Abstract
This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA–DNA processing and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 ml of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures. Although not discussed in this chapter, the same process can also be applied to integral membrane proteins (IMPs) with slight adaptations to the purification procedure.
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References
Ignoffo CM (1975) Entomopathogens as insecticides. Environ Lett 8:23–40
Invitrogen (2002) Guide to Baculovirus expression vector systems (BEVS) and insect cell culture techniques. Invitrogen Life Technologies, Carlsbad
Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575
Summers MD, Anderson DL (1972) Granulosis virus deoxyribonucleic acid: a closed, double-stranded molecule. J Virol 9:710–713
Matthews REF (1982) Classification and nomenclature of viruses. Intervirology 17:1–181
O’Reilly D ML, Luckow V (1992) Baculovirus expression vectors: a laboratory manual
Vaughn JL, Goodwin RH, Tompkins GJ et al (1977) The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 13:213–217
Granados RR, Guoxun L, Derksen ACG et al (1994) A new cell line from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis virus. J Invertebr Pathol 64:260–266
Smith GE, Summers MD, Fraser MJ (1983) Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol 3:2156–2165
Invitrogen (2010) Bac-to-Bac® Baculovirus expression system. Invitrogen Life Technologies, Carlsbad
Hanahan D, Jessee J, Bloom FR (1991) Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol 204:63–113
Shrestha B, Smee C, Gileadi O (2008) Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression. Methods Mol Biol 439:269
Invitrogen (2010) Growth and maintenance of insect cell lines. Invitrogen Life Technologies, Carlsbad
Roldao A, Oliveira R, Carrondo MJ et al (2009) Error assessment in recombinant baculovirus titration: evaluation of different methods. J Virol Methods 159:69–80
Acknowledgements
We would like to thank all the SGC scientists (past and present) who contributed towards the development of the methods. The SGC is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, Genome Canada, GlaxoSmithKline, Lilly Canada, the Novartis Research Foundation, Pfizer, Takeda, AbbVie, the Canada Foundation for Innovation, the Ontario Ministry of Economic Development and Innovation, and the Wellcome Trust.
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Mahajan, P., Strain-Damerell, C., Gileadi, O., Burgess-Brown, N.A. (2014). Medium-Throughput Production of Recombinant Human Proteins: Protein Production in Insect Cells. In: Chen, Y. (eds) Structural Genomics. Methods in Molecular Biology, vol 1091. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-691-7_6
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DOI: https://doi.org/10.1007/978-1-62703-691-7_6
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