Novel pdual expression vector for high-level expression in both mammalian and bacterial cells
Kerstien A. Padgett Joseph A. Sorge
Stratagene has developed the pdual expression vector,* which permits high-level expression of heterologous genes in both eukaryotic and prokaryotic systems. The vector contains the promoter and enhancer region of the human cytomegalovirus (CMV) immediate early gene for constitutive expression of cloned DNA inserts in mammalian cells. Expression of heterologous genes in bacteria is regulated by a hybrid T7/ lacO promoter. The pDual vector was engineered to carry the lac repressor gene (lacI). Expression is therefore inducible using IPTG in bacteria that contain the T7 RNA polymerase. ## In both bacterial and mammalian cells, the dominant selectable marker is the neomycin phosphotransferase gene under the dual control of the b-lactamase and SV40 promoters. A distinguishing feature of the pDual vector is the tandemly arranged bacterial Shine-Dalgarno1 and mammalian Kozak 2 consensus sequence. Each ribosome binding site is positioned at its optimal distance from the initiation codon of the target gene, which ensures efficient translation of mRNA generated in either system.
Mammalian expression vectors contain similar elements that may also be present in bacterial expression vectors. Yet mammalian and bacterial plasmid vectors vary considerably in their choice of promoters, the presence of splice signals and polyadenylation sites, as well as their respective translation initiation sequences. In fact, the differences between bacterial and mammalian expression vectors are so significant that, until now, these vectors could not be used interchangeably.
For expression in mammalian cells, eukaryotic genes are typically cloned first into 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.
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 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.
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 ect expression of the reporter in both systems. The presence of the CBP-tagged GFP fusion protein in bacterial and mammalian cell lysates was further verified by Western blot analyses using GFP antiserum (Figures 2C) and the Affinity CBP fusion detection kit (data not shown).
The hybrid T7/lacO phage promoter was tested by carrying out induction studies using IPTG. BL21(DE3) cells harboring the KPdual-1 construct were grown in LB broth supplemented with 50 mg/ml of kanamycin until the culture reached an OD600 of approximately 0.6. One-half of the culture was then induced by adding IPTG to a final concentration of 0.2 mM. The induced and uninduced cultures were harvested after an additional 3-hour growth period at 37C. Lysates were prepared by standard methods,6 and the total protein concentration was determined by Bradford assay. Equal amounts of protein from IPTG induced and uninduced bacterial lysates were resolved by SDS-PAGE, transferred to nitrocellulose and visualized by chemiluminescence using GFP antiserum. Figure 3 shows that there was no background expression of the GFP-CBP fusion protein in the absence of IPTG (lane 3), but expression of the fusion protein increased dramatically when cells were grown in the presence of the inducer (lan e 2).
The GFP-CBP fusion protein was purified from bacterial cell lysates using the Affinity purification system. The purification was performed using the batch method described in the manual supplied with the kit. The calmodulin affinity resin with adsorbed GFP-CBP fusion protein was applied to a disposable column, washed and then eluted at neutral pH as specified in the manual. Aliquots of the IPTG-induced and uninduced bacterial lysate and the eluted fraction containing the fusion protein were resolved by SDS-PAGE and visualized by staining with Coomassie brilliant blue dye (figure 4). The size of the GFP-CBP hybrid protein is approximately 32 kDa, compared to that of the untagged 28-kDa GFP. The results (figure 4, lane 3) indicate that the small CBP affinity tag, located on the C-terminus of the protein, provides an effective and convenient way to purify the protein of interest from bacteria. In addition, the thrombin protease cleavage site allows cleavage to occur in the presence of thrombin for applications where removal of the affinity tag is desired. A time-course experiment showed that successful cleavage was achieved when the purified GFP-CBP fusion protein was incubated at room temperature with as little as 2 ng of thrombin in the presence of calcium. A representative cleavage profile of the GFP-CBP fusion protein is shown in figure 4. Full-length fusion protein was present in an aliquot that was removed immediately after addition of thrombin (lane 4), approximately 50% of the CBP fusion tag was cleaved after a 6-hour incubation at room temperature (lane 5) and approximately 95% of the fusion tag was cleaved after 24 hours (lane 6).
The pDual expression vector was developed to allow simultaneous expression of proteins in prokaryotic and mammalian systems. The vector contains a hybrid bacterial promoter for inducible bacterial expression, the CMV promoter and enhancer region for constitutive mammalian expression and tandem consensus sequences for optimal translation initiation in both systems. The cloning site is compatible with the novel Seamless cloning technique.3,4 The CBP-affinity tag is located downstream of the cloning site, allowing simple detection and purification of a protein of interest. The CBP-affinity tag can be removed from the fusion protein via the thrombin cleavage site that immediately precedes the tag.
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* Patent pending