Australian funnel-web spiders: master insecticide chemists

Tedford, Hugo W.; Sollod, Brianna L.; Maggio, Francesco; King, Glenn F.

doi:10.1016/j.toxicon.2004.02.010

Cited by 132 publications

(107 citation statements)

References 125 publications

Supporting

Mentioning

105

Contrasting

Unclassified

Order By: Relevance

“…Previous alanine scanning mutagenesis studies by Tedford et al, [21] 2004 have identified 3 key functional residues (Pro10, Asn27, and Arg35) that determine specific binding to insect calcium channels. Here replacement of the 34th lysine residue in Hv1a with a glutamine residue by site directed mutagenesis was selected as glutamine is known to be present at this position of other members of the -ACTX-1 family ( [22] Tedford et al 2004) and was thus unlikely to disrupt biological function of the recombinant toxin.…”

Section: Analysis Of Biological Activity: Injection Bioassaysmentioning

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

View full text Add to dashboard Cite

. (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.

show abstract

Section: Analysis Of Biological Activity: Injection Bioassaysmentioning

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

View full text Add to dashboard Cite

show abstract

“…Many spider venoms contain a complex mixture of both neurotoxic and cytolytic toxins (see: www.arachnoserver.org). Virtually all insecticidal spider toxins contain a cystine-knot motif that provides them with chemical and biological stability (King et al, 2002;Tedford et al, 2004). These types of venoms contain acylpolyamines (from the Araneidae family), cytolytic toxins (from the Zodariidae family) and neurotoxic peptides (J-atratoxins), and neurotoxins (>10 kDa) and enzymes (~35 kDa) in the Sicariidae and Theridiidae families respectively (Vassilevski et al, 2009;Gunning et al, 2008).…”

Section: Typical Anti-insect Toxinsmentioning

confidence: 99%

Biological Activity of Insecticidal Toxins: Structural Basis, Site-Directed Mutagenesis and Perspectives

López-Pazos¹,

Cerón²

2013

Genetic Manipulation of DNA and Protein - Examples From Current Research

View full text Add to dashboard Cite

“…These peptides -isolated from venoms of spiders, snails, scorpions, snakes and other animals -are produced in a combinational manner that leads to a great diversity (1)(2)(3)(4). Most venom peptides are highly bridged proteins with multipairs of intrachain disulfide bonds.…”

Section: Introductionmentioning

confidence: 99%

Determination of disulfide bridges of two spider toxins: hainantoxin-III and hainantoxin-IV

Wang¹,

Liu²,

Qian³

et al. 2009

J. Venom. Anim. Toxins incl. Trop. Dis

View full text Add to dashboard Cite

Peptide toxins are usually highly bridged proteins with multipairs of intrachain disulfide bonds. Analysis of disulfide connectivity is an important facet of protein structure determination. In this paper, we successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. Both peptides are useful inhibitors of TTX-sensitive voltage-gated sodium channels and are composed of six cysteine residues that form three disulfide bonds, respectively. Firstly, the peptides were partially reduced by tris(2-carboxyethyl)-phosphine (TCEP) in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40°C for ten minutes. Subsequently, the partially reduced intermediates containing free thiols were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and alkylated by rapid carboxamidomethylation. Then, the disulfide bonds of the intermediates were analyzed by Edman degradation. By using the strategy above, disulfide linkages of HNTX-III and HNTX-IV were determined as I-IV, II-V and III-VI pattern. In addition, this study also showed that this method may have a great potential for determining the disulfide bonds of spider peptide toxins.

show abstract

Australian funnel-web spiders: master insecticide chemists

Cited by 132 publications

References 125 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

Biological Activity of Insecticidal Toxins: Structural Basis, Site-Directed Mutagenesis and Perspectives

Determination of disulfide bridges of two spider toxins: hainantoxin-III and hainantoxin-IV

Contact Info

Product

Resources

About