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Fig. 4 | BMC Biotechnology

Fig. 4

From: Lentiviral expression system for the purification of secreted proteins from human cell cultures

Fig. 4

Optimizing conditions for the purification of sCD4. a Monocistronic versus bicistronic vector design. Cells were transduced with the bicistronic vector LV-CMV-AAT-sCD4 or the monocistronic vector LV-CMV-M-AAT-sCD4 at a MOI of 50. 2 × 105 gene-modified 293 T cells in 1 ml media were plated in a 12-well plate and cultured for 4 days before culture supernatants were analyzed by ELISA. b Effect of stable versus transient expression on sCD4 secretion levels. 7 × 105 untransduced 293 T cells or 293 T transduced with LV-CMV-AAT-sCD4 were plated in a 12-well plate in 1 ml media and cultured for 1 day. Media were replaced with fresh media and unmodified 293 T cells were transfected with pLV-CMV-AAT-sCD4. On day 2 after plating, media were replaced with fresh media and the cells were cultured for 2 additional days before ELISA was performed on culture supernatants; *p < 0.05. c Effect of reducing serum concentration. Gene-modified 293 T cells were cultured and analyzed as in (b), but on day 2 after plating culture media were replaced with media with the indicated FBS concentration. d Effect of incubation time. Cells were cultured as in (c). Media were replaced on day 2 after plating with media containing 1 % FBS and supernatants were analyzed at the indicated time points by ELISA (left panel) or SDS-PAGE followed by Coomassie blue staining (right panel). All results are representative of 2 independent experiments performed in duplicates

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