Expression And Purification Of Bacillus Thuringiensis Cry8Ea1 And Cry8Ca2 Gene

The Cockchafer larvae are important underground pest that make damage in agriculture.Cry8Ea1and Cry8Ca2 protein expressed in Bacillus thuringiensis are toxic to Cockchafer larvae.In the studs; we investigated the expression and purification of these two proteins and the lethalconcentration 50 percent of both protein was also evaluated.HD8E and HBF-1 are Bt strains, with genes that encoding Cry8Ea1and Cry8Ca2 protoxinseperatly. The protoxin expressed by these two Bt strains were analysed by SDS-PAGE. One majorband with an estimated molecular mass of 130 kU was detected. Cry8Ea1 and Cry8Ca2 protoxinwere activated by trpsin or chymotrypsin and the toxin with molecular mass of 60kU were obtained.Both toxin was purified by size exclusion chromatography first and further by anion exchangechromatography. The purification method of Cry8Ea1 and Cry8Ca2 toxin were established in thisstudy. Great amount of pure toxin of Cry8Ea1 and Cry8Ca2 can be obtained by using this method.It was a great foundation for futher investigation on structure and function of these two protein.The insecidical activity of both pure toxin was tested by bioassays against scarab. Cry8Ea1protoxin and toxin are both toxic to Holotrichia parallela (scarab). The lethal concentration 50percent of of Cry8Ea1 protoxin was 9.46μg per gram soil, while that of the Cry8Ea1 toxin activatedby chymotrypsin was 35.39μg per gram soil. Cry8Ca2 protoxin and toxin are both toxic toAnomala carpulenta (scarab). The lethal concentration 50 percent of Cry8Ca2 protoxin was 0.47μgper gram soil, while that of Cry8Ca2 toxin activated by trypsin was 0.003μg per gram soil,Cry8Ca2 toxin activated by chymotrypsin was 0.08μg per gram soil.