Molecular Biotechnology. Bernard R. Glick
Figure 3.37 Construction of a recombinant bacmid. (A) An E. coli plasmid is incorporated into the AcMNPV genome by a double-crossover event (dashed lines) between DNA segments (5′ and 3′) that flank the polyhedrin gene to create a shuttle vector (bacmid) that replicates in both E. coli and insect cells. The gene for resistance to kanamycin (Kanr), an attachment site (att) that is inserted in frame in the lacZ′ sequence, and an E. coli origin of replication (oriE) are introduced as part of the plasmid DNA. (B) The transposition proteins encoded by genes of the helper plasmid facilitate the integration (transposition) of the DNA segment of the transfer vector that is bounded by two attachment sequences (attR and attL). The gene for resistance to gentamicin (Genr) and a gene of interest (GOI) that is under the control of the promoter (p) and transcription terminator (t) elements of the polyhedrin gene are inserted into the attachment site (att) of the bacmid. The helper plasmid and transfer vector carry the genes for resistance to tetracycline (Tetr) and ampicillin (Ampr), respectively. (C) The recombinant bacmid has a disrupted lacZ′ gene (*). The right-angled arrow denotes the site of initiation of transcription of the cloned gene after transfection of the recombinant bacmid into an insect cell. Cells that are transfected with a recombinant bacmid are not able to produce functional β-galactosidase.
Bacterial cells carrying a bacmid are cotransformed with the transfer vector and a helper plasmid that encodes the specific proteins (transposition proteins) that mediate recombination between the attachment sites on the transfer vector and on the bacmid and that carries a tetracycline resistance gene (Fig. 3.37B). After recombination, the DNA segment that is bounded by the two attachment sites on the transfer vector (the expression cassette carrying the target gene) is transposed into the attachment site on the bacmid, destroying the reading frame of the lacZ′ gene (Fig. 3.37C). Consequently, bacteria with recombinant bacmids produce white colonies in the presence of IPTG and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). White colonies that are resistant to kanamycin and gentamicin and sensitive to both ampicillin and tetracycline carry only a recombinant bacmid and no transfer and helper plasmids. After all of these manipulations, the integrity of the cloned gene can be confirmed by PCR. Finally, recombinant bacmid DNA can be transfected into insect cells, where the cloned gene is transcribed and the heterologous protein is produced.
The simultaneous expression of two or more cloned genes can lead to the formation of functional multimeric protein complexes. This can be accomplished by transfecting insect cells with a single recombinant baculovirus expressing multiple proteins. AcMNPV is particularly amenable to carrying large insertions, up to 38 kb, with several foreign genes due to its flexible envelope. In one study, the genes for three different envelope structural proteins from the human severe acute respiratory syndrome coronavirus (SARS-CoV) were expressed simultaneously at a high level from a single baculovirus vector (Fig. 3.38A). The proteins were found to assemble spontaneously and stably into virus-like particles (Fig. 3.38B). Virus-like particles, comprised of the assembled protein coat of a virus but without the nucleic acid genome, are the basis for some subunit vaccines (chapter 7).
Figure 3.38 Production of virus-like particles using a baculovirus-insect cell expression system. (A) Viral spike (S), membrane (M), and envelope (E) proteins, which comprise the envelope of the human SARS-CoV, are expressed in insect cells from a single recombinant baculovirus vector carrying all three viral genes. (B) Following expression, the S, M, and E proteins self-assemble to form a SARS-CoV virus-like particle that resembles the original virus but does not contain the viral genetic material. The virus-like particle is a candidate vaccine for protection against SARS. Pp, polyhedrin promoter; 10p, baculovirus p10 promoter.
Mammalian Glycosylation and Processing of Proteins in Insect Cells
Although insect cells can process proteins in a manner similar to that of other eukaryotes, some mammalian proteins produced in S. frugiperda cell lines are not authentically glycosylated. For example, insect cells do not normally add galactose and terminal sialic acid residues to N-linked glycoproteins. Where these glycans are normally added to mannose residues during the processing of some proteins in mammalian cells, insect cells will trim the oligosaccharide to produce paucimannose (Fig. 3.39). Consequently, the baculovirus system cannot be used for the production of several important mammalian glycoproteins. To ensure the production of “humanized” glycoproteins with accurate glycosylation patterns, insect cell lines have been constructed that express a combination of mammalian glycosyltransferases (Fig. 3.39).
Figure 3.39 N-glycosylation of proteins in the Golgi apparatus of insect, human, and “humanized” insect cells. While the sugar residues added to N-glycoproteins in the endoplasmic reticulum are similar in insect and human cells, further processing in the Golgi apparatus yields a trimmed oligosaccharide (paucimannose) in insect cells and an oligosaccharide that terminates in sialic acid in human cells. To produce recombinant proteins for use as human therapeutic agents, “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