Abstract
Baculovirus expression vector system (BEVS) in host insect cells is a powerful technology to produce recombinant proteins, as well as virus-like particles (VLP). However, BEVS is based on baculovirus infection, which limits the recombinant protein production by inducing insect cell death. Herein a new strategy to enhance cell life span and to increase recombinant protein production was developed. As baculovirus infection induces cellular oxidative stress, the ability of several antioxidants to inhibit cell death was tested during infection. The production of rotavirus structural proteins was used as model to analyse this new strategy. We found that only catalase is able to partially prevent cell death triggered by baculovirus infection and to inhibit lipid peroxidation. An increase in recombinant protein production was coupled with the partial cell death inhibition. In summary, the addition of catalase is a promising strategy to improve recombinant protein production in BEVS, by delaying insect cell death.
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Abbreviations
- Cat:
-
Catalase
- Sf-9:
-
Spodoptera frugiperda
- BEVS:
-
Baculovirus expression vector system
- DiOC6(3):
-
3,3′Dihexyloxacarbocyanine iodide
- ΔΨm :
-
Mitochondrial transmembrane potential
- MMP:
-
Mitochondrial membrane permeabilization
- PI:
-
Propidium iodine
- hpi:
-
Hours post-infection
- VLP:
-
Virus-like particles
- MOI:
-
Multiplitity of infection
- CCI:
-
Cell concentration at infection
References
al-Rubeai M, Singh RP (1998) Apoptosis in cell culture. Curr Opin Biotechnol 9(2):152–156
Nilou A, Betenbaugh MJ (2004) Life and death in mammalian cell culture: strategies for apoptosis inhibition. Trends Biotechnol 22(4):174–180
Jarvis DL (2003) Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production. Virology 310(1):1–7
Cruz PE et al (1998) Optimization of the production of virus-like particles in insect cells. Biotechnol Bioeng 60(4):408–418
Kroemer G, Reed JC (2000) Mitochondrial control of cell death. Nat Med 6(5):513–519
Lenaz G et al (1999) Mitochondria, oxidative stress, and antioxidant defences. Acta Biochim Pol 46(1):1–21
Wang Y, Oberley LW, Murhammer DW (2001) Antioxidant defense systems of two lipidopteran insect cell lines. Free Radic Biol Med 30(11):1254–1262
Wang Y, Oberley LW, Murhammer DW (2001) Evidence of oxidative stress following the viral infection of two lepidopteran insect cell lines. Free Radic Biol Med 31(11):1448–1455
Wang Y et al (2004) Effect of expression of manganese superoxide dismutase in baculovirus-infected insect cells. Appl Biochem Biotechnol 119(2):181–193
SaarinenMA, Murhammer DW (2002) The response of virally infected insect cells to dissolved oxygen concentration: recombinant protein production and oxidative damage. Biotechnol Bioeng 81(1):106–114
Clem RJ, Fechheimer M, Miller LK (1991) Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254(5036):1388–1390
Sahdev S et al (2003) Baculoviral p35 inhibits oxidant-induced activation of mitochondrial apoptotic pathway. Biochem Biophys Res Commun 307:483–490
Sah NK et al (1999) The baculovirus antiapoptotic p35 gene also functions via an oxidant dependent pathway. Proc Natl Acad Sci 96:4838–4843
Vieira HLA et al (2001) The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal. Oncogene 20(32):4305–4316
Mohan M et al (2003) Antioxidants prevent UV-induced apoptosis by inhibiting mitochondrial cytochrome c release and caspase activation in Spodoptera frugiperda (Sf-9) cells. Cell Biol Int 27:483–490
Crawford SE et al (1999) Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles. J Virol 73(6):4813–4822
Vieira HLA et al (2005) Triple layered rotavirus VLP assembly: kinetics of vector replication, mRNA stability and recombinant protein production. J Biotechnol 120:72–82
Mena J et al (2003) Titration of non-occluded baculovirus using a cell viability assay. BioTechniques 34:260–264
BuegeJA, Aust SD (1978) Microsomal lipid peroxidation. Meth Enzymol 52:302–310
Acknowledgments
This work was supported by European Commission (QLRT200101249), the Portuguese Fundação para a Ciência e Tecnologia (POCTI/BIO/55975/2004) and the Portuguese Fundação para a Ciência e Tecnologia, for HLAV’s SFRH/BPD/14575/2003 fellowship.
The authors express their gratitude to Prof. Polly Roy from the London School of Hygiene and Tropical Medicine, England, for providing the multigene baculovirus; to Dr. J. Cohen, Dr. D. Poncet and Dr. A. Charpilienne from CNRS-INRA, France, for providing the polyclonal anti-Rotavirus serum and the baculovirus coding for VP6; and to Dr. Matthias Haury from Instituto Gulbenkian para a Ciência, Portugal, for the help with the flow cytometry measurements.
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Vieira, H.L.A., Pereira, A.C.P., Carrondo, M.J.T. et al. Catalase effect on cell death for the improvement of recombinant protein production in baculovirus-insect cell system. Bioprocess Biosyst Eng 29, 409–414 (2006). https://doi.org/10.1007/s00449-006-0093-9
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DOI: https://doi.org/10.1007/s00449-006-0093-9