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Secretion of the Respiratory Syncytial Virus Fusion Protein From Insect Cells Using the Baculovirus Expression System

  • Boon-Huan Tan
  • Gaie Brown
  • Richard J. Sugrue
Part of the Methods in Molecular Biology book series (MIMB, volume 379)

Abstract

Sequences derived from the respiratory syncytial virus (RSV) fusion (F) protein were expressed in insect cells as recombinant glutathione-S-transferase (GST)-tagged proteins. The sequence covering the F2 subunit (GST-F2), and a truncated form of the F protein in which the transmembrane domain was removed (GST-F2/F1, were cloned into the baculovirus pAcSecG2T secretory vector. These virus sequences also had the endogenous virus signal sequence removed and replaced with a signal sequence derived from the baculovirus gp67 glycoprotein, which was present in pAcSecG2T. The recombinant RSV glycoproteins were successfully detected in expressing cells by immunofluorescence assay and in the tissue culture medium by western blot analysis. The secreted recombinant GST-F2/F1 protein was further analysed using glycosidases. Our results showed that the GST-F2/F1 protein were sensitive to peptide: N-glycosidase F (PNGase F) treatment, but not to Endoglycosidase H (EndoH) treatment. This indicates that the secreted recombinant proteins were modified by the addition of mature N-linked glycan chains.

Key Words

Respiratory syncytial virus Baculovirus GST-F2 protein GST-F2/F1 protein 

References

  1. 1.
    Kost, T. A., Condreay, J. P., and Jarvis, D. L. (2005) Baculovirus as a versatile vectors for protein expression in insect and mammalian cells. Nat. Biotech. 23,5, 567–573.CrossRefGoogle Scholar
  2. 2.
    Marz, L., Altmann, F., Staudacher, E., and Kubelka, V. (1995) Protein Glycosylation in Insects in Glycoproteins, vol. 29a (Montreuil, J., Vliegenthart, J. F. G., and Schachter, H. eds.)., pp. 543–563.Google Scholar
  3. 3.
    Marchai, I., Jarvis, D. L., Cacan, R., and Verbert, A. (2001) Glycoproteins in insect cells L sialylated or not? Biol. Chem. 382, 151–159.CrossRefGoogle Scholar
  4. 4.
    Altmann, F., Schwihla, H., Staudacher, E., Glossl, J., and Marz, L. (1995) Insect cells contain an unusual, membrane-bound β-N-acetylglucosaminidase probably involved in the processing of protein N-glycans. J. Biol. Chem. 270, 17,344–17,349.CrossRefPubMedGoogle Scholar
  5. 5.
    Aumiller, J. J., Hollister, J. R., and Jarvis, D. L. (2003) A transgenic lepidopteran insect cell line engineered to produce CMP-sialic acid and sialoglycoproteins. Glycobiology 13, 497–507.CrossRefPubMedGoogle Scholar
  6. 6.
    Hollister, J. R., Shaper, J. H., and Jarvis, D. L. (1998) Stable expression of mammalian beta 1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells. Glycobiology 8, 473–480.CrossRefPubMedGoogle Scholar
  7. 7.
    Hollister, J. and Jarvis, D. L. (2001) Engineering lepidopteran insect cells for sialoglycoprotein production by genetic transformation with mammalian \-1,4-galactosyltransferase and a 2, 6-sialyltransferase genes. Glycobiology 11, 1–9.CrossRefPubMedGoogle Scholar
  8. 8.
    Hollister, J., Grabenhorst, E., Nimtz, M., Conradt, H. O., and Jarvis, D. L. (2002) Engineering the protein N-glycosylation pathway in insect cells for production of biantennary, complex N-glycans. Biochemistry 41, 15,093–15,104.CrossRefPubMedGoogle Scholar
  9. 9.
    Arumugham, R. G., Seid, Jr., R. C., Doyle, S., Hildreth, S. W., and Parasio, P. R. (1989) Fatty acid acylation of the fusion glycoprotein of human respiratory syncytial virus. J. Biol. Chem. 264, 10,339–10,342.PubMedGoogle Scholar
  10. 10.
    Collins, P. L. and Mottet, G. (1991) Post-translational processing and oligomerization of the fusion glycoprotein of human respiratory syncytial virus. J. Gen. Virol. 72(pt12), 3095–3101.CrossRefPubMedGoogle Scholar
  11. 11.
    Whitford, M., Stewart, S., Kuzio, J., and Faulkner, P. (1989) Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus. J. Virol. 63(3), 1393–1399.PubMedGoogle Scholar
  12. 12.
    Stewart, L. M., Hirst, M., Lopez Ferber, M., Merryweather, A. T., Cayley, P. J., and Possess, R. D. (1991) Construction of an improved baculovirus insecticide containing an insect-specific toxin gene. Nature 352(6330), 85–88.CrossRefPubMedGoogle Scholar
  13. 13.
    Davies, A. H., Jowett, J. B., and Jones, I. M. (1993) Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins. BioTechnology 11(8), 933–936.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Boon-Huan Tan
    • 1
  • Gaie Brown
    • 2
  • Richard J. Sugrue
    • 2
  1. 1.Virology Group Defence Medical and Environmental Research InstituteDSO National LaboratoriesSingapore
  2. 2.MRC Virology UnitInstitute of VirologyGlasgowUK

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