Process options in hepatitis B surface antigen extraction from transgenic potato

The process conditions for recombinant hepatitis B surface antigen (HBsAg) extraction from transgenic potato were examined. The effects of temperature , the reducing agent P-mercaptoethanol (BME), and proteinase inhibitors on the level of antigenic activity of recovered HBsAg were determined. Sedimen tation profiles were performed to characterize HBsAg assembly into virus-li ke particles. Increasing the temperature of the sample for about 1 min incr eased the measured HBsAg antigenic activity. The optimum temperature was ar ound 50 degrees C. A 3-fold enhancement of the antigenic activity was obtai ned in extract from transgenic potato expressing HBsAg, when monoclonal ant ibodies were used to assay for HBsAg. When antigenic activity was determine d by polyclonal antibodies, no enhancement in the antigenic activity was ob tained. Temperature may affect the conformation of the a epitope to which t he monoclonal antibodies bind or alter the fluidity of surface lipid region s. BME increased the antigenic activity of HBsAg up to 4-fold when monoclon al antibodies directed against the a determinant were used, but there was n o increase with polyclonal antibodies. This observation suggests that BME a ffects the structure or presentation of the a epitope. In the presence of B ME and leupeptin, a proteinase inhibitor, higher antigenic activity was obt ained. Leupeptin might protect the antigen, which might become more suscept ible to proteolytic degradation after reduction, as a result of stimulation of sulfhydryl proteases. Although both temperature and BME increased the a ntigenic activity of HBsAg individually, when combined their interaction wa s antagonistic, resulting in reduced antigenic activity. Different proteina se inhibitors, including leupeptin, aprotinin, E-64, pefabloc, and pepstati n, had no significant effect on HBsAg from potato extract in a 2 h period i n the absence of BME. The sedimentation profile of potato-produced HBsAg wa s determined in 5-30% sucrose gradients. Yeast-derived recombinant HBsAg wa s used as a positive control. The HBsAg from transgenic potato showed sedim entation and density properties that are very similar to the yeast-produced antigen, indicating assembly into virus-like particles. BME treatment did not change the sedimentation profile.