Expression, purification, bioactivity, and partial characterization of a recombinant human bone morphogenetic protein-7 produced in human 293T cells.
Bone morphogenetic protein-7 (BMP-7) is a secreted multifunctional growth factor of the TGF-beta superfamily, which is predominantly known for its osteoinductive properties and emerging potential for treatment of kidney diseases. The mature 34-38 kDa disulfide-linked homodimer protein plays a key role in the differentiation of mesenchymal cells into bone and cartilage. In this study, the full-length sequence of hBMP-7 was amplified and, then, cloned, expressed, and purified from the conditioned medium of 293T cells stably transfected with a lentiviral vector. The mature protein dimer form was properly secreted and recognized by anti-BMP-7 antibodies, and the protein was shown to be glycosilated by treatment with exoglycosidase, followed by western blotting. Moreover, the activity of the purified protein was demonstrated both in vitro, by alkaline phosphatase activity in C2C12 cells, and in vivo by induction of ectopic bone formation in Balb/c Nude mice after 21 days, respectively. This recombinant protein platform may be very useful for expression of different human cytokines and other proteins for medical applications.
Bustos-Valenzuela
J C
JC
NUCEL - Cell and Molecular Therapy Center, University of São Paulo, São Paulo, Brazil.
Halcsik
E
E
Bassi
E J
EJ
Demasi
M A
MA
Granjeiro
J M
JM
Sogayar
M C
MC
D000818:Animals; D001681:Biological Assay / Q000379:methods; D055419:Bone Morphogenetic Protein 7 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D002846:Chromatography, Affinity; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D011994:Recombinant Proteins