Expression and characterization of murine osteoblast-specific factor 2 (OSF-2) in a baculovirus expression system.
Osteoblast-specific factor 2 (OSF-2) is a approximately 90-kDa protein selectively expressed in bone. OSF-2 cDNA was recently isolated from mouse and human cDNA libraries and shows limited sequence homology with fasciclin I, a cell adhesion protein expressed in insect nerve cells. Here we describe the expression of recombinant murine OSF-2 (rmOSF-2) in a baculovirus/insect cell system. Western blotting analysis employing polyclonal antiserum raised against a C-terminal synthetic OSF-2 peptide detected a protein of approximately 90-kDa as early as 2 days after infection of Sf9 cells with the recombinant virus. Tunicamycin treatment of infected cells resulted in a mobility shift of OSF-2 (approximately 90-kDa band) on Western blots. N-Glycanase digestion resulted in the same mobility shift of OSF-2, indicating that rmOSF-2 expressed in insect cells is N-glycosylated. However, OSF-2 was insensitive to endoglycosidase H digestion while a major fraction of this protein had affinity for concanavalin A. Finally, it was demonstrated that rmOSF-2 was able to bind to heparin. This finding suggests that OSF-2 might be associated with the bone extracellular matrix after secretion by osteoblasts and participate in cell adhesion and/or cell communication. The establishment of the baculovirus expression system with a high productivity of recombinant OSF-2 (around 40 micrograms/ml at maximum) and its heparin binding properties should allow us to obtain large amounts of rmOSF-2.
Sugiura
T
T
Laboratory of Molecular Biology, Hoechst Japan Limited, Saitama.
Takamatsu
H
H
Kudo
A
A
Amann
E
E
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003208:Concanavalin A / Q000378:metabolism; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism; D051379:Mice; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D014415:Tunicamycin / Q000494:pharmacology
D015815:Cell Adhesion Molecules; C512835:POSTN protein, human; C082838:Postn protein, mouse; D011994:Recombinant Proteins; D003208:Concanavalin A; D014415:Tunicamycin; D006493:Heparin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase