Abstract
Infecting insect cells with a baculovirus expression vector system (BEVS) is an increasingly popular method for the production of recombinant proteins. Due to the lytic nature of the system, however, determining the optimal harvest time is critical for maximizing protein yield. We found that measuring the change in average diameter during the progress of infection with an automated cell analysis system (Cedex HiRes, Innovatis AG) could be used to determine the time of maximum protein production and, thus, optimal harvest time. As a model system, we use insect cells infected with a baculovirus expressing enhanced green fluorescent protein (EGFP). We infected two commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-TN-5B1-4 (Hi5) with an Autographa californica nuclear polyhedrosis virus (AcNPV) encoding EGFP at various multiplicities of infection (MOI). We monitored the progress of infection with regard to viability, viable cell density and change in average cell diameter with a Cedex HiRes analyzer and compared the results to the EGFP produced. Peak protein production was reached one to two days after the point of maximum average diameter in all conditions. Thus, optimal harvest time could be determined by monitoring the change in average cell diameter during the course of an infection of a cell culture.
Similar content being viewed by others
Abbreviations
- BEVS:
-
baculovirus expression vector system
- Sf-9:
-
Spodoptera frugiperda
- Hi5:
-
Trichoplusia ni BTI-TN-5B1-4
- AcNPV:
-
Autographa californica nuclear polyhedrosis virus
- EGFP:
-
enhanced green fluorescent protein
- MOI:
-
multiplicity of infection
- Pfu:
-
plaque forming units
- RFI:
-
relative fluorescence intensity
- PI:
-
post-infection
References
Bédard C, Tom R, Kamen AA (1993) Growth, nutrient consumption, and end-product accumulation in Sf-9 and BTI-EAA insect cell cultures: insights into growth limitation and metabolism. Biotechnol Prog 9:615–624
Bonning BC, Roelvink PW, Vlak JM, Possee RD, Hammock BD (1994) Superior expression of juvenile hormone esterase and β galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters. J Gen Virol 75:1551–1556
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805
Cormack BP, Bertram G, Egerton M, Gow NA, Falkow S, Brown AJ (1997) Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiol 143:303–311
Hensler WT, Agathos SN (1994) Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells. Cytotechnology 15:177–186
Hill-Perkins M, Possee R (1990) A baculovirus expression vector derived from the basic protein promoter of Autographa californica nuclear polyhedrosis virus. J Gen Virol 71:971–976
Ho Y, Lo H-R, Lee T-C, Wu CPY, Chao Y-C (2004) Enhancement of correct protein folding in vivo by a non-lytic baculovirus. Biochem J 382:695–702
Ikonomou L, Schneider YJ, Agathos SN (2003) Insect cell culture for industrial production of recombinant proteins. Appl Microbiol Biotechnol 62:1–20
Jain D, Ramasubramanyan K, Gould S, Seamans C, Wang S, Lenny A, Silberklang M (1991) Production of antistasin using the baculovirus expression system. In: Hatch R, Gooche C, Moreira A, Alroy Y (eds) Expression systems and processes for rDNA products. ACS Symposium Series, No. 477. Amer Chem Soc, Washington pp 97–110
Jorio H, Tran R, Kamen A (2006) Stability of serum-free and purified baculovirus stocks under various storage conditions. Biotechnol Prog 22:319–325
Kamen AA, Bedard Ch, Tom R, Perret S, Jardin B (1996) On-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures. Biotechnol Bioeng 50:36–48
Kost TA, Ignar DM, Clay WC, Andrews J, Leray JD, Overton L, Hoffman CR, Kilpatrick KE, Ellis B, Emerson DL (1997) Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG in the baculovirus expression system. Gene 190:139–144
Lawrie AM, King AL, Ogden JE (1995) High level synthesis and secretion of human urokinase using a late gene promoter of the Autographa californica nuclear polyhedrosis virus. J Biotechnol 39:1–8
Li X, Zhao X, Fang Y, Jiang X, Duong T, Fan C, Huang C-C, Kain SR (1998) Generation of destabilized green fluorescent protein as a transcription reporter. J Bio Chem 273:34970–34975
March JC, Rao G, Bentley WE (2003) Biotechnological applications of green fluorescent protein. Appl Microbiol Biotechnol 62:303–315
Monsma SA, Scott M (1997) BacVector-3000: an engineered baculovirus designed for greater protein stability. Innovations 16–19
Õhman L, Alarcon M, Ljunggren J, Ramqvist A, Häggstrom L (1996) Glutamine is not an essential amino acid for Sf-9 insect cells. Biotechnol Lett 18:765–770
Olejnik AM, Czaczyk K, Marecik R, Grajek W (2004) Monitoring the progress of infection and recombinant protein production in insect cell cultures using intracellular ATP measurement. Appl Microbiol Biotechnol 65:18–24
Palomares LA, Ramírez OT (1996) The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture. Cytotechnology 22:225–237
Palomares LA, Pedroza JC, Ramírez OT (2001) Cell size as a tool to predict the production of recombinant protein by the insect-cell baculovirus expression system. Biotechnol Lett 23:359–364
Rhiel M, Mitchell-Logean CN, Murhammer DW (1997) Comparison of Trichoplusia ni BTI-Tn5B1-4 (High Five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures. Biotechnol Bioeng 55:909–920
Rosinski M, Reid S, Nielsen LK (2000) Osmolarity effects on observed insect cell size after baculovirus infection are avoided using growth medium for sample dilution. Biotechnol Prog 16:782–785
Suzuki T, Kanaya T, Okazaki H, Ogawa K, Usami A, Watanabe H, Kadono-Okuda K, Yamakawa M, Sato H, Mori H, Takahashi S, Oda K (1997) Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene. J Gen Virol 78:142–152
Taticek RA, Choi C, Phan SE, Palomares LA, Shuler ML (2001) Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture. Biotechnol Prog 17:676–684
Yang JD, Gecik P, Collins A, Czarnecki S, Hsu HH, Lasdun A, Sundaram R, Muthukumar G, Silberklang M (1996a) Rational scale-up of a baculovirus-insect cell batch process based on medium nutritional depth. Biotechnol Bioeng 52:696–706
Yang TT, Cheng L, Kain SR (1996b) Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res 24:4592–4593
Acknowledgements
The authors would like to thank Per-Erik Strömstedt and Philippe Cronet for their unfailing support of this project; Catherine Heddle for very kindly providing the recombinant EGFP-AcNPV baculovirus; and Thomas Becker for critically reading the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sander, L., Harrysson, A. Using cell size kinetics to determine optimal harvest time for Spodoptera frugiperda and Trichoplusia ni BTI-TN-5B1-4 cells infected with a baculovirus expression vector system expressing enhanced green fluorescent protein. Cytotechnology 54, 35–48 (2007). https://doi.org/10.1007/s10616-007-9064-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-007-9064-5