Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering.
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) congruent with 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.
Abdullah
Mohd Amir F
MA
The Protein Research Group, The Ohio State University, Columbus, Ohio, USA.
Alzate
Oscar
O
Mohammad
Marwan
M
McNall
Rebecca J
RJ
Adang
Michael J
MJ
Dean
Donald H
DH
D000330:Aedes; D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D001427:Bacterial Toxins; D001483:Base Sequence; D003465:Culex / Q000187:drug effects*; D017931:DNA Primers; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D006460:Hemolysin Proteins; D010572:Pest Control, Biological / Q000379:methods; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis