Expression and characterization of human bone morphogenetic protein-2 in silkworm larvae infected with recombinant Bombyx mori nuclear polyhedrosis virus.
Recombinant human bone morphogenetic protein-2 (rhBMP-2) was expressed in silkworm larvae, and a milligram quantity of the protein was purified and characterized. The expressed rhBMP-2 was biologically active in terms of induction of alkaline phosphatase activity in MC3T3-E1 cells and ectopic bone formation in mice. On SDS-polyacrylamide gel electrophoretic analysis, the purified protein showed a 16 kDa band under reducing conditions and a 30 kDa band under non-reducing conditions. The silkworm-expressed rhBMP-2 was glycosylated and susceptible to endo-beta-N-acetylglucosaminidase F (endo F) and endo H, but resistant to endo D. Deglycosylated rhBMP-2 treated with endo F retained its biological activity. These results suggest that rhBMP-2 exists as a dimer and disulfide bond(s) are responsible for the dimerization. Moreover, sugar chains have no direct effect on the biological activity of the protein. The availability of a quite large amount of rhBMP-2 has allowed us to study the biological function of this interesting factor in detail.
Ishida
N
N
Suntory Institute for Biomedical Research, Osaka.
Tsujimoto
M
M
Kanaya
T
T
Shimamura
A
A
Tsuruoka
N
N
Kodama
S
S
Katayama
T
T
Oikawa
S
S
Matsui
M
M
Nakanishi
T
T
D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D001846:Bone Development / Q000187:drug effects*; D019485:Bone Morphogenetic Proteins; D002460:Cell Line; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D006031:Glycosylation; D006133:Growth Substances / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000502:physiology*; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D019485:Bone Morphogenetic Proteins; D018076:DNA, Complementary; D006133:Growth Substances; D011506:Proteins; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase