Skip to main content
SpringerLink
Log in
Book cover

Protein Expression in Mammalian Cells pp 41–63Cite as

Expression of a Secreted Protein in Mammalian Cells Using Baculovirus Particles

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 801))

Abstract

There are many methods presently available to produce recombinant proteins in mammalian systems. The BacMam system is a simple straightforward method which overlaps two well-established technologies, namely the BEVS insect cell system and the transduction of mammalian cells in vitro. This chapter describes a method for the study of gene expression in mammalian cells in a series of simple steps. Protocols outlined include the design and construction of the recombinant baculovirus, cell culture techniques required to maintain both insect and mammalian cells, generation of baculovirus stocks, and methods to obtain maximal and reproducible gene expression in mammalian cells. Currently available statistical techniques using factorial design of experiment to optimize conditions for recombinant protein in vitro are outlined. Then details with respect to process scale-up in disposable bioreactors are included.

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

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Boyce FM, Bucher NLR. Baculovirus-mediated gene transfer into mammalian cells. Proceedings of the National Academy of Sciences of the United States of America 1996;93:2348–52.

    Google Scholar 

  2. Lehtolainen P, Tyynela K, Kannasto J, et al. Baculoviruses exhibit restricted cell type specificity in rat brain: a comparison of baculovirus- and adenovirus-mediated intracerebral gene transfer in vivo. Gene Therapy 2002;9:1693–9.

    Google Scholar 

  3. Grassi G, Kohn H, Dapas B, et al. Comparison between recombinant baculo- and adenoviral-vectors as transfer system in cardiovascular cells. Archives of Virology 2006;151:255–71.

    Google Scholar 

  4. Martyn JC, Dong X, Holmes-Brown S, et al. Transient and stable expression of the HCV envelope glycoproteins in cell lines and primary hepatocytes transduced with a recombinant baculovirus. Archives of Virology 2007;152:329–43.

    Google Scholar 

  5. Salminen M, Airenne KJ, Rinnankoski R, et al. Improvement in nuclear entry and transgene expression of baculoviruses by disintegration of microtubules in human hepatocytes. Journal of Virology 2005;79:2720–8.

    Google Scholar 

  6. Bilello JP, Cable EE, Myers RL, Isom HC. Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatopytes. Gene Therapy 2003;10:733–49.

    Google Scholar 

  7. Barsoum J, Brown R, Mckee M, Boyce FM. Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus G glycoprotein. Human Gene Therapy 1997;8:2011–8.

    Google Scholar 

  8. Sandig V, Hofmann C, Steinert S, et al. Gene transfer into hepatocytes and human liver tissue by baculovirus vectors. Human Gene Therapy 1996;7:1937–45.

    Google Scholar 

  9. Nicholson LJ, Philippe M, Paine AJ, et al. RNA interference mediated in human primary cells via recombinant baculoviral vectors. Molecular Therapy 2005;11:638–44.

    Google Scholar 

  10. Lee HP, Ho YC, Hwang SM, et al. Variation of baculovirus-harbored transgene transcription among mesenchymal stem cell-derived progenitors leads to varied expression. Biotechnology and Bioengineering 2007;97:649–55.

    Google Scholar 

  11. Ho YC, Chung YC, Hwang SM, et al. Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells. Journal of Gene Medicine 2005;7:860–8.

    Google Scholar 

  12. Airenne KJ, Hiltunen MO, Turunen MP, et al. Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery. Gene Therapy 2000;7:1499–504.

    Google Scholar 

  13. Ma L, Tamarina N, Wang Y, et al. Baculovirus-mediated gene transfer into pancreatic islet cells. Diabetes 2000;49:1986–91.

    Google Scholar 

  14. Kinnunen K, Kalesnykas G, Mähönen AJ, et al. Baculovirus is an efficient vector for the transduction of the eye: comparison of baculovirus- and adenovirus-mediated intravitreal vascular endothelial growth factor D gene transfer in the rabbit eye. J Gene Med 2009.

    Google Scholar 

  15. Spenger A, Ernst W, Condreay JP, et al. Influence of promoter choice and trichostatin A treatment on expression of baculovirus delivered genes in mammalian cells. Protein Expression and Purification 2004;38:17–23.

    Google Scholar 

  16. Gheshlaghi R, Scharer JM, Moo-Young M, Douglas PL. Medium optimization for hen egg white lysozyme production by recombinant Aspergillus niger using statistical methods. Biotechnology and Bioengineering 2005;90: 754–60.

    Google Scholar 

  17. Berger J, Hauber J, Hauber R, et al. Secreted Placental Alkaline-Phosphatase - A Powerful New Quantitative Indicator of Gene-Expression in Eukaryotic Cells. Gene 1988;66:1–10.

    Google Scholar 

  18. Condreay JP, Witherspoon SM, Clay WC, Kost TA. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proceedings of the National Academy of Sciences of the United States of America 1999;96:127–32.

    Google Scholar 

  19. Jardin BA, Montes J, Lanthler S, et al. High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: Comparison to a batch Sf-9-BEV system. Biotechnology and Bioengineering 2007;97 :332–45.

    Google Scholar 

  20. Bedard C, Tom R, Kamen A. Growth, Nutrient Consumption, and End-Product Accumu-lation in Sf-9 and Bti-Eaa Insect-Cell Cultures - Insights Into Growth Limitation and Metabolism. Biotechnology Progress 1993;9: 615–24.

    Google Scholar 

  21. Elias CB, Carpentier E, Durocher Y, et al. Improving glucose and gluamiue metabolism of human HEK 293 and Trichoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme. Biotechnology Progress 2003;19:90–7.

    Google Scholar 

  22. Zeiser A, Bedard C, Voyer R, et al. On-line monitoring of the progress of infection in Sf-9 insect cell cultures using relative permittivity measurements. Biotechnology and Bioengi-neering 1999;63:122–6.

    Google Scholar 

  23. Zeiser A, Elias CB, Voyer R, et al. On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process. Biotechnology Progress 2000;16:803–8.

    Google Scholar 

  24. Jardin BA, Zhao Y, Selvaraj M, et al. Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system. Journal of Biotechnology 2008;135:272–80.

    Google Scholar 

  25. Janakiraman V, Forrest WF, Chow B, Seshagiri S. A rapid method for estimation of baculovirus titer based on viable cell size. Journal of Virological Methods 2006;132:48–58.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, Artificial Cells and Organs Research Center, McGill University, Montreal, QC, Canada

    Barbara Ann Jardin & Satya Prakash

  2. Biomedical Technology and Cell Therapy Research Laboratory, Department of Physiology, Artificial Cells and Organs Research Center, McGill University, Montreal, QC, Canada

    Barbara Ann Jardin & Satya Prakash

  3. Bulk Manufacturing/Cells and Viral Media, Sanofi-Pasteur, Toronto, ON, Canada

    Cynthia B. Elias

Authors
  1. Barbara Ann Jardin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cynthia B. Elias
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Satya Prakash
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cynthia B. Elias .

Editor information

Editors and Affiliations

  1. SAIC-Frederick, Inc., Protein Expression Laboratory, National Cancer Institute, Frederick, Boyles Street 1050, Frederick, 21702, Maryland, USA

    James L Hartley

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Jardin, B.A., Elias, C.B., Prakash, S. (2012). Expression of a Secreted Protein in Mammalian Cells Using Baculovirus Particles. In: Hartley, J. (eds) Protein Expression in Mammalian Cells. Methods in Molecular Biology, vol 801. Humana Press. https://doi.org/10.1007/978-1-61779-352-3_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-352-3_4

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-351-6

  • Online ISBN: 978-1-61779-352-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation