Abstract
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 Thichoplusia 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.
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References
Smith, G.E., Summers, M.D. and Fraser, M.J. 1983. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 3: 2156–2165.
Pennock, G.D., Shoemaker, C. and Miller, L.K. 1984. Strong and regulated expression of Escherichia coli β-galactosidase in insect cells with a baculovirus vector. Mol. Cell. Biol. 4: 399–406.
Blissard, G.W. and Rohrmann, G.F. 1991. Baculovirus gp64 gene expression: Analysis of sequences modulating early transcription and transactivation by IE1. J. Virol. 65: 5820–5827.
Dickson, J.A. and Friesen, P.D. 1991. Identification of upstream promoter elements mediating early transcription from the 35k protein gene of Autographa californica nuclear polyhedrosis virus. J. Virol. 65: 4006–4016.
Luckow, V.A. 1991. Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors from Recombinant DNA Technology and Applications. Prokop, A., Baipai, R. and Ho, C. (Eds). McGraw-Hill, Inc., NY.
Millan, J.L. 1986. Molecular cloning and sequence analysis of human placental alkaline phosphatase. J. Biol. Chem. 261: 3112–3115.
Berger, J., Howard, A.D., Gerber, L., Cullen, B.R. and Udenfriend, S. 1987. Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells. Proc. Natl. Acad. Sci. USA 84: 4885–4889.
Berger, J., Hauber, J., Hauber, R., Geiger, R. and Cullen, B.R. 1988. Secreted placental alkaline phosphatase: A powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66: 1–10.
Wojchowski, D.M., Orkin, S.H. and Sytkowski, A.J. 1987. Active human erythropoietin expressed in insect cells using a baculovirus vector: A role for N-linked oligosaccharide. Biochim. Biophys. Acta 910: 224–232.
Jarvis, D.L. and Summers, M.D. 1989. Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells. Mol. Cell Biol. 9: 214–223.
Davidson, D.J., Fraser, M.J. and Castellino, F.J. 1990. Oligosaccharide processing in the expression of human plasminogen cDNA by Lepidop-teran insect (Spodoptera frugiperda) cells. Biochemistry 29: 5584–5590.
Kuroda, K., Geyer, H., Geyer, R., Doerfler, W. and Klenk, H. 1990. The oligosaccharide of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector. Virology 174: 418–429.
Ezra, E., Balcher, R. and Udenfriend, S. 1983. Purification and partial sequencing of human placental alkaline phosphatase. Biochem. Biophys. Res. Commun. 116: 1076–1083.
Takami, N., Ogata, S., Oda, K., Misumi, Y. and Ikehara, Y. 1988. Biosynthesis of placental alkaline phosphatase and its post-translational modification of glycophospholipid for membrane-anchoring. J. Biol. Chem. 263: 3016–3021.
Matsuura, Y., Possee, R.D. and Bishop, D.H.L. 1987. Expression of the S-coded genes of lymphocytic choriomeningitis arenavirus using a baculovirus vector. J. Gen. Virol. 76: 1515–1529.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. 1989. Current Protocols in Molecular Biology, Volumes 1 and 2, Greene Publishing Associates, NY.
Groebe, D.R., Chung, A.E. and Ho, C. 1990. Cationic lipid-mediated co-transfection of insect cells. Nucl. Acids Res. 18: 4033.
Wood, H.A. 1977. An agar overlay plaque assay method for Autographa californica nuclear-polyhedrosis virus. J. Invert. Path. 29: 304–307.
Sanger, F., Nicklen, S. and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467.
Wood, H.A. 1980. Isolation and replication of an occlusion body deficient mutant of the Autographa ca1ifornica nuclear polyhedrosis virus. Virology 105: 338–344.
McComb, R.B. and Bowers, G.N. 1972. Study of optimum buffer conditions for measuring alkaline phosphatase activity in human serum. Clin. Chim. Acta 18: 97–102.
Hausamen, T.U., Helger, R., Rick, W. and Gross, W. 1967. Optimal conditions for the determine of serum alkaline phosphatase by a new kinetic method. Clin. Chim. Acta 15: 241–245.
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Davis, T., Trotter, K., Granados, R. et al. Baculovirus Expression of Alkaline Phosphatase as a Reporter Gene for Evaluation of Production, Glycosylation and Secretion. Nat Biotechnol 10, 1148–1150 (1992). https://doi.org/10.1038/nbt1092-1148
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DOI: https://doi.org/10.1038/nbt1092-1148
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