Osteoinductive factors

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OSTEOINDUCTIVE FACTORS

פקטורים המשרים יצירת עצמות

GI5071D

NOVEE OSTEOINDUCTIVE FACTORS

The present invention relates to novel purified bone inductive factors which demonstrate in vivo activity in inducing cartilage and bone formation, and processes for obtaining such factors.

Background

Bone is a highly specialized tissue characterized by an extensive matrix structure formed of fibrous bundles of the protein collagen, and proteoglycans, noncollagenous proteins, lipids and acidic proteins. The processes of bone formation and renewal/repair of bone tissue, which occurs continuously throughout life, are performed by specialized cells. Normal embryonic long bone development is preceded by formation of a cartilage model. Bone growth is presumably mediated by osteoblasts (bone-forming cells), while remodeling of bone is apparently accomplished by the joint activities of bone-resorbing cells, called osteoclasts and osteoblasts. A factor which induces bone growth in circumstances where bone is not normally formed has application in the healing of bone fractures. An osteogenic preparation may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent would contribute to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also in cosmetic plastic surgery. Osteogenic factors may be also valuable in the treatment of periodontal disease, and in other tooth repair processes.

Repair of fractures, craniofacial defects, and periodontal defects are presently treated with a bone graft or a synthetic implant. An autograft has highest chance for success, but the trauma of surgery and the quantity of bone that can be harvested from the patient are major disadvantages. Allogenic bone grafts are also used. The availability of cadaver bone, the quality of the banked bone, and the potential spread of human disease are serious problems associated with this treatment. Ceramics, such as hydroxylapatite, or metals are also used, but these materials are only osteoconductive, i.e. new bone is generally only formed adjacent to the implant and the normal bone.

Physiologically acceptable chemical agents (hormones/ pharmaceuticals/growth factors) capable of inducing bone formation at a predetermined site are therefore desirable. Such agents could provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A variety of osteogenic, cartilage-inducing and bone inducing factors have been described. See, e.’g. European patent applications 148,155 and 169,016 for discussions of the work of others in this field. However, the procedures and techniques known in the art for obtaining putative osteogenic activities are prolonged and ill-defined and the factors have been only minimally identified and poorly characterized.

Thus there remains a need in the art for the accurate chemical and physiological characterization of a highly purified osteoinductive factor of a mammalian species for use in diagnosis, research and therapy of bone formation disorders.

Sampath and Reddi, PNAS 80:6591-6595 (1983) and Sampath et al., Exp. Cell Res. 143:4601982) 464־) disclose that there are protein containing fractions in partially purified bone extracts which induce bone.

Nathan US Patent No. 4,563,350 discloses an OF factor derived from bone further characterized as TGF-b proteins by Seyedin and Thomas EP 169,016.

Sin EP 148,155 discloses osteogenic factors which do not share any homology with the proteins of the present invention.

Urist, PNAS 81:3711984) 375־) discloses amino acid sequence which does not share homology with the amino acid sequences of the present invention.

Urist US Patent No. 4,455,256 discloses an activity found in an extract of demineralized bone tissue.

Urist US Patent No. 4,294,753 is understood to disclose a process of separating bone morphogenic protein form bone tissue.

Urist US Patent No. 4,619,989 discloses BMP compositions which do not share homology with the amino acid sequences of the proteins of the present invention. Physicochemical characteristics set forth in Table 3 of the '989 Patent differ from the physicochemical characteristics of the .83003/2 proteins of the present invention.

Urist, Science 220 (1983) discloses BMP protein which differs from the proteins of the present invention based, for example, on the amino acid composition analysis.

Brief Description of the Invention

The present invention relates to an osteoinductive factor substantially free from association with other proteinaceous materials and characterized by a peptide sequence comprising the same or substantially the same sequence as a continuous sequence selected from the group consisting of:

a) characterized by an amino acid sequence or a part thereof substantially as shown in Table V and Table VI,

b) characterized by an amino acid sequence or a part thereof substantially as shown in Table III, and Table VII,

c) characterized by an amino acid sequence or a part thereof substantially as shown in Table IVA and IVB, and the human counterpart thereof,

d) characterized by an amino acid sequence or a part thereof substantially as shown in Table VIII, or mixtures thereof.

Passages which are not in the ambit of the claims do not belong to the invention. The scope of protection is as defined in the claims, and as stipulated in the Patent Law (1968).

The present invention provides human and bovine bone growth factors substantially free from other human or bovine proteins and characterized by peptide sequences the same as or substantially homologous to the amino acid sequences illustrated in Tables II through VIII below. These factors are also characterized by certain bone growth factor biological and biochemical characteristics. For instance, the bone inductive factors of the invention are further characterized by biochemical and biological characteristics including activity at a concentration of 10 to lOOOng/gram of bone in an in vivo rate bone formation assay described below. The -DNA sequences for the bone growth factors of this invention may be encoded by the DNA sequences depicted in the Tables or sequences capable of hybridizing thereto and coding for polypeptides with bone growth factor biological properties or other variously modified sequences demonstrating such properties.

As one example of the bone growth factors of the invention is a human factor designated hBMP-1. hBMP-lis substantially free from association with other proteinaceous materials is characterized by a peptide sequence the same or substantially the same as that of amino acid #1 through amino acid #37 of Table V below which represents the genomic hBMP-1 and of amino acid #1 through amino acid #730 of Table VI which represents the cDNA hBMP-1. These peptide sequences are encoded by the 1 same or . substantially the same DNA sequence, as depicted in nucleotide #3440 through nucleotide #3550 of Table V and in nucleotide #36 through nucleotide #2225 of Table VI respectively. The coding sequence of Table V is flanked by approximately 28 nucleotides (a presumptive 5’ noncoding sequence) as well as approximately 19 nucleotides (a presumptive 3' noncoding sequence). These hBMP-1 polypeptides are further characterized by having at least one bone growth factor biological property.

One example of the bovine growth factors of the invention designated bBMP-1 is substantially free from association with other proteinaceous materials. It is characterized by a peptide sequence containing the same or substantially the same sequence as that of amino acid #1 through amino acid #37, of Table II below which represents the genomic bBMP-1. This peptide sequence is encoded by the same or substantially the same DNA sequence as depicted in nucleotide #294 through nucleotide #404 of Table

II. The bovine peptide sequence identified in Table II below is also 37 amino acids in length. In Table IT this coding sequence is flanked by approximately 21 nucleotides (a presumptive 5' noncoding sequence) as well as approximately 19 nucleotides (a presumptive 3' noncoding sequence). bBMP-1 is further characterized by having at least one bone growth factor biological property.

Another example of a human bone inductive protein composition of the invention is designated hBMP-2, Class I. It is substantially free from association with other proteinaceous materials and is characterized by a peptide sequence the same or substantially the same as that of amino acid #1 through amino acid #396 of Table VII which represents the cDNA bBMP-2, Class I. This peptide sequence is encoded by the same or substantially the same DNA sequence, as depicted in nucleotide #356 through nucleotide #1543 of Table VII. The human peptide sequence identified in Table VII is 396 amino acids in length. hBMP-2 Class I is further characterized by having at least one bone growth factor biological property.

A second example of a bovine factor of the invention designated bBMP-2, Class I has a DNA sequence identified in

Table III below which represents the genomic sequence. This bovine DNA sequence has s prospective 129 amino acid ceding sequence followed by approximately 205 nucleotides (a presumptive

3' non-coding sequence).

A further example of a human bone inductive protein composition of the invention is designated hBMP-2, Class II. It is substantially free from association with other proteinaceous materials and is characterized by a peptide sequence the same or substantially the same as that of amino acid #1 through amino acid #408 of Table VIII. This peptide sequence is encoded by the same or substantially the same DNA sequence as depicted in nucleotide #403 through nucleotide #1626 of Table VIII which represents the cDNA of hBMP-2 Class II. This factor is further characterized by having at least one bone growth factor biological property.

A further bovine bone inductive factor of the invention (bBMP-3) is substantially free from association with other human or bovine polypeptides and is characterized by the DNA sequence and amino acid sequence of Table IV A and B which represents the bovine genomic sequence. It is characterized by a peptide sequence the same or substantially the same as amino acid #1 through amino acid #175 of Table IV A and B. BMP-3 is substantially free from association with other human polypeptides and is further characterized by having at lease on bone growth factor biological property. This bovine factor maybe employed as a tool for obtaining an analogous human protein or other mammalian bone inductive protein. The proper characterization of this bovine bone inductive factor of the present invention provides the essential starting point for the method employing this sequence. The method, employing techniques known to those skilled in the art of genetic engineering, involves using the bovine DNA sequence as a probe to screen a human genomic or cDNA library; and identifying the DNA sequences which hybridize to the probes. A clone with a hybridizable sequence is plaque purified and the DNA isolated therefrom, subcloned and subjected to DNA sequence analysis. Thus as another aspect of this invention is a human protein hBMP-3, produced by this method.

Another aspect of the invention provides pharmaceutical compositions containing a therapeutically effective amount of one or more bone growth factor polypeptides according to the invention. These compositions may be employed in methods for treating a number of bone defects and periodontal disease. These methods according to the invention entail administering to a patient an effective amount of at least one polypeptide as described herein.

Still a further aspect of the invention are DNA sequences coding on expression for a human or bovine polypeptide having at least one bone growth factor biological property. Such sequences include the sequence of nucleotides in a 5' to 3' direction illustrated in Tables II through VIII. Alternatively a DNA sequence which hybridizes under stringent conditions with the DNA sequence of Tables II - VIII or a DNA sequence which hybridizes under non-stringent conditions with the illustrated DNA sequences and which codes on expression for a protein having at least one bone growth factor biological property are included in the present invention. Finally, allelic variations of the sequences of Tables II through VIII, whether such nucleotide changes result in changes in the peptide sequence or not, are also included in the present invention.

Still a further aspect of the invention is a vector containing a DNA sequence as described above in operative association with an expression control sequence. Such vector may be employed in a novel process for producing a bone growth factor polypeptide in which a cell line transformed with a DNA sequence encoding expression of a bone growth factor polypeptide in operative association with an expression control sequence therefor is cultured. This claimed process may employ a number of known cells as host cells for expression of the polypeptide. Presently preferred cell lines are mammalian cell lines and bacterial cells.

Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of preferred embodiments thereof.

Detailed Description of the Invention

The family of bone growth factors provided by the present invention are substantially free of association with other bovine or human proteins and are characterized by amino acid sequences the same as or substantially homologous to the sequences shown in Tables II - VIII below. The members of this novel family of growth factors are also characterized by demonstrating a biological property of a bone growth factor.

Also included in the present invention are synthetic polypeptides which wholly or partially duplicate continuous sequences of the amino acid residues of Tables II - VIII. These sequences, by virtue of sharing primary, secondary, or tertiary structural and conformational characteristics with bone growth factor polypeptides of Tables I - VIII may possess bone growth factor biological properties in common therewith. Thus, they may be employed as biologically active substitutes for naturallyoccurring primate bone growth factor polypeptides in therapeutic processes.

The bone growth factors provided herein also includes factors encoded by the sequences similar to those of Tables II - VIII, but into which modifications are naturally provided or deliberately engineered.

Other specific mutations of the sequences of the bone growth factors described herein involve modifications of one or both of the glycosylation sites. The absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at one or both of the asparagine-linked glycosylation recognition sites present in the sequences of the bone growth factors shown in Tables II - VIII. The asparagine-linked glycosylation recognition sites comprise tripeptide sequences which are specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either asparagine-X-threonine or asparagine-X-serine, where X is usually any amino acid. A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence.

The present invention also encompasses the novel DNA sequences, free of association with DNA sequences encoding other proteinaceous materials, and coding on expression for bone growth factors. These DNA sequences include those depicted in Tables II - VIII in a 5' to 3' direction and those sequences which hybridize under stringent hybridization conditions [see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to 389] to the DNA sequences of Tables II - VIII.

DNA sequences which hybridize to the sequences of Tables II - VIII under relaxed hybridization conditions and which code on expression for bone growth factors having bone growth factor biological properties also encode bone growth factors of the invention. For example, a DNA sequence which shares regions of significant homology, e.g. , sites of glycosylation or disulfide linkages, with the sequences of Tables II - VIII and encodes a bone growth factor having one or more bone growth factor biological properties clearly encodes a member of this novel family of growth factors, even if such a DNA sequence would not stringently hybridize to the sequence of Tables II - VIII.

Similarly, DNA sequences which code for bone growth factor polypeptides coded for by the sequence of Tables II - VIII, but which differ in codon sequence due to the degeneracies of the genetic code or allelic variations (naturally-occuring base changes in the species population which may or may not result in an amino acid change) also encode the novel growth factors described herein. Variations in the DNA sequences of Tables II -VIII which are caused by point mutations or by induced modifications to enhance the activity, half-life or production of the polypeptides encoded thereby are also encompassed in the invention.

Another aspect of the present invention provides a novel method for producing the novel osteoinductive factors. The method of the present invention involves culturing a fc suitable .cell or cell line, which has been transformed with a DNA sequence coding on expression for a novel bone growth factor polypeptide under the control of known regulatory Sequences. Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO) . The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature, 293 : 620-625 ( 198 1), or alternatively, Kaufman et al. Mol. Cell. Biol., 5 (7):1750-1759 (1985) or Howley et al, U.S. Patent 4,419,446. Another suitable mammalian cell line, which is described in the accompanying examples, is the monkey COS-1 cell line. A similarly useful mammalian cell line is the CV-1 cell line.

Similarly useful as host cells suitable for the present invention are bacterial cells. For example, the various strains of E. coli (e.g., HB101, MC1061 and strains used in the following examples) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al, Genetic Engineering, 8_:277-298 (Plenum Press 1986) and references cited therein.

Another aspect of the present invention provides vectors for use in the method of expression of these novel osteoinductive polypeptides.

Preferably the vectors contain the full novel DNA sequences described above which code for the novel factors of the invention. Additionally the vectors also contain appropriate expression control sequences permitting expression of the bone inductive factor sequences. Alternatively, vectors incorporating modified sequences as described above are also embodiments of the present invention and useful in the production of the bone inductive factors. The vectors may be employed in the method of transforming cell lines and contain selected regulatory sequences in operative association with the DNA coding sequences of the invention which are capable of directing the replication and expression thereof in selected host cells. Useful regulatory sequences for such vectors are known to one of skill in the art and may be selected depending upon the selected host cells. Such selection is routine and does not form part of the present invention.

A further aspect of the invention is a therapeutic method and composition for repairing fractures and other conditions related to bone defects or periodontal diseases. Such a composition comprises a therapeutically effective amount of one of the bone inductive factor protein compositions of the invention. The bone inductive factors according to the present invention may be present in a therapeutic composition in admixture with a pharmaceutically acceptable carrier or matrix. Further theraputic methods and compositions of the invention comprise a theraputic amount of a bone inductive factor of the invention with a theraputic amount of at least one of the other bone inductive factors of the invention. Additionally, a factor according to the present invention or a combination of the factors of the present invention may be co-administered with one or more different osteoinductive factors, with which it may interact. Further, the therapeutic composition may be combined with other agents beneficial to the treatment of the bone defect in question.

The therapeutic method includes locally administering the composition as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Preferably, the bone growth inductive factor composition would include a matrix capable of delivering the bone inductive factor to the site of bone damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of other materials presently in use for other implanted medical applications.

The bone inductive factor composition can also alternatively include other osteoconductive materials, such as hydroxylapatite, ceramics and the like. Further, the composition may desirably be encapsulated or injected in a viscous׳ form for delivery to the site of bone damage. The preparation of such physiologically acceptable protein compositions, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.

The dosage regimen will be determined by the attending physician considering various factors which modify the action of such a growth factor, e.g. amount of bone weight desired to be formed, the site of bone damage, the condition of the damaged bone, the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. Generally, the dosage regimen should be in the range of approximately 10 to 10⁶ nanograms of protein per gram of bone weight desired. Progress can be monitored by periodic assessment of bone growth and/or repair, e.g. x-rays. Such therapeutic compositions are also presently valuable for veterinary applications due to the lack of species specificity in bone inductive factors. Particularly domestic animals and thoroughbred horses are desired patients for such treatment with the bone inductive factor of the present invention.

The following examples illustrate practice of the present invention in recovering and characterizing the bovine bone inductive factors, employing the bovine bone inductive factors to recover human bone inductive factor, obtaining the human factors and in expressing the bone inductive factors via recombinant techniques.

EXAMPLE I

Isolation of Bovine Bone Inductive Factor

Ground bovine bone powder (20-120 mesh, Helitrex) is prepared according to the procedures of M. R< Urist et al., Proc. Natl Acad, Sci USA, 70:3511 (1973) with elimination of some extraction steps as identified below. Ten kgs of the ground powder is demineralized in successive changes of 0.6N HC1 at 4°C over a 48 hour period with vigorous stirring. The resulting suspension is extracted for 16 hours at 4 °C with 50 liters of 2M CaCl₂ and lOmM ethylenediamine- tetraacetic acid [EDTA], and followed by extraction for 4 hours in 50 liters of

0.5.1 ED1A. The residue is washed three times with distilled water before its resuspension in 20 liters of 4M guanidine hydrochloride [GuCl], 20mll Tris (pH 7.4), ImM N-ethylmaleimide,

InM rodoacetamide, M phenylmethylsulfonyl fluorine as described in Clin. Orthop, Rel.

171: 213 (1982). After 16 to 20 hours the supernatant is removed and replaced with another 10 liters of GuCl buffer. The residue is extracted for another 24 hours.

The crude GuCl extracts are combined, concentrated approximately 20 times on a Pellicon apparatus with a 10,000 molecular weight cut-off membrane, and then dialyzed in 50mlt Tris, 0.1M NaCl, 6M urea (pH7.2), the starting buffer for the first column. After extensive dialysis the protein is loaded a 4 liter DEAE cellulose column and the unbound fractions are collected.

The unbound fractions are concentrated and dialyzed against somli NaAc, 50mM NaCl (pH 4.6) in 6M urea. The unbound fractions are applied to a carboxymethyl cellulose column. Protein not bound to the column is removed by extensive washing with starting buffer, and the bone inductive factor containing material desorbed from the column by 50mM

NaAc, 0.25mM NaCl, 6M urea (pH 4.6). The protein from this step elution is concentrated 20- to 40- fold, then diluted 5 times with SOmM KPO₄ , 6M urea (pH6.0), The pH of the solution is adjusted to 6.0 with 500mM K₂HP0,. The sample is applied to an hydroxylapatite column (LKB) equilibrated in _{80raM KPOd 6M} urea (pH6.0) and all unbound protein is removed by washing the column with the same buffer. Bone inductive factor activity is eluted with lOOmM KPO₄ (pH7.4) and 6M urea.

The protein is concentrated approximately 10 times, and solid NaCl added to a final concentration of 0.15M. This material is applied to a heparin - Sepharose column equilibrated in 50mM KPO₄, 150mM NaCl, 6M urea (pH7.4). After extensive washing of the column with starting buffer, a protein with bone inductive factor activity is eluted by 50mM KPO₄, 700mM NaCl, 6M urea (pH7.4). This fraction is concentrated to a minimum volume, and 0,4ml aliquots are applied to Superose 6 and Superose 12 columns connected in series, equilibrated with 4M GuCl, 20mM Tris (pH7.2) and the columns developed at a flow rate of 0.25ml/min. The protein demonstrating bone inductive factor activity has a relative migration corresponding to approximately 30,000 dalton protein.

The above fractions are pooled, dialyzed against 50mM NaAc, 6M urea (pH4.6), and applied to a Pharmacia MonoS HR column. The column is developed with a gradient to 1.0M NaCl, 50mM NaAc, 6M urea (pH4.6). Active fractions are pooled and brought to pH3.0 with 10¾ trifluoroacetic acid (TFA). The material is applied to a 0.46 x 25cm Vydac C4 column in 0.1% TFA and the column developed with a gradient to 90% acetonitrile, 0.1% TFA (31.5% acetonitrile, 0.1% TFA to 49.5% acetonitrile, 0.1% TFA in 60 minutes at 1ml per minute). Active material is eluted at approximately 40-44% acetonitrile. Aliquots of the appropriate fractions are iodinated by one of the following methods:

P. J. McConahey et al, Int. Arch. Allergy, 29:185-189 (1966);

A. E. Bolton et al, B i o c h e m J , 133:529 (1973); and D. F. Bowen-Pope, J, Biol. Chem., 237:5161 (1982). The iodinated proteins present in these fractions are analyzed by SDS gel electro-phoresis and urea Triton X 100 isoelectric focusing. At this stage, the bone inductive factor is estimated to be approximately 10-50% pure.

EXAMPLE II

Characterization of Bovine Bone Inductive Factor

A. Molecular Weight

Approximately 20ug protein from Example I is lyophilized and redissolved in IX SDS sample buffer. After 15 minutes of heating at 37’C, the sample is applied to a 15% SDS polyacrylamide gel and then electrophoresed with cooling. The molecular weight is determined relative to prestained molecular weight standards (Bethesda Research Labs). Immediately after completion, the gel lane containing bone inductive factor is sliced into 0.3cm pieces. Each piece is mashed and 1.4ml of 0.1% SDS is added. The samples are shaken gently overnight at room temperature to elute the protein. Each gel slice is desalted to prevent interference in the biological assay. The supernatant from each sample is acidified to pH 3.0 with 10% TFA, filtered through a 0.45 micron membrane and loaded on a 0.46cm x 5cm C4 Vydac column developed with a gradient of 0.1%

TFA to 0.1% TFA, 90% CH3CN. The appropriate bone inductive factor - containing fractions are pooled and reconstituted with 20mg rat matrix. In this gel system, the majority of bone inductive factor fractions have the mobility of a protein having a molecular weight of approximately 28,000 - 30,000 daltons .

B. Isoelectric Focusing

The isoelectric point of bone inductive factor activity is determined in a denaturing isoelectric focusing system. The Triton X100 urea gel system (Hoeffer Scientific) is modified as follows: 1) 40% of the ampholytes used are Servalyte 3/10; 60% are Servalyte 7-9. 2) The catholyte used is 40mM NaOH. Approximately 20ug of protein from Example I is lyophilized, dissolved in sample buffer and applied to the isoelectrofocusing gel. The gel is run at 20 watts, 10 °C for approximately 3 hours. At completion the lane containing bone inductive factor is sliced into 0.5 cm slices. Each piece is mashed in 1.0ml 6M urea, 5mM Tris (pH 7.8) and the samples agitated at room temperature. The pH gradient of the gel is determined by soaking 5mm slices in 1.0ml water for over 2 hours. The samples are acidified, filtered, desalted and assayed as described above. The major portion of activity as determined in the assay described in Example III migrates in a manner consistent with a pl of 8.8 - 9.2.

C. Subunit Characterization

The subunit composition of bone inductive factor is also determined. Pure bone inductive factor is isolated from a preparative 15% SDS gel as described above. A portion of the sample is then reduced with 5mM DTT in sample buffer and re-electrophoresed on a 15% SDS gel. The approximately 30kd protein yields two major bands at approximately 20kd and ISkd, as well as a minor band at 30kd. The broadness of the two bands indicates heterogeneity caused most probably by glycosylation, other post translational modification, proteolytic degradation or carbamylation.

EXAMPLE III

Biological Activity of Bone Inductive Factor

A rat bone formation assay according to the general procedure of Sampath and Reddi, Proc. Natl, Acad, Sci. U.S.A., 80:6591-6595 (1983) is used to evaluate the osteogenic activity of the bovine bone inductive factor of the present invention obtained in Example I. This assay can also be used to evaluate bone inductive factors of other species. The ethanol precipitation step is replaced by dialyzing the fraction to be assayed against water. The solution or suspension is then redissolved in a volatile solvent, e.g. 0.1 - 0.2 % TFA, and the resulting solution added to 20mg of rat matrix. This material is frozen and lyophilized and the resulting powder enclosed in #5 gelatin capsules. The capsules are implanted subcutaneously in the abdominal thoracic area of 21 - 49 day old male long Evans rats. The implants are removed after 7 14 days. Half of each implant is used for alkaline phosphatase analysis [See, A. H. Reddi et al., Proc. Natl Acad Sci., 69:1601 (1972)] and half is fixed and processed for histological analysis. Routinely, lum glycolmethacrylate sections are stained with Von Kossa and acid fuschin to detect new bone mineral. Alkaline phosphatase, an enzyme produced by chondroblasts and osteoblasts in the process of matrix formation, was also measured. New cartilage and bone formation often correlates with alkaline phosphatase levels. Table I below illustrates the dose response of the rat matrix samples including a control not treatedediththobone inductive factor.

TABLE 1

Protein*

Implanted ug Cartilage Alk. Phos.u/l

7.5 2 Not done

2.5 3445.7

0.83 377.4

0.28 032.5

0.00 031.0 *At this stage the bone inductive factor is approximately 10-15% pure.

The bone or cartilage formed is physically confined to the space occupied by the matrix. Samples are also analyzed by SDS gel electrophoresis and isoelectric focusing as described above, followed by autoradiography. Analysis reveals a correlation of activity with protein bands at 28 - 30kd and a pl 9.0. An extinction coefficient of 1 OD/mg-cm is used as an estimate for protein and approximating the purity of bone inductive factor in a particular fraction. In the in vivo rat bone formation assays on dilutions as described above, the protein is active in vivo at 10 to 200ng protein/gram bone to probably greater than lug protein/gram bone.

EXAMPLE Bovine Bone Inductive Factor Protein Composition

The protein composition of Example IIA of molecular weight 28 - 30 kd is reduced as described in Example IIC and digested with trypsin. Eight tryptic fragments are isolated by standard procedures having the following amino acid sequences: Fragment 1: AAFLGDIALDEEDLG Fragment 2: AFQVQQAADL Fragment 3: NYQDMVVEG Fragment 4: STPAQDVSR Fragment 5: N Q E A L R Fragment 6: LSEPDPSHTLEE Fragment 7: F D A Y Y Fragment 8: LKPSN7ATIQSIVE

A less highly purified preparation of protein from bovine bone is reduced and electrophoresed on an acrylamide gel. The protein corresponding to the 18K band is eluted and digested with trypsin. Tryptic fragments are isolated having the following amino acid sequences: Fragment 9:SLKPSNHATIQS?V ^:

Fragment 10: SFDAYYCS7A Fragment 11: VYPNMTVESCA Fragment 12: VDFADI7W

Tryptic Fragments 7 and 8 are noted to be substantially the same as above Fragments 10 and 9, respectively.

A. bBMP-1

Probes consisting of pools of oligonucleotides (or unique oligonucleotides) are designed according to the method of

R. Lathe, J. Mol. Biol., 183 (1):1-12 (1985) and synthesized on an automated DNA synthesizer. One probe consists of a relatively long (32 nucleotides) guessmer [See J. J. Toole et al, Nature,

312:342-347 (1984)] of the following nucleotide sequence:

TCCTCATCCAGGGCAATGTCGCCCAGGAAGGC

Because the genetic code is degenerate (more than one codon can code for the same amino acid) , the number of oligonucleotides in a probe pool is reduced based on the frequency of codon usage in eukaryotes, the relative stability of G:T base pairs, and the relative infrequency of the dinucleotide CpG in eukaryotic coding sequences [see Toole et al., supra.]. The second set of probes consists of shorter oligonucleotides (17 nucleotides in length) which contain all possible sequences that could encode the amino acids. The second set of probes has the following sequences:

(a) A [A/G] [A/G] TC [T/C] TC [T/C] TC [A/G] TC [T/C] AA (b) A [A/G] [A/G] TC [T/C] TC [T/C] TC [A/G] TCNAG Bracketed nucleotides are alternatives. N means either A, T, C or G.

In both cases the regions of the amino acid sequence used for probe design are chosen by avoiding highly degenerate codons where possible. The oligonucleotides are synthesized on an automated DNA synthesizer; the probes are then radioactively labeled with polynucleotide kinase and ³²P-ATP.

These two sets of probes are used to screen a bovine genomic recombinant library. The library is constructed as follows: Bovine liver DNA is partially digested with the restriction endonuclease enzyme Sau 3A and sedimented through a sucrose gradient. Size fractionated DNA in the range of 15-30kb is then ligated to the bacteriophage Bam HI vector EMBL3 [Frischauf et al, J. Mol. Biol., 170:827-842 (1983)]. The library is plated at 8000 recombinants per plate. Duplicate nitrocellulose replicas of the plaques are made and amplified according to a modification of the procedure of Woo et al, Proc. Natl. Acad. Sei. USA, 75:3688-91 (1978).

The 32 mer probe is kinased with ³²P-gamma-ATP and hybridized to one set of filters in 5X SSC, 0.1% SDS, 5X Denhardts, lOOug/ml salmon sperm DNA at 45 degrees C and washed with 5X SSC, 0.1% SDS at 45 degrees C. The 17 mer probes are kinased and hybridized to the other set of filters in 3M tetramethylammonium chloride (TMAC), 0. IM sodium phosphate pH6.5, ImM EDTA, 5X Denhardts, 0.6% SDS, lOOug/ml salmon sperm DNA at 48 degrees C, and washed in 3M TMAC, 50mM Tris pH8.0 at 50 degrees C. These conditions minimize the detection of mismatches to the 17 mer probe pool [see, Wood et al, Proc. Natl , Acad . Sci, U.S.A., 82:1585-1588 (1985)]. 400,000 recombinants are screened by this procedure and one duplicate positive is plaque purified. DNA is isolated from a plate lysate of this recombinant bacteriophage designated lambda bP50. bP-50 is on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland under accession number 40295^ This bp-50 clone encodes the bovine *December 16, 1S86 growth factor designated bBMP-1.

The oligonucleotide hybridizing region of this BMP-1 clone is localized to an approximately SOObp Eco Rl fragment which is subcloned into M13 and sequenced by standard techniques. The partial DNA sequence and derived amino acid sequence of lambda bP-50 are shown below in Table IT.

The amino acid sequences corresponding to the tryptic fragments isolated from the bovine bone 28 to 30kd material are underlined in Table II. The first underlined portion of the sequence corresponds to tryptic Fragment 1 above from which the oligonucleotide probes are designed. The second underlined portion corresponds to tryptic Fragment 2 above. The predicted amino acid sequence indicates that tryptic Fragment 2 is preceded by a basic residue (R) as expected considering the specificity of trypsin. The nucleic acid sequence preceding the couplet CT at nucleotide positions #292-293 in Table II is presumed to be an intron (noncoding sequence) based on the presence of a consensus acceptor sequence (i.e., a pyrimidine rich tract, TCTCTCTCC, followed by AG) and the lack of a basic residue in the appropriate position of the derived amino acid sequence. The bBMP-1 peptide sequence from this clone is 37 amino acids in length and is encoded by the DNA sequence from nucleotide #294 through #404.

B. bBMP-2

Two probes consisting of pools of oligonucleotides are designed on the basis of the amino acid sequence of Fragment 3 and synthesized on an automated DNA synthesizer as described above.

Probe #1: ACNACCAT [A/G] T C [T/C] T G [A/G] A T Probe #2: C A [A/G] G A [T/C] ATGGTNGTNGA These probes are radioactively labeled and employed to screen the bovine genomic library constructed as described.in part A except that the vector is lambda JI Bam Hl arms [Mullins et al Nature 308: 856-858 (1984).] The radioactively labelled 17-mer Probe #1 is hybridized to the set of filters according to the method for the 17 mer probe described in part A.

400,000 recombinants are screened by the procedure described above. One duplicate positive is plaque purified and the DNA is isolated from a plate lysate of this recombinant bacteriophage designated lambda bP-21. Bacteriophage bP-21 is on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland under accession number ATCC 40310^ The bP21 clone encodes the bovine growth factor designated bBMP-2.

*March 6, 1987

TABLE II

280 290 (1) 308323

CCTTGCCTCT TCTCTCTCCA CCT GCC TTC CTT GGG GAC ATC GCC CTG GAC GAG GAG

Ala Phe Leu Gly Asp He Ala Leu Asp Glu Glu

338 353368

GAC TTG AGG GCC TTC CAA CTG CAG CAG GCT GCG GAC CTC AGA CAG GGT GCA ACC Asp Leu Arg Ala Phe Gin Val Gin Gin Ala Ala Asp Leu Arg Gin Arg Ala Thr

383 398 (37) 414424

CGC AGG TGT ICC ATC AAA GCT GCA GGTACACTGG GTACAGGCCA Arg Arg Ser Ser He Lys Ala Ala

The oligonucleotide hybridizing region of this bK-IP-2 clone is localized to an approximately 1.2 kb Sac I restriction fragment which is subcloned into M13 and sequenced by standard techniques. The partial DNA sequence and derived amino acid sequence of this Sac I fragment and the contiguous Hind IIISac I restriction fragment of bP-21 are she™ below in Table

III. The amino acid sequences corresponding to the tryptic fragment isolated from the bovine bone 28 to 30 kd material are underlined in Table III. Ihe underlined portion of the sequence corresponds to tryptic Fragment 3 above from which the oligonucleotide probes for bEMP-2 are designed. The predicted amino acid sequence indicates that tryptic Fragment 3 is preceded by a basic residue (K) as expected considering the specificity of trypsin. The arginine residue encoded by the CGT triplet is presumed to be the carboxy-terminus of the protein based on the presence of a stop coden (TAG) adjacent to it.

C. bEMP-3

Probes consisting of pools of oligonucleotides are designed on the basis of the amino acid sequences of the tryptic Fragments 9, 10 and 11, and synthesized on an automated DNA synthesizer.

TABLE TIT

<td colspan="2" rowspan="2"> (1) GGC CAC</td><td colspan="3" rowspan="2"> 15 GAT GGG AAA</td><td colspan="9"> 30</td><td rowspan="3"> 45 R</td>

<td rowspan="2"> GCA G</td><td rowspan="2"> CAC H</td><td colspan="3"> CCT CTC CAC</td><td colspan="3"> AGA AGA GAA</td><td rowspan="2"> AAG K</td>

<td> G</td><td> H</td><td> D</td><td> G</td><td> K</td><td> P</td><td> L</td><td> H</td><td> R</td><td> R</td><td> E</td>

<td> CAA</td><td> GCA</td><td> AAA</td><td> CAC</td><td> 60 AAA</td><td> CAG</td><td> CGG</td><td> AAA</td><td> CGC</td><td> 75 CTC</td><td> AAG</td><td> TCC</td><td> AGC</td><td> TGT</td><td> 90 AAG</td>

<td> Q</td><td> A</td><td> K</td><td> H</td><td> K</td><td> Q</td><td> R</td><td> K</td><td> R</td><td> L</td><td> K</td><td> s</td><td> S</td><td> C</td><td> K</td>

<td> AGA</td><td> CAC</td><td> CCT</td><td> TTA</td><td> 105 TAT</td><td> GIG</td><td> GcAC</td><td> TTC</td><td> AGT</td><td> 120 GAT</td><td> GTG</td><td> GvG</td><td> TGG</td><td> AAT</td><td> 135 GAC</td>

<td> R</td><td> H</td><td> P</td><td> L</td><td> Y</td><td> V</td><td> D</td><td> F</td><td> S</td><td> D</td><td> V</td><td> G</td><td> W</td><td> N</td><td> D</td>

<td> TGG</td><td> ATC</td><td> GTT</td><td> GCA</td><td> 150 COG</td><td> CCG</td><td> GGG</td><td> TAT</td><td> CAT</td><td> 165 GCC</td><td> TTT</td><td> TAC</td><td> TGC</td><td> CAT</td><td> 180 GGG</td>

<td> W</td><td> I</td><td> V</td><td> A</td><td> P</td><td> P</td><td> G</td><td> Y</td><td> H</td><td> A</td><td> F</td><td> Y</td><td> C</td><td> H</td><td> G</td>

<td> GAG</td><td> TGC</td><td> CCT</td><td> TTT</td><td> 195 CCC</td><td> CTG</td><td> GCC</td><td> GAT</td><td> CAC</td><td> 210 CTT</td><td> AAC</td><td> TCC</td><td> ACG</td><td> AAT</td><td> 225 GAT</td>

<td> .E</td><td> C</td><td> P</td><td> F</td><td> P</td><td> L</td><td> A</td><td> D</td><td> H</td><td> L</td><td> N</td><td> s</td><td> T</td><td> N</td><td> H</td>

<td> GCC</td><td> ATT</td><td> CTC</td><td> CAA</td><td> 240 ACT</td><td> CIG</td><td> GTC</td><td> AAC</td><td> TCA</td><td> 255 GTT</td><td> AAC</td><td> TCI</td><td> AAG</td><td> ATT</td><td> 270 CCC</td>

<td> A</td><td> I</td><td> V</td><td> Q</td><td> T</td><td> L</td><td> V</td><td> N</td><td> S</td><td> V</td><td> N</td><td> s</td><td> K</td><td> I</td><td> P</td>

<td> AAG</td><td> GCA</td><td> TGC</td><td> TGT</td><td> 385 GTC</td><td colspan="2"> CCA ACA</td><td> GAG</td><td> CTC</td><td> 300 AGC</td><td> GCC</td><td> ATC</td><td> TCC</td><td> ATG</td><td> 315 CTG</td>

<td> K</td><td> A</td><td> C</td><td> C</td><td> V</td><td> P</td><td> T</td><td> E</td><td> L</td><td> S</td><td> A</td><td> I</td><td> s</td><td> M</td><td> L</td>

<td> TAC</td><td> CTT</td><td> GAT</td><td> GAG</td><td> 330 AAT</td><td> GAG</td><td> AAG</td><td> GTG</td><td> GTA</td><td colspan="2"> 345 TTA AAG</td><td> AAC</td><td> TAT</td><td> CAG</td><td> 360 GAC</td>

<td> Y</td><td> L</td><td> D</td><td> E</td><td> N</td><td> E</td><td> K</td><td> V</td><td> V</td><td> L</td><td> K</td><td> N__</td><td> Y</td><td> _Q_</td><td> _D__</td>

<td> ATG</td><td> GTT</td><td> GTC</td><td> GAG</td><td> 375 GGT</td><td> TGT</td><td> GGG</td><td> TGT</td><td colspan="7"> (129) 397 407 CGT TAGCACAGCA AAATAAAATA</td>

<td> M</td><td> V</td><td> V</td><td> E</td><td> G</td><td> C</td><td> G</td><td> C</td><td> R</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 417 TAAATATATA</td><td> 427 TATATATATA</td><td> 437 TTAGAAAAAC</td><td> 447 AGCAAAAAAA</td><td> 457 TCAAGTTGAC</td>

<td> 467</td><td> 477</td><td> 487</td><td> 497</td><td> 507</td>

<td> ΑΟΠΤΑΑΤΑΤ</td><td> TTCCCAAIGA</td><td> AGACITTATT</td><td> TATGGAATGG</td><td> AATGGAGAAA</td>

<td> 517</td><td> 527</td><td> 537</td><td> 547</td><td> 557</td>

<td> AAGAAAAACA</td><td> CAGCTAITTT</td><td> GAAAACIATA</td><td> TTTATATCIA</td><td> CCGAAAAGAA</td>

<td> 567</td><td> 577</td><td> 587</td><td></td><td></td>

<td> GTIGGGAAAA</td><td> CAAATAITTT</td><td> aatgagigaa</td><td> TTATT</td><td></td>

Probe #1: ACNGTCAT [A/G] Τ Τ N G G [A/G] T A (based on Fragment 11)

Probe #2: C A [A/G] T A [A/G] T A N G C [A/G] T C [A/G] A A (based on Fragment 10)

Probe #3: T G [A/G/T] ATNGTNGC [A/G] T G [A/G] Τ T (based on Fragment 9)

A recombinant bovine genomic library constructed in EMBL3 is screened by the TMAC hybridization procedure detailed above in part A. 400,000 recombinants are screened in duplicate with Probe #1 which has been labeled with ³²P. All recombinants which hybridized to this probe are replated for secondaries. Triplicate nitrocellulose replicas are made of the secondary plates, and amplified as described. The three sets of filters are hybridized to Probes #1, #2 and #3, again under TMAC conditions. One clone, lambda bP-819, hybridizes to all three probes and is plaque purified and. DNA is isolated from a plate lysate. Bacteriophage lambda bP-819 is on deposit with the ATCC under accession number ן.';, wy.\

The region of bP-819 which hybridizes to Probe- #1 and #3 is localized and sequenced. The partial DNA and derived amino acid sequences of this region are shown in Table IV. A. The amino acid sequences corresponding to tryptic Fragments 9 and 11 are underlined. The arginine residue encoded by the AGA triplet is presumed to be the carboxy-terminus of the protein based on the presence of a stop codon (TAA) adjacent to it. The nucleic acid sequence preceding the couplet TC (positions 305-306) is presumed to be an intron (non-coding sequence) based on the presence of a consensus accepter sequence (i.e. a pyrimidine-rich stretch, TTCTCCCTTTTCGTTCCT, followed by AG) and the presence of a stop rather than a basic residue in the appropriate position of the derived amino acid sequence.

A different region of bP-819 which hybridizes to Probe 2 is localized and sequenced. The partial DNA and derived amino acid sequences of this region are shown in Table TV. B. The amino acid sequences corresponding to tryptic fragments 10 and 12 are underlined. bBMP-3 is therefore characterized by the DNA and amino acid sequence of Table IV A and Table IV B.

EXAMPLE

Human Bone Inductive Factors

A. hBMP-1

Because the bovine and human bone growth factor genes are presumed-to be significantly homologous, the bovine

BMP-1 DNA sequence of Table II (or portions thereof) was used as a probe to screen a human genomic library. The 800bp EcoRI fragment of the bovine genomic clone is labeled with ³²P by nick-translation. A human genomic library (Toole et al., supra) is plated on 20 plates at 40,000 recombinants per plate. Duplicate nitrocellulose filter replicas are made of each plate and hybridized to the nick-translated probe in 5 X SSC, 5 X Denhardt's, lOOug/ml denatured salmon sperm DNA, 0.1¾ SDS (the standard hybridization solution) at 50 degrees centigrade for approximately 14 hours. The filters are then washed in 1 X

TABLE IV. B.

3S3 393 403 413 (1) 428

QAGGAGGAAG CGGTCTAOGG GGGTCCTTCT GCCTCTGCAG AAC AAT GAG CTT CCT GGG GCA

Asn Asn Glu Leu Pro Gly Ala

443 458 473488

GAA TAT CAG TAC AAG GAG GAT GAA GTA TGG GAG GAG AGG AAG CCT TAC AAGACT

Glu Tyr Gin Tyr Lys Glu Asp Glu Vai Trp Glu Glu Arg Lys Pro Tyr LysThr

503 518533

CTT CAG ACT CAC CCC CCT GAT AAG AGT AAG AAC AAA AAG AAA CAG AGG AAGCCA

Leu Gin Thr Gin Pro Pro Asp Lys Ser Lys Asn Lys Lys Lys Gin Arg LysGly

548 563 578593

CCT CAG CAG AAG AGT CAG ACG CTC GAG TTT GAT GAA CAG ACC CTG AAG AAGGCA

Pro Gin Gin Lys Ser Gin Thr Leu Gin Phe Asp Glu Gin Thr Leu Lys LysAla

608 623638

AGA AGA AAG CAA TGG ATT GAA CCC CGG AAT TCT GCC AGA CGG TAC CTT AAACTG

Arg Arg Lys Gin Tip He Glu Pro Arg Asn Cys Ala Arg Arg Tyr Leu LysVai

653 668 683698

GAC TTC GCA GAT ATT GGC TGG AGC GAA TGG ATT ATT TCC CCC AAG TCC TTCGAT

Asp Phe Ala Asp He Gly Trp Ser Glu Trp He He Ser Pro Lys Ser PheAsp

GCC TAT TAC TCC TCC GGA GCG

Ala Ί\τ Tyr Cys Ser Gly Ala

728 743 (Hl) 756766

TGC CAG TIC CCC ATG CCA AAG GTAGCCATIG TTTHTCTCC Cys Gin Phe Pro MET Pro Lys

776 ’786

TCTCCTICCC AITTCCATAG

TABLE IV.A.

2S4

CTCTCCTGTG TTCTCCCTTT TCGTTCCTAG (112) 319

TCT TTG AAG CCA TCA ΆΑΤ CAC GCT ACC

Ser Leu Lys Pro Ser Asn His Ala Thr

334 349 364379

ATC CAG ACT ATA CTG AGA GCT GTG GGG GTC GTC CCT GGA ATC CCC GAG CCTTGC lie Gin Ser lie Vai Arg Ala Vai Gly Vai Vai Pro Gly He Pro Glu ProCys

394 409 424439

TCT GTG CCA GAA AAG ATG TCC TCA CTC AGC ATC TIA TTC ITT GAT GAA AACAAG

Cys Vai Pro Glu Lys LET Ser Ser Leu Ser He Leu Phe Phe Asp Glu AsnLys

454 469 484(175)

AAT CTG GTA CTT AAA CTA TAT CCA AAC ATG ACA GTA CTG TCT TGT GCT TGCAGA

Asn Vai Vai Leu Lys Vai Tyr Pro Asn MET Thr Vai Glu Ser Cys Ala CysArg

503 513 523533

TAACCTGGTG AAGAACTCTT CTGCTTGCTT AACTCAATCG

SSC, 0.1% SDS at 50 degrees centigrade and subjected to autoradiography. Five duplicate positives are isolated and plaque purified. DNA is obtained from a plate lysate of one of these recombinant bacteriophage, designated LP-H1. The hybridizing region of LP-H1 is localized to a 2.5kb Xbal/Hindlll restriction fragment. The partial DNA sequence and derived amino acid sequence of lambda LP-H1 are shown below in Table V. LP־־H1 has been deposited with the American Type Culture Collection under accession number 40311.* This clone encodes the human factor called hBMP-1.

Because the size of coding regions and the positions of noncoding regions is generally conserved in homologous genes of different species, the locations of the coding and noncoding regions of the bone inductive factor genes may be identified. Regions of homology between the two species' genes, flanked by RNA processing signals at homologous sites, indicate a coding region.

A probe specific for the human coding sequence given in Table V is used to identify a human cell line or tissue which synthesizes bone inductive factor. The probe is made according to the following method. Two oligonucleotides having the following sequences:

(a) GGGAATTCTGCCTTTCTTGGGGACATTGCCCTGGACGAAGAGGACCTGAG (b) CGGGATCCGTCTGAGATCCACAGCCTGCTGTACCTGGAAGGCCCTCAGG are synthesized on an automated synthesizer, annealed, extended using the Klenow fragment of E. coli DNA polymerase I, digested with the restriction enzymes Eco Rl and Bam HI, and inserted into an M13 vector. A single-stranded ³²P-labeled probe is then made off a template preparation of this subclone by standard techniques. Polyadenylated RNAs from various cell and tissue sources are electrophoresed on formaldehyde-agarose gels and

TABLE V

3419 3429 3439

CAGCCCIGGC TTCITCITIT GTCTTTAGCT (1) 3454

GCC TTT CTT GGG GAC ATT GCC CIG GAC

Ala Phe Leu Gly Asp lie Ala Leu Asp

3469 3484 34993514

GAA GAG GAC CIG AGG GCC TTC CAG GTA CAG CAG GCT GTG GIT CTC AGA CGGGAC

Glu Glu Asp Leu Arg Ala Phe Gin Vai Gin Gin Ala Vai Asp Leu Arg ArgHis

3529 3544 (37) 35603570

AGA GCT CGT AAG TCC TCC ATC AAA GCT GCA GGTAAGCCGG GTGCCAATGG

Thr Ala Arg Lys Ser Ser lie Lys Ala Ala transfered to nitrocellulose by the method of Toole et al., supra. The probe is then hybridized to the nitrocellulose blot in 50% formamide, 5 X SSC, 0.1% SDS, 40 mM sodium phosphate pH 6.5, 100 ug/ml denatured salmon sperm DNA, and 5 mM vanadyl ribonucleosides at 42° C overnight and washed at 65° C in 0.2 X SSC, 0.1% SDS. Following autoradiography, the lane containing RNA from the human osteosarcoma cell line U-2 OS contains hybridizing bands corresponding to RNA species of approximately

4.3 and 3.0 kb.

cDNA is synthesized from U-2 OS polyadenylated RNA and cloned into lambda gtlO by established techniques (Toole et al., supra). 20,000 recombinants from this library are plated on each of 50 plates. Duplicate nitrocellulose replicas are made of the plates. The above described oligonucleotides are kinased with ³²P-gamma-ATP and hybridized to the two sets of replicas at 55° centigrade in standard hybridization solution overnight. The filters are then washed in 1 X SSC, 0.1% SDS at 55° centigrade and subjected to autoradiography. One duplicate positive, designated lambda U2OS-1, is plaque purified. Lambda U2OS-1 is on deposit with the ATCC under accession number 40343 -J use 16,

Tile entire nucleotide sequence and derived amino acid sequence of the insert of lambda U2OS-1 is given in Table VI. This cDNA clone is presumed to contain all the sequence necessary to encode the entire precursor protein because it encodes a Met followed by a hydrophobic leader sequence characteristic of a secreted protein, and contains a stop codon at positons 222622 28. This clone contains an open reading frame of 2190 pb, encoding a protein of 730 amino acids. This clone contains sequence identical to the coding region given in Table V, and thus is a cDNA for hBMP-1 corresponding to human gene fragment contained in the genomic sequence lambda LP-H1.

B. hBMP-2: Class I and II

The Hindlll-SacI bovine genomic bBMP-2 fragment described in Example IV. B. is subcloned into an M13 vector. A ³²Plabeled single-stranded DNA probe is made from a template preparation of this subclone. This probe is used to sc-een polyadenylated RNAs from various cell and tissue sources as described above in part A. A hybridizing band corresponding to an mRNA species of approximately 3.8 kb is detected in the lane containing RNA from the human cell line U-2 OS. The HindlllSacl fragment is labeled with ³²P _nick translation and used to screen the nitrocellulose filter replicas of the abovedescribed U-2 OS cDNA library by hybridization in standard hybridization buffer at 65° overnight followed by washing in 1 xssc, 0.1% SDS at 65°. Twelve duplicate positive clones are picked and replated for secondaries. Duplicate nitrocellulose replicas are made of the secondary plates and both sets hybridized to the bovine genomic probe as the primary screening was performed. One set of filters is then washed in 1 X SSC, 0.1% SDS; the other in 0.1 X SSC, 0.1% SDS at 65°.

Two classes of hBMP-2 cDNA clones are evident based on strong (4 recombinants) or weak (7 recombinants) hybridization signals under the more stringent washing conditions (0.1 X SSC, 0.1-״ SDS) . All 11 recombinant bacteriophage are plaque purified, small scale DNA preparations made from plate lysates of each^ and the inserts subcloned into pSP65 and into M13 for sequence analysis. Sequence analysis of the strongly hybridizing clones (Class I) indicates that they have extensive sequence homology with the sequence given in Table III. _TheSe clones are therefore CDNA encoding the human equivalent of the protein encoded by e gene whose partial sequence is given in Table III. Sequence analysis of the weakly hybridizing recombinants (Class II) indicates that they are also quite homologous with the sequence given m Table II! at the 3׳ end of their coding regions, but ess so m the. more 5- regions. Thus they encode a human protein of similar, though not identical, structure to that above.

Full length hBMP-2 Class I cDNA clones are obtained in the following manner. The 1.5 kb insert of one of the class II subclones (II-10-1) is isolated and radioactively labeled by

TABLE VI

20 30(!)

CIAGAGGCCG CITCCCICGC CGCCGCCCCG CCAGC ATG

MET

CCC GGC GTG GCC CGC CIG CCG

Pro Gly Vai Ala Arg Leu Pro

80 95110

CIG CIG CTC GGG CIG CIG CIG CIC CCG CGT CCC GGC CGG CCG CIG GAC TIG GCC

Leu Leu leu Gly Leu Leu leu Leu Pro Arg Pro Gly Arg Pro Leu Asp Leu Ala

125 140155

GAC TAC ACC TAT GAC CIG GOG GAG GAG GAC GAC TCG GAG CCC CIC AAC TACAAA

Asp Tyr Ihr lyr Asp Leu Ala Glu Glu Asp Asp Ser Glu Pro Leu Asn TyrLys

170 185 200215

GAC CCC TGC AAG GCG GCT GCC TTT CTT GGG GAC ATI GCC CIG GAC GAA GAGGAC

Asp Pro Cys Lys Ala Ala Ala Phe Leu Gly Asp lie Ala Leu Asp Glu GluAsp

230 245 260275

CIG AGG GCC TIC CAG GTA CAG CAG GCT GIG GAT CTC AGA CGG CAC AGA GCT CGT leu Arg Ala Phe Gin Vai Gin Gin Ala Vai Asp leu Arg Arg His Thr Ala Arg

290 305320

AAG TCC TCC AIC AAA GCT GCA GTT CCA GGA AAC ACT TCT ACC CCC AGC TGC CAG

Lys Ser Ser He Lys Ala Ala Vai Pro Gly Asn Thr Ser Thr Pro Ser Cys Gin

335 350 3653S0

AGC ACC AAC GGG CAG OCT CAG AGG GGA GCC TGT GGG AGA TGG AGA GGT AGA TCC

Ser Thr Asn Gly Gin Pro Gin Arg Gly Ala Cys Gly Arg Trp Arg Gly Arg Ser

395 410425

CGT AGC CGG CGG GCG GCG ACG TCC OGA CCA GAG CGT GIG TGG CCC GAT GGG GTC

Arg Ser Arg Arg Ala Ala Thr Ser Arg Pro Glu Arg Vai Trp Pro Asp Gly Vai

440 455 470485

ATC CCC TTT GTC AIT GGG GGA AAC TTC ACT GGT AGC CAG AGG GCA GTC ITC OGG

He Pro The Vai lie Gly Gly Ten Phe Thr Gly Ser Gin Arg Ala Vai Phe Arg

500 515 530545

CAG GCC ATG AGG CAC TGG GAG AAG CAC ACC TGT GTC ACC TTC CIG GAG CGC ACT

Gin Ala MET Arg His Trp Glu Lys His Thr Cys Vai Thr Phe Leu Glu Arg Thr

560 575590

GAC GAG GAC AGC TAT ATT GIG TIC ACC TAT CGA OCT TGC GGG TGC TGC TCCTAC

Asp Glu Asp Ser Tyr He Vai Phe Thr Tyr Arg Pro Cys Gly Cys Cys SerTyr

605 620 635650

GIG GGT CGC CGC GGC GGG GGC CCC CAG GCC ATC TCC ATC GGC AAG AAC TGT GAC

Vai Gly Arg Arg Gly Gly Gly Pro Gin Ala He Ser He Gly Lys Asn Cys Asp

665 6S0 655

AAG TIC GGC ATT GTG GTC CAC GAG CTG GGC CAC GTC GTC GGC TTC TGG CAC GAA

Lys Phe Gly He Val Val His Glu Leu Gly His Val Val Gly Phe Trp His Glu

710 725 740755

CAC ACT OGG CCA GAC CGG GAC CGC CAC GTT TCC ATC GTT CGT GAG AAC ATC CAG

His Thr Arg Pro Asp Arg Asp Arg His Val Ser lie Val Arg Glu Asn He Gin

770 785 800815

CCA GGG CAG GAG TAT AAC TTC CTG AAG ATG GAG CCT CAG GAG GTG GAG TCC CTG

Pro Gly Gin Glu Tyr Asn Phe Leu Lys MET Glu Pro Gin Glu Val Glu Ser Leu

830 845860

GGG GAG ACC TAT GAC TTC GAC AGC ATC ATG CAT TAC GCT CGG AAC ACA TTC TCC

Gly Glu Thr Tyr Asp Phe Asp Ser lie MET His Tyr Ala Arg Asn Thr Phe Ser

875 890 905920

AGG GGC ATC TTC CTG GAT ACC ATT GTC CCC AAG TAT GAG GIG AAC GGG GTGAAA

Arg Gly He Phe Leu Asp Thr Lie Val Pro Lys Tyr Glu Val Asn Gly ValLys

935 950965

CCT CCC ATT GGC CAA AGG ACA CGG CTC AGC AAG GGG GAC ATT GCC CAA GCCCGC

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

980 995 10101025

AAG CTT TAC AAG TGC CCA GCC TGT GGA GAG ACC CIG CAA GAC AGC ACA GGCAAC

Lys Leu Tyr Lys Cys Pro Ala Cys Gly Glu Thr Leu Gin Asp Ser Thr GlyAsn

1040 1055 107010S5

TIC TCC TCC CCT GAA TAC CCC AAT GGC TAC TCT GCT CAC ATG CAC TGC GTGTGG

Rae Ser Ser Pro Glu Tyr Pro Asn Gly Tyr Ser Ala His MET His Cys ValTrp

1100 11151130

CGC ATC TCT GTC ACA CCC GGG GAG AAG ATC ATC CIG AAC TTC ACG TCC CTGGAC

Arg lie Ser Val Thr Pro Gly Glu Lys He He Leu Asn Phe Thr Ser LeuAsp

1145 1160 11751190

CIG TAC CGC AGC CGC CIG TGC TGG TAC GAC TAT GTG GAG GTC CQA GAT GGCTTC

Leu Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp GlyPhe

1205 12201235

TGG AGG AAG GCG CCC CTC CGA GGC CGC TTC TGC GGG TCC AAA CTC CCT GAGCCT

Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe Cys Gly Ser Lys Leu Pro GluPro

1250 1265 12801295

ATC GTC TCC ACT GAC AGC CGC CTC TGG GTT GAA TIC CGC AGC AGC AGC AATTGG

He Val Ser Thr Asp Ser Arg Leu Trp Val Glu Phe Arg Ser Ser Ser AsnTrp

1310 1325 13401355

GTT GGA AAG GGC TTC TTT GCA GTC TAC GAA GCC ATC TGC GGG GGT GAT GIGAAA

Val Gly Lys Gly Rae Rae Ala Val Tyr Glu Ala He Cys Gly Gly Asp ValLys

1370 13S5 1400

AAG GAC TAT GGC CAC ATT CAA TCG CCC ?AC TAC CCA GAC GAT TAC CGG CCC AGC

Lys Asp Tyr Gly His lie Gin Ser Pro Asn Tyr Pro Asp Asp Tyr Arg Pro Ser

1415 1430 14451460

AAA GTC TGC ATC TGG CGG ATC GAG GTG TCT AAG GGC TTC CAC GTG GGC CTCACA

Lys Vai Cys He Trp Arg He Gin Vai Ser Glu Gly Phe His Vai Gly LeuThr

1475 14901505

TTC CAG TCC TTT GAG ΑΠ' GAG CGC CAC GAC AGC TCT GCC TAC GAC TAT CTGCAG

Phe Gin Ser Phe Glu He Glu Arg His Asp Ser Cys Ala Tyr Asp Tyr LeuGlu

1520 1535 15501565

GTG CGC CAC GGG CAC ACT CAG AGC AGC ACC CTC ATC GGG CGC TAC TGT GGCTAT

Vai Arg Asp Gly His Ser Glu Ser Ser Thr Leu He Gly Arg Tyr Cys GlyTyr

1580 1595 16101625

CAG AAG CCT CAT CAC ATC AAG AGC ACG TCC AGC CGC CTC TGG CTC AAG TTCGTC

Glu Lys Pro Asp Asp Lie Lys Ser Thr Ser Ser Arg leu Trp Leu Lys PheVai

1640 16551670

TCT GAC GGG TCC ATT AAC AAA GCG GGC TTT GCC GTC AAC TTT TTC AAA CAGGTG

Ser Asp Gly Ser He Asn Lys Ala Gly Phe Ala Vai Asn Phe Phe Lys GluVai

1685 1700 17151730

GAC GAG TGC TCT CGG CCC AAC CGC GGG GGC TGT GAG CAG CGG TGC CTC AACACC

Asp Glu Cys Ser ?eg Pro Asn Arg Gly Gly Cys Glu Gin Arg Cys Leu AsnThr

1745 17601775

CTG GGC AGC TAC AAG TGC AGC TGT GAC CCC GGG TAC GAG CTC GCC CCA GACAAG leu Gly Ser Tyr Lys Cys Ser Cys Asp Pro Gly Tyr Glu Leu Ala Pro AspLys

1790 1805 18201835

OGC OGC TCT GAG GCT GCT TCT GGC GGA TTC CTC ACC AAG CTC AAC GGC TCCATC

Arg Arg Cys Glu Ala Ala Cys Gly Gly Phe Leu Thr Lys Leu Asn Gly Serlie

1850 1865 18801895

ACC AGC CCG GGC TCG CCC AAG GAG TAC CCC CCC AAC AAG AAC TCC ATC TCGCAG

Thr Ser Pro Gly Trp Pro Lys Glu Tyr Pro Pro Asn Lys Asn Cys He TrpGin

1910 19251940

CTC GTC GCC CCC ACC CAG TAC CGC ATC TCC CTC CAG TTT GAC TTC TTT GAGACA

Leu Vai Ala Pro Thr Gin Tyr Arg He Ser Leu Gin Phe Asp Phe Phe GluThr

1955 1970 19852000

GAG GGC AAT GAT GTG TCC AAG TAC GAC TTC GTC GAG GTC CGC AGT GGA CTCACA

Glu Gly Asn Asp Vai ¢¾⁷5 Lys Tit Asp Phe Vai Glu Vai Arg Ser Gly LeuThr

2015 20302045

GCT GAC TCC AAG CTC CAT GGC AAG TTC TCT GGT TCT GAG AAG CCC GAG GTCATC

Ala Asp Ser Lys Leu His Gly Lys Phe Cys Gly Ser Glu Lys Ho Glu Vailie

2060 2075 20902105

ACC TCC CAG TAC AAC AAC ATG CGC GTG GAG TTC AAG TCC GAC AAC ACC GTGTCC

Thr Ser Gin Tyr Asn Asn MET Arg Vai Glu Phe Lys Ser Asp Asn Thr VaiSer

2120 2135 21502165

AAA AAG GGC TTC AAG GCC CAC TTC TTC TCA GAA AAG AGG CCA GCT C1G CAGCCC

Lys Lys Gly Phe Lys Ala His Phe Phe Ser Glu Lys Arg Pro Ala Leu GinPro

2180 21952210

CCT CGG GGA CGC CCC CAC CAG CTC AAA TTC CCA GTG CAG AAA AGA AAC CGGACC

Pro Arg Gly Arg Pro His Gin Leu Lys Phe Arg Vai Gin Lys Arg Asn AugThr (730)

2225 2235 2245 2255 2265 227522S5

CCC CAG TCAGGCCIGC CAGGCCTCCC GGACCCCTTG TTACTCAGGA ACCICACCTT GCACGGAATG

Pro Gin

2295 2305 2315 2325 23352345

GGATGGGGGC TTCGGTGCCC ACCAACCCCC CACCICCACT CTGCCATTCC GGCCCACCTC

2355

CCTCTGGCCG

2365 2375 2385 2395 2405 24152425

GACAGAACIG CTGCTCTCTT CTCCCCACTG TGCCOGTCCG CGGACCGGGG ACCCTTCCCC GTGCCCTACC

2435

2445

CCCTCCCATT TTGATCGTGT

2455 2465 2475 2485 2495

CTGTCACATT TCCTCTTGTG AAGTAAAACA GGCACCCCIG CGTCCTGCCT

CTAGA nick-translation. One set of the nitrocellulose replicas of the U-2 OS cDNA library screened above (50 filters, corresponding to 1,000,000 recombinant bacteriophage) are rehybridized with this probe under stringent conditions (hybridization at 65° in standard hybridization buffer; washing at 65° in 0.2 X SSC, 0.1% SDS). All recombinants which hybridize to the bovine genomic probe which do not hybridize to the Class II probe are picked and plaque purified (10 recombinants). Plate stocks are made and small scale bacteriophage DNA preparations made. After subcloning into M13, sequence analysis indicates that 4 of these represent clones which overlap the original Class I clone. One of these, lambda U2OS-39, contains an approximately 1.5 kb insert and is on deposit with the ATCC under , accession number «MS.

DNA sequence (compiled from lambda U20S-39 and several other Class I cDNA recombinants) and derived amino acid sequence are shown below in Table VII. Lambda U205-39 is expected to contain all of the nucleotide sequence necessary to encode the entire human counterpart of the protein encoded by the bovine gene segment whose partial sequence is presented in Table III. This cDNA contains an open reading־frame of 1188 bp, encoding a protein of 396 amino acids, preceded by a 5’ untranslated region of 342 bp with stop codons in all frames.

Full-length Class II human cDNA clones are obtained in the following manner. The 200 bp EcoRl-SacI fragment from the 5' end of the Class II recombinant II-10-1 is isolated from its plasmid subclone, labeled by nick-translation, and hybridized to a set of duplicate nitrocellulose replicas of the U-2 OS cDNA library (25 filters/set; representing 500,000 recombinants). Hybridization and washing are performed under stringent conditions as described above. 16 duplicate positives are

TABLE VII

20 30 40 50 60 70

CTCGACTCTA GAGTCTGTGT CAGCACTTGG CTGGGGACTT

CTIGAACTTG CAGGGAGAAT AACTTGCGCA

100 110 120 130 140

CCCCACTTTG CGCCGGTGCC TTTGCCCCAG CGGAGCCIGC TTCGCCATCT CCGAGCCCCA CCGCCCCTCC

150 160

170 180 190 200 210

ACTCCTCGGC CTIGCCOGAC ACTGAGACGC TGTTCCCAGC CTGAAAAGAG AGACTGCGOG GCCGGCACCC

220 230 240 250 260 270280

GGGAGAAGGA GGAGGCAAAG AAAAGGAACG GACATICGGT CCTTGCGCCA GGTCCTTTGA CCAGAGITTT

290 300 310 320 330 340350

TCCATGIGGA OGCTCTTTGA ATGGACCTGT CCCCGOGIGC TTCTTAGAOG GACTGCGGTC TCCTAAAGGT (1) 370 385400

CGACC ATG GTG GCC GGG ACC OGC TCT CTT CTA GCG TIG CTG CTT CCC CAGGTC

MET Vai Ala Gly Thr Arg־ Cys Leu Leu Ala Leu Leu Leu Pro GinVai

415 430445

CIC CIG GGC GGC GCG GCT GGC CTC GTT CCG GAG CIG GGC OGC AGG AAG TTCGCG

Leu Leu Gly Gly Ala Ala Gly Leu Vai Pro Glu Leu Gly Arg Arg Lys PheAla

460 475 490505

GCG GCG TCG TOG GGC CGC CCC TCA TCC CAG CCC TCT GAC GAG CTC CTG AGCGAG

Ala Ala Ser Ser Gly Arg Pro Ser Ser Gin Pro Ser Asp Glu Vai Leu SerGlu

565־ 550 535520

TTC GAG TIG CGG CIG CTC AGC ATG TTC GGC CIG AAA CAG AGA CCC ACC CCCAGC

Phe Glu Leu Arg Leu Leu Ser MET Phe Gly Leu Lys Gin Arg Pro Thr ProSer

580 595610

AGG GAC GCC CTG GTG CCC CCC TAC ATG CTA GAC CTG TAT CGC AGG CAC TCGGCT

Arg Asp Ala Vai Vai Pro Pro Tyr MET Leu Asp Leu Tyr Arg Arg His SerGly

625 640 655670

CAG CGG GGC TCA CCC GCC CCA GAC CAC CGG TIG GAG AGG GCA GCC AGC CGAGCC

Gin Pro Gly Ser Pro Ala Pro Asp His Arg Leu Glu Arg Ala Ala Ser ArgAla

685 700715

AAC ACT GTG OGC AGC TTC CAC CAT GAA GAA TCT TIG GAA GAA CIA CCA GAA ACG

Asn Thr Vai Arg Ser Phe His His Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr

AGT GGG ALA ACA ACC CGG AGA TIC TTC TTT AAT TEA AGT TCT ATC CCC ACG GAG

Ser Gly Lys Thr Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Lie Pro Thr Glu

790 805 820835

GAG TTT ATC ACC TCA GCA GAG CTT GAG GTT TTC CGA GAL CAG ATG CAL GATGOT

Glu Phe Lie Thr Ser Ala Glu Leu Gin Vai Phe Arg Glu Gin MET Gin AspAla

850 865860

TTA GGA ALC AAT AGC AGT TIC CAT CAC CGA ATT ALT ATT TAT GAL ATC ATAAAA

Leu Gly Asn Asn Ser Ser Phe His His Arg He Asn He Tyr Glu He HeLys

895 910 925940

CCT GCA ACA GCC ALC TCG AAA TTC CCC GTC ACC AGT CTT TIG GAC ACC AGGTTG

Pro Ala Thr Ala Asn Ser Lys Phe Pro Vai Thr Ser Leu Leu Asp Thr ArgLeu

955 9709S5

GTG ALT CAG AAT GCA AGC AGG TGG GAA AGT TIT GAT GTC ACC CCC GCT GTGATG

Vai Asn Gin Asn Ala Ser Arg Trp Glu Ser Phe Asp Vai Thr Pro Ala VaiMET

1000 1015 10301045

CGG TCG ACT GCA CAG GGA CLC GCC ALC CAT GGA TTC GTG GTG GAL GTG GCCCAC

Arg Trp Thr Ala Gin Gly His Ala Asn His Gly Phe Vai Vai Glu Vai AlaHis

1060 1075 10901105

TTG GAG GAG AAA CAA GGT GTC TCC ALG AGA CAT GTT AGG ATA AGC AGG TCTTTG

Leu Glu Glu Lys Gin Gly Vai Ser Lys Arg His Vai Arg He Ser Arg SerLeu

1120 11351150

CAC CLA GAT GAA CAC AGC TGG TCA CAG ATA AGG CCA TTG CIA GTA ACT TTTGGC

His Gin Asp Glu His Ser Trp Ser Gin He Arg Pro Leu Leu Vai Thr PheGly

H65 1180 11951210

CAT GAT GGA AAA GGG CAT CCT CTC CAC AAA AGA GAA AAA OCT CAA GCC AAACAC

His Asp Gly Lys Gly His Pro Leu His Lys Arg Glu Lys Arg Gin Ala LysHis

1225 12401255

AAA CLG CGG AAA CGC CTT AAG TCC AGC TCT AAG AGA CAC CCT TIG TAC GTGGAC

Lys Gin Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg His Pro Leu Tyr VaiAsp

1270 1285 13001315

TTC AGT GAC GTG GGG TGG AAT GAC TCG ATT GTC GCT CCC CCG GGG TAT CACGCC

Phe Ser Asp Vai Gly Trp Asn Asp Trp He Vai Ala Pro Pro Gly Tyr HisAla

1330 1345 13601375

TTT TAC TGC CAC GGA GAL TCC CCT TTT CCT CIG GCT GLT CLT CIG AAC TCCACT

Phe T\r Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn SerThr

1390 1405!420

AAT CAT GCC ATT GTT CAG ACG TTG GTC AAC TCT GTT AAC TCT AAG ATT CCTAAG

Asn His Ala lie Vai Gin Thr Leu Vai Asn Ser Vai Asn Ser Lys Lie ProLys

1435

1450

1465

1480

GCA TGC TGT GTC CCG ACA GAA CTC AGT GCT ATC TCG ATG CIG TAG CTT GAG GAG

Ala Cys Cys Vai Pro Thr Glu Leu Ser Ala lie Ser MET Leu Tyr Leu Asp Glu

1495 1510 1525

AAT GAA AAG GTT GTA ΊΊΑ AAG AAC TAT CAG GAC ATG GIT GTG GAG GGT TGT GGG

Asn Glu Lys Vai Vai Leu Lys Asn Tyr Gin Asp MET Vai Vai Glu Gly Cys Gly

1540 ¢396) 1553 1563 1573 15S3 1593 1603

TGT CGC TAGTACAGCA AAATTAAATA CATAAATATA TATATATATA TATATTTIAG AAAAAAGAAA Cys Arg

ΑΑΑλ picked and replated for secondaries. Nitrocellulose filter replicas of the secondary plates are made and hybridized to an oligonucleotide which was synthesized to correspond to the sequence of II-10-1 and is of the following sequence:

CGGGCGCTCAGGATACTCAAGACCAGTGCTG

Hybridization is in standard hybridization buffer AT 50° C with washing at 50° in 1 X SSC, 0.1% SDS. 14 recombinant bacteriophage which hybridize to this oligonucleotide are plaque purified. Plate stocks are made and small scale bacteriophage DNA preparations made. After sucloning 3 of these into M13, sequence analysis indicates that they represent clones which overlap the original Class II clone. One of these, U2OS-3, contains an approximately 1.8 kb insert and is on deposit with the ATCC under accession number/1c-42?׳_^“^unilfe jp'Sirtial DNA sequence and ״ / ־*־ derived amino acid sequence of U20S-3 are shown below in Table VIII. This clone is expected to contain all of the nucleotide sequence necessary to encode the entire human protein. This cDNA contains an open reading frame of 1224 bp, encoding a protein of 408 amino acids, preceded by a 5' untranslated region of 394 bp with stop codons in all frames, and contains a 3' untranslated region of 308 bp following the in-frame stop codon.

The sequences of BMP-2 Class I and II, as well as BMP-3 as shown in Tables III, IV, VII and VIII have significant homology to the beta (B) and beta (A) subunits of the inhibins. The inhibins are a family of hormones which are presently being investigated for use in contraception. See, A. J. Mason et al, Nature, 318:659-663 (1985). To a lesser extent they are also homologous to Mullerian inhibiting substance (MIS), a testicular glycoprotein that causes regression of the Mullerian duct during development of the male embryo and transforming growth factor-beta (TGF-b) which can inhibit or stimulate

TABLE VIII

20 30 40 50 60 70

CTCTAGAGGG CAGAGGAGGA GGGAGGGAGG GAAGGAGCGC GGAGCCCGGC CCGGAAGCTA GGIGAGTGTG

90 100 110 120 130140

GCATCCGAGC TGAGGGACGC GAGCCTGAGA CGCCGCTGCT GGTCCGGCTG AGTATCIAGC TTGTCICCCC

150 160 170 180 190 200210

GATGGGAITC CCGTCCAAGC TATCICGAGC CTGCAGCGCC ACAGTCCCCG GCCCIOGCCC AGGTICACIG

220 230 240 250 260 270280

CAACCGTICA GAGGTCCO2A GGAGCTGCIG CTGGCGAGCC CGCTACTGCA GGGACCTAIG GAGCCAITCC

290 300 310 320 330 340350

GTAGTGCCAT CCCGAGCAAC GCACIGCIGC AGCTTCCCIG AGCCTTTCCA GCAAGTITCT TCAAGATTGG

360 370 380 390 400(1)

CTGTCAAGAA TCAIGGACTG TTATTATATG CCTICITTTC TGTCAAGACA CC ATG ATT CCT

MET lie Pro

417 432 447462

GGT AAC CGA ATG CIG ATG GTC GTT TTA TTA TGC CAA GTC CTG CIA GGA GGC GOG

Gly Asn Arg MET Leu MET Vai Vai Leu Leu Cys Gin Vai Leu Leu Gly Gly Ala

477 492507

AGC CAT GCT AGT TTG ATA CCT GAG ACG GGG AAG AAA AAA GTC GCC GAG ATT CAG

Ser His 'la Ser Leu lie Pro Glu Thr Gly Lys Lys Lys Vai Ala Glu lie Gin

522 537 552567

GGC CAC GOG GGA GGA CGC CGC TCA GGG CAG AGC CAT GAG CTC CIG CGG GAC TTC

Gly His Ala Gly Gly Arg Arg Ser Gly Gin Ser His Glu Leu Leu Arg Asp Phe

582 597 612627

GAG GCG ACA CTT CIG CAG ATG ITT GGG CIG CGC CGC CGC COG CAG CCT AGC AAG

Glu Ala Thr Leu Leu Gin MET Phe Gly Leu Arg Arg Arg Pro Gin Pro Ser Lys

642 657672

AGT GCC GTC ATT CCG GAC TAC ATG CGG GAT CTT TAC CGG CTT CAG TCT GGGGAG

Ser Ala Vai lie Pro Asp Tyr MET Arg Asp Leu Tyr Arg Leu Gin Ser GlyGlu

687 702 717732

GAG GAG GAA GAG CAG ATC CAC AGC ACT GGT CTT GAG TAT CCT GAG CGC CCGGCC

Glu Glu Glu Glu Gin He His Ser Thr Gly Leu Glu Tyr Pro Glu Arg Pro Ala

747 762 777

AGC CGG GCC AAC ACC GTC AGC AGC TIC CAC CAC GAA GAA CAT CTG CAG AAC ATC

Ser Arg Ala Asn Thr Vai Arg Ser Phe His His Glu Glu His Lsu Glu Asn lie

792 807 822837

CCA GGG ACC ACT CAA AAC TCT GCT ITT CGT TTC CTC ITT AAC CTC AGC AGCATC

Pro Gly Thr Ser Glu Asn Ser Ala Phe Arg Phe Leu Phe Asn Leu Ser Serlie

852 867 882897

CCT CAG AAC CAG GCG ATC TCC TCT GCA GAG CTT CGG CTC TTC CGG CAG CAGGTG

Pro Glu Asn Glu Ala lie Ser Ser Ala Glu Leu Arg Leu Phe Arg Glu GinVai

912 927942

GAC CAG GGC CCT CAT TGG GAA AGG GGC TIC GAC CGT ATA AAC ATT TAT GAGGIT

Asp Gin Gly Pro Asp Trp Glu Arg Gly Phe His Arg He Asn He Tyr GluVai

957 972 9871002

ATG AAG CCC CCA GCA GAA GIG GTG CCT GGG CAC CTC ATC ACA CCA CIA CTGGAC

MET Lys Pro Pro Ala Glu Vai Vai Pro Gly His Leu He Thr Arg Leu LeuAsp

1017 10321047

ACG AGA CTG GTC CAC CAC AAT GTG ACA CGG TGG GAA ACT ITT GAT GTG AGCCCT

Thr Arg Leu Vai His His Asn Vai Thr Arg Trp Glu Thr Phe Asp Vai SerPro

1062 1077 10921107

GCG GTC CIT CGC TGG ACC CGG GAG AAG CAG CCA AAC TAT GGG CIA GCC ATTGAG

Ala Vai Leu Arg Trp Thr Arg Glu Lys Gin Pro Asn Tyr Gly Leu Ala HeGlu

H22 1137 11521167

GTG ACT CAC CTC CAT CAG ACT CGG ACC CAC GAG GGC CAG CAT GTC AGG ATTAGC

Vai Thr His Leu His Gin Ihr Arg Ihr His Gin Gly Gin His Vai Arg HeSer

1182 11971212

CGA TCG TTA CCT CAA GGG AGT GGG AAT TGG GCC CAG CTC CGG CCC CTC CIGGTC

Arg Ser Leu Pro Gin Gly Ser Gly Asn Trp Ala Gin Leu Arg Pro Leu LeuVai

1227 !242 12571272

ACC ITT GGC CAT GAT GGC CGG GGC CAT GCC TTC ACC CGA CGC CGG AGG GCCAAG

Thr Phe Gly His Asp Gly Arg Gly His Ala Leu Thr Arg Arg Arg Arg AlaLys

1287 13021317

OGT AGC CCT AAG CAT CAC TCA CAG OGG GCC AGG AAG AAG AAT AAG AAC TGCCGG

Arg Ser Pro Lys His His Ser Gin Arg Ala Arg Lys Lys Asn Lys Asn CysArg

1332 1347 13621377

CGC CAC TCG CIC TAT GTG GAC TTC AGC GAT GTG GGC TGG AAT GAC TGG ATTGTG

Arg His Ser Leu Tyr Vai Asp Phe Ser Asp Vai Gly Trp Asn Asp Trp HeVai

1392 1407 14221437

GCC CCA CCA GGC TAC CAG GCC TTC TAC TCC CAT GGG GAC TCC CCC ΊΤΤ CCA CTG

Ala Pro Pro Gly Tyr Gin Ala Phe Tyr Cys His Gly Asp Cys Pro Phe Pro Leu

1452 14671482

GCT GAC CAC CTC AAC TCA ACC AAC CAT GCC ATT GTG CAG ACC CTG GTC AATTCT

Ala Asp His Leu Asn Ser Tnr Asn His Ala He Vai Gin Thr Leu Vai AsnSer

1497 1512 15271542

GTC AAT TCC AGT ATC CCC AAA GCC TGT TGT GTG CCC ACT GAA CIG AGT GCCATC

Vai Asn Ser Ser He Pro Lys Ala Cys Cys Vai Pro Thr Glu Leu Ser AlaHe

1557 15721587

TCC ATG CTG TAC CTG GAT GAG TAT GAT AAG GTG GTA CIG AAA AAT TAT CAGGAG

Ser MET Leu Tyr Leu Asp Glu Tyr Asp Lys Vai Vai Leu Lys Asn Tyr GinGlu

1602 1617 (394) 1636 1646 16561666

ATG GTA GTA GAG GGA TGT GGG TGC CGC TGAGATCAGG CAGTCCTTGA GGATAGACAG ATATACACAC

MET Vai Vai Glu Gly Cys Gly Cys Arg

1676 16S6 1696 1706 1716 17261736

CACACACACA CACCACATAC ACCACACACA CACGITCCCA TCCACTCACC CACACACTAC AGAGACTGCT

1746

1756 1766 1776 1786 17961806

TCCTTATAGC TGGACTHTA ΊΤΤΑΑΑΑΑΑΑ AAAAAAAAAA AAIGGAAAAA ATCCCTAAAC ATTCACCTIG

1816 1826 1836 1846 1856 18661876

ACCTTATTIA TGACTTTACG TGCAAATGTT TTGACCATAT TGATCATATA TITIGACAAA ATATATTTAT

1886 1896 1906 1916 1926 19361946

AACTACGTAT TAAAAGAAAA AAATAAAATG AGTCATTATT ΊΤΑΑΑΑΑΑΑΑ AAAAAAAACT CTAGAGTCGA

OGGAATTC growth of cells or cause them to differentiate. Furthermore, the sequence of Table VII has significant homology to the Drosophila decapentaplegic (DPP-C) locus transcript. See, J. Massague, Cell, 49 : 437-438 (1987); R. W. Padgett et al, Nature, 325:81-84 (1987); R. L. Cate et al, Cell 45: 685-698 (1986). It is considered possible therefore that BMP-2 Class II is the human homolog of the protein made from this transcript from this developmental mutant locus.

C. BMP-3

Because bovine and human bone growth factor genes are presumed to be significantly homologous, oligonucleotide probes which have been shown to hybridize to the bovine DNA sequence of Table IV are used to screen a human genomic library. A human genomic library (Toole et al., supra) is screened using these probes, and presumptive positives are isolated and DNA sequence obtained as described above. Evidence that this recombinant encodes a portion of the human bone inductive factor molecule relies on the bovine/human protein and gene structure homologies.

Once a recombinant bacteriophage containing DNA encoding a portion of the human BMP-3 molecule is obtained the human coding sequence is used as a probe as described in example V A to identify a human cell line or tissue which synthesizes BMP-

3. mRNA is selected by oligo (dT) cellulose chromatography and cDNA is synthesized and cloned in lambda gtlO by established techniques (Toole et al., sucra).

Alternatively, the entire gene encoding this human bone inductive factor can be identified and obtained in additional recombinant clones if necessary. Additional recombinants containing further 3' or 5 ' regions of this human bone inductive factor gene can be obtained by identifying unique DNA sequences at the end(s) of the original clone and using these as probes to rescreen the human genomic library. The gene can then be reassembled in a single plasmid by standard molecular biology techniques and amplified in bacteria. The entire human BMP-3 factor gene can then be transferred to an appropriate expression vector. The expression vector containing the gene is then transfected into a mammalian cell, e.g. monkey COS cells, where the human gene is transcribed and the RNA correctly spliced. Media from the transfected cells are assayed for bone inductive factor activity as described herein as an indication that the gene is complete. mRNA is obtained from these cells and cDNA synthesized from this mRNA source and cloned. This procedure has been described for the cloning and expression of erythropoietin by Lin et al., supra.

The procedures described above may similarly be employed to isolate other species' bone inductive factor of interest by utilizing the bovine bone inductive factor and/or human bone inductive factor as a probe source. Such other species¹ bone inductive factor may find similar utility in, inter alia, fracture repair.

EXAMPLE

Expression of Bone Inductive Factors.

In order to produce bovine, human or other mammalian bone inductive factor, the DNA encoding it is transferred into an appropriate expression vector and introduced into mammalian cells by conventional genetic engineering techniques.

One skilled in the art can construct mammalian expression vectors by employing the sequence of Tables II-VIII or other modified sequences and known vectors, such as pCD [Okayama et al., Mol. Cell BigL., 2:161-170 (1982)] andpJL3, pJL4 [Gough et al., EMBO J. , 4:645-653 (1985)]. The transformation of these vectors into appropriate host cells can result in expression of osteoinductive factors. One skilled in the art could manipulate the sequences of Tables II-VIII by eliminating or replacing the mammalian regulatory sequences flanking the coding sequence with bacterial sequences to create bacterial vectors for intracellular or extracellular expression by bacterial cells. For example, the coding sequences could be further manipulated (e.g. ligated to other known linkers or modified by deleting non-coding sequences there-from or altering nucleotides therein by other known techniques). The modified bone inductive factor coding sequence could then be inserted into a known bacterial vector using procedures such as described in T. Taniguchi et al., Proc. Natl Acad. Sci. USA, 77:5230-5233 (1980). This exemplary bacterial vector could then be transformed into bacterial host cells and bone inductive factor expressed thereby. For a strategy for producing extracellular expression of bone inductive factor in bacterial cells., see, e.g. European patent application EPA 177,343.

Similar manipulations can be performed for the construction of an insect vector (See, e.g. procedures described in published European patent application 155,476] for expression in insect cells. A yeast vector could also be constructed employing yeast regulatory sequences for intracellular or extracellular expression of the factors of the present invention by yeast cells. [See, e.g., procedures described in published PCT application W086/00639 and European patent application EPA 123,289] .

A method for producing high levels of an osteoinductive factor of the invention from mammalian cells involves the construction of cells containing multiple copies of the heterologous bone inductive factor gene. The heterologous gene can be linked to an amplifiable marker, e.g. the dihydrofolate reductase (DHFR) gene for which cells containing increased gene copies can be selected for propagation in increasing concentrations of methotrexate (MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol., 159:601-629 (1982). This approach can be employed with a number of different cell types.

For example, a plasmid containing the bovine bone inductive factor gene of Table II in operative association with other plasmid sequences enabling expression thereof and the DHFR expression plasmid pAdA26SV(A)3 [Kaufman and Sharp, Mol. Cell. Biol., 2:1304 (1982)] can be co-introduced into DHFR-deficient CHO cells, DUKX-BII, by calcium phosphate coprecipitation and transfection. DHFR expressing transformants are selected for growth in alpha media with dialyzed fetal calf serum, and subsequently selected for amplification by growth in increasing concentrations of MTX (sequential steps in 0.02, 0.2, 1.0 and 5uM MTX) as described in Kaufman et al., Mol Cell Biol. , 5:1750 (1983). Transformants are cloned, and biologically active bone inductive factor expression is monitored by rat bone formation assay. Bone inductive factor expression should increase with increasing levels of MTX resistance. Similar procedures can be followed to produce other bone inductive factors.

Alternatively, the human gene is expressed directly, as described- above. Active bone inductive factor may be produced in bacteria or yeast cells. However the presently preferred expression system for biologically active recombinant human bone inductive factor 'is stably transformed CHO cells.

As one specific example, to produce the human bone inductive factor (hBMP-1) of Example V, the insert of U2OS-1 is released from the vector arms by digestion with Sal I and subcloned into the mammalian expression vector pI-IT2CX digested with Xho I. The mammalian expression vector pMT2 Cla-Xho (pMT₂ CX) is a derivative of p91023 (b) (Wong et al., Science 228:810-815, 1985) differing from the latter in that it contains the ampicillin resistance gene in place of the tetracycline resistance gene and further contains a Xhol site for insertion of cDNA clones. The functional elements of pMT2 Cla-Xho have been described (Kaufman, R.J., 1985 , Proc. Natl. Acad. Sci. USA 82.:689-693) and include the adenovirus VA genes, the SV40 origin of replication including the 72 bp enhancer, the adenovirus major late promoter including a 5' splice site and the majority of the adenovirus tripartite leader sequence present on adenovirus late mRNAs, a 3' splice acceptor site, a DHFR insert, the SV40 early polyadenylation site (SV40), and pBR322 sequences needed for propagation in E. coli.

Plasmid pMT2 Cla-Xho is obtained by EcoRI digestion of pMT2-VWF, which has been deposited with the American Type Culture Collection (ATCC), Rockville, ND (USA) under accession number ATCC 67122. EcoRI digestion excises the cDNA insert present in pMT2-WF, yielding pMT2 in linear form which can be ligated and used to transform E. coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2 DNA can be prepared by conventional methods. pMT2CX is then constructed by digesting pMT2 with Eco RV and Xbal, treating the digested DNA with Klenow fragment of DNA polymerase I, and ligating Cla linkers (NEBiolabs, CATCGATG). This removes bases 2266 to 2421 starting from the Hind III site near the SV40 origin of replication and enhancer sequences of pMT2. Plasmid DNA is then digested with EcoRI, blunted as above, and ligated to an EcoRI adapter, 5' PO4-AATTCCTCGAGAGCT 3' 3 ׳ GGAGCTCTCGA 5' digested with Xhol, and ligated, yielding pMT2 Cla-Xho, which may then be used to transform E. coli to ampicillin resistance. Plasmid pMT2 Cla-Xho DNA may be prepared by conventional methods. Plasmid DNA from this subclone is transfected into COS cells by the DEAE-dextran procedure. [Sompayrac and Danna PNAS 73:75757578 (1981); Luthman and Magnusson, Nucl,Acids Res. 11: 12951308 (1983)] Serum-free 24 hr. conditioned medium is collected from the cells starting 40 - 70 hr. post-transfection.

Example

Biological Activity of Expressed Bone Inductive Factor

A. BMP-1

To measure the biological activity of the expressed bone inductive factor (hBMP-1) obtained in Example VI above. The factor is partially purified on a heparin sepharose column. 4 ml of transfection supernatant from 1 100 mm dish is concentrated approximately 10 fold by ultrafiltration on a YM 10 membrane and then dialyzed against 20mM Tris, 0.15 M NaCL, pH 7.4 (starting buffer) . This material is then applied to a 1.1 ml Heparin Sepharose column in starting buffer. Unbound proteins are removed by an 8 ml wash of starting buffer, and bound proteins, including BMP-1, are desorbed by a 3-4 ml wash of 20 mM Tris, 2.0 M NaCL, pH 7.4.

The proteins bound by the Heparin column are concentrated approximately 10-fold on a Centricon 10 and the salt reduced by diafiltration with 0.1% trifluoroacetic acid. The appropriate amount of this solution is mixed with 20 mg of rat matrix and then assayed for in vivo bone and cartilage formation as previously described in Example III. A mock transfection supernatant fractionation is used as a control.

The implants containing rat matrix to which specific amounts of human BMP-1 have been added are removed from rats after seven days and processed for histological evaluation. Representative sections from each implant are stained for the presence of new bone mineral with von Kossa and acid fuschin, and for the presence of cartilage-specific matrix formation using toluidine blue. The types of cells present within the section, as well as the extent to which these cells display phenotype are evaluated.

Addition of human BMP-1 to the matrix material resulted in formation of cartilage-like nodules at 7 days post implantation. The chondroblast-type cells were recognizable by shape and expression of metachromatic matrix. The amount of activity observed for human BMP-1 was dependent upon the amount of human BMP-1 protein added to the matrix. Table IX illustrates the dose-response relationship of human BMP-1 protein to the amount of bone induction observed.

Table IX

<td> IMPLANT NUMBER</td><td colspan="2"> AMOUNT USED</td><td> HISTOLOGICAL SCORE</td>

<td></td><td colspan="2"> (equivalent of ml transfection media)</td><td></td>

<td> 876-134-1</td><td> 10</td><td> BMP-1</td><td> C+2</td>

<td> 876-134-2</td><td> 3</td><td> BMP-1</td><td> C+l</td>

<td> 876-134-3</td><td> 1</td><td> BMP-1</td><td> c +/-</td>

<td> 876-134-4</td><td> 10</td><td> MOCK</td><td> c -</td>

<td> 876-134-5</td><td> 3</td><td> MOCK</td><td> c -</td>

<td> 876-134-6</td><td> 1</td><td> MOCK</td><td> c -</td>

Activity was scored on a scale from 0(-) to 5.

Similar levels of activity are seen in the Heparin sepharose fractionated COS cell extracts. Partial purification is accomplished in a similar manner as described above except that 6 M urea was included in all the buffers.

Theraputic amounts of each of the bone inductive factors of the present invention, including variants thereof as described above, may be used individually for repairing fractures and other conditions related to bone defects or peridontal diseases. Furthermore, each of the bone inductive factors of the present invention may be used in combination with one or more of the other bone inductive factors of the invention. For instance hBMP-1 may be used in combination with either hBMP-2 Class I, Class II, BMP-3, or any combination of these. Similarly, hBMP-2 Class I, hBMP-2, or BMP-3 may be used with any combination of bone inductive factors of the invention. Bone inductive factors or combinations thereof of the invention may be coadministered with other osteoinductive factors or other theraputic agents.

The bone inductive factors of the present invention may be used in conjunction with a variety of biomaterials which provide a theraputically suitable enviroment for the induction of bone growth.

Preferably the bone growth inductive factor compositions of the invention are used in conjunction with a matrix capable of delivering the factor composition to the site of bone damage, providing a structure for the developing bone and cartilage. It is preferred that the matrix be capable of being resorbed into the body. There are presently many different materials that are used for both hard and soft tissue replacement. The type of material used will depend on several factors and the choice is within the knowledge of those having ordinary skill in the art. The choice of material is based on, for example, biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. Similarly, the application of the osteoinductive factor will define the appropriate formulation. Potential matrices for osteoinductive factor may be biodegradable and chemically defined, such as but not limited to calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyanhydrides; biodegradable and biologically well defined, such as bone or dermal collagen, other pure proteins or extracellular matrix components; nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics; or combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics might also be altered in composition, such as in calcium-aluminate-phosphate and processing to alter for example, pore size, particle size, particle shape, and biodegradability.

As described above the dosage regimen will be determined by the attending physician considering various factors which modify the action of such a growth factor. The amount of bone that is formed is dependent on the amount and type of factor used and the type of matrix in the final compositon. The dosage regimen should be in the range of 10 -10⁶ng per gram of bone weight desired. The dosage may vary with the type of matrix

5S used in the reconstitution and the composition of BMP's. The addition of other known growth factors, such as IGF 1 (insulin like growth factor 1), to the final composition, may also effect the dosage.

The foregoing descriptions detail presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are believed to be encompassed within the claims appended hereto.