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Expression of neuroparsin cDNA in insect cells using baculovirus vectors.

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26
Girardie et al.
Archives of Insect Biochemistry and Physiology 46:26–35 (2001)
This article originally published in Volume 36
Archives of Insect Biochemistry and Physiology 36:11–23 (1997)
Expression of Neuroparsin cDNA in
Insect Cells Using Baculovirus Vectors
J. Girardie,1* H. Chaabihi,2 B. Fournier,1 M. Lagueux,3 and A. Girardie1
1
Laboratoire de Neuroendocrinologie, URA CNRS 1138, Université Bordeaux I,
Talence Cedex, France
2
Protéine Performance, St. Christol-les-Alès, France
3
UPR CNRS 9022 Réponse Immunitaire et Développement chez les Insectes,
Strasbourg Cedex, France
The cDNA encoding neuroparsin A, a polytropic neurohormone
of the locust, Locusta migratoria, was inserted into the genome
of Autographa californica nuclear polyhedrosis virus such that
transcription was under control of the p10 promoter. A polypeptide having the same charge and the same apparent molecular
weight as the authentic neuroparsin A and that was reactive
against neuroparsin immune serum was produced in recombinant virus-infected lepidopteran cell lines but not in control
virus-infected cells. The baculovirus-expressed polypeptide was
purified by two steps of liquid chromatography (anion exchange and reversed phase) which were previously used to
purify the natural neuroparsin. The purified baculovirus-expressed polypeptide enhanced fluid reabsorption of everted
rectum preparations, as did the natural neuroparsin. Thus,
this gene expression system produced a polypeptide identical to authentic neuroparsin. Arch. Insect Biochem. Physiol.
36:11–23, 1997. © 1997 Wiley-Liss, Inc.
Key words: insect; Locusta migratoria; neuroparsin; gene expression;
baculovirus-insect cells
INTRODUCTION
Neuroparsin is a polytropic neurohormone
with a distinctive amino acid sequence isolated
from the neural lobes of the corpora cardiaca of
Locusta migratoria (Girardie et al., 1989, 1990).
It is produced as an 83 residue polypeptide
(neuroparsin A, molecular mass = 8,759 daltons,
calculated pl = 6.71) by the A1-type cell bodies of
the pars intercerebralis (Bourême et al., 1987) and
is partially enzymatically transformed into a less
basic form of 78 residues (neuroparsin B, molecular mass = 8,188 daltons, calculated pl = 5.98) dur© 2001 Wiley-Liss, Inc.
ing storage at the axonal endings located in the
corpora cardiaca (Girardie et al., 1987b). Both
Acknowledgments: We are grateful to Mrs. Sophie Daburon
and Miss Catherine Cêtre for their technical assistance. The
authors gratefully thank Dr. Thomas Durkin for linguistic
review of the manuscript. This work was partly supported
by the Conseil Régional d’Aquitaine, Pôle Médicament.
*Correspondence to: J. Girardie, Laboratoire de Neuroendocrinologie, URA CNRS 1138, Avenue de Facultés,
Université Bordeaux I, 33405 Talence Cedex, France. E-mail:
j.girardie@invertebre.u-bordeaux.fr
Received 29 October 1996; Accepted 8 February 1997.
Baculovirus-Expressed Neuroparsin
forms (A and B) trigger antijuvenile (Girardie
et al., 1987a), antidiuretic (Fournier and Girardie,
1988), hypertrehalosemic, and hyperlipemic
(Moreau et al., 1988) effects in the locust. Only
the antidiuretic effect is well studied at the target organ (Girardie and Fournier, 1993; Fournier
et al., 1994; Nogaro et al., 1995). A large quantity of neuroparsin is needed to characterize its
receptor(s). The relative simplicity of the neuroparsin cDNA sequence, composed only of the
neurohormone A and 22 residue signal peptide
(Lagueux et al., 1992), makes the synthesis of
neuroparsin in a gene expression system feasible.
To our knowledge, only four insect neurohormones — the diuretic hormone of tobacco hornworm,
the eclosion hormone and the prothoracotropic hormone of silkworm, and the pheromone biosynthesis activating neuropeptide of corn earworm — have
been successfully produced by recombinant DNA
techniques. Three expression systems have been
used: a vector plasmid expressed in Escherichia coli
(Kono et al., 1990), a shuttle plasmid expressed in
yeast (Hayashi et al., 1990), and a baculovirus vector expressed either in whole insect (Maeda, 1989;
O’Reilly et al., 1995) or in insect cells (Eldridge et
al., 1991; Vakharia et al., 1995). Each of the expression systems led to the production of biologically active neurohormones. The levels of expression
reported were different and were dependent both
on the neurohormone and the particular expression
system.
In the present study, we have attempted to
express a cloned neuroparsin cDNA in insect cells
using a baculovirus vector. The expression system produced a protein whose charge, apparent
molecular weight, and reactivity to neuroparsin
immune serum were similar to those of authentic neuroparsin A. This protein, purified by two
steps of liquid chromatography used for the natural neuroparsin, triggered a similar antidiuretic
activity as the authentic neuroparsin.
MATERIALS AND METHODS
Generation of Recombinant Baculoviruses
Expressing Neuroparsin cDNA
Full-length neuroparsin cDNA was purified
from plasmid pTZ-neurocDNA (Lagueux et al.,
1992) as a 360 bp BsaH I–Pvu II fragment. The
BsaH I site is located 12 nucleotides upstream of
27
the ATG start codon of neuroparsin gene, and the
Pvu II site is located 22 nucleotides downstream
of the stop codon. Following blunt-ending with
Klenow polymerase, the BsaH I–Pvu II fragment
was ligated to plasma pGm16 (Blanc et al., 1993)
previously digested with Bgl II and blunt-ended.
The transfer vector pGm16 bears the p10 promoter followed by a Bgl II site, for cloning of foreign genes, and baculovirus-flanking sequences
for recombination at the p10 locus. Spodoptera
frugiperda Sf9 cells (CRL 1711, ATCC; Rockville,
MD) were cotransfected with DNA of the generated vector carrying the neuroparsin gene under
control of the p10 promoter (pGm16-NP) and genomic DNA of the baculovirus AcSLP10 (Chaabihi
et al., 1993). For this cotransfection, the lipofection method (Felgner and Ringold, 1989) was
followed using the DOTAP reagent (BoehringerMannheim, Germany). Because AcSLP10 is a
modified Autographa californica polyhedrosis virus, which possesses only one strong late promoter
(the p10) with the polyhedrin coding sequence inserted downstream, unrecombined AcSLP10 has
an occlusion body-positive phenotype. Consequently, screening and purification of recombinant
baculoviruses were carried out by plaque assay
as described by Summers and Smith (1987).
Three selected recombinant viruses expressing the neuroparsin gene under p10 promoter control were used in the present study and designated
bPP1-08-1111, bPP1-08-2111, and bPP1-08-5111.
Production of Neuroparsin in Insect Cells
Sf9 cells were grown in 25 cm2 Falcon flasks
with 5 ml of TC 100 medium (Gibco BRL, Gaithersburg, MD) supplemented with 5% fetal calf serum. Cell cultures with a density of 4 × 106 cells
per flask were infected with either one of the three
recombinants (bPP1-08-1111, bPP1-08-2111, bPP108-5111) or with AcSLP10 as control (two flasks
for each virus type). Sf9 cells were also grown in
75 cm2 Falcon flasks with 15 ml of medium, and
cell cultures with a density of 20 × 106 cells per
flask were infected with either the recombinant
bPP1-08-1111 (three flasks) or with AcSLP10 as
control (one flask).
Infected cells were harvested 4 days postinfection by centrifugation at 3,000 rpm (Jouan
CR 312) for 10 min at 4°C. Cell pellets were
washed twice with phosphate buffered saline. Cell
28
Girardie et al.
pellets and the clarified supernatant (culture medium) were stored at –80°C until used.
Extraction of the Recombinant Neuroparsin
Extraction of the putative recombinant neuroparsin was attempted from both clarified supernatants (culture medium) and pellets of cells
infected with the three recombinant viruses.
Clarified supernatants and pellets of cells infected
with the ACSLP10 control virus were treated in
the same way.
All supernatants were dialyzed overnight at
4°C against distilled water using 6,000 dalton cutoff tubing since neuroparsin is conserved using
this procedure (Fournier and Girardie, 1988).
Even after dialysis, the culture medium was sometimes not clear (chiefly for the largest volume),
and insoluble material was then separated by centrifugation at 3,000 rpm. Soluble and insoluble
fractions were separately freeze-dried and stored
at –80°C until extraction.
Two extraction media were tested both on cell
pellets and on soluble and insoluble freeze-dried
dialyzed supernatants: Tris-HCl buffer (sample
buffer of PAGE or SDS-PAGE or solvent of anion
exchange chromatography) and 70% methanol (extraction medium or natural neuroparsin). Proteins
extracted by the buffer were analyzed directly by
electrophoresis or chromatography, while proteins
extracted by methanol were analyzed following
total evaporation of the extraction medium and
redissolution in adequate buffers.
Electrophoresis Analysis
The extracted proteins were analyzed by either 7.5% native PAGE according to Laemmli
(1970) or 12% SDS-PAGE according to Schägger
and Von Jagow (1987). Electrophoresis was performed with the Bio-Rad Mini-Protean II dual
Slab Cell (Bio-Rad, Richmond, CA) at constant
voltage according to the manufacturer’s protocols.
Gels were either stained in a fixative of 50%
methanol solution containing 10% acetic acid and
0.1% Coomassie brilliant blue R250 or directly
electrotransferred for immunodetection with antineuroparsin immune serum.
Western Blotting Procedures
Following native or 12% SDS-PAGE, proteins
were transferred onto Immobilon-P (Millipore, St.
Quentin, Yvelines, France) using the Bio-Rad Mini
Trans-Blot Electrophoretic Transfer Cell according to the manufacturer’s protocols (25 mM Trisglycine buffer, pH 8.3, with 20% v/v methanol).
The transfer was carried out at a constant voltage (100 v) and a current of 180 mA at the beginning and 250 mA at the end. The efficiency of
sample transfer was evaluated by Coomassie blue
staining of the gel.
Immunostaining
Blots were treated for 30 min at room temperature in buffer A (0.2 M Tris-HCl buffer, pH
7.2, 3% bovine serum albumin [BSA]) to saturate
nonspecific reactive sites, followed by a 16 h incubation at 4°C with anti-neuroparsin serum (1/
2,000) in the same buffer. After three washes,
blots were incubated for 2 h at room temperature with a peroxidase-labelled goat anti-rabbit
Fab (1/1,000) in buffer A. Following buffer washes,
the peroxidase reaction was carried out for 5 min
at room temperature with 0.05% diaminobenzidine as chromogen (DAB; Sigma, St. Quentin
Fallavier, France) and 0.06% hydrogen peroxidase
as substrate.
Purification of the Recombinant Neuroparsin
Anion-exchange liquid chromatography.
The extracted proteins were injected onto a Mono
Q HR 5/5 column (Pharmacia, Gaithersburg MD)
and a Beckman (Fullerton, CA) gradient system
(model 333) with a variable wavelength detector
(model 165). A 33 min linear gradient from 0–50%
of 1 M NaCl and then a 2 min linear gradient from
50–100% were employed at a flow rate of 1 ml/min
at room temperature. Fractions were collected
manually.
Reversed-phase chromatography, desalting, and purification. Fractions collected
from anion-exchange chromatography were loaded
onto a reversed-phase C8 Pro RPC 5/10 column
(Pharmacia) and eluted with a linear gradient
from 28–50% of CH3CN in 25 min at a flow rate
of 0.5 ml/min. Peak fractions were collected manually and tested for antidiuretic activity.
In Vitro Rectal Test
Antidiuretic activity of the putative neuroparsin peak was measured using the everted
rectal sac preparation as previously described
Baculovirus-Expressed Neuroparsin
(Fournier and Girardie, 1988). Following a prior
incubation of 1 h in chloride-free standard saline
for equilibration, recta were emptied and refilled
with 10 µl of standard saline (Hanrahan and
Phillips, 1982) containing either natural neuroparsin or putative biosynthetic neuroparsin or no
neuroparsin. The biological response (fluid reabsorption) was then measured after a 1 h incubation.
RESULTS
Electrophoretic Analysis
Natural native neuroparsins A and B are
unique polypeptide chains having 12 cysteine residues. Neuroparsin A (calculated pl = 6.71) and
neuroparsin B (calculated pl = 5.98) can be easily
distinguished by their charge using native 7.5%
PAGE (Girardie et al., 1987a). In spite of the 12
cysteines forming disulfide bonds, denaturation of
neuroparsin using SDS induces self-association of
two, three, or four molecules (Girardie et al., 1985;
Girardie and Fournier, 1993). In the presence of
a reducing agent (β-mercaptoethanol), the monomer is the major form and often the only one visualized when Coomassie blue is used to stain the
gels (Fig. 3A). However, when sensitive methods
of detection are used, such as immunodetection,
the monomeric form is always the major one; however, self-association of molecules is generally observed (Fig. 1A).
Extracts of cells and their culture media upon
electrophoresis separated into several protein
bands following the Coomassie blue staining (Fig.
3). Some of these protein bands had the same
electrophoretic mobility as natural neuroparsin.
However, only specific detection with the antineuroparsin immune serum was capable of confirming the expression of either neuroparsin or
neuroparsin-like molecules by the baculovirus-cell
system.
Whatever the extraction medium and the
type of electrophoretic separation used, protein
mixtures from equivalent amounts of either media
or cells infected by the three distinct recombinant
viruses (1111, 2111, 5111) contained immunoreactive band(s), while no band was detected from the
culture medium or the cells infected by the control virus (Fig. 1). Following 7.5% PAGE, only one
immunoreactive band, having the same charge as
neuroparsin A, was detected both in cell and me-
29
dium extracts but chiefly in cell extracts (Fig. 2).
This result indicates that the translation product
of the neuroparsin cDNA composed of a 22 residue peptide signal containing two basic arginins
and neuroparsin A is rapidly processed into
neuroparsin A in the cells. A more acidic band (putative neuroparsin B) was never detected either
in the cell or in the medium extracts. Following
SDS-PAGE, pure natural neuroparsin A was detected for the most part as a monomer (the lowest
molecular weight band) and also as two self-associated forms: a dimer and tetramer (Fig. 1A). In
cell extracts, several slightly immunostained
bands were sometimes visualized (cross-reactivity), and two bands were strongly immunostained.
One band (the lowest molecular weight) had the
same apparent molecular weight as the monomeric natural neuroparsin A, and the other appears to comigrate with the tetramer (Fig. 1C).
In extracts of the medium, one or two reactive
bands were immunodetected (Fig. 1M). The lowest molecular weight band had the same apparent molecular weight as the dimer of natural
neuroparsin. The other band, which was always
less reactive, had an apparent molecular weight
between that of the monomer and the tetramer
and could represent self-association of three molecules (trimer) of neuroparsin (Fig. 1M).
The quantity of expressed neuroparsin extracted from cells, evaluated by the intensity of
its immunoreactivity, appeared to be higher than
the quantity of neuroparsin contained in the culture medium of the cells (Figs. 1, 2), indicating
that neuroparsin A is either mainly present in
cells or is more easily extracted from the cells.
SDS-PAGE of pellets, obtained with both extraction solutions from either media or cells and
reextracted with the sample buffer, showed numerous bands, but a band having the same apparent molecular weight as neuroparsin was
observed only in the pellet of extracted cells (Fig.
3, lanes 1–4). This band was also immunostained
with anti-neuroparsin immune serum (data not
shown). Thus, it appears that the expressed
neuroparsin is localized mostly in cells rather than
in the culture medium. Moreover, this result also
indicates that the extraction of the expressed
neuroparsin was not complete.
No significant difference in the quantity of
extracted neuroparsin was observed between the
30
Girardie et al.
Fig. 1. Immunoblot analysis of natural neuroparsin A (A)
purified from neural corpora cardiaca from 50 locusts and of
proteins extracted by 70% methanol from 106 cells (C) and
from the entire volume of the corresponding culture medium
(M) following infection by either 0111 AcSLP10 control virus
(lane 0) or 1111 (lane 1), 2111 (lane 2), and 5111 (lane 5)
AcSLP10 recombinant viruses. Following 12% SDS-PAGE,
proteins were electroblotted and probed with anti-neuroparsin
immune serum. Three bands were revealed for the natural
neuroparsin (A): the lowest form (major band = true monomeric neuroparsin) and two heavier forms induced by SDS
(dimer and tetramer). Bands were present in extracts of cells
and of their culture media only when cells were infected with
recombinant viruses (C, lanes 1, 2, 5; M, lanes 1, 2, 5). In
cells (C, lanes 1, 2, 5), the most immunoreactive band (the
lowest molecular weight and the largest) had the same apparent molecular weight as the true monomeric neuroparsin.
Bands corresponding to dimeric and tetrameric neuroparsin
were not easily distinguishable among several slightly stained
bands revealed by cross-reactivity. In media (M, lanes 1, 2,
5) two bands were revealed. The most immunoreactive and
the largest band had the same apparent molecular weight as
the SDS-induced dimer of neuroparsin. The other band,
heavier than the dimer and lighter than the tetramer, probably corresponds to the SDS-induced trimeric neuroparsin.
Fig. 2. Immunoblot analysis of natural neuroparsin A (lane
A) purified from 20 corpora cardiaca and of proteins extracted
by Tris-HCl from 2 × 106 cells infected with the 1111 recombinant virus and from the entire volume of the corresponding culture medium (lane M). Following 7.5% PAGE, proteins
were electroblotted and probed with anti-neuroparsin immune
serum. Only one band was revealed clearly in cells (lane C)
and poorly in medium (lane M). This band had the same
charge as natural neuroparsin A.
Fig. 3. Effect of the SDS-PAGE sample buffer on the 70%
methanol insoluble proteins (pellet) from cells infected with
the 5111 recombinant virus and their culture medium. Following 12% SDS-PAGE, proteins were stained with Coomassie
blue. The sample buffer extracted numerous proteins from
the cell pellet as well as the medium pellet, giving an idea of
the number of total protein bands present but not immunodetected on the Western blot of the Fig. 1. A protein (the
lowest) having a similar apparent molecular weight to neuroparsin A was present only in the cell pellet. A: Neuroparsin A
purified from neural corpora cardiaca from 50 locusts. C:
(lanes 4, 2, 1): Three serial half dilutions of protein extracts
from cells. M: (lanes 8, 4): Two serial half dilutions of protein
extracts from culture medium.
Baculovirus-Expressed Neuroparsin
three recombinant viruses. Quantitative variations between distinct batches of the same recombinant virus were sometimes more evident than
between the three recombinant viruses (data not
shown).
The level of neuroparsin expression was
evaluated on immunoblots by measuring the optical density of the major band obtained with expressed neuroparsin and comparing it to that of
monomeric natural neuroparsin. The band intensity of monomeric neuroparsin expressed by 106
infected insect cells was always around half as
much as the band intensity of natural monomeric
neuroparsin purified from neural corpora cardiaca
of 50 locusts (Figs. 1, 2). So, the level of neuroparsin expression was quite low, at most 3 µg and
even lower per 106 infected cells since the neural
corpora cardiaca of one locust contains about 120
ng of neuroparsin A.
Chromatographic Purification
Proteins extracted from the culture media
from either the experimental recombinant virus
or the control virus transfected cells were fractionated by anion-exchange chromatography and
were eluted as very poorly defined peaks of very
high UV absorbance irrespective of the two solvents used (buffer or 70% methanol). No difference was observed between the chromatograms
of proteins extracted from the media of either the
experimental or control cells. Because of this result and those of the electrophoretic analysis, no
further attempt was made to purify neuroparsin
from media.
Proteins extracted from the experimental
and control infected cells eluted as sharp peaks
in anion-exchange chromatography. However, the
profile obtained with each of the two extraction
media was somewhat different with respect to the
number of peaks and the intensity of comparable
peaks. The most surprising observation was the
presence of a peak having the same elution time
(same concentration of NaCl) as neuroparsin A
in extracts of cells infected either by recombinant
or control viruses (Fig. 4A). Consequently, fractions eluted at the time of putative neuroparsin
(7–8 min), 2 min before, and 2 min after were
reinjected separately onto the reversed-phase column. Only the fractions originating from cells infected with recombinant virus and eluted just
31
before (data not shown) and chiefly at the time
of neuroparsin A gave a sharp major peak. This
peak was eluted at the same time (6–7 min) as
natural neuroparsin A (Fig. 4B). A comparison of
the area of peaks with reversed-phase peaks of
natural neuroparsin A indicated that 20 × 106 infected cells were equivalent to about 200 locusts,
and consequently 106 infected cells were equivalent to ten locusts. So, the level of expression of
purified neuroparsin was approximately 1.2 µg
of expressed neuroparsin per 106 infected cells.
Biological Activity
The HPLC fraction eluted at the time of the
putative neuroparsin and originating from cells
infected by the control virus, the purified expressed neuroparsin, and natural neuroparsin
were tested in parallel for their antidiuretic activity. The fraction eluted from cells infected with
control virus was tested without dilution even in
the absence of the UV peak in the fraction. Since
we anticipated that the biological activity of the
expressed neuroparsin would be somewhat lower
than that of natural neuroparsin, we used three
serial half dilutions, all of which induced maximal effects in the case of natural neuroparsin
(plateau of the dose-response curve in Fournier
and Girardie, 1988). The fraction from the cells
infected by the control virus did not increase the
spontaneous rectal fluid reabsorption measured
in controls, whereas both neuroparsins increased
it to a similar degree (Fig. 5). However, because
the conditions of bioassay were not quantitative,
we can only deduce that the expressed neuroparsin is biologically active, like authentic neuroparsin, within the limitations of the quantification
used. Nevertheless, the existence of a maximal
effect with each of the three serial half-dilutions
supports our estimation of the quantity of purified expressed neuroparsin.
DISCUSSION
Using the plasmid pTZ-neuroparsin fulllength cDNA (Lagueux et al., 1992), we generated vectors carrying the neuroparsin gene under
the control of the p10 promoter of AcSLP 10 virus for infecting Sf9 insect cells. Three recombinant viruses expressing the neuroparsin gene
were selected and were observed to produce a bio-
32
Girardie et al.
Fig. 4. A: Anion-exchange liquid chromatography profiles of
25 mM Tris-HCl (pH 8) extracts of 10 × 106 cells infected by
either the 0111 control virus (CV) or the 1111 recombinant
virus (RV). Extracts were applied onto a Pharmacia Mono Q
HR 5/5, 10 mm; buffer A was 25 mM Tris-HCl, pH 8; buffer B
was 1 M NaCl in buffer A. Elution was conducted by linear
gradient from 0–0.5 M NaCl for 33 min at a flow rate of 1
ml/min. Detection was monitored by absorbance at 225 nm.
No striking differences were observed between the two profiles, and in both cases a peak (arrow) was eluted at the elution time of neuroparsin A (7–8 min). B: Reversed-phase liquid
chromatography profile of the fraction eluted between 7 and
8 min during anion-exchange liquid chromatography of the
extracts of 20 × 106 cells infected either with the recombinant virus (RV) or with the control virus (CV). The fraction
was applied onto a ProRPC C1/C8 300 Å column (5 × 10 mm);
buffer A was 0.08% TFA in 90% water and 10% CH3CN; buffer
B was 0.08% TFA in CH3CN. Elution was conducted by linear gradient from 22–54% B for 25 min at a flow rate of 0.5
ml/min. Detection was monitored by absorbance at 220 nm.
A sharp peak (arrow) was eluted at the same time as natural
neuroparsin (8–9 min) only in the fraction originating from
cells infected with the recombinant virus.
Baculovirus-Expressed Neuroparsin
33
Fig. 5. Fluid reabsorption of everted rectum preparations with the incubation medium alone (C) and the
same medium complemented with the total 8–9 min
HPLC fraction from cells infected with the control virus (CV) or three increasing doses (0.5, 1, and 2 corpora cardiaca equivalents) of either the 8–9 min HPLC
fraction from cells infected with the recombinant virus (sharp peak) or purified natural neuroparsin. Data
are expressed as the mean +/– SE. The number of
recta tested is given in parentheses. No stimulation
was obtained with the fraction from cells infected with
the control virus. Similar stimulation was obtained
with either authentic or baculovirus-expressed neuroparsins.
synthetic molecule having the same charge, the
same apparent molecular weight, the same denaturation-induced self-associations, and the
same reactivity against neuroparsin immune serum as the authentic neuroparsin. The baculovirus-expressed molecule was isolated by the
same two steps of liquid chromatography previously used to purify the natural neuroparsin
(Girardie et al., 1989). The purified molecule increased in vitro rectal water reabsorption as did
the natural neuroparsin (Fournier and Girardie,
1988). Thus, a neuroparsin which was identical
in structure and function to natural neuroparsin
was successfully expressed in the baculovirus-insect cell system.
Baculovirus-expressed neuroparsin was detected mainly in the infected cells but not in the
culture medium, in contrast to the eclosion hormone (Eldridge et al., 1991) and the pheromone
biosynthesis activating peptide (Vakharia et al.,
1995) which, in such expression systems, were
rapidly secreted from the cells. However, the expressed neuroparsin appeared to be stable in the
medium as well as in the cells, since no immunoreactive molecule with either a charge less basic or an apparent molecular weight lower than
the monomeric neuroparsin A was detected. Thus,
the cleavage of neuroparsin A into a less basic
neuroparsin B, which occurs during storage in the
axonal endings of the neurosecretory cells of
Locusta, did not occur in the expression system.
The signal peptide of neuroparsin has two
arginines which normally might induce a more basic net charge of the translation product than
neuroparsin A. Since the net charge of the translation product extracted either from the cells or from
the medium was the same as neuroparsin A, it appears that the signal peptide of the translation product was correctly cleaved intracellularly. This was
also the case for the purified eclosion hormone expressed in yeast (Hayashi et al., 1990) but not for
all the forms of the purified Escherichia-expressed
eclosion hormone, some of them having molecular
weights slightly lower than the natural neurohormone (Kono et al., 1990). In addition, recombinant
neuroparsin A seems to be correctly folded since it
was biologically active.
Although a portion of baculovirus-produced
neuroparsin was secreted into the culture medium, cells appeared to be the most abundant
source of neuroparsin for purification because of
the absorbance of the culture medium and its volume. The quantity of purified expressed neuroparsin per 106 cells was approximately equal to
the quantity of natural neuroparsin that can be
purified from ten locusts. It corresponds to one of
the lower levels (about 1 µg per 106 cells) reported
for other heterologous proteins synthesized in insect cells using the baculovirus expression vector
system (Luckow and Summers, 1988; Miller,
1988). Concerning the biologically estimated yield
of the two unpurified insect neurohormones ex-
34
Girardie et al.
pressed in a baculovirus vector, the amount of the
purified expressed neuroparsin was low compared
to that of the eclosion hormone (Eldridge et al.,
1991) but similar to that of the pheromone biosynthesis activating neuropeptide (Vakharia et
al., 1995). The observed poor secretion of expressed neuroparsin might be due to a perturbation of a certain stage of the secretory pathway.
The use of alternative expression systems in order to obtain a better level of expression will require further investigation.
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