Abstract
Drosophila melanogaster Schneider 2 (S2) cells have been increasingly used as a suitable expression system for the production of different recombinant proteins, and the employment of bioreactors for large-scale culture is an important tool for this purpose. In this work, Drosophila S2 cells producing the rabies virus glycoprotein RVGP were cultivated in bioreactor, employing a serum-free medium, aiming an improvement in cell growth and in glycoprotein production. To overcome cell growth limitation commonly observed in stirred flasks, different experiments in bioreactor were performed, in which some system modifications were carried out to attain the desired goal. The study showed that this cell line is considerably sensitive to hydrodynamic forces, and a high cell density (about 16.0 × 106 cells mL−1) was only obtained when Pluronic F68® percentage was increased to 0.6% (w/v). Despite ammonium concentration affected RVGP production, and also cell growth, an elevated amount of the target protein was obtained, attaining 563 ng 10−7 cells.
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Astray RM, Augusto EFP, Yokomizo AY, Pereira CA (2008) Analytical approach for extraction of recombinant membrane viral glycoprotein from stably transfected Drosophila melanogaster cells. Biotechnol J 3(1):98–103
Augusto EFP, Oliveira MS (2001) Processos com Células Animais. In: Biotecnologia Industrial, 1st edn, vol 3. Edgard Blücher, São Paulo
Bachmann AS, Corpuz G, Hareld WP, Wang G, Coller BA (2004) A simple method for the rapid purification of copia virus-like particles from Drosophila Schneider 2 cells. J Virol Methods 115:159–165
Banks DJ, Hua G, Adang M (2003) Cloning of a Heliothis virescens 110 kDa aminopeptidase N and expression in Drosophila S2 cells. Insect Biochem Mol 33:499–508
Bovo R, Galesi ALL, Jorge SAC, Piccoli RAM, Moraes AM, Pereira CA, Augusto EFP (2008) Kinetic response of a Drosophila melanogaster cell line to different medium formulations and culture conditions. Submitted to Cytotechnology
Chisti Y (2000) Animal-cell damage in sparged bioreactors. Trends Biotechnol 18:420–432
Deml L, Wolf H, Wagner R (1999a) High level expression of hepatitis B virus surface antigen in stably transfected Drosophila Schneider-2 cells. J Virol Methods 79:191–203
Deml L, Schirmbeck R, Reimann J Wolf H, Wagner R (1999b) Purification and characterization of hepatitis B virus surface antigen particles produced in Drosophila Schneider-2 cells. J Virol Methods 79:205–217
Freshney RI (1994) Culture of animal cells: a manual of basic technique, 3rd edn. Wiley-Liss, New York
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(11):1399–1407
Gaudin Y (1997) Folding of rabies virus glycoprotein: epitope acquisition and interaction with endoplasmic reticulum chaperones. J Virol 71(5):3742–3750
Gaudin Y, Ruigrok RWH, Knossow M, Flamand A (1993) Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion. J Virol 67(3):1365–1372
Gibson KR, Vanek PG, Kaloss WD, Collier GB, Connaughton JF, Angelichio M, Livi GP, Fleming PJ (1993) Expression of dopamine-β-hydroxylase in Drosophila Schneider 2 cells – evidence for a mechanism of membrane-binding other than uncleaved signal peptide. J Biol Chem 268:9490–9495
Ikonomou L, Schneider YJ, Agathos SN (2003) Insect cell culture for industrial production of recombinant proteins. Appl Microbiol Biotechnol 62:1–20
Jeon HK, Chang KH, Kim KI, Chung IK (2003) Functional expression of recombinant tumstatin in stably transformed Drosophila melanogaster S2 cells. Biotechnol Lett 25:185–189
Kirkpatrick RB, Ganguly S, Angelichio M, Griego S, Shatzman A, Silverman C, Rosenberg M (1995) Heavy chain dimers as well as complete antibodies are efficiently formed and secreted from Drosophila via a BiP-mediated pathway. J Biol Chem 270(34):19800–19805
Lee JM, Park JH, Park JO, Chang KH, Chung IS (2000) Expression of recombinant erythropoietin in stably transformed Drosophila melanogaster S2 cells. In Vitro Cell Dev Biol Anim 36:348–350
Maillard AP, Gaudin Y (2002) Rabies virus glycoprotein can fold in two alternative, antigenically distinct conformations depending on membrane-anchor type. J Gen Virol 83:1465–1476
Millar NS, Baylis HA, Reaper C, Bunting R, Mason WT, Sattelle DB (1995) Functional expression of a cloned Drosophila muscarinic acetylcholine receptor in a stable Drosophila cell line. J Exp Biol 198:1843–1850
Palomares LA, González M, Ramírez OT (2000) Evidence of Pluronic F68® direct interaction with insect cells: impact on shear protection, recombinant protein, and baculovirus production. Enzyme Microb Technol 26:324–331
Perret BG, Wagner R, Lecat S, Brillet K, Rabut G, Bucher B, Pattus F (2003) Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of g-protein coupled receptors. Protein Expr Purif 31:123–132
Swiech K, da Silva CS, Arantes MK, dos Santos AS, Astray RM, Pereira CA, Suazo CAT (2008) Characterization of growth and metabolism of Drosophila melanogaster cells transfected with the rabies-virus glycoprotein gene. Biotechnol Appl Biochem 49:41–49
Torfs H, Shariatmadari R, Guerrero F, Parmentier M, Poels J, Van Poyer W, Swinnen E, De Loof A, Akerman K, Van den Broeck J (2000) Characterization of a receptor for insect tachykinin-like peptide agonists by functional expression in a stable Drosophila Schneider 2 cell line. J Neurochem 74:2182–2189
Tota MR, Xu L, Sirotina A, Strader CD, Graziano MP (1995) Interaction of [fluorescein-Trp25]glucagon with the human glucagon receptor expressed in Drosophila Schneider-2 cells. J Biol Chem 270:26466–26472
Valle MA, Kester MB, Burns AL, Marx SJ, Spiegel AM, Shiloach J (2001) Production and purification of human menin from Drosophila melanogaster S2 cells using stirred tank reactor. Cytotechnology 35:127–135
van der Pol L, Tramper J (1998) Shear sensitivity of animal cells from a culture-medium perspective. Trends Biotechnol 16:323–328
Van den Broeck J, Vulsteke V, Huybrechts R, De Loof A (1995) Characterization of a cloned locust tyramine receptor cDNA by functional expression in permanently transformed Drosophila S2 cells. J Neurochem 64:2387–2395
Yang JD, Gecik P, Collins A, Czarnecki S, Hsu HH, Lasdun A, Sundaram R, Muthukumar G, Silberklang M (1996) Rational scale-up of a baculovirus-insect cell batch process based on medium nutritional depth. Biotechnol Bioeng 52:696–706
Yokomizo AY, Jorge SAC, Astray RM, Fernandes I, Filho ORG, Horton DSPQ, Tonso A, Pereira CA (2007) Rabies virus glycoprotein expression in Drosophila S2 cells. I. Functional recombinant protein in stable co-transfected cell line. Biotechnol J 2(1):102–109
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This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil), Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP Grant Nos. 02/09482-3 and 03/00675-6, São Paulo, Brazil), Instituto Butantan (São Paulo, Brazil) and Instituto de Pesquisas Tecnológicas (São Paulo, Brazil).
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Ângela M. Moraes is recipient of a CNPq fellowship.
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Galesi, A.L.L., Aguiar, M.A., Astray, R.M. et al. Growth of recombinant Drosophila melanogaster Schneider 2 cells producing rabies virus glycoprotein in bioreactor employing serum-free medium. Cytotechnology 57, 73–81 (2008). https://doi.org/10.1007/s10616-008-9139-y
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DOI: https://doi.org/10.1007/s10616-008-9139-y