A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders.
Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the "carrier" snowdrop lectin (GNA) to confer oral toxicity. The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th - 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide.
School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK.
Sheng
S
Yang
School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK.
Prashant
P
Pyati
Fera (Food and Environment Research Agency), Sand Hutton, York, UK.
Elaine
E
Fitches
School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK. Electronic address: J.A.Gatehouse@durham.ac.uk.
John A
JA
Gatehouse
D000595:Amino Acid Sequence; D000818:Animals; D019476:Insect Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007313:Insecta / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D007306:Insecticides / Q000378:metabolism / Q000633:toxicity*; D008969:Molecular Sequence Data; D010572:Pest Control, Biological; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D026941:Sodium Channel Blockers / Q000378:metabolism / Q000633:toxicity*; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism; D013111:Spider Venoms / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D013112:Spiders / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D019476:Insect Proteins; D007306:Insecticides; D011993:Recombinant Fusion Proteins; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D013111:Spider Venoms
Biotechnology; Crop protection; Insecticide; Neurotoxin; Protein transport; Recombinant protein expression system