831 Q. Feng et al. Insect Biochemistry and Molecular Biology 30 2000 829–837
Expression System
from Life
Technologies Gaithersburg, MD, USA according to the manufac-
turer’s protocol. The cDNA insert was first cloned into the mini-Tn7 element of a pFASTBAC donor plasmid
at the sites of EcoR I and Xho I. The recombinant plas- mid was then transformed into DH10BAC cells contain-
ing helper plasmid and Autographa californica multicap- sid nucleopolyhedrovirus AcMNPV genomic DNA.
The mini-Tn7 element carrying the cDNA insert was transposed into the AcMNPV genome, resulting in a
recombinant virus. The recombinant baculovirus DNA was selected by disruption of the lacZ
9 gene and the recombinants were confirmed by PCR followed by
Southern blotting. Spodoptera frugiperda cells SF-21, Vaughn et al.,
1977 were cultured in Grace’s medium Grace, 1962 in six-well plates at a concentration of 10
6
cellswell. The cells were incubated for 5 h at 28
° C with a transfec-
tion mixture containing 300 ng of the recombinant DNA and 10
µ l of CellFectin
reagent Life Technologies.
After removal of the transfection mixture, the cells were cultured at 28
° C for 4 days. The recombinant baculo-
virus was harvested and the titer was determined. The virus was then used at an MOI of 0.04 to infect SF-21
cells cultured in 15-ml flasks. The infected cells were harvested at 0, 1, 6, 12, 24, 48, 72, and 96 h post-inocu-
lation h.p.i. for analysis of mRNA and protein.
2.8. JH binding assay SF-21 cells inoculated with the recombinant virus
were homogenized
in the
homogenization buffer
described in Section 2.5. The homogenate was centri- fuged at 1000g for 10 min to pellet cell debris. The
supernatant was centrifuged again at 30 000g for 60 min to pellet cell membranes. The membrane pellet was sus-
pended in 20 mM Tris–HCl buffer, pH 7.5. For specific binding, the binding mixture contained 50 mM Tris–HCl
pH 7.5, 0.1 mgml membrane protein, 15 nM [
3
H]-JH III in a total volume of 200
µ l. For determination of non-
specific binding, unlabeled JH III at 1.5 µ
M was added to the above mixture. The binding reaction was perfor-
med at 28 °
C for 60 min. The mixture was centrifuged
at 30 000g for 30 min to pellet the membranes. The pel- let was rinsed twice with 500
µ l of 50 mM Tris–HCl
binding buffer. The top portion of the tubes was cut off right above the membrane pellet and the bottom portion
was dropped into 10 ml of scintillation cocktail and the radioactivity was counted.
3. Results
3.1. Cloning of 25K cDNA A cDNA library was constructed using mRNA iso-
lated from locust vitellogenic ovaries and fat bodies and screened using the polyclonal antibodies raised against
the 35-kDa JH III membrane receptor Sevala et al., 1995. Ten positive clones were isolated after screening
3.2 ×
10
4
recombinant λ
phage plaques. Restriction enzyme analysis and partial sequencing revealed that
eight out of the 10 clones contained a 0.8-kb identical insert. Three of the eight clones containing an identical
insert were sequenced completely on both strands. Sequence analysis showed that the three clones had an
identical sequence of 766 nucleotides. The nucleotide sequence and deduced amino acid sequence are shown
in Fig. 1. The longest open reading frame ORF begin- ning with ATG started at nucleotide 14 and ended at
nucleotide 688. This ORF encodes a 225-amino acid protein with a predicted molecular mass of 24.688 kDa.
A putative polyadenylation signal, AATAAA, was found between nucleotide 731 and 736, followed by a
polyA
18
sequence Fig. 1. The deduced amino acid sequence contained a putative signal peptide 1–16
amino acid residues, preceding a putative cleavage site between Ser-16 and Arg-17. Since this cDNA concep-
tually translates into a 24.69-kDa protein, we named it 25K cDNA.
3.2. Sequence comparison Comparison of the 25K cDNA sequence with the
sequences in GenBank showed that the deduced amino acid sequence of 25K cDNA was similar to the amino
acid sequence of the 21-kDa juvenile hormone-induced protein identified in the locust hemolymph 21K, Zhang
et al., 1993. The amino acid identity was 42.3 between these two proteins. This 25K cDNA also shared
an 11.6 amino acid identity with a 19-kDa protein 19K, which was also cloned from the locust Kanost
et al., 1988. The amino acid sequences of the N-ter- minal ends of these three proteins show considerable
similarity, whereas the sequences from the amino acid residue 111 to the C-terminal end were different Fig.
2A. The deduced amino acid sequence of the 25K cDNA contained seven repeat elements near the C-ter-
minal end Fig. 2B and C. Each repeat consists of 10 amino acids, nine of which are highly conserved. Seven
of the nine highly conserved amino acids in the repeat elements were polar amino acids, which makes the con-
served sequence highly hydrophilic Fig. 2C. No such repeat elements were present in the deduced amino acid
sequences of either 21K or 19K cDNA. A GenBank search revealed that no other protein identified so far had
a similar repeat sequence.
3.3. Developmental expression of 25K mRNA To determine where and when the mRNA for 25K
protein is expressed during adult maturation, total RNA was extracted from the fat body of female and male
832 Q. Feng et al. Insect Biochemistry and Molecular Biology 30 2000 829–837
Fig. 1. Nucleotide and deduced amino acid sequences of 25K cDNA cloned from L. migratoria. The putative signal peptide is underlined. The
stop codon TGA is marked with asterisks and the putative polyadenylation signal AATAAA is double-underlined. The seven repeat elements are shaded with black. The numbers on the left refer to nucleotides and those on the right refer to amino acid residues.
Fig. 2. A Alignment of the deduced amino acid sequence of the 25K protein this study with the 21K protein Zhang et al., 1993 and the
19K protein Kanost et al., 1988 using the Clustal Alignment Program Higgins and Sharp, 1988. The residues that match the 25K protein exactly are shaded in solid black. The consensus residues are given in lines below the alignment panels when three residues match, otherwise they are
shown as a period. B Amino acid sequence matrix of the 25K protein, showing the internal repeats. C Alignment and hydrophilicity of repeat elements of the 25K protein. The consensus amino acid residues are shaded in black when at least four out of seven residues match.
833 Q. Feng et al. Insect Biochemistry and Molecular Biology 30 2000 829–837
Fig. 3. Developmental expression of 25K mRNA in the fat bodies and ovarian follicle cells of adult locust. The top panel shows the Northern
blot containing 10 µ
g of total RNA hybridized with a 25K cDNA probe. The bottom panel shows ribosomal RNA stained with ethidium bromide, indicating equal loading of RNA.
adults and from the ovaries on days 0–8 after the insects had molted into adults. Expression of the mRNA for the
25K protein in these tissues was examined using North- ern blots hybridized with the 25K cDNA as a probe. A
1.2-kb mRNA was detected in the fat body of mated female adults Fig. 3. The mRNA started to appear on
day 4 after adult emergence. By day 6 the mRNA level increased in abundance and continued to increase up to
day 8. This mRNA was absent in the fat body of adult males day 8, and no mRNA was detected in the ovaries
up to 8 days. These results indicated that the synthesis of 25K mRNA is tissue-, development- and sex-specific.
3.4. In vitro translation of 25K cDNA In vitro translation of the 25K cDNA was performed
using a reticulocyte lysate system to characterize the peptide encoded by this cDNA. The translation product
migrated at 32 kDa when resolved on SDS–PAGE Fig. 4, instead of the predicted 25 kDa. We examined five
independent clones and all of them produced the ident- ical protein with an apparent molecular mass of 32 kDa.
Fig. 4. In vitro translation of 25K cDNA in reticulocyte lysate system
in the presence of [
35
S]-methionine. Lane C, control, pBluscript vector alone without any insert; Lanes 1–5, various clones with the 25K
cDNA insert. The translation product was separated in a 12 SDS– PAGE gel.
3.5. Expression of 25K cDNA in a baculovirus expression system
To express the 25K cDNA, a recombinant AcMNPV expressing the 25K cDNA AcMNPV-25K was con-
structed and used to infect SF-21 cells. RNA and pro- teins were isolated from the infected cells at different
times post-inoculation. Northern blots hybridized with a 25K cDNA probe showed that 1.2-kb mRNA started to
appear at 24 h.p.i. in the cells inoculated with AcMNPV- 25K Fig. 5A, left. The mRNA level increased by 48
h.p.i. This mRNA was not detected in the control cells that were inoculated with another recombinant baculo-
virus expressing a 35-kDa protein cDNA AcMNPV- 35K Fig. 5A, right.
Proteins in the cells inoculated with AcMNPV-25K were analyzed using SDS–PAGE followed by Western
blotting. The expressed protein was detected in both the membrane fraction and the cytoplasm of the cells inocu-
lated with the recombinant virus. The protein profile of SDS–PAGE showed a 32-kDa protein starting to appear
at 48 h.p.i., 1 day after the appearance of the mRNA Fig. 5B. The expressed protein was recognized
immunologically by the antibodies used for screening the cDNA library Fig. 5C. Thus, the 25K cDNA we
cloned was expressed both in the baculovirus expression system and in the in vitro translation system as a protein
with an apparent molecular mass of 32 kDa.
3.6. JH binding assay To examine whether JH binds to the expressed protein
from AcMNPV-25K, we conducted a preliminary JH binding assay using the membrane fractions prepared
834 Q. Feng et al. Insect Biochemistry and Molecular Biology 30 2000 829–837
Fig. 5. Expression of 25K cDNA in SF-21 cells inoculated with the recombinant baculovirus expressing the 25K cDNA AcMNPV-25K. A
mRNA expression in the SF-21 cells inoculated with AcMNPV-25K left or inoculated with a recombinant baculovirus carrying a 35-kDa protein cDNA as control right. The top panel shows the Northern blot containing 10
µ g of total RNA hybridized with a 25K cDNA probe. The bottom
panel shows ribosomal RNA stained with ethidium bromide, showing equal loading of RNA. B SDS–PAGE of the 25K protein expressed in SF- 21 cells inoculated with AcMNPV-25K. Five micrograms of cell proteins were loaded in each lane. SDS–PAGE gel was stained with Coomsasie
Blue R250. C Western blot was immunostained with the polyclonal antibodies 1:2000 produced against the JHMR, followed by sheep anti- rabbit antibodies conjugated with alkaline phosphatase 1:5000.
from the cells infected with AcMNPV-25K. The results showed no specific binding of [
3
H]-JH III to the mem- brane protein data not shown. This was consistent with
the results of protease cleavage assay with Protease K and JH III that also showed that JH did not bind to the
protein expressed from the in vitro translation system data not shown.
4. Discussion
