SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture

摘要

A transient transfection process was established using a novel 'in-house' developed transfection reagent, Ro-1539. It allows rapid production of large quantities of various recombinant proteins. Here we describe the transient expression of the secreted human placental alkaline phosphatase (SEAP) by HEK293EBNA and CHO cells in serum-free suspension culture. Unexpectedly, high expression levels of SEAP (150 μg/ml) were found 3–4 days post-transfection when placental alkaline phosphatase (AP) was used as the reference enzyme. To confirm these data, an SDS–PAGE analysis was performed and the visible SEAP protein band (MW of 65 kDa) was compared with co-migrated purified placental AP protein as reference. The scanning analysis of the gel showed that SEAP, a truncated form of AP, has a higher specific activity than the purified placental AP. A correction factor was introduced permitting a direct comparison of placental AP activity with the expression levels of SEAP. Scale-up of the transfection system from spinner flask to bioreactor was simple and straightforward, resulting in similar yields of SEAP. Finally, the effectiveness of Ro-1539 was compared to that of other transfection reagents.