Skip to main content
Log in

A new Trichoplusia ni cell line for membrane protein expression using a baculovirus expression vector system

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

A new cell line, MSU-TnT4 (TnT4), was established from Trichoplusia ni embryos for use with baculovirus expression vectors and evaluated for its potential for membrane protein production. To evaluate membrane protein synthesis, recombinant baculoviruses were constructed to express the human neurotensin receptor 1 as an enhanced green fluorescent protein (GFP) fusion. TnT4 cells had a doubling time of 21 h and expressed the membrane-GFP fusion protein at approximately twice the level as Sf21 cells from the p10 promoter, as evaluated by GFP intensity. Expression of secreted alkaline phosphatase (SEAP) was similar to that of Sf21 cells. Expression of membrane-GFP fusion proteins in recombinant baculoviruses provides a rapid method for evaluating the potential of new cell lines for the production of membrane proteins using a baculovirus expression vector system (BEVS).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.

Similar content being viewed by others

References

  • Akermoun M.; Koglin M.; Zvalova-Iooss D.; Folschweiller N.; Dowell S. J.; Gearing K. L. Characterization of 16 human G protein-coupled receptors expressed in baculovirus-infected insect cells. Protein Expr. Purif. 44: 65–74; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Becker G. W.; Miller J. R.; Kovacevic S.; Ellis R. M.; Louis A. I.; Small J. S.; Stark D. H.; Roberts E. F.; Wyrick T. K.; Hoskins J.; Chiou X. G.; Sharp J. D.; McClure D. B.; Riggin R. M.; Kramer R. M. Characterization by electrospray mass-spectrometry of human Ca2+-sensitive cytosolic phospholipase A2 produced in baculovirus-infected insect cells. Bio-Technol. 12: 69–74; 1994.

    CAS  Google Scholar 

  • Chai H.; AlRubeai M.; Chua K. L.; Oh S. K. W.; Yap M. G. S. Insect cell line dependent gene expression of recombinant human tumor necrosis factor-beta. Enzyme Microb. Technol. 18: 126–132; 1996.

    Article  CAS  Google Scholar 

  • Chen C. J.; Thiem S. M. Differential infectivity of two Autographa californica nucleopolyhedrovirus mutants on three permissive cell lines is the result of lef-7 deletion. Virology 227: 88–95; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Clem R. J.; Fechheimer M.; Miller L. K. Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254: 1388–1390; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Cullen B. R.; Malim M. H. Secreted placental alkaline-phosphatase as a eukaryotic reporter gene. Methods Enzymol. 216: 362–368; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Davis T. R.; Trotter K. M.; Granados R. R.; Wood H. A. Baculovirus expression of alkaline-,phosphatase as a reporter gene for evaluation of production, glycosylation and secretion. Bio-Technol. 10: 1148–1150; 1992.

    CAS  Google Scholar 

  • Davis T. R.; Wickham T. J.; McKenna K. A.; Granados R. R.; Shuler M. L.; Wood H. A. Comparative recombinant protein-production of 8 insect-cell lines. In Vitro Cell. Dev. Biol.-Anim. 29A: 388–390; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Gaw Z. -Y.; Liu N. T.; Zia T. U. Tissue culture methods for cultivation of virus grasserie. Acta Virol. 3Suppl.: 55–60; 1959.

    PubMed  Google Scholar 

  • Grace T. Establishment of four strains of cells from insect tissues grown in vitro. Nature (London) 195: 788–789; 1962.

    Article  CAS  Google Scholar 

  • Granados R. R.; Li G. X.; Derksen A. C. G.; McKenna K. A. A new insect-cell line from Trichoplusia ni (BTI-TN-5B1-4) Susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis-virus. J. Invertebr. Pathol. 64: 260–266; 1994.

    Article  Google Scholar 

  • Grisshammer R.; Tate C. G. Overexpression of integral membrane-proteins for structural studies. Q. Rev. Biophys. 28: 315–422; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Grunewald S.; Haase W.; Reilander H.; Michel H. Glycosylation, palmitoylation, and localization of the human D-2S receptor in baculovirus-infected insect cells. Biochemistry 35: 15149–15161; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Hink W. F. A serum-free medium for the culture of insect cells and production of recombinant proteins. In Vitro Cell. Dev. Biol. 27: 397–401; 1991.

    Article  Google Scholar 

  • Hollister J.; Grabenhorst E.; Nimtz M.; Conradt H.; Jarvis D. L. Engineering the protein N-glycosylation pathway in insect cells for production of biantennary, complex N-glycans. Biochemistry 41: 15093–15104; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Kuchler R. J. Biochemical methods in cell culture and virology. Dowden, Hutchinson & Ross, Stroudsburg; 1977.

    Google Scholar 

  • Lu A.; Miller L. K. Species-specific effects of the hcf-1 gene on baculovirus virulence. J. Virol. 70: 5123–5130; 1996.

    PubMed  CAS  Google Scholar 

  • Lynn D. E. Development and characterization of insect cell lines. Cytotechnology 20: 3–11; 1996.

    Article  Google Scholar 

  • Lynn D. E. Novel techniques to establish new insect cell lines. In Vitro Cell. Dev. Biol., Anim. 37: 319–321; 2001.

    CAS  Google Scholar 

  • Massotte D. G protein-coupled receptor overexpression with the baculovirus-insect cell system: A tool for structural and functional studies. Biochim. Biophys. Acta, Biomembr. 1610: 77–89; 2003.

    Article  CAS  Google Scholar 

  • McCusker E. C.; Bane S. E.; O'Malley M. A.; Robinson A. S. Heterologous GPCR expression: A bottleneck to obtaining crystal structures. Biotechnol. Prog. 23: 540–547; 2007.

    Article  PubMed  CAS  Google Scholar 

  • McIntosh A. H.; Grasela J. J.; Matteri R. L. Identification of insect cell lines by DNA amplification fingerprinting (DAF). Insect Mol. Biol. 5: 187–195; 1996.

    Article  PubMed  CAS  Google Scholar 

  • McKenna K. A.; Hong H. Z.; vanNunen E.; Granados R. R. Establishment of new Trichoplusia ni cell lines in serum-free medium for baculovirus and recombinant protein production. J. Invertebr. Pathol. 71: 82–90; 1998.

    Article  CAS  Google Scholar 

  • Ng G. Y. K.; George S. R.; Zastawny R. L.; Caron M.; Bouvier M.; Dennis M.; Odowd B. F. Human serotonin 1B receptor expression in Sf9 cells—Phosphorylation, palmitoylation, and adenylyl-cyclase inhibition. Biochemistry 32: 11727–11733; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Ng G. Y. K.; Odowd B. F.; Caron M.; Dennis M.; Brann M. R.; George S. R. Phosphorylation and palmitoylation of the human D2L dopamine-receptor in Sf9 cells. J. Neurochem. 63: 1589–1595; 1994.

    Article  PubMed  CAS  Google Scholar 

  • O'Reilly, D. R.; Miller, L. K.; Luckow, V. Baculovirus expression vectors. A laboratory manual. New York: Freeman; 1992.

    Google Scholar 

  • Ogonah O. W.; Freedman R. B.; Jenkins N.; Patel K.; Rooney B. C. Isolation and characterization of an insect cell line able to perform complex N-linked glycosylation on recombinant proteins. Bio-Technol. 14: 197–202; 1996.

    CAS  Google Scholar 

  • Palomares L. A.; Joosten C. E.; Hughes P. R.; Granados R. R.; Shuler M. L. Novel insect cell line capable of complex N-glycosylation and sialylation of recombinant proteins. Biotechnol. Prog. 19: 185–192; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Sarramegna V.; Talmont R.; Demange P.; Milon A. Heterologous expression of G-protein-coupled receptors: Comparison of expression systems from the standpoint of large-scale production and purification. Cell. Mol. Life Sci. 60: 1529–1546; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Smith G. E.; Ju G.; Ericson B. L.; Moschera J.; Lahm H. W.; Chizzonite R.; Summers M. D. Modification and secretion of human interleukin-2 produced in insect cells by a baculovirus expression vector. Proc. Natl. Acad. Sci. U. S. A. 82: 8404–8408; 1985.

    Article  PubMed  CAS  Google Scholar 

  • Vaughn J. L.; Goodwin R. H.; Tompkins G. J.; McCawley P. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro Cell. Dev. Biol.: Plant 13: 213–217; 1977.

    Article  CAS  Google Scholar 

  • Weyer U.; Knight S.; Possee R. D. Analysis of very late gene-expression by Autographa californica nuclear polyhedrosis virus and the further development of multiple expression vectors. J. Gen. Virol. 71: 1525–1534; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Wickham T. J.; Davis T.; Granados R. R.; Shuler M. L.; Wood H. A. Screening of insect cell-lines for the production of recombinant proteins and infectious virus in the baculovirus expression system. Biotechnol. Prog. 8: 391–396; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Zavodzky P.; Cseh S. Production of multidomain complement glycoproteins in insect cells. Cytotechnology 20: 279–288; 1996.

    Article  CAS  Google Scholar 

  • Zhao Y.; Sane D. C. Expression of a recombinant baculovirus for vitronectin in insect cells—Purification, characterization of posttranslational modifications and functional-studies of the recombinant protein. Arch. Biochem. Biophys. 304: 434–442; 1993.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgement

This work was supported by Michigan State University Grant REF03-016.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Suzanne M. Thiem.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, F., Manzan, M.A., Peplinski, H.M. et al. A new Trichoplusia ni cell line for membrane protein expression using a baculovirus expression vector system. In Vitro Cell.Dev.Biol.-Animal 44, 214–223 (2008). https://doi.org/10.1007/s11626-008-9095-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-008-9095-z

Keywords

Navigation