WO1991008749A1

WO1991008749A1 - Novel procedure for obtaining proteins starting from an extract of non-collagenous proteins from bony material and its use for the preparation of a novel protein inhibiting the proliferation of osteoblasts

Info

Publication number: WO1991008749A1
Application number: PCT/EP1990/002295
Authority: WO
Inventors: Patrick Mahy; Hugo Van Heuverswyn
Original assignee: Innogenetics NV SA
Current assignee: Fujirebio Europe NV SA
Priority date: 1989-12-20
Filing date: 1990-12-20
Publication date: 1991-06-27
Anticipated expiration: 1992-06-20
Also published as: AU7066091A

Abstract

The invention relates to a procedure for obtaining a protein purified to a satisfactory homogeneous state, starting from an extract of non-collagenous proteins of bone matrix. Another subject of the invention is a device for the implementation of the procedure of the invention, a protein inhibiting the proliferation of osteoblasts likely to be obtained by the above-mentioned procedure. The procedure described in the present invention is characterized in that it comprises the following steps: the extract of non-collagenous proteins, solubilized beforehand in a solvent, is subjected to a preparative chromatography in the liquid phase on a column of gel with the aid of an elution buffer and an electric field, under conditions such that the desired protein is eluted, and is found, during or at the end of the chromatography, to be present in solution in the elution buffer, alone and free from contaminants, in at least one of the fractions collected at the exit of the column, the fraction containing the desired protein in solution in the elution buffer is recovered, the elution buffer is removed and the desired protein is recovered which, if necessary, is subjected to a renaturation step and/or a reactivation step.

Description

NOVEL PROCEDURE FOR OBTAINING PROTEINS STARTING FROM AN EXTRACT OF NON-COLLAGENOUS PROTEINS FROM BONY MATERIAL, AND ITS USE FOR THE PREPARATION OF A NOVEL PROTEIN INHIBITING THE PROLIFERATION OF OSTEOBLASTS

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The subject of the invention is a novel procedure for obtaining and purifying proteins starting from an extract of non-collagenous proteins from bone matrix.

Another subject of the invention is a device for the implementation of the procedure of the invention.

Another subject of the invention is an inhibitory protein of the proliferation of osteoblasts likely to be obtained by the above-mentioned procedure.

Another subject of the invention is pharmaceutical compositions containing as active substance the above-mentioned protein, intended in particular for the treatment of Paget's disease, osteosarcomas.

Another subject of the invention is the utilization of the protein for the diagnosis of a disease likely to be correlated with a concentration located outside the range of values usually attributed to a healthy individual.

Bone is an extremely specialized tissue with unique mechanical properties which are derived from its special matrix structure. Although it is composed above all of mineral substances, the small amount of protein which it contains is, however, indispensable for its growth, its maintenance including its permanent remodeling and its repair. Among the constituents of bone may be mentioned collagen, the activity of which is to ensure the resistance of bone to tension, including proteoglycans, non-collagenous proteins, lipids and the acidic proteins associated with the mineral part constituted of hydroxyapatite crystals. cells. Osteogenesis is conducted by the osteoblasts, the cells forming bone. The tissue remodeling which bone undergoes apparently results from the interaction between the cells resorbing bone, i.e. the osteo-

Clasts, and the osteoblasts. These specialized cells are responsible for this particular process. They produce specific substances

(hormones) which-ιare involved qualitatively and quantitatively in the structure of bone.

Bone diseases as well as physical stresses (fracture) cause bone hyperactivity at a rate higher than that which a healthy

individual can withstand. It is thus important to identify the chemical agents (hormones, pharmaceutical substances, growth factors) which induce bone formation but. also which limit it, control it and which prevent any dysregulation. Such substances may Selectively bone forming cells , stimulate the seine cells, or be responsible for the differentiation of suitable progenitor cells as well as for the monitoring of these same processes with or without

the support of resorbtive cells.

Urist, Huo, Brownell et al. have demonstrated the presence of non-collagenous proteins associated with the bone matrix,

have isolated them by acting on demineralized bone by means of a

succession of dissociative treatments. This has made it possible for thera to fractionate the non-collagenous proteins with the aid of

repetitive chromatographiy and to characterize an osteogenic protein, the morphogenetic protein of bone (BHP) (Proc. Natl. Acad. Sci. USA, vol. 81, pp. 371-375, January 1984, M.R. Urist et al.).

More precisely, in the article previously cited, it is

pointed out that this protein was isolated from a non-collagenous extract (derived from demi r li ed bone) insoluble in Triton X-100 and in water, to electrophoresis on

polyacrylamide gel in the presence of sodium dodecylsulfate (SDS-PAGE), which gives bands, each of which corresponds to the visualization of an individual protein the acrylamide gel wasthencut into slices in order to separate the individual bands.

A preparative electrophoresis on gel was then carried out:

the slices of the gel were extracted with 6M urea in order to isolate More precisely, in the article previously cited, it is pointed out that this protein was isolated from a non-collagenous extract (derived from demineralized bone) insoluble in Triton X-100 and in water, the extract of which was subjected to electrophoresis on polyacrylamide gel in the presence of sodium dodecylsulfate (SDS-PAGE), which gives bands, each of which corresponds to the visualization of an individual protein; the acrylamide gel was then cut into slices in order to separate the individual bands.

A preparative electrophoresis on gel was then carried out: the slices of the gel were extracted with 6M urea in order to isolate the individual proteins from their support. They were subjected again to an electrophoresis on slab gels in order to confirm their molecular weight and their purity (absence of contaminants). This has made it possible in particular to separate a protein of molecular weight of 18, 500 daltons which has been designated as "BMP".

It is known to the person skilled in the art that the procedure previously described impairs the yield and the osteo-inducing biological activity of the proteins sought.

That is the reason why another preparative procedure was envisaged in order to obtain biologically active osteo-inducing proteins (BMP).

In the PNAS article previously cited, the authors provide another method for obtaining the protein. This procedure is the following: the above-mentioned protein has also been isolated from a non-collagenous extract insoluble in Triton X-100 and in water, derived from demineralized bone, the extract of which was subjected to chromatography on a hydroxyapatite column in order to recover the fraction containing three proteins of molecular weight ranging from 17,000 to 18,000 daltons separated from the proteins of molecular weight were then separated from the gel slices by electroelution (Development and Diseases of Cartillage and Bone Matrix, p. 149-176, 1987, Alan R. Liss, Inc.; M.R. Urist et al.).

Furthermore, several groups have demonstrated osteogenic activities for different proteins of different molecular weights and isoelectric points (3).

In the same way, Urist et al. have also described osteogenesis. as a classical biological system containing osteo-inducing substances (demonstrated) (cf. above) and inhibitory substances (not characterized) which can act on the mitoses of osteoblasts, for example, or on the differentiation of osteo-progenitor cells (1).

Furthermore, Brownell et al. have described a substance OIP "osteoblast inhibitor protein" molecular weight of about 38000d biological activity of which is to inhibit

the proliferation of osteoblast cells (ROS 17/2.8) of an osteosarcoma linerby40%to70%This protein shows a complete sequence homology with osteonectin (2)

When wundeiung the state of the artmethodfor obtaining proteins isolated and purified starting from a set of non-collagenous proteins, derived from demineralized bone tissue, the presently available techniques one much then:

- require several chromatographies and hence imply a loss of material,

- or make it possible to work only with small amounts of starting materials, and hence to obtain amounts of isolated proteins which are sometimes difficult to detect;

- or also lead to the denaturation and loss of biological activity of the desired protein;

- or finally involve the removal of dyes classically used in the SDS-PAGE and which, in any case, lead to the recovery of denatured proteins.

In this respect, it should be mentioned that it is difficult to/ remove (completely dyes used for staining proteins which one wishes to reveal by SDS-PAGE. In addition, the staining interferes with the Furthermore, Brownell et al. have described a substance OIP "osteoblast inhibitor protein" (molecular weight of about 38,000 daltons), the biological activity of which is to inhibit the proliferation of osteoblast cells (ROS 17/2.8) of an osteo-sarcoma line by 40% to 70%. This protein shows a complete sequence homology with osteonectin (2).

When considering the state of the art methods for obtaining proteins isolated and purified starting from a set of non-collagenous proteins, derived from demineralized bone tissue, the presently available techniques are such that they:

- require several chromatographies and, hence, imply a loss of material,

- or make it possible to work only with small amounts of starting materials, and, hence, to obtain amounts of isolated proteins which are sometimes difficult to detect;

In this respect, it should be mentioned that it is difficult to completely remove dyes used for staining proteins which one wishes to reveal by SDS-PAGE. In addition, the staining interferes with the evaluation of the amount of proteins assayed by spectrophotometry. Moreover, in the biological assays, nothing proves that traces of dyes can induce an induction or an inhibition of the biological activity of the proteins obtained.

The subject of the present invention is to provide a novel procedure making it possible to separate and purify individual proteins in a satisfactory state of homogeneity and biologically active, starting from an extract of non-collagenous proteins of bone matrix. Another subject of the present invention is a procedure for renaturation and/or reactivation of proteins obtained starting from an extract of noncollagenous proteins of bone matrix.

Another subject of the invention is a novel preparative chromatography column likely to be implemented in the procedure of the invention.

Another subject of the invention is a novel protein likely to be obtained by the procedure of the invention and possessing interesting biological properties of inhibition of the proliferation of osteoblasts.

Another subject of the invention is pharmaceutical compositions containing as active substance the protein of the invention.

Another subject of the invention is a novel diagnostic procedure, involving the protein of the invention and/or antibodies, in particular polyclonal antibodies and especially monoclonal antibodies directed against this protein.

The procedure of the invention for obtaining a protein purified to a satisfactory state of homogeneity starting from an extract of non-collagenous proteins of bone matrix is characterized in that it comprises the following steps:

- the extract of non-collagenous protein, dissolved beforehand in a solvent, is subjected to a preparative chromatography in the liquid phase on a gel column with the aid of an elution buffer and an electric field under conditions such that the desired protein is eluted and is found, during or at the end of the chromatography, to be present in solution in the elution buffer, alone and free of contaminants, in at least one of the fractions recovered at the exit of the column, - the fraction containing the sought protein in solution in the elution buffer is recovered, the elution buffer is removed and the desired protein is recovered and, if necessary, is subjected to a renaturation step and/or a reactivation step.

An advantageous procedure of the invention of a protein purified to a satisfactory homogeneous state, starting from an extract of non-collagenous proteins of bone matrix, is characterized in that it comprises the following steps:

- the extract of non-collagenous proteins, dissolved beforehand in a solvent, is subjected to a preparative chromatography in the liquid phase on a gel column likely to separate the proteins in an order corresponding to their molecular weight,

- with the aid of an elution buffer, the introduction of which takes place in the neighbourhood of the top of the column, in particular in an upper reservoir situated at the top of the column, and the exit of which occurs in the neighbourhood of the base of the column, in particular in a lower reservoir situated at the base of the column, this elution buffer being such that it confers on the proteins a relatively uniform charge, and

- with the aid of an electric field created between the introduction of the elution buffer and the exit of the elution buffer, under conditions such that the desired protein is eluted and is found, during or at the end of the chromatography, to be present in solution in the elution buffer, alone and free from contaminants, in at least one of the fractions recovered at the exit of the column,

- the fraction containing the desired protein in solution in the elution buffer is recovered, the elution buffer is removed and the desired protein is recovered, and, if necessary, is subjected to a renaturation step and/or a reactivation step.

The procedure of the invention makes it possible to separate proteins starting from a set of non- collagenous proteins of bone matrix and to separate them individually in purified form and biologically active and in the same way makes it possible to obtain a specific protein in a purified form and biologically active.

The procedure of the invention presents the advantage of limiting the number of fractionations, hence, consequently, of limiting the loss of the product.

Furthermore, it presents the advantage of being able to work with amounts of extract of non-collagenous proteins of bone matrix (starting extract) such that the desired protein is obtained in sufficient quantity to be detected and in sufficient quantity for the biological assays to be carried out.

Furthermore, it presents the advantage of giving rise to purified proteins, i.e. free from contaminants, which, if they were present, would be likely to modify the biological activity.

In addition, it presents the advantage of giving rise to proteins, the biological properties of which are not irreversibly denatured.

By "satisfactory homogeneous state" is to be understood a protein which is homogeneous with respect to one or several characteristics of purity or homogeneity normally used by the person skilled in the art in the area of the chemistry of proteins. For example, a protein in a satisfactory state of homogeneity shows constant and reproducible characteristics within standard experimental deviations with respect to parameters which are the following: amino acid analysis, spectrum of bands in standard electrophoresis on polyacrylamide gel (PAGE) or another chromatographic technique, molecular weight, isoelectric point, biological properties and other parameters.

This term does not mean that it is necessary to exclude the presence of minor impurities which do not interfere with the biological activities of the protein and which may be present, for example, on the grounds of adequate purification.

In this respect, the expression "purified to a satisfactory homogeneous state" means in particular that the protein is free of contaminants which, if they were present, would modify the biological properties of the protein.

The expression "extract of non-collagenous proteins of a bone matrix" designates a set, for example, an aggregate of non-collagenous proteins extracted from a bony tissue, advantageously demineralized.

As demineralized bony tissue, mention may be made of bones, dentine or osteosarcomas.

The extract of non-collagenous proteins of a bone matrix may be obtained by treating a bone in the following manner:

Typically, it is possible to start from long bones (diaphysis of long bones) from a mammal (for example bovine tibia) or from the diaphyses derived from femurs or tibias of rats.

These bones are treated and demineralized by using the well-known conventional standard techniques such as those described by Urist in the patent

Nº 4,294,753.

A practical method of treatment for demineralization of a bone is the following: the periosteal layer surrounding the bone (the bone is preferably taken from a young mammal) is taken from a young mammal. The bones taken immediately after slaughter of the animal are cleaned. The soft tissues adhering to the bone are resected, for example manually (with the aid of a scalpel).

The bone marrow is removed by successive washings with cold water. The bone is then cut into small pieces of about 2 cm, soaked in liquid nitrogen then powdered in fine particles (I 500 μm) with the aid of a grinder (Wiley).

The homogenized particles are washed with a saline solution or a buffer, such as Tris-HCl buffer (pH 7.0) or 0.15 M NaCl and water, then with an ethanol/ether solution or a chloroform/methanol solution in a volume/volume ratio equal to 1. The washed homogenized particles are then dried in a vacuum or in air: this bone powder may be stored at -80°C for prolonged periods of time. In order to demineralize and extract the proteins efficaciously, the bone powder is sieved in order to obtain particles having a range of size of about 75 to 500 μ in diameter. The demineralization (i.e. the removal of calcium phosphate from the bone matrix) is ensured by repeated washings with a solution of HCl, for example by stirring the bone powder for 1 h with about 10 to 15 ml of 0.5 N HCl per gram of dry weight of bone powder, decantation of the liquid, then repetition of this procedure 3 or 4 times.

The demineralized bone powder is then washed with deionised distilled water until the pH approaches neutrality. The demineralized bone powder may be stored at very low temperatures, for example from -20 to -80°C.

The demineralization of the bone powder may also be carried out by using other known standard procedures, for example by utilizing a chelating agent such as ethylenediaminetetraacetic acid. In order to determine whether the bone powder is sufficiently demineralized after the treatment with HCl in order to be suitable for extraction of the proteins, the powder, rinsed with water, may be tested for its mineral content, for example by the method of Von Kossa, see J. Von Kossa, Ziegler ' S Beitr. 29 , 163 (1901) . When the Von Kossa spot is negative, the treated bone powder is sufficiently demineralized to be suitable for the extraction of the proteins.

As far as the extract of non-collagenous proteins is concerned, it may be extracted from the demineralized bone tissue according to the method of Urist (1).

Generally speaking, one may proceed as follows:

- one starts from demineralized bone powder, ground as indicated previously, and defatted by the action of a chloroform-ethanol mixture,

- the non-collagenous proteins are extracted by dissolution of the bone powder in a solution of 4 M GuHCl/0.5 M CaCl₂ or 6 M urea/0.5 M CaCl₂ and 1.0 mM of N-ethylmaleimide (NEM) 0.1 mM of benzamidine-HCl per liter, and are stirred at room temperature for 24 h, which gives rise to a suspension,

- the above-mentioned suspension is filtered through a Whatman filterpaper, the filtered volume obtained is transferred to an ultrafiltration device with 10 K hollow fibres (Amicon), which makes it possible to obtain a final volume to be dialysed less than or equal to 10% of the filtered volume and makes possible an easier and more efficacious dialysis.

- it is dialysed against water fo 3 days at 4°C; the content of the dialysis membranes is heated to ± 35ºC each time a gelification is produced;

- when the content has been dialysed 3 times, it is heated to 35ºC and the precipitate is centrifuged; the precipitate is washed with cold water and is redissolved in 4 MGuHCl/0.05 M CaCl₂;

- it is centrifuged in order to remove the gelatinous peptides and the supernatant is dialysed in 11 volumes of 0.25 M sodium citrate buffer (pH 3.1); after the dialysis, the content of the dialysis membrane is heated to about 35°C, the precipitate is centrifuged at 30-35ºC and washed 3 times with cold water; in order to remove the lipoproteins and associated lipids, the washed precipitate is extracted with a chloroform- methanol mixture 1/1; the solvent is filtered, for example, through a Bϋchner funnel and the product is left to dry at room temperature;

- the proteins, defatted and soluble in 4M GuHCl, are dissolved in 100 volumes of GuHCl; the insoluble material is centrifuged; the supernatant is dialysed against one volume of cold water which decreases the GuHCl concentration from 4 to 1.5 M; this separates the insoluble 24 K protein from the proteins soluble in 1.5 M GuHCl, by differential precipitation; the solution is centrifuged at 9000 rpm for 30 min. at 4ºC;

- the precipitate is washed 3 times with cold water and lyophilized, then the soluble proteins are dialysed in 1.5 M GuHCl in order to produce a precipitate; the proteins insoluble in water and soluble in 1.5 M GuHCl are centrifuged and washed 3 times with cold water;

- the moist proteins are redissolved in 1 1 of 4 M GuHCl/0.5 M CaCl₂ and dialysed against an equal volume of 4 M GuHCl containing 0.10 M of Trizma-HCl, pH 7.2 and 0.2% or Triton X-100 (0.2 g per 100 ml) (ref. 1); the protein of 34 K is dissolved;

- the precipitate insoluble in Triton X-100 is centrifuged; an extract of non-collagenous proteins is obtained free from proteins of 6 K, 24 K and 34 K; it is dialysed and extracted against deionized water at

4ºC until there is precipitation, and lyophilized; it is also possible to recover the proteins soluble in Triton X-100 and lyophilize them;

- the precipitate obtained by dialysis against water at 4ºC is then taken up again and redissolved in 6 M urea and is purified by ultrafiltration; when it is dialysed again against water, most of the content of the dialysis membrane does not precipitate and the soluble portion is lyophilized and recovered.

In the foregoing and in what follows, Gu represents guanidine.

As far as the starting extract of non-collagenous proteins of the bone matrix used as starting material in the procedure of the invention is concerned, it is advantageously insoluble in water, and is either insoluble in Triton X-100, or is soluble in Triton X-100.

The extract of non-collagenous proteins of bone matrix is advantageously insoluble in water and insoluble in Triton X-100.

By preparative chromatography, is specified a chromatography on a column of gel, which is advantageously constituted of meshes. For a given dimension of meshes, the largest proteins are obstructed and the others pass through the gel as a function of their molecular weight, the smallest proteins being eluted first. In order to make the proteins move forward, they are electrically charged in an essentially uniform manner owing to the elution buffer and a current is applied such that the direction of the current and the charge of the proteins propel the proteins through the gel. The fractions issuing from the column containing the eluted proteins are recovered in a fraction collector which contains tubes.

The current is advantageously applied between an upper reservoir (situated in the neighbourhood of the top of the column and which feeds the column with elution buffer) and a lower reservoir (situated at the base of the column) in which the column rests and in which the elution buffer is found after passage through the gel.

For the elution, the two parameters to take into account are the density of the gel (i.e. the size of the meshes) and the intensity of the current: if the gel is too dense compared with the size of the proteins to be eluted, it is necessary to increase the current on account of the retention caused by the density of the gel, which leads to too high a temperature, likely to denature the proteins. Advantageously, the intensity of the current should not exceed about 70 mA, and the voltage (U) should not exceed about 150 V.

Conversely, if the gel is not sufficiently dense in relation to the size of the proteins to be eluted, the proteins risk being eluted too quickly, which diminishes the specificity of the separation to the extent that there is a risk of not recovering in at least one of the tubes of the collector only the desired protein. In fact, there is a risk of recovering at least two proteins per tube.

The gel may be selected from among acrylamide- bisacrylamide gel and may be composed of different concentrations of acrylamide, for example about 20% - about 12% - about 3%, or formed of a linear or non linear gradient of about 25% to about 3%, and advantageously from about 20% to about 3%.

According to an advantageous embodiment of the procedure of the invention, the gel is a polyacrylamide gel, the density of which is selected as a function of the size of the desired protein. To give an idea, - when the desired protein has a molecular weight higher than 100 kD, the density of the acrylamide gel used is less than or equal to 10%; - when the desired protein has a molecular weight included between about 40 kD to about 100 kD, the density of the acrylamide gel used is higher than or equal to about 10%;

- when the desired protein has a molecular weight lower than about 40 kD, the density of the acrylamide gel used is higher than or equal to about 15%;

- when the desired protein has a molecular weight lower than about 20 kD, the density of the acrylamide gel used is higher than or equal to about 20%.

The polyacrylamide gel which fills the column may advantageously be constituted by an initial layer of gel of low density (about 3%) (concentration gel) and a second layer of gel of higher density (about 12% to about 17%) (resolution gel). The part of the gel corresponding to the resolution may itself be constituted of two different densities, for example a layer of about 12% to about 15% , and a second layer of about 15% to about 17%.

In order to first solubilize the starting extract of non-collagenous proteins of bone matrix, recourse may be had as solvent to a buffer solution containing urea and more particularly a solution containing

- about 0.05 to about 0.1 M of Tris, and advantageously about 0.0625 M of Tris

- about 0.1 to about 1% of SDS, in particular 0.2% of SDS , and

- about 1 M to about 8 M of urea, in particular 2 M of urea,

- for a total volume of 100 ml (depending on the experimental requirements) in distilled water (preferably deionized) at pH of about 6.8 (possibly going from a pH of about 6 to about 8) determined by the addition of concentrated HCl and in the absence of β-mercaptoethanol from the solution. In the elution buffer, the element responsible for the essentially uniform charge conferred on the proteins is advantageously selected from among in particular SDS which charges the proteins negatively. An advantageous solution buffer is constituted by:

- about 0.02 M to about 0.1 M, in particular about 0.4 M Tris, and 0.1% SDS, from about 0.01 to about 1%, in particular about 0.1% of SDS and from about 0.2 to about 1 M of glycine, in particular about 0.384 M glycine, at pH from about 7 to about 9, in particular at pH 8.3 (concentrated HCl).

It is necessary that the solvent of the starting extract of non-collagenous proteins of bone matrix be compatible with the elution buffer of the preparative chromatography.

At the end of the preparative chromatography, the proteins contained in the starting extract are separated and each individual protein is found alone in at least one tube of the collector, and may be found alone in several tubes. The set of tubes is such that in principle the number of tubes in which a specific protein is found depends on the rate of elution.

However, when it is desired to obtain a specific protein, it may be recovered during chromatography to the extent that it is known to which peak of the tracing of the chromatogram it corresponds.

In the procedure of the invention, the elution buffer of the preparative chromatography is removed in order to recover the desired protein.

According to another embodiment of the procedure of the invention, when the protein is recovered it is possible to carry out a renaturation and/or reactivation step.

According to another advantageous embodiment of the procedure of the invention, the step for the removal of the elution buffer may be carried out under conditions such that there is at the same time renaturation and/or reactivation of the sought protein, in particular in the case in which the elution buffer contains elements likely to denature the proteins, such as sodium dodecylsulfate.

In order to remove the elution buffer and renature and/or reactivate the sought protein, it is possible to have recourse to at least one of the following two steps in any order, namely:

* dialysis of the fraction containing the desired protein in solution in the elution buffer of the preparative chromatography against a mixture of PBS, BSA and Tween-20, and

* dialysis against water, advantageously distilled water.

An advantageous mixture of PBS, BSA and Tween-20 is constituted by:

- PBS: about 0.15 M,

- BSA: about 5 μg/ml,

- Tween-20: about 0.5 mg/ml.

When the two dialysis steps take place, the precipitate or advantageously the content of the dialysis membrane (i.e. the precipitate and the supernatant) obtained at the end of that one of the two dialyses which constitutes the first step, is solubilized in order to be dialysed.

According to an advantageous embodiment of the procedure of the invention, at the end of the preparative chromatography step, are first carried out

- an initial dialysis of the fraction containing the desired protein in solution in the elution buffer of the preparative chromatography against a mixture of PBS, BSA and Tween-20, which leads to the formation of a precipitate and a supernatant,

- the precipitate or advantageously the whole of the content of the dialysis membrane (i.e. the precipitate and the supernatant) is recovered in order not to lose the proteins which might have passed into the supernatant, and advantageously the whole of the content of the dialysis membrane is then lyophilized, which makes it possible to store the lyophilizate obtained,

- the redissolution of the lyophilizate is then carried out, in particular with the aid of GuHCl of molarity of about 4 M to about 8 M, in particular 6 M or of urea of about 4 M to about 8 M, in particular 6 M,

- a second dialysis of the dissolved lyophilizate is carried out against water.

It is also possible to consider taking the content of the membrane after the first dialysis and, instead of lyophilizing it, dissolving it progressively in a solvent (GuHCl or urea), to the extent that the volume of solvent added is such that the total volume (eluted volume from the preparative chromatography plus volume of solvent) does not exceed, for example, about 200 ml.

In order to obtain complete solubilization, the volume of solvent depends on the amount of material to be dissolved and the capacity of this material to be soluble in the GuHCl solution.

When SDS is used in the elution buffer it is advantageous, on the one hand, to remove it to the extent that it denatures the proteins, i.e. causes them to loose their biological properties, and, on the other hand, to the extent that it is toxic for the cells, both in vivo and in vitro.

Furthermore, in the case in which, at the end of the preparative chromatography, one must resort to the two dialyses described above, separated by a redissolution, it is also advantageous to remove the redissolution agent, in particular when it is GuHCl or urea, which denature the proteins. The preparative chromatography of the invention is advantageously carried out with a system such that the elution buffer is added continuously in order that there is no limitation of the elution by the volume of the elution buffer. It is possible to make provision for a continuous feeding device or indeed provide an upper reservoir situated above the column, which reservoir is placed in contact with a sufficient quantity of elution buffer, which makes it possible to replenish the upper reservoir as the elution solvent is used up.

In order to determine which is the fraction after preparative chromatography which contains the sought protein, it is possible to carry out electrophoresis on polyacrylamide gel in the presence of SDS (SDS-PAGE). When the desired protein is found alone in several tubes, the content of the tubes is then pooled.

The preparative chromatography makes it possible to utilize considerable quantities of starting extract of non-collagenous proteins of bone matrix since it is not limited by the elution volume. As an indication, the extract used may vary from 20 to 100 mg, this being a function of the percentage which the desired protein represents in relation to the extract.

According to another preferred embodiment of the invention, the preparative chromatography step is preceded by a preliminary step of fractionation of all of the non-collagenous proteins, making it possible to obtain a fraction enriched in the desired protein, which enriched fraction is then subjected to the preparative chromatography step.

The preliminary step may be constituted by any separation which makes it possible to obtain a fraction enriched in the sought protein. For example, it is possible to have recourse to a Sephacryl filtration, to a DEAE or to a chromatography on hydroxyapatite.

According to another advantageous embodiment of the procedure of the invention, after the preliminary step, the buffer is removed, in particular when this latter is different from the solubilization buffer and from the elution buffer used in the preparative chromatography, and the fraction enriched in the desired protein is redissolved in a solubilization buffer compatible with the elution buffer of the preparative chromatography, the solubilization buffer containing in particular urea, and being preferably constituted by a mixture: urea, Tris-HCl, and SDS.

In another preferred embodiment of the invention, the preliminary step is constituted by a chromatography on hydroxyapatite, designated hereafter by HAP chromatography.

The HAP chromatography is performed as indicated for example in the following documents: Proc. Natl. Acad. Sci. USA, vol. 81, pp. 371-375, January 1984, M.R. Urist et al.; Development and Diseases of Cartillage and Bone Matrix, p. 149-176, 1987, Alan R. Liss, Inc.

In order to solubilize the extract of non- collagenous proteins in order to subject them to the HAP chromatography, it is possible to advantageously use the buffer 0, the composition of which is the following:

- buffer 0: 6 M urea + 0.01 M of phosphate (600 ml of stock urea solution + 10 ml of stock phosphate solution plus 390 ml of distilled water for 1 liter of buffer).

The stock phosphate solution is obtained in the following manner:

- preparation of 100 ml of 1 M phosphate solution (Merck), that is 51% of Na₂HPO₄ and 49% of NaH₂PO₄. The Na₂HPO₄ and NaH₂PO₄ are dissolved in 50 ml of distilled water, then the NaH₂PO₄ is mixed with Na₂HPO₄, until a solution of pH 6.8 is obtained.

The stock urea solution is obtained in the following manner:

- preparation of a stock solution of 1 liter of 10 M urea, that is 600 g dissolved in 1 liter of distilled water.

The elution buffer advantageously used in HAP chromatography is the same as that used to solubilize the extract of non-collagenous proteins.

The HAP chromatography progresses such that it is possible to collect fractions (each usually containing several proteins), which fractions are eluted as a function of the affinity of the proteins which they exhibit towards hydroxyapatite.

The HAP chromatography is advantageously done at a concentration of 10 mM to 500 mM of phosphate buffer, and advantageously of 10 mM to 250 mM of phosphate buffer.

The fraction containing the desired protein is determined, for example, by SDS-PAGE electrophoresis.

After the hydroxyapatite chromatography, it is necessary, on the one hand, to remove the elution buffer, and, on the other, to dissolve the fractions obtained in the elution buffer of the preparative chromatography.

In order to do this, it is possible to dialyse the fraction enriched in the desired protein, in particular against water, advantageously distilled water, the purpose of which is to precipitate the proteins contained in the said fraction. The precipitate is recovered, with or without the supernatant. It is lyophilized and the precipitate is redissolved, in particular in a solubilization buffer containing urea and constituted in particular by a mixture: urea. Tris-HCl and SDS, as indicated above, in order to subject it to preparative chromatography.

Usually, as one works with relatively large quantities, it is possible to ignore the supernatant and take only the precipitate.

Instead of the lyophilization of the precipitate obtained after the dialysis step, it is also possible to concentrate the precipitate obtained after the dialysis by centrifugation, and after the concentration step, it is possible to pass over a membrane of the Amicon type, for example, and redissolve the concentrate obtained in the solubilization buffer of the preparative chromatography to the extent to which the volume of the solubilization buffer does not lead to too large a volume which does not exceed the capacity of the preparative column.

The resolubilization of the lyophilized precipitate obtained after the HAP chromatography may take place in a solubilization buffer containing per 100 ml:

- SDS: about 200 mg,

- Tris-HCl: about 756 mg,

urea: about 12 g, adjusted to a pH of 6.8 (concentrated HCl).

The procedure of the invention comprising the preliminary step of fractionation makes it possible to enrich the starting extract in a ratio such that by starting from about 4 g of starting extract, a fraction enriched in protein is obtained containing about 215 mg of sought protein, which makes it possible to obtain about 0.350 mg of the sought protein after the preparative chromatography. If one starts with the same amount of starting extract, treated directly in preparative chromatography for about 100 mg of starting extract, about 0.250 mg of sought protein is obtained after the preparative chromatography. Consequently, generally speaking, the preliminary step of fractionation makes it possible to improve the yield of the quantity of desired protein by a factor of about 20 fold, and the sum of the preliminary fractionation step and the preparative column chromatography make it possible to improve the yield of the quantity of desired protein by a factor of about 400 fold.

It should be noted that, in the case in which recourse is had in the procedure of the invention to a preliminary step of HAP chromatography, what is subjected to the preparative chromatography is not the starting extract of non-collagenous proteins of bone matrix, but a fraction enriched in the desired protein.

In the case in which recourse is had to the preliminary fractionation step, the preparative chromatography thus takes place on a fraction enriched in the desired protein and it occurs as was described with regard to the preparative chromatography carried out on an extract of non-collagenous protein of bone matrix.

In the case of the recovery of a specific protein, in order to locate which is the fraction obtained after the HAP chromatography which contains the sought protein in order to subsequently subject this fraction to the preparative chromatography , it is possible to subject each of the fractions obtained after the HAP chromatography to a SDS-PAGE electrophoresis .

The HAP chromatography makes it possible to treat about 10 mg of proteins per g of HAP. In the case of large quantities of proteins to be purified, this ratio may be increased to about 20 mg of proteins/g of HAP.

A particularly advantageous procedure for obtaining a protein of the invention is characterized in that - a demineralized, ground and defatted extract of non- collagenous proteins of bone matrix is subjected to a chromatography on hydroxyapatite, the extract of the non-collagenous proteins having been solubilized beforehand, preferably in the same buffer as the elution buffer of the HAP chromatography, in particular the buffer 0,

- a fraction enriched in the sought protein in solution in the elution buffer of the HAP chromatography is recovered,

- the elution buffer of the HAP chromatography is removed, in particular by dialysis against distilled water and the precipitate obtained is recovered, possibly lyophilized, containing a fraction enriched in the desired protein,

- the fraction enriched in the desired protein is subjected to a preparative chromatography after having been dissolved in a solvent advantageously containing urea, the acrylamide gel of the preparative column having a density such that it makes possible the elution of the desired protein with the aid of an elution buffer advantageously containing SDS, and with the aid of an electric field, and the conditions of chromatography being such that the desired protein is found alone in solution in the elution buffer, and free of contaminants in at least one of the tubes of the collector corresponding to the fractions collected in parallel to the chromatogram,

- the protein is recovered, if necessary reactivated and/or renaturated by removing the elution buffer of the preparative chromatography, in particular

* by a first dialysis of the protein in solution in the elution buffer of the preparative chromatography against PBS, BSA and Tween-20, after which the precipitate, and possibly the supernatant, obtained is recovered, the precipitate and possibly the supernatant containing the sought protein being possibly lyophilized, the SDS being partially removed after this first dialysis,

* by a solubilization of the above-mentioned precipitate, and possibly of the supernatant, containing the sought protein, with the aid of a solubilization agent, in particular with the aid of urea of about 4 M to about 8 M, in particular about 6 M, or of GuHCl of about 4 M to about 8 M, in particular about 6 M,

* and by a second dialysis of the solution previously obtained against distilled water, the precipitate, and possibly the supernatant, obtained containing the desired protein may be lyophilized, the SDS being completely removed as well as the above- mentioned solubilization agent after this second dialysis,

- the sought protein is recovered.

According to an advantageous embodiment of the invention, after the first dialysis and the second dialysis, which follow the preparative chromatography, the content of the dialysis membrane, i.e. the precipitate and the supernatant, is recovered in order to avoid losing the desired protein.

According to another embodiment of the procedure of the invention, when, after the preparative chromatography step, the two dialyses are carried out, separated by a dissolution and when the elution buffer of the preparative chromatography is a mixture of SDS, glycine and Tris-HCl, and the solubilization agent between the two dialyses is urea or GuHCl, the purpose of the initial dialysis is to partially remove SDS as well as Tris-HCl and glycine, whereas the purpose of the second dialysis is to remove the SDS as well as the urea or GuHCl completely. The advantage of the procedure of the invention is to be able to obtain a protein of molecular weight of about 27 kD, possessing inhibitory properties towards the proliferation of osteoblasts, this protein being obtained by the procedure comprising the following steps:

- a non-collagenous fraction of bone matrix insoluble in Triton X-100 and in water is subjected to a chromatography on hydroxyapatite,

- the fraction eluted at a concentration of 10 mM of phosphate buffer is recovered in solution in the elution buffer and containing a fraction enriched in the sought protein,

- the buffer is removed in particular by dialysis against distilled water in order to obtain a precipitate, possibly lyophilized,

- the above-mentioned precipitate is resolubilized with the aid of an appropriate solvent,

- this solubilized fraction enriched in the desired protein is subjected to a preparative chromatography with the aid of an elution buffer, the function of which is also to charge the proteins to be eluted with negative charges, which buffer contains in particular SDS, and the protein of molecular weight of about 27 kD is collected in solution in the elution buffer,

- the above-mentioned protein in solution in the elution buffer of the preparative chromatography is subjected to two dialyses: namely, a first dialysis against a mixture of PBS, BSA and Tween-20, and a second dialysis against distilled water, these two dialyses being carried out in any order, and these two dialysis steps being separated by the recovery of the content of the dialysis membrane, or of the precipitate resulting from the dialysis, which content or precipitate is completely solubilized in urea or GuHCl,

- the desired protein is recovered. Another subject of the invention is a device for preparative chromatography in the liquid phase.

This device consists of

- a column filled with gel,

- an upper reservoir to which the upper outlet of the column is connected, this reservoir fitted with a negative electrode being intended to receive the elution liquid, and being fed almost continuously in order that the level of the elution liquid in the reservoir is practically constant,

- a porous disk, advantageously plastic (called here "first disk") which maintains the gel in the column, and a ring situated under the above-mentioned disk which determines the volume of the elution chamber,

- a membrane situated at the base of the elution chamber, designated hereafter as intermediate membrane,

- a collar of larger diameter than the diameter of the elution chamber, at the interior of which the first disk and the collar determining the volume of the elution chamber are situated, which collar accommodates all of the upper parts of the device, including the column, the lower end of which fits into the said collar,

- advantageously a second disk, preferably of plastic, situated under the intermediate membrane, this second disk being itself placed on a third disk, in particular of plexiglass, which makes possible the passage of the current and which seals the base of the column by means of fastenings such as screws situated on the perimeter of the third disk and defining a ring, the second and third disks contiguous to the intermediate membrane and the intermediate membrane being advantageously pierced at their center by a small orifice allowing the passage of a capillary connecting the elution chamber to the fraction collector, in particular via a pump, - a lower reservoir in which the base of the column with the elution chamber is immersed, this reservoir containing a volume at least equal to that of the upper reservoir and advantageously having dimensions identical with those of the upper reservoir, this lower reservoir containing a positive electrode as well as the same buffer as that of the upper reservoir, the volumes of the reservoirs being maintained constant by the flow from the upper reservoir into the lower reservoir which is emptied as a function of the speed of rotation of the pump which pumps the proteins in the buffer of the elution chamber towards the collector while filling the upper reservoir at the same speed.

More precisely, this device consists of a column filled with acrylamide-bisacrylamide gel, the upper outlet of the column being connected to an upper reservoir (for example by the intermediary of a joint situated under the reservoir), intended to receive the elution liquid. This reservoir fitted with the negative electrode (for example platinum wire) is itself replenished almost continuously in order that the level of the elution liquid in the reservoir is practically constant.

The gel is maintained in the column by a porous disk, advantageously plastic (called here "first disk") and a collar situated under the above-mentioned disk, determines the volume of the elution chamber. At the base of the elution chamber is a membrane, for example Amicon 10 kD (called here "intermediate membrane") (which allows proteins of molecular weight less than 10 kD to pass) and which retains in the elution chamber the proteins of molecular weight equal to or greater than 10 kD.

The first disk and the collar determining the volume of the elution chamber are situated at the interior of a collar of larger diameter than the diameter of the elution chamber, which collar accommodates all of the upper part of the device, including the column, the lower end of which fits into the said collar.

This device may advantageously include a second disk, situated under the intermediate membrane. This porous disk, preferably of plastic, provides a stability to the system.

The second disk is itself placed on a third disk, in particular of plexiglass, which makes possible the passage of the current and which seals the base of the column by means of fastenings such as screws, situated on the perimeter of the third disk and defining a ring.

The second and third disks contiguous to the membrane and the membrane are advantageously pierced in their center by a small orifice allowing the passage of a fine capillary connecting the elution chamber to the fraction collector via a pump. In fact, the capillary opens into the upper part of the elution chamber and stops under the first disk which it does not cross.

The fact that the capillary discharges in the neighbourhood of the center of the first disk has the advantage of a direct and effective recovery of the proteins. In fact, the proteins, after the passage through the gel, are found at the base of the gel, but in view of the properties of the system, the proteins have a tendency to be found at the center of the acrylamide in relation to the transverse plane of the column, following upon the suction generated by the pump.

Hence, this makes it possible to avoid losing the proteins which have passed through the column.

The base of the column with the elution chamber is immersed in a lower reservoir which can contain a volume at least equal to that of the upper reservoir and having advantageously dimensions identical to those of the upper reservoir, this lower reservoir containing a positive electrode (for example, platinum wire) as well as the same buffer as that of the upper reservoir. The volumes of the reservoirs are maintained constant by the flow from the upper reservoir into the lower reservoir which is emptied as a function of the speed of rotation of the pump (for example with 4 channels) which pumps the proteins in the buffer of the elution chamber towards the collector while filling the upper reservoir at the same speed. The whole may form a closed circuit if the buffer is recycled or if it is a matter of a fresh buffer supplied to the upper reservoir at the same speed as it is pumped into the lower reservoir.

To give an idea, the dimensions of the device of the invention may be the following:

- the upper reservoir may accommodate a volume of about

1 1,

- the column has a height of about 18 cm and a diameter of about 4 cm,

- the elution chamber has a volume of about 6 ml, that is, a height of about 0.5 cm and a diameter of about 2 cm,

- the collar of larger diameter than that of the elution chamber has a diameter of about 4 cm to about 5 cm,

- the capillary has an internal diameter of about 10 μ to about 20 μ,

- the lower reservoir contains a volume at least equal to that of the upper reservoir.

Another subject of the invention is a peptide, polypeptide or protein comprising at least one of the following peptide sequences: S1 :

H₂N-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr- Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln- Ala-Ser-Thr-Tyr

S2:

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-

Ile-Ser-Pro-Tyr

S3:

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg

Another subject of the invention is a peptide, polypeptide or protein, comprising in its peptide sequence one of the sequences defined below, or constituted by one of the sequences defined below:

S1S2

H₂N-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-

Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-

Ala-Ser-Thr-Tyr-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-

Gly-Pro-Thr-Ile-Ser-Pro-Tyr

S1S3

H₂N-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr- Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln- Ala-Ser-Thr-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg

S2S1

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-

Ile-Ser-Pro-Tyr-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-

Phe-Gln-Tyr-Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-

Tyr-Ile-Gln-Ala-Ser-Thr-Tyr

S3S1

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Arg-Pro-Gly-Tyr- Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-Gly-Leu-Pro-Asp-Leu-Val- Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-Ala-Ser-Thr-Tyr S3S2

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Thr-Gly-His-His-

Ala-Tyr-Ala-Ser-Gly-Pro-Thr-Ile-Ser-Pro-Tyr

S2S3

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-

Ile-Ser-Pro-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg

S1S2S3

H₂N-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-

Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-

Ala-Ser-Thr-Tyr-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-

Gly-Pro-Thr-Ile-Ser-Pro-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg

S1S3S2

H₂N-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-

Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-

Ala-Ser-Thr-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Thr-

Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-Ile-Ser-Pro-Tyr

S2S1S3

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-

Ile-Ser-Pro-Tyr-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-

Phe-Gln-Tyr-Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-

Tyr-Ile-Gln-Ala-Ser-Thr-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg

S2S3S1

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-

Ile-Ser-Pro-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Arg-

Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-Gly-Leu-Pro-

Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-Ala-Ser-Thr-Tyr

S3S1S2

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Arg-Pro-Gly-Tyr-

Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-Gly-Leu-Pro-Asp-Leu-Val-

Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-Ala-Ser-Thr-Tyr-Tyr-Thr-

Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-Ile-Ser-Pro-Tyr S3S2S1

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Thr-Gly-His-His-

Ala-Tyr-Ala-Ser-Gly-Pro-Thr-Ile-Ser-Pro-Tyr-Tyr-Arg-

Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-Gly-Leu-Pro-

Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-Ala-Ser-Thr-Tyr

Among these peptides, polypeptides and proteins defined above, an advantageous group is constituted by those in which the amino acid composition is given in the following Table I:

More particularly, the subject of the invention is a peptide, polypeptide or protein inhibitory to the proliferation of osteoblasts comprising at least one of the peptide sequences S1, S2, S3, S1S2, S1S3, S2S1, S3S1, S2S3, S3S2, S1S2S3, S1S3S2, S2S1S3, S2S3S1, S3S1S2 or S3S2S1, defined above, exhibiting a molecular weight of about 27 kD (27,000 daltons), determined by SDS-PAGE electrophoresis (one-dimensional or two- dimensional).

The subject of the invention is a peptide, polypeptide or protein (inhibitory) of the proliferation of osteoblasts comprising at least one of the peptide sequences S1, S2, S3, S1S2, S1S3, S2S1, S3S1, S2S3, S3S2, S1S2S3, S1S3S2, S2S1S3, S2S3S1, S3S1S2 or S3S2S1, defined above, exhibiting a molecular weight of about 27 kD, determined by one-dimensional SDS-PAGE electrophoresis and an isoelectric point of about 4.0 to about 5.5, measured by two-dimensional SDS-PAGE electrophoresis according to the technique of O'Farell (5).

Another advantageous group of peptides, polypeptides and proteins of the invention is constituted by those of amino acid composition defined in Table I, which represent inhibitors of the proliferation of osteoblasts and which exhibit a molecular weight of about 27 kD measured by one- dimensional SDS-PAGE electrophoresis, an isoelectric point of about 4.0 to 5.5, measured by two-dimensional SDS-PAGE electrophoresis according to the technique of O'Farrell.

Another subject of the invention is any peptide or polypeptide contained in one of the following polypeptide sequences: S1, S2, S3, S1S2, S1S3, S3S1, S2S1, S2S3, S1S2S3, S1S3S2, S2S1S3, S2S3S1, S3S1S2 or S3S2S1, exhibiting properties of inhibition of the proliferation of osteoblasts.

It goes without saying that the free reactive functions which are present in certain of the amino acids which form part of the constitution of the chain sequence of the peptides, polypeptides and proteins of the invention, in particular the free carboxyl groups which are borne by the glutamine groups or by the C- terminal amino acid, on the one hand, and/or the free groups borne by the N-terminal amino acid, or by an amino acid in the interior of the peptide chain, for example lysine, on the other, may be modified to the extent that this modification does not impair the properties of inhibition of the growth of osteoblasts of the peptides, polypeptides and proteins of the invention.

The molecules which are thus modified naturally form part of the invention. The carboxyl groups mentioned above may be acylated or esterified.

Other modifications also form part of the invention. In particular, the amine or ester functions or the two terminal amino acids may themselves be implicated in linkage with other amino acids. For example, the N-terminal amino acid may be linked to a sequence comprising from one to several amino acids corresponding to a part of the C-terminal region of another peptide.

Furthermore, any peptide sequence resulting from the modification by substitution and/or by addition and/or by suppression of one or several amino acids of the peptides, polypeptides and proteins according to the invention form part of the invention to the extent that this modification does not impair the properties of the polypeptides and proteins mentioned. The polypeptides and proteins according to the invention may or may not be glycosylated, particularly in certain of their sites of glycosylation of the type Asn-X-Ser or Asn-X-Thr, X representing any amino acid.

Another subject of the invention is one of the peptide sequences S1, S2 or S3 defined previously.

Another subject of the invention is an inhibitory protein of the proliferation of osteoblasts likely to be obtained by the following steps:

- demineralization and defatting of ground bone tissues, - treatment of the thus demineralized and defatted bone tissue under aqueous conditions with a soluble neutral salt in water and a solubilizing agent of a composition containing the sought protein, the said agent being selected from the couple constituted by urea and guanidine thus leading to the conversion of bone collagen into gelatin and

- the extraction of a composition containing the sought protein in the said solution of the solubilization agent,

- the separation of the solubilization agent and the neutral salt from the said solution leading to the precipitation of the composition containing the desired protein in the aqueous medium,

- the dissolution of the precipitate of the composition containing the sought protein in a solubilization agent such as that defined above, and the treatment of this composition in order to obtain a non-collagen fraction of bone matrix containing the sought protein insoluble in Triton X-100 and water,

- the chromatography on hydroxyapatite of the above- mentioned non-collagenous fraction of bone matrix in soluble Triton X-100 and in water,

the recovery of the fraction eluted at the concentration of 10 mM of phosphate, in solution in the elution buffer and containing a fraction enriched in the sought protein,

- the removal of the elution buffer of the HAP chromatography, in particular by dialysis in distilled water, in order to obtain a precipitate, possibly lyophilized,

the resolubilization of the above-mentioned precipitate with the aid of a suitable solvent containing urea in particular,

- the preparative chromatography of this solubilized fraction enriched in the sought protein with the aid of an elution buffer, the function of which is also to charge the proteins to be eluted with negative charges, which buffer contains in particular SDS, and the recovery of the protein of molecular weight of about 27 kD in solution in the elution buffer,

two dialyses of the above-mentioned protein in solution in the above-mentioned elution buffer: namely, a first dialysis against a mixture of PBS, BSA and Tween-20 and a second dialysis against distilled water, these two dialyses being carried out in any order and these two dialysis steps being separated by the recovery of the content of the dialysis membrane or of the precipitate obtained after dialysis, which content or precipitate is completely solubilized in urea or GuHCl,

- the recovery of the desired protein.

Another subject of the invention is the protein defined above purified by HPLC, for example after trypsinization.

The protein of the invention is characterized by the following properties:

- acidic hydrophobic protein

- soluble in an aqueous medium only under dissociating conditions: i.e. in an aqueous medium containing a concentration of at least 6 M urea, or a concentration of at least 6 M GuHCl,

- insoluble in alcohol,

- insoluble in acetone,

- insoluble in the mixture chloroform-methanol (1/1), which binds weakly to the OH- group of the hydroxyapatite given that it is diluted by the 10 mM fraction of phosphate buffer on HAP chromatography, and which is such that it is cleaved by trypsin in the form of small peptides. The invention also relates to the nucleic acids coding for the peptides, polypeptides and proteins defined above.

The invention also relates to a procedure for the preparation of a peptide, polypeptide or protein according to the invention comprising the following steps:

- the culture in a suitable medium of a cell host which has previously been transformed by a suitable vector containing a nucleic acid coding for the polypeptide or protein of the invention, and

- the recovery of the polypeptide produced by the above transformed cell host from the above-mentioned culture medium.

The polypeptides and proteins of the invention may be prepared according to standard techniques in the field of peptide synthesis.

The synthesis may be carried out in homogeneous solution or on a solid phase.

For example, the method of synthesis in homogeneous solution which may be utilized is that described by Houbenweyl in the monograph entitled "Methode der Organischen Chemie" (Method of Organic Chemistry) edited by E. Wunsch, vol. 15-1 and II. THIEME, Stuttgart 74.

The polypeptides and proteins of the invention may also be prepared according to the method described by R.D. Merrifield in the article entitled "Peptide synthesis on a solid phase" (J. Am. Chem. Soc, 45, 2149-2154).

The invention also relates to the antibodies produced starting from the peptides, polypeptides or proteins of the invention and obtained by immunization of an animal with the aid of a polypeptide or a protein of the invention. It is obvious that this production is not limited to polyclonal antibodies. It also relates to any monoclonal antibody produced by any hybridoma likely to be formed according to standard methods starting from spleen cells of an animal, in particular a mouse or a rat, immunized against a purified peptide or a purified polypeptide or a purified protein of the invention, on the one hand, and cells of a myeloma line, on the other, and selected for its ability to produce the monoclonal antibodies recognizing the peptides, polypeptides or proteins utilized initially for the immunization of the animal.

The invention also relates to any antibody of the invention labelled by a suitable marker of the enzymatic, fluorescent, chemiluminescent or radioactive type.

The invention relates to a diagnostic procedure, in particular for detection, identification and quantitation in vitro of antibodies directed against one of the proteins or one of the polypeptides or peptides of the invention and correlatable with a disease, these antibodies being present in a biological sample containing them, this procedure comprising:

- the placing in contact of the biological sample with a peptide, polypeptide or protein of the invention under conditions allowing an immunological reaction in vitro between the said peptide, polypeptide or the said protein and the antibodies which are possibly present in the biological sample and

- the in vitro detection of the antigen/antibody complex which may be formed.

In a preferred manner, the biological medium is constituted by serum.

The detection may be carried out by any standard procedure. As an example, a preferred method makes use of an immuno-enzymatic procedure according to a ELISA or immunofluorescent or radio-immunological RIA or equivalent technique.

The invention also relates to a diagnostic procedure, in particular of detection, identification and quantitation in vitro of a protein, a polypeptide or a peptide of the invention in a human biological sample likely to contain them, this procedure comprising:

- the placing in contact of the biological sample with a suitable antibody according to the invention under conditions allowing an in vitro immunological reaction between the said antibody and the antigen which may be present in the biological sample and the in vitro detection of the antigen/antibody complex which may be formed.

Preferably, the biological medium is constituted by human serum.

In order to implement the in vitro diagnostic methods to determine a peptide, polypeptide or protein of the invention, it is possible to use a kit comprising:

- an antibody of the invention,

- reagents for making a medium suitable to bring about an immunological reaction,

reagents making it possible to detect the antigen/antibody complexes which have been produced by the immunological reaction, the said reagents possibly having a marker or being capable of being known by a labelled reagent, more particularly in the case in which the antibody mentioned above is not labelled.

In order to demonstrate the in vitro diagnosis of the presence of antibodies of the invention correlatable with a disease, it is possible to utilize the following kit comprising: - a peptide, a polypeptide of protein of the invention,

- reagents to make a medium suitable for the appearance of an immunological reaction,

reagents making it possible to detect the antigen/antibody complexes which have been produced by the immunological reaction, the said reagents possibly having a marker or being capable of being recognized by a labelled reagent, more particularly in the case in which the above polypeptide or protein is not labelled.

The invention also relates to a pharmaceutical composition containing a polypeptide or protein of the invention in combination with a pharmaceutically acceptable vehicle.

The invention will be better understood by the following examples which form preferred embodiments but in no case constitute a limitation.

Description of the schemas and figures

Figure 1 corresponds to the standard profile obtained after hydroxyapatite chromatography eluted at 1 ml/min in a solution of 10 mM of phosphate. The profile obtained shows a peak at 10 mM of phosphate buffer, a peak at 50 mM of phosphate buffer and several peaks obtained with the gradient (from 50 to 250 mMPi) of phosphate buffer. In this respect, it is observed that a protein is eluted at 200 mM of phosphate buffer: it is the "BMP" protein, already mentioned above (1) and pinpointed by the arrow bearing the mention "osteo-inducing fraction".

Figure 2 depicts the chromatography device of the invention.

More precisely, Figure 2a represents the chromatography device in its entirety; Figure 2b represents a breakdown of the lower part of the column and of the supporting device and Figure 2c represents a bottom view of the reference 16, the significance of which will be given later. In Figure 2a is shown in 1 the upper reservoir intended to receive the eluant and fitted with means to supply the buffer continuously such as a pipette 4 conveying the buffer through the intermediary of a pump.

The buffer overflow runs off through the means of evacuation such as the small cylinder 3.

In 2 is represented the negative electrode (which may be constituted by a platinum wire).

In 6, is represented the column, in the interior of which the gel is located. The upper outlet of the column is in contact with the upper reservoir, for example through the intermediary of a joint 5 located beneath the reservoir 1.

In 7 is represented the elution chamber receiving the column and in 8 a conduit, such as a capillary, connected to the fraction collector via a pump and in 9 the lower reservoir into which the column is immersed.

In Figure 2b, is represented in 6 the lower end of the column, in 11 the first porous disk of plastic, in 12 the collar determining the volume of the elution chamber, in 13 the collar of larger diameter than that of the elution chamber and which accommodates all of the upper part of the device, including the column, the lower end of the column fitting into this collar.

In 14 is represented the Amicon PM10 membrane pierced at its center by a capillary connected in the elution chamber to the collector.

In 15, is shown a second disk of plastic, pierced at its center, allowing the passage of the capillary.

In 16, is shown a third disk of plexiglass, in which the Amicon membrane and the second disk of plastic are maintained, for example, by screws and accommodate all of the upper part of the device, including the column, and the disposition of the means of fastening, for example the screws of which defines a ring.

The third disk 16 seals the base of the column.

Figure 3 shows the profile obtained after chromatography on the preparative column. The first peaks correspond to the proteins of low molecular weight, i.e. lower than about 20 kD. Hence, it is necessary to wait for the tenth fraction in order to detect the protein of about 27 kD of the invention.

Figure 4a represents a SDS-PAGE of fractions collected immediately from the preparative column.

The fractions analysed correspond to those containing an individual protein and are located by following the profile of the chromatogram.

Two of these analysed fractions contain the protein of 27 kD of the invention.

The first line corresponds to a bovine albumin control, the second to the molecular weight standard, the third to the starting material, that is the non- collagenous protein insoluble in Triton X-100.

The next four lines correspond to individual proteins, each one corresponding to a peak on the profile of the chromatogram.

The lines 9 and 10 (pinpointed by arrows on Figure 4a) correspond to the protein of 27 kD of the invention. The analysis is carried out on a discontinuous gel in the presence of SDS with a gel of concentration of 0.3% of A-AA (acrylamide- bisacrylamide) and a resolution gel of 12.6% of A-AA. A current of about 150 V and of 15 mA is applied when the proteins pass through the concentration gel (i.e. during about 2 h) in order to be subsequently increased to about 200 V and 25 mA, when the proteins pass through the resolution gel i.e. during about 4 hours. The protein bands are visualized by staining with silver nitrate. The standard (Pharmacia) which contains different markers including phosphorylase at 94,400 daltons, bovine serum albumin at 67,000 daltons, ovalbumin at 43 , 000 daltons , carbonic anhydrase at 30,000 daltons, the soya trypsin inhibitor at 20,100 daltons and ribonuclease at 14,400 daltons.

Figure 4b shows the result of a SDS-PAGE carried out on the protein of the invention at variable concentrations in the presence or not of reducing agent at variable concentrations in order to determine the influence of reduction on the protein of the invention. The first line corresponds to the starting material (non-collagenous proteins insoluble in Triton X-100), the second line to the molecular weight standard, the third line to the protein of the invention of 27 kD in the presence of mercaptoethanol, the fourth line to the protein of the invention without reducing agent, the fifth line to the protein of the invention in the presence or mercaptoethanol two times less concentrated, the sixth line to the protein of the invention without reducing agent and two times less concentrated, the seventh line to the molecular weight standard. The bands are revealed by staining with silver nitrate.

In view of the result read off on this gel, it may be deduced that the protein of the invention is not affected by reduction with mercaptoethanol.

Figure 5 corresponds to a gel in two dimensions stained with silver nitrate. The molecular weight standard is the same as that utilized for the one- dimensional gel. The first dimension relates to the isoelectric point. It is a matter of the standard method in the presence of servalites (Serva, W-Germany) which form a pH gradient from 3.5 to 7, reducing agent (mercaptoethanol, Biorad) and a dissociating agent, that is 9.5 M urea (Sigma). It is possible to distinguish on this gel a long band which corresponds to the protein of the invention and a very short band which corresponds to an impurity. This being so, the biological assays were carried out with the protein of the invention obtained after the preparative chromatography and the results obtained show that this impurity does not modify the biological properties of the protein of the invention. It may be deduced from this that the procedure of the invention makes it possible to obtain a protein in a satisfactory state of purity.

However, the HPLC analysis of the protein of the invention was carried out on the protein free from the impurity detected on two-dimensional SDS-PAGE.

Figure 6 shows the inhibitory biological activity of the protein of 27 kD on the spontaneous proliferation (i.e. in the absence of any mitogen) of osteoblasts derived from rat bone, after 72 hours and incorporation of tritiated thymidine 20 hours before the stopping of the culture in comparison with non- stimulated cells. More precisely, the cells are maintained in a culture medium containing BSA instead of FCS during an assay of 72 hours. The values represent the mean ± SD for n = 7. On the abscissa are indicated the amount of proteins of the invention (ng of proteins), and on the ordinate the measurement of the inhibition of the proliferation of osteoblasts is given in counts per minute corresponding to the detection of the tritiated thymidine.

The statistical significance is obtained by non- paired small samples for non-parametric values (i.e. the statistical test of Mann and Whitney).

One star (*) signifies that p is less than 0.05, two stars (**) signify that p is less than 0.01, three stars (***) signify that p is less than 0.005, four stars (****) signify that p is less than 0.001. The statistical significance results from using the Mann and Whitney test.

The control corresponds to the blank column and the incorporation of the protein of the invention corresponds to the hatched columns.

The inhibition is statistically significant.

Figure 7 shows an inhibitory activity of the protein of 27 kD on the proliferation of ROS 17/2.8 cells for doses of 10 μg/ml (column with the widely spaced hatchings), 1 μg/ml (column with the closely spaced hatchings), 0.1 μg/ml (column with the dots) in comparison with identical doses containing:

- a non-collagenous fraction soluble in Triton X-100 and water extracted from demineralized bone represented in the figure by SNCP (soluble non-collagenous proteins),

- osteonectin of 38 kD (2).

The control is constituted by BSA. "NS" signifies that the result is not significant; ** correspond to p < 0.01; *** correspond to p < 0.005 and **** correspond to p < 0.001. The values represent the mean ± SD. However, in the case of the protein of the invention of 27 kD, the column with widely spaced hatchings corresponds to a dose of 3.5 μg/ml, that with closely spaced hatchings to a dose of 0.35 μg/ml and that with dots to a dose of 0.035 μg/ml. On the ordinates, the measurement of the inhibition of the proliferation of the ROS 17/2.8 cells is represented in counts per minute (cpm) corresponding to the detection of tritiated thymidine. The inhibition is significant for the protein of the invention at doses lower than those used for known inhibitors.

Figure 7, 4 x n = 3 signifies that the experiments were repeated 4 times in triplicate, the number of cultures n being equal to 3, the number of experiments being equal to 12. EXAMPLE: PREPARATION OF THE PROTEIN OF 27 kD OF THE

INVENTION:

a) Extraction and separation of the proteins starting from demineralized and powdered bone

The method followed to extract the non-collagenous proteins from bone is that described by Urist et al.

(1).

1 - The pulverized material is demineralized for 72 hours in a solution of 0.6 N HCl (Ferak) at 4ºC.

2 - The material collected after filtration (that is ± 800 g for 5 g of bone) is dissolved in a solution (± 4 L/kg of bone powder) of 6 M urea (Sigma) /0.5 M CaCl₂ (Ferak), containing protease inhibitors such as 2 mM N-ethylmaleimide (NEM, Aldrich-Chemie), 0.1 mM benzamidine-HCl (Sigma) and 2 mM sodium azide (antibacterial, Merck), at room temperature and for 24 hours.

3 - The solution is passed through Whatman-1 filter papers (Whatman Int., UK).

4 - The filtered solution is concentrated with the aid of a Amicon cell of 200 cc (Amicon division, Ultrafiltration Cell, USA) fitted with a Diaflo membrane (MW 10, Diaflo Ultrafilters, Amicon division, USA). The final volume represents about one tenth of the starting solution.

5 - The concentrated solution is then dialysed against distilled water (dH₂O), in the cold room. Dialysis membranes with a retaining power of 6 - 8 kD (Spectra- Por, USA) are used. The water is changed 3 times per day during 3 days.

6 - When the dialysis is finished, a sort of gel may form, thus it is necessary to heat the membrane gently to 35°C. Its content is centrifuged at 9000 rpm for 30 minutes at 30-35°C. One collects the precipitate which will be washed several times with dH₂O and it is redissolved in 6 M urea / 0.5 M CaCl₂. 7 - When the precipitate obtained in 6. is completely dissolved, the solution is centrifuged at 9000 rpm for 30 minutes, but this time the temperature is brought to about 4°C, the insoluble material is discarded and the supernatant is then dialysed against 11 volumes of citric acid (0.25 M) at pH 3.1 (buffered with concentrated NaOH (Merck)) and this overnight. This removes a part of the gelatinous proteins.

8 - The next day, the content of the dialysis membrane is brought to 35°C, it is centrifuged at 9000 rpm for 30 minutes at 30-35°C. The supernatant is discarded and the precipitate is washed abundantly with dH₂O so as to remove all traces of citric acid. The precipitate is resolubilized in about 2 L of chloroform/methanol 1/1 (Ferak), at room temperature overnight on a magnetic stirrer. In this way the lipids and the lipoproteins are removed.

9 - The next day, at room temperature, the solution is passed through a Bύchner filter and evaporated in a hood.

10 - The dry material retained on the filter is defatted in about 2 L of 6 M urea / 0.5 M CaCl₂, then it is centrifuged at 9000 rpm, 30 minutes at 4°C. Only the supernatant is collected, the precipitate is discarded.

11 - The supernatant is dialysed about 24 hours at 4°C, against an equal volume of 6 M urea containing 0.1 M Trizma-HCl (Ferak, W-Germany), at pH 7.2 and 0.2% of Triton X-100 (0.2 g/ 100 ml, Sigma, USA).

12 - The dialysis in distilled water in the cold room takes about 72 hours. The content of the dialysis membrane is then centrifuged at 9000 rpm at 48ºC, for 30 minutes, the precipitate is lyophilized, it is a matter of the non-collagenous proteins insoluble in the Triton X-100 (that is 2.5 g). The supernatant, soluble in Triton X-100 and in distilled water, represents mainly the proteins of 6, 24, 34 kD (that is 6 g).

b) Hydroxyapatite (HAP) chromatography:

HAP is selected as means of purification, given the high affinity which the non-collagenous proteins show for this substrate. 1 gr of HAP (Bio-Gel HTP, BioRad) binds about 10 mg (about 7 to about 20 mg) of proteins and occupies about 3.5 cm³. In this case, 40 g of HAP are utilized in order to absorb 400 mg of non- collagenous proteins. A chromatography column (Pharmacia) of a volume of about 200 cm³ is used, however its volume is reduced to about 160 cm³ with the aid of a piston, given that 40 gr of HAP only occupy 140 cm³. The HAP is immersed in the buffer 0 and may never become dry, all of the chromatography takes place at room temperature.

The buffer 0 requires:

- The preparation of 100 ml of 1 M phosphate solution (Merck), that is 51% of Na₂HPO₄ and 49% of NaH₂PO₄. Na₂HPO₄ and NaH₂PO₄ are dissolved in 50 ml of distilled water, then NaH₂PO₄ is mixed with Na₂HPO₄ until a solution of pH 6.8 is obtained.

- the preparation of a stock solution of 1 L of 10 M urea, that is 600 gr dissolved in 1 L of distilled water.

The buffer 0 has the following composition: 6 M urea + 0.01 M of phosphate (600 ml stock sol. of urea + 10 ml stock sol. of phosphate + 390 ml dH₂O for 1 L of buffer).

The HAP is washed with the buffer 0 by shaking gently by hand. It is allowed to settle for one hour, the supernatant is then discarded, the HAP is rewashed with the buffer 0 for a half hour by operating in the same manner. After this, the supernatant is discarded and replaced by fresh buffer 0, the HAP is shaken more vigorously and poured into the column in one movement. In this case, a short and wide column is preferred to a long and narrow column in contrast to the columns intended for chromatographies by gel filtration (cf. a Sephadex S-200). After 24 hours, the column is placed in closed circuit and washed with the buffer 0, its flow rate is thus identical to what will be the rate of elution of the chromatography, that is 1 ml/min, this being defined because of a pump, the speed of rotation of which is adjustable. While the column becomes stabilized during the night, the 400 mg of non- collagenous proteins are dissolved in 40 ml of buffer 0 in a Erlenmeyer on a magnetic plate at 37ºC during the same period of time. They are then centrifuged at 20,000 rpm, 15 min. The supernatant is chromatographed. The lower end of the column is connected to a U.V. detector (reading at 280 nm, LKB, Sweden), itself connected to a graphic recorder (LKB): The chromatography takes place under the following conditions:

- elution speed: 1 ml/min.

- graphic recorder: v = 3 cm/hr and impedance = 10 mV

- each of the fractions is collected in a Erlenmeyer.

In fact, by following the chromatogram, a fraction is recovered at 10 mM of phosphate buffer, another fraction at 50 mM of phosphate buffer and in the gradient of phosphate buffer a fraction is recovered which emerges at 200 mM of phosphate buffer (and which contains the BMP protein).

More precisely, the fraction emerging at 10 mM of phosphate buffer is recovered when a peak is observed, obtained when about 3 times the volume of the column of HAP (that is 420 cc) of buffer 0 have passed through, and the fraction collected is dialysed in water, then lyophilized.

The HAP chromatography made it possible to obtain a fraction emerging at 10 mM of phosphate buffer, which fraction contains a set of proteins of molecular weight of about 15 , 000 to about 40 , 000 daltons, and contains in particular the sought protein,

c) Chromatography on a preparative column (7):

This involves a type of uninterrupted electrophoresis in a column of polyacrylamide gel. Although the principle of this chromatography is maintained, several important modifications were introduced in order to increase the efficiency (in this respect, reference should be made in particular to Figure 2). This instrument made of plexiglass consists mainly of an upper reservoir, sides 12 cm/12 cm square and 9 cm high, comprising a negative electrode (platinum wire), a cylindrical point making it possible to maintain a constant level in the reservoir and a central outlet equipped for example with a thread on which a column 4 cm in diameter and 18 cm high is connected. The lower end of the column comes to fit partially into a second wider cylinder about 3 cm high at the bottom of which a collar 0.5 cm high defines the volume of the elution chamber. A disk of porous plastic is placed between the base of the column, in direct contact with the acrylamide gel, and the internal collar. A Amicon membrane (Diaflo, MW 10, Amicon Corp., USA) covers a second porous disk, itself placed on a disk of plexiglass with spokes allowing the passage of current and which seals the base of the column, for example by means of screws. The disks contiguous to the membrane and the membrane are pierced at their center with a small hole allowing the passage of a capillary connecting the elution chamber to the fraction collector via a pump (LKB). The base of the column with the elution chamber is immersed in a reservoir of dimensions preferably identical to those of the upper reservoir which contains a positive electrode (platinum wire) as well as the same buffer. The volumes of the reservoirs are maintained constant by the flow from the upper reservoir into the lower which is emptied as a function of the speed of rotation of the pump (with 4 channels) which pumps the proteins in the buffer of the elution chamber towards the collector while replenishing the upper reservoir at the same rate.

The buffer used is the same as that intended for the electrophoreses: 0.04 M of Tris (Ferak) + 0.1% of SDS (BioRad) + 0.384 M of glycine (Ferak) in distilled water at pH 8.3 (Tris-HCl).

The column is composed of a 3% concentration gel of acrylamide-bisacrylamide (A-AA BioRad) which occupies 15% of the volume of the column, which rests on a resolution gel of 12% A-AA which occupies 30% of the column which itself is superposed on a second resolution gel of 17% A-AA and which also occupies 30% of the column. This procedure stops the migration of the proteins of high molecular weight and increases distance between the proteins which have penetrated the gel, thus facilitating their separation.

The conditions of chromatography are the following: the electrodes are connected to a current generator (Consort E 554) which delivers 70 mA, the pump which turns at 0.5 ml/min connects the elution chamber to the U.V. detector (reading at 180 nm) linked to a graphic recorder (the paper speed which is 3 cm/hr and the impedance of which is 10 mV) and to the fraction collector which collects 3 ml/tube every 6 minutes. The purification, which takes place at room temperature, lasts about 72 hours.

d) Immediate analysis of the fractions by one- dimensional and two-dimensional electrophoresis:

Following the profile of the preparative chromatography, 100 μl derived from the samples corresponding to the peaks are taken, applied to an electrophoresis on polyacrylamide gel in the presence of sodium dodecylsulfate (SDS-PAGE) in order to evaluate the quality of the separation and to select the interesting fraction most judiciously (Figures 4a and 4b). The fraction or fractions containing only the protein of 27 kD are recovered and dialysed in order to remove the elution buffer of the preparative chromatography which contains SDS. The gels show the migration of the protein under dissociating and reducing conditions, according to the method described by Laemmli U.K. (4). This protein obtained and purified to an essentially homogeneous state starting from bovine bone according to the procedure of the invention has an apparent molecular weight of 27,000 daltons and an isoelectric point (Fig. 5) of about 4.0 to about 5.5 according to the technique of O'Farrell (5).

e) Dialysis and renaturation of the proteins

The immediate electrophoresis on the fractions collected after the preparative column makes it possible to group the fractions containing only the protein of 27 kD, to concentrate them in a Amicon cell (Amicon Ultrafiltration Cell, M-52, Amicon Corp., USA) equipped with a filtration membrane (Diaflo, PM 10, Amicon Corp., USA) so as to have no more than very small volumes to dialyse. These quantities which vary between 1 and 10 ml are dialysed (dialysis membranes of retaining power of 6-8000 d, Spectra/Por, USA) in a 100 fold larger volume of 0.15 M of phosphate buffer (PBS) containing 5 μg/ml of BSA (Sigma) and 0.5 mg/ml of Tween-20 (Merck).

After a dialysis of 3 days in the cold room, the content of the dialysis membranes (precipitate and supernatant) are frozen, then lyophilized. They are resolubilized in 6 M GuHCl (Aldrich-Chemie, W-Germany), and redialysed in distilled water in the cold room for a further 3 days at least. After being freed from GuHCl and salts of SDS, both of which are denaturants, during the second dialysis, proteins are obtained which have recovered their biological activity.

The protein of 27 kD is trypsinized, the separated on High Pressure Liquid Chromatography (HPLC). The sequence of amino acids is carried out on the major peaks thus obtained with the aid of a sequence analyser (Beckman 121) according to the standard method.

A first subunit, S1 corresponds to:

The second subunit, S2 corresponds to:

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr- Ile-Ser-Pro-Tyr

The third subunit, S3 is composed of:

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg

Amino acid composition

These values represent approximately the number of residues of the amino acids indicated per mole of respective proteins (subunits), the molecular weight of which is estimated by the method conventionally used such as a SDS-PAGE. The values for these protein subunits of the invention include the experimental error of the type of analysis of the composition of the amino acids; ND or "not determined" signifies there was no determination for the amino acids indicated.

Biological assays

Culture of osteoblasts derived from long bones of the rat (6):

The long bones of the rat (Sprague-Dawley) namely the femurs furnish osteoblast cells. These bones, withdrawn in a sterile manner, are washed abundantly in phosphate buffer (PBS) composed of 160 g NaCl (Merck) + 4 g KCl (Merck) + 23 g Na₂HPO₄ (Merck) + 4 g KH₂HPO₄ (Merck) per 2 L of distilled water giving a 10 fold concentrated solution of 0.15 M of PBS.

Starting from the epiphyses, 1 cm is measured, the latter are removed and the extremity of the bone is sawed transversally at a length of about 0.7 cm. The pieces are placed in a Petri dish (Falcon, Becton Dickinson) containing PBS (sterile) and the inner bone surface is scraped with a scalpel as far as the epiphysial cartilage (clearest and densest part). The small bony debris and the cells collected in this way are washed 4 times in PBS. After washing, the entire material is transferred to large Petri dishes containing about 15 ml of culture medium which is changed every 3 days.

This medium is mainly composed of: modified Eagle medium (MEM, Gibco), enriched with 10% of heat- inactivated FCS (Gibco), 1% of L-Glu (Gibco), 100 U/ml of penicillin (Gibco), 100 μg/ml of streptomycin (Gibco) and 1% of non-essential amino acids (NEAA, Gibco). The cells are stored in an incubator at 37ºC and 5% CO₂. A single layer of cells entirely covers the interior of the Petri dish after 4 to 5 weeks. There are then about 2 times 10⁶ cells per dish. The cells at confluence are trypsinized (Trypsin solution/EDTA, Gibco) in the following manner:

1 - when the cells stick to the plastic of the Petri dish, the culture medium is removed.

2 - 2 ml of trypsin are added per dish and shaken gently.

3 - 3 ml of trypsin are added per dish for one minute.

4 - the trypsin is removed.

5 - the dishes are placed for 10 minutes in the incubator, the cells are completely detached from the plastic.

6 - the dishes are rinsed several times in a little medium so as to obtain a final volume of about 10 ml after several rinsings.

7 - it is transferred into 15 ml "Falcon" tubes (Becton Dickinson), the cells are centrifuged 5 minutes at

200 g (that is, about 1200 to 1400 rotations).

8 - the cells are collected and pooled (the supernatant is discarded).

9 - the cells are centrifuged a second time by adding medium if necessary under the same conditions as in 7.

10 - the supernatant is discarded and the cells are counted in a hemocytometer.

11 - the cells are transferred to Falcon culture dishes with 24 wells at a density of 10⁴ cells/cm² containing the medium + 0.1% of BSA (Sigma, USA) or 2% of FCS passed over charcoal so as to have a medium depleted in serum and growth factors. It is a question of the first "subculture".

Thirty-five, 3.5 and 0.35 ng of the inhibitory protein of the invention are tested for 72 hours on 10⁴ cells/cm² and their proliferation is measured by the incorporation of tritiated thymidine (³H-TdR, Amersham, U.K.) into the DNA, added to the culture during the last 20 hours. After this, the medium is aspirated and the cells are washed in 500 μl of 10% trichloroacetic acid (TCA, Sigma) on crushed ice. This operation is repeated twice for 10 minutes. A last washing, not on ice, is done in 500 μl of ethanol/ether 3:1 (Ferak). The dishes are dried. Five hundred μl of 0.2 N NaOH (Merck) are added per well for 10 minutes at 90ºC; the 500 μl aliquots are then transferred to scintillation vials in 9.5 ml of instagel (scintillation fluid), counted in a β counter (liquid spectrometry) and expressed as counts per minute (cpm). If the solution of Instagel is cloudy, 50 μl of 0.3 N HCl (Merck) or 100 μl of 0.5 N acetic acid (Merck) are added.

The synthesis of alkaline phosphatase by the osteoblast cells obtained from the long bones of the rat is assayed in order to confirm their phenotypic character (CBR, Boehringer Diagnostica, W-Germany). This enzyme is a specific marker for osteoblasts and reveals the homogeneity of the culture.

By using the protein of the invention, and/or an active polypeptide such as defined above, and/or an immunologically related part of the above protein or the above polypeptide, with or without a pharmaceutical vehicle in the case of in vivo administration, an inhibitory activity is observed in vitro with regard to a healthy osteoblast cell line but also in the case of osteoblasts of a malignant cell line derived from rat osteosarcoma (ROS 17/2.8). The protein of the invention is different from the inhibitor of 38 kD (Figure 7) by its sequence, given that it was confirmed that this inhibitor of 38 kD shares an entire sequence homology with osteonectin (2). The doses of the protein of the invention to be administered depend on the in vitro or in vivo model selected, on the target cells and/or influential external circumstances such as certain diseases of the bone like Paget's disease or the developmental stage of a bone tumor, for example.

Each active preparation may include a judicious bioactive material such as growth factors, chemotactic agents, steroids, antibiotics, anti-inflammatory agents and other similar substances.

BIBLIOGRAPHY

1. Urist M.R., Chang J.J., Lietze A., Huo Y.K., Brownell A.G. and DeLange R.J.: Preparation and bioassay of bone morphogenetic protein and polypeptide fragments, In Barnes, D, and Sirbaska, D.A. (eds): Methods of Enzymol., 146; 294, 1987.

2. Brownell A.G. and Torres X., Osteogenesis inhibitory protein decreases DNA synthesis in ROS 17/2.8 osteoblast-like cells, Calcif. Tissue Int., Suppl. 44, G4-S37, 1989.

3. Hauschka, P.V., Mavrakos, A.E., Iafrate, M.D., Doleman, S.E., and Klagsbrun, M.: Growth factors in bone matrix. J. Biol. Chem., 261; 165, 1986.

4. Laemmli, U.K., Nature, 227; 680, 1970.

5. O'Farrell Ph, High resolution two dimensionel electrophoresis of proteins, Biol. Chem., 250; 4007, 1975.

6. Maurizi M., L. Binaglia, E. Donti, F. Ottaviani, G. Paludetti, and Venti Donti G. Morphological and functional characteristices of human temporal-bone cell cultures. Cell Tissue Res., 229; 505, 1983.

7. Briggs M.R., J.T. Kadonaga, S.P. Bell, and Tjian R., Purification and biochemical characterization of the promoter-specific transcription factor, Sp 1, Science, 234; 47, 1986.

Claims

1. Procedure for the preparation of a protein purified to a satisfactory homogeneous state, starting from an extract of non-collagenous proteins of bone matrix, characterized in that it comprises the following steps:

- the extract of non-collagenous proteins, solubilized beforehand in a solvent, is subjected to a preparative chromatography in the liquid phase on a column of gel with the aid of an elution buffer and an electric field under conditions such that the sought protein is eluted, and is found, during or after chromatography, to be present in solution in the elution buffer, alone and free from contaminants, in at least one of the fractions collected at the exit of the column,

the fraction containing the desired protein in solution in the elution buffer is recovered, the elution buffer is removed and the desired protein is recovered which, if necessary, is subjected to a renaturation step and/or a reactivation step.

2. Procedure according to Claim 1, characterized in that it comprises the following steps:

- the extract of non-collagenous proteins, solubilized beforehand in a solvent, is subjected to a preparative chromatography in the liquid phase on a column of gel likely to separate the proteins in an order corresponding to their molecular weight,

- with the aid of an elution buffer, the introduction of which occurs in the neighbourhood of the top of the column, in particular in an upper reservoir situated at the top of the column, and the outflow of which takes place in the neighbourhood of the base of the column, in particular in a lower reservoir situated at the base of the column, this elution buffer being such that it confers on the proteins a relatively uniform charge, and

- with the aid of an electric field generated between the introduction of the elution buffer and the outflow of the elution buffer under conditions such that the sought protein is eluted, and is found, during or after the chromatography, to be present in solution in the elution buffer, alone and free from contaminants, in at least one of the fractions collected at the exit of the column,

- the fraction containing the desired protein in solution in the elution buffer is recovered, the elution buffer is removed and the sought protein is recovered, which, if necessary, is subjected to a renaturation step and/or a reactivation step.

3. Procedure according to Claim 1, in which after its elution, the desired protein is found in solution in the elution buffer of the preparative chromatography and is subjected to at least one of the following two steps in any order, namely: dialysis against PBS, BSA and Tween-20, and dialysed against water and when these two steps take place, the precipitate obtained after that one of the dialyses which constitutes the first step, is solubilized.

4. Procedure according to any one of the Claims 1 to 3, in which is first carried out

- a first dialysis of the fraction containing the sought protein in solution in the elution buffer of the preparative chromatography against a mixture of PBS, BSA and Tween-20, which leads to the formation of a precipitate and a supernatant,

- the precipitate or, advantageously, the whole of the content of the dialysis membrane (i.e. the precipitate and the supernatant) is recovered,

- the whole of the content of the dialysis membrane is then lyophilized, and a lyophilizate is obtained, - the redissolution of the lyophilizate is then carried out, in particular in GuHCl, at a molarity of about 4 to about 8 M, in particular 6 M, or in urea at a molarity of about 4 M to about 8 M, in particular 6 M, and

- a second dialysis of the dissolved lyophilizate is carried out against water.

5. Procedure according to any one of the Claims 1 to 4, in which the preparative chromatography step is preceded by a preliminary fractionation of the extract of non-collagenous proteins of bone matrix, making it possible to obtain a fraction enriched in the sought protein, the enriched fraction of which is subjected to the step of preparative chromatography.

6. Procedure according to any one of the Claims 1 to 5, in which the preliminary step is constituted by a chromatography on hydroxyapatite.

7. Procedure according to one of the Claims 1 to 6, in which after the preliminary step, the buffer is removed, in particular when the latter is different from the solubilization buffer and the elution buffer used in the preparative chromatography, and the fraction enriched in the sought protein is redissolved in a solubilization buffer compatible with the elution buffer of the preparative chromatography, the solubilization buffer containing in particular urea, and being preferably constituted by a mixture of urea, Tris-HCl and SDS.

8. Procedure for obtaining a protein according to one of the Claims 1 to 7, characterized in that:

- an extract of non-collagenous proteins of demineralized, ground and defatted bone matrix is subjected to a chromatography on hydroxyapatite, the extract of non-collagenous proteins having been solubilized beforehand preferably in the same buffer as the elution buffer of the HAP chromatography, in particular in buffer 0,

- a fraction enriched in the desired protein in solution in the elution buffer of the HAP chromatography is recovered,

- the elution buffer of the HAP chromatography is removed, in particular by dialysis against distilled water and the precipitate obtained, possibly converted into a lyophilizate, containing a fraction enriched in the sought protein is recovered,

- the fraction enriched in the desired protein is subjected to a preparative chromatography after having been dissolved in a solvent advantageously containing urea, the acrylamide gel of the preparative column having a density such that it allows the elution of the desired protein with the aid of an elution buffer advantageously containing SDS and with the aid of an electric field, the conditions of chromatography being such that the desired protein is found alone in solution in the elution buffer, and free of contaminants, in at least one tube of the fraction collector,

- the protein is recovered, and if necessary reactivated and/or renatured by removing the elution buffer of the preparative chromatography, in particular

* by a first dialysis of the protein in solution in the elution buffer of the preparative chromatography against PBS, BSA and Tween-20 after which the precipitate obtained, and possibly the supernatant, are recovered, the precipitate and possibly the supernatant containing the sought protein being possibly lyophilized, the SDS being partially removed at the end of this first dialysis,

* by a solubilization of the above-mentioned precipitate, and possibly of the supernatant, containing the sought protein with the aid of a solubilization agent, in particular with the aid of urea of about 4 M to about 8 M, in particular of about 6 M, or of GuHCl of about 4 M to about 8 M, in particular of about 6 M,

* and by a second dialysis of the solution previously obtained against distilled water, the precipitate obtained, and possibly the supernatant, containing the sought protein being lyophilized, the SDS as well as the above-mentioned solubilization agent being completely removed at the end of this second dialysis,

- the sought protein is recovered.

9. Procedure for obtaining a protein of molecular weight of about 27 kD, according to Claim 1, possessing properties inhibitory towards the proliferation of osteoblasts, according to Claim 1, characterized in that it comprises the following steps:

- the buffer is removed, in particular by dialysis, in distilled water in order to obtain a precipitate, possibly lyophilized,

- this solubilized fraction enriched in the sought protein is subjected to a preparative chromatography with the aid of an elution buffer, the function of which is also to charge the proteins to be eluted with negative charges, which buffer contains in particular SDS, and the protein of molecular weight of about 27 kD in solution in the elution buffer is recovered, - the above-mentioned protein in solution in the elution buffer of the preparative chromatography is subjected to two dialyses: namely, a first dialysis against a mixture of PBS, BSA and Tween-20 and a second dialysis against distilled water, these two dialyses being carried out in any order, and these two dialysis steps being separated by the recovery of the content of the dialysis membrane, or of the precipitate resulting from the dialysis, which content or precipitate is completely solubilized in urea or GuHCl,

- the sought protein is recovered.

10. Device for preparative chromatography in the liquid phase comprising:

- a column filled with gel,

- a porous disk, advantageously plastic (called below "first disk") which maintains the gel in the column, and a collar situated beneath the above-mentioned disk which determines the volume of the elution chamber,

- a membrane situated at the base of the elution chamber, hereafter designated as intermediate membrane,

- a collar of larger diameter than the diameter of the elution chamber, at the interior of which the first disk and the collar determining the volume of the elution chamber are situated, which collar accommodates all of the upper part of the device including the column, the lower extremity of which fits into the said collar,

- advantageously a second disk preferably of plastic, situated beneath the intermediate membrane, this second disk being itself placed on a third disk, in particular of plexiglass, which allows the passage of current and which seals the base of the column by means of fastenings such as screws, situated on the perimeter of the third disk and defining a ring, the second and third disks contiguous to the intermediate membrane and the intermediate membrane being advantageously pierced at their center by a small orifice allowing the passage of a capillary connecting the elution chamber to the fraction collector, in particular via a pump,

- a lower reservoir in which the base of the column with the elution chamber is immersed, this reservoir containing a volume at least equal to that of the upper reservoir and having advantageously dimensions identical with those of the upper reservoir, this lower reservoir containing a positive electrode as well as the same buffer as that of the upper reservoir, the volumes of the reservoirs being maintained constant by the flow from the upper reservoir into the lower reservoir which is emptied as a function of the speed of rotation of the pump which pumps the proteins in the buffer of the elution chamber toward the collector while replenishing the upper reservoir at the same rate.

11. Peptide, polypeptide or protein comprising in its peptide chain sequence at least one of the following peptides sequences:

S1:

S2:

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr- Ile-Ser-Pro-Tyr S3:

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg

12. Peptide, polypeptide or protein according to Claim 11, comprising in its peptide sequence one of the sequences defined below or constituted by one of the sequences defined below:

S1S2

H₂N-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-

Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-

Ala-Ser-Thr-Tyr-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-

Gly-Pro-Thr-Ile-Ser-Pro-Tyr

S1S3

S2S1

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr- Ile-Ser-Pro-Tyr-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr- Phe-Gln-Tyr-Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr- Tyr-Ile-Gln-Ala-Ser-Thr-Tyr

S3S1

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Arg-Pro-Gly-Tyr- Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-Gly-Leu-Pro-Asp-Leu-Val- Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-Ala-Ser-Thr-Tyr

S3S2

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Thr-Gly-His-His-

Ala-Tyr-Ala-Ser-Gly-Pro-Thr-Ile-Ser-Pro-Tyr

S2S3

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-

Ile-Ser-Pro-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg S1S2S3

H₂N-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-

Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-

Ala-Ser-Thr-Tyr-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-

Gly-Pro-Thr-Ile-Ser-Pro-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg

S1S3S2

H₂N-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-

Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-

Ala-Ser-Thr-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Thr-

Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-Ile-Ser-Pro-Tyr

S2S1S3

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr- Ile-Ser-Pro-Tyr-Tyr-Arg-Pro-Gly-Tyr-Gly-Thr-Gly-Tyr- Phe-Gln-Tyr-Gly-Leu-Pro-Asp-Leu-Val-Pro-Asp-Pro-Tyr- Tyr-Ile-Gln-Ala-Ser-Thr-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg

S2S3S1

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-

Ile-Ser-Pro-Tyr-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Arg-

Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-Gly-Leu-Pro-

Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-Ala-Ser-Thr-Tyr

S3S1S2

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Arg-Pro-Gly-Tyr-

Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-Gly-Leu-Pro-Asp-Leu-Val-

Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-Ala-Ser-Thr-Tyr-Tyr-Thr-

Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr-Ile-Ser-Pro-Tyr

S3S2S1

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg-Tyr-Thr-Gly-His-His-

Ala-Tyr-Ala-Ser-Gly-Pro-Thr-Ile-Ser-Pro-Tyr-Tyr-Arg-

Pro-Gly-Tyr-Gly-Thr-Gly-Tyr-Phe-Gln-Tyr-Gly-Leu-Pro-

Asp-Leu-Val-Pro-Asp-Pro-Tyr-Tyr-Ile-Gln-Ala-Ser-Thr-Tyr

13. Peptide, polypeptide or protein according to one of the Claims 11 or 12, in which the amino acid composition is the following:

14. Peptide, polypeptide or protein according to Claim 11 to 13, modified by substitution and/or by addition and/or by suppression of one or several amino acids of the peptides, polypeptides and proteins defined in Claims 11 to 13, to the extent to which the peptides, polypeptides and proteins thus modified exhibit properties inhibitory of the proliferation of osteoblasts.

15. Peptide, polypeptide or protein according to one of the Claims 11 to 14, inhibitory of the proliferation of osteoblasts characterized by a molecular weight of 27 kD determined by one-dimensional SDS-PAGE electrophoresis, an isoelectric point of about 4.0 to about 5.5, measured according to the method of O'Farrell described in J. Biol. Chem. 250, 4007, 1975.

16. Peptide sequence S1 of formula:

17. Peptide sequence S2 of formula:

H₂N-Tyr-Thr-Gly-His-His-Ala-Tyr-Ala-Ser-Gly-Pro-Thr- Ile-Ser-Pro-Tyr

18. Peptide sequence S3 of formula:

H₂N-Val-Leu-Arg-Phe-Pro-Gln-Arg

19. Peptide, polypeptide or protein inhibitory of the proliferation of osteoblasts, characterized in that it contains in its peptide chain sequence a peptide sequence contained in one of the following sequences: S1, S2, S3, S1S2, S2S1, S1S3 , S3S1, S2S3, S3S2, S1S2S3, S1S3S2, S2S1S3, S2S3S1, S3S1S2 or S3S2S1, the sequences S1, S2 and S3 having the meanings indicated in Claim 11.

20. Protein inhibitory of the proliferation of osteoblasts obtained by the following steps:

- demineralization and defatting of ground bone tissue,

- treatment of the bone tissue thus demineralized and defatted under aqueous conditions with a neutral salt soluble in water and a solubilization agent of a composition containing the desired protein, the said agent being selected from the couple constituted by urea or guanidine thus leading to the conversion of the bone collagen into gelatin,

- the extraction of a composition containing the desired protein in the said solution of solubilization agent,

- the dissolution of the precipitate of the composition containing the desired protein in a solubilization agent such as defined above, and the treatment of this composition in order to obtain a non-collagenous fraction of bone matrix containing the desired protein insoluble in Triton X-100 and water,

- the chromatography on hydroxyapatite of the above- mentioned non-collagenous fraction of bone matrix insoluble in Triton X-100 and water,

the recovery of the fraction eluted at the concentration of 10 mM of phosphate in solution in the elution buffer and containing a fraction enriched in the desired protein,

- the removal of the elution buffer of the HAP chromatography, in particular by dialysis, in distilled water in order to obtain a precipitate, possibly lyophilized,

the resolubilization of the above-mentioned precipitate with the aid of a suitable solvent containing in particular urea,

- the preparative chromatography of this solubilized fraction enriched in the desired protein with the aid of an elution buffer, the function of which is also to charge the proteins to be eluted negatively, which buffer contains in particular SDS, and the recovery of the protein of molecular weight of about 27 kD in solution in the elution buffer,

- two dialyses of the above-mentioned protein in solution in the above-mentioned buffer: namely, a first dialysis against a mixture of PBS, BSA and Tween-20 and a second dialysis against distilled water, these two dialyses being carried out in any order, and these dialysis steps being separated by the recovery of the content of the dialysis membrane or of the precipitate obtained after the dialysis, which content or precipitate is completely solubilized in urea or GuHCl,

- the recovery of the sought protein.

21. Sequence of nucleic acids coding for any one of the peptides, polypeptides or proteins according to Claims 11 to 20.

22. Antibody, in particular monoclonal, directed against any one of the polypeptides of proteins according to the Claims 11 to 19.

23. Diagnostic procedure in particular for the detection, identification and quantitation in vitro of antibodies directed against one of the proteins or polypeptides or peptides according to any one of the Claims 11 to 19 and correlatable with a disease, these antibodies being present in a biological sample containing them, this procedure comprising

- the placing in contact of the biological sample with a peptide, polypeptide or a protein of the invention under conditions allowing an in vitro immunological reaction between the said peptide, polypeptide or protein and the antibodies which are possibly present in the biological sample and

- the in vitro detection of the antigen/antibody complex which may be formed.

24. Diagnostic procedure, in particular for the detection, identification and quantitation in vitro of a protein, polypeptide or peptide according to any one of the Claims 11 to 19 in a human biological sample likely to contain them, this procedure comprising:

25. Kit for the implementation of the procedure according to Claim 23, characterized in that it comprises: - a peptide, polypeptide or protein of the invention,

- reagents for making a medium suitable for the appearance of an immunological reaction,

reagents making it possible to detect the antigen/antibody complexes which have been produced by the immunological reaction, the said reagents possibly having a marker or being capable of being recognized by a labelled reagent more particularly in the case in which the above polypeptide or protein is not labelled.

26. Kit for the implementation of the procedure according to Claim 24, characterized in that it comprises:

- an antibody of the invention,

- reagents for making a medium suitable in order to cause an immunological reaction,

reagents making it possible to detect the antigen/antibody complexes which have been produced by the immunological reaction, the said reagents possibly having a marker or being capable of being known by a labelled reagent, more particularly in the case in which the above-mentioned antibody is not labelled.

27. Pharmaceutical composition containing as active substance any one of the peptides, polypeptides or proteins according to Claims 11 to 20.