Pesticidal fusion protein improvements

Abstract

Methods of increasing the biological activity of toxins. Methods of increasing the biological activity of pesticide toxins through the incorporation of pro-regions into nucleic acid constructs for the production of said toxins.

1 - 39 . (canceled) 
     
     
         40 . A method of increasing the biological activity of a recombinant toxin, the method comprising:
 providing a nucleic acid construct comprising:
 a toxin sequence, or a fragment or variant thereof, linked to 
 (ii) a pro-region, or fragment or variant thereof; and 
   optionally expressing the recombinant toxin.   
     
     
         41 . The method according to  claim 40  wherein the toxin is an arthropod toxin. 
     
     
         42 . The method according to  claim 40  wherein the toxin is ω-ACTX-Hv1a, or a fragment or variant thereof. 
     
     
         43 . The method according to  claim 40  wherein the toxin is δ-amaurobitoxin-PI1a, or a fragment or variant thereof. 
     
     
         44 . The method according to  claim 40  wherein the pro-region is a sequence between a signal peptide sequence and a mature protein N-terminus, optionally wherein the pro-region is identifiable by the steps of:
 comparing the sequence determined for a protein isolated from its normal source with the sequence predicted by the gene encoding it; 
 identifying N-terminal sequences not present in the final protein product; 
 identifying a signal peptide sequence in the N-terminal sequence not present in the final protein product; and 
 identifying a pro-region sequence between the signal peptide sequence and the mature protein N-terminus, optionally by removal of the signal peptide sequence from the N-terminal sequence not present in the final protein product. 
 
     
     
         45 . The method according to  claim 40  wherein the pro-region is rich is acidic amino acid residues. 
     
     
         46 . The method according to  claim 40  wherein the pro-region comprises the amino acid sequence EDTRADLQGGEAAEKVFRR, or a fragment or variant thereof. 
     
     
         47 . The method according to  claim 40  wherein the pro-region comprises the amino acid sequence ISYEEGKELFQKER, or a fragment or variant thereof. 
     
     
         48 . The method according to  claim 40  wherein the pro-region comprises the amino acid sequence ISYEEGKELFQKER, or a fragment or variant thereof. 
     
     
         49 . The method according to  claim 48  wherein the carrier protein is a plant lectin selected from any one or more of the following: snowdrop lectin (GNA), garlic lectin  Allium sativum , pea lectin  Pisum sativum  (P-lec), peanut lectin  Arachis hypogaea , french bean lectin (PHA, Phytohaemagglutinin), or a fragment or variant thereof. 
     
     
         50 . A nucleic acid construct comprising:
 (i) a pro-region, or fragment or variant thereof; and   (ii) a site adjacent to the pro-region into which a toxin gene sequence, or fragment or variant thereof may be inserted.   
     
     
         51 . The nucleic acid construct according to  claim 50  further comprising the toxin gene sequence or fragment or variant thereof inserted into the site adjacent to the pro-region. 
     
     
         52 . The nucleic acid construct according to  claim 50  wherein the nucleic acid construct is an expression construct. 
     
     
         53 . The nucleic acid construct according to  claim 50  wherein the nucleic acid constructs further comprise a sequence which encodes an affinity tag. 
     
     
         54 . A pesticide composition comprising a toxin protein produced according to the method described in  claim 40 . 
     
     
         55 . The pesticide composition according to  claim 54  wherein the composition comprises a toxin protein in an amount of between 0.001% and 99% by weight preferably between 0.5% and 98% by weight, more preferably between 1.0% and 95% by weight. 
     
     
         56 . A process for the preparation of a pesticide composition which comprises the admixture of an amount of toxin protein produced according to one of the methods described in  claim 40  with one or more suitable carriers, diluents, adjuvants, preservatives, dispersants, solvents, emulsifying agents in effective pesticidal amount. 
     
     
         57 . A method of preventing or treating a pest infection of a plant comprising applying a quantity of toxin protein produced according to the methods described in  claim 40  to the plant or its locus of growth. 
     
     
         58 . A molluscicide bait composition comprising a toxin protein produced according to the methods described in  claim 40 . 
     
     
         59 . A transgenic plant or progeny thereof comprising a nucleic acid construct as described in  claim 51  capable of expressing a toxin with increased biological activity.