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 induct ion of apoptosis during infection of Sf2l cells by Autographa californica m ulticapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apo ptosis in a broad range of cells and circumstances. In this study, we exami ned the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV i nfected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-Lagged P3 5 protein were generated using a double selection technique, involving sele ction in the antibiotic G418, followed by a second round of selection by ex posure 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) baculov irus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9(P35AcV5-1) and Sf9(P35AcV5-3) ) showed increased resistance to actinomycin D-induced apoptosis and a prof ound resistance to nutrient deprivation. When these cell lines were infecte d with a recombinant baculovirus expressing a secreted glycoprotein (secret ed alkaline phosphatase), expression of the glycoprotein from these cells e xceeded that from the parental Sf9 cells and was comparable to expression l evels obtained from Tn5B1-4 cells, the best available cell line for high-le vel expression. Increased levels of protein secretion in Sf9(P35AcV5-1) and Sf9(P35AcV5-3) cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.