Enzyme kinetics and glycan structural characterization of secreted alkaline phosphatase prepared using the baculovirus expression vector system

Secreted human alkaline phosphatase (SEAP, a model protein containing a single N-glycan chain) was expressed in Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines infected with recombinant Autographa californica multiple nuclear polyhedrovirus expressing SEAP under control of the polyhedrin promoter. SDS-PAGE showed that both systems expressed fairly pure rSEAP products. The rSEAP expression level was 7.0 U/mL in Tn-5B1-4, higher than the 4.1 U/mL produced by Sf-9. Kinetic analysis showed that V_max and K_m of human placental SEAP were approx 10-fold higher than that of rSEAP, whereas the V_max and K_m of rSEAP prepared using both insect cell lines were comparable. To characterize the recombinant SEAP (rSEAP) glycosylation, the purified rSEAP was digested with PNGase F to release the N-glycan chains. Glycan analysis showed the presence of oligomannose-type N-linked glycans (i.e., Man_2-8GlcNAc₂ and FucMan_{3 or 4}GlcNAc₂) in rSEAP from Sf9 and Tn-5B1-4 cell lines. The proportions of these oligosaccharide structures were different in the two cell lines. Man₄GlcNAc₂ and FucMan₄GlcNAc₂ were the major rSEAP N-glycans produced in Sf-9 cells, while Man₂GlcNAc₂ was the major rSEAP N-glycan produced in Tn-5B1-4 cells.