Crystallographic Analysis Of Insecticidal Crystal Proteins Cry51Aa1 And Cry6Aa2 From Bacillus Thuriengensis

Bacillus thurigiensis (Bt) is a ubiquitous gram-positive bacterium in the environment, which can produce a variety of insecticidal crystal proteins. Some insecticidal genes of Bt have been successfully used in genetically modified crops, which are playing a prominent role in agricultural production field. Bt insecticidal crystal proteins are grouped into three major families:Cry, Cyt and Parasporin. Cry and Cyt toxins can kill specific insects, while the parasporins are toxic to some cancer cells. Cry51Aal and Cry6Aa2 can kill hemiptera insects and plant-parasitic nematodes, respectively. In order to further understand the activity mode of these two proteins. Our project focuses on solving the three dimension structures of Cry51Aal and Cry6Aa2.According to the biology information, we conclude that Cry51Aal belongs to the minor group Mtx of Cry protein family. The second structure of Cry51Aal mainly consists of (3 sheets, the homology modeling results show that β sheets domain the three dimension structures of Cry51Aal when the 2D42 and 2ZTB are acted as the models. However, the amino acid sequence of Cry6Aa is unique and quite distinct from other Cry proteins.Cry51Aal was expressed in BMB171. The two-step purification of ion exchange chromatography and gel filtration chromatography were used to get large scale of Cry51Aal protein. Gel filtration chromatography and analytical ultracentrifugation indicted that Cry51Aal was a dimer in the solution. Crystal growth was used the method of hanging drop vapor diffusion and crystal screen was taken the crystal screening kits. Optimizing the conditions of Cry51Aal crystal growth, we obtained high quality crystals with reservoir solution containing 16% PEG2000,100 mmol/L critic acid pH 3.0,140 mmol/L (NH4)2SO4,3 mmol/L glycyl-glycyl-glycyl and 1-2 mg/mL protein at 16℃. And we also obtained the gold derivative crystals in the same growth condition. In the SSRF BL17U beamline, we got the native crystal and gold derivative crystal X-ray diffraction datas. The conditions collecting the X-ray diffraction were -73℃, the 12.6 keV energy of the light,0.9792 A wave and the MX225 CCD. We used the mosflm program to process our data. The crystals of Cry51Aal had the space group P412112 and unit-cell parameters were a=b=54.5, c=212.2, α=β=γ=90°.Cry6Aa2 was expressed in BMB171. However, the full length of Cry6Aa2 was unstable. Treated by the trypsin, we got a stable fragment of 43 kDa protein. After ion exchange chromatography, Cry6Aa2 was digested by trypsin, and then loaded onto the gel filtration chromatography. The result of gel filtration chromatography showed the Cry6Aa2 was a monomer. The truncated Cry6Aa2 performed four different shape crystals (needle, plate, trapezoid and snowflake) during the crystal screening steps. However, when doing the father opitimization, needle shape crystals just grew longer, plate shape crystals couldn’t been repeated, trapezoid shape crystals proved to be salt crystals and the snowflake crystals were optimized into rod crystals. Rod crystals were promisingly optimized to improve the quality of crystals in the future.