Insecticidal Fusion Proteins For The Control of Coleopteran Pests

Fusion proteins containing a toxin fused to a carrier domain which directs transport across the insect gut epithelium have been shown to be effective orally active insecticides. Expression of functional recombinant fusion proteins comprising of snowdrop lectin (Galanthus nivalis agglutinin; GNA) fused to toxins from Indian red scorpion (Mesobuthus tamulus toxin; ButaIT) and Blue Mountains funnel-web spider (Hadronyche versuta toxins; ω-ACTX-Hv1a (ω-ACTX); κ-ACTX-Hv1c (κ-ACTX)) was carried out in both yeast (Pichia pastoris) and plant (Arabidopsis thaliana) expression systems. Addition of purification tags, altering the design of assembly of the fusion protein and point mutation of toxin sequence were all investigated to improve yield and reduce proteolytic cleavage during expression and purification. Recombinant proteins were assayed for oral toxicity against T. castaneum as a model coleopteran species. Fusion proteins incorporating ButaIT and ω-ACTX toxins showed toxicity ranging from complete mortality when fed at 1mg g-1 ((his)6-GNA-ω-ACTX and ω-ACTX-GNA-(his)6) to 65% mortality when fed at 2mg g-1 (ButaIT-GNA-(his)6). Fusion proteins incorporating κ-ACTX and GNA were shown to be non-toxic despite individual components being functional. Lack of toxicity was due to high proteolytic cleavage in the insect gut environment. Data was obtained to support the use of Tribolium as a model for wireworm (Agriotes spp.), serious pests of potatoes in the UK. Selected fusion proteins were expressed in transgenic Arabidopsis. Expression for ButaIT-GNA as a fusion polypeptide was readily detectable in transformants with estimated levels of expression of approx. 0.15% total soluble protein in leaf tissue. When plants expressing ButaIT-GNA fusion protein were fed to larvae of the tomato moth (Lacanobia oleracea) the fusion protein was shown to be fully functional with levels of toxicity comparable to that seen in previous artificial diet bioassays. ω-ACTX based constructs expressed in Arabidopsis were subject to high levels of proteolytic cleavage in planta and so were not assayed for toxicity.