Purification of Water-Soluble Bone-Inductive Protein from Bovine Demineralized Bone Matrix

概要

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The water-soluble fraction containing bone-inductive activity was purified from guanidine-hydrochloride extracts of bovine demineralized bone. The purification steps include ultrafiltration, dialysis, affinity chromatography on heparin-Sepharose and gel chromatography on Sephacryl S-200. Combination of these steps was proven to be an effective and rapid method for the purification of this protein.Subcutaneous implantation of the water-soluble protein with type I collagen was carried out in the thorax of rats. When alkaline phosphatase activity and calcium content in implants were used as indices for purification, the water-soluble bone-inductive protein was purified >600-fold according to the enzyme activity and 64-fold according to the calcium content. A morphological examination revealed that many chondrocyte and osteoblast cells were seen in the location of the implanted material.Sodium dodecyl sulfate/gel electrophoresis of the protein produced in this way under non-reducing conditions revealed four protein bands of 18,16,14 and 11 kDa. None of the separated bands had any biological activity. This result suggests that the water-soluble bone-inductive activity depends on an associated form of various proteins in the range of 18 to 11 kDa.
社団法人日本薬学会の論文
1993-05-15