Efficient expression of a heterodimer of bone morphogenetic protein subunits using a baculovirus expression system.
Recombinant baculoviruses as expression vectors for Xenopus bone morphogenetic protein (xBMP)-2, 4 and 7 were generated. The conditioned medium of insect cells infected with the virus for xBMP-2 or 4 showed strong alkaline phosphatase-inducing activity in a mouse osteoblastic cell line, MC3T3-E1, although a large portion of the activity remained in the infected cells. In contrast, xBMP-7 was preferentially secreted into the medium, but had only weak activity. Conditioned media following simultaneous inoculation with the viruses for xBMP-7 and for xBMP-2 or 4 showed a remarkably increased level of activity. The increased activity was clearly separated from other peaks derived from single infection on a cation-exchange column and was found to arise from the disulfide-linked heterodimer consisting of xBMP-4 and 7 subunits by immunoblot analysis. The heterodimer also augmented osteocalcin production and parathyroid hormone-sensitivity more strongly than the homodimers. These results suggest that our expression system provides a convenient source of heterodimeric BMP with high osteogenic differentiation-inducing activity.
Hazama
M
M
Pharmaceutical Research Division, Takeda Chemical Industries Ltd., Osaka, Japan.
Aono
A
A
Ueno
N
N
Fujisawa
Y
Y
D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001681:Biological Assay; D019485:Bone Morphogenetic Proteins; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006133:Growth Substances / Q000096:biosynthesis; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects; D010281:Parathyroid Hormone / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D018411:Spodoptera; D014162:Transfection; D014981:Xenopus
D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D006133:Growth Substances; D046911:Macromolecular Substances; D010281:Parathyroid Hormone; D011506:Proteins; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase