Molecular Biotechnology: Principles and Applications - Glick, B. R., & Pasternak, J. J. 2002

Fundamentals of Molecular Biotechnology
Production of Recombinant Proteins Using Eukaryotic Systems
Expression Vectors for Mammalian Cell Culture

Extrachromosomal mammalian expression vectors are used to study the Functions and regulation of mammalian genes. In addition, they can be employed to produce authentic recombinant Proteins with potential medical Applications in treating various human diseases. Although a great variety of mammalian expression vectors have already been constructed, they all share similar properties and resemble other eukaryotic expression vectors.

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Fig. 7.13. General Structure of a mammalian expression vector. The polylinker (PL) and selectable marker (SM) are under the control of a eukaryotic promoter (p) and a polyadenylation signal (pa). Vector Replication in E. coli and mammalian Cells is driven by the replication initiation sites ori^E and ori^euk, respectively. The ampicillin resistance Gene (Amp^r) is used for the Selection of transformed E. coli cells.

The vector shown in Fig. 7.13 contains a eukaryotic replication origin from an animal virus (such as simian virus 40 [SV40]). The promoters of the cloned and selectable marker genes, as well as their METABOLISM/31.html">Transcription termination signals (polyadenylation signals), must be derived from Eukaryotic cells. Typically, these utilize regulatory DNA sequences from animal Viruses (e.g., human cytomegalovirus, SV40, or HSV) or mammalian genes (e.g., the ß-Actin, metallothionein, thymidine kinase, or bovine Growth Hormone genes). Strong promoters and efficient polyadenylation signals are preferred. The sequences required for the selection and Amplification of the mammalian expression vector in E. coli are derived from a standard E. coli cloning vector (such as the plasmid pBR322).

Selectable Marker Genes

To select for transfected mammalian cells, the bacterial Neo^r gene, which encodes neomycin phosphotransferase, is frequently used. This system employs the toxic compound geneticin (G-418), which blocks Translation in non-transfected mammalian cells. In transfected cells, however, G-418 is phosphorylated by neomycin phosphotransferase and thereby inactivated. Consequently, only cells synthesizing the Neo^r gene product survive and proliferate.

Another selection system for transfected mammalian cells is based on the gene encoding the enzyme Dihydrofolate Reductase (DHFR). This system utilizes cells with a defective DHFR gene, meaning cells that do not synthesize functional DHFR. Following the transfection of DHFR^- cells with a mammalian expression vector carrying a functional DHFR gene, methotrexate is added to the culture medium. Non-transfected cells fail to grow in its presence, whereas cells synthesizing dihydrofolate reductase survive. After initial selection for the DHFR gene, the concentration of methotrexate in the medium is increased to isolate cells containing a high copy number of the vector, which consequently yield high amounts of the recombinant protein.

Other dominant-marker selection schemes have also been developed, such as one utilizing the enzyme Glutamine Synthetase (GS), which confers resistance to the cytotoxic effects of Methionine sulfoximine. This system uses a vector carrying the GS gene, which is introduced into mammalian Cell culture, followed by increasing the concentration of methionine sulfoximine in the medium to select for cells harboring a high copy number of the vector. The host cells must also possess endogenous GS, as only multiple copies of the introduced GS gene can confer resistance to methionine sulfoximine. This strategy offers certain advantages over the one described above.

A wide variety of protein genes have already been cloned into mammalian expression vectors and successfully expressed in host cells. In some cases, product yields were enhanced by inserting an intron between the promoter and the cloned gene. The mechanism behind this phenomenon remains unclear; however, it is possible that the primary transcript of the cloned gene contains cryptic splice sites that could lead to the excision of part of the coding region, whereas the presence of an additional intron suppresses splicing at these sites.

High levels of cloned Gene Expression have been achieved by coordinating its expression with that of a selectable marker gene. For instance, the DHFR gene can be inserted in close proximity to the cloned gene so that both genes are driven by the same promoter, share a common polyadenylation signal, and have the DHFR gene flanked by intron splice sites. DHFR and the recombinant protein are then translated from the primary transcript and the spliced mRNA, respectively (Fig. 7.14).

Expression of Two Cloned Genes in a Single Mammalian Cell

Some commercially valuable proteins in their active form consist of different polypeptide chains. For example, human thyroid-stimulating hormone is a heterodimer, and Hemoglobin is a tetramer consisting of two subunits with two copies each (a₂ß₂). To obtain an active multimeric protein, one can attempt to clone the gene or cDNA of each subunit, synthesize and purify the subunits, and then mix them in a test tube. However, this approach successfully yields only a few multimeric proteins because proper folding of polypeptide chains rarely occurs in vitro. In contrast, the assembly of dimeric and tetrameric proteins in vivo proceeds very efficiently. Therefore, strategies have been developed to synthesize two different recombinant proteins within a single cell.

Fig. 7.14. Coordinated expression of the dihydrofolate reductase (DHFR) gene and a recombinant protein. The DHFR gene is inserted between the donor and acceptor splice sites of an intron (dots), upstream of the target gene (gene a). Both the DHFR gene and the cloned gene are under the control of a single eukaryotic promoter (p) and share a common polyadenylation signal (pa). DHFR is translated from the unspliced (primary) transcript, whereas the heterologous protein (protein a) is translated from the processed (spliced) transcript.

To achieve this, host cells were simultaneously transfected with two mammalian expression vectors, each carrying the gene or cDNA for one of the subunits along with different selectable marker genes (Fig. 7.15). The transfected cells underwent double selection, and consequently, the surviving cells carried both vectors. Two-vector systems have been successfully used to synthesize authentic dimeric and tetrameric recombinant proteins. Unfortunately, doubly transfected cells frequently lose one of the two vectors. Moreover, the copy number of each vector is not always equal, meaning one subunit may be synthesized in greater quantities than the other, which can reduce the final product yield. To overcome these problems, vectors containing both cloned genes were constructed. In some cases, these genes were placed under the control of independent promoters and polyadenylation signals (Fig. 7.16). Furthermore, to guarantee the synthesis of recombinant proteins in equal amounts, so-called bicistronic vectors were created, in which the cloned genes are separated by a DNA segment containing an internal ribosome entry site. Such sites have been discovered in the genomes of mammalian viruses; they allow the simultaneous translation of different proteins from a polycistronic mRNA. Transcription of the gene—internal ribosome entry site—gene construct is regulated by a single promoter and a single polyadenylation signal. A single transcript with two genes is synthesized, and translation initiates from both the 5'-end of the mRNA and the internal site, resulting in the Synthesis of the subunits of the dimeric protein a and ß (Fig. 7.17).

Fig. 7.15. Dual-vector expression system. The cloned genes (a and ß) encode the subunits of a dimeric protein (aß). Following simultaneous transfection of a cell with two Plasmids, both subunits are synthesized within The Cell and assemble into a functional dimeric protein. Both vectors carry origins of replication functional in E. coli (ori^E) and mammalian cells (ori^euk); a marker gene (Amp^r) for selecting transformed E. coli cells; and a eukaryotic promoter (p) and polyadenylation signal (pa) that regulate the expression of the selectable marker gene (SM) and each of the cloned genes.

In summary, mammalian expression vectors are just as versatile and efficient as vectors for other eukaryotic expression systems when it comes to producing authentic recombinant proteins for research and medical purposes. However, industrial-scale synthesis of recombinant proteins using modified mammalian cells is prohibitively expensive. In this case, less expensive expression systems are preferred, except in situations where the authenticity of the recombinant protein can only be achieved using mammalian cell culture.

Fig. 7.16. Expression vector with two independently transcribed genes. The cloned genes (a and ß) encode the subunits of a dimeric protein (aß). Each gene is inserted into the vector as part of a separate transcription unit and is under the control of a eukaryotic promoter (p) and a polyadenylation signal (pa). Each subunit is translated from its own mRNA; upon association, the subunits form a functional dimeric protein (aß). The vectors contain origins of replication functional in E. coli (ori^E) and mammalian cells (ori^euk); a marker gene (Amp^r) for selecting transformed E. coli cells; and a selectable marker gene (SM) under the control of a eukaryotic promoter (p) and polyadenylation signal (pa).

Fig. 7.17. Bicistronic expression vector. The cloned genes (a and ß) encode the subunits of a dimeric protein (aß). They are separated by a DNA segment that, after transcription at the mRNA level, functions as an internal ribosome entry site. Each gene is under the control of a eukaryotic promoter (p) and a polyadenylation signal (pa). Introduction/27.html">Translation of the mRNA initiates from the 5'-end and from the internal site (angled arrows). The synthesized subunits associate to form a functional dimeric protein. The vector contains origins of replication functional in E. coli (ori^E) and mammalian cells (orieuk); a selectable marker gene (Amp^r) for selecting transformed E. coli cells; and a selectable marker gene (SM) under the control of a eukaryotic promoter (p) and polyadenylation signal (pa).