Vectors for dual expression of target genes in bacterial and mammalian cells.

For protein production and purification, many different cell systems ranging from bacteria to mammalian cell lines are commonly used. High-level expression of recombinant proteins in E. coli is often achieved using the strong yet tightly regulatable T7 promoter system (23). Many convenient T7 promoter-based bacterial expression vectors are commercially available from different sources and are widely used. For the expression of genes in mammalian cells, many different expression systems also exist, with the cytomegalovirus (CMV) immediate early enhancer/ promoter-based expression vectors (4) being among the most common. Some of these mammalian expression vectors also carry a T7 promoter to facilitate the in vitro transcription/translation of inserted genes. To generate vectors with the capacity to overcome the species barrier and to function in bacterial and mammalian cells, expression systems have been developed that use a T7 promoter in combination with a modified T7 RNA polymerase that is targeted to the nucleus of mammalian cells (2,12). Besides bacterial expression of target genes from these vectors, co-transfection of the expression vector and the vector carrying the modified T7 RNA polymerase gene leads to a strong expression of target genes in mammalian cells. A related strategy that relies on a combination of a phage promoter and an internal ribosomal binding site (IRES) derived from the encephalomyocarditis virus (EMCV) has also been used to express target genes in E. coli and mammalian cells using a single vector (8). Forman and Samuels (5) have combined the Rous sarcoma virus long terminal repeat (RSV-LTR) and the T7 promoter to generate a family of expression vectors (pEXPRESS) that allows expression of target genes in both eukaryotic and prokaryotic cells (independent of a modified T7 RNA polymerase), as well as translation of these genes in a cell-free system. Additionally, a single expression plasmid has been developed that allows protein expression in mammalian cell lines and in the yeast Pichia pastoris (16). To combine the T7 promoter-based bacterial expression system with one of the strongest and most widely used CMV promoter/enhancer-based mammalian expression systems (15), we have established an easy procedure for the generation of expression vectors with the capacity to function in both bacterial and mammalian cells. Our strategy relies on the generation of a strong bacterial ribosomal binding site (rbs) downstream of the T7 promoter of a mammalian expression vector, in combination with a Kozak consensus sequence (9–11) for optimal translation initiation in mammalian cells. To illustrate this strategy, we have established a dual expression system amplifying the EGFP gene (Clontech Laboratories, Palo Alto, CA, USA) using PCR (1) and inserted it into the NheI and XhoI sites of the CMV promoter-based mammalian expression vector pCI (Promega, Madison, WI, USA) downstream of its T7 promoter (Figure 1B). pCI also contains an intron sequence for increased expression levels in mamBenchmarks