Abstract
Recombinant protein production has become an indispensable tool for various research directions and biotechnological applications in the past decades. Among the numerous reported expression systems, insect cells provide the possibility to produce complex target proteins that require posttranslational modifications. Stable expression in Drosophila S2 cells represents an attractive alternative to the widely used baculovirus expression system, offering important advantages in particular for difficult-to-express proteins, e.g., membrane proteins or heavily glycosylated multi-domain proteins that are stabilized by a complex disulfide pattern. Here we present the methodology that is required for the generation of stable Drosophila S2 cell transfectants and for production of recombinant proteins using those transfectants.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Naggie S, Bentley W (1998) Appearance of protease activities coincides with p10 and polyhedrin-driven protein production in the baculovirus expression system: effects on yield. Biotechnol Prog 14:227–232
Ellgaard L, Helenius A (2001) ER quality control: towards an understanding at the molecular level. Curr Opin Cell Biol 13:431–437
Kleizen B, Braakman I (2004) Protein folding and quality control in the endoplasmic reticulum. Curr Opin Cell Biol 16:343–349
Jarvis D, Fleming J, Kovacs G et al (1990) Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells. Biotechnology 8:950–955
Brazzoli M, Helenius A, Foung S et al (2005) Folding and dimerization of hepatitis C virus E1 and E2 glycoproteins in stably transfected CHO cells. Virology 332:438–453
Land A, Zonneveld D, Braakman I (2003) Folding of HIV-1 envelope glycoprotein involves extensive isomerization of disulfide bonds and conformation-dependent leader peptide cleavage. FASEB J 17:1058–1067
Krey T, d’Alayer J, Kikuti C et al (2010) The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule. PLoS Pathog. doi:10.1371/journal.ppat.1000762.t002
Kwong P, Wyatt R, Robinson J et al (1998) Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648–659
Li B, Tsing S, Kosaka A et al (1996) Expression of human dopamine beta-hydroxylase in Drosophila Schneider 2 cells. Biochem J 313:57–64
Schneider I (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol 27:353–365
Brillet K, Pereira C, Wagner R (2010) Expression of membrane proteins in Drosophila melanogaster S2 cells: production and analysis of a EGFP-fused G protein-coupled receptor as a model. Methods Mol Biol 601:119–133
Bunch T, Grinblat Y, Goldstein L (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res 16:1043–1061
Chung Y, Keller E (1990) Positive and negative regulatory elements mediating transcription from the Drosophila melanogaster actin 5C distal promoter. Mol Cell Biol 10:6172–6180
Angelichio M, Beck J, Johansen H et al (1991) Comparison of several promoters and polyadenylation signals for use in heterologous gene expression in cultured Drosophila cells. Nucleic Acids Res 19:5037–5043
Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533
Arnau J, Lauritzen C, Petersen G et al (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48:1–13
Kimple M, Sondek J (2004) Overview of affinity tags for protein purification. Curr Protoc Protein Sci 36:9.9.1–9.9.19
Malhotra A (2009) Tagging for protein expression. Methods Enzymol 463:239–258
Iwaki T, Figuera M, Ploplis V et al (2003) Rapid selection of Drosophila S2 cells with the puromycin resistance gene. Biotechniques 35(482–484):486
Johansen H, van der Straten A, Sweet R et al (1989) Regulated expression at high copy number allows production of a growth-inhibitory oncogene product in Drosophila Schneider cells. Genes Dev 3:882–889
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Backovic, M., Krey, T. (2016). Stable Drosophila Cell Lines: An Alternative Approach to Exogenous Protein Expression. In: Murhammer, D. (eds) Baculovirus and Insect Cell Expression Protocols. Methods in Molecular Biology, vol 1350. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3043-2_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3043-2_17
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3042-5
Online ISBN: 978-1-4939-3043-2
eBook Packages: Springer Protocols