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Mammalian and Bacterial Expression in One Vector

ect expression of the reporter in both systems. The presence of the CBP-tagged GFP fusion protein in bacterial and mammalian cell lysates was further verified by Western blot analyses using GFP antiserum (Figures 2C) and the Affinity CBP fusion detection kit (data not shown).

Figures 2

Inducible Expression of GFP-CBP Fusion Protein in BL21(DE3) Cells

The hybrid T7/lacO phage promoter was tested by carrying out induction studies using IPTG. BL21(DE3) cells harboring the KPdual-1 construct were grown in LB broth supplemented with 50 mg/ml of kanamycin until the culture reached an OD600 of approximately 0.6. One-half of the culture was then induced by adding IPTG to a final concentration of 0.2 mM. The induced and uninduced cultures were harvested after an additional 3-hour growth period at 37C. Lysates were prepared by standard methods,6 and the total protein concentration was determined by Bradford assay. Equal amounts of protein from IPTG induced and uninduced bacterial lysates were resolved by SDS-PAGE, transferred to nitrocellulose and visualized by chemiluminescence using GFP antiserum. Figure 3 shows that there was no background expression of the GFP-CBP fusion protein in the absence of IPTG (lane 3), but expression of the fusion protein increased dramatically when cells were grown in the presence of the inducer (lan
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