Purification of monocomponent bovine bone morphogenetic protein in a water-soluble form.
Noncollagenous protein material was extracted from HCl-demineralized bovine bone particles in 4 M guanidinium hydrochloride. Water- and citrate buffer-insoluble material was collected, solubilized in 6 M urea and fractionated by preparative isoelectric focusing using a running voltage 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 citrate buffer-insoluble material contained mainly high-molecular-weight protein complexes which were osteoinductive, and < 5% of the material was osteoinductive monocomponent bone morphogenetic protein. The Tris buffer-soluble material contained only two polypeptides: an osteoinductive peptide of molecular weight 18,500 and a non-osteoinductive peptide of molecular weight 8,000. The very high voltage used during the isoelectric focusing caused a slow break-down of the urea-soluted protein complexes, which significantly increased the yield of monocomponent bone morphogenetic protein. By the present method it is possible to prepare Tris buffer solution containing up to 2 mg/ml of pure monocomponent bone morphogenetic protein.
Jortikka
L
L
Department of Clinical Medicine, University of Tampere, Finland.
Marttinen
A
A
Lindholm
T S
TS
D000818:Animals; D001681:Biological Assay; D001857:Bone Matrix / Q000737:chemistry; D019485:Bone Morphogenetic Proteins; D001842:Bone and Bones / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D051379:Mice; D010012:Osteogenesis / Q000502:physiology; D011506:Proteins / Q000302:isolation & purification* / Q000502:physiology; D012995:Solubility
D019485:Bone Morphogenetic Proteins; D011506:Proteins