Abstract
Insect-selective neurotoxins are useful agents for developing recombinant baculoviral insecticides. AaHIT, one such toxin, was expressed in AcMNPV and had a dramatic effect on pest insects. However, the toxin production was relatively low. In order to identify reasons for the low production of recombinant AaHIT (rAaHIT) in insect cells, we analyzed the expression efficiency using Northern blotting, enzyme-linked immunosorbent assay, Western blotting, and a bioassay using Sarcophaga falculata larvae. The levels of rAaHIT mRNA and protein in Sf21 cells were comparable with those of another secreted protein, recombinant juvenile hormone esterase (rJHE), which was expressed in the same baculovirus system. However, the secretion efficacy of rAaHIT was significantly lower than that of rJHE. In addition, the toxic activity in the medium was approximately 1% of the total secreted rAaHIT. Western blot analyses suggested that rAaHIT aggregated and accumulated in cells. We attempted to increase the amount of soluble rAaHIT in cells using a co-expressed chaperonin, BiP. Although co-expressed BiP apparently increased the amount of soluble rAaHIT in cells, no improvement of active toxin production in the medium was detected. These results suggest that secretion and folding of rAaHIT are insufficient in insect cells.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 225-232 |
| Number of pages | 8 |
| Journal | Applied Entomology and Zoology |
| Volume | 37 |
| Issue number | 2 |
| State | Published - May 2002 |
Fingerprint
Keywords
- AaHIT
- BiP
- Insect selective scorpion neurotoxin
- Recombinant baculovirus
ASJC Scopus subject areas
- Insect Science
Cite this
Expression efficiency of a scorpion neurotoxin, AaHIT, using baculovirus in insect cells. / Taniai, Kiyoko; Inceoglu, Ahmet B.; Hammock, Bruce D.
In: Applied Entomology and Zoology, Vol. 37, No. 2, 05.2002, p. 225-232.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Expression efficiency of a scorpion neurotoxin, AaHIT, using baculovirus in insect cells
AU - Taniai, Kiyoko
AU - Inceoglu, Ahmet B.
AU - Hammock, Bruce D.
PY - 2002/5
Y1 - 2002/5
N2 - Insect-selective neurotoxins are useful agents for developing recombinant baculoviral insecticides. AaHIT, one such toxin, was expressed in AcMNPV and had a dramatic effect on pest insects. However, the toxin production was relatively low. In order to identify reasons for the low production of recombinant AaHIT (rAaHIT) in insect cells, we analyzed the expression efficiency using Northern blotting, enzyme-linked immunosorbent assay, Western blotting, and a bioassay using Sarcophaga falculata larvae. The levels of rAaHIT mRNA and protein in Sf21 cells were comparable with those of another secreted protein, recombinant juvenile hormone esterase (rJHE), which was expressed in the same baculovirus system. However, the secretion efficacy of rAaHIT was significantly lower than that of rJHE. In addition, the toxic activity in the medium was approximately 1% of the total secreted rAaHIT. Western blot analyses suggested that rAaHIT aggregated and accumulated in cells. We attempted to increase the amount of soluble rAaHIT in cells using a co-expressed chaperonin, BiP. Although co-expressed BiP apparently increased the amount of soluble rAaHIT in cells, no improvement of active toxin production in the medium was detected. These results suggest that secretion and folding of rAaHIT are insufficient in insect cells.
AB - Insect-selective neurotoxins are useful agents for developing recombinant baculoviral insecticides. AaHIT, one such toxin, was expressed in AcMNPV and had a dramatic effect on pest insects. However, the toxin production was relatively low. In order to identify reasons for the low production of recombinant AaHIT (rAaHIT) in insect cells, we analyzed the expression efficiency using Northern blotting, enzyme-linked immunosorbent assay, Western blotting, and a bioassay using Sarcophaga falculata larvae. The levels of rAaHIT mRNA and protein in Sf21 cells were comparable with those of another secreted protein, recombinant juvenile hormone esterase (rJHE), which was expressed in the same baculovirus system. However, the secretion efficacy of rAaHIT was significantly lower than that of rJHE. In addition, the toxic activity in the medium was approximately 1% of the total secreted rAaHIT. Western blot analyses suggested that rAaHIT aggregated and accumulated in cells. We attempted to increase the amount of soluble rAaHIT in cells using a co-expressed chaperonin, BiP. Although co-expressed BiP apparently increased the amount of soluble rAaHIT in cells, no improvement of active toxin production in the medium was detected. These results suggest that secretion and folding of rAaHIT are insufficient in insect cells.
KW - AaHIT
KW - BiP
KW - Insect selective scorpion neurotoxin
KW - Recombinant baculovirus
UR - http://www.scopus.com/inward/record.url?scp=0036018808&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0036018808&partnerID=8YFLogxK
M3 - Article
AN - SCOPUS:0036018808
VL - 37
SP - 225
EP - 232
JO - Applied Entomology and Zoology
JF - Applied Entomology and Zoology
SN - 0003-6862
IS - 2
ER -