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little influence on the downstream processing strate-
gy, except for special designs where the insect cells
are physically retained. Examples are stationary beds
(Kompier et al., 1991), microcapsules (King et al.,
1989) or production of secreted proteins in perfusion
reactors. In these cases, obviously the cell separation
is built into the culture system.
The choice of the subsequent steps depends on the
localisation of the protein (cell associated or secret-
ed). For cell associated products, the cell paste usu-
ally goes through one or several freeze/thaw cycles
to facilitate lysis. Cells are then mechanically bro-
ken or lysed in detergent, and/or hypotonic buffers.
Cell debris is eliminated by another solid-liquid sep-
aration and chromatography follows. Membrane pro-
teins represent a special class of proteins which require
specific attention. Expression levels are usually much
lower than soluble proteins and purification from the
membrane may irreversibly damage the native struc-
ture. This may result in a pure but modified protein.
For proteins secreted on large scale, the conditioned
medium is concentrated 10–50 fold by ultrafiltration
or precipitation induced by salts or pH. The concen-
trate is then processed by chromatographic techniques.
In some cases, the concentration step is omitted and
non-concentrated supernatant is directly processed by
immuno-affinity binding (Kikuchi et al., 1994; Hur-
witz et al., 1991).
Chromatography is based on separation by charge,
hydrophobicity, size or affinity for a ligand (molecu-
lar recognition). Since the protein is normally properly
folded and immunoreactive, many purification strate-
gies include an immuno-affinity step. It is quite often
used very early in the purification process. Epitopes
used for the affinity capture may be from the protein
itself (Greenfield et al., 1988; Pochon et al., 1992;
Singer et al., 1994), from a small tag (Furlong et
al., 1988; Clark et al., 1992; Herren & Pech, 1992;
Haubruck et al., 1993; Wang et al., 1994) or a fusion
partner (Cooke et al., 1994; Peng et al., 1993). This
allows the use of the same generic method for the
recovery of many different proteins. Depending on the
final target purity, immuno-affinity purification may be
the only necessary step (Clark et al., 1992).
Some enzymes are expressed at such high levels in
the baculovirus/insect cell system, that they were puri-
fied to homogeneity and crystallised: bovine inositol
polyphosphate 1-phosphatase (York et al., 1994), rat
acid phosphatase (Vihko et al., 1993), human purple
acid phosphatase (Hayman & Cox, 1994).
Expression of cytoplasmic or trans-membrane pro-
teins can lead to aggregation, insolubilisation and con-
sequent inactivation. Some proteins are made with only
a small proportion soluble: human 5 lipoxygenase
(Denis et al., 1991), phosphatase I subunit (Berndt
& Cohen., 1990), glucocorticoid receptor (Alnemri
et al., 1991), protein kinase C (Rankl et al., 1994),
antiporter (Fafournoux et al., 1991). Thirty
percent of the transferrin receptor had non-native disul-
fide bridges (Domingo & Trowbridge, 1988). These
non-active or insoluble species can only be restored
active after solubilisation into denaturants and refold-
ing, which renders the whole process very inefficient.
Both cytoplasmic and secreted proteins are subject to
proteolytic degradation(McGlynn et al., 1992; Jäger et
al., 1992; Grabenhorst et al., 1992) which complicates
the purification.
Some recombinant proteins can also be produced
in larvae instead of cells in culture. Purification
from larvae is not specifically addressed here. It has
been published for cytoplasmic proteins such as v-
sis (Morishita & Maeda, 1991), adenosine deaminase
(Medin et al., 1990) and secreted proteins such as
human (Maeda & Kawai, 1985), mouse
interleukin–3 (Miyajima et al., 1987), feline inter-
feron (Nakamura et al., 1992) and apolipoprotein E
(Gretch et al., 1991). For secreted proteins, down-
stream processing of the larvae hemolymph is identical
to that of the cell culture supernatant.
Wild type (or recombined with a toxic gene) bac-
ulovirus is also produced at large scale for use as