Processing of Delta Endotoxin from Bacillus thuringiensis Subspp.Kurstaki HD-1 and HD-73 by Immobilized Trypsin and Chymotrypsin

Indrasith, Leslie S.; Ogiwara, Katsutoshi; MlNAMI, Masayoshi; Iwasa, Tomoko; Maruyama, Takeshi; Suzuki, Nobukazu; Asan, Shoji; Sakanaka, Kazunobu; Hori, Hidetaka

doi:10.1303/aez.26.485

Cited by 21 publications

(7 citation statements)

References 10 publications

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“…Protoxin (130 kDa) prepared as described earlier was activated with trypsin (41), and the activated toxin (60 kDa) was labeled with biotin using an ImmunoProbe Biotinylation Kit (Sigma). Midgut BBMV were prepared from C2 and Rin strains as described by Wolfersberger et al (42).…”

Section: Methodsmentioning

confidence: 99%

Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori

Atsumi¹,

Miyamoto²,

Yamamoto

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

197

158

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Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93 , which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis.

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Section: Methodsmentioning

confidence: 99%

Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori

Atsumi¹,

Miyamoto²,

Yamamoto

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

197

158

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“…The 130-kDa protoxins were activated with trypsin immobilized on Sepharose 4B as described previously (16). The activated 60-kDa Cry1A toxins were fractionated by DEAE-Sepharose column chromatography (30-ml bed volume) in Tris-HCl (pH 8.3) using a linear gradient of 50 to 400 mM NaCl over 300 ml at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…SDS-PAGE was performed as described previously (25,16), and the proteins were visualized by staining with silver or Coomassie brilliant blue (CBB). Native PAGE was performed as described by Kishimoto et al (20).…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of Bombyx mori , That Binds to Cry1A Toxins

Hossain

Shitomi

Moriyama

et al. 2004

Appl Environ Microbiol

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We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and K d constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc.

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“…Cry1Ab was obtained from the transformed E. coli JM109 harboring plasmid pYD4.0 encoding cry1Ab (a gift from K. Kanda, Saga University) cultured in LB broth containing 50 g of ampicillin/ml (19). The insecticidal crystal proteins obtained as above were solubilized and activated with immobilized trypsin as described previously (18) and purified as according to Hossain et al (15).…”

Section: Methodsmentioning

confidence: 99%

Bombyx mori Midgut Membrane Protein P252, Which Binds to Bacillus thuringiensis Cry1A, Is a Chlorophyllide-Binding Protein, and the Resulting Complex Has Antimicrobial Activity

Pandian

Ishikawa

Togashi

et al. 2008

Appl Environ Microbiol

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The epithelial cell membrane 252-kDa protein (P252) isolated in our laboratory from Bombyx mori midgut was shown to bind strongly with Cry1Aa, Cry1Ab, and Cry1Ac toxins of Bacillus thuringiensis (15). In the current paper, P252 was shown to bind with chlorophyllide (Chlide) to form red fluorescent protein (RFP) complex, termed Bm252RFP, with absorbance and fluorescence emission peaks at 600 nm and 620 nm, respectively. P252 at a concentration of 1 M is shown to bind with about 50 M Chlide in a positively cooperative reaction to form Bm252RFP under aerobic conditions and in the presence of light at 37°C. Various parameters influencing this reaction have been optimized for efficient in vitro chemical synthesis of Bm252RFP. Circular dichroism spectra revealed that P252 is composed of a ␤-structure (39.8% ؎ 2.2%, based on 5 samples) with negligible contribution of ␣-helix structure. When bound to Chlide, the ␤-structure content in the complex is reduced to 21.6% ؎ 3.1% (n ‫؍‬ 5). Since Chlide had no secondary structure, the observed reduction suggests significant conformational changes of P252 during the formation of Bm252RFP complex. Bm252RFP had antimicrobial activity against Escherichia coli, Serratia marcescens, B. thuringiensis, and Saccharomyces cerevisiae with 50% effective concentrations of 2.82, 2.94, 5.88 M, and 21.6 M, respectively. This is the first report ever to show clear, concrete binding characteristics of the midgut protein to form an RFP having significant antimicrobial activity.The actual insecticidal mechanism of Cry toxins of Bacillus thuringiensis and the receptor protein that leads to pore formation are yet to be fully clarified. However, it has generally been accepted that B. thuringiensis Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins bind with putative receptor molecules, aminopeptidase N (APN) (21,33,40), and/or cadherinlike proteins (17,28,39). It is believed that after binding with these receptors, hydrophobic ␣-helices in domain I of Cry1A toxins penetrate into the brush border membrane (BBM) of midgut epithelial cells. Then, by small-pore formation on the BBM, these cells lose their homeostasis, ultimately leading to the insect's death (7,22,45). To understand the complete insecticidal mechanism, we have been interested in elucidating the interaction between epithelial cell membrane proteins and Cry toxins.During the search for the Cry toxin binding proteins, we found 252-kDa proteins in the membrane of epithelial cells from the Bombyx mori midgut (14-16). The protein (P252) was purified from a Triton X-100 soluble fraction of BBM vesicles (BBMVs) of B. mori, and it was purified to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. It was shown to have a molecular mass of 252 kDa by SDS-PAGE. But the molecular size of about 985 kDa obtained from gel filtration chromatography suggests that the protein could be a homotetramer, and this P252 was shown to bind strongly with Cry1Aa, Cry1Ab, and Cry1Ac toxins of B. thurin...

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Processing of Delta Endotoxin from Bacillus thuringiensis Subspp.Kurstaki HD-1 and HD-73 by Immobilized Trypsin and Chymotrypsin

Cited by 21 publications

References 10 publications

Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori

Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori

Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of Bombyx mori , That Binds to Cry1A Toxins

Bombyx mori Midgut Membrane Protein P252, Which Binds to Bacillus thuringiensis Cry1A, Is a Chlorophyllide-Binding Protein, and the Resulting Complex Has Antimicrobial Activity

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