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Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
Published on: January 3, 2012
Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in
E A Cowles1, H Yunovitz, J F Charles
1Department of Entomology, University of California, Riverside 92521, USA.
Abstract:
The binding proteins, or receptors, for insecticidal Bacillus thuringiensis subsp. kurstaki delta-endotoxins are located in the brush border membranes of susceptible insect midguts. The interaction of one of these toxins, CryIA(c), with proteins isolated from Heliothis virescens larval midguts was investigated. To facilitate the identification of solubilized putative toxin-binding proteins, a solid-phase binding assay was developed and compared with toxin overlay assays. The overlay assays demonstrated that a number of proteins of 170, 140, 120, 90, 75, 60, and 50 kDa bound the radiolabeled CryIA(c) toxin. Anion-exchange fractionation allowed the separation of these proteins into three toxin binding fractions, or pools. Toxin overlay assays demonstrated that although the three pools had distinct protein profiles, similar-size proteins could be detected in these three pools. However, determination of toxin affinity by using the solid-phase binding assay showed that only one of the three pools contained high-affinity binding proteins. The Kd obtained, 0.65 nM, is similar to that of the unsolubilized brush border membrane vesicles. Thus, the solid-phase binding assay in combination with the toxin overlay assay facilitates the identification and purification of high-affinity B. thuringiensis toxin-binding proteins from the insect midgut.
Insights
Researchers identified high-affinity Bacillus thuringiensis (Bt) toxin-binding proteins in insect midguts. A novel solid-phase binding assay, combined with toxin overlay assays, aids in discovering these crucial Bt toxin receptors.
Area of Science:
- Insect toxicology
- Molecular biology
- Biochemistry
Background:
- Insecticidal Bacillus thuringiensis subsp. kurstaki (Bt) delta-endotoxins bind to receptors in susceptible insect midgut brush border membranes.
- Understanding these interactions is key to developing effective Bt-based pest control strategies.
Purpose of the Study:
- To investigate the interaction between the CryIA(c) Bt toxin and proteins from Heliothis virescens larval midguts.
- To develop and validate a solid-phase binding assay for identifying and purifying high-affinity Bt toxin-binding proteins.
Main Methods:
- Radiolabeled CryIA(c) toxin overlay assays were used to detect toxin-binding proteins.
- Anion-exchange fractionation separated proteins into different pools.
- A solid-phase binding assay was developed to determine toxin-binding affinity (Kd).
Main Results:
- Toxin overlay assays identified multiple binding proteins (170, 140, 120, 90, 75, 60, and 50 kDa).
- Anion-exchange fractionation yielded three distinct binding pools.
- The solid-phase binding assay revealed high-affinity binding proteins (Kd = 0.65 nM) in only one pool.
Conclusions:
- The combination of solid-phase binding and toxin overlay assays effectively identifies and purifies high-affinity Bt toxin-binding proteins.
- This methodology advances the understanding of Bt toxin-insect interactions and aids in the development of novel insecticides.
