Abstract
The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421
Brondyk WH (2009) Selecting an appropriate method for expressing a recombinant protein. Methods Enzymol 463:131–147
Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575
Rosser MP, Xia W, Hartsell S et al (2005) Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expr Purif 40:237–243
Aricescu AR, Assenberg R, Bill RM et al (2006) Eukaryotic expression: developments for structural proteomics. Acta Crystallogr D Biol Crystallogr 62(Pt 10):1114–1124
Nettleship JE, Assenberg R, Diprose JM et al (2010) Recent advances in the production of proteins in insect and mammalian cells for structural biology. J Struct Biol 172:55–65
Vijayachandran LS, Viola C, Garzoni F et al (2011) Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells. J Struct Biol 175:198–208
Assenberg R, Wan PT, Geisse S et al (2013) Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol 23:393–402
Harrap KA (1972) The structure of nuclear polyhedrosis viruses. I. The inclusion body. Virology 50:114–123
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
Ayres MD, Howard SC, Kuzio J et al (1994) The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202:586–605
Fraser R, Heslop VR, Murray FE et al (1986) Ultrastructural studies of the portal transport of fat in chickens. Br J Exp Pathol 67:783–791
Smith GE, Fraser MJ, Summers MD (1983) Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene. J Virol 46:584–593
Roy P, Noad R (2012) Use of bacterial artificial chromosomes in baculovirus research and recombinant protein expression: current trends and future perspectives. ISRN Microbiol 2012:628797
Luckow VA, Lee SC, Barry GF et al (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67:4566–4579
Hitchman RB, Possee RD, Crombie AT et al (2010) Genetic modification of a baculovirus vector for increased expression in insect cells. Cell Biol Toxicol 26:57–68
Zhao Y, Chapman DA, Jones IM (2003) Improving baculovirus recombination. Nucleic Acids Res 31:E6–6
Abdulrahman W, Uhring M, Kolb-Cheynel I et al (2009) A set of baculovirus transfer vectors for screening of affinity tags and parallel expression strategies. Anal Biochem 385:383–385
Walls D, Loughran ST (2011) Tagging recombinant proteins to enhance solubility and aid purification. Methods Mol Biol 681:151–175
Waugh DS (2005) Making the most of affinity tags. Trends Biotechnol 23:316–320
Perrakis A, Musacchio A, Cusack S et al (2011) Investigating a macromolecular complex: the toolkit of methods. J Struct Biol 175:106–112
Berger I, Blanco AG, Boelens R et al (2011) Structural insights into transcription complexes. J Struct Biol 175:135–146
Sokolenko S, George S, Wagner A et al (2012) Co-expression vs. co-infection using baculovirus expression vectors in insect cell culture: Benefits and drawbacks. Biotechnol Adv 30:766–781
Wasilko DJ, Lee SE, Stutzman-Engwall KJ et al (2009) The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus. Protein Expr Purif 65:122–132
Lynn DE (2007) Routine maintenance and storage of lepidopteran insect cell lines and baculoviruses. Methods Mol Biol 388:187–208
Li MZ, Elledge SJ (2012) SLIC: a method for sequence- and ligation-independent cloning. Methods Mol Biol 852:51–59
Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256
Cha HJ, Gotoh T, Bentley WE (1997) Simplification of titer determination for recombinant baculovirus by green fluorescent protein marker. Biotechniques 23(782–4):786
Hopkins R, Esposito D (2009) A rapid method for titrating baculovirus stocks using the Sf-9 Easy Titer cell line. Biotechniques 47:785–788
Hitchman RB, Siaterli EA, Nixon CP et al (2007) Quantitative real-time PCR for rapid and accurate titration of recombinant baculovirus particles. Biotechnol Bioeng 96:810–814
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
Kool M, Voncken JW, van Lier FL et al (1991) Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties. Virology 183:739–746
Artimo P, Jonnalagedda M, Arnold K et al (2012) ExPASy: SIB bioinformatics resource portal, in. Nucleic Acids Res W597–603
Biegert A, Mayer C, Remmert M et al (2006) The MPI Bioinformatics Toolkit for protein sequence analysis. Nucleic Acids Res 34(Web Server issue):W335–W339
Mooij WT, Mitsiki E, Perrakis A (2009) ProteinCCD: enabling the design of protein truncation constructs for expression and crystallization experiments. Nucleic Acids Res 37(Web Server issue):W402–W405
Dosztanyi Z, Csizmok V, Tompa P et al (2005) IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21:3433–3434
Linding R, Russell RB, Neduva V et al (2003) GlobPlot: Exploring protein sequences for globularity and disorder. Nucleic Acids Res 31:3701–3708
Yang ZR, Thomson R, McNeil P et al (2005) RONN: the bio-basis function neural network technique applied to the detection of natively disordered regions in proteins. Bioinformatics 21:3369–3376
Prilusky J, Felder CE, Zeev-Ben-Mordehai T et al (2005) FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21:3435–3438
Weyer U, Possee RD (1991) A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells. J Gen Virol 72:2967–2974
Belyaev AS, Roy P (1993) Development of baculovirus triple and quadruple expression vectors: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells. Nucleic Acids Res 21:1219–1223
Berrow NS, Alderton D, Sainsbury S et al (2007) A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res 35:e45
Vijayachandran LS, Thimiri Govinda Raj DB, Edelweiss E et al (2013) Gene gymnastics: Synthetic biology for baculovirus expression vector system engineering. Bioengineered 4:279–287
Acknowledgments
This work was funded by the CNRS, the INSERM, the Université de Strasbourg (UdS), the Alsace Region, and the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01 Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI) and supported by national member subscriptions. It benefited from grants ANR-12-BSV8-0015-01 from the Agence Nationale de la Recherche, INCA-2008-041 from the Institut National du Cancer, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale (FRM) (ING20101221017), and La Ligue contre le Cancer (fellowship to LR).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Osz-Papai, J. et al. (2015). Insect Cells–Baculovirus System for the Production of Difficult to Express Proteins. In: García-Fruitós, E. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 1258. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2205-5_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2205-5_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2204-8
Online ISBN: 978-1-4939-2205-5
eBook Packages: Springer Protocols