Protein Cry34Ab1 is an important constituent of the aegerolysin protein family and one of representatives within the group of insect-specific Cry toxins. It usually acts in concert with his protein partner Cry35Ab1, a member of the toxin_10 family of proteins. Together, they form a binary membrane complex that permeabilizes the plasmalemma of the epithelial midgut cells of western corn rootworm larvae (Diabrotica virgifera virgifera). Forementioned toxins are synthesized by the bacterium Bacillus thuringiensis in parasporal crystalline inclusions. In our work we have successfully produced and isolated recombinant proteins Cry34Ab1 and Cry35Ab1 from bacterium Escherichia coli, which were stable at pH values 3, 5.75 and 9. We have found that the protein complex Cry34Ab1_Cry35Ab1 binds to artificial lipid membranes made of commercial lipids phosphatidylethanolamine_phosphatidylglycerol_cholesterol and phosphatidylethanolamine_phosphatidylglycerol/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine. Protein complex binds to lipid membranes reconstituted from total lipids extracted from insect Sf9 cell line in vitro or from western corn rootworm larvae. In all tested vesicles, repetitive strong binding of the protein complex Cry34Ab1_Cry35Ab1 was documented. In contrast to weaker binding of separate components, this finding suggests that the proteins act in a synergistic way. Protein complex Cry34Ab1_Cry35Ab1 successfully permeabilized only vesicles composed of phosphatidylethanolamine_phosphatidylglycerol_cholesterol and Sf9 total lipids. We obtained similar results with cryo-electronic microscopy, where formation of 13-fold pores, like those found in other aegerolysin proteins, were observed. Using the MTT test, we confirmed that the protein complex Cry34Ab1_Cry35Ab1 is indeed cytotoxic for the Sf9 cell line. Results obtained in our work might insinuate on the existence of a lipid receptor or lipid domain, which is a binding receptor for the protein complex Cry34Ab1_Cry35Ab1.
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