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Stable cell lines expressing baculovirus P35: resistance to apoptosis and nutrient stress, and increased glycoprotein
1Boyce Thompson Institute at Cornell University, Ithaca, New York 14853-1801, USA.
Abstract:
The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells by Autographa 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.
Insights
Constitutive expression of baculovirus P35 protein enhances cell resistance to apoptosis and nutrient deprivation. This improves recombinant protein production in insect cells by prolonging the infection cycle.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- Baculovirus P35 protein inhibits apoptosis.
- Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infection induces apoptosis in Sf21 cells.
- P35 protein's role in cellular stress response and protein production is not fully understood.
Purpose of the Study:
- To investigate the effects of constitutive P35 expression on cellular stress response.
- To evaluate the impact of P35 on protein production in AcMNPV-infected cells.
Main Methods:
- Generated Sf9 cell lines with constitutive expression of AcMNPV P35 using a double selection technique.
- Assessed apoptosis resistance to actinomycin D and nutrient deprivation.
- Measured baculovirus-mediated expression of intracellular and secreted proteins.
Main Results:
- P35-expressing cell lines demonstrated increased resistance to actinomycin D-induced apoptosis and nutrient deprivation.
- Recombinant secreted glycoprotein expression in P35-expressing cells exceeded parental Sf9 cells.
- Protein secretion levels were comparable to high-expression cell lines (Tn5B1-4).
Conclusions:
- Constitutive P35 expression confers enhanced cellular resistance to apoptosis and stress.
- P35 expression improves baculovirus-mediated protein production, likely due to a prolonged infection cycle.
