( A gene of in...)An Epitope Tagging Vector for Gene Expression in Mammalian Cells

A gene of interest can be efficiently cloned into the pCMV-Tag1 vector for terminal tagging with either the FLAG or c-myc epitopes or internal tagging with the FLAG epitope. With these choices, researchers can optimize the tagging position for each protein they study. The cloning strategies for various tagging choices are summarized in figure 2.

Expression in the pCMV-Tag1 Vector

The firefly luciferase gene was cloned into the Bgl II and Xho I sites of the pCMV-Tag1 vector such that the luciferase protein was tagged with the FLAG epitope at the N-terminus and the c-myc epitope at the C-terminus. We chose the luciferase gene because it can be assayed both enzymatically and immunologically. This construct was transiently transfected into Chinese hamster ovary (CHO) cells, and the cell lysates were assayed for luciferase activity. The results of these luciferase assays (figure 3) demonstrate that luciferase tagged with both FLAG and c-myc is biologically active. Control transfections, with the reagents alone or the pCMV-Tag1 vector without the luciferase insert, show low background levels.

Figure 3

To demonstrate the easy detection of the epitope tags, we performed Western blot analyses of cell lysates derived from cells transfected with the pCMV-Tag1 vector and the pCMV-Tag1 vector with the luciferase insert (figure 4). Aliquots of these samples were simultaneously loaded and electrophoresed in three separate gels.

Figure 4

The three gels were then probed individually with either anti-luciferase, anti-FLAG or anti-c-myc antibodies. The results indicate that the fusion p
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