PURIFICATION OF MONOCOMPONENT BOVINE BONE MORPHOGENETIC PROTEIN IN A WATER-SOLUBLE FORM

Noncollagenous protein material was extracted from HCl-demineralized b ovine bone particles in 4 M guanidinium hydrochloride. Water- and citr ate buffer-insoluble material was collected, solubilized in 6 M urea a nd fractionated by preparative isoelectric focusing using a running vo ltage of 5000 V. The material removed from the area between pH 4.7 and 5.7 of the isoelectric focusing gel was osteoinductive (identified by its capacity to induce bone development). This was solubilized in 6 M urea and dialyzed against 0.2 M Tris buffer. The Tris buffer-soluble material was fractionated by HPLC gel filtration. The water- and citra te buffer-insoluble material contained mainly high-molecular-weight pr otein complexes which were osteoinductive, and < 5 % of the material w as osteoinductive monocomponent bone morphogenetic protein. The Tris b uffer-soluble material contained only two polypeptides: an osteoinduct ive peptide of molecular weight 18,500 and a non-osteoinductive peptid e of molecular weight 8,000. The very high voltage used during the iso electric focusing caused a slow break-down of the urea-soluted protein complexes, which significantly increased the yield of monocomponent b one morphogenetic protein. By the present method it is possible to pre pare Tris buffer solution containing up to 2 mg/ml of pure monocompone nt bone morphogenetic protein.