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, t o a brush border membrane (BBM) isolated from midguts of the diamondback mo th Plutella xylostella was examined by surface plasmon resonance (SPR)-base d biosensor. BBM was mixed with 1,3-ditetradecylglycero-2-phosphocholine (P C14), a neutral charged artificial lipid, and was reconstructed to a monola yer on a hydrophobic chip for the biosensor. The binding of Cry1Ac to the r econstructed monolayer was analyzed by a two-state binding model, and it wa s shown that Cry1Ac bound to the monolayer in the first step with an affini ty 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 aff inity to Cry1Ac, providing an insect-free system for rapid and large-scale screening of B. thuringiensis insecticidal proteins by the SPR-based biosen sor technology. (C) 2001 Elsevier Science B.V. All rights reserved.