Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion

Fitches, Elaine; Edwards, Martin G.; Mee, Christopher; Grishin, Eugene V.; Gatehouse, Angharad M. R.; Edwards, John; Gatehouse, John A.

doi:10.1016/j.jinsphys.2003.09.010

Cited by 153 publications

(139 citation statements)

References 18 publications

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“…1b). This has been reported previously for a fusion protein encoding a venom peptide SFl1, from the spider Segestria florentina, linked to GNA ( [5] Fitches et al, 2004). In the case of SFI1/GNA, expression by X33 cells was found to produce a 1:1 ratio of intact to cleaved protein, whereas predominantly intact SFI1/GNA was obtained using the protease deficient strain SMD1168H.…”

Section: Construct Modification and Expression Analysessupporting

confidence: 68%

“…Snowdrop lectin (Galanthus nivalis agglutinin; GNA) has been used as a "carrier" domain in the production of recombinant insecticidal fusion proteins, where it confers oral toxicity on peptide toxins from scorpions and spiders ( [5][6][7][8][9] . Oral toxicity is achieved via delivery of attached toxins, by GNA, to the central nervous system of target pests.…”

Section: Introductionmentioning

confidence: 99%

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Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA inPichia pastoris:sequence modifications and a simple method for the generation of multi-copy strains

Pyati

Fitches

Gatehouse

2014

Journal of Industrial Microbiology and Biotechnology

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. (2014) 'Optimising expression of the recombinant fusion protein biopesticide -hexatoxin-Hv1a /GNA in Pichia pastoris : sequence modi cations and a simple method for the generation of multi-copy strains.', Journal of industrial microbiology biotechnology., 41 (8). pp. 1237-1247. Further information on publisher's website:http://dx.doi.org/10.1007/s10295-014-1466-8Publisher's copyright statement:The nal publication is available at Springer via http://dx.doi.org/10.1007/s10295-014-1466-8. Additional information:Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. AbstractA recombinant construct encoding for the expression of an insecticidal fusion protein comprised ofcontaining the spider-venom peptide -hexatoxin-Hv1a (Hv1a), from the venom of the funnel web spider Hadronyche versuta, linked to the snowdrop lectin (Galanthus nivalis agglutinin; (GNA) has been shown to be an effective oral insecticide against lepidopteran larve. The construct for producing the Hv1a/GNA fusion protein in P. pastoris has been modified to improve levels of intact recombinant protein recoverable from fermented culture supernatants. by sSite directed mutagenesis to remove a potential Kex2 cleavage site at the C-terminus of the Hv1a peptide. The modifed construct resulted in markedly increased levels of intact fusion protein expressed in wild type, but not protease deficient, P. pastoris strains, improving levels of intact recombinant protein recoverable from culture supernatants. indicative of increased resistance to yeast proteases. Injection assays of purified fusion protein in lepidopteran larvae demonstrated that sequence modification did not affect the insecticidal activity of the recombinant toxin. The incorporation of a C-terminal (histidine) 6 tag in the modified construct enabled a single step purification of the fusion protein from fermenter supernatants derived from bench-top fermentation. A straightforward method for producing multicopy expression plasmids for production of recombinant proteins in P. pastoris is described, which does not rely on multiple integrations to give clones of P. pastoris containing high copy numbers of the introduced gene. The method has been used to increase production of the secreted recombinant fusion protein in a pilot scale laboratory fermentation system by almost 10-fold on a per litre of culture basis, providing a means to allow evaluation as a commercial biopesticide.

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Section: Construct Modification and Expression Analysessupporting

confidence: 68%

Section: Introductionmentioning

confidence: 99%

Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA inPichia pastoris:sequence modifications and a simple method for the generation of multi-copy strains

Pyati

Fitches

Gatehouse

2014

Journal of Industrial Microbiology and Biotechnology

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“…Recombinant fusion proteins have been reported to be effective in the development of oral vaccines (20,21). However, this is the first demonstration, to our knowledge, that Tf moiety in Tf-based recombinant technology enhances the GI absorption of a therapeutic protein drug.…”

Section: Discussionmentioning

confidence: 89%

“…Conceivably, such a short linker may decrease the chance for immunogenicity of the fusion protein; but it may also block the accessibility of the functional moiety for its receptor binding. It is noteworthy that a recombinant nerve growth factor-Tf fusion protein has been produced previously, in which a linker of IgG 3 hinge region was inserted between the two protein moieties to retain the in vitro biological activity (20).…”

Section: Discussionmentioning

confidence: 99%

Recombinant granulocyte colony-stimulating factor-transferrin fusion protein as an oral myelopoietic agent

Bai

Ann

Shen

2005

Proc. Natl. Acad. Sci. U.S.A.

116

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An expression construct harboring granulocyte colony-stimulating factor (G-CSF)-transferrin (Tf) fusion protein (G-CSF-Tf) was engineered by fusing human cDNAs encoding G-CSF and Tf to explore the feasibility of using Tf as a carrier moiety for oral delivery of therapeutic proteins. The recombinant protein, G-CSF-Tf, was harvested from protein-free, conditioned medium of transfected HEK293 cells. The in vitro studies demonstrated that the purified G-CSF-Tf fusion protein possesses the activity of both Tf receptor (TfR) binding in Caco-2 cells and G-CSF-dependent stimulation of NFS-60 cell proliferation. Subcutaneous administration of G-CSF-Tf fusion protein to BDF1 mice demonstrated a pharmacological effect comparable to the commercial G-CSF on the increase of absolute neutrophil counts (ANC). However, the fusion protein elicited a significant increase in ANC upon oral administration to BDF1 mice, whereas G-CSF had no effect. This study also showed that orally administered G-CSF-Tf elicits a sustained myelopoietic effect up to 3 days, whereas the s.c. administered G-CSF or G-CSF-Tf lasts only 1 day. Furthermore, coadministration of free Tf abolished the increase of ANC by orally delivered G-CSF-Tf, suggesting that the recombinant protein is absorbed via a TfR-mediated process in the gastrointestinal tract. Taken together, we conclude that the Tf-based recombinant fusion protein technology represents a promising approach for future development of orally effective peptide and protein drugs. myelopoiesis ͉ oral delivery ͉ protein drug

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“…The effectiveness of this approach has been demonstrated for other toxins that work in the insect hemocoel, including allatostatin and insect-specific toxins from spider venom (Fitches et al, 2002;Fitches et al, 2004;Rachel E Down, 2006).…”

Section: Fig 11mentioning

confidence: 99%

Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens

Tang

Lei

et al. 2007

Tissue and Cell

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Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion

Cited by 153 publications

References 18 publications

Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA inPichia pastoris:sequence modifications and a simple method for the generation of multi-copy strains

Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA inPichia pastoris:sequence modifications and a simple method for the generation of multi-copy strains

Recombinant granulocyte colony-stimulating factor-transferrin fusion protein as an oral myelopoietic agent

Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens

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