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Modifying secretion and post-translational processing in
insect cells
Eric Ailor and Michael J Betenbaugh*
The baculovirus–insect cell system is a valuable tool for the
expression of heterologous proteins. Due to limitations in the
intracellular processing environment, however, heterologous
secreted and membrane proteins are often insoluble, poorly
processed, or contain ‘non-human’ modifications. Recent
attempts to modify the insect cell secretory pathway by
overexpressing processing factors have demonstrated the
potential to overcome these limitations.
Addresses
Department of Chemical Engineering, Johns Hopkins University, 3400
North Charles Street, Baltimore, MD 21218, USA
*e-mail: beten@jhu.edu
Current Opinion in Biotechnology 1999, 10:142–145
http://biomednet.com/elecref/0958166901000142
© Elsevier Science Ltd ISSN 0958-1669
Abbreviations
BEVS baculovirus expression vector system
BiP immunoglobulin heavy chain binding protein
ER endoplasmic reticulum
Glc glucose
GlcNAc N-acetylglucosamine
IgG immunoglobulin G
Man mannose
PDI protein disulfide isomerase
SP signal peptidase
TGF transforming growth factor
Introduction
The baculovirus expression vector system (BEVS) is one
of the major recombinant DNA expression systems used
today for the production of a wide variety of heterologous
proteins [1•]. One of the major advantages of BEVS is
that it can be used to produce relatively large quantities
of post-translationally modified heterologous proteins
[2•]. These proteins are used in a number of applications,
including diagnostics, structure-function studies, and
vaccines. A large fraction of the recombinant protein pro-
duced in insect cells using BEVS, however, can
sometimes be poorly processed and accumulate as aggre-
gates [3••]. The production of very high levels of
polypeptide with the strong baculovirus polyhedrin pro-
moter and the shut down of host protein synthesis
following baculovirus infection may lead to a limitation in
the supply of secretory assistance factors [4,5]. In addi-
tion, although post-translational processing in insect cells
is more similar to mammalian cells than bacteria and
yeast, it is not always identical and, for applications such
as therapeutic proteins, this may be critical [6]. Improper
secretory processing can be especially problematic at sev-
eral days postinfection when the host cell’s
post-translational processing machinery has deteriorated.
An innovative approach to overcome these limitations in
the baculovirus–insect cell system is to engineer the
secretory pathway by supplementing secretory process-
ing proteins absent or limited in supply in the host insect
cell intracellular environment. The focus of this paper is
to review secretory processing in insect cells and high-
light recent attempts to improve secretion and
post-translational modifications in insect cells by overex-
pressing heterologous proteins involved in these
processing steps.
The secretory pathway
The eukaryotic secretory pathway is a complex multi-
organelle system which provides for the folding, assembly,
post-translational modification, and transport of newly
translated polypeptides. Associated with each of these
compartments is a collection of cellular proteins which
facilitate the secretion process.
Cytosolic processing and chaperones
Secreted proteins are initially translated from ribosome-
bound mRNA in the cytosol prior to translocation across
the endoplasmic reticulum (ER) membrane. Polypeptide
translocation across the ER membrane can occur either
by a cotranslational or post-translational mechanism [7].
Transport in mammalian cells is primarily cotranslational,
though both post-translational and cotranslational routes
are used in yeast. The predominant mechanism in insect
cells is not currently known.
The cytosolic chaperone hsp70 is believed to assist in
the translocation process by maintaining cytosolic
polypeptides in a translocation-competent form. In addi-
tion, this cytosolic chaperone may suppress aggregation
of these polypeptides by preventing nonspecific inter-
molecular hydrophobic interactions [8].
In coexpression studies of human hsp70 and murine
immunoglobulin G (IgG) in insect cells using BEVS,
hsp70 was shown to bind to IgG chains in soluble cell
fractions and enhance solubility of the IgG light chain
precursor [9••]. As a result of higher soluble levels of the
light chain precursor, subsequent processing of the light
chain precursor into its mature form led to an increase in
secreted levels of IgG.
Eukaryotic cytosolic hsp70 functions in concert with a col-
lection of cofactors, including hsp40 [10], Hip [11], and
Hop [12], in a cycle of binding and release of target
polypeptides which enhances the chaperoning efficiency
of hsp70. Coexpression of these cofactors may, therefore,
be necessary to optimize hsp70 function.
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Modifying secretion and post-translational processing in insect cells Ailor and Betenbaugh
ER processing
Following translocation, the polypeptides fold, assem-
ble, and may undergo additional processing in the ER.
As this environment includes very high concentrations of
unfolded polypeptides, chaperones, foldases, and other
enzymes are necessary to assist folding and limit aggre-
gation of the polypeptides [13].
Signal peptide processing
For secreted and many membrane-associated proteins, a
signal peptide at the amino-terminus is responsible for
targeting the polypeptide to the ER [14]. In some cases,
however, the signal peptide may remain attached to the
polypeptide indicating improper processing in the secre-
tory pathway. Tessier et al. [15] noted the accumulation
of prepropapain aggregates in the cytoplasm of infected
insect cells and modified the prepropapain to include a
insect-cell-derived signal peptide from bee melittin.
The replacement of the heterologous prepropapain sig-
nal peptide with the honeybee melittin signal peptide
resulted in a fivefold increase in propapain secretion.
The use of alternative signal peptides including bee
melittin, cecropin B, and baculovirus structural 64K gly-
coprotein, however, does not ensure the proper
processing of recombinant proteins [16]. For example,
aggregates of precursor single chain antibody fragments
(scFv) using the bee melittin signal peptide can accu-
mulate following expression in insect cells [17••].
Cleavage of the amino-terminal signal peptide in the ER is
accomplished by signal peptidase (SP). SP is a single mem-
brane protein in bacteria and a protein complex in
eukaryotes; however, both bacterial and eukaryotic SPs
can cleave signal peptides from either source interchange-
ably [18]. A recent study demonstrated that overexpression
of a bacterial signal peptidase enhanced the processing of
a single chain antibody fragment precursor [17••].
ER chaperones and foldases
As in the cytosol, chaperones act to improve protein fold-
ing and assembly in the ER by binding to regions of
nascent polypeptides that would otherwise form improp-
er associations and aggregate. Immunoglobulin heavy
chain binding protein (BiP) is an ER-resident chaperone
of the hsp70 protein family that complexes with a num-
ber of polypeptides destined for secretion [19] and may
also be involved in assisting translocation of polypep-
tides into the ER [20]. As extensive aggregation of
immunoglobulin was observed when expressed in insect
cells [21], recombinant BiP was coexpressed in insect
cells with IgG to determine if BiP could alleviate the
aggregation problem [3••]. Binding studies revealed that
the recombinant BiP associated with the immunoglobu-
lins. In addition, Western blot analysis, radiolabeling
studies, and enzyme-linked immunosorbent assays
(ELISA) demonstrated that coexpression of BiP signifi-
cantly increased the soluble and secreted IgG levels
obtained from Trichoplusia ni cells.
143
Like cytosolic hsp70, BiP functions optimally in the
presence of cofactors in a similar binding and release
cycle. Modeling studies have indicated that BiP’s activi-
ty is not yet optimized when the chaperone is expressed
independently [22•]. Altering the kinetics of BiP release
from a target polypeptide by coexpressing a cofactor
could reduce polypeptide aggregation and further
increase secreted protein yields.
There are a number of additional chaperones, including
calnexin and calreticulin, within the ER compartment
that act independently or in concert with other factors to
facilitate processing of both secreted and membrane
proteins along the correct folding pathway. Calnexin has
been identified as a membrane-bound chaperone which
associates with a number of membrane proteins [23] and
secreted glycoprotein folding intermediates [24] prior to
their folding and transport from the ER. Calreticulin is
the ER lumen homolog of calnexin and exhibits many of
the same binding characteristics as calnexin. Calnexin
appeared to be instrumental in facilitating the assembly
of class I histocompatability molecule heavy chains in
Drosophila melanogaster cells [25]. Recent research indi-
cates that overexpression of calnexin and calreticulin
using BEVS can also assist folding and assembly of
membrane proteins in Spodoptera frugiperda cells
(CG Tate, EM Whiteley and MJ Betenbaugh, unpub-
lished data).
Whereas chaperones prevent intermediates from follow-
ing unproductive folding pathways, catalytic enzymes in
the ER can accelerate folding along the proper pathway.
In this way, these cellular enzymes collaborate with
chaperones to maximize production of functional pro-
teins. Disulfide bond formation occurs as polypeptides
pass into the ER, which is a more oxidizing environment
than the cytosol. The ER enzyme protein disulfide iso-
merase (PDI) is known to catalyze oxidization,
reduction, and isomerization of disulfide bonds in vitro
[26]. A recent study has demonstrated that overexpres-
sion of PDI increases the folding and secretion of
heterologous IgG from insect cells [27]; however, a
mutation in the PDI carboxyl-terminus catalytic site can
actually reduce protein solubility in vivo in an anti-chap-
eroning role [28•]. The presence of the mutations may
destabalize the PD1 molecule and cause it to aggregate
with other polypeptides in the ER.
Another enzyme, peptidyl-prolyl cis-trans isomerase, cat-
alyzes the isomerization of the cis-trans X–Pro (where
X is any amino acid) peptide bond in polypeptides [29].
This step can be rate-limiting for the folding of some
polypeptides. An increase in the functional yield of a
recombinant membrane protein, human dopamine trans-
porter, was observed when expressed in an Sf-9 cell line
stably transformed with peptidyl-prolyl cis-trans iso-
merase as compared to similarly infected native Sf-9
cells [30••].
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144 Biochemical engineering
Oligosaccharide processing
In addition to folding and assembly, polypeptides may be
subject to other post-translational modifications in the ER,
including the addition of carbohydrate groups. N-glycosyla-
tion begins in insect and mammalian cells with the transfer of
the oligosaccharide Glc3Man9GlcNAc2 (where Glc, Man and
GlcNAc refer to glucose, mannose and N-acteylglucosamine,
respectively) from a lipid complex to an asparagine residue on
the polypeptide chain in the ER [31]. As the glycoprotein
passes through the ER and Golgi apparatus, enzymes trim
and add different sugars to this N-linked glycan. These mod-
ification steps can vary in mammalian and insect hosts leading
to differences in the structures of the final attached carbohy-
drates. These structural differences may have a significant
impact on the in vivo clearance rates of the glycoproteins from
mammalian and insect cell hosts [6].
Initial studies of N-linked glycans attached to heterologous
and homologous glycoproteins from insect cells indicated the
presence of primarily either high-mannose (Man9–5GlcNAc2)
or truncated, paucimannosidic oligosaccharides (Man3–1
GlcNAc2) [31]. Recent studies, however, have indicated the
presence of low levels of partially elongated hybrid and com-
plex structures terminating in GlcNAc or galactose [32•,33].
The ability to produce low levels of hybrid and complex
N-linked oligosaccharides was further supported by the find-
ing that these cells contained low levels of the enzymes
responsible for some of these processing steps [34].
The presence of paucimannosidic N-glycans in insect
cells may be explained, at least in part, by the presence
of an N-acetylglucosaminidase that cleaves GlcNAc
attached to the α(1,3) Man branch [35,36]. Chemicals
have been added in an attempt to inhibit this glycosidase
activity, but significant levels of paucimannosidic struc-
tures remain even in the presence of these inhibitors
[37]. These paucimannosidic structures are not common-
ly found on mammalian glycoproteins and represent a
glycoform that may be rapidly cleared in vivo.
Insect cells have also been genetically engineered to
enhance the extent of complex glycosylation. Wagner et al.
[38] coexpressed GlcNAc transferase I to increase the
number of recombinant glycoproteins with oligosaccha-
rides containing GlcNAc on the α(1,3) Man branch. The
introduction of a mammalian β(1,4)-galactosyltransferase
into insect cells using viral vectors [39] or stably-trans-
formed cell lines [40••] demonstrated that both approaches
can provide extended glycosylation of foreign glycopro-
teins expressed in insect cells.
Prosequence processing
In addition to signal-sequence processing, a number of
secreted proteins require cleavage at another site within
the polypeptide chain to obtain functional activity.
Heterologous proprotein processing may be absent or
severely limited in insect cells [41], whereas mammalian
cells possess a family of enzymes referred to as proprotein
convertases [42]. The most characterized proprotein con-
vertase to date is furin, which is localized to the trans-Golgi
apparatus in mammalian cells [43]. When coexpressed in
insect cells with transforming growth factor (TGF) β1,
furin was able to increase the functional yield of TGFβ1
7.8-fold when compared to TGFβ1 expressed alone [44••].
Conclusions
Due to its ability to produce heterologous proteins rapidly in
an eukaryotic environment, BEVS has been used to produce
a number of complex proteins. Many secreted and mem-
brane proteins produced in insect cells, however, frequently
form insoluble aggregates or are improperly processed.
Recent developments in engineering the protein processing
pathway of insect cells may be a viable method for over-
coming these limitations. Coexpression of chaperones,
peptidases, foldases, and glycosylating enzymes have
proven effective in enhancing secretion, processing, and gly-
cosylation of several heterologous proteins expressed in
insect cells. An improved understanding of the secretory
pathway and the role of helper proteins should provide even
greater tools to optimize the production of recombinant
secreted and membrane proteins from insect cells.
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