilibrated 
with 6M urea/50mM Tris-hydrochloride (pH 7.0). The column was washed 
with the same buffer, whereafter the elution was carried out with 30ml 
of the same buffer but containing 0.5M sodium chloride. The eluate was 
concentrated to about 5ml by ultrafiltration using a centricon. 
dialyzed against deionized water and freeze-dried. Subsequently, the 
freeze-dried proteins were dissolved in 1% sodium deoxycholate/50mM 
Tris-hydrochloride buffer (pH 8.0) and then the solution was loaded to 
a ConA-Sepharose column (200 At 1. Pharmacia) preliminarily equilibrated 
with 1% sodium deoxycholate/50mM Tris-hydrochloride buffer. The column 
was washed with the same buffer, whereafter the elution was carried out 
with 1ml of the same buffer but containing 0.5M methyl- a-D-mannoside. 

As a control, SRD-hBlP vector not containing the cDNA encoding 
the human bone format ion- inducing protein was transfected into COS-1 
cells and treated in the same manner as described above. 
Assay for the bone formation-inducing activity 

The bone formation-inducing activities were measured for the 
above-described fractions, i.e., ConA-bound fraction and ConA-unbound 
fraction by the method of Sarapath et al (Sampath et al., Proc. Natl. 
Acad. Sci. USA., 80, 6591. 1983). 

First, a matrix residue was prepared from a rat-decalcifi«d bone 
by removing the bone formation-inducing active ingredient with 4M 
guanidine hydrochloride, then a pellet was prepared by copr^cipitating 
25mg of the matrix residue and the protein sample contained in the ConA- 
bound fraction. Subsequently, this pellet was implanted subcutaneously 

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in the abdominothoracic part of a rat 21-28 days old and removed from 
the rat after 12 days. For this pellet, alkaline phosphatase activity 
and amount of calcium therein were measured. These procedures were 
repeated also on the ConA-bound fraction of the control. These results 
are shown in Table 1 below. 



Table 1 



Sample 


Alkaline phosphatase activity 
unit/implant 


Calcium 
mg/implant 


ConA-bound fraction 


27.5 


9.0 


ConA-unbound fraction 


0.76 


0.24 


ConA-bound fraction 
(Control) 


2.98 


0.0 



As can be seen from Table 1. the ConA-bound fraction containing 
the protein of the present invention exhibited very high bone formation- 
inducing activity in comparison with the control. It is evident from 
these data that the protein of the present invention has high bone 
formation-inducing activity. 



Example 4 

Expression of the human bone formation-inducing protein in 293T cell 
To demonstrate that the human bone format ion- inducing protein of 
the present invention expressed in 293T (J. Gen. Virology, 36. 59-72 
(1977)) cell has bone formation-inducing activity, the transient 
expression vector constructed in Example 3 was introduced into 293T 
cells to produce the protein. For the protein secreted in the 
supernatant of the medium used, the activity was measured. 
Transfection with the expression vector for the human bone formation - 
inducing protein and production of the protein 

The transient expression vector constructed in Example 3 (SRD- 

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hBIP) was transfected into 293T cells to produce the human bone 
formation-inducing protein. 

A 100ml culture dish containing medium (10ml of MEM containing 
10 \/\% fetal calf serum) was inoculated with ixio* of 293T cells, 
incubated at 37'C for 18 hours with BJK COj gassing and then 20 //g of a 
precipitate of calcium phosphate which was prepared using the SRD-hBIP 
constructed in Example 3 was added thereto. After incubating the cells 
for 18 hours with 5% CDs gassing, the medium was removed. 10ml of MEM 
containing 2 v/\% fetal calf serum was added thereto and then the cells 
was incubated at 37 'C with 5« CO2 gassing. After 3 days, the cells 
were removed from the medium by centrifugation at 10.000 rpm at 4'C for 
10 minutes to obtain the conditioned medium. 

To 1.400ml of the conditioned medium.- urea and Tris- 
hydrochloride (pH 7.0) was added to yield a final concentration of 6M 
urea and 50n4I Tris-hydrochloride (pH 7.0). The mixture was loaded to a 
Heparin-Sepharose column (20ml. Pharmacia) preliminarily equilibrated 
with 6M urea/50mM Tris-hydrochloride (pH 7.0). The column was washed 
with the same buffer, whereafter the elution was carried out with 30ml 
of the same buffer but containing 0.5M sodium chloride. The eluate was 
concentrated to about 5ml by ultrafiltration using a centricon. 
dialyzed against deionized water and then freeze-dried. Subsequently, 
the freeze-dried proteins were dissolved in 1% sodium deoxycholate/50raM 
Tris-hydrochloride buffer (pH 8.0) and then the solution was loaded to a 
ConA-Sepharose column (200 u\. Pharmacia) preliminarily equilibrated 
with \% sodium deoxycholate/50mM Tris-hydrochloride buffer. The column 
was washed with the same buffer, whereafter the elution was carried out 
with 1ml of the same buffer but containing 0.5M methyl- a -D-mannoside. 

As a control, pcDL-SRD vector not containing the cDNA encoding 
the human bone formation-inducing protein was transfected into 293T 

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cells and treated in the same manner as described above. 
Assay for the bone format ion- inducing activity 

The bone formation-inducing activities were measured in the same 
manner as Example 3. 

Namely, a pellet was prepared by coprecipitating 25mg of the 
matrix residue and the protein sample obtained above, then implanted 
subcutaneously in the abdominothoracic part of a rat 21-28 days old. 
After 12 days, the pellet was removed from the rat and alkaline 
phosphatase activity and amount of calcium therein were measured. These 
procedures were repeated also on the ConA-bound fraction of the control. 
The results are shown in Table 2 below. 



Table 2 



Sample 


Alkaline phosphatase activity 
unit/implant 


Calcium 
iQg/implant 


ConA-bound fraction 


10.3 


0.94 


ConA-unbound fraction 


0.4 


0.04 


ConA-bound fraction 
(Control) 


2.24 


0.25 


ConA-unbound fraction 
(Control) 


0.33 


0.0 



As can be seen from Table 2. the ConA-bound fraction containing 
the protein of the present invention exhibited very high bone formation- 
inducing activity in comparison with the control. It is evident from 
this data that the protein of the present invention expressed by 293T 
cells also has a high bone format ion- inducing activity. 



Example 5 

Expression of the rat bone formation-inducing protein in COS-1 cell 
To demonstrate that the rat bone formation-inducing protein of 

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the present invention has bone formation-inducing activity, the cDNA 
obtained in Example 2 was ligated to a transient expression vector and 
the ligated vector was introduced into COS-1 cells to produce the 
protein. For the protein secreted in the conditioned medium, the 
activity was measured. 
Construction of the expression vector 

Similarly to in Example 3. pcDL-SRD obtained by modifying pcDL- 
SR a296 reported by Takebe et al was used as the transient expression 
vector. 

Namely, this vector was obtained by digesting v€ctor pcDL-SRa 
296 by restriction enzymes Kpnl and Pst I. blunting with DNA polymerase I 
and inserting EcoRI linker thereinto. 

Initially, this vector was digested by restriction enzyme EcoRI, 
blunted using DNA polymerase I and then treated with alkaline 
phosphatase to dephosphorize. 

On the other hand, non-coding regions of the cDNA coding for the 
bone formation-inducing protein of the present invention were removed 
by the method illustrated in Fig. 3. 

First, the following two sets of primers for PCR. which 
correspond to the N-terminus (ATG; initiation codon) and the C-terminus 
(TAA: stop codon) of the protein of the present invention respectively, 
were synthesized: 
N-terrainus primer 

N-1 5' -GATATCATGGCTCCAGGTCTTGC-3' 
N-2 5* -GCACGGAAGCTTCGGAeG-3' 
C-terminus primer 

C-1 5' -GATATCTTACCGACAGGCACAGGT-3' 
C-2 5' -CCAGGAGGTACCTGAAGG-3' 
The PCR reactions were carried out using luU of Primer N-1 and 

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1 fiU of Primer N-2 using RB45 as a template in the following solution 
for 30 cycles to amplify 5' -terminus DNA fragment: 
Solution for PCR: 

lOmM Tris-hydrochloride (pH 8.3). 50mM KCl. 1.5mM MgCh. 0.0015K 

gelatin. 200 /iM dATP. 200 uU dCTP. 200 uli dGTP. 200 uU dTTP. 

5 units AmpliTaq (Perkin Elmer Cetus). 500pg RBplasmid DNA/50/zl 
Condition for PCR: 

94 "C for 1 minute. 45 "C for 2 minutes and 72 'C for 2 minutes 

After the reactions, the DNAs were electrophoresed on agarose 
gel to separate them from the reaction solution and then DNA fragment of 
interest was isolated and purified using the glass adsorption method. 

On the other hand. 3' -terminus DNA fragment was amplified using 
1 uU of Primer C-1 and 1 fiU of Primer C-2 under the same reaction 
condition as the 5' -terminus DNA fragment, and the DNA fragment was 
isolated and purified in the same manner as the 5' -terminus DNA fragment. 

The thus-obtained EcoRV-Hindlll (EV-H3) fragment, which is DNA 
on the 5' "terminus side, and KpnI-EcoRV (Kpn-EV) fragment, which is DNA 
on the 3' -terminus side, were individually ligated to T-vector 
described in Example 2 and then the base sequence thereof was determined 
in the same manner as Example 1. 

The plasmid DNA ligated with the EV-H3 fragment, which is DNA on 
the 5' -terminus side, was digested by restriction enzymes EcoRI and 
Hindi n to isolate the EV-H3 fragment, then the isolated EV-H3 fragment 
was inserted into BlueScriptSKlIi plasmid between restriction enzyme 
recognition sites EcoRV and Kpnl together with Hindlll-Kpnl (H3-Kpn) 
fragment isolated by digesting RB45 with restriction enzymes Hindlll 
and Kpnl. Further, the plasmid DNA thus-obtained was digested by 
restriction enzymes EcoRV and Kpnl to isolate EV-Kpn fragment. 

On the other hand, the plasmid DNA ligated with the Kpn-EV 

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fragment was digested by restriction enzymes Kpnl and EcoRV to isolate 
Kpn-EV fragment. 

Both fragments thus-obtained were ligated to the restriction 
enzyme EcoRI recognition site of the above-mentioned pcDL-SRD vector, 
the terminus of which had been blunted, to construct an expression 
vector for the bone format ion- inducing protein. A restriction map of 
the plasmid thus-obtained is shown in Pig. 4. 

Transfection with the expression vector for the rat bone formation - 
inducing protein and production of the protein 

The vector thus-obtained was transfected into COS-1 cells to 
produce the rat bone formation-inducing protein. 

A lOOn culture dish containing medium (10ml of DMBU containing 
10 v/v5K fetal calf serum) was inoculated with 1 xio* of COS-1 cells, 
incubated at 37 *C for 18 hours with 5% C02 gassing and then washed with 
10ml of phosphate buffered saline (PBS(-)) twice. To this, 2ui of 
mixture of the above-mentioned vector and DEAE dextran was added. 
After incubating the cells at room temperature for 15 minutes, 5ml of 
DMEM containing 10 vM fetal calf serum and \00uU of chloroquine was 
added thereto. After culturing the cells at 37'C for 3.5 hours with 55K 
C02 gassing, the medium was removed and then 900 At 1 of 10 v/\% DUEM 
containing fetal calf serum and 10% dimethyl sulfoxide (DMSO) was added 
thereto. The cells were incubated at room temperature for 2 minutes 
without CO: gassing, washed with 5ml of PBS(-) and 5ml of DUEM 
containing 10 v/v% fetal calf serum in this order and then 10ml of DMEM 
containing 10 v/v% fetal calf serum was added thereto. After incubating 
the cells at 37'C for 24 hours with 5% CO2 gassing, the medium was 
removed and then 10ml of DMEM containing 2% fetal calf serum was added 
thereto to further incubate at 37*C with 5% CO2 gassing. After 5 days, 
the cells were removed from the medium by centrifugation at 10,000 rpm 

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at 4V for 10 minutes to obtain the conditioned medium. 

To 700ml of the conditioned medium, urea and Tris-hydrochloride 
(pH 7.0) was added to yield a final concentration of 6M urea and 50mM 
Tris-hydrochloride (pH 7.0). The mixture was loaded to a Heparin- 
Sepharose column (20mL Pharmacia) preliminarily equilibrated with 6M 
urea/50mM Tris-hydrochloride (pH 7.0). The column was washed with the 
same buffer, whereafter the elution was carried out with 30ml of the 
same buffer but containing 0.5M sodium chloride. The eluate was 
concentrated to about 5ral by ultrafiltration using a centricon, 
dialyzed against deionized water, freeze-dried and then dissolved in 1% 
sodium deoxycholate/50mM Tris-hydrochloride buffer (pH 8.0), 
Subsequently, this solution was loaded to a ConA-Sepharose column (200 
III, Pharmacia) preliminarily equilibrated with said buffer. The column 
was washed with the same buffer, whereafter the elution was carried out 
with 1ml of the same buffer but containing 0.5M methyl- a -D-mannoside. 

As a control, pcDL-SRD vector not containing the cDNA encoding 
the human bone formation-inducing protein was transf^cted into COS-1 
cells and treated in the same manner as described above. 
Assay for the bone format ion- inducing activity 

The bone formation-inducing activities were measured for the 
above-described fractions, i.e., ConA-bound fraction and ConA-unbound 
fraction in the same manner as Example 3. 

First, a pellet was prepared by coprecipitating 25mg of the 
matrix residue, which was prepared from a rat-decalcified bone by 
removing the bone formation-inducing active ingredient with 4M €uanidine 
hydrochloride, and the obtained protein. The pellet was then implanted 
subcutaneously in the abdominothoracic part of a rat 21-28 days old. 
After 12 days, the pellet was removed from the rat and the alkaline 
phosphatase activity and amount of calcium therein were measured. 

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These procedures were repeated also on the control. These results are 
shown in Table 3 below. 



Table 3 



Sample 


Alkaline phosphatase activity 
unit/implant 


Calcium 
mg/implant 


ConA-bound fraction 


23.9 


8.9 


ConA-unbound fraction 


0.81 


0.06 


Control 


1.25 


0.13 



As can be seen from Table 3. the ConA-bound fraction containing 
the protein of the present invention exhibited very high bone formation- 
inducing activity in comparison with the control. It is evident from 
these data that the protein of the present invention has high bone 
formation-inducing activity. 

Figs. 5 to 10 show the histological observations of the pellets 
12 days after they were implanted. Pigs. 5 to 7 are for the pellet 
prepared from the rat bone formation-inducing protein contained in the 
ConA-adsorbed fraction, which was partially purified, and the matrix 
residue of rat bone. Pigs. 8 to 10 are for the pellet prepared from the 
protein contained in the supernatant of t^e culture medium for the 
control, which was partially purified with Heparin-Sepharose. and the 
matrix residue of rat bone. Figs. 5 and 8 are microphotographs ( xi50) 
of slices of the pellet stained with henatoxylin and eosin. Figs. 6 and 
9 are microphotographs (x200) of slices stained with PAS and alcian 
blue, and Pigs. 7 and 10 are microphotographs ( x.200) of slices stained 
with von Kossa. 

Fig. 5 shows that the interstitial cells markedly proliferated in 
the area among the implanted collagen matrix particles. In this area, 
osteoclasts and fibloblasts can be also observed and. around the matrix 

- 3 1 - 



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particles, cells which are considered to be osteoblasts can be also 
observed. Further, a proliferation of cartilage matrix containing 
chondrocyte can be observed. On the other hand, in the collagen matrix, 
it can be observed that a marked calcification occurred. The 
calcification can be observed in the cartilage matrix also. 

In Fig. 6, the cartilage cells stained with alcian blue are 
observed. Although the cells were weakly stained, there are strongly- 
stained cells scattered among the weakly-stained ones. Also, calcified 
substance stained with PAS can be observed. 

From Fig. 7. it is observed that the calcified substance is 
present in the collagen matrix and the cartilage matrix. 

On the other hand. Figs. 8 to 10 show that the cells scarcely 
proliferated in the area among the implanted collagen matrix particles. 

It is considered that the cells present in this area are only 
fibloblasts. No cartilage matrix, osteoclast, osteoblast or calcified 
substance is observed. 

Thus, it is also evident from the above figures that the protein 
of the present invention has an ability of inducing the bone formation. 

Example 6 

Expression distribution of mRNA of the rat bone format ion- inducing 
protein 

Poly(A)RNAs were extracted from various tissues of rat and the 
expression distribution thereof for mRNA of the rat bone formation- 
inducing protein was determined by the Northern hybridization method. 

First, RNAs were individually extracted cerebellum, costa, 
costal cartilage, trachea, blood vessel. spUen, thymus, muscle and 
bone marrow of SD rats 10-15 weeks old from and femur and calvaria of 
neonatal SD rats by the acid guanidine tiocyanate/phenol/chloroform 

-32- 



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extraction method and poIy(A)RNAs were extracted therefrom respectively 
with oligo-dT latex. 

lOuZ of every poly(A)RNAs was denatured in IH glyoxal/5051) 
dimethylsulfoxide/lOmM sodium phosphate buffer (pH 7.0) at 50*C for 1 
hour, then subjected to 1^ agarose gel (lOmM sodium phosphate buffer 
(pH 7.0)) electrophoresis. RNAs of known molecular weights (GIBCO BRL) 
and treated in the same manner as described above were used as markers. 

Next, the poly(A)RNAs thus-isolated were transferred from the 
agarose gel to a nylon membrane (High bond N. Amarsham). The filter was 
prehybridized at A2'C for 3 hours in a hybridization solution (40^ 
formamide. 6xSSPE. 5 xDenhardt's. 0A% SDS. 100 jug/ml salmon sperm 
DMA) and then was hybridized with a rat bone formation-inducing protein 
probe, which was labeled with ( a-"P)dCTP by a multiprime-labeling kit. 
at 42 'C for 20 hours in the above hybridization solution. The rat bone 
formation-inducing protein probe was a DNA fragment having a total 
length of 2.2 kb of cDNA for the rat bone formation-inducing protein 
which was produced by digestion of plasmid RB12 with restriction enzyme 
Not I. Subsequently, after washing this filter in 0. IxSSC. 0.\% SDS 
solution at 50*C. an autoradiography of Northern blot analysis was 
conducted to determine the expression distribution and amount of mKNA 
of the rat bone formation-inducing protein for every tissue on the 
basis of signal intensity. 

In carrying out this autoradiography, hybridizations were 
carried out also for BifP-2 and BMP-3 using rat BMP-2 cDNA and rat BMP-3 
cDNA as probes to determine the expression distribution and amount 
thereof. BMP-2 and BMP-3 are bone morphogenetic proteins described in 
J. P. KOHYO Nos. Hei 2-500241 and Hei 3-503649. 



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Preparation of probe for rat BMP-2 

DNA probe (5' -ACTAATCATGCCATTGTTCAGACGTTGGTCAACTCTGTTAAC-3' ) 
was synthesized based on the base sequence of the human BMP-2 cDNA 
described in J. P. KOHYO No. Hei 2-500241. The human BiiP-2 cDNA was 
cloned from a human placenta cDNA library using this probe. 
Subsequently, using a cDNA fragment containing the whole coding 
region for human ffllP-2 as a probe, rat BMP-2 cDNA was cloned from a 
rat placenta cDNA library. The thus-obtained cDNA fragment 
containing the whole coding region for rat BMP-2 was used as a 
probe for the Northern hybridization. 
Preparation of probe for rat BMP-3 

Primer DNA was synthesized based on the base sequence of the 
human BMP-3 cDNA described in J. P. KOHYO No. Hei 2-500241. Using 
this primer, a DNA fragment containing ISObp of maturation region 
was amplified from the human placenta chromosome DNA by the PGR 
method according to the method described in Examples 1 and 2 and 
then subcloned into plasmid BlueScriptllSK-i-. Using this DNA 
fragment as a probe, rat BMP-3 cDNA was cloned from a rat femur 
cDNA library. The thus-obtained cDNA fragment containing whole rat 
BMP-3 cDNA was used as a probe for the Northern hybridization. 
Pigs. IIA to HE show the autoradiograms thus obtained. Pigs. IIA 
and IIB are for the bone formation-inducing protein of the present 
invention (BIP). Fig. 110 is for BMP-2 and Figs.llD and HE are for BMP-3. 
Lanes A to K are the autoradiograms for the following tissues: 



A 



cerebellum 



mature SD rat 



B 



costa 



ditto 



C 



costal cartilage 



ditto 



D 



trachea 



ditto 



E 



blood vessel 



ditto 



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PCr/JP93/00952 



F spleen ditto 

G thymus ditto 

H muscle ditto 

I bone marrow ditto 

J femur neonatal SD rat 

K calvaria ditto 



It can be seen from Figs. IIA and IIB that the mRNAs for the rat 
bone formation-inducing protein of the present invention are 4. Ikb and 2. 
8kb in size and distributed to femur (lane J) and calvaria (lane K) of 
the neonatal SD rats and cerebellum (lane A), costal cartilage (lane C). 
trachea (lane D) and blood vessel (lane E) of the oatured rat in high 
density. 

On the other hand. Fig.llC shows that rat BMP-2 mRNAs are 3.8kb 
and 2.5kb and Figs. IID and HE show that rat BlfP-S mRNAs are 6.7kb. 
4. 7kb and 2. 6kb in size. Thus, the mRNAs for the rat bone formation- 
inducing protein of the present invention are different from those of 
rat BMP-2 and -3 in size. This means that the gene of the bone 
formation-inducing protein of the present invention is different from 
that of either m-2 or -3. 

The expression distribution data also indicate that there is a 
great difference between the rat BMPs and the rat bone formation- 
inducing protein of the present invention. That is, the rat HIIP-2 mRNAs 
are distributed to trachea (lane D) in high density but to cerebellum 
(lane A), costal cartilage (lane C) and blood vessel (lane E) in low 
density, and also the rat BMP-3 mRNAs are distributed to trachea (lane 
D) in high density but to cerebellum (lane A) and costal cartilage 
(lane C) in low level. 

Thus, the rat bone format ion- inducing protein of the present 
invention is distinctly different from BMP-2 and -3. Because of the 

-3 5- 



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fact that the mRNAs for the bone formation-inducing protein of the 
present invention distribute to cerebellum and costal cartilage in high 
density, it can be expected that the protein would play an important 
part in the nervous system and chondrogenesis. 

Exanple 7 

Replacement of the process site of the human bone formation-inducing 
protein with the consensus sequence 

In order to cleave the maturation protein from the precursor 
protein of the present invention efficiently, the following original 
DNA sequence encoding the amino acid sequence containing the process 
sequence of the precursor protein: 

5' -TG CAG AAA GCC CGG AGG AAG CAG TGG GAT GAG CCG AGG GTG TGC TCC 
3' -C TTT CGG GCC TCC TTC GTC ACC CTA CTC GGC TCC CAC ACG AGG 
Glu Lys Ala Arg Arg Lys Gin Trp Asp Gin Pro Arg Val Cys Ser 
CGG AGG TAC-3' 
GCC TC-5' 
Arg Arg Tyr 

which is shown in SEQ. ID No:l as nucleotides 1171 to 1226 will be 
replaced with the following synthesized DNA containing the consensus 
sequence as described above: 
(Human BMP-2 type) 

5' -TG CAC AAA CGC GAG AAG AGG CAG TGG GAT GAG CCG AGG GTG TGC TCC 
3' -G nr GCG CTC nC TCC GTC ACC CTA CTC GGC TCC CAC ACG AGG 
His Lys Arg Gin Lys Arg 
CGG AGG TAC-3' 
GCC TC-5* 

First, as shown in Fig. 12. SK+/H3 A.Kpn will be digested by 
restriction enzymes Bbsl and Kpnl and then li gated with the synthesized 

-3 6- 



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PCT/JP93/009S2 



DNA to obtain the modified SK+/H3A.Kpn. Subsequently, the modified 
SK+/H3A,Kpn will be digested by restriction enzymes EcoRV and Kpnl to 
obtain the EcoRV-Kpnl fragment containing the precursor region and the 
modified portion. 

On the other hand, SK+/EV as shovsfn in Fig. 12 will be digested by 
restriction enzymes Kpnl and EcoRV to obtain the KpnI-EcoRV fragment 
containing the C-terminus portion of the maturation region of the human 
bone format ion- inducing protein of the present invention. 

These two DNA fragments will be inserted into the restriction 
enzyme EcoRV recognition site of BlueScriptI 1SK+. Through these 
procedures, the base sequence corresponding to the process site of the 
human bone formation-inducing protein of the present invention, which is 
contained in EcoRV fragment in the plasmid DNA thus obtained, will be 
replaced with that corresponding to the consensus sequence. 

Also, the following synthesized DNA will be used for this 
technique: 

(Human proactivin A type) 

5' -TG CAG AAA CGC COG AGO AGG CAG TGG GAT GAG CCG AGG GTG TGC TCC 
3' -C TTT GCG CCC TCC TCC GTC ACC CTA CTC €GC TCC CAC ACG AGG 
Gin Lys Arg Arg Arg Arg 
CGG AGG TAC-3' 
GCC TC-5' 

Fig. 12 illustrates the above procedures. 
Example 8 

Establishment of the cell producing the human bone formation-inducing 
protein 

A stably-producing cell was established using human 293 cell as 
host cell. 

"37" 



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Construction of stable expression vector 

Restriction enzyme EcoRV recognition site of vector pEVMT was 
digested by restriction enzyme EcoRV. whereafter the 5' terminus thereof 
was dephosphorized with bacterial alkaline phosphatase. To this DNA. a 
DMA fragment isolated by digesting the plasmid BlueScriptllSKVEV. which 
was prepared in Example 3. by restriction enzyme EcoRV was ligated 
using T4 DNA ligase to construct a stable expression vector pEVMT(hBIP). 

The same procedures were repeated using vector pEVCMV to 
construct a stable expression vector pEVCMV(hBlP). 
Transformation of human 293 cell and isolation of stably-producing cell 

Calcium phosphate coprecipitates with 20 ug of the stable 
expression vector pEVMT(hBIP) DNA and 2 jt/g of pSV2neo were prepared 
according to the method of Chen et al (C. Chen & H. Okayama. Mol. Cell 
Biol., 7^ 12745-12752 (1987)). These coprecipitate were added dropwise 
to a 100mm culture dish which had been inoculated with 2 xio* of human 
293 cells per 1ml of medium (90JK MEM and 103! fetal calf serum) and 
incubated at 37 'C for one evening with 5% CO2 gassing. After keeping 
this temperature for one evening, the cells were ripped off from the 
dish using 0.0253; trypsin/0. 015K EDTA and then diluted 1:10 with a 
selection medium i%% MEM + 10% fetal calf serum + Smg of €418). The 
diluted cells were inoculated in ten lOOmra dishes, and then incubated in 
the same medium while freshening the medium until visible colonies were 
formed. 

Each colony thus formed was subjected to a magnification culture 
in a medium and then measured for amount of the human bone formation- 
inducing protein secreted in the conditioned medium by an enzyme 
immunoassay method (EIA) to isolate a producing cells for the protein 
in large quantities. 

The same procedures were repeated using the stable expression 

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vector pEVCMV(hBIP) to isolate a producing cells for the protein in 
large quantities. 

Fig. 13 illustrates the above procedures. 
If the resulting cells are gradually domesticated to a high 
concentration of methotrexate (MTX) in the medium, a highly-expressing 
clones will be isolated. 



-39- 



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SEQUENCE LISTING 

SEQ. ID NO: 1 

SEQUENCE LENGTH: 1560 nucleotides 

SEQUH>ICE TYPE: nucleotide with corresponding protain 

STRANDEDNESS: double 

TOPOLOGY: linear 

MOLECULE TYPE: cDNA to mRNA 

ORGANISM: human 

GCCGCGAGGT CAGTCCGCAG CCTCCGGTGC GCCAGCGCTC GCCTTCCTCC TCCTGGACTT 60 
CGGCCCTTTG CCGCCCTCAC CACGCC ATG GCT CAT GTC CCC GCT CGG ACC AGC 113 



Met Ala His Val Pro Ala Arg Thr Ser 
-365 -360 



CCG GGA CCC GGG CCC 


CAG 


CTG 


CTG CTG CTG 


CTG 


CTG CCG TTG TTT 


CTG 


161 


Pro Gly Pro Gly Pro 


Gin 


Leu 


Leu Leu Leu 


Leu 


Leu Pro Leu Phe 


Leu 




-355 






-350 




-345 






CTG TTG CTC CGG GAT 


GTG 


GCC 


GGC AGC CAC 


AGG 


GCC CCC GCC TGG 


TCC 


209 


Leu Leu Leu Arg Asp 


Val 


Ala 


Gly Ser His 


Arg 


Ala Pro Ala Trp 


Ser 




-340 






-335 




-330 






GCA CTG CCC GCG GCC 


GCC 


GAC 


GGC CTG CAG 


GGG 


GAC AGG GAT CTC 


CAG 


257 


Ala Leu Pro Ala Ala 


Ala 


Asp 


Gly Leu Gin 


Gly 


Asp Arg Asp Leu 


Gin 




-325 






-320 




-315 






CGG CAC OCT GGG GAC 


GCG 


GCC 


GCC ACG TTG 


GGC 


CCC AGC GCC CAG 


GAC 


305 


Arg His Pro Gly Asp 


Ala 


Ala 


Ala Thr Leu 


Gly 


Pro Ser Ala Gin 


Asp 




-310 




-305 






-300 






ATG GTC GCT GTC CAC 


ATG 


CAC 


AGG CTC TAT 


GAG 


AAG TAC AGC CGG 


CAG 


353 


Met Val Ala Val His 


Met 


His 


Arg Leu Tyr 


GIu 


Lys Tyr Ser Arg 


Cln 




-295 


-290 






■285 




-280 





-4 0- 



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PCr/JP93/00952 



GGC GCG CGG CCG 
Gly Ala Arg Pro 



CTG GAA 
Leu Glu 

ATG CAA 
Met Gin 

GAG CCG 
Glu Pro 
-230 
GCC AAG 
Ala Lys 
-215 
CAG CAC 
Gin His 



GTG GTC 
Val Val 
-260 
GAC TCG 
Asp Ser 
■245 

CCT CGG 
Pro Arg 

AAC GCT 
Asn Ala 

CTG CTC 
Leu Leu 



CTA CTC CGC GGG 
Leu Leu Arg Gly 
-180 

CAG GCC AAG GAC 
Gin Ala Lys Asp 

-165 
GAG CTG CTC CTC 
Glu Leu Leu Leu 
-150 



GGA GGG GGC AAC ACG GTC CGC AGC TTC AGG GCC AGG 401 

Gly Gly Gly Asn Thr Val Arg Ser Phe Arg Ala Arg 

■275 -270 -265 

GAC CAG AAG GCC GTG TAT TTC TTC AAC CTG ACT TCC 449 

Asp Gin Lys Ala Val Tyr Phe Phe Asn Leu Thr Ser 

-255 -250 
GAA ATG ATC CH ACG GCC ACT TTC CAC HC TAC TCA 497 
Glu Met He Leu Thr Ala Thr Phe His Phe Tyr Ser 

-240 -235 
TGG CCT CGA GCG CTC GAG GTG CTA TGC AAG CCG GGG 545 
Trp Pro Arg Ala Leu Glu Val Leu Cys Lys Pro Arg 

-225 -220 
TCA GGC CGC CCG CTG CCC CTG GGC CCG CCC ACA CGC 593 
Ser Gly Arg Pro Leu Pro Leu Gly Pro Pro Thr Arg 
-210 -205 -200 

nC CGC AGC CTC TCG CAG AAC ACG GCC ACA CAG GGG 641 
Phe Arg Ser Leu Ser Gin Asn Thr Ala Thr Gin Gly 
195 -190 -185 

GCC ATG CCC CTG GCG CCC CCA CCG CGC GGC CTG TGG 689 
Ala Met Ala Leu Ala Pro Pro Pro Arg Gly Leu Trp 

-175 -170 
ATC TCC CCC ATC GTC AAG GCC GCC CGC CGG GAT GGC 737 
He Ser Pro He Val Lys Ala Ala Arg Arg Asp Gly 

-160 -155 
TCC GCC CAG CTG GAT TCT GAG GAG AGG GAC CCC «GGG 785 
Ser Ala Gin Leu Asp Ser Glu Glu Arg Asp Pro Cly 

-145 -140 



-4 1- 



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GTG CCC CGG CCC AGC CCC TAT GCG CCC TAC ATC CTA GTC TAT GCC AAC 833 

Val Pro Arg Pro Ser Pro Tyr Ala Pro Tyr He Leu Val Tyr Ala Asn 

-135 -130 -125 -120 

GAT CTG GCC ATC TCG GAG CCC AAC AGC GTG GCA GTG ACG CTG CAG AGA 881 

Asp Leu Ala lie Ser Glu Pro Asn Ser Val Ala Val Thr Leu Gin Arg 

-115 -110 -105 

TAC GAC CCC TTC CCT GCC GGA GAC CCC GAG CCC CGC GCA GCC CCC AAC 929 
Tyr Asp Pro Phe Pro Ala Gly Asp Pro Glu Pro Arg Ala Ala Pro Asn 

-100 -95 -90 

AAC TCA GCG GAC CCC CGC GTG CGC CGA GCC GCG CAG GCC ACT GGG CCC 977 
Asn Ser Ala Asp Pro Arg Val Arg Arg Ala Ala Gin Ala Thr Gly Pro 

-85 -80 -75 

CTC CAG GAC AAC GAG CTG CCG GGG CTG GAT GAG AGG CCG CCG CGC GCC 1025 
Leu Gin Asp Asn Glu Leu Pro Gly Leu Asp Glu Arg Pro Pro Arg Ala 

-70 -65 -60 

CAC GCA CAG CAC TTC CAC AAG CAC CAG CTG TGG CCC AGC CCC TTC CGG 1073 
His Ala Gin His Phe His Lys His Gin Leu Trp Pro Ser Pro Phe Arg 
-55 -50 -45 -40 

GCG CTG AAA CCC CGG CCA GGG CGC AAA GAC CGC AGG AAG AAG GGC CAC 1121 
Ala Leu Lys Pro Arg Pro Gly Arg Lys Asp Arg Arg Lys Lys Gly Gin 

-35 -30 -25 

GAG GTG TTC ATG GCC GCC TCG CAG GTG CTG GAC TH GAC GAG AAG ACG 1169 
Glu Val Phe Met Ala Ala Ser Gin Val Leu Asp Phe Asp Glu Lys Thr 

-20 -15 -10 

ATG CAG AAA GCC CGG AGG AAG CAG TGG GAT GAG CCG AGG <iTQ TGC TCC 1217 
Met Gin Lys Ala Arg Arg Lys Gin Trp Asp Glu Pro Arg Val Cys Ser 
-5 1 5 



-4 2- 



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PCT/JM3/009S2 



CGG AGG TAG CTG AAG GTG GAG TTC GCA GAG ATC GGC TGG AAT GAA TGG 1265 
Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp lie Gly Trp Asn Glu Trp 
10 15 20 25 

ATA ATC TCA CGG AAA TCT TTT GAT GCC TAG TAG TGG GGG GGA GCA TGT 1313 
lie He Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ala Gly Ala Cys 

30 35 40 

GAG TTC CCC ATG CCT AAG ATC GTT CGT CCA TCC AAC CAT GCC ACC ATC 1361 
Glu Phe Pro Met Pro Lys lie Val Arg Pro Ser Asn His Ala Thr He 

45 50 55 

GAG AGC m GTC AGG GCT GTG GGC ATC ATC CCT GGC ATC CCA GAG CCC 1409 
Gin Ser He Val Arg Ala Val Gly He He Pro Gly He Pro Glu Pro 

60 65 70 

TGG TGT GTT CCC GAT AAG ATG AAC TCC CTT GGG CTC CTC TTC CTG GAT 1457 
Cys Cys Val Pro Asp Lys Met Asn Ser Leu Gly Val Leu Phe Leu Asp 

75 80 85 

GAG AAT CGG AAT GTG GTT CTG AAG GTG TAG CCC AAC ATG TCC GTG GAG 1505 
Glu Asn Arg Asn Val Val Leu Lys Val Tyr Pro Asn Met Ser Val Asp 
90 95 100 105 

ACC TGT GCC TGC CGG TGAGACCACT CCAGGGTGGA AAGAAGCCAC GCCCAGCAGA 1560 
Thr Cys Ala Cys Arg 
110 



-4 3- 



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SEQ. ID NO: 2 

SEQUENCE LENGTH: 182 nucleotides 
SEQUENCE TYPE: nucleotide 
STRANDEDNESS: double 
TOPOLOGY: linear 

MOLECULE TYPE: Other nucleic acid 

AGCCATCAAA TCATGCTACC ATCCAGAGTA TAGTGAGAGC TGTGGGGGTC GTTCCTGGGA 
TtCCTGAGCC HGCTGTGTA CCAGAAAACA TGTCCTCACT CAGTATTTTA TTCTTTGATG 
AAAATAAGAA TGTAGTGCTT AAAGTATACC CTAACATGAC AGTAGAGTCT TGCGCHGCA 
GA 



SEQ. ID NO: 3 

SEQUENCE LENGTH: 1497 nucleotides 
SEQUENCE TYPE: nucleotide with corresponding protain 
STRANDEDNESS: double 
TOPOLOGY: linear 
MOLECULE TYPE: cDNA to mRNA 
ORGANISM: rat 

CACTGAGCCT TCCCTGTCTG CCCTCCTGGG CTCAGACCCT TCACCACTGT CACTCAGCC 59 
ATG GCT CCA GGT CH GCT CGG ATC AGC TTG AGG TCT GAG CTG CTG CCC 107 
Met Ala Pro Gly Leu Ala Arg He Ser Leu Arg Ser Gin Leu Leu Pro 

-365 -360 -355 

TTG GTG CCG CTG CTC CTG CTA CTG CGG GGC OCA GGC TGC GGC CAC AGA 155 
Leu Val Pro Leu Leu Leu Leu Leu Arg Gly Ala Gly Cys Gly His Arg 
-350 -345 -340 -335 



80 
120 
180 
182 



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PCT/JP93/009S2 



GTC CCC TCA TGG TCC TCA CTG CCT TCA OCA OCT GAC AGT GTG CAG AGG 203 
Val Pro Ser Trp Ser Ser Leu Pro Ser Ala Ala Asp Ser Val Gin Arg 

-330 -325 -320 

GAC AGG GAC CTC GAG CAG TCA CCC GGG GAC GCT GCA GCC GCT CTG GGT 251 
Asp Arg Asp Leu Gin Gin Ser Pro Gly Asp Ala Ala Ala Ala Leu Gly 

-315 -310 -305 

CCA GGC GCC CAG GAC ATA GTC GCT GTC CAC ATG CTC AGG CTC TAT GAG 299 
Pro Gly Ala Gin Asp lie Val Ala Val His Met Leu Arg Leu Tyr Glu 

-800 -295 -290 

AAG TAC AAC CGG AGA GGC GCT CCA CCA GGA GGA GGC AAC ACC GTC CGA 347 
Lys Tyr Asn Arg Arg Gly Ala Pro Pro Gly Gly Gly Asn Thr Val Arg 

-285 -280 -275 

AGC TTC GGT GCC CGG CTG GAT GTG ATC GAC CAG AAG CCT GTG TAT TTC 395 
Ser Phe Arg Ala Arg Leu Asp Val lie Asp Gin Lys Pro Val Tyr Phe 
-270 -265 -260 -255 

TTC AAC TTG ACT TCC ATG CAA GAC TCA GAA ATG ATC CTC ACA GCC ACC 443 
Phe Asn Leu Thr Ser Met Gin Asp Ser Glu Met lie Leu Thr Ala Thr 

-250 -245 -240 

TTC CAC nC TAC TCA GAA CCT CCA CGG TGG CCC CGG GCT CGT GAG GTA 491 
Phe His Phe Tyr Ser Glu Pro Pro Arg Trp Pro Arg Ala Arg Glu Val 

-285 -230 -225 

TTC TGC AAG CCC CGA GCT AAG AAT GCA TCC TGC CGG CTC CTG ACC CCA 539 
Phe Cys Lys Pro Arg Ala Lys Asn Ala Ser Cys Arg Leu Leu Thr Pro 

-220 -215 -210 

GGT CTG CCT GCT CGC TTG CAC CTA ATC HC CGC AGT CTC TCG CAG AAC 587 
Gly Leu Pro Ala Arg Leu His Leu He Phe Arg Ser Leu Ser Gin Asn 
-205 -200 -195 



wo 94/01557 



PCr/JP93/009S2 



ACT GCC 
Thr Ala 
-190 
CCA CGT 
Pro Arg 

GCC CGA 
Ala Arg 

GAG AAG 
Glu Lys 

Cn GTC 
Leu Val 
-125 
GTG ACG 
Val Thr 
-110 
GGA GCA 
Gly Ala 

CAG GTA 
Gin Val 

AGA CCA 
Arg Pro 



ACT CAG 
Thr Gin 

GGC CTC 
Gly Leu 

AGG GAT 
Arg Asp 
-155 
GAT CTC 
Asp Leu 
■140 

TAT GCA 
Tyr Ala 

CTA CAG 
Leu Gin 

GCC CCC 
Ala Pro 

TCC AAA 
Ser Lys 
-75 
GCG CCT 
Ala Pro 
-60 



GGG CTG 
Gly Leu 
-185 
TGG CAG 
Trp Gin 
■170 

GGA GAA 
Gly Glu 

GGA GTG 
Gly Val 

AAT GAC 
Asn Asp 

AGA TAC 
Arg Tyr 
-105 
AAC AGC 
Asn Ser 
-90 

CCC CTG 
Pro Leu 

GCC CTG 
Ala Leu 



CTC CGC 
Leu Arg 

GCC AAG 
Ala Lys 

CTT CTT 
Leu Leu 

CCA CGG 
Pro Arg 
-135 
CTG GCC 
Leu Ala 
120 

GAC CCA 
Asp Pro 

TCA GCG 
Ser Ala 

CAA GAC 
Gin Asp 

CAC GCC 
His Ala 
-55 



GGG GCC 
Gly Ala 

GAC ATC 
Asp He 
-165 
CTC TCT 
Leu Ser 
•150 

CCC AGT 
Pro Ser 

ATC TCG 
He Ser 

TTT CCA 
Phe Pro 

GAT CCC 
Asp Pro 
-85 
AAT GAA 
Asn Glu 
-70 

CAG CAT 
Gin His 



ATG GCC 
Met Ala 
■180 

TCC TCA 
Ser Ser 

GCT CAG 
Ala Gin 

TCC CAC 
Ser His 

GAG CCC 
Glu Pro 
-115 
GCT GGA 
Ala Gly 
100 

CGC GTG 
Arg Val 

cn CCA 
Leu Pro 

TTC CAC 
Phe His 



CTG ACA 
Leu Thr 

ATC ATC 
He He 

CTG GAT 
Leu Asp 
-145 
ATG CCC 
Met Pro 
-130 
AAC AGT 
Asn Ser 

GAC TTT 
Asp Phe 

C<;C AGG 
Arg Arg 

GGG CTG 
Gly Leu 
-65 
AAG CAC 
Lys His 
-50 



CCT CCA 
Pro Pro 
-175 
AAG GCT 
Lys Ala 
-160 
TCT GGA 
Ser Gly 

TAT ATC 
Tyr He 

GTA GCA 
Val Ala 

GAG CCT 
Glu Pro 
-95 
GCG GCA 
Ala Ala 
-80 

GAC GAA 
Asp Glu 

GAG TTC 
Glu Phe 



635 



683 



731 



779 



827 



875 



923 



971 



1019 



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TGG TCC ACT CCT TTC CGG GCA CTG AAA CCC CGC ACC GGG CGC AAA GAC 1067 
Trp Ser Ser Pro Phe Arg Ala Leu Lys Pro Arg Thr Gly Arg Lys Asp 

-45 -40 -35 

CGC AAG AAG AAA GAC CAG GAT ACA TTC ACC CCC TCC TCC TCG CAG GTG 1115 
Arg Lys Lys Lys Asp Gin Asp Thr Phe Thr Pro Ser Ser Ser Gin Val 
-30 -25 -20 -15 

CTG GAC TTT GAT GAG AAG ACG ATG CAG AAA GCC AGG AGG CGG CAG TGG 1163 
Leu Asp Phe Asp Glu Lys Thr Met Gin Lys Ala Arg Arg Arg Gin Trp 

-10 -5 1 

GAT GAG CCT CGG GTC TGC TCC ACG AGG TAC CTG AAG GTG GAT TTT GCA 1211 
Asp Glu Pro Arg Val Cys Ser Arg Arg Tyr Leu Lys Val Asp Phe Ala 

5 10 15 

GAC ATC GGG TGG AAT GAA TGG ATC ATC TCA CCC AAA TCC TTT GAT GCC 1259 
Asp lie Gly Trp Asn Glu Trp He He Ser Pro Lys Ser Phe Asp Ala 

20 25 30 

TAC TAC TGT GCT GGA GCC TGC GAG TO CCC ATG CCC AAG ATC GTC CGC 1307 
Tyr Tyr Cys Ala Gly Ala Cys Glu Phe Pro Met Pro Lys He Val Arg 
35 40 45 50 

CCG TCC AAC CAT GCC ACC ATC CAG AGC ATC GTC AGA GCT GTG tJGC ATT 1355 
Pro Ser Asn His Ala Thr He Gin Ser He Val Arg Ala Val Gly He 

55 60 65 

GTC CCT GGC ATC CCG GAG CCA TGC TGT GTC CCA GAC AAG ATG AAC TCC 1403 
Val Pro Gly He Pro Glu Pro Cys Cys Val Pro Asp Lys Met Asn Ser 

70 75 80 

CTT GGA GTC CTT TO CTG GAC GAC AAT CGG AAT GTG m CTG AAG GTG 1451 
Leu Gly Val Leu Phe Leu Asp Glu Asn Arg Asn Val Val Leu Lys Val 
85 90 95 



-4 7- 



wo 94/01557 



PCT/JP93/00952 



TAC CCC AAT ATG TCC GTA GAG ACC TGT GCC TGT CGG TAAGGTGGCT 1497 
Tyr Pro Asn Met Ser Val Glu Thr Cys Ala Cys Arg 
100 105 110 

TCAAGATGGA AGGCAGACCT CCTOACCCC TGCTGTGCAG AGTGGCATTC TTGGAGCCAG 1557 



-4 8- 



wo 94/01557 



PCr/JP93/009S2 



Claims 

L A protein comprising an amino acid sequence of amino acids 1 to 
110 in SEQ. ID No:l or analogous sequences thereto. 

2. The protein according to claim 1, wherein the protein is derived 
from human. 

3. The protein according to claim 1. wherein the protein is derived 
from rat. 

4. A DNA encoding a protein comprising an amino acid sequence of 
amino acids 1 to 110 in SEQ. ID No:l or analogous sequences thereto. 

5. The DNA according to claim 4, wherein the protein is derived 
from human. 

6. The DNA according to claim 4. wherein the protein is derived 
from rat. 

7. The DNA according to claim 4. wherein the DNA comprises base 
sequence of nucleotides 1191 to 1520 in SEQ. ID No:l or analogous 
sequences thereto. 

8. The DNA according to claim 7, wherein the DNA comprises base 
sequence of nucleotides 87 to 1520 in SEQ. ID Noel or analogous 
sequences thereto. 

9. A method for producing a protein comprising an amino acid 
sequence of amino acids 1 to 110 in SEQ. ID No:l or analogous sequences 
thereto, which comprises: 

(a) transforming a cell with a DNA encoding said protein which 
further comprises expression-controlling sequences; and 

(b) culturing said transformant. 

10. A pharmaceutical composition comprising a protein which 
comprises an amino acid sequence of amino acids 1 to 110 in SEQ. ID No:l 
or analogous sequences thereto or active fragment thereof as a active 

-4 9" 



wo 94/01557 PCT/JP93/009S2 

ingredient together with pharmaceutically-acceptable carriers. 

11. The pharmaceutical composition according to claim 10, wherein 

the composition is for implantation. 



"5 0- 



wo 94/01557 



PCr/JP93/00952 



F i g. 1 





NcoA-Sal fragment 



Sal-Kpn fragment 



C-terminus primer ( C 
PGR template: HE 24 




I 



PGR 



Kp n E V 




EV-Kpn fragment 



L 



Sequencing 
Kpn-EV fragment 



T 



Ligation 



£ V : E c oR V 

N c o : N c o I 

H 3 : H i n d I I I 

K p n : K p n t 

S a I : S a I I 

£ I : £ c o R I 

S K-f- : B I u e S c r i p t I t S 



Expression vector 




> « 

UJ CO 



EV fragment 



1/13 



wo 94/01557 



PCr/JP93/00952 



F i g. 2 




hBIP 



2/13 



wo 94/01557 



PCr/JP93/00952 



F i g. 3 



5 '-side 
PGR tenplate: RB 45 
N-terminos primer (N-l,N-2) 
EV H3 





Sequencing 
EV-K3 fragaent 



H3-Kpn fragment 




EV-Kpn fragoent 



3 '-side 
PGR tenplate: RB 45 
C-terminus primer {C-l,C-2) 



Kpn 



EV 




Sequencing 
Kpn-EV fragment 



BodRV 
HindllT 

ECXSRI 



Kpn EV^ 




Expression Vector 



3/13 



wo 94/01SS7 



PCT/JP93/009S2 



Fig. 4 




rBIP 



4/13 



wo 94/01557 



PCr/JP93/00952 



F i g. 5 




5/13 



wo 94/01557 



PCr/JP93/00952 



F i g. 6 




6/13 



wo 94/01SS7 



PCr/JP93/00952 



F i g. 7 




7/13 



wo 94/01557 



PCr/JP93/00952 



F i g. 8 




8/13 



Wb 94/01SS7 



PCr/JP93/00952 



Fig. 9 




9/13' 



wo 94/01SS7 



PCT/JP93/009S2 



Fig. 10 




I0/i"3 



wo 94/01557 



PCr/JP93/009S2 



Fig. 11 



B 



B I P 



ABCDEFGHI 

L-. I . I I I I I 




4.4 — ? 
2.37 — 



BMP-2 



7.46 — 



B C D E F G H 

' ' I I I I I 



4.4 — 



2.37 



v:,v-.c."-x- 



1.35 — 



fc^v^ ^^^^^^ ^i '-^^-^^- 



AB CDEFGHI 

L ' ' !._„J r I t I 



BMP-3 



7.46 — 
4.4 — 

2.37 — 
1.36 — 



1 



J K 



7.46- 
4.4 - 

2.37- 
1.35* 



11/13 



wo 94/01SS7 



PCr/JP93/00952 




EcoRV 



I 



Bbs! Kpnl 

Bbsl & Kpnt digest 




EcoRV 



Bbsl Kpnl 



F i g, 1 2 



hBiP 

TG CA6 AM Ga CGG AGG AAG CAG TG6 GAT GAG COj AGG CTG TGC TCC CGG AGG TA^ 
C TTT CG6 GCC TCC nC GTC ACC QA ac GGC TCC CAC ACG AGG GCC TC 

Gin Lys Ala Arg Arg Lys Gin Trp Asp 61 u Pro Arg Val Cys Ser Arg Tyr 



hBMP-2 type 

TG CAC AAA GAG AAG AGG CAG TGG GAT GAG CCG AQG tSTG TGC TCC CG6 AGG TAC 
6 TTT GCG ac ffc Ta GTC ACC m aC GGC T{X CAC ACG AGG GCC TC 

His Lys AfQ 61 u Lvs Arg 



h proactivin-A type 

TGCAGAAACGCCOGAGGAGGCAG TG6 6AT GAG-CCG AGG 6TG T6C TCC ^ AGG TaC 
C TTT GCG GCC TCC TCC 6TC ACC CTA aC 6GC TCC CAC ACG A6G «X TC 

Gin Lys Arg Arg Arg Arg Gin 




1 



Bbsl 

EcoRV & Kpnl 
digest 





Kpnl & EcoRV 
digest<Short) 



Kpnl EcoiRV 



12/13 



wo 94/01557 



PCT/JP93/009S2 



Fig. 13 



EcoRV 



BanHI 




EeoB? 




INTERNA'nONAL SEARCH REPORT 

PCT/JP 93/00952 

immatioitti Application No rwi/ur jo/uu^at 



I. CXASSIFICATION OF SUBJECT MATTER (if sevoaJ dassttotioD symboU apply, todicatc all)' 



According to Inteniational Patent Qasstfication (IPQ or to both National Qasstficatioo and IFC 

Int. CI . 5 C12N15/12; A61K37/02; A61K9/22 



U. FIELDS SEARCHED 



Mtttimiim Doatracntatlon Searched^ 



Classification Systcn 


Classification Symbols 


Int.Cl. 5 


C07K ; C12N ; A61K 



Documeotation Scarcbed otber than Minimum Docnmcntation 
to the Extent that such Doegments are Indnded in the Fields Searched* 



m. DOCUMENTS CONSIDERED TO BE REIXVANT^ 



Catcgoiy" 


Citation of Docomcat, with indicatioa, wticrc appropriate, of the idcvant passages^ 


Rdennt to aaim No." 


A 


wo, A, 8 800 205 (GENETICS INSTITUTE, INC.) 


1-11 




14 January 1988 






see page 8, line 31 - page 13, line 1 






see page 25 - page 28 






see claims 




A 


PROCEEDINGS OF THE NATIONAL ACADEMY OF 


1-11 




SCIENCES OF USA 






vol. 87, no. 24, December 1990, WASHINGTON 






US 






pages 9843 - 9847 






CELESTE, A. ET AL. 'Identification of 






transforming growth factor beta family 






members present in bone- inductive protein 






purified from bovine bone' 






see the whole document 






-/-- 





" Spcdal categories of dted docnmcDts : 

'A' document defining the general state of the art wfaicfa is not 
considered to be of particular rdevanoe 

V earlier docnmcBt but published on or ato the intcmatiottal 
filingdate 

t' document which may throw doubts on priority daim(s) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

"O* document referring to an oral disclosure, use, eadiibition or 
other means 

document published prior to the international fiUog date but 
later than the priority date daimed 



*T* later docnmem published iftci the imematiooal filing date 
or priority date and not in conflict with the application but 
dted to understand the prindplc or theory onderiying the 
toventios 

docufflcnl of paiticnhr rdevancc; the claimed invention 
cannot be coasida-cd novel or cannoi be considered to 
taivolve an Inventive step 

•Y* docummt of partknlar rdevanoe; the claimed Inventioa 
cannot be considered to involve an inventive step when the 
document is combined with one or more other such docu- 
mented such coablnatiDn bdng obvious to a person skilled 
Intheart 

*A* document member of the same patent family 



IV. CERTinCATION 



Date of the Actual Completion of the International Search 

25 OCTOBER 1993 



Due of Mafliog of this latenatioaal Sevcb Report 

0 5 -11- 1993 



International Searching Anthority 

EUROPEAN PATENT OFFICE 



Sipuuure of Authorized Officer 

ANDRES S.M. 



Font PCT/JSA/210 ttccoAd Umh Umtry |«I5) 



International Applicatlott No 



PCT/JP 93/00952 



ni. DOCUMENTS CONSIDERED TO BE RELEVANT 



(CONTINUED FROM THE SECOND SHEET) 



Category* 



Citation of Documeci, with Indication, where appropriate, of the relevant passages 



Relevant to Qaim No. 



TRENDS IN GENETICS 

vol. 8, no. 3, March 1992, UK 

pages 97 - 102 

ROSEN, V. & THIES, S. 'The BMP proteins 1n 
bone formation and repair' 
see the whole document 



1-11 



ram rCT/ISA/2tO lean tkBtl Ijatny MS) 



^E5J£,T^EJ^'"^ATIONAL SEARCH REPORT 
ON INTERNATIONAL PATENT APPLICATION NO. JP 9300952 

SA 76087 

TWs Bimn lists the patent bmily meadim rdatiDg to the patent docnmentt dted in the above^entioned imemMional awcb report. 
Hie members arc as contained in the European Patent Office EDP file «n mm report. 

Hie European Patent Office b in no way liable for ifaese paitieulars wfaicfa are merely given for the porposc of information. 25/10/93 



Patent document 
dted in search report 



Publication 
date 



WO-A-8800205 



14-01-88 



Patent ftuntly 



Publication 



US-A- 
AU-B- 
AU-A- 
EP-A- 
JP-T- 
US-A- 
US-A- 
US-A- 
US-A- 
US-A- 
US-A- 
US-A- 



4877864 
613314 
7783587 
0313578 
2500241 
5013649 
5166058 
5187076 
5116738 
5106748 
5108922 
5141905 



31-10-89 
01-08-91 
29-01-88 
03-05-89 
01-02-90 
07-05-91 

24- 11-92 
16-02-93 
26-05-92 
21-04-92 
28-04-92 

25- 08-92 



For more details about tUs annex : tee Official Journal of the European Patent Office, No. 12/82 



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