Expression in Insect Cells

Baculoviruses are rod-shaped viruses that infect insects and insect cell lines. They have double-stranded circular DNA genomes in the range of 90-180 kbp (Ayres et al., 1994). Viral infection results in cell lysis, usually 3-5 d after the initial infection, and the subsequent death of the infected insect. The nuclear polyhedrosis viruses are a class of baculoviruses that produce occlusion bodies in the nucleus of infect cells. These occlusion bodies consist primarily of a single protein, polyhedrin, which surrounds the viral particles and protects them from harsh environments. Most viruses of this type need to be eaten by the insect before infection will occur, and the occlusion body protects the viral particles from degradation in the insect gut. The polyhedrin gene is transcribed at very high levels late in the infection process (2-4 d post-infection). In cultured insect cells, the production of inclusion bodies is not essential for viral infection or replication. Consequently, the polyhedrin promoter can be used to drive target gene expression.

The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has become a popular tool of the production recombinant proteins in insect cells (Fraser, 1992). It is used in conjunction with insect cell lines derived from the moth Spodoptera frugiperda. These cell lines (e.g. Sf9 and Sf21) are readily cultured in the laboratory, and a scheme for constructing baculovirus recombinants is shown in Figure 8.6. The size of the baculoviral genome generally precludes the cloning of target genes directly onto it. Instead, the target gene is cloned downstream of the polyhedrin promoter in a transfer plasmid (Lopez-Ferber, Sisk and Possee, 1995). The transfer plasmid also contains the sequences of baculovirus genomic DNA that flank the polyhedrin gene, both upstream and downstream. To produce recombinant viruses, the recombinant transfer plasmid is co-transfected with linearized baculovirus vector DNA into insect cells. The flanking regions of the transfer plasmid participate in homologous recombination with the viral DNA sequences and introduce the target gene into the baculovirus genome. The recombination process also results in the repair of the circular viral DNA and allows viral replication to proceed through the re-formation of ORF1629 (a viral capsid associated protein that is essential for the production of viral particles). Recombinant viral infection can be observed microscopically by viewing viral plaques on a lawn of insect cells. Plaques containing recombinant virus will be unable to form occlusion bodies due to the lack of a functional polyhedrin protein (Smith, Summers and Fraser, 1983). Screening plaques this way is, however, technically difficult. Therefore, the transfer plasmids also usually contain the lacZ gene, or another readily observable reporter gene, which allows for the visual identification of recombinant plaques by their blue appearance after staining with X-Gal (Figure 8.6). Following transfection and plaque purification to remove any contaminating parental virus, a high-titre virus stock is prepared, and used to infect large-scale insect cell culture for protein production. The infected cells undergo a burst of target protein production, after which the cells die and may lyse.

Protein production in baculovirus infected insect cells has the advantage that very high levels of protein can be produced relative to other eukaryotic expression systems, and that the glycosylation pattern obtained is similar, but not identical, to that found in higher eukaryotes (Possee, 1997; Joshi et al., 2000). Baculoviruses also have the advantage that multiple genes can be expressed from a single virus. This allows the production of protein complexes whose individual components may not be stable when expressed on their own (Roy et al., 1997). The main disadvantages of producing proteins in this way is that the construction and purification of recombinant baculovirus vectors for the expression of target genes in insect cells can take as long as 4-6 weeks, and that the cells grow slowly (increasing the risk of contamination) in expensive media. An alternative approach to recombinant viral genome production uses f-

Baculoviral genome P

ORF'O^1 polyH g oRneagy

Overcome Shyness 101

Overcome Shyness 101

You can find out step by step what you need to do to overcome the feeling of being shy. There are a vast number of ways that you can stop feeling shy all of the time and start enjoying your life. You can take these options one step at a time so that you gradually stop feeling shy and start feeling more confident in yourself, enjoying every aspect of your life. You can learn how to not be shy and start to become much more confident and outgoing with this book.

Get My Free Ebook

Post a comment