Hinchliffe, Gareth (2007) Expression of novel insecticidal proteins in the yeast pichia pastoris. Masters thesis, Durham University.
The development of Fusion Protein Technology during the search for new environmentally benign pesticides has prompted further investigation into novel insecticidal proteins from predatory and parasitic insects. Eulophus pennісоrnis is a parasitoid wasp used in biological pest control which causes developmental arrest in its host. E. pennicornis venom gland cDNAs were isolated and sequenced. Searches for sequence similarity in the global databases revealed a splicing factor, serine protease and a neprilysin-like protein as key components besides an already isolated reprolysin-like metalloprotease and juvenile hormone-inducible protein. Attempts were made to express the reprolysin-like metalloprotease in Pichia pastoris as a fusion protein with GNA and with structural truncations. Constructs for the fusion and truncations were produced using recombinant DNA techniques but unfortunately, little success was achieved with the expression. The results presented point towards reprolysin-like metalloprotease perhaps being toxic to the p. pastoris yeast cells. Avidin, another protein with the potential for exploitation in Fusion Protein Technology, was also expressed as a recombinant protein in p. pastoris and successfully purified to almost 100% homogeneity. In this study, bioassays with Mamestra brassicae (cabbage moth) larvae confirmed its transport to the haemolymph and insecticidal activity when delivered orally above a threshold concentration via artificial diet. Fluorescent labelling of recombinant avidin also highlighted the mechanics involved in its transport within M. brassicae larvae and confirmed its suitability for use in Fusion Protein Technology. Avidin-antibody coupling experiments gave inconclusive results for transport to the haemolymph of Sitobion avenae (cereal aphid), but revealed a more permeable gut structure in M. brassicae larvae, potentially opening up new avenues for the control of the larvae of pest lepidopterans using lethal antibodies.
|Item Type:||Thesis (Masters)|
|Award:||Master of Science|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||08 Sep 2011 18:30|