All mammalian expression vectors tend to have similar features and are not very different in design from other eukaryotic expression vectors. A typical mammalian expression vector (Fig. 3.40) contains a eukaryotic origin of replication, usually from an animal virus, such as SV40. The promoter sequences that drive expression of the cloned gene(s) and the selectable marker gene(s), and the transcription termination sequences (including polyadenylation signals), must be eukaryotic and are frequently taken from either human viruses or mammalian genes. Strong constitutive promoters and efficient polyadenylation signals are preferred. Inducible promoters are often used when continuous synthesis of the heterologous protein is toxic to the host cell. Expression of a gene of interest may be increased by placing the sequence for an intron between the promoter and the multiple cloning site, within the transcribed region. The sequences that are required for selection and propagation of a mammalian expression vector in E. coli are derived from a standard E. coli cloning vector.
Figure 3.40 Generalized mammalian expression vector. The multiple cloning site (MCS) and selectable marker gene (SMG) are under the control of eukaryotic promoter (p), polyadenylation (pa), and termination of transcription (TT) sequences. An intron (I) enhances the production of heterologous protein. Propagation of the vector in E. coli and mammalian cells depends on the origins of replication oriE and orieuk, respectively. The ampicillin resistance (Ampr) gene is used for selecting transformed E. coli.
For the best results, a gene of interest must be equipped with translation control sequences (Fig. 3.41). Initiation of translation in higher eukaryotic organisms depends on a specific sequence of nucleotides surrounding the start (AUG) codon in the mRNA called the Kozak sequence (e.g., GCCGCC(A or G)CCAUGG) in vertebrates. The corresponding DNA sequence for the Kozak sequence is placed at the 5′ end of the gene of interest, often followed by a signal sequence to facilitate secretion, a protein sequence (tag) to enhance the purification of the heterologous protein, and a proteolytic cleavage sequence that enables the tag to be removed from the heterologous protein. A stop codon is required for translation to cease at the correct location. Finally, the sequence content of the 5′ and 3′ untranslated regions (UTRs) is important for efficient translation and mRNA stability. Either synthetic 5′ and 3′ UTRs or those from the human β-globin gene are used in mammalian expression vectors. The codon content of the gene of interest may also require modification to suit the translational preferences of the host cell.
Figure 3.41 Translation control elements. A gene of interest can be fitted with various sequences that enhance translation and facilitate both secretion and purification, such as a Kozak sequence (K), signal sequence (S), protein affinity tag (T), proteolytic cleavage site (P), and stop codon (SC). The 5′ and 3′ UTRs increase the efficiency of translation and contribute to mRNA stability.
The majority of mammalian cell expression vectors carry a single gene of interest that encodes a functional polypeptide. However, the active form of some commercially important proteins consists of two different protein chains. For example, human thyroid-stimulating hormone is a two-chain protein (heterodimer), and both hemoglobin and antibodies are tetramers with two copies of each subunit, α2β2 and H2L2, respectively. It is possible to clone the gene or cDNA for each subunit of a multimeric protein, synthesize and purify each subunit separately, and then mix the subunits together in a test tube. Unfortunately, relatively few multisubunit proteins are properly assembled in vitro. By contrast, in vivo assembly of dimeric and tetrameric proteins is quite efficient. Consequently, various strategies have been devised for the production of two different recombinant proteins within the same cell.
To produce hetero-dimeric or -tetrameric proteins, single vectors that carry two cloned genes have been developed. The two genes are placed under the control of independent promoters and polyadenylation signals (double-cassette vectors) (Fig. 3.42A). Alternatively, to ensure that equal amounts of the recombinant proteins are synthesized, “bicistronic” vectors have been constructed with the two cloned genes separated from each other by a DNA sequence that contains an internal ribosomal entry site (IRES) (Fig. 3.42B). IRESs are found in mammalian virus genomes, and after transcription, they allow simultaneous translation of different proteins from a polycistronic mRNA molecule. Transcription of a “gene α–IRES–gene β” construct is controlled by one promoter and polyadenylation signal. Under these conditions, a single “two-gene” (bicistronic) transcript is synthesized, and translation proceeds from the 5′ end of the mRNA to produce one of the subunits (subunit α) and internally from the IRES element to produce the second subunit (subunit β) (Fig. 3.42B).
Figure 3.42 Expression vectors for production of hetero-dimeric or -tetrameric proteins in mammalian cells. (A) Two-gene expression vector. The cloned genes (gene α and gene β) encode subunits of a protein dimer (αβ). The cloned genes are inserted into a vector and are under the control of different eukaryotic promoter (p), polyadenylation (pa), and termination of transcription (TT) sequences. Each subunit is translated from a separate mRNA, and a functional protein dimer (αβ) is assembled. (B) Bicistronic expression vector. Each cloned gene (gene α and gene β) is inserted into a vector on either side of a sequence for an IRES. The two genes and the IRES sequence form a transcription unit under the control of a single eukaryotic promoter. Translation of the mRNA occurs from the 5′ end and internally (right-angled arrows). Both subunits (α and β) are synthesized and assembled into a functional protein dimer (αβ). Both expression vectors carry origins of replication for E. coli (oriE) and mammalian cells (orieuk), a selectable marker (Ampr) for selecting transformed E. coli, and a selectable marker gene (SMG) that is under the control of eukaryotic promoter (p), polyadenylation (pa), and termination of transcription (TT) sequences.
Selectable Markers for Mammalian Expression Vectors
For the most part, the systems that are used to select transfected mammalian cells are the same as those for other eukaryotic host cells (Table 3.15). A number of bacterial marker genes have been adapted for eukaryotic cells. For example, the bacterial neo gene, which encodes neomycin phosphotransferase, is often used to select transfected mammalian cells. In eukaryotic cells, G-418 (Geneticin), which is phosphorylated and thereby inactivated by neomycin phosphotransferase, replaces neomycin as the selective agent because neomycin is not an effective inhibitor