Efficient production of protein complexes in mammalian cells using a poxvirus vector
Fig 2
Protein production in suspension cell cultures.
A. BHK 21 suspension cells (1.2 106 cells/ml) were infected in 50 ml cell culture medium with MVA-T7 encoding β glucuronidase at increasing MOIs. After 24 hours infection cells were pelleted and enzymatic activity determined in duplicate. B. BHK 21 suspension cells (106 cells/ml) were infected in a 100 ml cell culture medium with an MVA-T7 virus encoding a GST-tagged HIV-1 integrase at 0.1 PFU/cell. After 48 hours infection in the absence of IPTG, 1 ml, 2 ml, 5 ml, 10 ml or 20 ml of infected cells were added to 50 ml of uninfected cells (106 cells/ml). Cell culture medium was added so that the culture volumes were identical in all samples and protein expression was induced by the simultaneous addition of IPTG. Control infections were conducted with 4PFU/cell of the MVA-T7 virus encoding GST-integrase in the presence or absence of IPTG. 24 hours later 1.5ml of the infected cell cultures were recovered for analysis of total protein by PAGE. U uninfected cells.–and + indicate the presence or absence of IPTG in the infected cell cultures. C. 45 ml samples of the infected BHK 21 suspension cultures in panel B were pelleted, sonicated and the GST-integrase purified and examined by PAGE. U uninfected cells.–and + indicates the absence or presence of IPTG in the infected cell cultures.