Production,Secretion,and Stability of Human Secreted Alkaline Phosphatase in Tobacco NT1 Cell Suspension Cultures

摘要

Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production,secretion,and stability in plant cell cultures.The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP).During batch culture,the highest level of active SEAP in the culture medium (0.4 U/mL,corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase.Although the level of active SEAP decreased during the stationary phase,the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation.The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase.Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture.Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation.Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis.We also show for the first time that the factors promoting activity loss are heat labile at 95 deg C but not at 70 deg C,and they are not inactivated after a 5 day incubation period under normal culture conditions (27 deg C).In addition,there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation,indicating that these factors were unimportant.