Transient expression of osteopontin in HEK 293 cells in serum-free culture

As a highly phosphorylated glycoprotein, OPN has important implications in the pathogenesis of many types of cancers and autoimmune diseases. With the green fluorescent protein (GFP) as a reporter, the parameters to transfect HEK 293-F cells were optimized. These included the concentrations of plasmid DNA and linear 25 kDa PEI, the reaction time for DNA/PEI complex formation and the time to transfect the cells. When the cloned full-length human osteopontin (OPN-a) gene was transiently expressed, a limited growth of cells and excessive accumulations of lactate and ammonium were observed in shaker flask culture. However, when compared to 27 μg/mL in shaker flask culture, the yield of OPN-a reached 49 μg/mL in a 5 L bioreactor as a result of the doubled maximal viable cell density in the presence of 1 g/L protein hydrolysates in the post-transfection growth medium. The purified OPN-a was proven to induce the cell adhesion after chromatographic purification.