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Mammalian and Bacterial Expression in One Vector

a bacterial vector and then subcloned into a vector suitable for eukaryotic expression. Each step involves isolation and characterization of clones containing the gene of interest, requiring a significant investment of time and biological reagents. Stratagene has now eliminated the need to subclone from one vector system to another by combining the essential features of both in a dual expression vector. The novel pDual vector (figure 1) can achieve high-level expression of heterologous genes in both mammalian and bacterial cells.

figure 1

The pDual expression vector can be used in conjunction with the seamless cloning technique.3,4 PCR amplification of the gene of interest, using primers that contain the Eam1104 I restriction sites and a minimal flanking sequence, permits rapid and efficient cloning in the presence of the type IIS Eam1104 I restriction enzyme. After digesting the PCR product with Eam1104 I, the fragment can be inserted via a three nucleotide 5 overhang that encodes the genes own ATG. The use of the type IIS Eam1104 I restriction enzyme eliminates any primer-related residual nucleotides that are generally present when regular restriction enzyme recognition sites are encoded by the PCR primer sequences.

Taking advantage of the versatility of PCR cloning, the pDual vector offers two options for expressing a target gene. The reverse primer can be designed to contain a stop codon, which results in the target gene product being expressed in its native form. Alternatively, by designing a reverse primer that eliminates the genes natural termination codon, researchers can fuse their target gene to the small calmodulin binding peptide (CBP) affinity tag located downstream of the cloning site. The 4-kDa CBP fusion tag, expressed on the C-term
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