Evolution and Genetic Engineering - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 728

precursors added (e.g. amino sugars) to improve processing. Cell lysis can be reduced by
adding genes (e.g. bel 2) to the host cell that reduces apotosis. An engineering solution is
to remove the product from the medium as it is formed. For example, perfusion (i.e. chemo-
stat or CSTR) systems with an integrated product capture step can be used.

14.7.5. Insect Cell-Baculovirus System
A popular alternative for protein production at small ( 100 L) or laboratory scale is the

insect cell-baculovirus system. This system is particularly attractive for rapidly obtaining bio-
logically active protein for characterization studies. Typical host cell lines come from the fall
armyworm ( Spodoptera frugiperda ) and the cabbage looper ( Trichoplusia ni ). The baculovirus,
Autographa californica nuclear polyhedrosis virus (Ac NPV), is used as a vector for insertion of
recombinant DNA into the host cell. This virus has an unusual biphasic replication cycle in
nature. An insect ingests the occluded form, in which multiple virus particles are embedded
in a protein matrix. The protein matrix protects the virus when it is on a leaf from environ-
mental stresses (e.g. UV radiation). This protein matrix is from the polyhedrin protein. In the
midgut of the insect, the matrix dissolves, allowing the virus to attack the cells lining the
insect's gut; this is the primary infection. These infected cells release a second type of virus;
it is nonoccluded (no polyhedrin matrix) and buds through the cell envelope. The nonoc-
cluded virus (NOV) infects other cells throughout the insect (secondary infection).
In insect cell culture, only NOVs are infectious, and the polyhedrin gene is unnecessary.
The polyhederin promoter is the strongest known animal promoter and is expressed late
in the infection cycle. Replacing the polyhederin structural gene with the gene for a target
protein allows high-level target protein production (up to 50% of cellular protein). Proteins
that are secreted and glycosylated are often made at much lower levels than nonsecreted
proteins.
In addition to high expression levels, the insect-baculovirus system offers safety advan-
tages over mammal-retrovirus systems. The insect cell lines derived from ovaries or embryos
are continuous but not transformed. The baculovirus is not pathogenic toward either plants
or mammals. Thus, the insect-baculovirus system offers potential safety advantages. Another
important advantage is that the molecular biology and high-level expression of correctly
folded proteins can be achieved in less than a month. This system also has the cellular
machinery to do almost all the complex posttranslational modifications that mammalian cells
do. However, even when the machinery is present, at least some of the proteins produced in
the insect cell-baculovirus system are not processed identically to the native protein. In some
cases, their slight variations may be beneficial (e.g. increased antigenic response in the devel-
opment of an AIDS vaccine), while in others they may be undesirable. While complex glyco-
forms (including silaic acid) have been made, it is more common to observe only simple
glycoforms. Production of complex forms requires special host cell lines and is sensitive to
culture conditions.
The insect cell-baculovirus system is a good system to illustrate an overall schematic
perspective on heterologous protein production. Any bioprocess for protein production is
complex, consisting of the nonlinear interaction of many subcomponents. Thus, the optimal
process is not simply the sum of individually optimized steps. Fig. 14.14 presents a schematic
view for the insect cell-baculovirus system. Because of the viral component, this system is

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