Purification and characterization of mosquitocidal Bacillus sphaericus BinA protein.
Certain strains of Bacillus sphaericus produce a highly toxic mosquito-larvicidal binary toxin during sporulation. The binary toxin is composed of toxic BinA (41.9kDa) and receptor binding BinB (51.4kDa) polypeptides and is active against vectors of filariasis, encephalitis and malaria. The toxin has been tested with limited use for the control of vector mosquitoes for more than two decades. The binA gene from a local ISPC-8 strain of B. sphaericus that is highly toxic to Culex and Anopheles mosquito species was cloned into pET16b and expressed in Escherichia coli. The purified BinA protein differs by one amino acid (R197M) from BinA of the highest toxicity strains 1593/2362/C3-41. Majority of the expressed protein was observed in inclusion bodies. BinA inclusions alone from E. coli did not show toxic activity, like reported previously. However, the active form of BinA could be purified to homogeneity from the soluble fraction of E. coli cell lysate, grown at reduced temperature after isopropyl beta-d-thiogalactopyranoside induction. The purified BinA protein with and without poly-histidine tag showed LC(50) dose of 82.3 and 66.9ngml(-1), respectively, at 48h against Culex quinquefasciatus larvae. The secondary structure of BinA is expected to be mainly beta strands as estimated using far-UV circular dichroism. The estimates matched well with the secondary structure predictions using amino acid sequence. This is the first report of large-scale purification and accurate toxicity estimation of soluble B. sphaericus BinA. This can help in design and synthesis of improved bacterial insecticide.
Bhabha Atomic Research Centre, Mumbai, India.
Ramesh S
RS
Hire
Ashok B
AB
Hadapad
Tanaji K
TK
Dongre
Vinay
V
Kumar
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification*; D003001:Cloning, Molecular; D003465:Culex / Q000254:growth & development*; D004926:Escherichia coli / Q000235:genetics; D007306:Insecticides / Q000302:isolation & purification*; D007814:Larva; D007928:Lethal Dose 50; D008969:Molecular Sequence Data; D009032:Mosquito Control / Q000379:methods; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D001427:Bacterial Toxins; D007306:Insecticides; C046134:binB protein, Bacillus sphaericus