COMPARISON OF TOXIN OVERLAY AND SOLID-PHASE BINDING ASSAYS TO IDENTIFY DIVERSE CRYIA(C) TOXIN-BINDING PROTEINS IN HELIOTHIS-VIRESCENS MIDGUT

The binding proteins, or receptors, for insecticidal Bacillus thuringi ensis subsp. kulstaki delta-endotoxins are located in the brush border membranes of susceptible insect midguts. The interaction of one of th ese toxins, CryIA(c), with proteins isolated from Heliothis virescens larval midguts was investigated. To facilitate the identification of s olubilized putative toxin-binding proteins, a solid-phase binding assa y was developed and compared with toxin overlay assays. The overlay as says demonstrated that a number of proteins of 170, 140, 120, 90, 75, 60, and 50 kDa bound the radiolabeled CryIA(c) toxin. Anion-exchange f ractionation allowed the separation of these proteins into three toxin binding fractions, or pools. Toxin overlay assays demonstrated that a lthough the three pools had distinct protein profiles, similar-size pr oteins could be detected in these three pools. However, determination of toxin affinity by using the solid-phase binding assay showed that o nly one of the three pools contained high-affinity binding proteins. T he K-d obtained, 0.65 nM, is similar to that of the unsolubilized brus h border membrane vesicles. Thus, the solid-phase binding assay in com bination with the toxin overlay assay facilitates the identification a nd purification of high-affinity B. thuringiensis toxin-binding protei ns from the insect midgut.