Abstract
Historically, it has been proved difficult to adapt the traditional baculovirus expression systems to an automated platform because of the complexity of the processes involved. One of the major bottlenecks is the selection of recombinant from parental viruses. We have developed a bacmid vector (flashBAC™) that does not require any form of selection pressure to separate recombinant virus from nonrecombinant parental virus. The method relies on homologous recombination in insect cells between a transfer plasmid containing the gene of interest and a replication-deficient bacmid. The gene of interest replaces the bacterial replicon at the polyhedrin locus, simultaneously restoring a virus gene essential for replication, and as only recombinant virus can replicate, no further separation techniques are required. This chapter describes methods for producing and expression testing multiple recombinant baculoviruses on automated platforms using the flashBAC system.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
McCall, E. J., Danielsson, A., Hardern, I. M., et al., (2005) Improvements to the throughput of recombinant protein expression in the baculovirus/insect cell system. Protein Expr.Purif.42, 29–36.
Sarafanov, A., and Saenko, E. (2004) High-throughput optimization of protein expression in the baculovirus system based on determination of relative expression efficiency of viral stocks. Anal. Biochem.328, 98–100.
Redaelli, L., Zolezzi, F., Nardese, V., et al., (2005) A platform for high-throughput expression of recombinant human enzymes secreted by insect cells. J. Biotechnol.120,59–71.
Schlaeppi, J. M., Henke, M., Mahnke, M., et al., (2006) A semi-automated large-scale process for the production of recombinant tagged proteins in the baculovirus expression system.Protein. Expr. Purif.50,185–195.
Shrestha, B., Smee, C., and Gileadi, O. (2008) Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression. Methods Mol. Biol.439, 269–289.
Karkkainen, H. R., Lesch, H. P., Maatta, A. I., et al., (2009) A 96-well format for a high-throughput baculovirus generation, fast titering and recombinant protein production in insect and mammalian cells. BMC. Res. Notes. 2, 63.
Yao, L., Sun, J., Xu, H., et al., (2010) A novel economic method for high throughput production of recombinant baculovirus by infecting insect cells with Bacmid-containing diminopimelate-auxotrophic Escherichia coli. J. Biotechnol.145, 23–29.
Possee, R. D., Hitchman, R. B., Richards, K. S., et al., (2008) Generation of baculovirus vectors for the high-throughput production of proteins in insect cells. Biotechnol. Bioeng.101, 1115–1122.
Summers, M. D., and Smith, G. E. (1987) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agric. Exp. Stn. Bull. 1555.
Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., et al.,(1977) The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 13, 213–217.
Bahia, D., Cheung, R., Buchs, M., et al., (2005) Optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen. Protein. Expr. Purif.39, 61–70.
Chambers, S. P., Austen, D. A., Fulghum, J. R., et al., (2004) High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells. Protein. Expr. Purif. 36, 40–47.
Hitchman, R. B., Siaterli, E. A., Nixon, C. P., et al., (2007) Quantitative real-time PCR for rapid and accurate titration of recombinant baculovirus particles. Biotechnol. Bioeng.96, 810–814.
Acknowledgments
The authors would like to thank Elisabetta Locanto and Adam Chambers for technical assistance in developing the 24 deep-well block method described in this chapter.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hitchman, R.B., Possee, R.D., King, L.A. (2012). High-Throughput Baculovirus Expression in Insect Cells. In: Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 824. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-433-9_33
Download citation
DOI: https://doi.org/10.1007/978-1-61779-433-9_33
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-432-2
Online ISBN: 978-1-61779-433-9
eBook Packages: Springer Protocols