3.36). With this system, over 90% of the baculovirus plaques are recombinant.
Figure 3.36 Production of recombinant baculovirus. Single Bsu36I sites are engineered into gene 603 and a gene (1629) that is essential for AcMNPV replication. These genes flank the polyhedrin gene in the AcMNPV genome. After a baculovirus with two engineered Bsu36I sites is treated with Bsu36I, the segment between the Bsu36I sites is deleted. Insect cells are cotransfected with Bsu36I-treated baculovirus DNA and a transfer vector with a gene of interest under the control of the promoter (p) and terminator (t) elements of the polyhedrin gene and the complete sequences of both genes 603 and 1629. A double-crossover event (dashed lines) generates a recombinant baculovirus with a functional gene 1629. With this system, almost all of the progeny baculoviruses are recombinant.
To eliminate the need to use plaque assays to identify and purify recombinant viruses, several methods have been developed that introduce the target gene into the baculovirus genome at a specific nucleotide sequence by recombination, either in an intermediate bacterial host, such as E. coli, or in vitro outside of a host cell. Transfection of insect cells is required only for the production of the heterologous protein. AcMNPV DNA can be maintained in E. coli as a plasmid known as a bacmid, which is a baculovirus–plasmid hybrid molecule. In addition to AcMNPV genes, the bacmid contains an origin of replication for propagation in E. coli, a kanamycin resistance gene for selection of the bacmid, and an integration site (attachment site) that is inserted into the lacZ′ gene without impairing its function (Fig. 3.37A and B). Another component of this system is the transfer vector that carries the gene of interest cloned between the polyhedrin promoter and a terminator sequence. In the transfer vector, the target gene expression unit (expression cassette) and a gentamicin resistance gene are flanked by DNA attachment sequences that can bind to the attachment site in the bacmid (Fig. 3.37B). An ampicillin resistance gene lies outside the expression cassette for selection of the transfer vector.
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
