Engineering of Bacillus thuringiensis Cry Proteins to Enhance the Activity against Western Corn Rootworm

A novel Bacillus thuringiensis Cry protein, Cry8Hb, active against Diabrotica virgifera virgifera (Western corn rootworm, WCRW) was discovered. Unexpectedly, the anti-rootworm activity of the Cry8Hb toxin was enhanced significantly by fusing Escherichia coli maltose binding protein (MBP) to this Cry toxin. While the exact mechanism of the activity enhancement remains indefinite, it is probable that the enhancement is a result of increased solubility of the MBP-Cry8Hb fusion in the rootworm midgut. This hypothesis was examined using a synthetic Cry3 protein called IP3-1, which was not soluble at a neutral pH like Cry8Hb and marginally active to WCRW. When IP3-1 was fused to MBP, its anti-WCRW activity was enhanced 13-fold. To further test the hypothesis, DNA shuffling was performed on IP3-1 to increase the solubility without MBP. Screening of shuffled libraries found six new IP3 variants showing very high anti-WCRW activity without MBP. Sequence and 3D structure analysis of those highly active, shuffled IP3 variants revealed several charge-altering mutations such as Lys to Glu on the putative MBP-attaching side of the IP3 molecule. It is likely that those mutations make the protein acidic to substitute the functions of MBP including enhancing the solubility of IP3 at a neutral pH.