( An Epitope Tagging Vector for Gene Exp...)An Epitope Tagging Vector for Gene Expression in Mammalian Cells

Tanya Hosfield Quinn Lu
Stratagene

The pCMV-Tag1 vector is an epitope tagging vector designed for gene expression in mammalian cells. A target gene inserted into the pCMV-Tag1 vector can be tagged with the FLAG epitope (N-terminal, C-terminal or internal tagging), the c-myc epitope (C-terminal) or both the FLAG (N-terminal) and c-myc (C-terminal) epitopes. Tagged constructs generated in the pCMV-Tag1 vector can be transfected into mammalian cells, and the tagged gene product can be easily characterized using commercially available, tag-specific antibodies.

The epitope tagging technique involves fusion of a protein of interest to a peptide-epitope that is recognized by a readily available antibody. In this technique, expression of the fusion protein is monitored using a tag-specific antibody, allowing a new protein to be studied without generating a new, specific antibody to that protein. Epitope tagging can be used to localize gene products in living cells, identify associated proteins, track movement of fusion proteins within the cell, or characterize new proteins by immunoprecipitation.^1-3

pCMV-Tag1 Vector for Epitope Tagging

Stratagenes pCMV-Tag1 vector is a general mammalian expression vector that contains the FLAG and c-myc epitopes positioned for either terminal or internal tagging of a target protein. The pCMV-Tag1 vector (figure 1) is derived from the pCMV-Script vector⁴ and contains sequences for the FLAG and c-myc epitopes. These specific epitope tags are small, not interfering with the function of the target protein, and highly immunoreactive. The FLAG epitope is a synthetic epitope that consists of eight amino acid residues (DYKDDDDK).⁵ The c-myc epitope is derived from the human c-myc gene and contains 10 amino acid residues (EQKLISEEDL).⁶ In addition to the epitope tag sequences, the pCMV-Tag1 vector contains features for expression of fusion proteins in eukaryotic cells. The cytomegalovirus (CMV) promoter allows constitutive expression of the cloned DNA in a wide variety of mammalian cell lines. The neomycin-resistance gene is under control of both the prokaryotic b-lactamase promoter to provide kanamycin resistance in bacteria and the SV40 early promoter to provide G418 resistance in mammalian cells. The multiple cloning site (MCS) of the pCMV-Tag1 vector is arranged to allow a variety of cloning strategies to be used, resulting in C-terminal, N-terminal or internal tagging of the protein of interest. A Kozak consensus sequence of GCC(A or G)CCATGG⁷ provides optimal expression of the fusion protein when the N-terminal FLAG epitope tag is used. Other cloning options, which require fusion proteins to include their own translational start sequence, are also possible.

Cloning Strategies for Epitope Tagging

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 protein, composed of FLAG-luciferase-c-myc, is easily detected by Western blot analysis.

In order to demonstrate G418 resistance in the pCMV-Tag1 vector, we used Stratagenes Mammalian Transfection Kit to obtain stable CHO cell lines containing either the pCMV-Tag1 vector or the pCMV-Tag1 vector with the luciferase insert. For the cells transfected with the pCMV-Tag1 vector containing the luciferase gene, the luciferase assay was used to verify the presence of the fusion protein (data not shown).

Conclusions

The pCMV-Tag1 expression vector incorporates the small and highly immunoreactive FLAG and c-myc epitopes into constructs for N-terminal, C-terminal and internal tagging. These tags eliminate the need for raising specific antisera to study a target gene. The epitope tags can be easily detected in transfected cells using well-characterized, commercially available antibodies. The pCMV-Tag1 vector offers a fast, versatile and reliable method for analyzing the function of gene products in vivo.

Acknowledgments

We would like to thank Denise Wyborski, Cathy Chang, Xu Li, Chao-Feng Zheng, Wei-Ping Yang, Phyllis Frosst and the members of the Gerace lab at TSRI for suggestions, discussion and materials.

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