Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells.
Bone is a dynamic tissue that responds to many factors including vitamin D, parathyroid hormone, estrogen, calcitonin, and bone morphogenetic proteins (BMPs). The ability to stimulate new bone growth would permit novel therapies for situations where bone mass has been lost due to accident or disease. Purified BMP-2, in conjunction with a suitable matrix, is sufficient to stimulate the synthesis of new bone (Wang et al., 1990). We have expressed recombinant human BMP-2 at high levels in Chinese hamster ovary cells using methotrexate-mediated gene amplification. Several forms of BMP-2 are secreted from CHO cells: (1) an amino-terminal propeptide of 40-45 kDa, (23) a mature active 30 kDa homodimer consisting of 18-22 kDa subunits, and (3) a small amount of uncleaved 60 kDa precursor protein. The mature, active protein is predominantly a 30 kDa homodimer consisting of subspecies of 18 and 22 kDa which differ by proteolytic processing at their amino termini. Mature BMP-2 and propeptide contain high mannose and complex N-linked oligosaccharides, respectively. The molar amount of secreted, processed propeptide is approximately 5-fold higher than mature BMP-2 in conditioned medium. BMP-2 associates with both the extracellular matrix and the surface of CHO cells, which may in part account for the unequal levels of extracellular propeptide and mature forms of the molecule in the conditioned medium. Recombinant BMP-2 can be expressed in sufficient quantities to assess its therapeutic potential for bone regeneration.
Israel
D I
DI
Genetics Institute, Cambridge, MA 02140.
Nove
J
J
Kerns
K M
KM
Moutsatsos
I K
IK
Kaufman
R J
RJ
D000818:Animals; D019485:Bone Morphogenetic Proteins; D016466:CHO Cells; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional; D006133:Growth Substances / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D019485:Bone Morphogenetic Proteins; D006133:Growth Substances; D011506:Proteins; D011994:Recombinant Proteins