“humanized” insect cells have been engineered to express several enzymes that process human glycoproteins accurately. Blue squares, N-acetylglucosamine; red circles, mannose; green squares, galactose; orange squares, sialic acid.
Further improvements to prevent undesirable processing of heterologous proteins in insect cells are the removal of the genes encoding chitinase and the protease v-cathepsin from the AcMNPV genome. v-Cathepsin is normally produced late in the infection cycle to facilitate the release of new virions from the insect host. It also reduces the yield of heterologous proteins through proteolytic cleavage. Chitinase is produced in conjunction with v-cathepsin and is thought to function in the proper folding of v-cathepsin and in the degradation of the host exoskeleton. It is secreted at very high levels from baculovirus-infected insect host cells and can compete with secreted target proteins for the secretory apparatus, thereby reducing yields of the target protein. Coexpression of chaperones to ensure proper folding of the target protein has also resulted in increased yields of functional heterologous proteins.
Mammalian Cell Expression Systems
Currently, about half of the commercially available therapeutic proteins are produced in mammalian cells. Chinese hamster ovary (CHO) cells are most commonly used because they produce proteins with human-like glycans and have been adapted for growth in high-density suspension cultures in serum-free medium, which not only reduces costs but also facilitates purification of the target protein and reduces the risk of contamination with animal-derived material. They are receptive to transfection and can achieve long-term (stable) gene expression and high yields of heterologous proteins. Other host cell lines are derived from mouse myelomas, baby hamster kidney (BHK), and human embryo kidney (HEK 293). Although mammalian cells have been used for some time to produce therapeutic proteins, especially antibodies, and vectors carrying suitable expression signals have been developed, current efforts are aimed at improving productivity through the development of high-producing cell lines, increasing the stability of production over time, and increasing expression by manipulating the chromosomal environment in which the recombinant genes are integrated.
Vector Design
Most cloning vectors constructed for the expression of heterologous genes in mammalian cells are based on the genomes of viruses that infect mammalian cells. Many vectors are derived from a simian virus (simian virus 40 [SV40]) that can replicate in several mammalian species. The genome of this virus is a double-stranded DNA molecule of 5.2 kb that carries genes expressed early in the infection cycle that function in the replication of viral DNA (early genes) and genes expressed later in the infection cycle that function in the production of viral capsid proteins (late genes). For use as a cloning vector, some of the early and late genes are removed and replaced with a target gene under the control of appropriate mammalian expression signals. Although many cloning vectors are based on SV40 DNA, its use is restricted to small inserts because only a limited amount of DNA can be packaged into the viral capsid. Other vectors that can accommodate larger amounts of cloned DNA are derived from adenovirus; bovine papillomavirus, which can be maintained as a multicopy plasmid in some mammalian cells; and adeno-associated virus, which can integrate into specific sites in the host chromosome. Baculovirus delivery systems have also been developed to express target proteins in mammalian cells. Although baculoviruses cannot replicate in mammalian cells, they can be transduced into these cells with a transduction efficiency reaching 100% in some cases, where they enter the nucleus and transiently express heterologous genes that were inserted in the viral genome. In addition, stable artificial chromosome expression systems have been developed for some mammalian cell lines. These carry specific sequences for integration of one or more copies of a target gene by recombination.
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
