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Kinetic studies of recombinant rabies virus glycoprotein (RVGP) cDNA transcription and mRNA translation in Drosophila melanogaster S2 cell populations

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Abstract

Recombinant rabies virus glycoprotein (RVGP) was expressed in cell membranes of stably transfected Drosophila S2 cells using constitutive and inducible promoters. Although with quantitative differences of RVGP expression in both systems, the cDNA transcription, as evaluated by relative RVGP mRNA levels measured by qRT-PCR, sustained the amount of RVGP producing cells and the RVGP volumetric (ΠRVGP) productivity. At the transition to the stationary cell growth phase, once the cell culture slowed down its rate of multiplication, an accumulation of RVGP mRNA and RVGP was clearly observed in both cell populations. Nevertheless, cell cultures performed under sub-optimal temperatures indicated that an envisaged increase in the RVGP production is not only dependent on cell growth rate, but essentially on optimal cell metabolic state.

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Abbreviations

RVGP:

Rabies virus glycoprotein

qRT-PCR:

Quantitative reverse transcriptase-polimerase chain reaction

X:

Cell concentration (cell/mL)

Pc :

Rabies virus glycoprotein cell content (μg/107 cells)

μ:

Specific cell growth rate (h−1)

ΠRVGP :

Volumetric productivity (ng mL−1 h−1)

References

  • Astray RM, Augusto E, Yokomizo AY, Pereira CA (2008) Analytical approach for the extraction of recombinant membrane viral glycoprotein from stably transfected Drosophila melanogaster cells. Biotechnol J 3:98–103

    Google Scholar 

  • Augusto EF, Moraes AM, Piccoli RA, Barral MF, Suazo CA, Tonso A, Pereira CA (2010) Nomenclature and guideline to express the amount of a membrane protein synthesized in animal cells in view of bioprocess optimization and production monitoring. Biologicals 38:105–113

    Google Scholar 

  • Batista FR, Pereira CA, Mendonça RZ, Moraes AM (2008) Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells. Cytotechnology 57:11–22

    Google Scholar 

  • Batista FR, Moraes AM, Büntemeyer H, Noll T (2009) Influence of culture conditions on recombinant Drosophila melanogaster S2 cells producing rabies virus glycoprotein cultivated in serum-free medium. Biologicals 37:108–118

    Google Scholar 

  • Batista FR, Greco KN, Astray RM, Jorge SA, Augusto EF, Pereira CA, Mendonça RZ, Moraes AM (2011) Behavior of wild-type and transfected S2 cells cultured in two different media. Appl Biochem Biotechnol 163:1–13

    Google Scholar 

  • Brillet K, da Conceição MM, Pattus F, Pereira CA (2006) Bioprocess parameters of cell growth and human mu opioid receptor expression in recombinant Drosophila S2 cell cultures in a bioreactor. Bioprocess Biosyst Eng 28:291–293

    Google Scholar 

  • Butler M (2004) Animal cell culture and technology, 2nd edn. Taylor & Francis, New York

    Book  Google Scholar 

  • Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 162:156–159

    Article  CAS  Google Scholar 

  • Chung YT, Keller EB (1990a) Positive and negative regulatory elements mediating transcription from the Drosophila melanogaster actin 5C distal promoter. Mol Cell Biol 10:6172–6180

    CAS  Google Scholar 

  • Chung YT, Keller EB (1990b) Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter. Mol Cell Biol 10:206–216

    CAS  Google Scholar 

  • Dietzschold B (1977) Oligosaccharides of the glycoprotein of rabies virus. J Virol 23:286–293

    CAS  Google Scholar 

  • Dinnis DM, James DC (2005) Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature? Biotechnol Bioeng 91:180–189

    Article  CAS  Google Scholar 

  • Doyle A, Griffths JB (1998) Cell and tissue culture laboratory procedures in biotechnology. Wiley, New York

    Google Scholar 

  • Fujii-Taira I, Yamaguchi S, Iijima R, Natori S, Homma KJ (2009) Suppression of the ecdysteroid-triggered growth arrest by a novel Drosophila membrane steroid binding protein. FEBS Lett 583:655–660

    Google Scholar 

  • Galesi ALL, Pereira CA, Moraes AM (2007) Culture of transgenic Drosophila melanogaster Schneider 2 cells in serum-free media based on TC100 basal medium. Biotechnol J 2:1399–1407

    Article  CAS  Google Scholar 

  • Galesi AL, Aguiar MA, Astray RM, Augusto EF, Moraes AM et al (2008) Growth of recombinant Drosophila melanogaster Schneider 2 cells producing rabies virus glycoprotein in bioreactor employing serum-free medium. Cytotechnology 57:73–81

    Google Scholar 

  • Gaudin Y (1997) Folding of rabies virus glycoprotein: epitope acquisition and interaction with endoplasmic reticulum chaperones. J Virol 71:3742–3750

    CAS  Google Scholar 

  • Gaudin Y, Ruigrok RWH, Tuffereau C (1992) Rabies virus glycoprotein is a trimer. Virology 187:627–632

    Article  CAS  Google Scholar 

  • Jennings NS, Smethurst PA, Knight CG, O’Connor MN, Joutsi-Korhonen L, Stafford P, Stephens J, Garner SF, Harmer IJ, Farndale RW, Watkins NA, Ouwehand WH (2006) Production of calmodulin-tagged proteins in Drosophila Schneider S2 cells: a novel system for antigen production and phage antibody isolation. J Immunol Methods 316:75–83

    Google Scholar 

  • Johansson DX, Drakenberg K, Hopmann KH, Schmidt A, Yari F, Hinkula J, Persson MA (2007) Efficient expression of recombinant human monoclonal antibodies in Drosophila S2 cells. J Immunol Methods 318:37–46

    Google Scholar 

  • Jorge SA, Hera C, Spina AM, Moreira RC, Pinho JR, Menck CF (1996) Expression of the hepatitis B virus surface antigen in mammalian cells using an Epstein–Barr-virus-derived vector. App Microbiol Biotechnol 46:533–537

    Google Scholar 

  • Kim YK, Shin HS, Tomiya N, Lee YC, Betenbaugh MJ, Cha HJ (2005) Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells. Biotechnol Bioeng 20:452–461

    Google Scholar 

  • Lee JM, Jeon HB, Sohn BH, Chung IS (2007) Functional expression of recombinant canstatin in stably transformed Drosophila melanogaster S2 cells. Protein Expr Purif 52:258–264

  • Lemos MA, Santos AS, Astray RM, Pereira CA, Jorge SA (2009) Rabies virus glycoprotein expression in Drosophila S2 cells. I. Design of expression/selection vectors, subpopulations selection and influence of sodium butyrate and culture medium on protein expression. J Biotechnol 143:103–110

    Google Scholar 

  • Li HC, Huang CC, Chen SF, Chou MY (2005) Assembly of homotrimeric type XXI minicollagen by coexpression of prolyl 4-hydroxylase in stably transfected Drosophila melanogaster S2 cells. Biochem Biophys Res Commun 336:375–385

    Google Scholar 

  • Lim HJ, Kim YK, Hwang DS, Cha HJ (2004) Expression of functional human transferrin in stably transfected Drosophila S2 cells. Biotechnol Prog 20:1192–1197

    Google Scholar 

  • Mallender WD, Yager D, Onstead L, Nichols MR, Eckman C, Sambamurti K, Kopcho LM, Marcinkeviciene J, Copeland RA, Rosenberry TL (2001) Characterization of recombinant, soluble beta-secretase from an insect cell expression system. Mol Pharmacol 59:619–626

    Google Scholar 

  • McCarrol L, King LA (1997) Stable insect cell cultures for recombinant protein production. Curr Opin Biotechnol 8:590–594

    Article  Google Scholar 

  • Mendonça RZ, Greco KN, Sousa AP, Moraes RH, Astray RM, Pereira CA (2008) Enhancing effect of a protein from Lonomia obliqua hemolymph on recombinant protein production. Cytotechnology 57:83–91

    Google Scholar 

  • Mendonça RZ, Greco KN, Moraes RH, Astray RM, Barral M (2009) Study of kinetic parameters for the production of recombinant rabies virus glycoprotein. Cytotechnology 60:143–151

    Google Scholar 

  • Moraes AM, Jorge SA, Astray RM, Suazo CA, Calderón Riquelme CE, Augusto EF, Tonso A, Pamboukian MM, Piccoli RA, Barral MF, Pereira CA (2012) Drosophila melanogaster S2 cells for expression of heterologous genes: from gene cloning to bioprocess development. Biotechnol Adv 30:613–628

  • Nie L, Wu G, Zhang W (2006) Correlation between mRNA and protein abundance in Desulfovibrio vulgaris: a multiple regression to identify sources of variations. Biochem Biophys Res Commun 339:603–610

    Article  CAS  Google Scholar 

  • Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 3:1559–1582

    Article  Google Scholar 

  • Palomares LA, Estrada-Moncada S, Ramírez OT (2004) Production of recombinant proteins: challenges and solutions. In: Balbás P, Lorence A (eds) Recombinant gene expression: reviews and protocols. Humana Press, Totowa, pp 15–52

    Chapter  Google Scholar 

  • Perrin P, Thibodeau L, Sureau P (1985) Rabies immunosome (subunit vaccine) structure and immunogenicity. Pre- and post-exposure protection studies. Vaccine 3:325–332

    Article  CAS  Google Scholar 

  • Perrin P, Lafon M, Sureau P (1996) Enzyme linked immuno-sorbent assay (ELISA) for the determination of glycoprotein content of rabies vaccines. In: Meslin FX, Kaplan MM, Koprowski H (eds) Laboratory techniques in rabies. WHO, Geneva, pp 383–388

    Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007

    Article  Google Scholar 

  • Scotter AJ, Kuntz DA, Saul M, Graham LA, Davies PL, Rose DR (2006) Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells. Protein Expr Purif 47:374–383

    Google Scholar 

  • Southon A, Burke R, Norgate M, Batterham P, Camakaris J (2004) Copper homoeostasis in Drosophila melanogaster S2 cells. Biochem J 383:303–309

    Google Scholar 

  • Swiech K, Rossi N, Astray RM, Suazo CA (2008a) Enhanced production of recombinant rabies virus glycoprotein (rRVGP) by Drosophila melanogaster S2 cells through control of culture conditions. Cytotechnology 57:67–72

    Google Scholar 

  • Swiech K, Rossi N, Silva BG, Jorge SA, Astray RM, Suazo CA (2008b) Bioreactor culture of recombinant Drosophila melanogaster S2 cells: characterization of metabolic features related to cell growth and production of the rabies virus glycoprotein. Cytotechnology 57:61–66

    Google Scholar 

  • Swiech K, da Silva CS, Arantes MK, dos Santos AS, Astray RM, Pereira CA, Suazo CA (2008c) Characterization of growth and metabolism of Drosophila melanogaster cells transfected with the rabies virus glycoprotein gene. Biotechnol Appl Biochem 49:41–49

    Google Scholar 

  • Ventini DC, Astray RM, Lemos MA, Jorge SA, Riquelme CC, Suazo CA, Tonso A, Pereira CA (2010) Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter. J Biotechnol 146:169–172

    Google Scholar 

  • Wagner RR (1991) Rhabdoviridae and their replication. In: Fields BN, Knipe DM (eds) Fundamental virology. Raven Press, New York, pp 493–496

    Google Scholar 

  • Washburn MP, Koller A, Oshiro G (2003) Protein pathway and complex clustering of correlated mRNA and protein expression analyses in Saccharomyces cerevisiae. Proc Nat Acad Sci USA 100:3107–3112

    Article  CAS  Google Scholar 

  • World Health Organization (2007) Weekly epidemiological report, vol 82. World Health Organization, Geneva, pp 425–436

    Google Scholar 

  • Yokomizo AY, Jorge SA, Astray RM, Fernandes I, Ribeiro OG, Horton DS, Tonso A, Tordo N, Pereira CA (2007) Rabies virus glycoprotein expression in Drosophila S2 cells. I. Functional recombinant protein in stable co-transfected cell line. Biotechnol J 2:102–109

    Google Scholar 

  • Yoon SK, Hong JK, Choo SH, Song JY, Park HW, Lee GM (2006) Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production. J Biotechnol 122:463–472

    Google Scholar 

  • Zhang F, Ma W, Zhang L, Aasa-Chapman M, Zhang H (2007) Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected Drosophila cell line. Virol J 4:17

    Google Scholar 

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Acknowledgments

We thank Dr. Jorge Mário da Costa Ferreira Jr., Laboratório de Imunoquímica, Instituto Butantan, São Paulo, SP, Brazil, for flow cytometry analysis. This work was financially supported by grants from FAPESP (2011/08331-0), CNPq and Butantan Foundation. C. A. Pereira is recipient of a CNPq 1A senior fellowship.

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Author notes

  1. A. Y. Yokomizo

    Present address: Cristália Produtos Químicos e Farmacêuticos Ltda, Itapira, Brazil

Authors and Affiliations

  1. Laboratório de Imunologia Viral, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, 05503-900, Brazil

    R. M. Astray, S. A. C. Jorge, M. A. N. Lemos, A. Y. Yokomizo, V. L. L. Boldorini, A. L. P. Puglia & C. A. Pereira

  2. Laboratório de Imunogenética, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, 05503-900, Brazil

    O. G. Ribeiro

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Astray, R.M., Jorge, S.A.C., Lemos, M.A.N. et al. Kinetic studies of recombinant rabies virus glycoprotein (RVGP) cDNA transcription and mRNA translation in Drosophila melanogaster S2 cell populations. Cytotechnology 65, 829–838 (2013). https://doi.org/10.1007/s10616-012-9522-6

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  • DOI: https://doi.org/10.1007/s10616-012-9522-6

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