We have devised a simple and efficient baculovirus expression vector system to evaluate insect tissue culture cells for their capacity to express, glycosylate and secrete foreign proteins. A truncated placental alkaline phosphatase (SEAP) gene was inserted into the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the transcriptional control of the polyhedrin gene promoter. Production levels, glycosylation, and secretion of the recombinant protein were examined in Trichoplusia ni (BTI-TN-5B1-4) and Spodoptera frugiperda (Sf9) cell lines. The assay for SEAP activity, which is fast, inexpensive, and quantitative to concentrations of 20 picograms per milliliter, was used to assess cell-associated and secreted SEAP activity. The proportion of SEAP which is modified with N-linked oligosaccharide can also be determined due to the difference in mobilities during SDS-PAGE between the glycosylated and nonglycosylated forms of the protein.

The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharides and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.

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