Affinity purification of secreted alkaline phosphatase produced by baculovirus expression vector system

Human secreted alkaline phosphatase (SEAP) was produced in a stably-transfo rmed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infec tion with a recombinant Autographa californica multiple nuclear polyhedrovi rus containing the SEAP gene under control of the polyhedrin promoter. An a ffinity chromatographic column prepared by linking 4-amino-benzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell s upernatant following removal of cells and virus and 10-fold concentration t hrough ultrafiltration. We found that the binding of SEAP on the affinity m atrix follows the Langmuir isotherm model. In addition, either recycling SE AP sample through the column for 24 h or loading high SEAP concentrations r esulted in a high-purity product. Some nonspecific binding of protein on th e matrix occurred when low concentrations of SEAP sample were loaded. Final ly, we found that SEAP binding occurs rapidly, i.e., within 30 min of addin g the SEAP sample to the affinity matrix.