Bone protein purification process

Claim 2

A process of purifying bone matrix proteins to obtain a cold-water-soluble 30 to 32k dalton protein capable of stimulating cartilage formation in mesenchymal-like cells comprising the steps of: (a) preparing a guanadinium chloride extract of demineralized, defatted bone; (b) dialyzing the guanidinium chloride soluble extract against decreasing ionic strength buffers down to water until it is substantially salt-free; (c) separating the cold-water-soluble proteins from the cold-water-insoluble proteins present in the retentate; (d) adsorbing the cold-water-soluble proteins in the retentate with an anionic exchanger at about pH 8.0 and desorbing by eluting with a substantially linear salt gradient; (e) assaying fractions eluted by the salt gradient in undifferentiated mesenchymal-like cell cultures to identify fractions having the greatest chondrogenic activity; (f) passing only those identified fractions having the greatest chondrogenic activity over a molecular sieve column; (g) assaying fractions passed over the molecular sieve in undifferentiated mesenchymal-like cells to identify fractions having the greatest chondrogenic activity; (h) repeating steps (f) and (g); (i) passing only those identified protein fractions having the greatest chondrogenic activity over concanavalin--A coupled gel; (j) collecting the unbound protein from step (i) in one fraction; and (k) passing the unbound protein from step (j) over a molecular sieve column to isolate a single 30 to 32k dalton protein.

Claim 3

The purified protein produced by the process of claim 1.

Claim 4

A process of purifying bone matrix proteins to obtain a cold-water-soluble 30 to 32k dalton protein capable of stimulating cartilage formation in embryonic cells comprising the steps of: (a) preparing a guanadinium chloride extract of demineralized, defatted bone; (b) dialyzing the guanidinium chloride soluble extract against decreasing ionic strength buffers down to water until is is substantially salt-free; (c) separating the cold-water-soluble proteins from the cold-water-insoluble proteins present in the retentate; (d) absorbing the cold-water-soluble proteins in the retentate with an anionic exchanger at about pH 8 and desorbing by eluting with a substantially linear salt gradient; (e) assaying fractions eluted by the salt gradient in undifferentiated embryonic limb bud mesenchymal cell culture to identify fractions having the greatest chondrogenic activity; (f) passing only those identified fractions having the greatest chondrogenic activity over a molecular sieve column; (g) assaying fractions passed over the molecular sieve in undifferentiated embryonic limb bud cells to identify fractions having the greatest chondrogenic activity; (h) repeating steps (f) and (g); (i) passing only those identified protein fractions having the greatest chondrogenic activity over concanavalin--A coupled gel; (j) collecting the unbound protein from step (i) in one fraction; and (k) passing the unbound protein from step (j) over a molecular sieve column to isolate a single 30 to 32k dalton protein.

Claim 5

A process of purifying bone matrix proteins to obtain a cold-water-soluble 30 to 32k dalton protein capable of stimulating cartilage formation in embryonic cells comprising the steps of: (a) preparing a guanadinium chloride extract of demineralized, defatted bone; (b) dialyzing the guanidinium chloride soluble extract against decreasing ionic strength buffers down to water until is is substantially salt-free; (c) separating the cold-water-soluble proteins from the cold-water-insoluble proteins present in the retentate; (d) absorbing the cold-water-soluble proteins in the retentate with an anionic exchanger at about pH 8 and desorbing by eluting with a substantially linear salt gradient; (e) assaying fractions eluted by the salt gradient in undifferentiated embryonic limb bud mesenchymal cell culture to identify fractions having the greatest chondrogenic activity; (f) passing only those identified fractions having the greatest chondrogenic activity over a molecular sieve column; (g) assaying fractions passed over the molecular sieve in undifferentiated embryonic limb bud cells to identify fractions having the greatest chondrogenic activity; (h) repeating steps (f) and (g); (i) passing only those identified protein fractions having the greatest chondrogenic activity over concanavalin--A coupled gel; (j) collecting the unbound protein from step (i) in one fraction; and (k) passing the unbound protein from step (j) over a molecular sieve column to isolate a single 30 to 32k dalton protein.

Claim 6

The highly purified protein extracted from bone matrix by the process of claim 1 having the following characteristics: (a) molecular weight of 30-32k daltons as assessed by electrophoresis; (b) solubility in substantially pure water at a temperature of 4° C.; (c) affinity for anionic exchangers at about pH 8.0; (d) non-affinity for concanavalin--A; and (e) activity as a stimulator of chondrogenesis in undifferentiated cells.

Claim 7

The highly purified protein extracted from bone matrix by the process of claim 3 having the following characteristics: (a) molecular weight 30-32k daltons as assessed by electrophoresis; (b) solubility in substantially pure water at a temperature of 4° C.; (c) affinity for anionic exchangers at about pH 8.0; (d) non-affinity for concanavalin--A; and (e) activity as a stimulator of chondrogenesis in undifferentiated cells.