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Insoluble Proteins pp 181–205Cite as

Insect Cells–Baculovirus System for the Production of Difficult to Express Proteins

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1258))

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.

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References

  1. Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421

    Article  CAS  PubMed  Google Scholar 

  2. Brondyk WH (2009) Selecting an appropriate method for expressing a recombinant protein. Methods Enzymol 463:131–147

    Article  CAS  PubMed  Google Scholar 

  3. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. 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

    Article  CAS  PubMed  Google Scholar 

  5. 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

    Article  CAS  PubMed  Google Scholar 

  6. 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

    Article  CAS  PubMed  Google Scholar 

  7. 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

    Article  CAS  PubMed  Google Scholar 

  8. 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

    Article  CAS  PubMed  Google Scholar 

  9. Harrap KA (1972) The structure of nuclear polyhedrosis viruses. I. The inclusion body. Virology 50:114–123

    Article  CAS  PubMed  Google Scholar 

  10. 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

    CAS  PubMed Central  PubMed  Google Scholar 

  11. Ayres MD, Howard SC, Kuzio J et al (1994) The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202:586–605

    Article  CAS  PubMed  Google Scholar 

  12. 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

    CAS  PubMed Central  PubMed  Google Scholar 

  13. 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

    CAS  PubMed Central  PubMed  Google Scholar 

  14. 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

    Article  PubMed Central  PubMed  Google Scholar 

  15. 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

    CAS  PubMed Central  PubMed  Google Scholar 

  16. 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

    Article  CAS  PubMed  Google Scholar 

  17. Zhao Y, Chapman DA, Jones IM (2003) Improving baculovirus recombination. Nucleic Acids Res 31:E6–6

    Article  PubMed Central  PubMed  Google Scholar 

  18. 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

    Article  CAS  PubMed  Google Scholar 

  19. Walls D, Loughran ST (2011) Tagging recombinant proteins to enhance solubility and aid purification. Methods Mol Biol 681:151–175

    Article  CAS  PubMed  Google Scholar 

  20. Waugh DS (2005) Making the most of affinity tags. Trends Biotechnol 23:316–320

    Article  CAS  PubMed  Google Scholar 

  21. Perrakis A, Musacchio A, Cusack S et al (2011) Investigating a macromolecular complex: the toolkit of methods. J Struct Biol 175:106–112

    Article  CAS  PubMed  Google Scholar 

  22. Berger I, Blanco AG, Boelens R et al (2011) Structural insights into transcription complexes. J Struct Biol 175:135–146

    Article  CAS  PubMed  Google Scholar 

  23. 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

    Article  CAS  PubMed  Google Scholar 

  24. 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

    Article  CAS  PubMed  Google Scholar 

  25. Lynn DE (2007) Routine maintenance and storage of lepidopteran insect cell lines and baculoviruses. Methods Mol Biol 388:187–208

    Article  PubMed  Google Scholar 

  26. Li MZ, Elledge SJ (2012) SLIC: a method for sequence- and ligation-independent cloning. Methods Mol Biol 852:51–59

    Article  CAS  PubMed  Google Scholar 

  27. Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256

    Article  CAS  PubMed  Google Scholar 

  28. Cha HJ, Gotoh T, Bentley WE (1997) Simplification of titer determination for recombinant baculovirus by green fluorescent protein marker. Biotechniques 23(782–4):786

    Google Scholar 

  29. Hopkins R, Esposito D (2009) A rapid method for titrating baculovirus stocks using the Sf-9 Easy Titer cell line. Biotechniques 47:785–788

    Article  CAS  PubMed  Google Scholar 

  30. 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

    Article  CAS  PubMed  Google Scholar 

  31. 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

    Article  CAS  PubMed  Google Scholar 

  32. 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

    Article  CAS  PubMed  Google Scholar 

  33. Artimo P, Jonnalagedda M, Arnold K et al (2012) ExPASy: SIB bioinformatics resource portal, in. Nucleic Acids Res W597–603

    Google Scholar 

  34. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. 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

    Article  CAS  PubMed  Google Scholar 

  37. Linding R, Russell RB, Neduva V et al (2003) GlobPlot: Exploring protein sequences for globularity and disorder. Nucleic Acids Res 31:3701–3708

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. 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

    Article  CAS  PubMed  Google Scholar 

  39. 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

    Article  CAS  PubMed  Google Scholar 

  40. 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

    Article  CAS  PubMed  Google Scholar 

  41. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. 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

    Article  PubMed Central  PubMed  Google Scholar 

  43. 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

    Article  PubMed Central  PubMed  Google Scholar 

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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).

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Authors and Affiliations

  1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS, BP 163, Illkirch, Cedex 67404, France

    Judit Osz-Papai, Laura Radu, Wassim Abdulrahman, Isabelle Kolb-Cheynel, Nathalie Troffer-Charlier, Catherine Birck & Arnaud Poterszman

Authors
  1. Judit Osz-Papai
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  2. Laura Radu
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  3. Wassim Abdulrahman
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  4. Isabelle Kolb-Cheynel
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  5. Nathalie Troffer-Charlier
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  6. Catherine Birck
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  7. Arnaud Poterszman
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Correspondence to Arnaud Poterszman .

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Editors and Affiliations

  1. Departament de Genètica i de Microbiologia, CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) and Universitat Autònoma de Barcelona, Bellaterra (Barcelona), Spain

    Elena García-Fruitós

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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

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  • DOI: https://doi.org/10.1007/978-1-4939-2205-5_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2204-8

  • Online ISBN: 978-1-4939-2205-5

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