Introduction of Culex Toxicity into Bacillus thuringiensis Cry4Ba by Protein Engineering

Abstract

ABSTRACT Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC 50 ], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti , Anopheles quadrimaculatus , C . quinquefasciatus , and C . pipiens . Putative loop 1 and 2 exchanges eliminated activity towards A . aegypti and A . quadrimaculatus . Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C . quinquefasciatus (LC 50 ≅ 114 ng/ml) and C . pipiens (LC 50 ⩬ 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or Aedes . These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B . thuringiensis Cry toxins.