CA2017466A1 - Bone calcification factor - Google Patents

Bone calcification factor

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Publication number
CA2017466A1
CA2017466A1 CA 2017466 CA2017466A CA2017466A1 CA 2017466 A1 CA2017466 A1 CA 2017466A1 CA 2017466 CA2017466 CA 2017466 CA 2017466 A CA2017466 A CA 2017466A CA 2017466 A1 CA2017466 A1 CA 2017466A1
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Grant status
Application
Patent type
Prior art keywords
bcf
dna
sequence
cells
bone
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA 2017466
Other languages
French (fr)
Inventor
Michael C. Kiefer
Frank R. Masiarz
Philip J. Barr
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chiron Corp
Original Assignee
Michael C. Kiefer
Frank R. Masiarz
Philip J. Barr
Chiron Corporation
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Abstract

ABSTRACT

The isolation, identification and production by recombinant methods of bone calcification factor, a 22KD polypeptide, are disclosed. The peptide has calcification-inducing activity when implanted with matrix Gla protein into mammals.

Description

2~7~5~

BONE CALCIFICATION FArTOR

This invention relates to a cla~s of mature native mammalian proteins which initiates calcification and which is named herein as bone calc~fication factor (BCF). Representative of this class are human and bovine BCF, for which the full lenqth coding seguences are provided herein. The BCF is provided by isolation from bone sources and by synthesis using recombinant DNA technigues.

BACKGROUND OF THE INVENTION

It is known that demi~eralized bone matrix induces new bone formation when implanted in the soft ti6sue by a process generally designated as matrix induced bone formation ~see Urist, M.R., Science, 150: 893-899 (1965)). There have been nu~erous efforts to extract and identify the active material (or materials) which induces this procQss, and it has been generally referred to in the l$terature ag bone morphogenetic protein~s) (BMP). It $~ uncertain whether BMP iB a single material or a mixture o~
materials, and there does not ~ppear to be agreement among the investigators a8 to which ~ateri~l, iP any, is the bone morphogenetic protein.

The therapeutic use of BMP offers considerable advantages over use of traditional ~one graft '~17~g materials. While not intended to be limited by any theory, one hypothesis assumes that BMP transforms tissue cells into o~teoblasts (cells that manufacture bone). During a process that replicates normal human fetal development, ~MP-induced osteoblasts form cartilage which, over a period of several months, evolve into solid ~one. Ihus ~MP may be useful for replacing bone that has ~een destroyed ~y disease or accident, for use in treatment of scoliosis victims, for treatment of mal- or mis-formed bone, for use in healing of a fracture, etc.

It is thus an abject of the present invention to produce a functional bone calcification factor or a component thereof, which i8 a 22 XD protein identified by its entire amino acid sequence, which initiates calcification.

It is another object of the present invention to produce this biologically active 22 XD protein by recombinant DNA technology.

It is yet another object of the present invention to construct nucleic ac~d screening probes for isolation of the gene comprising the 22 XD BCF.

~t is yet another object of the present invention to provide an amino acid seguence of mature 22 KD BCF
which can be thus prepared by direct biochemical synthesis or from constituent amino acids by peptide synthesis, for example as by the Merrifield method, and particularly ~q ~e of automated peptide synthesis technDlogy.

These and other ob~ects of the invention will be apparent from the following description of the 2017~

preferred embodiments and from practice of the invention.

~UMMARY OF THE INVE~TION

The present invention provides a class of mature native mammalian proteins (the class is termed herein as BCF), represented by native human and bovine BCF
described herein, which initiate calcification ~n ~ivo which is important for formation of bone. The human and/or bovine BCF can be used to identify and isolate other mammalian BCF proteins which may or may not be homologous (in their nucleotide and amino acid sequences) to human or bovine BCF and which exhibit the BCF biological activity. It is recognized that there may be al.elic variations in BCF within a species, and such allelic variants are also within the scope of the class of proteins provided by the present invention.

The present invention further provides polypeptides which are analogs of BCF, such as BCF muteins, fusion proteins, comprising BCF or BCF domains, and BCF
fragments. The term fusion protein includes a protein comprising ~ complete 8CF seguence or a BCF
domain, and a heterologous N- or C-terminal seguence (such as a signal sequence or seguence which protects the protein from degradation). A BCF mutein is a protein substantially homologous to a nat$ve BCF
6eguence (e.~., a minimum of about 75%, 85%, 90% or 95% homologous) wherein at least on~ amino acid i~
different. A BCF fragment or domain i8 an amino acid seguence of sufficient length from a BCF protein such that it is identif~able a8 having been derived from such BCF protein. The origin of a particular peptide can be determined, for example, by comparing its sequence to those in public databases.

' The present invention further provides a 22 KD bone calcification factor having the human and bovine amino acid sequences shown in FIG. 1. The present invention also provides methods of preparing the 22 XD bone calcification factor (BCF) by recombinant DNA
technigues.

~he present invention pr~vides ~he DNA 6equence encoding BCF, which may be used to construct vectors for expression in host systems hy recombinant DNA
techniques.

The present invention also prov$des therapeutic compositions comprising BCF and matrix Gla protein (MGP) for initiating calcification and ~ethods for inducing calcification in vertebrates by introducing in vivo at the desired ~ite an effective calcifica~ion initiating amount of BCF and MGP. The identity of MGP was first reported by Price, Urist and Otawara in ~iochem. Biophys. Res. Comm. 117:765-~71 ~1983).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the DNA seguences and encoded amino acid sequences of human and bovine BCF including their ~ignal sequences;

FIG. lA depicts the ~mino acid sequence of human BCF
(hBCF) without its signal peptide, FIG. lB depicts the amino acid sequence of bovine BCF (bBCF) without its 6igDal peptide:

FIG. lC depicts the DNA sequence encod$ng hBCF
without its signal sequence;

FIG. lD depicts the DNA seguence encoding bBCF
without its signal ~eguence;

FIG. 2 illustrates t~e seguence of human BCF tryptic fragment no. 41, a t~o-fold degenerate 45-mer oligonucleotide probe (probe A), and a ~econd probe B, designed therefrom, consisting of 64 18-mers which are complementary to all possible codons shown.

FIG. 3 illustrates probe C (derived from clone 0st 3-7) and four BCF cDNA clones isolated from a bovine cDNA library (bbl.1-7) and two human osteosarcoma cDNA libraries (Ost 1-7, 0st 3-7 and 0st 3-17). The length, coding region (boxed) and partial restriction map of the clones is included. The NcoI and SpeI
~ites (~) are only present in the human BCF
sequences.

FIG. 4 illustrates oligonucleotide adapters (boxed) used to prepare BCF expression vectors which are secreted from yeast using the alpha-factor signal peptide.

FIG. 4A illustrates the junction between the ~CF-encoding DNA and promoter in an expression vector used to express unsecreted BCF in yeast.

FIGS. 5, 6 ~nd 7 are photomicrographs of the quadriceps pouches of ~ice 21 days after implantation of a composite of recombinant hBCF and MGP, showing initiation of calcification.

DETAILED DESCRIPTI~N OF THE INVE~IIQN

The BCF according tv the present ~nvention may be obtained, free of other osteoinductive associated factors, directly ~rom bone sources, by preparative 201r~L6~

peptide synthesis using chemical methods (such as the Merrifield synthesis method) or by recombinant DNA
technology.

As more particularly described in Example 1, BCF may be obtained ~y purification from human, bovine, or other vertebrate bone from partially purified extracts ~.g., U.S. Patent 4,795,804 and references cited therein) by preparative gel electrophoresi6 and electroelution of the 22 X protein.

BCF may also be obtained by recombinant DNA methods, such as by screening reverse transcripts of mRNA, or by screening genomic libraries from any cell. T~e DNA may also be obtained by simply synthesizing the DNA using commonly available techniques and DNA
synthesizing apparatus. Synthe~is may be advantageous because unique restriction sites may be introduced at the time of preparing the DNA, thereby facilitating the use of the gene in vectors containing restriction sites not otherwise present in the native source. Furthermore, any desired site modification in the DNA may be introduced by synthesis, without the need to further modify the DNA
by mutagenesis.

In general, DNA encoding BCF may be obtained fro~
human, bovine or other sources by constructing a cDNA
library froo mRNA lgolated from bones of the vertebrate; and screening with labeled DNA probes encoding portions of the human or chains in order to detect clones in the cD~A library that contain homologou~ ~eguences; or ~y polymerase chain reaction (PCR) amplification of the cDNA ~fro~ ~RNA) and 6ubcloning and screen~ng with l~beled DNA probe3; ~nd then analyzing the clones by restriction enzyme analysis and nucleic acid ~equencing so as to .

20~ 7~66 ~7-identify full-length clones and, if full-length clones are not present in the library, recovering appropriate fragments from the various clones and ligating them at restriction ~ites common to the clones to assemble a clone encoding a full-length molecule. Particularly preferred DNA probes are set forth in the accompanying example~. Any sequences missing from the library may be obtained by the 3' extension on the complementary mRNA of synthetlc oligodeoxynucleotides identified by screening cDNA ~n the library ~so-called primer extension), or homologous sequences may be 6up~1ied from known cDNAs derived from human or bovine seguences as shown ~n FIG. 1.

The practice of the present invention will employ, unless otherwise indicated, conventional molecular biology, microbiology, and recombinant DNA technigues within the 6kill of the art. Such techniques are explained fully in the literature. See e.g., Maniati~, Frit~ch ~ Sambrook, ~Molecular Cloning: A
~aboratory Manual~ (1982); ~DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985);
"Oligonucleotide Synthesis~ (M.J Gait ed. 1984);
"Nucleic Acid Hybridization" (B.D. Rames ~
S.J. Higgins eds. 1985); ~Transcription And Translation" (B.D. Hames ~ S.J. Higgins eds. 1984);
"Animal Cell Culture" (R.I. Freshney ed. 1986);
"Immobilized Cells And Enzymes~ (IRL Press, 1986);
B. Perbal, ~A Practical Gu~de ~o Molecular Cloning~
(1984).

In describing the pre6ent invention, the following terminology will be u6ed in accordance with the definitions 6et out below.

2~:~74~

The term "osteoinductive associated factors"
includes factors known in the art which are present in mammalian bone or other ma~malian tissue and tend to co-purify with BMP ~r ~XP activity. Such factors include proteins which have been isolated from bone having reported molecular weights of 34 RD, 24 XD, 18.5 KD, 17.5 KD, 17 XD, 16.5 RD, 14 RD (as cited in U.S. Patent No. 4,761,471), and 6 RD (reported by Price, P.A., et al., from ~NAS, 73, pp. 1447-1451, 1976).

A ~replicon" is any genetic element (~.~., plasmid, chromoso~e, virus) ~ha~ f~nctions as an aut~nomous unit of DNA replication in vivo; ~.e., capable of replication under its own control.

A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of th~ attached segment.

A "double-stranded DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its normal, double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter ~li~, in linear DNA molecules (Q.g., restr~ction fragments), viruses, plasmids, and chromosomes. In d~scussing the structure of part~cular double-stranded DNA molecules, ~equence~ ~ay be described herein according to t~e ~ormal convention of g$ving only the 6eguence in tn~ ~' to 3' dir~ction along the nontranscribed strand of DNA (~.e., the strand having a seguence h~mologous to the ~RNA).

A DNA "coding sequence" is that portion of a DNA
~equence, the transcript of which is translated into a polypeptide ~n v vo when placed under the control of appropriate regulatory ~equences. The complementary DNA strand will be understood to be that strand which is transcribed. The boundaries of the coding seguence are determined by a start codon at the 5' (amino) terminus and a translat$on stop codon at the 3' (carboxy) terminus. A coding seguence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA seguences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA 6eguences. A
polyadenylation signal and transcription termination seguence will usually be located 3' to the coding seguence.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding ~eguence. For purposes of defining the present invention, the promoter seguence is bounded at it~ 3' terminus by the translation ~tart codon of a coding seguence and extend~ up~tream (S' direction) to include the minimum number of base~ or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease Sl), as well as protein bind$ng domains (consensus 6equences) responsible for the binding of RNA polyme~se. Eucaryotic promoters will often, but not alway~, contain ~TATA~ boxes and ~CAT~ boxes.
Procaryctic promoters contain Shine-Dalgarno seguences $n addition to the -10 and -35 consensus 6eguences.

A coding sequence is "under the control" of the promoter ~equence in a cell when RNA polymerase which binds the promoter 6equence transcribes the coding sequence into mRNA which is then in turn translated S into the protein encoded by the coding sequence.

A cell has been "transformed" by exogenous DNA when such exogenou~ DNA has been introduced into the cell membrane. Exogenous DNA may or may not be integrated ~covalently linked) to chromosomal DNA making up the genome of the cell. In procaryotes and yeast, for example, the exogenous DNA may be maintained on an episomal element such as a plasmia. ~ith respect to eucaryotic cells, a stably transformed cell is one in which the exogenous DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells contain~ng the exogenous DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A ~cell line" is a clon~-of a primary cell that is capable of stable growth in vitro for many generation6.

Two DNA sequences are "substantially homologous~ when at least about 85S (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides match over the defined length of the DNA
~equences. Sequences that are 6ubstant$ally homologous can be identified in a Southern hybridization experiment under, for ex~mple, stringent conditions a~ defined for that particular system. Defining appropriatQ hybridization conditions i8 within the skill of the art. See, e.g., Maniati~ et al., ~upra: DNA Cloning, Vols. I
II, supra; Nucleic Acid Hybridization, ~E~.

2~17~S

A "heterologous" reqion of the DNA construct is an identifiable segment of DNA within a larger DNA
molecule that is not found in association with the larqer molecule in nature. Thus, when the heterologous region encodes a ~ammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the qenome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself i~ not found in nature (e.g., a cDNA
where the genomic coding sequence contains introns, or 6ynthetic 6equences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

A composition comprising ~A" (where "A" is a single protein, DNA molecule, vector, etc.) is ~ubstantially frQe of "B" (where "B" comprises one or more contaminating proteins, DNA molecules, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the c~mposition i6 ~An.
Preferably, ~A" comprises at least about 90~ by we$ght of the A+B species in the composition, most preferably ~t least about 99% by weight. It i8 also preferred that a composition, which i8 6ubstantially free of contamination, contain only a singlQ
molecular weight ~pecies having the activity or characteristic of the species ~f interest.

As more particularly described in the following exa~ples, human bnd bovine cDNA l$brar~es were initially probed for sequences encoding BCF sequences using labeled oligodeoxynucleotides whose seguences were based on a partial am~no a~id sequence determined from analysis of purified protein samples 2~7~

derived from bone described herein. However, it is realized that once being provided with non-chromosomal DMA encoding human and bovine BCF and their leades seguences as described ~erein, one of ordinary 6kill in the art vould recognize that other precisely hybridiz~ng probes may be prepared from the descri~ed sequence6 i~ order to readily obtain the remainder of ~he desired human or ~vine gene.

The non-chromosomal DNA provided by t~e pre ent $nvention is novel, since it is ~elieved that the naturally-occurring human and bovi~e ~enes (chromosomal) contain in~ron6 (transcri~ed sequences, the corresponding amino acids of which do not appear in the mature protein). Hence, the term "non-chromosomal n excludes the DNA sequence~ whichnaturally occur in the chromo~omes of human or bovine cells. The present invention ~lso en~ompasses the non-chromosomal cDNA sequenoes deri~a~le from the DNA
sequences disclo6ed herein.

Vectors are used to amplify the DNA which encodes the chains, either in order to p¢epare quantities of DNA
for further processing (clon~ vectnrs) or for expression of the chains te~pn~ssion vectors).
Vectors comprise plasmids, ~in~ses (~ncluding phage), and integr~table DNA fragme~*~ ~--, fragments that are integratable into the h~st genome ~y recombination. Cloning vectors need ~t contain expression control 6eguences. However, control seguences in ~n expression vector include a transcriptional pr~moter, ~n option~l Dperator sequence to control tr~nscription, a ~equence encoding suitabl~ rRNA ribosomal bind$ng sites (for prokaryotic expression), Dnd seguence~ which control termination of transcripkion and translation. The expression yector ehould pre~erably include a ?

selection gene to facilitate the stable expression of BCF and/or to identify transformants. However, the selection qene for maintaining expression can be 6upplied by a 6eparate vector in cotransformation systems using eukaryotic host &ells.

Suitable vectors generally will cont~in replicon (origins of replication, for use in non-integrative vectors) and control 6equences which are derived from species compatible with the intended expression host.
By the term "replicable" vector as used herein, it is intended to encompass vectors containing such replicons as well as vectors which are replicated by integration into the host genome. Transformed host cells are cells which have been transformed or transfected with vectors containing BCF encoding DNA.
The expressed BCF will be deposited intracellularly or secreted into either the periplasmic space or the culture supernatant, depend$ng upon the host cell selected and the presence of suitable proces~ing signals in the expressed peptide, ~.g. ho~ologous or heterologous signal sequences.

Suitable host cells are prokaryotes or eukaryotic cells. Prokaryotes include Gram negative or Gram positive organisms, for example ~. ÇQli or bacilli.
Eukaryotic cells include yeast, higher eukaryotic cells such as established cell lines of mammalian origin, or insect cells. Expression in insect cells may be accompll6hed using host cells and in~ect expression vectors as disclosed by luckow, Y.A., and Summers, M.B., Biotechnolooy 6:47-55 (1976).

Expre6sion vector~ for ho~t cell~ or~narily lnclude an origin of replication, a promoter located upstream from the BC~ coding sequence, together with a ribosome binding ~ite, a polyadenylation 6ite, and a transcriptional termination se~uence. Those o~
ordinary skill will appreciate that certain of these oeguences are not reguired for expression in certa~n hosts. An expression vector fGr use with microbes need only contain an origin of replicat~on recognized by the host, a promoter whic~ will function in the host and a selection gene.

An expression vsctor is constructed according to the present invention so that the ~CF coding sequence is located in the vector with the appropriate regulatory 6equences, the positioning and orientat~on of the coding seguence with respect to the control sequences being such that the coding sequ~n~e is transcribed under the "cont~ol~ of the control ~eguences (~--~
RNA polymerase which binds to the DNA molecule at thecontrol sequences transcribes the coding 6equence).
The control sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding seguence can be cloned directly into an expression vector which already contains the control seguences and an appropriate restriction site. For expression of ~CF in procaryotes and yeast, the control sequences will necessarily be heterologou~ to the coding sequence. If the host cell is a procaryote, it is also necessary that the coding seguence be free of introns (~.g., cDNA). If the selected host cell is ~ mammalian oell, the control seguences can be heterologous or homologous to the BMP coding sequence, and the coding sequence can either be genomic DNA containing ~ntrons or cDNA.
Either genomic or cDNA cod~ng ~quences can be expressed in yeast.

Expression vectors must contain ~ promoter which is recognized by the host organism. Promoters commonly known and available which are used in recombinant DNA
construction include the ~-lactamase (penicillinase) and lactose pr~ot~r systeme~ a tryptophan (trp) promoter syste3 and the tac promoter. While these are commonly used, other ~nown microbial promoters are ~uitable.

In addition to prokaryotes, eukaryotic cells ~uch as yeast are transformed ~ith BCF encoding vector6.
Saccharomyce$ cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other 6trains are commonly avallable ~a useful herein. Yeast vectors generally will contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding BCF, seguences for polyadenylation and transcription termination, and a selection gene.

Suitable promoting sequences in yeast vectors include the promoters for the glycolytic enzymes such as enolase, 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, ph~sphofructokinase, glucose-6-phosphate isomeras2, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate i~omerase, phosphoglucose isomeraee, and glucokinase.

Other yeast promoters, wh~ch have the additional advantage Or transcription controlled by growth conditions are the pro~oter regions for alcohol dehydrogenase 1 or 2, isocytochrom~ C, ac~d phosphatase,-~6 wQll ~s enzy~es responsible for maltose an~ gal~ctose utilizat$on.

Higher eukaryot~c ~ell cultures may be used, whether from vertebrate or invertebrate cells, including 6 ~

insects, and the procedures of propagation thereof are known. See, for example, Tissue Culture, Academic Press, Rruse and Patterson, editors (1973).

Suitable host cells for expressing BCF in higher eukaryotes include: ~onXey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651): baby hamster kidney cells (BHX, bTCC CRL 10); chine6e hamster ovary-cells-DHFR (described by Urlaub and Chasin, PNAS (USA) 77: 4216 ~1980)); mouse sertoli cells (TM4, Mather, J.P., Biol. Reprod. ~: 243-251 (1980)); monkey ~idney cells (CVI ATCC CCL 70);
african green monkey kidney cells (VER0-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL
2); canine kidney cells (MDCX, ATCC CCL 34): buffalo lS rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells ~Hep G2, HB 8065): mouse mammary tumor (MMT 060652, ATCC CCL
51); rat hepatoma cell~ (HTC, M1, 54, Baumann, M., et al., J. Cell Biol. 85: 1-8 (1980)) and TRI cells (Mather, J.P., et al., Annals N.Y. Acad. Sci. 383:
44-68 (1982)). Co nonly used promoters are derived from polyoma, ~denovirus 2, and Simian Virus 40 (SV40). It will be appreciated that when expressed in mammalian tissue, the recombinant BCF may h~e higher molecular weight due to glycosylation. It is therefore intended that partially or completely glycosylated forms of BCF having ~olecular weights greater than that provided by the amino acid backbone are within the ~cope of this inventlon.

A number of ~rocaryotic expression vectors are ~nown in the art. See, e.g., U.S. Patent Nos. 4,440,859:
4,436,815; 4,431,740; 4,431,739; 4,~28,941;
4,425,437; 4,418,149; 4,411,994; 4,366,246;
4,342,832; ~ç~ Q U.R. Pub. Nos. GB 2,121,054;
GB 2,008,123; GB 2,007,675; and European Pub.

2Q~746~

No. 103,395. Preferred procaryotic expression systems are in E. ÇQli- Other preferred expression vectors are those for use in eucaryotic systems. An exemplary eucaryotic expression system is that employing vaccinia virus, which i well-~nown in the art. See. e.g., Macket et ~1. (1984) J. virQl.
49:857; "DNA Cloning," Vol. II, pp. 191-211, ~upra;
PCT PUb. No. WO 86/07593. Yeast exprecsion vectors are known in the art. See, ~.g., U.S. Patent Nos. 4,446,235; 4,443,539: 4,430,428: see also European Pub. Nos. 103,409; 100,561: 96,491. Another expression system is vector pHSl, which transforms Chinese hamster ovary cells. The use of the vector is described in PCT Pub. No. WO 87/02062, the disclosure of which is incorporated herein by reference.

Mammalian tissue can be cotransformed with DNA
encoding a 6electable marker such as dihydrofolate reductase (DHFR) or thymidine kinase ~nd DNA encoding BCF. If wild type DHFR protein i5 employed, it is preferable to select a host cell which is deficient in DHFR, thus permitting the use of the DHFR coding ~eguence a~ marker for successful transfection in hgt~ medium, which lacks hypoxanthine, glycine, and 2S thymidine. An appropriate host cell in t~is case is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasin, 1980, roc. Nat.
Acad. Sci. ~SA) 77: ~216.

Recently, expression vectors derived ~rom Bacclovirus for use in insect cells have become ~nown in the art.

Dependinq on the expression system ~nd host selected, BCF i6 produced by growing host cells transformed by an expression vector described above 20~7466 under conditions whereby the BCP protein is expressed. The enzyme protein is then isolated from the host cells and purified~ If the expression system secretes the enzyoe i~to growth media, the protein can be purified directly ~rom cell-~ree media. If the BCF protein i~ not secreted, it $s isolated from cell lysates. the selection of the approprlate growth conditions and recovery methods are within the skill of the art.

The recombinantly made BCF is recovered from transformed cell~ in accordance with p~ 6e ~nown procedures. Preferably, an expression vector will be used which provides for secretion of BCF from the transformed cells, thus the cells may be separated by centrifugation. The BCF generally is purified by general protein purification techniques, including, but not limited to, size exclusion, ion-exchange chromatography, HPLC, and the like.

Once a coding seguence for BCF has been prepared or isolated, it can be cloned into any suitable vector or replicon and thereby ~ainta~ned in a composition which is ~ubstantially free of vectors that do not contain a BCF coding sequence ~ ., free of other library clones). Numerous cloning vectors are known to those of skill in the art. Examples of recombinant DNA vectors for cloning and host cells which they can tranQform $nclude the various bacteriophage lambda vectors ~E. ÇQ10 ~ pBR322 (E. ÇQl~), pACYC177 (~. ÇQlO , pKT230 (gram-negat$ve bacterla), pGV1106 (gra~-negative bacteria), pLaFRl (gram-negative bacter$a), pME290 ~non-E. ÇQli qra~-negative ~acterla), pflV14 ~. ÇQl~ and Baclllus subtilis), pBD9 (BacilluQ), pIJ61 (Streptomyces), pUC6 (Streptomyces), actinophage, ~C31 (Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces), and bovine papillo~a virus (mammalian cells). See generallv, DNA Cloning:
Vols. I ~ upra: T. Maniatis et ~1-, EYE~;
B. Perbal, supra.

It i8 further intended that calcification-initiating fragments of BCF are within the scope of the present invention. Such active fragments may be prGduced, for example, ~y pepsin digestion o~ BCF. The active fragments may be identified by the ln ViYo and/or ~n y~5~Q assays described hereinbelow.

Alternatively the BCF may be made by conventional peptide synthesis using the principles of the Merrifield synthesis and preferably using commercial automatic apparatus designed to employ the methods of ~5 the Nerrifield synthesis. Peptides prepared using the Merrifield synthesis may be purified using conventional a~finity chromatography, gel filtration and/or RP-~PLC.

FIG. 1 shows the aligned nucleotide and deduced amino acid ~equences for both bovine and human BCF for maximum amino acid seguence identity. Amino acids not conserved in both species are boxed. The put~tive initiation codon i6 located at position -~7 followed by a ~tretch of amino acids sbowing strong hydrophobicity characteristics of signal peptides. A
putati~e signal peptide cle~vage ~ite iB indicated by the arrow. The putative ~ature proteins begin at positi~n l(Gln), and contain 183 amino ~cids, of which 96.2~ ~re identical. The derived molecular weight of human BCF i8 21,967 and for bovine BCF iB
21,984. The underlined ~equences are those from which the oligonucleotide probes are deriv~d.

2 0 ~ 6 ~

Substantially pure BCF, higher molecular glycosylated forms thereof, or active fragments thereof, or the nontoxic salts thereof, combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, may be administered to mammals, including humans, e~ther intravenously, subcutaneously, percutaneously, intramuscularly or orally.

Such proteins are often a~ministered in the form of pharmaceutically acceptable nontoxic salts, ~uch as acid addition salt6 or metal complexes, .g., with zinc, iron or the li~e twhich are considered as salts for purposes of this appli~ation). Illustrative of such acid addition salts are hydrochlor~de, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succin~te, malate, ascorbate, tartrate and the like. If the active ingrediQnt is to be administered in tablet form, the tablet may contain a binder, such a~ tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.
If administration in liguid form i~ desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.

Pharmaceutical compositions will usually contain an effective amount of BCF in con~unction with a conventional, pharmaceutically acceptable carrier.
Ihe dosage w~ll vary depending upon the specific purpose for which the protein i8 being administered, and dosage levels in the range of ~bout 0.1 pg to about 100 milligrams per Kg. of body weight may be used.

2~-~ 7~66 Implants of recombinant BCF, when mixed with matrix Gla protein (MGP) (see Example 7), will initiate calcification. The BCF may ~e either the human or bovine form or mixtures thereof. Similarly, the MGP
may be any mammalian form thereof, preferably human, bovine or mixtures thereof. ~atrix Gla protein (MGP) may be isolated in the course of preparation vf bone msrphogenetic protein (BMP) from de~ineralized gelatinized bovine cortical bone ~y the methods of Urist et ~1. [See: Price et al., proc. Natl. Acad.
Sci. USA 73:1447, 1976; Urist, M.R., Huo, Y.g.
Brownell, A.G., Hohl, W.M. Buyske, J., Lietze, A~o Tempst, P., Humkapillar, M., and ~e~ange, R.J.:
Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc. Natl. Acad.
~Çi. 81:371-375, 1984 and Urist, M.R., Chang, J.J., Lietze, A., Huo, Y.K., Brownell, A.G., and DeLange, R.J.: Methods of preparation and bioassay of bone morphogenetic protein and polypeptide fragments. In:
Barnes, D., and S~rbaska, D.A. (eds.): Methods in Enzvmolooy, vol. ~46. New York, Academic Press, 1987, pp. 294-312]. A preparation containing HGP is first separated from other bone matrix prote$n by hollow fiber ultrafiltration through a 10 K pore-size filter. Under dissociated conditions in 6M urea and 0.02 M edetic acid (EDTA), the MGP assumes an elongated structure in which proteins with 14 to 15 R
molecular weiqht (Mr) pass through ~ 10 R filter.
The MGP iB further purified by ion exchange chro~atography (Berg, R.A. In: Methods in Enzymology, 1982, vol. 82:372-398).

Furthermore, to initiate calcificatlon BCF and ~GP
may be mixed with any combination of one or more other proteins, particularly, with one or more other proteins derived from bone. Such mixtures may not ~7~

only initiate calcification, but may 8160 induce cartilage formation and bone growth.

Implants of mixture~ of 8C~ and MGP induce calcification in the quadriceys compart~ent. The BCF
cDNA may al80 be utili2ed in a diagnostic test for identifying subjects having defective ~CF-genes, defective BCF or autoantibodies directed against BCF:
or to detect levels of BCF, ~hich ~ay be an indication of osteoporosis.

Preparations of BCF may be as6ayed ~n yivo according to the method ~es~ri~ed by ~rist et ~1., Methods in Enzymolo~y (D. Barnes and D.A. Sirbaska, Eds.), vol. 146, pp. 294-312, Academic Press, N.Y. (1987), and in Yitro by the ~ethod of Sato and Urist, Clin.
Orthop., 183:180-187 (1984) as modified by Kawamura and Urist, ev. Biol., 130:435-442 (19~8), all of which are incorporated by ref~ence herein.

It is preferred ~hat the BCF ~e admixed with matrix Gla protein ~MGP) t~ for~ a de~lvery system comprising the6e two proteins. The amount of MGP in the composition is not believed ~o ~e critical and, for convenience, equal porti~ Df BCF and MGP may be used in dosages $n the range o~ about 0.1 ~g (combined weight of BCF and ~ to 100 mg/Kg. body weight.

The BCF and MGP ~ay be implant~a as a t~me-rolease composition encapsulated, for instance, in liposomes or other time-selea~e m~hranes, natur~l or ~ynthetic, which are a~sorbable by the host ~ub~oct.
m e purification protocol~, described in detail below, allow for the f~r~t time the purification of native BCF $n su~ficient quantity and at a high enough purity to permit accurate amino acid ~17~

sequencing. The ~mino acid sequences derived from the purified BCF ~llow for the design of probes to aid in the isolation of native BCF nucleic ac$d 6equence, or the design of synthetic nucleic acid seguences encoding the amino acid seguence of BCF.

Specific anti-sera or monoclonal antibodies (described below) can be made to a synthet~c or recombinant BCF peptide havinq the sequence or fragments of the seguence of amino acid residues, ~uch as those shown in Figures lA or lB. An example is the tryptic fragment shown in FIG. 2, and antibodies thereto can be used to immunoprecipitate any BCF present in a selected tissue, cell extract, or body fluid. Purified BCF from this source can then be sequenced and used as a basis for designing specific probes as described above. Antibodies to other regions that diverge from ~nown BCF can also be used. Also useful as antigens are purified native or recombinant BCF.

As mentioned above, a DNA sequence encoding BCF can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the BCF amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
The complete sequence i~ assembled from overlapping oligonucleotides prepared by ~tandard methods ~nd asse~bled into a complete coding seguence. ~Ç, ~.g., Edge (1981) Nature ~92:7S6; Nambair, et ~1.
(1984) Science ~ 1299; Jay et ~1. (1984) ~. Biol.
Ç~ L:6311.

Synthetic DNA sequences allow convenient construction of genes which will express BCF analogs or "muteins~.
Alternatively, DNA encoding muteins can be made by &

site-directed mutagenesis of native BCF genes or cDNAs, and muteins can be made directly using conventional polypeptide synt~esi~. ~uteins altered, for example, by the substitutio~ ~f acidic residues ~e.g., Glu or Asp) could have reduced activity toward membrane-bound or complex substrates or have anti-~ense therapeutic uses for overproduction of BCF.

Site-directed mutagenesis i8 conducted using a primer synthetic oligonucleotide complementary to a sin~le stranded phage DNA to be mutagen~zed except for limited mismatching, representing ~he desired mutation. ~riefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage.

Theoretically, 50% of the new plagues will contain the phage hav$ng, as a single strand, the mutated form: 50% will have the original 6equence. The resulting plagues are hybridized ~ith kinased synthetic primer at a temperature which permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plague~ which hybridize with the probe are then picked, cultured, and the DNA
recovered.

Native, recombinant or synthetic B~F peptide- (~ull length or ~ubunits) can be u~ed to produce both polyclonal and monoclonal hntibodies. IY polyclonal antibodies ~re desired, purified 8CF peptide is used to i~munize a selected mammal (e.~., mouse, rabbit, goat, horse, etc.) and serum from the immunized 2 ~

animal later collected and treated according to known procedures. Compositions containing polyclonal antibodies to a variety of anti~en~ in addition to BCF can be made ~ubstantially free of antibodies which are not anti-BCF by ~mmunoaffinity chromatography.

Monoclonal anti-BCF antibodies can also be readily produced by one skilled in the art form the disclosure herein. The general me~hodology for making monoclonal antibodies by hybridomas is well known. I~mortal, antibody-producing cell lines can also be created by techniques ot~er t~an fusion, such as direct transformation of B lymphocytes w~tb oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et ~1-, "~ybridoma Techniques" (1980); Bammerling et ~1., "Honoclonal Antibodies And T-cell Hybridomas" (1981): Xennett ç~
~1., "Monoclonal Antibodies" ~1980): Ç~ al80 U.S.
Patent Nos. 4,341,761: 4,399,121: 4,427,783:
4,444,887; 4,451,570: 4,466,917; 4,472,500:
4,491,632: 4,493,890.

Panels of monoclonal ant$bodies produced against BCF
peptides can be ~creened for various propertie~:
i.~., isotype, epitope, affinity, etc. Of particular interest are monoclonal antibodies that ne~tralize the activity of BCF. Such monoclonals can be readily identified in ~CF activity assays. High affinity antibodies are also useful ~n immunoaffinity purification of native or recombinant BCF.

Antibodies to ~CF forms de~ribed herein (both polyclonal and monoclonal~ ~ay b4 u~ed to inhibit or to reverse arterial calc~ficatio~. An ~ppropriate therapeutic method would be to treat the patient with an e~fective dose of anti-BCF antibodies through a ~7~

conventional intravenous route. In the treatment of local, acute inflammation, treatment with anti-BCF
antibody wo~ld ~e indicated, perhaps by intramuscular injection. The~e compssitions may also be useful in targeting ~a~ious forms of tumors, since tumors are known to sQ~eti~s calcify, suggesting the presence of BCF. 2CF antagonists, such as BCF muteins, could also be used in place of an~ibodies.

The determination of the appropriate treatment regi~en (~.e~, dosage, freguency of administration, systemic YS. lor~l, etc.) is within the ~kill of the art. For administration, the antibodies will be formulated in a unit dosage in;ectable form (solution, suspension, emulsion, etc.) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are usually nontox$c and non~herapeutic. Examples of such vehicles are water, saline, Ringer'~ ~olution, dextrose ~olution, and Hank's solution. Nonagueo~s vehicles ~uch as fixed oils and ethyl oleate may also be used. A preferred vehicle is ~% ~w/w) human albumin in saline. The vehicle ~ay contain minor amounts of additives, ~uch as substances that enhance isotonicity and chemical stability, ~.g., buffers and preservatives. The antibody is typically formulated in such vehicle~ at concentrations of abcut 1 ~g~ml to 10 mg/ml.

Anti-9CF antibodies will also be useful ln d~agnoctic applications. The present invention contemplate~ ~
method, particularly a diagnostic method, ln which a ~ample from a human (or other mammal) iB provided, and the amount of BCF i8 quantitatively measured in an assay. For example, employing anti-BCF
antibodies in a quantitative ~mmunoassay could be used to detect genetic deficiency in BCF. Antibody specific for BCF could be formulated into any ~7~6 conventional immunoassay format: e.g., homogeneous or heterogeneous, radioimmunoassay or ELISA. The various formats are well known to tho~e skilled in the art. See, e.a., "Immunoassayh A Practical Guide" (D.W. Chan and M.T. Perlstein, eds. 1987) the di~clo~ure of which i6 incorporated herein by reference.

In general, recombinant production of B~F can provide co~positions of that BCF ~ubstantially free of other proteins having osteoinductive associated functions.
The ability to obtain high levels of purity i8 a result of recombinant expression systems which can produce ~CF in substantial quantities vis-a-vis ~n vivo sources. Thus, by applying conventional techniques to recombinant cultures, BCF compositions can be produced that are substantially ~ore pure than the compositions available from bone sources.

Purified BCF will be particularly useful as a tool in the design and screening of calcification inhibitors. First, milligram amounts of the material are obtainable according to the present invention.
Milligram amounts are capable of crystallization to permit three dimensional studies using ~-ray diffraction and computer analysis. Thi~ may permit deduction concerning the shape of the mDlecule, thus defining proper shapes for substances u~able as inhibitors of the activity normally exhibited by BCF.
Generally, antagonists have been ~peptides" whose interactions with a factor which i~ inhibited are stabilized by ~odification of the ~esidues~
participating in the peptide bond so as to enhance the ability of the "peptide" to interact specifically with converting factor. ~hus the peptide bond ~oins specifically chosen carboxylic acids and amines (not necessarily amino ~cids).

- 2 ~ ~ r~

These "peptides~ are configured in a three dimensional array 60 ~ to complement the contour~ of *he intended target, converting enzyme. A similar lock and ~ey spatial arrangement may result from ~olecules designed c~mplementary to the surface contours of the BCF of t~e invention. It i8 understood tbat "surface~ includes convolutions which may face inward, and specifically includes the active site. Furthermore, "complementary" is understood to mean that, in addition to ~patial conformations which ~fit", interactions between the protein and the molecule which matches its surface contour~ are attractive and positive. T~ese interactions may be hydrogen bonding, ionic, or hydrophobic affinity.

Accordingly, the invention contemplates peptide antagonists or agonists (2-15 amino acid6) to BCF
which are characterized by three dimensional contours complementary to the three dimens~onal contours on the surface of recombinant BCF. By peptide in this context i6 meant that the antagonist or agonist contains carboxylic acid amide bonds correspondinq to one less than the number of resldues. The carboxylic acid and amine participants need not be ~-amino acids.

Second, even without the assistance of a three dimensional structure determination, purified BCF of the invention is of significance as a reagent in screening BCF inhibitor~ ~n vitro as an 8~ hQQ
approach to evaluation. lmpure BCF preparation6 currently available yield confusing data due to the impact of the impurities on the test results. For example, cont2~inants ~hich turn out to be themselves inhibitors, activators, or substrates for BCF may interfere with the evaluation. Thu8, a substantial 3S improvement in current screening techn$ques for BCF

6 ~

inhibitors would be effected by the availability of the purified BCF protein.

It will be understood that this description and disclosure of the invention i8 intended to cover all S changes and m~difications of the invention which are within the spirit and scope of the invention. It is within the knowledge of the art to insert, delete or substitute amino acids within the amino acid sequence o~ a BCF without fiubstantially affecting the calcification and bone growth inducing activ~ty of the molecule. The invention i~ expressly ~tat~d to be broad enough to include intentional deletions, add$tions or substitutions. Furthermore, lt is recognized that one skilled in the art could lS recombinantly produce such modified proteins.

Native, reco~inant or synthetic BCF peptides (full length or subunits~ can be further used to produce both polyclonal and monoclonal antibodies. If polyclonal ~ntibodies are desired, purified BCF is used to i~munize a selected mammal ~e.g., mouse, rabbit, goat, horse, etc.) and serum from the immunized animal later collected and treated according to known procedures. Compos$tions containing polyclonal antibodies to a variety of ant$gens in addition to BCF can be made ~ubstantially free of antibodies which are not anti-BCF by immunoaffinity chromatography.

Monoclonal ant~-BCF antlbodies can also be readily produce~ by one skilled ln t~e art from the disclosure herein. The general methodology for ~ak$nq monoclonal antibodies by hybridomas i8 well known. Immortal, antibody-producing cell lines can also be created by techn~ques other than fusion, such as direct transformation of B lymphocytes with 2 ~

oncogenic DNA, or transfection with Epstein-Barr virus. See, ~.q., M. Schreier et al., ~Hybridoma Techniques" (1980); Hammerling et ~1-, "Monoclonal Antibodies And T-cell Hybridomas" (1981); Kennett et ~1., ~Monoclonal Antibodies" (1980); see ~l~Q U.S.
Patent Nos. 4,341,761; 4,399,121; 4,427,783;
4,444,887, 4,451,570; 4,466,917; 4,472,500;
4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against BCF
peptides can be screened for various properties;
.e., isotype, epitope, affinity, etc. Of particular interest are monoclonal antibodie~ that neutralize the activity of BCF. Such monoclonals can be readily identified in BCF activity assays. High affinity antibodies are also useful in immunoaffinity purification of native or recombinant BCF.

Anti-BCF antibodies will also be useful in diagnostic application~. For example, bone isolated ~rom osteoporosis patients may show that it i6 deficient in BCF. Thus, the present $nvention contemplates a method, particularly a diagnostic method, in which a bone 6ample from a human tor other mammal) is provided, and the amount of BCF i~ guantitatively measured in an assay. Antibody ~pecific ~or BCF
could be formulated into any conventional immunoassay format; e.g., homogeneous or heterogeneous, radioimmunoassay or ELISA. Tho variou~ formats are well known to tho~e ~killed in tho art. Seo, ~.g., ~ unoassay: A ~ractical Gu~de~ (D.W. Chan and M.T. Perl~tein, eds. 1987) the di6closure of wh~ch iB
incorporated herein by reference. Quantitative assays other than immunoassays could al~o bo u~ed to measure the relative levels of BCF compared to a s$andard or prior observed BCF level in a patient.

, ~7~

rhe following examples are provided by way of illu~tration but are not intended to limit the invention in any way.

~equence Analysis of BCF

The 22K proteins of interest, partially puri~ied from human and bovine sources as descr~bed by Urist, et ~1-, Proc. Nat. Acad. Sci. ~a, ~1, 3?1-375 (1984), were further purified to homogene~ty by preparative gel electrophoresis and electroelution (M.W.
Hunkapiller, E. ~ujan, F. Ostrander and L.E. Hood, Methods in EnzYmolooy, ~1: 227-236 (1983)). This purification showed that the initial partially purified sample6 contained, in addition to the 22R
BCF, other mammalian proteins at 34K, l9K, 14R and 6X. After precipitation with acetone (W. H.
Xonigs~erg and L. Henderson, Methods in Enzymology, ~: 254-259 ~1983)) and quantitation by amino acid analysis (B~A. Bidlingmeyer, S.A. ~ohen and T.~.
Tarvin, JournaL Qf Chromat~araphv, 336: 93-104 (lg84)), the material was reduced under denaturing conditions with 2-mercaptoethanol and cysts~ne residues were derivatized with 4-vinyl-pyridine (H.
Friedman, L.G. Rrull and J.F. Cavins, Journal of Bioloaical Chem~6trv, ~ 3868-3871 11970)). After exhaustive dialysi6 to remove the ~enaturant, proteln recovery was a~sessed by a repetition o~ ~mino acld analysi6. The protein~ were digested with TPCR-trypsin ln the pre~ence o~ 2H ~re~ to gensrat~
unblocked peptide fragment~ ~u$tab1e for ~equ~nce analys$~ (G. Allen, Sequencina o~ Proteins ~nd Peptides, page~ 51-62 (1981), Elsevier/North Nolland Publishing Company, Amsterdam, Holland). Product~ of the digestion were resolved by reverse-phas0 high performance liquid chromatography using gradient~ of 2 ~

~cetonitrile or acetcnitrile/isopropanol in aqueous trifluoroacetic acid tJ.E. Shlvely, Met~ods of Protein Microcharacterization, pages 41-87 (1986), Humana Press, Cl~fton, New Jer6ey). Peptide fractions were sub~ec~ed to automated E~m~n degradation using an kpplied Biosystems ~70A protein 6eauencer (M.W. Hunkapiller, ~.M. ~ewick, W.J. Dreyer and L.E. Hood, Methods in Enzvmolooy, ~1: 399-413 ~1983)). The phenylthiohydantoin amino ~cid derivatives were identified by chromatography on an Applied Biosystems 120A PIH analyzer (M.W.
Hunkapiller, Applied Biosvstems, User Bulletin Number 14 (1~85), Applied Biosystems, Foster City, California). The hBCF sequence determined by this method is confirmed by the ~equence deduced from the human cDNA in FIG. 1.

RNA I601ation mRNA was isolated ~rom fresh 7-month old calf bones (obtained from Rancho Veal Meat Packers, Petaluma, CA) or from human osteosarcoma cells.

Calf femur mids~afts were scraped free of connective tissue and ~arrow, broken into cQarse fragments, and frozen at -80'C. ~uman osteosarcoma cells were also frozen ~t -80'C. RNA was i601ated ~ro~ both the frozen ti~sues by the guanidinium thiocyanate/C6Cl method (Maniat~s, T., Fr$tscb, E.F. and 8ambrooX, J.
~olecular Clonina: A Laboratorv Manual (Cold Spring Harbor Lab., Cold Spring Harb~r, NY ~1982); Freeman, G.J., Clayberger, ~., DeKruyf~, R., Rosenblum, D.S.
and Cantor, H. Proc Natl. Ac~d. Sci.: ~SA, 80: ~094-4098 (1983)). An Qsterizer was used to pulverize the bovine tissue directly in the guanidinium thiocyanate extraction solution. Poly~A)+ RNA was .. : ' ' .

~01746S

purified by a 6ingle fractionation over oligo~dT?-Cellulose ~Maniatis, ~ al., supra).

Construction of the cDNA Libraries Fir~t strand cDNA was synthesized from bovine matrix or human osteosarcoma poly(A)+ RNA using conditions similar to Okayama and Berg (Okayama, H. and Berg, P.
Holec. and Cell Biol. l: 4094-4098 (1983)). About 5~g of poly(A)+ RNA in 20~1 5mM tris-hydrochloride (pH 7.5) was heated to 65-C for 3 min, then quick cooled on wet ice and immediately ad~usted (at room temperature) to contain 50mM Tris-hydrochloride (pH
8.3 at 42-C), 8mM ~gC12, 30mM XCl, lOmM
dithiothreitol, 2mM each of dATP, dGTP, dTTP ~nd t~-32P]dCTP(-300cpm/pmol), 60 units RNasin, and 2.5 ~g oligo (dT)12-18 (total volume 40 ml). The reaction was initiated by the addition of 50-60 units of cloned moloney murine leukemia virus reverse transcriptase and continued for 60 min at 42-C.
Double-stranded [ds) cDNA synthesis and EcoRI linker addition were performed by two different methods.
Initially, the second cDNA strand was synthesized by the method of Wickens et ~1. (Wickens, H.P., Buell, G.N. and Schi~ke, R.T. J. Biol. Chem. ~ 2483-2495 (1978)). The hairpin loop was removed from the ds cDNA by SI nuclease, methylated ~ith EcoRI methylase, made blunt-ended with T4 DNA polymeraQe, ligated to phosphorylated EcoRI llnkers and flnally digested with Eco~I (Maniatis, et ~1., su~ra). Later, the ~econd cDNA strand was synthesized by the method o~
Gubler and Hoffman (Gubler, U. and Hoffman, B.J. Gene 2S: 263-269 (1983)) as modified by Aruffo and Seed (Aruffo, A. and Seed, B. Proc. Natl. Acad. 8ci.: USA
74: 8573-8577 (1987)). The ds cDNA was then ligated to asymmetrically (hemi) pho~phorylated EcoRI
adapters (see oligonucleotide synthesis) as described 2 Q ~

by Aruffo and Seed, supra, phosphorylated with T4 polynucleotide kinase (Maniati6, et ~1., supra), adjusted to 0.5M NaCl, 25mM EDTA ~nd heated at 75-C
for 15 ~in to inactivate the polynucleotide kinase.
The ds cDNA prepared by both procedures was separated from unligated linkers/adapters ~y chromatography on Biogel A-15m and recovered by ethanol precipitation.
cDNA was ligated to ~ZAP arms (Str?tagene) with T4 DNA ligase (New England ~iolabs) as described by supplier, but included 15% polyethylene glycol (PEG) 8000 ~Sigma), a modification described by Pfeiffer and Zimmerman (Pheiffer, B.H. and Zim~er~an, S.B.
Nucl. Acids Res. 11: 7853-7871 (1983)). The ligated DNA was recovered by centrifugation (12,000 xg), washed with chloroform, dried, resuspended in 4~1 water and incubated with an in vitro packaging extract (Stratagene) according to supplier.
Reco~b~nant phage were propagated in ~. Qli BB4 (Stratagene).

~xn~hçsls of Oliaonucleot$des Oligonucleotides were synthesized by the phosphoramidite method with an Applied Biosystems (Foster City, CA) model 380A synthesizer, pur$fied by polyacrylamine gel electrophoresis and desalted on a Waters SEP-PAK (C18) cartridge. A 10-mer oligonucleot$de (5'CCGAATTCGG3') was synthesized and u6ed as the EcoRI l$nker for cDNA library construction. Prior to ligation, the linker was phosphorylated with T4 polynucleotide k$nase (Naniati~, T., Frit~ch, E.F. ~nd Sambrook, J., Molecular Clon$ng: A Laboratory ~anual (Cold Sprinq Harbor Lab., Cold Spring Harbor, NY, ~982)). A 14-mer oligonucleotide (5'CCTGTAGATCTCCG3') and a 18-mer oligonucleotide (5'AATTCGGAGATCTACAGG3') were 2Q~7~6 synthesized and used as the EcoRI adapters. The 14-mer was phosphorylated (Maniatis, ç~ ~1., upra) and subsequently heated to 95'C for 15 min to inactivate polynucleotide kinase, prior to annealing with the 18-mer. These asymmetrically phosphorylated adapters also contained an internal ~glll restriction enzyme site. Based on the amino acid ~e~uence of the human ~CF trypt~c fragment, a two-iold degenerate 45-mer oligonucleotide probe was designed (FIG. 2, probe A) following the rules of ~athe (~a~e, R.~, Mol.
Biol. 183: 1-12 (1985)). The two oligonucleotide probes (A and B) were synthesized based on the ~m~no acid seguence of a purified ~ryptlc peptide of the human bone calcificati~ factor (hBCF) (FIG. 2~.

~x~NpLE 4 Screenina of the cDNA Librar~es a. Human osteosarcoma l~braries.

Approximately 300,000 recQmbinant phage were plated (SO,OOO phage/137mm dia. platet ~n ~. ~gli BB4, grown for S-6 h ~t 37-C, transferred to nitrocellulose filters (Hillipore, HATF 137)j prooessed accord~ng to Benton and Davis (Benton, W.D. and Davis, R.~., ~ç~çn~ 180 (1977)1 and soreened ~ith oligonucleotide probe ~. Eighteen putative hBCF cDNA
clones from 300,000 recombinants were identified.
Southern blot analyses (described below) of the cDNA
inserts ~ere performed u~ing probe A and also probe B
(FIG~ 2), a fully de~enerate 18-~er oligonucleotide oontained withln probe A. Most of tho clone~
hybr~dized to both probes. The probe wa8 ~nd-labeled with T~ polynucleotide ~a~e ~nd 1~32-P]ATP
(Maniatis, et ~1., supra) to a speclfic activity of 1-2x108 cpm/~g. The Pilter~ were prehybrid~zed for 1-2 h at 37-C in 20% (vol/vol) formamide, 5xSSC

21~7~6 (lxSSC = 0.15 ~ sodium chloride/O.l~M sodium citrate, pH 7), 5x Denhardt'~ solution (lx Denhardt's solution - O.02~ polyvinylpyrrolidone/0.02% Ficoll/0.02%
bovine serum ~lbumin), 10% dextran sulfate, 50mM
~odium phosphate pH 6.8, 1 ~M sodiu~ pyrophosphate, 0.1% NaDcdS04 a~d 50pg/ml denatured salmon sperm DNA.
Labeled probe was added to a concentration o~
106cpm/~1 and hybridization was continued overnight at 37-C with gentle 6haking. The filter~ were washed twice (20 mln/wash) in 2X5SC, 0.1% NaDodS04 at 55-C
and expo~ed to Rodak XAR-2 film with ~ Dupont ~ightning Plu~ intensifying ~creen overnight at -80-C. Areas of plagues giving signals on duplicate filters were pic~ed, replated and rescreened as above until pure plagues were obtained.

Two of the double positive clones (Ost 3-7 and 0st 3-17, FIG. 3) were sequenced and shown to contain identical overlapping sequences as well as a region encoding the tryptic fragment (FIG. 1 underlined and FIG. 2). 0st 3-17 contains the complete mature protein cod~ng seguence, but not the complete signal peptide, as ~6 evident from the bBCF cDNA shown in FIG. 1.

An additional 300,000 reco~binant phages fro~ two different osteosarcoma cDNA libraries were later plated and screened a8 above but ~ith the following changes: (1) The hybridization mix contained 40%
~ormamide, 5xSSC, 5X Denhardt'~ solution, 10% PEG
8000, 50mM sodium phosphate pH 6.8, 0.5% NaDodS04 and 50~g/ml denatured; (2) the filters washed at 65-C ln 2xSSC, 0.1% NaDodS04; and (3) the probe wa~ ~ 240bp.
DNA ~ragment obta$ned by digesting cDNA clone 0st 3-7 with ~glII and A~p718 (probe C, FIG 3). The probe was purified and labeled by the oligo~primer method (Feinberg, A.P. and Vogelste~n, ~., Anal. Biochem.

137: 266-267 (1984)) to a specific activity of >1 x 109 cpm/~g. Approximately 20 clones from each library gave ~trong hybridization signal6 and restriction enzyme analysis of these clones identified several that were longer than 0st 3-17.
One of these, O~t 1-7 ~FIG. 3~, was ~eguenced and ~udged to be full length based on its homology to t~e bovine BCF cDNA clone, described below.

b. 80vine bone matrix cDNA library.

Approximately 300,000 recombinants from the bovine bone matrix cDNA library were screened with probe C
(FIG. 3), a 240b.p. BglII - Asp 718 gNA fragment from 0st 3-7, under the conditions described for probe A
except that formamide was om~tted fro~ the hybridization solution. The filters were washed at 55-C in 2xSSC, 0.1% NaDodS04. Twenty-four positive plaques were ldentified. Clone bbl.l-7 (FIG. 4), which conta$ned the largest insert, was sequenced and ~hown to contain sequences homologous to hBCP. The deduced amino acid seguence of the bBCF cDNA
indicates that amino acid -4 is not the initiation codon due to the presence of a Val at this position.
The most likely initiation codon i~ located at a-ino acid position -17 which is 28 nucleotides beyond the 5' end of the hBCF 0st 3-17 clone. The Het at position -17 iB also preceded by an acceptable ribosome binding s1te.

~g Subclonina. Sequenc~ng and Analv~i~

Recomb~nant pla6~ids were relea~ed in the 81uescr~pt SK(-)vector from ~ZAP by the M13 rescue/exci~ion protocol described by the supplier (Stratagene). The plasmids were propagated in E. çQl~ BB4, and plasmid r~

DNA was isolated by the alkaline lysis method (Maniat$s, çt al., SUDra). cDNA inserts were excised with either EcoRI or BqlII restriction enzymes ~Boehringer-Mannhe$m), pur~fied by polyacrylamide gel electrophoresis (MAniatis, ~ ~1., supra) and passage over an Elutip-d colu~n (Schle$cher and Schuell) and subcloned into the ~13 seguencing vector~ (Yanisch-Perron, C., Viera, J. ~nd Messing, J., Gene 33: 103-119 (198g)). DNA sequencing was performed by the dideoxy chain termination method (Sanger, F. Nicklen, S. and Coulson, A.R., ~roc. Natl. Acad. Sci. USA 74:
5463-67 (1977)) using M13 primers as well as ~pecific internal primers. Ambiguous reg$ons were resolved 7-deaza-2-deoxyguanidine-triphosphate (Barr, P.J., Thayer, R.M., Laybourn, P., Najarian, R.C., Seela, F., and ~olan, D., ~otechniques 4: 428-32 (1986~)-and sequenase (U.S. Biochemicals).

. Northern Blot Poly(A)~ RNA was fractionated on a 1.4S agarose gel in the presence of formaldehydé (Lehrach, H., Diamond, D., Wozney, J.~. and Boedtker, H., ~iQQhemistrv ~: 4743-51 (1977)) ~nd directly transferred to nitrocellulose according to Thomas (Thomas, P., Proc. Natl. Acad. Sci. USA 77: 5201-5 (1980)). Filters were hybridized with probe C as previously descr$bed (EXAHPL~ ~, Screening of the cDNA ~ibrarie~) in the ~0% formamide containing hybridizat$on solution and were washed at 55-C in 2xSSC, 0.1% NaDodS04 And O.lxSSC, 0.1% NaDodS04 with autoradiography following each set of washings.

RNA transfer blot analysi~ demonstrate~ the presence of two mRNA forms of ~0.9 and 1.8kB in human osteosarcoma tissue. The two forms were also observed in human placenta but were absent in a human - 2 0 ~

liver cell line, HEPG2. The two forms were al~o observed in bovine bone matrix cells ~ith the larger form predominating. A trace of the large 6pecies could also be seen in boviDe bone marrow. The two mRNAs are ~ost likely generated by dif$erential polyadenylation at the 2 site~ (AATAAA) found in the 3' untranslated region.

b. Southern Blot 1. Genomic lO~g o~ geno~ic DNA ~lontech~ was digested with EcoRI, fracti~nated on 0.7S ~garose gels ~nd transferred to nitrocellulose according to Maniatis, et al., supra. Hybridization and wa~hing conditions were identical to those described in a. above.

Genomic DNA transfer bl~ analysis suggest6 that hBCF and bBCF are elngle copy ~or low copy) genes due to the few bands seen in ~he EcoRI digest.

2. cDNA Clon DNA from c~NA clone~ were ~igested with EcoRI or BglII, fractionated on 1.0% agarose gels, transferred to nitrocellulo6e (Maniatis~ ç~ u~ra) and hybridized with probe A as previously described or with probe B in a tetramethylammonium chlorid-contain~ng hybridization solution under conditions described by Wood (Wood, W.I., Git~chier, J., LaskQy, L. and Law~, R., p~gc. Natl. A~ad. Sci. ~BA 82: 1585-88 (1985~).

;` 2~7~6~

ExAMpLE 6 Expression of hBCF in Yeast Yeast expression vectors were constructed for intracellular production or secretion of hBCF from the ADH2/GAPDH regulatable promoter. Since the first cDNA clone did not contain DNA encoding a signal peptide, methionine-4, was used as the N-terminal amino acid for these constructions. Su~sequent cDNA
analyses, and identification o~ a classical ~ignal pept~de, and a ~ignal peptidase cleavage ~te, allowed construction of a yeast ~-factor/hBCF fusion with glutamine ~1 as the N-terminal amino acid of recombinant hBCF. For clonlng into expression vectors, natural Nco-l, Bgl-l and Spe-1 sites were used together with the synthetic oligonucleotide adapters shown (FIG. 4). Since Spe-l and Xba-l give the same restriction enzyme overhang, the initial construction w~s simply an in~ertion of the Nco-l/
SPE-l hBCF geno into Nco-l/Xba-l digested pBSlOOhaFGF, a vector contain~ng ADH2/GAPDH promoter and GAPDH terminator elements flanking a synthetic human acidic fibroblast growth factor (FGF) gene.
The haFGF gene contains an Nco-l and unique Xba-l sites. The resulting plasmid, pBSlOOhBCF, was used for further constructions. Thus, the Nco-l/Sal-l fragment containing the hBCF gene was cloned together with BAMHl~Nco-l fragments encoding the GAPDH and ADH2/~APDH promoters lnto BamHl/Sal-l dig~sted pBS24.1. The resulting plasmids; pBS24A/GhBCF~Q ~-4 to 183) and pBS24 GAPhBCF (-3 to 183), were u~ed to direct intracellul~r expression of hBCF. For ~ecretion, Bgl-l/S~ ragments were exci~ed and cloned into pBS24.1 together with BamHl/Xba-l ~ragment3 encoding the ADH2/GAPDH promoter, th~ yeast n-factor secretory ~$gnal/leader sequence, Dnd the ~ynthstic linker~ shown $n FIG. 4 (boxed), ~or ~7~:6 expression of hBCF(-4 to 183) and hBCF(l-lB3). Yeast cells transformed with expression plasmids encoding ADH2/GAPDH promoter-hBCF(-4-183) and GAPDH promoter-hBCF(-4-183) fusions, as analyzed by SDS-PAGE and Coomass~e blue 6taining of total proteins, 6howed no expresgion a8 compared wit~ control yeast cells.
Therefore, to study secretion ~ystems, the pBS24 plasmids containing ~-factor leader-hBCF (-3-183) and hBCF (1-183) fusions were constructed. In each case, transcription was driven by the ADH2/GAPDH promoter.
Yeast 6train ABllO was transformed with the yeast expression plasmids, and yeast supernatants were analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE. ~i~h level6 of expression of the approximately 22kD product was observed by Coomassie blue ~taining, when compared with control yeast ceils transformed with the parent yeast vector pBS24. The transformant ABllO (pBS24.1 22kQ) is deposited under accession number ATCC 20948.
The active lot of yeast cell~ comprised a pool of two lot~, KQ-2 and KQ-3, from which the recombinant BCF waæ isolated and purified as follows.

Lot K0-2 The cells were removed by centrifugation from the fermentation medium, and the medium concentrated using a YM10 Amicon ~piral cartridge. The concentrate was diafiltered $nto water, tben 20 ~M
Tris-Cl, 1 ~M EDTA, 3 M NaCl, pH 7.5. This was passed over a column of prep grado Superose 12 ~t 4-C, then at room temperature. The 22XBCF did not stlck to either column. The ~lowthrou~h wa~ purified by adsorption onto SuRerose 12 ~R 10/30, and eluted with the same buffer but with 1 ~ NaCl.

-42- 2 ~
Lot K0-3 The cells were ~emoved and the medium concentrated as described ~b~ve. The concentrate was diafiltered against water, then 20 mM Tri~-Cl, 1 mM EDTA, pH 7.5.
This was loaded onto a Mono-Q HR 10/10 column, washed, and eluted with a gradient to 0.5 M ~aCl.
The 22KBCF-cDntaining fractions were identified by gel electrophoresis, pooled, ad~usted to 3 M NaCl with solid NaCl, and loaded onto a Superose 12 HR
10/30 column. The 22K~CF was eluted with buffer containing 1 H NaCl.

L~t X0-2/3 ~he Superose eluates (from Lots KQ-2 and XQ-3) were pooled, concentrated using YM 10 membrane in Amicon 6tirred cell, dialyzed versus water, and lyophilized.

Alternatively, the recombinant BCF may be isolated and purified as follows.

The media is removed from the cells and concentrated approximately ten fold. The pH is adjusted to 7.5, the concentrate $~ diluted to a conductivity below 5 mS/cm, then applied to Fast Flow Q ion-exchange resin pre-eguilibrated with 50mM Tris/Hcl, lmM EDTA, lmM PNSF, pH 7.5. The column i~ washed with 1 column volume of the above buffer and eluted u~ing a 0-lM
~aCl ~alt gradient in the above buffer. ~he 22R~CF
i~ eluted at a conductivity o~ 20-30 mSfcm, which i8 confirmed using SDS-PAGE. The 22KBCF conta$ning fractions are pooled, t~e pH maintained at 7.5 ~nd made 4M with sespect to Urea, concentrated, and run over ~ S-100 ~izing column in 4N Urea, lOOmN
Tri6/HCl, lmM EDTA, lmM PMSF at pH 7.5. The 22RBCF
containing fractions are ident~ied by SDS-PAGE, 20~6~

pooled, concentrated, and dialysed aga~nst lOmM
ammonium bicar~onate pH 7.8. The 22X~CF is then lyophilized and may be stored dry at 4-C.

EXa~PLE 7 A ~ample of purified recombinant human ~CF(rhBCF) expressed as in Example 6, 1 ~g tlot XQ 2/3), was dissolved in water containing 5 mg of buman fibrin.
The rBCF-fibrin composite was lyophilized and implanted in the mouse thigh muscle pouch. In another mouse, bovine matrix Gla protein was dissolved in 6M urea containing 1 mg of human rhBCF
and dialyzed again~t water. The precipitate and 6upernatant were lyophilized to prepare a co~posite of MGP and hBCF protein~. The composite was implanted in the guadriceps pouch. For controls, the contralateral thiqhs were implanted with bovine matrix Gla protein plus albumin in one nouse: in the other mouse the control con~i~ted of 5 ng of human fibrin and 1.0 mg o~ albumin.

Microradiographs of the r~CF-matrix Gla protein composite showed areas of calcified tissue.
Histological section6 6howed small round cells, multinucleated cells, macrophages with hyperplasia and hypertrophy of mesenchymal type cell6. There were plates of calcified ground 6ubstance but no cartilage or bone cell~. FIG. S is a photomicrograph showing hyperplasi~ and hypertrophy of connective tissue cells on the surface of a composite i~plant ~indicatad by P) of reco~binant protein hBCF ~ot KQ
2/3, 1 ~gt ~nd 1 ~g of human ~atrix Gla protein.
Calcificatlon of the implanted proteins i8 indicated by CP in the guadriceps pouch of a ~ouse on day 21.
The fibrous connective tissue envelope i~ indicated by F.

FIG. 6 is a photomicrograph showing islands of calcified protein (CP) induced by a composite of recombinant hBCF and biological human matrix Gla protein. Note the large foam cell (arrow). The entire implant is enclosed in a fibrous capsule (F) by day 21.

FIG. 7 is a photomicrograph showing ~ co~posite of recombinant h~CF and ~atrix Gla protein on day 21.
Note the highly vascular interior of the l~plant including small round cells, multinucleated cells, macrophages, calcifying intercellular substance (CP) and large foam cells (arrow). All of the implanted protein and reactive tissue was enclosed in a fibrous envelope (F) on day 21.

Claims (37)

1. A composition comprising a polypeptide selected from the group consisting of mammalian bone calcification factor and analogs thereof substantially free of other osteoinductive associated factors.
2. A composition according to Claim 1, wherein said mammalian bone calcification factor is human BCF.
3. A composition according to Claim 1, wherein said mammalian bone calcification factor is bovine BCF.
4. A composition according to Claim 2 wherein said polypeptide comprises an amino acid sequence as shown in FIG 1A or fragment thereof.
5. A composition according to Claim 3 wherein said polypeptide comprises an amino acid sequence as shown in FIG. 1B or a fragment thereof.
6. A composition according to Claim 4, wherein said polypeptide comprises an amino terminus selected from the group consisting of the signal peptide sequence met-asp-lou-ser-leu-leu-trp-val-leu-leu-pro-leu-vaal-thr-mst-ala-trp-gly and sequences of said signal peptide sequence derived by deletion of one or more amino acids from the amino terminus of said signal peptide sequence.
7. A composition according to Claim 5, wherein said polypeptide comprises an amino terminus selected from the group consisting of the signal peptide sequence met-asp-leu-thr-leu-leu-trp-val-leu-leu-pro-leu-vaal-thr-val-ala-trp-gly and sequences of said signal peptide sequence derived by removal of one or more amino acids from the amino terminus of said signal peptide sequence.
8. Non-chromosomal DNA as shown in FIG 1C.
9. DNA complementary to the sequence shown in FIG. 1C.
10. Non-chromosomal DNA as shown in FIG 1D.
11. DNA complementary to the sequence shown in FIG. 1D.
12. Non-chromosomal DNA according to Claim 8 further comprising the 5' sequence selected from the group consisting of the leader sequence ATG GAC CTC AGT CTT
CTC TGG GTA CTT CTG CCC CTA GTC ACC ATG GCC TGG GGC
and sequences derived by removal of one or more deoxynucleotides from the 5'-terminus of said leader sequence.
13. Non-chromosomal DNA according to Claim 10 further comprising the 5' sequence selected from the group consisting of the leader sequence ATG GAC CTC
ACT CTT CTG TGG GTG CTT CTG CCA CTG GTC ACC GTG GCT
TGG GGA and sequences derived by removal of one or more deoxynucleotides from the 5'-terminus of said leader sequence.
14. A method comprising the steps of (a) constructing a vector which includes the DNA sequence shown in FIG. 1C or a fragment thereof, (b) transforming a host cell with said vector, and (c) culturing the resultant transformed cell under conditions to express the peptide encoded by said DNA sequence or fragment thereof.
15. A method comprising the steps of (a) constructing a vector which includes the DNA sequence shown in FIG. 1D or a calcification-initiating fragment thereof, (b) transforming a host cell with said vector, and (c) culturing the resultant transformed cell under conditions to express the peptide encoded by said DNA sequence or fragment thereof.
16. A method according to Claim 14 wherein said DNA
sequence further comprises a 5' sequence selected from the group consisting of the leader sequence ATG
GAC CTC AGT CTT CTC TGG GTA CTT CTG CCC CTA GTC ACC
ATG GCC TGG GGC and any sequence derived by removal of one or more deoxynucleotides from the 5'-terminus of said leader sequence.
17. A method according to Claim 15 wherein said DNA
nucleotide sequence further comprises a 5' sequence selected from the group consisting of the leader sequence ATG GAC CTC ACT CTT CTG TGG GTG CTT CTG CCA
CTG GTC ACC GTG GCT TGG GGA and any sequence derived by removal of one or more deoxynucleotides from the 5'-terminus of said leader sequence.
18. A method according to any of Claims 14 through 17 wherein said host cell is eukaryotic.
19. A method according to Claim 18 wherein said host comprises yeast.
20. A method according to Claim 18 wherein said vector comprises a GAPDH promoter which controls expression of said peptide.
21. A method according to Claim 18 wherein said promoter comprises an ADH2/GAPDH promoter which controls expression of said peptide.
22. A replicable vector comprising DNA according to Claim 8.
23. A replicable vector comprising DNA according to Claim 10.
24. A replicable vector comprising DNA according to Claim 12.
25. A replicable vector comprising DNA according to Claim 13.
26. A host cell transformed with a replicable vector according to Claim 22.
27. A host cell transformed with a replicable vector according to Claim 23.
28. A host cell transformed with a replicable vector according to Claim 24.
29. A hot cell transformed with a replicable vector according to Claim 25.
30. A composition for inducing calcification, comprising an effective calcification-inducing amount of a mixture of mammalian bone calcification factor or an analog thereof, and matrix Gla protein.
31. A composition according to Claim 30 wherein said factor comprises human bone calcification factor.
32. A composition according to Claim 30 wherein said factor comprises bovine bone calcification factor.
33. A composition according to Claim 31 wherein said factor has an amino acid sequence as shown in FIG. 1A, or a fragment thereof.
34. A composition according to Claim 32 wherein said factor has an amino acid sequence as shown in FIG. 1B, or a fragment thereof.
35. A method for inducing calcification for the formation of bone in a vertebrate, comprising the step of administering to said vertebrae in a pharmaceutically effective manner an effective calcification-inducing amount of a composition according to any one of Claims 3- to 34.
36. A diagnostic method comprising the steps of providing a bone sample from a mammal and measuring the amount of mammalian bone calcification factor in said sample in a quantitative assay.
37. A composition comprising antibodies recognizing an epitope unique to mammalian bone calcification factor.
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US5366875A (en) * 1986-07-01 1994-11-22 Genetics Institute, Inc. Methods for producing BMP-7 proteins
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US5543394A (en) * 1986-07-01 1996-08-06 Genetics Institute, Inc. Bone morphogenetic protein 5(BMP-5) compositions
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US5939388A (en) * 1986-07-01 1999-08-17 Rosen; Vicki A. Methods of administering BMP-5 compositions
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US6034062A (en) * 1997-03-13 2000-03-07 Genetics Institute, Inc. Bone morphogenetic protein (BMP)-9 compositions and their uses
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US6177406B1 (en) 1986-07-01 2001-01-23 Genetics Institute, Inc. BMP-3 products
US5939388A (en) * 1986-07-01 1999-08-17 Rosen; Vicki A. Methods of administering BMP-5 compositions
US5688678A (en) * 1990-05-16 1997-11-18 Genetics Institute, Inc. DNA encoding and methods for producing BMP-8 proteins
US7378392B1 (en) 1990-05-16 2008-05-27 Genetics Institute, Llc Bone and cartilage inductive proteins
US5661007A (en) * 1991-06-25 1997-08-26 Genetics Institute, Inc. Bone morphogenetic protein-9 compositions
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US5866364A (en) * 1991-11-04 1999-02-02 Genetics Institute, Inc. Recombinant bone morphogenetic protein heterodimers
US6190880B1 (en) 1991-11-04 2001-02-20 Genetics Institute Recombinant bone morphogenetic protein heterodimers, compositions and methods of use
US6593109B1 (en) 1991-11-04 2003-07-15 Genetics Institute, Inc. Recombinant bone morphogenetic protein heterodimers, compositions and methods of use
US5703043A (en) * 1993-05-12 1997-12-30 Genetics Institute, Inc. Bone morphogenetic protein-10 (BMP-10) compositions
US5932216A (en) * 1993-05-12 1999-08-03 Celeste; Anthony J. Antibodies to bone morphogenetic protein-10 (BMP-10)
US5637480A (en) * 1993-05-12 1997-06-10 Genetics Institute, Inc. DNA molecules encoding bone morphogenetic protein-10
US5658882A (en) * 1993-12-07 1997-08-19 Genetics Institute, Inc. Methods of inducting formation of tendon and/or ligament tissue comprising administering BMP-12, BMP-13, and/or MP-52
US6719968B2 (en) 1993-12-07 2004-04-13 Genetics Institute, Llc. Tendon-inducing compositions
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US6958223B2 (en) 1996-06-03 2005-10-25 United States Surgical Corporation Methods for producing extracellular matrix proteins
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