Production of bone morphogenetic protein-7 using pET expression system

Abstract

Abstract The polypeptide representing the mature part of human bone morphogenetic protein-7 (BMP-7) was efficiently expressed in E. coli BL21 (DE3). The rhBMP-7 is a disulfide-bonded homodimeric protein with an apparent MW of 29,000 as shown by the SDS-PAGE and gel chromatography. rhBMP-7 stimulates ALP specific activity in a dose-dependent manner. In a kinetic experiment, ALP activity in both the rhBMP-7 (100 ng/ml)-treated and control cultures increases gradually, and activity in the rhBMP-7-treated culture is consistently higher throughout the culture period. We also quantitated the specific osteoblastic marker osteocalcin in rhBMP-7-treated culture medium. When serum-starved MC3T3-E1 cells are treated with medium containing various concentrations of rhBMP-7, the production of osteocalcin is increased about 7.2-fold in a dose-dependent manner up to 320 ng rhBMP-7/ml. In summary, we established an E. coli expression system for a high level of rhBMP-7 production and inductive effects of osteogenic differentiation markers by rhBMP-7 in MC3T3-E1 cells were evaluated. rhBMP-7 is strongly mitogenic for MC3T3-E1 cells, showing dose-dependent induction of ALP activity and osteocalcin production. Thus our expression system provides a convenient source of rhBMPs for in vitro or in vivo study.