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Mammalian and Bacterial Expression in One Vector

inus of the protein, can then be used for easy detection and purification of the fusion protein using Stratagenes Affinity CBP fusion detection kit and Calmodulin Affinity Resin, respectively. The new pDual expression vector is offered either in supercoiled form or predigested with Eam1104 I.

Prokaryotic and Eukaryotic Expression of the Green Fluorescent Protein

We chose the green fluorescent protein5 (GFP) gene as the reporter because its expression can be visualized easily in bacterial colonies, using a long-wave UV light source, and in transfected tissue culture cells, using standard fluorescence microscopy. Stratagenes Seamless cloning kit4 was used to amplify the GFP gene from the original TU585 construct using high-fidelity Pfu DNA polymerase with primers that contained the Eam1104 I recognition site. The digested vector and insert were ligated in the presence of the Eam1104 I restriction enzyme, eliminating the need for gel purification of the restriction fragments. Epicurian Coli XL1-Blue supercompetent bacteria were transformed with the ligation product, and 18 colonies were screened by PCR for the presence of the reporter gene. One hundred percent of the screened colonies contained the desired insert, and one of the clones, designated KPdual-1, was chosen for further studies. To test the ability of the pDual vector to express the GFP reporter in both prokaryotes and eukaryotes, we introduced the KPdual-1 construct into BL21(DE3) competent cells, which express the T7 RNA polymerase, and into mammalian tissue culture cells, respectively. Figures 2A and 2B show that the pDual vector is able to dir
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