Abstract
Receptor binding is one of the crucial steps to exhibit the insecticidal activity of Cry toxins. In addition, binding to receptors is a determining step for the specificity of toxins. In this work, receptor binding domain II was cloned from the full-length Cry4Ba toxin and heterologously expressed in Escherichia coli. The 21 kDa purified protein was characterized as Cry4Ba domain II using Western blotting and tandem mass spectrometry coupled to liquid chromatography. Circular dichroism revealed the correct folding of the isolated domain II fragment, similar to that found in the Cry4Ba protein. Binding analysis using an enzyme-linked immunosorbent assay revealed that the purified Cry4Ba-domain II had bound to the 54 kDa alkaline phosphatase cloned from Aedes aegypti (Aa-mALP) with a dissociation constant of approximately 116.27 ± 11.09 nM. The binding affinity of Cry4Ba-domain II to Aa-mALP was comparable to that of Cry4Ba domain III, suggesting that both domains II and III of the Cry4Ba contributed equally in binding to the Aa-mALP protein. Our findings should provide more valuable insight on the molecular mechanisms in the toxin-receptor interaction of the Cry4Ba toxin.