Insect Molecular Biology (2003) 12(5), 415–425
Expression of functional Anopheles merus α-amylase in
Blackwell Publishing Ltd.
the baculovirus/Spodoptera frugiperda system
P. C. Effio*, A. V. Folgueras-Flatschart, W. R. Montor,
F. M. Pernasetti, M. T. Pueyo and M. C. Sogayar
Instituto de Quimica, Universidade de São Paulo,
São Paulo, Brazil
Abstract
The Anopheles merus (Diptera, Nematocera, Culicoi-
dea) α-amylase gene (AmerAmy, GenBank Accession
Number U01210) was amplified with its own or with
the Zabrotes subfasciatus α-amylase signal peptide
(ZsAmerAmy, GenBank Accession Number AY270183)
by PCR, using designed primers. The AmerAmy gene
was sequenced from its promotor to the TGA codon.
As a positive control, the Z. subfasciatus α-amylase gene
with its own signal peptide (ZsAmy, GenBank Acces-
sion Number AF255722) was also amplified by PCR.
These three sequences were inserted into the baculo-
virus genome using the Bac-to-Bac™ system. Recom-
binant baculovirus preparations were used to infect
Sf 9 Spodoptera frugiperda insect cells. The A. merus
α-amylase was successfully expressed as an active
enzyme detected mainly in cell culture supernatants.
Keywords: alpha amylase, Anopheles merus,
expression, Baculovirus/Spodoptera frugiperda.
Introduction
Different α-amylase (EC 3.2.1.1) genes from archaebacteria,
eubacteria, fungi, animal and plant sources have been
cloned and sequenced (Vihinen & Matsala, 1989). These
enzymes hydrolyse alpha-1,4 glycosidic bonds present in
oligosaccharides and starch (Steup, 1988), playing a key
role in the overall metabolism of these organisms. From
animals to bacteria, α-amylases display a wide variety of
Received 16 December 2002; accepted after revision 25 April 2003. Corre-
spondence: Drs Manuel Troyano Pueyo and Mari Cleide Sogayar, Instituto
de Quimica, Universidade de São Paulo, CP 26077, São Paulo 05513–970,
SP, Brazil. E-mail: matpueyo@ig.com.br and mcsoga@iq.usp.br
*On leave from the Universidad Nacional Pedro Ruiz Gallo, Lambayeque,
Perú.
© 2003 The Royal Entomological Society
kinetic parameters, optimum pH and substrate specificity
(Bertoft et al., 1984; Akazawa et al., 1988; Knutson, 1993).
The α-amylases are widely used by insects in their larval
stage, as has previously been reported (Terra & Ferreira,
1994). Closely linked amylase genes have been detected in
the housefly, Musca domestica (Ogita, 1968), silkworm,
Bombyx mori (Kikkawa, 1953), in several species of the
flour beetle, Tribolium (Pope et al., 1986), and in the yellow
fever mosquito, Aedes aegypti (Grossman et al., 1997). To
date, the only functional recombinant α-amylases to be
reported are those from Zabrotes subfasciatus (Grossi de Sá
& Chrispeels, 1997) and Diabrotica virgifera virgifera
(Titarenko & Chrispeels, 2000). In Culicoidea, the expres-
sion of an α-amylase gene was determined at the mRNA
level in the salivary glands of Ae. aegypti (Grossman &
James, 1993). However, that protein was not detected as an
active enzyme as was accomplished for an α-glucosidase
(Marinotti & James, 1990) in these organs. The results
on Anopheles merus presented here evinced for the first
time the expression, using the baculovirus/Spodoptera
frugiperda cell system, of a Culicoidea α-amylase recom-
binant gene as a functional enzyme. These results strongly
suggest that this protein could also be an active enzyme of
the digestive secretion in these Diptera.
Results
Generation of recombinant α-amylase constructs
The coding sequence for A. merus α-amylase (AmerAmy)
was obtained and subcloned into the baculovirus-based
pFastBac1 plasmid vector as described in Experimental
procedures. Two different constructs were sought, namely
pFastBac1-AmerAmy (contains the Ameramy gene plus
the sequence corresponding to its own signal peptide) and
pFastBac1-ZsAmerAmy (contains the AmerAmy gene plus
the sequence corresponding to the signal peptide of the
amylase gene of Z. subfasciatus). A construct made with
the Z. subfasciatus α-amylase gene (pFastBac1-ZsAmy)
was used as a control. Transformation of plasmid DNA into
E. coli DH10Bac allowed the production of the following
bacmid recombinants: Bd-AmerAmy, Bd-ZsAmerAmy and
Bd-ZsAmy.
415
416 P. C. Effio et al.

Figure 1. Lugol test of starch hydrolysis by the supernatant samples of Sf9 cells infected with the recombinant baculoviruses Bv-AmerAmy, Bv-ZsAmerAmy
and Bv-ZsAmy. 1, uninfected Sf9 cells; 2, Sf9 cells infected with baculovirus lacking insert. The following holes correspond repectively to cells infected with
recombinant baculovirus: 3, Bv-AmerAmy; 4, Bv-ZsAmerAmy; 5, Bv-ZsAmy. The extreme right hole corresponds to a negative control: 6, lugol in Milli-Q water.
Starch substrate (480 µl) was assayed for hydrolysis with 20 µl of supernatant of Sf9 cell cultures infected with recombinant baculoviruses. After 12 h at 37 °C,
10 µl was removed and mixed with 200 µl of lugol. Nonhydrolysed starch yields a blue colour (Sf9, Bv and Milli-Q water). Hydrolysis of starch to glucose and
maltose yields a yellow colour (Bv-ZsAmy). Hydrolysis to oligosaccharides yields a purple colour (Bv-AmerAmy, Bv-ZsAmerAmy).

Figure 2. Chromatographic pattern of starch
cleavage by A. merus α-amylase. Starch was
dissolved to 0.5% in activity buffer (100 mM
phosphate buffer, pH 6.0, 20 mM NaCl, 0.1 mM
CaCl2). To determine the cleavage pattern, a
series of four Eppendorf tubes containing 480 µl
of starch were prepared. To each of those tubes,
20 µl of the vacuum-concentrated supernatant
from Sf9 cultures infected with the recombinant
baculoviruses, Bv-AmerAmy, Bv-ZsAmerAmy and
Bv-ZsAmy, was added. Tubes were incubated at
37 °C for 24 h and centrifuged at 13 000 g for
10 min at 4 °C. The supernatant was dried at
60 °C and redissolved in 25 µl of Milli-Q water.
A total of 1 µl (five applications of 0.2 µl) of each
sample was applied. The supernatant from the
Sf9 culture infected with baculovirus lacking insert
was used as negative control. Lane 1: glucose
plus maltose. Supernatant samples from Sf9
cultures infected with recombinant baculoviruses
are in: Lane 2, Bv-ZsAmy; Lane 4, Bv-AmerAmy;
Lane 5, Bv-ZsAmerAmy. Lane 3, negative control.
Figure 3. Activity assay of AmerAmy in blue starch gel. Supernatants from Sf9 cell cultures infected
with the recombinant baculoviruses Bv-AmerAmy, Bv-ZsAmerAmy and Bv-Zs were precipitated with
cold acetone and redissolved in 10% of the initial volume with Milli-Q water. After electrophoretic
separation in PAGE-SDS, the gel was washed three times with Milli-Q water for 30 min. Another
wash with activity buffer was made in order to reactivate the enzymatic activity as described in
Experimental procedures. An agar gel containing blue starch was layered over the polyacrylamide gel.
The set was wrapped in a PVC film and incubated at 37 °C for 12 h. Active amylases separated in the
polyacrylamide gel below are detected in samples from culture supernatants as clear bands in the agar/
blue starch gel. Electrophoretic conditions were 100 V/20 mA. Samples of 5 and 0.5 µg were applied in
lanes containing A. merus and Zabrotes proteins, respectively. Both pellet and supernatant samples
were analysed in lanes labelled as follows: 1 and 5, Sf9 from noninfected culture; 2 and 6, Bv from culture
infected with baculovirus lacking insert; 3, Bv-AmerAmy, supernatant from culture infected with
baculovirus carrying sequences corresponding to A. merus α-amylase and to its own signal peptide;
4, Bv-ZsAmerAmy, supernatant from culture infected with baculovirus carrying sequences
corresponding to the Zabrotes signal peptide and A. merus α-amylase; 7, Bv-AmerAmy, pellet from
culture infected with baculovirus carrying sequences corresponding to its own signal peptide and
A. merus α-amylase; 8, Bv-ZsAmerAmy, pellet from culture infected with baculovirus carrying
sequences corresponding to the Zabrotes signal peptide and A. merus α-amylase; 9, Bv-ZsAmy,
supernatant from culture infected with baculovirus carrying sequences corresponding to Zabrotes’s own
signal peptide and α-amylase.

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
 Expression of the Anopheles merus α-amylase gene
Transfection of Sf9 insect cells; production of baculoviruses
carrying the AmerAmy, ZsAmerAmy and ZsAmy genes
Sf 9 insect cells were transfected with the Bd-AmerAmy, Bd-
ZsAmerAmy and Bd-ZsAmy bacmid constructs for primary
baculovirus particle production. After transfection, baculo-
virus production was confirmed by two different methods,
based on the work we previously reported (Folgueras-
Flatschart et al., 2000), namely the observation of the
cytopathic effect that occurs upon subculturing of trans-
fected cells and amplification of specific DNA fragments,
by PCR, using culture supernatants potentially containing
baculovirus particles, as a template and specific primers for
AmerAmy-F, ZsAmerAmy-F and AmerAmy-R. Baculovirus
master stocks were amplified for Sf9 infection and protein
production. After 96 h of massive infection [multiplicity of
infectivity (MOI) = 10; numbers are relationships between
viral particles and cells], Sf9 cultures displayed typical
cytophathic effects, i.e. low cell density and poor adherence
to the substrate, indicating that virus production was taking
place. The nontransfected Sf9 cultures as well as those
infected with empty baculoviruses displayed a normal cell
monolayer.
Protein determination in cell supernatants
The total protein content of cell supernatants was deter-
mined as described in Material and methods. Cells trans-
fected with Bv lacking insert as a control gave a mean value
(three determinations) of 670 µg/ml. This figure corres-
ponds in general to proteins of the bovine serum used in
the culture medium. Thus, all determinations accomplished
in the supernatants of cells transfected with baculoviruses
carrying inserted sequences were corrected by subtracting
670 µg/ml from the actual data. Bv-ZsAmy transfected cells
were included as an internal control. Typical protein content
values obtained when the insect cells were transfected with
Bv-AmerAmy, Bv-ZsAmerAmy and Bv-ZsAmy are 7.5, 8.5
and 19.0 µg/ml, respectively.
Detection of α-amylase activity
Amylase activity was detected both in culture supernatants
and in cell extracts using the lugol method, as described in
Material and methods. The results, shown in Fig. 1, indicate
that only amylases from culture supernatants obtained
from cells infected with recombinant baculovirus Bv-
AmerAmy and Bv-ZsAmerAmy were able to degrade the
starch substrate to dextrins displaying a number of glucose
residues sufficient to give a purple-coloured lugol solution.
The Z. subfasciatus α-amylase, produced by Bv-ZsAmy-
infected Sf9 cells, degrades starch to products that have a
number of glucose residues not enough to give a colour
complex with l 3− (Bailey & Whelan, 1961). The products
obtained upon starch hydrolysis by recombinant α-
amylases were analysed by paper chromatography. The
© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
417
chromatographic pattern shown in Fig. 2 indicates that the
amylases present in culture supernatants from cells infected
with Bv-AmerAmy and Bv-ZsAmerAmy yield a cleavage
pattern that is typical of α-amylase, with relatively large
amounts of oligosaccharides and small amounts of glucose
and maltose, whereas Bv-ZsAmy culture supernatants
degraded starch mainly to glucose and maltose and small
amounts of oligosaccharides, as shown in Fig. 1.
The α-amylase activity present in Sf9 culture super-
natants was also analysed by the blue starch overlay method
and by Western blotting. The results shown in Figs 3 and
4 indicate that proteins present in supernatants obtained
from Sf9 cells infected with Bv-AmerAmy and Bv-ZsAmerAmy
display amylase activity. The Coomassie-blue-stained
SDS-PAGE of proteins pelleted from culture medium of
infected cells revealed a single protein band of approxim-
ately 62 kDa, in both ZsAmy and ZsAmerAmy prepara-
tions (Fig. 4A). However, the band corresponding to ZsAmy
is much more intense than that of ZsAmerAmy. When the
gel was silver stained, a second (faint) band, of approxim-
ately 52 kDa, was observed in ZsAmy supernatants (data
not shown). Western blotting showed that two α-amylases
were secreted by cells infected with the ZsAmy baculovirus.
The protein(s) present in the 62 kDa band reacted weakly
with the Z. subfaciatus anti-(α-amylase) antibody, whereas
the second band (≈ 52 kDa) reacted strongly with this
antiserum. In the case of A. merus, only one band, cor-
responding to the secreted protein that exhibits an apparent
MW = 62 kDa, was detected in culture supernatants ob-
tained from cells infected with the ZsAmerAmy baculovirus
(Fig. 4B).
Sequence analysis
The promotor of the amylase gene is likely to be CCCAT-
CAACT with 97% confidence (Fig. 5).
The conceptual translation product of the α-amylase
genomic sequence is also presented in Fig. 5. Features of
this product are: (a) MKLLVRLAPILLLALTGRPVAA, a plau-
sible signal peptide with 90% confidence, suggesting that
the peptide is secreted; (b) a mature peptide consisting of
492 residues starting with the blocking N-terminal glutamine,
an indication that the peptide may be in contact with digest-
ive secretion (Strobl et al., 1997); (c) 54.8 kDa, the mole-
cular weight calculated for the unmodified mature peptide.
Comparison with other amino acid sequences reveals that
a glutamic acid (E) and two aspartic acid (D) residues that
are conserved in the α-amylase family (Janecek, 1997) are
known to be involved in catalysis at the active sites of por-
cine pancreatic (Qian et al., 1994) and Tenebrio molitor
(Strobl et al., 1998) α-amylases. They are found in Amer-
Amy as D215, D318 and E252. Three conserved histidine
residues, which are thought to be involved in substrate
binding (Qian et al., 1994; Strobl et al., 1998), are also
present in AmerAmy as H125, H219, H317. By comparison
418 P. C. Effio et al.

Figure 4. SDS-PAGE and Western blot of proteins present in culture supernatants from Sf9 cells infected with recombinant Bv-AmerAmy and Bv-ZsAmy
baculoviruses. A: Coomasie-blue staining of proteins fractionated by SDS-PAGE. Proteins recovered from supernatants of cell cultures transfected with
Bv-ZsAmy and Bv-ZsAmerAmy are in lanes 1 and 3, respectively. The 62 kDa band corresponding to ZsAmy is more intense than that of ZsAmerAmy. In
lane 2 are the molecular weight markers (Gibco). B: Western blot of proteins recovered from supernatants of cell cultures transfected with Bv-ZsAmy and
Bv-ZsAmerAmy are in lanes 1 and 3, respectively. Two bands of 62 and 52 kDa of different intensities are separated in lane 1. A unique band of 62 kDa is
observed in lane 3. A sketch of the molecular weight markers is shown in lane 2. Arrows point to α-amylase.

with previous data (Strobl et al., 1998), other conserved
residues can be related to the chloride (R213, N316, R351)
and calcium (N124, R176, D185, H219) binding sites in the
A. merus enzyme (Fig. 5).
The similarity and identity of AmerAmy with other α-amylases
is shown in Table 1. AmerAmy has 51–59, 49–51, 47, 11, 24
and 8–23% identity with α-amylases from, respectively,
insects, mammals, birds, plants, fungi and bacteria.
AmerAmy does not have any putative N-glycosylation
site (consensus sequence Asn-X-Ser/Thr). Instead, Amer-
Amy has the sites T131, S141, T142 and T359, which could
be O-glycosylated, with probabilities of 86.32, 96.33, 94.14
and 74.57%, respectively (Table 2).
Discussion
We chose to express the AmerAmy sequence in the baculo-
virus/insect cell system not only because it is a versatile
and reliable system for the production of recombinant
proteins (Possee, 1997; Grossi de Sá & Chrispeels, 1997;
Titarenko & Chrispeels, 2000) but also because it allows
expression of eukaryotic genes in a eukaryotic cell system,
taking advantage of their protein synthesis machinery,
thereby facilitating folding and post-translational modifications
(O’Reilly et al., 1992). These include N- and O-glycosilation,
acylation, proper proteolysis and oligomerization of the
protein product via processes that are often identical to that
occurring into the original cells. Therefore, proteins may be
secreted from the cell or be directed to other subcellular
structures. In addition, the insect cytoplasmic environment
is less reducing than that of the E. coli cells, thus enabling
proper disulphide bridge (–S–S–) assembly. This feature is
important in the case of the A. merus α-amylase because
it displays 11 cystein residues, seven of which are concen-
trated between amino acids 384 and 506 (Fig. 5). The number
of –S–S– bonds in this protein is not known but the high
frequency of cystein residues probably enhances the com-
plexity of folding. Note that the α-amylase nucleotide
sequence used throughout this work is of genomic origin.
The lack of introns (Pernasetti, 1991) is a valuable feature
for the expression of this gene as an active α-amylase that
can now be used to study structural, kinetic and inhibitory
behaviour in the presence of insect plant amylase inhibitors.
This could reveal clues for future mosquito control strategies.
We previously used the baculovirus/insect cell expres-
sion system to produce active mouse Fos and Jun proteins
(Corvello et al., 1995), bovine Herpesvirus glycoproteins
(Folgueras-Flatschart et al., 2000), the alpha7 subunit of
acetylcholine nicotinic receptor (Aztiria et al., 2000) and
human prolactin (Pereira et al., 2001). Here we describe
the results showing that, for the first time, it was possible to
express the A. merus α-amylase in the active form.
The attempt to express the A. merus α-amylase gene
attached to the sequence that codes for the signal peptide
of α-amylase from Z. sufasciatus stems from the fact that
this enzyme was previously successfully expressed in the
baculovirus/insect cell system (Grossi de Sá & Chrispeels,
1997). Signal peptide sequences derived from insect protein

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
 Expression of the Anopheles merus α-amylase gene 419

Figure 5. Nucleotide sequence of the AmerAmy gene and its conceptual translation. The vertical arrow indicates the probable cleavage site of the signal
peptide. Open boxes indicate conserved residues of the active site, and shaded boxes are conserved residues involved in substrate binding. Conserved residues
of the chloride and calcium binding sites are, respectively, outlined letters and letters into bold outlined boxes. The shaded bordered sequence corresponds to
that conserved in the C terminal region of several amylases. The wavy bordered sequence corresponds to the typical sequence of the AmerAmy C terminal
region. See Discussion for further details.

have been used with variable results in the baculovirus/
insect cell system. Previous studies on the expression of
a plant protein, papain (Tessier et al., 1991), have shown
that the use of an insect-derived signal peptide enhanced
the secretion five-fold over that detected when using the
native signal peptide. However, other studies have found that
insect-derived signal peptide sequences were not optimal
(Jarvis et al., 1993; Dupuis et al., 1997; Dahl et al., 2000) or
were not linked to the secretion of heterologous proteins in
the baculovirus/insect cell system (Tessier et al., 1991;
Dupuis et al., 1997). The level of secretion of A. merus α-
amylase and Z. subfasciatus reported here (7.5, 8.5 and
19.0 µg/ml) are lower than those reported for other hetero-
logous proteins: GRP78/BJP (50–75 µg/ml), hepatitis B
surface antigen (90 µg/ml; Lanford et al., 1989) and plant
phaseolin (90 µg/ml; Bustos et al., 1988). Although a large
difference between secretion of AmerAmy (7.5 µg/ml) and
ZsAmerAmy (8.5 µg/ml) was not detected in the results
reported here, ZsAmy was two-fold (19.0 µg/ml) higher
(see Fig. 4 for comparative purposes). Our data as well as
those referred to above suggest that the recognition and
the processing of signal peptides from heterologous pro-
teins are not the only factors involved in the secretion level
when the baculovirus/insect cell system is used. It is likely
that, among other factorss, disulphide bond formation and
folding could be rate-limiting steps.
The identity of AmerAmy with its mammalian, insect
and bird counterparts is relatively high (59, 51 and 47%,
respectively), as shown in Table 1.
We identified three amino acids (D215, E252, D318) that
are known to be important for catalysis and three histidine
residues (H125, H219, H317) that are known to be involved
in substrate binding (Qian et al., 1994). Residues R213,
N316 and R351 were identified as belonging to the chloride
binding site, whereas residues N124, R176, D185, H219
were related to the calcium binding site (Strobl et al., 1998).

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
420 P. C. Effio et al.

Table 1. Identity and similarity of A. merus

Amino acid Amino acid α-amylase residues when compared with other
Organism Accession no. identity (%) similarity (%) known α-amylases

Insects
Anopheles gambiae L04753 98 100
Tenebrio molitor S75702 59 71
Drosophila melanogaster AB043051 55 68
Drosophila simulans D11734 55 70
Culex tarsalis U01211 55 67
Aedes aegyptis O02652 56 67
Tribolium castaneum U04271 56 69
Diabrotica virgifera virgifera AF208002 53 65
Phaedon cochleriae Y17902 52 64
Lutzomyia longipalpis AF132512 51 66

Mammals
Mus musculus J00360 51 66
Homo sapiens M24895 49 65
Rattus norvegicus J00703 49 64
Sus scrofa AF06472 49 64

Birds
Gallus gallus U63411 47 63

Plants
Hordeum vulgare X15226 11 not determined
Oriza sativa X52240 11 not determined
Vigna mungo X73301 11 not determined

Fungi
Aspergillus orizae X12726 24 43

Bacteria
Xanthomonas campestris M85252 23 35
Pirococcus woesei AF240464 11 not determined
Geobacillus stearothermophilus AF032864 08 not determined

These findings indicate that, as with other amylases, AmerAmy
has a canonical composition and distribution of residues
in order to attain its function. The amino acid C-terminal
sequences of α-amylases from organisms from taxonomic
classes Mammalia, Insecta (orders Diptera, Hymenoptera,
Lepidoptera, Coleoptera), Arachnida, Nematoda, Osteich-
thyes (order Teleostea), Bivalvia and Crustaceae were
compiled (not shown) in order to contrast them with the
corresponding sequence of A. merus α-amylase deter-
mined in this work. As a general rule, the C terminal tail
starting at D comprises 12 amino acids. An example of
this terminal C sequence is D G V L A I H V N A K L from
T. molitor (Strobl et al., 1997). According to our analysis, it
can be noted as the widely conserved DGX1X2AIH sequence.
In addition, the similarity of residues between DG and A
and after H was noted. In most of the sequences collected,
the two amino acids preceding AIH are hydrophobic: VL, IL,
VI, VV, FV or FI. The amino acids immediately following A I
H are hydrophobic (A, V, I, G) and hydrophilic (KXK, NXK,
NXR, SXK, EXK, DXK, DXR) with lysine being highly con-
served, or in a few examples substituted by arginine or
glutamine. However, this tail is not observed in sequences
that are currently available from bacteria, fungi, arachnida,
nematoda or plant amylases. It seems likely that the C-
terminal region, as described before, is a well-defined feature
of certain α-amylases. The significance and the extent of
its distribution in living organisms remains to be ascert-
ained. Interestingly, the sequence determined for the C-
terminal region in A. merus amylase displays DGVIAIH as
expected, but does not follow the rule of similarity or length
after it. Instead, a stretched SEVSVCVCCRMRSDGRV
sequence was detected. This seems to be restricted to the
amylase of A. merus and in part to the same protein of
A. gambiae (accession number L04753). A part of this
sequence, VSVCVC, is also found in proteins that are not
related to amylases of several organisms and viruses,
including an inner region of agCP8155 (accession number
EAA13780.1), an unidentified protein from A. gambiae.
Thus, concerning its C-terminal portion, the A. merus pro-
tein seems to be unique within the amylases from several
higher organisms. Nevertheless, it is possible that the odd
C-terminal portion described may be present only in the
conceptual translation of the genomic sequence, but not in
the functional protein itself.
O-glycosylation is expected to occur in AmerAmy at the
T138, T142, and S141 residues and in ZsAmy at the T425
residue (Table 2). AmerAmy does not display classical
N-glycosylation sites (Asn-X-Ser/Thr) whereas ZsAmy has
one starting at N442, close to the C-terminus (Grossi de Sá
& Chrispeels, 1997).

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
 Expression of the Anopheles merus α-amylase gene 421

Table 2. Prediction of O-glycosylation sites on α-amylases from different of the relationship between the intensity of the ZsAmy and
insects based on protein and DNA sequence data
ZsAmerAmy 62 kDa bands reveals that it is inverted in
O-glycosylation Fig. 4A and Fig. 4B. To a smaller mass of ZsAmerAmy cor-
Insect Residue probability (%) responds a stronger antibody reaction. This may suggest
that the hypothetical modification at the mosquito protein
Aedes aegypti, Amy 1 T90 84.45
S23 75.63 does not interfere with the antibody cross-reaction and that
S93 73.06 it is not probably placed at the amylase epitope. Together,
S149 69.06 these two observations suggest that the epitope of these
S703 71.20
Aedes aegypti, Amy 2 T64 71.30 amylases is located near the C terminus, the domain C
Anopheles merus T138 86.32 (Strobl et al., 1998) of these proteins.
T142 94.14 The very faint band below the main band of Anopheles
T359 74.57
S141 96.33 amylase in Fig. 4(B), lane 3, (Western blot) is not distin-
Apis melifera None guishable from the sharp band of Fig. 4(A), lane 3
Culex tarsalis T63 97.96 (stained). The faint band is probably due to the lesser
T138 77.00
S460 73.06 O-glycosylation of T359 (see Table 2) and seems to be
Dermatophagoides pteronyssimus None the product of a residual event.
Diabrotica virgifera virgifera T427 93.66 The unique band of Anopheles amylase in Fig. 4(A,B),
Drosophila melanogaster, Amy distal T130 98.09
T358 59.83 lane 3, has a unique band of activity in Fig. 3, lanes 3 and 4.
S129 97.53 This suggests that the putative O-glycosylation of this protein
S135 99.85 is compatible with enzymatic activity. The two bands detected
S457 79.19
Drosophila melanogaster, Amy prox. T131 97.65 for the amylase of Zabrotes in Fig. 4A,B, lane 1, also have
T359 59.83 a unique activity band in Fig. 3, lane 9. Clearly, only one of
S130 81.06 the bands in Fig. 4A,B, lane 1, retains activity. This is con-
S136 99.90
S458 79.19 sistent with previous results (Grossi de Sá & Chrispeels,
Euroglyphus maynei None 1997). As judged by the slightly faster migration of the band
Lutzomyia longipalpis S357 94.95 in Fig. 3, lane 9, the activity is probably on the glycosylated
Phaedon cochleriae S136 78.50
T355 95.83 band (62 kDa) of Zabrotes α-amylase. If this interpretation
Tenebrio molitor None is correct, in Z. subfasciatus only the α-amylase modified
Tribolium castaneum None by a putative N-glycosylation retains enzymatic activity.
Zabrotes subfasciatus T425 83.24
The exact role played by glycosylation in protein function
is as yet not completely understood, but the data available
point towards variable roles for different proteins. Questions
on how N- or O-linked glycosylation might affect protein
In AmerAmy, the S141 T142 site has a high (94–96%) function are still to be adressed. A systematic compilation
probability of being O-glycosylated (Table 2). It occurs near of data on glycosylation regarding protein nature and func-
the consensus region I (DIIINH), into the B domain of tion, cell type, subcellular localization and secretion may
the enzyme. The results in Fig. 4 showed a single 62 kDa prove to be useful in establishing the importance of this
band in AmerAmy supernatant that cross-reacts with the post-translational modification.
ZsubAmy anti-amylase antibody. Considering that the The colour of the iodine–iodide complex with starch is
expected molecular weight for AmerAmy is 54.8 kDa, this dependent on the number of glucose residues in the chain
indicates that the enzyme was post-translationally modified, (Bailey & Whelan, 1961). A chain of less than 10 glucose
probably by glycosylation, increasing its molecular weight residues does not yield any colour. However, as the number
up to an apparent value of 62 kDa as occurs with ZsAmy of residues increases, a change from red to red–purple,
(Grossi de Sá & Chrispeels, 1997; Fig. 4B). purple and blue is observed. Thus, Fig. 1 indicates that the
The Western blot exihibited in Fig. 4(B) suggests that the Zabrotes enzyme produces mainly oligosaccharides with
putative glycosylation is a different event for these enzymes few glucose residues. Instead, the sugars released by the
in Spodoptera cells. The bruchide amylase may or may not mosquito amylase may have a higher number of glucose
be modified whereas that of the mosquito was entirely mod- residues. In accordance with these observations are the
ified. The strong cross-reaction observed for the 52 kDa results presented in Fig. 2. This shows a quite different
band of Zabrotes contrasts with the relatively faint 62 kDa pattern of extensive (12 h) starch hydrolysis for the two
band (Fig. 4B). This may be an indication that the modifica- enzymes. That of Zabrotes releases mainly glucose and
tion is a disturbance factor in the antibody attachment to maltose and relatively smaller amounts of oligosaccha-
the amylase epitope, which is probably located at the C- rides. The mosquito enzyme produces low levels of glucose
terminus and contains the N442 residue. Visual inspection and maltose and relatively higher levels of oligosaccharides.

© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
422 P. C. Effio et al.
Figures 1 and 2 strongly suggest that the bruchide and
the mosquito amylases act on starch by different
mechanisms. The former enzyme resembles a glucosi-
dase, which uses oligosaccharides formed by a few
glucose units as a substrate, yielding glucose and maltose
(Gray et al., 1979). This raises the question of how these
oligosaccharides are produced because glucosidases
do not attack starch (Gray et al., 1979). The Anopheles
enzyme displays the normal α-amylase pattern of starch
degradation.
Mosquitoes transmit more human parasitic diseases
than any other arthropod group. The Anophelinae, mem-
bers of the gambiae complex that comprises several mor-
phologically indistinguishable species (White, 1985), were
responsible for nearly 500 million clinical cases of malaria
each year in the 1980s (Sturchler, 1989). Knowledge of the
feeding habits of these diptera is of utmost importance in
order to design control strategies. Studies on the physiolo-
gical role of the yellow fever mosquito (Ae. aegypti) salivary
glands showed that these organs are sexually dimorphic. In
females, they express different gene products from unique
regions of the gland enabling the female to adopt two dif-
ferent feeding modes based on blood or sugar meals. By
contrast, the male is only able to use the sugar feeding mode.
The results presented here suggest that the genomic
sequence of A. merus α-amylase may be expressed
as a fully active enzyme during the mosquito’s life cycle,
in accordance with previous data demonstrating the expres-
sion of α-amylase mRNA in salivary glands of Ae. aegypti
(Grossman & James, 1993). Together, these point towards
a sugar feeding mode in both Culicinae and Anophelinae
mosquitoes, which probably use starch as the primary
target of α-amylases. However, a starch diet requires
both α-amylase and glucosidase acting as part of digest-
ive secretion in order to achieve the ultimate goal of the
polysaccharide metabolism: D-glucose. In addition to α-
amylase (Grossman & James, 1993), Ae. aegypti also
expresses an active glucosidase (Marinotti & James, 1990)
in compliance with the above requirement. This suggests
that a glucosidase may also be present in Anophelinae,
thus rendering starch an efficient and widespread substrate
for a sugar feeding mode that is decisive for both male and
species survival. The observation that salivary glands are
not able to hydrolyse starch (Marinotti & James, 1990) is
not consonant with these assumptions but it suggests an
alternative hypothesis. The α-amylase could be partially
secreted, remaining insoluble and attached to the external
surface of the cell membrane. It would be interesting to
assess the role of the atypical C terminus discussed above
in a putative anchoring of α-amylase. Nevertheless, the
data presented here and previously (Grossman & James,
1993) may indicate that the number of mosquitoes feeding
on flowers and nectar (Van Handel et al., 1972) would be
more accurate if other carbohydrates, such as starch and
© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
other α-amylase substrates, were included in the diet, even
if at a smaller ratio.
Experimental procedures
Cells and bacterial strains
Spodoptera frugiperda Sf 9 cells were obtained from the American
Type Culture Collection (ATCC, CRL-1711, Manassas, VA, USA).
E. coli DH5alpha and E. coli JM103 were purchased from New
England Biolabs Inc. (Beverly, MA, USA). E. coli DH 10B was
purchased from Gibco-BRL (Rockville, MD, USA).
Isolation of the α-amylase gene from A. merus
The α-amylase gene from A. merus (Diptera, Nematocera, Culi-
coidea; GenBank accession number U01210) was isolated from a
genomic library prepared by Frank H. Collins (Center for Infectious
Diseases, Atlanta, Georgia, USA) in lambda phage EMBL 3. A
recombinant phage was separated by probing the library with a
0.6 kbp Pvu II fragment of a Drosophila melanogaster α-amylase
gene (Boer & Hickey, 1986). The cloned fragment was isolated,
restricted with HindIII and ligated to the plasmid pIBI24 (Interna-
tional Biotechnologies Inc.). The ligation mixture was used to
transform E. coli JM 103 competent cells. By employing the same
0.6 kbp PvuII probe, a recombinant plasmid containing a 1.7 kbp,
HindIII fragment was isolated, characterized by standard restric-
tion analysis and partially sequenced (Pernasetti, 1991). The
1.7 kbp fragment contains a complete α-amylase gene from
A. merus (AmerAmy) that was studied throughout this work.
Cloning the AmerAmy gene DNA into the pGEM-T plasmid
The AmerAmy gene was amplified by PCR from the pIBI24-
AmerAmy construct, using two distinct forward primers (F) in order
to obtain AmerAmy with its proper signal peptide or, alternatively, the
AmerAmy coding sequence plus the Z. subfasciatus α-amylase
signal peptide (ZsAmerAmy). The same reverse primer (R) was
used in both amplifications.
The primers used are shown below:
AmerAmy-F. 5′-TCGGGATCCATGAAACTTTTAGTGCGCCTAG-
CACCG-3′
ZsAmerAmy-F. 5′-TCGGGATCCATGAAGTTAGGAGTAGTGCA-
GTTGATCCTCGGTTTGGCCGTGGGGTTCACCCAGCACGAT-
CCCCACTTTGTCCGC-3′
AmerAmy-R. 5′-CCGCTCGAGAAGCTTGAATTCTCACACCCG-
GCCATCAGACCTCATTCG-3′
PCR was performed under the following conditions: 200 m M
Tris-HCl, pH 8.0; 50 mM KCl; 0.2 mM of each dNTP; 1.5 mM
MgCl2; 0.5 µM Forward primer; 0.5 µM Reverse primer; 0.1 µg DNA
template (pIBI24-AmerAmy construct); 1 µl (5 U) Taq DNA-
Polymerase; Milli-Q water to a final volume of 200 µl. Amplification
was achieved upon twenty-eight cycles of 45 s at 95 °C, 30 s at
55 °C, 2 min at 72 °C and an extension step, at 72 °C, for 10 min
The PCR products were extracted from the agarose gel using a
recovering DNA kit (Amersham Biosciences, Uppsala, Sweden)
and cloned into the T/A cloning plasmid pGEM-T (Promega, Madison,
 Expression of the Anopheles merus α-amylase gene
WI, USA). As a control, the amylase gene from Z. subfasciatus
(ZsAmy) was also amplified by PCR and cloned into the pGEM-T
plasmid. To amplify ZsAmy, a recombinant plasmid containing
the sequence that codes for Z. subfasciatus α-amylase was
used as template in a PCR reaction with specific primers ZsAmy-F
and ZsAmy-R (see below). The template DNA and primers were
gently provided by Dr Fatima Grossi de Sá (EMBRAPA, Brazilia,
Brazil).
ZsAmy-F. 5′-CGCGGAATCAAACGATGAAGTTAGGAGTAGTGC-3′
ZsAmy-R. 5′-CCGCTCGAGAAGCTTGCGCCCGCTTACAGTT-
TGGAAGTGAG-3′
Subcloning of the α-amylase genes into the Bac-to-Bac™ system
The resulting pGEM-T, constructs containing the amylase DNAs,
were digested with Bam HI and Hin dIII restriction enzymes in order
to insert the DNA, in frame, into the pFastBacl transfer vector of
the Bac-To-Bac™ Baculovirus Expression System (Life Techno-
logies, Rockville, MD, USA). The recombinant plasmids obtained
(pFastBac1-AmerAmy, pFastBac1-ZsAmerAmy and pFastBac1-
ZsAmy) were used to transform competent E. coli DH10 Bac (con-
taining the wild-type baculovirus genome in the bacmid form) to
generate the corresponding recombinant bacmids upon trans-
position, namely Bd-AmerAmy, Bd-ZsAmerAmy and Bd-ZsAmy.
Production of recombinant baculovirus
Spodoptera frugiperda Sf 9 cells were transfected with recom-
binant bacmid DNA for production of the baculovirus particles.
Cells were cultured at 28 °C in TNM-FH medium (Life Technolo-
gies) supplemented with 50 U/ml ampicilin and 50 µg/ml strepto-
mycin. For transfection, 9 × 105 cells were plated in 35-mm tissue
culture dishes and incubated for 1 h in 2 ml Sf900-II SFM (Life
Technologies) without antibiotics to allow adhesion of the cells to
the dish. The medium was then changed to 1 ml serum-free
Sf900-II without antibiotics, containing recombinant bacmid DNA
(5 µl of a standard mini-preparation of plasmid DNA) that had
been pre-incubated for 30 min at room temperature with CellFectin
(6 µl) (Life Technologies). Cells were incubated with the liposome–
DNA complex at 27 °C for 5 h. The transfection medium was
removed and 2 ml of TNF-FH medium, containing antibiotics,
was added. Bd-AmerAmy and Bd-ZsAmerAmy DNA were trans-
fected into Sf 9 cells and nonrecombinant bacmid (Bd) DNA and
Bd-ZsAmy were used as, respectively, negative and positive con-
trols. Transfected cells were incubated at 27 °C for 72 h allowing
baculovirus production and release into the culture medium. The
culture medium from each transfection was collected, clarified
(500 g for 5 min) and stored at 4 °C as a master virus stock. Trans-
fection efficiency, recombinant baculovirus (Bv-AmerAmy, Bv-
ZsAmerAmy and Bv-ZsAmy) and nonrecombinant baculovirus
(Bv) production were monitored by visualization of the cytopathic
effect displayed by transfected cells within 48 h after subculturing,
under a phase contrast microscope and assaying the presence of
baculovirus DNA through PCR analysis (Folgueras-Flatschart
et al., 2000). To this end, baculovirus present in 50 µl of infected
culture supernatant was sedimented at 12 000 g for 10 min in a
microcentrifuge tube, and a volume (25 µl) of proteinase K buffer
(10 mM Tris-HCl, pH 7.8; 5 mM EDTA; 0.5% SDS) containing
50 µg/ml of proteinase K (Sambrook et al., 1989) was added to the
© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
423
pellet to digest viral proteins during 1 h at 56 °C. An additional
heating at 95 °C for 20 min was done in order to inactivate the
enzyme before proceeding to the PCR step. Viral DNA amplifica-
tion was carried out using 2 µl of this DNA preparation as the tem-
plate at the same conditions and primers described above. This
procedure was used to obtain the AmerAmy, ZsAmerAmy and
ZsAmy DNAs.
Recombinant baculovirus stocks
Samples of Bv-AmerAmy and Bv-ZsAmerAmy have been properly
deposited in the Cell and Molecular Biology Laboratory, Depart-
ment of Biochemistry, Institute of Chemistry, University of São Paulo,
under the care of M.C.S. They are available upon request.
Amplification of baculovirus stocks
For amplification of baculovirus master stocks, 1 × 106 Sf 9 cells
were plated in a 25-cm2 flask and incubated for 1 h with 10 µl of
baculovirus master stock in 1 ml of TNM-FH medium containing
antibiotics (corresponding to an MOI of 0.01– 0.1). After this
period, the medium was completed to 4.5 ml and the infected cells
were incubated for 48 h at 27 °C. The culture medium was col-
lected, clarified (500 g for 5 min) and stored at 4 °C as viral stocks
for recombinant protein production.
Recombinant protein production
For massive infection and recombinant protein production, 25-cm2
culture flasks containing 1 × 106 Sf 9 cells, plated and grown over-
night in TNM-FH medium, at 27 °C, were infected with 500 µl of
amplified baculovirus stock (corresponding to MOI of 10) and
incubated at 27 °C for 96 h or until detection of cytopathic effect in
90–100% under an inverted phase contrast Nikon microscope.
Clarified culture medium (supernatant) and cells (pellet) were col-
lected after centrifugation (500 g for 5 min).
Amylase purification from culture supernatants
Amylase secreted to the culture medium by baculovirus-infected
cells was concentrated to 10% of the initial volume by precipitating
proteins with three volumes of cold acetone (stored at −20 °C) for
3 h and centrifugation at 12 000 g at 4 °C for 30 min The pellet
was redissolved in 100 µl of Milli-Q water and stored at −20 °C for
analysis of extracellular activity of the expressed α-amylase.
Protein determination
Quantification of proteins present in supernatants of pelleted cells
was accomplished by using the methods described previously
(Bradford, 1976; Smith et al., 1985; Morton & Evans, 1992). Egg
albumine was used as the standard protein.
Preparation of baculovirus-infected cell extracts
Infected cells were lysed essentially as described (Grossi de Sá
& Chrispeels, 1997), for 1 h with lysis buffer (10 mM Tris-HCl;
130 mM NaCl; 0.1% SDS; 5 mM phenylmethylsulphonil fluoride,
pH 7.5). To pellet cellular debris, the lysate was clarified by
centrifugation at 12 000 g at 4 °C for 10 min. The supernatant
was stored at −20 °C for analysis of intracellular activity of the
expressed amylase.
424 P. C. Effio et al.
α-amylase assays and paper chromatography
α-amylase activity was detected by the lugol (I2 + KI) procedure
(Silva, 1989) using soluble starch as substrate (in 100 mM phos-
phate, pH 6.0; 20 mM NaCl; 0.1 mM CaCl2). Samples (20 µl) of
supernatants or infected cell extracts were added to 480 µl of the
substrate solution. The reaction mixture was incubated for 12 h at
37 °C. A sample (10 µl) of this reaction was then mixed with 200 µl
of lugol in 0.05 N HCl. A nearly complete starch hydrolysis pro-
duces a clear yellowish solution. The hydrolysed substrate (480 µl)
was dried at 60 °C, redissolved with 25 µl of Milli-Q water and
assayed by paper chromatography. To a 12 × 6-cm strip of Whatman
1MM paper, 1 µl of each sample was applied. The mobile phase
was a mixture of isopropanol/acetic acid/water (7 : 1 : 2). Chroma-
tography was ascendent up to 10 cm. Reducing sugars were
detected by precipitation of metalic silver (Trevelyan et al., 1950;
Robyt & French, 1963).
SDS-PAGE and amylase activity
Proteins (5 µg) present in supernatants or extracts of infected
cell culture were fractionated by SDS-PAGE in 20 × 20-cm 15%
polyacrylamide-SDS gels (Silva, 1989) at 100 V/20 mA. Owing to
the high activity observed in previous analysis, only 0.5 µg of ZsAmy
was analysed. Upon fractionation, the gel was washed three times
with Milli-Q water for 30 min and once with activity buffer (100 mM
phosphate buffer, pH 6.0; 20 mM NaCl; 0.1 mM CaCl2) in order to
remove the SDS and allow the enzyme to renature. A layer of agar
containing 1% blue starch in the activity buffer was cast at 40 °C
on to the polyacrylamide gel to function as the indicator gel. Active
amylase was detected on the indicator gel as clear bands resulting
from the higher diffusion rate of the hydrolysis products in a dark
blue background.
SDS-PAGE and Western blotting
Protein samples from culture supernatants (10 µg) or extracts
(20 µg) of cells infected with recombinant or wild-type baculovirus
were separated by SDS-PAGE in 10% gels (20 × 20 cm) according
to Sambrook et al. (1989). Two gels were prepared: one to detect
the protein bands after Coomasie blue (Sambrook et al., 1989) or
silver staining (Silver Stain Plus kit, BIO-RAD, Hercules, CA, USA);
the other was used for Western blotting. Proteins were transfered
to nitrocellulose membrane by a Transblot-SD Semi-Dry Transfer
Cell. Rabbit anti-Z. subfasciatus α-amylase antibody (kindly pro-
vided by Dr Fatima Grossi de Sá), diluted 1 : 10 000, was used for
immunodetection. Western blotting was performed as described
previously (Sambrook et al., 1989).
Sequence analysis
The nucleotide sequence of A. merus α-amylase (accession
number U01210, GenBank) was processed using the Clone 4
software to obtain the conceptual translation product. This was
then processed in the http://www.up.univ-mrs.fr/∼wabim/d_abim/
compo-p.html server to calculate its molecular weight (Mr). The
promoter sequence was predicted from the http://www.fruitfly.org/
seq-tools/promoter.html server (Waibel et al., 1989), the signal
peptide from http://www.cbs.dtu.dk/services/signalp/ (Nielsen
et al., 1997) and glycosylation sites from http://www.cbs.dtu.dk/
services/netoglyc/ (Hansen et al., 1998). Sequence alignments of
α-amylases were obtained by using the CLUSTAL W program
(Thompson et al., 1994).
© 2003 The Royal Entomological Society, Insect Molecular Biology, 12, 415–425
Acknowledgements
The expert technical assistance of Irenice Cairo da Silva,
Sandra de Souza, Sirlei Mendes de Oliveira and Zizi de
Mendonça is greatly appreciated. This work was supported
by FAPESP, CNPq, FINEP, ICGEB and PRP-USP. P.C.E.
was the recipient of a predoctoral Fellowship from CNPq.
This work is a partial fulfilment of a Doctoral thesis require-
ments (P.C.E.).
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