EP0489062A1 - Proteine specifique aux os - Google Patents

Proteine specifique aux os

Info

Publication number
EP0489062A1
EP0489062A1 EP90912603A EP90912603A EP0489062A1 EP 0489062 A1 EP0489062 A1 EP 0489062A1 EP 90912603 A EP90912603 A EP 90912603A EP 90912603 A EP90912603 A EP 90912603A EP 0489062 A1 EP0489062 A1 EP 0489062A1
Authority
EP
European Patent Office
Prior art keywords
leu
asn
ile
lys
protein
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.)
Withdrawn
Application number
EP90912603A
Other languages
German (de)
English (en)
Other versions
EP0489062A4 (en
Inventor
Hanne Bentz
Ranga Nathan
David M. Rosen
James R. Dasch
Saeid M. Seyedin
Karl A. Piez
Yasushi Ogawa
William H. Andrews
Frank H. Stephenson
Joel Hedgpeth
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.)
Celtrix Pharmaceuticals Inc
Original Assignee
Celtrix Pharmaceuticals Inc
Celtrix Laboratories Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Celtrix Pharmaceuticals Inc, Celtrix Laboratories Inc filed Critical Celtrix Pharmaceuticals Inc
Publication of EP0489062A1 publication Critical patent/EP0489062A1/fr
Publication of EP0489062A4 publication Critical patent/EP0489062A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to protein chemistry. More particularly, it relates a protein that occurs specifically in bone, antibodies to such proteins, and immunoassays for detecting such proteins.
  • BMP-1, BMP-2 Class I, BMP-2 Class II and BMP-3 that are alleged to have osteogenic activity by themselves or in combination with other f ctors. Sequences are provided for each of these proteins which show no homology to the sequence (see below) of the protein of the present invention.
  • TGF-beta cartilage- inducing factor
  • U.S. Patent No. 4,627,982 concerns a partially purified bone-inducing factor present in the CMC-bound fraction of U.S. 4,434,094 that elutes in the portion of the NaCl gradient below that in which the major portions of TGF-betal and TGF-beta2 elute (i.e., below about 150 mM NaCl).
  • the present invention relates to the identification of an ingredient of that fraction.
  • One aspect of the invention is a substantially pure polypeptide that is found in bone and has the following sequence:
  • R 1 is Ala or Thr
  • R2 is Asn or His
  • R3 is Thr or
  • Ser, R 4 is Ala or Thr, R5 is lie or Val, R6 is Val or lie
  • R is Phe or lie, and substantially pure polypeptides that are and substantially homologous thereto.
  • recombinant materials i.e., recombinant DNA, recombinant vectors, and recombinant cells or microorganisms
  • processes for producing the polypeptides of the invention antibodies specific to the polypeptides, and immunoassays for the polypeptides.
  • Figure 1 is a flow chart of the process that was used to isolate the bovine species of the bone-specific protein of the invention from demineralized bovine bone.
  • Figure 2 is a graph of the optical densities (absorbances at 280 nm) of the gel filtration fractions of the gel filtration fractions of the example (IfC) .
  • Figure 3 is a graph of the optical densities (absorbances at 280 nm) of eluate fractions from the preparative ion exchange chromatography of the example (1ID).
  • Figure 4 is a graph of the optical densities (absorbances at 280 nm) of eluate fractions from the cross-linked ConA chromatography step of the example (HE);
  • Figure 5 is a graph of the optical densities (absorbances at 280 nm) of eluate fractions from the heparin-sepharose chromatography step of the example (1IF);
  • Figure 6 is a graph of the optical densities (absorbances at 230 nm) of the gradient fractions from the C18-RP-HPLC chromatography step of the example ' (1.G) ;
  • Figure 7 is a table showing results of amino acid sequencing of the bovine isolate of the invention and locations of the sequenced fragments in the overall sequence.
  • Figure 8 is a photograph of an autoradiograph of SDS-PAGE analyses of the purified bovine protein that are described in the example ( ⁇ H) (lanes A and C show glycosylated protein; lanes B and D show enzymatically deglycosylated protein) .
  • Figure 9 is a schematic diagram illustrating the structure of the bovine gene that encodes the mature bovine species of the protein of the invention. Various restriction sites are indicated.
  • Figure 10 is a restriction map of the region of the human gene that encodes the human species of the protein.
  • Figure 11 in parts A-E, shows the DNA sequence and deduced amino acid sequence of the mature human spe ⁇ cies of the protein of the invention in comparison with the sequences of the bovine gene and protein.
  • Part A shows the preliminary unconfirmed sequence of a putative "amino terminal" precursor exon of the human gene for the prepropolypeptide. The extent of this exon has not been determined. Bases of uncertain identity in the DNA sequence are represented by numbers. The signal sequence indicated in the figure is putative and was identified by the von Heijne algorithm.
  • Part B shows the sequence of another precursor exon, designated -1, that is downstream of the amino terminal exon and upstream of the exon that encodes the amino terminal of the mature protein.
  • Parts C-E show the sequences of exons 1-3 which encode the mature protein. Asterisks designate points of identity between the bovine and human DNA sequences. Amino acid numbering is relative to the mature sequence with the first amino acid thereof designated "1". Consensus 3' and 5' exon splice sites are indicated by YYYYYYYYYYNYAG and JAGGTRAGT, respectively.
  • Figure 12 is a schematic diagram of the mammalian expression vector phOIFl ⁇ which contains the human gene that encodes the human species of the invention protein.
  • proteins from different mammalian species will have substantially homologous amino acid sequences that vary from the bovine or human proteins described herein, if at all, in one or more amino acid residue additions, deletions or substitutions and/or substantially similar glycosylation patterns.
  • the amino acid sequences of "substantially homologous" proteins will usually be at least 50% identical, more usually at least 80% identical, and preferably at least 90% identical to the bovine/human amino acid sequence described herein.
  • Such proteins may be derived from bone or other tissues of diverse mammalian origin or synthesized using recombinant DNA procedures.
  • the term is intended to include muteins or analogs of the native protein that are altered- in manners known in the art, such as by substitution of cysteines with neutral (uncharged) amino acids to avoid improper disulfide bonding, by substitution or elimination of residues in the asparagine-linked glycosylation sites of the proteins to alter glycosylation patterns, by substitution of methionines to make the molecules less susceptible to oxidation, by conservative substitution of other residues, by chemical modification of one or more residues, by substitution with nonnatural amino acids or by elimination or alteration of side-chain sugars.
  • the source of protein prepared by purification from native sources is advantageously porcine or bovine long bone because of its ready availability.
  • the process for isolating the protein from bone is as follows.
  • the bone is first cleaned using mechanical or abrasive techniques, fragmented, and further washed with, for example, dilute aqueous acid preferably at low temperature.
  • the bone is then demineralized by removal of the calcium phosphates in their various forms, usually by extraction with stronger acid. These techniques are understood in the art, and are disclosed, for example, in U.S. 4,434,094.
  • the resulting preparation, a demineralized bone is the starting material for the preparation of the protein from native sources.
  • the initial extraction is designed to remove the nonfibrous (e.g., noncollagenous) proteins from the demineralized bone.
  • chaotropic agents such as guanidine hydrochloride (at least about 4 molar), urea (8 molar) plus salt, or sodium dodecylsulfate (at least about 1% by volume) or such other chaotropic agents as are known in the art (Termine et al., J Biol Chem (1980) 255:9760-0772; and Sajera and Hascall, J Biol Chem (1969) 244 ⁇ :77-87 and 2384-2396).
  • the extrac ⁇ tion is preferably carried out at reduced temperatures to reduce the likelihood of digestion or denaturation of the extracted protein.
  • a protease inhibitor may be added to the extractant, if desired.
  • the pH of the medium depends upon the extractant selected. The process of extraction generally takes on the order of about 4 hr to 1 day.
  • the extractant may be removed by suitable means such as dialysis against water, preceded by concentration by ultrafiltration if desired. Salts can also be removed by controlled electrophoresis, or by molecular sieving, or by any other means known in the art. It is also preferred to maintain a low temperature during this process so as to minimize denaturation of the proteins.
  • the extractant chaotropic agent need not be removed, but rather the solution need only be concentrated, for example, by ultrafiltration.
  • the extract, dissolved or redissolved in chaotropic agent, is subjected to gel filtration to obtain fractions of molecular weight in the range of about 20,000 to 36,000 daltons.
  • Gel sizing is done using standard techniques, preferably on a Sephacryl S-200 column at room (10 C-25 C) temperature.
  • the sized fraction is then subjected to ion exchange chromatography using CMC at approximately pH 4.5- 5.2 preferably about 4.8, in the presence of a nonionic chaotropic agent such as 6M urea.
  • a nonionic chaotropic agent such as 6M urea.
  • Other cation exchangers may be used, including those derived from polyacrylamide and cross-linked dextran; however cellulosic cation exchangers are preferred.
  • the solution must be freed of competing ions before application to the column.
  • the protein is adsorbed on the column and is eluted in an increasing salt concentration gradient in the range of about 10 mM to about 150 mM. This fraction is designated "CMB-1" for convenience.
  • CMB-1 is lyophilized and the dry CMB-1 is dis ⁇ solved in aqueous sodium deoxycholate (DOC), pH 8.0.
  • DOC sodium deoxycholate
  • This solution is affinity chromatographed on an equilibrated column of ConA cross-linked to resin.
  • the ConA-bound material is eluted from the resin with aqueous DOC containing a displacement carbohydrate. This fraction is designated "CAB-1" for convenience.
  • CAB-1 is reequilibrated for heparin-sepharose chromatography by desalting on a GH-25 column equilibrated on heparin-sepharose buffer, 6M urea, 0. IM NaCl, 50 mM Tris-HCl pH 7.2.
  • the desalted fraction is loaded onto a heparin-sepharose column. After washing, bound material is eluted from the column using the same buffer at a 0.5M NaCl salt concentration. The resulting eluate is designated "HSB-1" for convenience.
  • HSB-1 is diluted and adjusted to pH 2 and loaded onto a C18-RP-HPLC column. Bound proteins were gradient eluted from the column using a solvent consisting of 90% acetonitrile in 0.1% aqueous TFA (Solvent B) . The protein of the invention elutes at approximately 47-50% of solvent B (42-45% acetonitrile) by volume.
  • Proteins eluted by the C18 chromatography were iodinated by the chloramine-T method. Analysis of the fraction by SDS-PAGE and autoradiography shows a major broad band at 20,000 to 28,000 daltons comprising the protein. The "smearing" of the protein is believed to mainly be the result of heterogeneity in the glycosylation of the molecule or perhaps variable post-translational modification or proteolytic degradation. After enzymatic or chemical deglycosylation, SDS-PAGE analysis of the pre ;.ein gives a single band of approximately 10,000 daltons . Reduction of the deglycosylated protein with dithiothreitol does not affect its migration.
  • the initial amino acid (Lys) in the above sequence is nearest to the N-terminal. Initially, the nature of the signal obtained for the residue designated X did not permit this residue to be identified.
  • Repeated sequencing of the entire peptide and sequencing of oligopeptides generated from endoproteinase Lys-C (an enzyme that cleaves proteins at Lys residues) and endoproteinase Glu-C (an enzyme that cleaves proteins at Glu residues) digests have revealed that the above sequence is preceded by an Ala residue which is the N-terminus, that the residue designated X is Leu, that the second Thr residue (the 26th residue in the above sequence) was incorrect and that this residue is actuallv a Leu residue, and that the isolate consists of a protein of approximately 106 amino acids.
  • Figure 7 provides a summary of these sequence analyses.
  • the symbol “CHO” designates a carbohydrate substitueat.
  • the symbol “COOH” represents a carboxyl group and designates the carboxy terminus.
  • the first column (on the left) provides the sequence analysis of the N-terminal fragment described above.
  • the second, fourth, and sixth columns give the sequences of three major Lys-C fragments of the isolate.
  • the third and fifth columns give the sequences of two Glu-C fragments.
  • sequence for the native bovine protein is as follows:
  • the sequence of the corresponding human protein was determined by obtaining the human gene using DNA probes based on the bovine DNA sequence, sequencing the human gene and deducing the amino acid sequence of the protein encoded thereby.
  • the sequence of the human protein was found to be as follows .
  • a comparison of the human sequence with the bovine sequence shows that there are seven differences at positions 15, 20, 23, 54, 84, 85 and 105 of the sequence. Accordingly, at least the residues at those positions may be interchanged. It is possible, of course, that sequences of other mammalian or. avian species may exhibit other differences.
  • the genes for the mature bovine and human proteins it was discovered that the genes each encode a precursor segment. Portions of the precursor segments for the bovine and human proteins are shown in Figure 11, parts A and B. Accordingly, it is believed that the protein occurs as a prepropolypeptide and is processed into the mature protein defined by the sequences indicated above. Polypeptides comprising the mature protein sequence and including a portion or all of the precursor segments are intended to be within the scope of the invention.
  • the invention provides the protein in substantially pure form in which it is essentially free of other molecules with which it is associated in nature.
  • the term “substantially pure” intends a composition containing less than about 30% by weight contaminating protein, preferably less than about 10% contaminating protein, and most preferably less than about 5% by weight contaminating protein.
  • the term “substantially pure” is used relative to proteins with which the protein is associated in nature and is not intended to exclude compositions in which the protein is admixed with nonproteinaceous carriers or vehicles, or proteinaceous carriers or vehicles, provided other protein(s) with which it is associated naturally are absent.
  • the invention also provides the protein in novel partially glycosylated or totally deglycosylated forms (both of which are referred to herein as "deglycosylated”) .
  • oligonucleotide probes which contain the codons for a por- tion or all of the determined amino acid sequences are prepared and used to screen DNA libraries for substantially homologous genes that encode related proteins.
  • the homologous genes may be from other species of mammals or animals (e.g., avians) or may represent other members of a family of related genes.
  • the basic strategies for preparing oligonucleotide probes and DNA libraries, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, e ⁇ . , DNA CLONING: VOLUME I (D.M. Glover ed. 1985); NUCLEIC ACID HYBRIDIZATION (B.D.
  • a DNA library is prepared. Since the initially identified protein was bovine, it was logical to probe a bovine library first, find full length clones and use the full length bovine clones to probe libraries of other mammalian species to identify the protein gene (and thus the amino acid sequences) of other species.
  • the library can consist of a genomic DNA library. Bovine and human genomic libraries are known in the art. See, e.g. , Lawn et al., Cell (1978) 15:1157-1174. DNA libraries can also be constructed of cDNA prepared from a poly-A RNA (mRNA) fraction by reverse transcription. See, e.g. , U.S. Patent Nos.
  • the ⁇ iRNA is isolated from an appropriate cell line or tissue that expresses the factor. Libraries from cells involved in bone formation (e.g., osteoblasts) or from osteotumors (e.g., osteosarcoma lines) are likely sources to probe for the nucleic acids that encode the protein.
  • cDNA or genomic DNA is cloned into a vector suitable for construction of a library.
  • a preferred vector is a bacteriophage vector, such as phage lambda. The construction of an appropriate library is within the skill of the art.
  • oligonucleo- tides to probe the library are prepared and used to isolate the desired genes.
  • the oligonucleotides are synthesized by any appropriate method.
  • the particular nucleotide sequences selected are chosen so as to correspond to the codons encoding the known amino acid sequences of the protein. Since the genetic code is redundant, it will often be necessary to synthesize several oligonucleotides to cover all, or a reasonable number, of the possible nucleotide sequences which encode a particular region of a protein. Thus, it is generally preferred in selecting a region upon which to base the probes, that the region not contain amino acids whose codons are highly degenerate.
  • probes containing codons that are rare in the mammal from which the library was prepared may not be necessary, however, to prepare probes containing codons that are rare in the mammal from which the library was prepared.
  • one of skill in the art may find it desirable to prepare probes that are fairly long, and/or encompass regions of the amino acid sequence which would have a high degree of degeneracy in corresponding nucleic acid sequences, particularly if this lengthy and/or degenerate region is highly characteristic of the protein.
  • Probes covering the complete gene, or a substantial part of the genome may also be appropriate, depending upon the expected degree of homology. Such would be the case, for example, if a cDNA of a bovine protein was used to screen a human gene library for the corresponding human protein gene.
  • probes or sets of probes
  • Automated oligonucleo- tide synthesis has made the preparation of large families of probes relatively straightforward. While the exact length of the probe employed is not critical, generally it is recognized in the art that probes from about 14 to about 20 base pairs are usually effective. Longer probes of about 25 to about 60 base pairs are also used.
  • the selected oligonucleotide probes are labeled with a marker, such as a radionucleotide or biotin using standard procedures.
  • the labeled set of probes is then used in the screening step, which consists of allowing the single-stranded probe to hybridize to isolated ssDNA from the library, according to standard techniques. Either stringent or permissive hybridization conditions could be appropriate, depending upon several factors, such as the length of the probe and whether the probe is derived from the same species as the library, or an evolutionarily close or distant species.
  • the selection of the appropri ⁇ ate conditions is within the skill of the art. See gener ⁇ ally, NUCLEIC ACID HYBRIDIZATION, supra.
  • hybridization conditions be of suf- ficient stringency so that selective hybridization occurs; i.e., hybridization is due to a sufficient degree of nucleic acid homology (e.g., at least about 75%), as op ⁇ posed to nonspecific binding.
  • a DNA coding sequence for a protein can be prepared synthetically from overlapping oligonucleotides whose sequence contains codons for the amino acid sequence of the protein.
  • oligonucleotides are prepared by standard methods and assembled into a complete coding sequence. See, e.g. , Edge, Nature (1981) 292:756; Na bair et al., Science (1984) 223:1299; Jay et al., J Biol Chem (1984) 259:6311. Accordingly recombinant polynucleotides that encode the polypeptides may be prepared and isolated by one or more of the above described techniques .
  • recombinant polynucleotide denotes a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin which, by virtue of its origin or manipulation (1) is not associated with all or a portion of the nucleic acid with which it is associated in nature or in the form of a library, (2) is linked to a polynucleotide to which it is not linked in nature or in a library, or (3) is not found in nature or in a library.
  • the DNA sequence coding for the protein can be cloned in any suitable vector, identified, isolated, and thereby maintained in a composition substantially free of vectors that do not contain the coding sequence of the protein (e.g., other library clones).
  • cloning vectors are known to those of skill in the art, ' and the selection of an appropriate cloning vector is a matter of choice.
  • Examples of recombinant DNA vectors for cloning and the host cells which they transform include bacteriophage lambda (E. coli) , pBR322 (E. coli) , pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E.
  • the coding sequence for gene encoding the protein is placed under the control of a promoter, ribosome binding site (for bacterial and eucaryotic expression), and optionally an operator (collectively referred to herein as "control" sequences) , so that the DNA sequence encoding the protein (referred to herein as the "coding" sequence) is transcribed into RNA and the RNA translated into protein in the host cell transformed by the vector.
  • the coding sequence may or may not contain a signal peptide or leader sequence. The determination of the point at which the precursor protein begins and the signal peptide ends is easily determined from the N-terminal amino acid sequence of the precursor protein.
  • the protein can also be expressed in the form of a fusion protein, wherein a heterologous amino acid sequence is expressed at the N-terminal. See, e.g. , U.S.. Patents Nos. 4,431,739; 4,425,437.
  • the recombinant vector is constructed so that the protein coding sequence is located in the vector with the appropriate control sequences, the positioning and orientation of the protein coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the control of the control sequences (i.e., by RNA polymerase which attaches to the DNA molecule at the control sequences) .
  • the control sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequence and an appropriate restriction site downstream from control sequences.
  • the control sequences will be heterologous to the coding sequence.
  • the selected host cell is a mammalian cell
  • the control sequences can be heterologous or homologous to the protein coding sequence
  • the coding sequence can be genomic DNA, cDNA or synthetic DNA. Either genomic or cDNA coding sequence may be expressed in yeast.
  • sequences such as the yeast alpha factor signal sequence or other sequences that direct secretion are included in the control sequence.
  • the gene may be expressed in yeast or mammalian cells (COS, CHO, or CV-1 cells) using vectors and procedures known in the art.
  • a number of procaryotic expression vectors are known in the art. See, e.g. , U.S. Patent Nos. 4,440,859;
  • Recombinant protein can be produced by growing host cells transformed by the expression plasmid described above under conditions whereby the protein is produced. The protein is then isolated from the host cells and purified. If the expression system secretes protein into growth media, the protein can be purified directly from cell-free media. If the recombinant protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art or are apparent from the recovery methods used to isolate the native proteins. The recombinant protein may be recovered by affinity chromatography using the antibodies produced in accordance with the invention. Recombinant protein may be unglycosylated or have a different glycosylation pattern than the native molecule depending upon the host that is used to produce it. The proteins are useful for making antibodies that recognize sequential epitopes of the protein, and are useful as bone markers.
  • Either native, deglycosylated, or synthetic (recombinant) protein can be used to produce antibodies, both polyclonal and monoclonal.
  • antibody is intended to include whole Ig of any isotype or species as well as antigen binding fragments, chimeric constructs and single chain antibodies. If polyclonal antibodies are desired, purified protein is used to immunize a selected 0 mammal (e.g., mouse, rabbit, goat, horse, etc.) and serum from the immunized animal later collected and treated according to known procedures.
  • compositions containing polyclonal antibodies to a variety of antigens in addition to the protein can be made substantially free of anti- c bodies which do not bind specifically to the protein bodies by passing the composition through a column to which protein has been bound. After washing, polyclonal antibodies to the protein are eluted from the column.
  • Monoclonal anti-protein antibodies can also be readily n produced by one skilled in the art. The general methodol ⁇ ogy for making monoclonal antibodies by hybridomas is well known. Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or c . transfection with Epstein-Barr virus. See, e.g. , Schreier, M.
  • Antibodies which recognize an epitope in the binding region of the protein can be readily identified .in competition assays between antibodies and protein. Antibodies which recognize a site on the protein are useful, for example, in the purification of the protein from cell lysates or fermentation media, in characterization of the protein and in identifying immunologically related proteins . Such immunologically related proteins (i.e., that exhibit common epitopes with the protein) are another aspect of the invention.
  • the anti-protein antibody is fixed (immobilized) to a solid support, such as a column or latex beads, contacted with a solution containing the protein, and separated from the solution. The protein, bound to the immobilized antibodies, is then eluted.
  • Antibodies to the protein may be used to identify osteoblasts and osteocytes by conventional immunoassay procedures . Such identification may be used to follow bone and/or cartilege turnover.
  • Bovine metatarsal bone was obtained fresh from the slaughterhouse and transported on ice. Bones were cleaned of all periosteum and marrow with high pressure water, crushed into fragments using a liquid-nitrogen- cooled grinder and pulverized into powder using a liquid- nitrogen-cooled mill . The pulverized bone was washed four times for 20 minutes in 4 C deionized water (8 liters/kg). The bone was then washed overnight with the same volume of deionized water at 4 C. The bone powder was demineralized for 5 hr in 0.5 N HC1 (21 liter/kg) at 4°C. The acid was decanted, and the demineralized bone powder was washed several times with 4 C deionized water until the wash reached a pH>3. The excess water was removed on a suction filter.
  • Demineralized bone powder was extracted with 4M guanidine-HCl, 10 mM EDTA pH 6.8 (2 liters/kg bone powder) for 16 hr at 4 C.
  • the suspension was suction-filtered to recover the guanidine-HCl-soluble fraction and concentrated at least 5-fold by ultrafiltration using a 10,000 dalton cut-off membrane (S10Y10 Amicon spiral cartridge) .
  • Figure 2 constitutes a low molecular weight (LMW, 10,000- 30,000 daltons) protein fraction possessing the greatest activity. This fraction was pooled and dialyzed against 6 changes of 180 volumes of deionized water and lyophilized. All operations except lyophilization and dialysis (4 C) were conducted at room temperature.
  • LMW low molecular weight
  • Proteins eluted with 10-150 mM NaCl were collected and dialyzed against 6 changes of 110 volumes of deionized water for 4 days and lyophilized. All of the foregoing operations were conducted at room temperature except dialysis (4°C).
  • step D above was enriched by affinity chromatography using concanavalin A (ConA)-Sepharose 4B (Pharmacia).
  • ConA concanavalin A
  • Sepharose 4B Puracia
  • the resin was cross-linked with-glutaraldehyde essentially as described by K.P. Campbell, D.H. MacLennan, J Biol Chem (1981) 256:4626. Briefly, resin was pelleted (500 x g, 5 min) and washed twice with 4 volumes of 250 mM NaHCO.,, pH 8.8. The resin was then equilibrated in the same buffer for 6-8 hrs at 4 C.
  • the resin was cross-linked by the addition of 4 volumes of 250 mM NaHCO-,, pH 8.8, 250 mM methyl-alpha-D-mannopyranoside (alpha-MM), 0.03% glutaraldehyde with gentle mixing for 1 hr at room temperature.
  • the reaction was quenched by washing the resin twice in IM Tris-HCl, pH 7.8.
  • the resin was stored in the same buffer containing 0.01% Thimersol at 4°C until use.
  • the bound fraction eluted from the ConA column was reequilibrated by chromatography on a GH-25 column (Pharmacia) equilibrated in 6M urea, 0.IM NaCl, 50 mM Tris-HCl pH 7.2 heparin-sepharose buffer. Approximately 80 mg (1 mg/ml) were loaded on a 25 ml large heparin sepharose column (Pharmacia) . The column was washed of all unbound material. Then bound proteins were eluted with the same equilibrating buffer but containing 0.5M NaCl as shown in Figure 5. About 5-8 mg of heparin- sepharose bound proteins were recovered.
  • Glycopeptidase F cleaves N-linked oligo- saccharides at the innermost N-acetylglucosamine residue. High mannose, hybrid and complex oligosaccharides are susceptible to the enzyme. Protein was iodinated by the chloramine-T method. Labeled protein was digested for 12- 15 hours with 6.7 units/ml glycopeptidase F (Boehringer Mannheim) in 0.IM Tris-HCl, pH 7.4, 10 mM EDTA, at 37°C. Both the glycosylated and deglycosylated forms were analyzed by sodium dodecyl sulfate/15% polyacrylamide slab gels prepared according to standard methods. Figure 8 is a photograph of the autoradiograph.
  • the protein is designated "OIF" in the drawings.
  • the following four 20-mer oligonucleotide probes were synthesized using a Biosearch 8600 DNA synthesizer. The sequences of these probes were derived from the amino acid sequence of the bovine protein that was isolated from bone.
  • A is adenine
  • C is cytosine
  • G is guanine
  • T is thymine
  • N is A, C, G or T
  • Q is A, C, or T
  • R is A or G
  • W is A, G or T
  • Y is C or T.
  • the DNA was concentrated to 1 mg/ml and 1 ug was mixed with 1 ug EMBL3 DNA.
  • the mixture was treated with DNA ligase as described by the supplier and packaged via the "gigapack kit” (Stratagene) to. make a library stock. Approximately 10,000 viable phage were plated on each of 60 plates (150 mm) (see Molecular Cloning: A Laboratory Manual, Mania is, Fritsch and Sambrook, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1983)).
  • Phage plaques were transferred to nitrocellulose filters (4 replicates per plate) . Absorbed DNA was de ⁇ natured by treatment with 0.5M NaOH, 1.5M NaCl. Filters were neutralized in 0.5M Tris, pH 8.0, 1.5M NaCl, washed with 2XSSC, air dried, and baked for 2 hr at 80°C under vacuum.
  • Phage containing the gene were identified by hybridizing 32P-labeled oligonucleotides with filters containing DNA transferred from phage plaques. Plaques hybridizing in duplicate to at least two of the four oligonucleotides were purified and further characterized.
  • a bovine cancellous bone mRNA library was prepared using the polymerase chain reaction (PCR) with primers selected from within Exons 1 and 3.
  • PCR polymerase chain reaction
  • a clone containing the bOIF cDNA sequence was isolated and the nucleotide sequence determined. The resulting sequence confirmed the exon splicing contemplated by Figures 9 and 11.
  • phage 41 Only one phage from the EMBL3 library (phage 41) hybridized to both DNA fragments and a second phage (phage 28) from the gene library hybridized with only the exon 1 fragment upon fescreening. All other phage did not hybridize with the radioactive probes upon rescreening, indicating that their original identification was either an artifact of hybridization or that the human protein cognate DNA was lost upon replication during isolation of the phage.
  • Phage 41 and 28 were subjected to restriction site mapping and Southern blot analysis to locate the sequences for the human probes and the DNA sequence of these regions was determined.
  • FIG. 12 is a schematic diagram of plasmid phOIFl ⁇ .
  • COS-7 cells were transfected with phOIFl ⁇ (see Feigner, P.L., et al. , Proc Natl Acad Sci (USA) (1967) ⁇ 4:7413). After transfection the cells were allowed to grow in medium with or without serum (5%). In these experiments only the cells grown in serum-containing media synthesized the protein.
  • Polyclonal antibodies to (1) a synthetic 30-mer polypeptide having a sequence corresponding to the amino acids 1-30 of Figure 7 except for a Leu > Asn substitution at position 25 and (2) the native protein purified from bone as described above were prepared and characterized as follows.
  • Antiserum to the 1-30-mer was raised in a rabbit by injecting the rabbit with 500 ug of the polypeptide in complete Freund's adjuvant (CFA) , followed by boosts of 500 ug of the polypeptide in incomplete Freund's adjuvant (ICFA) at approximately three week intervals. The antiserum was obtained after the fourth boost and had a titer as measured by ELISA of >1:10,000. Rabbit antiserum to the native protein was raised similarly using an initial injection of 50 ug protein in CFA followed by boosts of 50 ug protein in ICFA. This antiserum had a titer of >1:10,000 by ELISA.
  • the antiserum to the 1-30-mer was tested in Western blots on the purified native protein, deglycosylated native protein, and on crude native protein (Con-A bound material), all fixed post-blotting with 0.2% glutaraldehyde.
  • the antiserum detected the purified native protein at _>1 ug and also recognized the deglycosylated protein and the crude protein.
  • the antiserum to the native protein recognized the native protein at ⁇ 100 ng in Western blots.
  • Murine monoclonal antibodies to the purified native protein were prepared as follows. From two fusions 25 positive wells were identified by immunoprecipitation. A group of female Balb/c mice was injected intraperitoneally (IP) with 10-20 ug of purified native protein in CFA. The animals were boosted with 10-20 ug of protein in ICFA. Following the third boost, the mice were bled and serum antibody titers against the protein checked by ELISA. Two animals were found to have titers of ⁇ >1:40,000. They were given a final intravenous (IV) injection of 20 ug protein four days prior to the fusion.
  • IP intraperitoneally
  • IV intravenous
  • the antibody also picked up the protein in a crude fraction (total Con-A bound) and was found to recognize the C-terminal peptide (76-105) but not the N- terminal peptide (1-30).
  • Clones 3B2.17 and 2C11-6 were subcloned by limiting dilu ⁇ tion and were found to be stable and to be IgG isotype. These clones have been deposited in the American Type Culture Collection (ATCC) on 5 April 1989 under the provi ⁇ sions of the Budapest Treaty. Their ATCC designations are, respectively, HB10099 (3B2.17) and HB10098 (2C11.6). M.3.
  • Rat fetuses (19 days old) and 3 day old rats were used for tissue sections. Tissue was fixed in 10% formalin and 5u sections were prepared in paraffin. The sections were treated with xylene (deparaffinized) .and washed with Tris-buffered saline (TBS), pH 7.6, three times. The washed sections were then contacted with a mixture of TBS, 0.05% Tween, 0.5% bovine serum albumin (BSA), 10% normal mouse serum (NMS), for 1 hr at 25°C or overnight at 4°C.
  • TBS Tris-buffered saline
  • NMS normal mouse serum
  • Monoclonal antibody 3B2 (see M.2) at 5 ug/ml in TBS/Tween/BSA/NMS was added and the sections were incubated for 1 hr at room temperature. The sections were then washed three times in TBS/-Tween and contacted with biotinylated goat anti-mouse antibody for 10 min at room temperature. The sections were then washed again three times with TBS/Tween and contacted with streptavidin- horseradish peroxidase for 10 min at room temperature. Thereafter, the sections were washed a final three times with TBS/Tween and contacted with substrate.

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Abstract

Protéines ayant la séquence (I), dans laquelle R1 représente Ala ou Thr, R2 représente Asn ou His, R3 représente Thr ou Ser, R4 représente Ala ou Thr, R5 représente Ile ou Val, R6 représente Val ou Ile et R7 représente Phe ou Ile. Ces protéines sont présentes dans les ostéoblastes et dans les ostéocytes, et peuvent être utilisées comme marqueurs afin de suivre le métabolisme des os et/ou du cartilage.
EP19900912603 1989-08-21 1990-08-21 Bone-specific protein Withdrawn EP0489062A4 (en)

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US5939388A (en) * 1986-07-01 1999-08-17 Rosen; Vicki A. Methods of administering BMP-5 compositions
US5366875A (en) * 1986-07-01 1994-11-22 Genetics Institute, Inc. Methods for producing BMP-7 proteins
US6150328A (en) 1986-07-01 2000-11-21 Genetics Institute, Inc. BMP products
US5187076A (en) * 1986-07-01 1993-02-16 Genetics Institute, Inc. DNA sequences encoding BMP-6 proteins
US5013649A (en) * 1986-07-01 1991-05-07 Genetics Institute, Inc. DNA sequences encoding osteoinductive products
US6432919B1 (en) 1986-07-01 2002-08-13 Genetics Institute, Inc. Bone morphogenetic protein-3 and compositions
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US6586388B2 (en) 1988-04-08 2003-07-01 Stryker Corporation Method of using recombinant osteogenic protein to repair bone or cartilage defects
US5670336A (en) * 1988-04-08 1997-09-23 Stryker Corporation Method for recombinant production of osteogenic protein
US5258494A (en) * 1988-04-08 1993-11-02 Stryker Corporation Osteogenic proteins
US5324819A (en) * 1988-04-08 1994-06-28 Stryker Corporation Osteogenic proteins
US5284756A (en) * 1988-10-11 1994-02-08 Lynn Grinna Heterodimeric osteogenic factor
US7378392B1 (en) 1990-05-16 2008-05-27 Genetics Institute, Llc Bone and cartilage inductive proteins
US5688678A (en) * 1990-05-16 1997-11-18 Genetics Institute, Inc. DNA encoding and methods for producing BMP-8 proteins
US5650276A (en) * 1991-03-11 1997-07-22 Creative Biomolecules, Inc. Morphogenic protein screening method
US5707810A (en) * 1991-03-11 1998-01-13 Creative Biomolecules, Inc. Method of diagnosing renal tissue damage or disease
DK0592562T3 (da) * 1991-06-25 1999-08-30 Genetics Inst BMP-9-præparater
US6287816B1 (en) 1991-06-25 2001-09-11 Genetics Institute, Inc. BMP-9 compositions
EP0601129B1 (fr) * 1991-08-30 2000-11-15 Creative Biomolecules, Inc. Procede de triage de proteines morphogeniques
ATE238417T1 (de) * 1991-11-04 2003-05-15 Inst Genetics Llc Rekombinante knochenmorphogenetische protein heterodimere, zusammensetzungen und verfahren zur verwendung
US6492508B1 (en) 1996-06-03 2002-12-10 United States Surgical Corp. A Division Of Tyco Healthcare Group Nucleic acids encoding extracellular matrix proteins
US5928940A (en) * 1996-09-24 1999-07-27 Creative Biomolecules, Inc. Morphogen-responsive signal transducer and methods of use thereof
US6034062A (en) * 1997-03-13 2000-03-07 Genetics Institute, Inc. Bone morphogenetic protein (BMP)-9 compositions and their uses

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JPH05500503A (ja) 1993-02-04
AU632160B2 (en) 1992-12-17

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