Screening of the Bacillus thuringiensis Cry1Ac delta-endotoxin on the artificial phospholipid monolayer incorporated with brush border membrane vesicles of Plutella xylostella by optical biosensor technology.
The binding of Cry1Ac, an insecticidal protein of Bacillus thuringiensis, to a brush border membrane (BBM) isolated from midguts of the diamondback moth Plutella xylostella was examined by surface plasmon resonance (SPR)-based biosensor. BBM was mixed with 1,3-ditetradecylglycero-2-phosphocholine (PC14), a neutral charged artificial lipid, and was reconstructed to a monolayer on a hydrophobic chip for the biosensor. The binding of Cry1Ac to the reconstructed monolayer was analyzed by a two-state binding model, and it was shown that Cry1Ac bound to the monolayer in the first step with an affinity constant (K(1)) of 508 nM, followed by the second uni-molecular step with an equilibrium constant (K(2)) of 0.472. The overall affinity constant K(d) was determined to be 240 nM. The binding was markedly inhibited by N-acetyl-D-galactosamine (K(i)=8 mM). The monolayer was shown to retain a high affinity to Cry1Ac, providing an insect-free system for rapid and large-scale screening of B. thuringiensis insecticidal proteins by the SPR-based biosensor technology.
Okumura
S
S
Fukuoka Industrial Technology Center, 1465-5 Aikawa, Kurume, 839-0861, Fukuoka, Japan.
Akao
T
T
Mizuki
E
E
Ohba
M
M
Inouye
K
K
D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins; D018826:CD13 Antigens / Q000378:metabolism; D004064:Digestive System / Q000378:metabolism; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins; D066298:In Vitro Techniques; D019476:Insect Proteins; D008567:Membranes, Artificial; D008854:Microscopy, Electron; D008871:Microvilli / Q000378:metabolism; D009036:Moths / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D020349:Surface Plasmon Resonance
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C095416:Cry toxin receptors; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D008567:Membranes, Artificial; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens