Stable cell lines expressing baculovirus P35: Resistance to apoptosis and nutrient stress, and increased glycoprotein secretion
The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells byAutographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9P35AcV5-1 and Sf9P35AcV5-3) showed increased resistance to actinomycin D-induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9P35AcV5-1 and Sf9P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.
Key wordsInsect baculovirus AcMNPV Sf9 Apoptosis P35
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- Berry, S. J. Insect nucleic acids. Comprehensive Insect Physiol. Biochem. Pharmacol. 10:219–253; 1985.Google Scholar
- O'Reilly, D. R.; Miller, L. K.; Luckow, V. A. Baculovirus expression vectors, a laboratory manual. New York: W. H. Freeman; 1992.Google Scholar
- Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning. Cold Spring, Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.Google Scholar
- Summers, M. D.; Smith, G. E. A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Bulletin No. 1555; 1987.Google Scholar
- Wickham, T. J. Baculovirus-insect cell interactions in producing heterologous proteins: attachment, infection, and expression in different cell lines. Ithaca, NY: Department of Chemical Engineering, Cornell University; 1991:208.Google Scholar
- Zhou, Q.; Krebs, J. F.; Snipas, S. J.; Price, A.; Alnemri, E. S.; Tomaselli, K. J.; Salvesen, G. S. Interaction of the baculovirus anti-apoptotic protein p35 with caspaes. Specificity, kinetics, and characterization of the caspase/p35 complex. Biochemistry 37:10,757–10,765; 1998.Google Scholar