Versatile Mammalian Expression Vectors: A Review

Many options for expressing proteins in mammalian cells

Versatile Mammalian Expression Vectors:A Review

Quinn Lu • Tanya Hosfield • Mary Buchanan • Kerstein Padgett
Stratagene

We describe Stratagene’s pcmv-script®, pCMV-Tag1 through 5, and pdual® expression vectors* and present data to illustrate their features. These vectors offer convenient options, such as high-fidelity PCR cloning, epitope tagging, or dual expression in prokaryotic and eukaryotic systems. All three vector systems are designed specifically for high-level expression of gene products in mammalian cells. By evaluating the unique features of each expression vector, it is simple to select the one best suited for a specific experimental system.

The demand for versatile mammalian expression vectors is growing, since many researchers study eukaryotic gene transcription and translation. In many instances, achieving posttranslational modifications of a gene product of interest is necessary to obtain an active eukaryotic protein. Vectors with convenient options, such as the capacity for high-fidelity PCR cloning and epitope tagging for characterization studies, have become integral to sophisticated research inquiries.

Stratagene offers versatile mammalian expression vectors that provide a variety of options for functional studies of gene products. These options include the following: potential for gene expression in both bacterial and mammalian cells; availability of antibiotic selection for both bacterial and/or mammalian cells; inclusion of regulatory sequences for high expression levels; ability to clone PCR fragments generated from a variety of PCR enzymes; possibility for classical cloning method, seamless® cloning method* or PCR-Script^™ cloning method; and presence of epitope and affinity tags in variable positions for in vivo studies of gene products. Hence, it is easy to choose an expression vector (Table 1) that best fits the research parameters.

Table 1
Mammalian Expression Vectors

Efficient Cloning of PCR Fragments

Stratagene’s pcmv-script® PCR cloning kit is an expression system that is a mammalian adaptation of the PCR-Script cloning strategy.¹ This method for cloning PCR-generated fragments is popular because it is efficient, easy to use, and superior to other available methods.² In the PCR-Script cloning method, PCR products are incubated with the predigested PCR-Script vector, Srf I restriction enzyme, and T4 DNA ligase. The Srf I enzyme recognizes the rare oligonucleotide sequence 5¢-GCCCGGGC-3¢ in the vector and recircularizes any vector that ligates to itself without an insert. Once an insert has ligated to the vector, the site is destroyed.

By including the Srf I enzyme in the ligation reaction, the PCR-Script cloning method maintains high concentrations of digested vector DNA and allows the use of nonphosphorylated, unmodified PCR products. This method ensures a low background of recircularized vector and increased ligation efficiency for blunt-ended DNA fragments by the simultaneous, opposing reactivity of the Srf I enzyme and T4 DNA ligase on nonrecombinant vector DNA.

Figure 1

The PCR-Script method is used in the pCMV-Script PCR cloning kit, which features the pCMV-Script vector (Figure 1), a mammalian expression vector that uses the CMV promoter ^### for constitutive expression in a wide variety of cell lines. The pCMV-Script vector shares many of the essential characteristics of the PCR-Script vector: the multiple cloning site (MCS), capacity to clone inserts generated with any PCR enzyme, high efficiency, and 1-hour ligation. However, the pCMV-Script vector also has many distinctive features. It contains the neomycin-phosphotransferase gene under dual control of the b-lactamase and SV40 promoters, which selects for kanamycin resistance in bacteria and G418 resistance in mammalian cells. Classical cloning strategies are also possible with the pCMV-Script vector’s MCS; Xho I and EcoR I sites are included to construct directional cDNA libraries.³

Expression in the pCMV-Script Vector

In order to demonstrate the cloning efficiency of the pCMV-Script PCR cloning kit, a number of PCR products were cloned into the pCMV-Script vector.¹ One of these constructs, the firefly luciferase gene, was used to transfect Chinese hamster ovary (CHO) cells. To compare the expression level of this reporter gene inserted into the pCMV-Script vector with the expression level in another available vector product, we also inserted the luciferase gene into the pcDNA 3.1 vector (Invitrogen, San Diego, CA). Each construct and appropriate controls were used to transfect CHO cells. Forty-eight hours following transfection, CHO cells were harvested, luciferase activity was quantitated (Figure 2), and the protein of the correct molecular weight was detected by Western blot analysis (data not shown). Luciferase activity and expression from the pCMV-Script vector were equivalent to that of the pcDNA 3.1 vector.

Figure 2

Use Tag-Specific Antibodies to Study Gene Products

Stratagene offers another option to clone and express mammalian genes by applying the useful epitope- tagging technique: A protein of interest is fused to a peptide-epitope that is recognized by a commercially available antibody. Epitope tagging can be used to localize gene products in living cells, identify associated proteins, track movement of fusion proteins within the cell, characterize new proteins or immunoprecipitate fusion proteins. Epitope tagging makes functional studies of a target protein possible while eliminating the cost, labor, and time required to generate a specific antibody to that protein.

Figure 3

Stratagene’s pCMV-Tag1 through 5 vectors (Figure 3) are a series of epitope-tagging vectors designed for gene expression in mammalian cells that contain either the FLAG^® coding sequence^§§ and/or c-myc epitope-coding sequences. Because the FLAG and c-myc epitopes are small, they usually do not interfere with the function of the target protein. They are also highly immunoreactive, allowing detection with commercially available antibodies. The FLAG epitope is a synthetic peptide that consists of eight amino acid residues (DYKDDDDK).⁴ The c-myc epitope is derived from the human c-myc gene and contains 10 amino acid residues (EQKLISEEDL).⁵

For the pCMV-Tag1 vector, a variety of tagging positions are possible. 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.⁶ The pCMV-Tag2 vector contains sequences for N-terminal tagging with the FLAG epitope, and the pCMV-Tag3 vector is used for N-terminal tagging with the c-myc epitope. For constructing C-terminal fusion proteins, the pCMV-Tag4 vector includes the sequence for the FLAG epitope, and the pCMV-Tag5 vector features the sequence for the c-myc epitope. Each of the pCMV-Tag2 through 5 vectors offers specific tagging positions and is available as a kit that includes vectors in all three reading frames to simplify subcloning. All constructs generated in the pCMV-Tag vectors can be transfected into mammalian cells, and fusion proteins can be easily characterized using commercially available, tag-specific antibodies.

The pCMV-Tag1 through 5 vectors feature not only optimized propagation and expression of fusion proteins in eukaryotic cells, but also attributes conferred by their pCMV-Script vector backbone. Thus, the pCMV-Tag1 through 5 vectors include constitutive expression of the cloned DNA in a wide variety of mammalian cell lines and the capability of selecting stable transformants by kanamycin resistance in bacteria and G418 resistance in mammalian cells. The Kozak consensus sequence of CC(A or G)CCATGG provides optimal expression of the fusion protein for N-terminal epitope tags.

Expression and Detection of Epitope Tags

Figure 4

To demonstrate how easy it is to detect epitope tags, we performed Western blot analyses of cell lysates derived from CHO cells transfected with the pCMV-Tag2 through 5 vectors containing the luciferase gene insert (Figure 4). Aliquots of these lysates were fractionated on three separate protein gels and transferred to nitrocellulose. The membranes were probed individually with anti-luciferase, anti-FLAG, or anti-c-myc antibodies. Results indicate that the fusion protein, with either FLAG or c-myc at N-terminal or C-terminal positions, is easily detected by Western blot analysis (Figure 4).

To demonstrate that luciferase tagged terminally with either FLAG or c-myc is biologically active, we cloned the luciferase gene into the pCMV-Tag2 through 5 vectors to create constructs with all possible tagging options: N-terminal tagging with FLAG, N-terminal tagging with c-myc, C-terminal tagging with FLAG and C-terminal tagging with c-myc. Assays of cell lysates made from transient transfections of these constructs into CHO cells and COS cells showed significant luciferase activity, whereas lysates from control transfections showed only background levels of luciferase activity (data not shown).

Dual Expression in Bacterial and Mammalian Cells

Figure 5

Stratagene also offers the unique pDual vector (Figure 5), which features high-level expression of heterologous genes in both mammalian and bacterial systems. The regulatory features of vectors for mammalian and bacterial expression vary considerably; each type of vector offers different choices of promoters, splice signals, and polyadenylation sites, as well as their respective translation initiation sequences. Oftentimes, a eukaryotic gene must first be cloned into a bacterial vector, then the gene must be subcloned into a vector suitable for eukaryotic expression. However, Stratagene’s pDual expression vector⁸ eliminates the need to subclone from one vector system into another.

The pDual vector features constitutive expression of cloned DNA in a wide variety of mammalian cell lines and the capability of selecting stable transformants by G418 resistance in mammalian cells. For bacterial systems, the pDual vector contains the hybrid T7/lacO promoter^‡‡, which provides inducible protein expression with IPTG. The T7 RNA polymerase promoter provides the highest levels of protein expression in E. coli. The presence of the lac repressor gene (lacI) on the plasmid confers tight repression of the system. The pDual vector has tandemly arranged bacterial Shine-Dalgarno⁹ and mammalian Kozak⁷ consensus sequences. These tandem consensus sequences ensure optimal translation initiation in both bacterial and mammalian systems, because each ribosome binding site is positioned at its optimal distance from the initiation codon of the cloned target gene. When using the pDual vector, researchers may option to express the protein in its native form or fuse their target gene to the 4-kDa calmodulin-binding peptide (CBP) affinity tag. This CBP tag allows the fusion protein to be easily detected with Stratagene’s Affinity^™ CBP fusion detection kit and purified with Calmodulin Affinity Resin.⁸

Seamless Cloning

The pDual expression vector can be used in conjunction with the Seamless cloning kit.¹⁰ For the Seamless cloning technique, the target gene is PCR-amplified using primers that contain the Eam1104 I restriction sites and a minimal flanking sequence, which 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 target gene’s own ATG. The type IIS Eam1104 I restriction enzyme eliminates any primer-related residual nucleotides that are generally present when regular restriction enzyme recognition sites are specified by the PCR primer sequences.

Conclusions

Together, the pCMV-Script, pCMV-Tag1 through 5, and pDual vectors offer a variety of options to express gene products in mammalian cells. Choose among these versatile vectors to optimize gene cloning for mammalian expression in any experimental system. For the pCMV-Script vector, there is high-level constitutive expression from the CMV promoter, cloning using either classical or PCR-Script cloning technology, and stable transfection via the neomycin-resistance gene. For the pCMV-Tag1 through 5 vectors, target genes can be inserted for tagging with FLAG and/or c-myc epitopes. When these constructs are transfected into mammalian cells, functional studies of the tagged-fusion proteins are possible using commercially available tag-specific antibodies. The pDual vector provides expression in both bacterial and mammalian systems and features a hybrid bacterial promoter for inducible bacterial expression. This vector can be used in conjunction with the Seamless cloning technique.

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* Patent pending.