Biomedical Engineering Reference
In-Depth Information
5.1.3
Insect Cells
The production of heterologous proteins in lepidopteran insect cells using bacu-
lovirus expression vector has several highly desirable attributes. The process
development time for primary production of a recombinant protein is short,
high yield is possible (
30 mg l -1 ) and the system has the ability to carry out a
wide array of post-translational processing [190].
Insect cells lack the capacity to process carbohydrate moieties to “complex”
type containing Gal and terminal SA residues, express highly mannosylated
proteins, and also are reported to attach xylose [191-193]. They do, however,
accomplish the most important stage in the overall folding and quality control
of glycoproteins involving glucosidase II and glucosyl transferase [194]. There
have been some reports indicating the formation of complex glycosylated
recombinant proteins in baculovirus infected insect cells [193]. In addition,
there has been a report of successful O -linked glycosylation on pseudorabies
protein (gp50) following its expression in insect cells [195].
Recombinant baculoviruses, especially Autographa californica nuclear poly-
hedrosis virus (AcNPV), are widely used to express heterologous proteins in a
eukaryotic processing environment. They are particularly useful for the high-
level expression of eukaryotic proteins with fastidious co- or post-translational
processing requirements [194]. The virus infects Autographa californica and
30 other insect species.The most commonly used cell line is derived from the fall
armyworm , Spodoptera frugiperda . Examination of N -glycosylation processing
of an alternative cell line ( Estigmena acrea ) indicated significant differences in
the glycosylation pattern. Except for ST, Ea4 cells possess most of the enzymes
involved in production of hybrid and complex N -glycans [196]. Traditionally,
very late viral promoters (driving expression of polyhedrin or p10) have been
used in expression vectors. However, post-translational events such as secretion
and glycosylation appear to occur more efficiently when early promoters are
used [197-199]. Recently, a new type of baculovirus vector has been developed
that can express foreign genes immediately after infection under the control of
the promoter from the viral immediately early (ie1) gene. These vectors have
been used to modify the N -glycan processing capabilities of insect cells by direct-
ing the expression of a heterologous processing enzyme (
>
1,4-GalT) during the
early phase of infection. The enzyme then functions as part of insect cell machi-
nery and contributes to the production of a foreign glycoprotein synthesized
later in infection with more extensively processed N -glycans [200]. Interestingly,
GPI membrane anchors are efficiently produced in the baculovirus system [201].
b
5.1.4
Plants
Transgenic plants are emerging as an important expression system for foreign
genes. Their major attractions are the potential for protein production on an
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