Solubility enhancement of Cry2Aa crystal through carboxy-terminal extension and synergism between the chimeric protein and Cry1Ac
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It was reported that the highly conserved C-terminal region of Bacillus thuringiensis Cry1A protoxins was very important for parasporal crystal formation and solubility feature in alkaline environment. In order to improve the solubilization efficiency of Cry2Aa crystal, the coding sequences of Cry2Aa protein and the C-terminal half of Cry1Ac were fused seamlessly through Red/ET homologous recombination and expressed in an acrystalliferous B. thuringiensis strain under the control of the cry1Ac promoter and terminator. Microscopic observation revealed that the recombinant strain containing the chimeric gene cry2Aa-1Ac produced distinct parasporal inclusion with semispherical to approximately cuboidal shape during sporulation. SDS-PAGE analysis showed that this strain expressed stable 130-kDa Cry2Aa-1Ac chimeric protein, which was confirmed to be the correctly expressed product by LC-MS/MS. The chimeric protein inclusion could be effectively dissolved at pH 10.5 and activated by trypsin like the parental Cry1Ac crystal. While, the parental Cry2Aa crystal exhibited very low solubility under this condition. Bioassays against third-instar larvae of Helicoverpa armigera proved that the chimeric protein was more toxic than Cry2Aa. Additionally, synergistic effect was clearly detected between the chimeric protein and Cry1Ac against H. armigera, while there was only additive effect for the combination of wild Cry2Aa and Cry1Ac. These results indicated that the developed chimeric protein might serve as a potent insecticidal toxin used in the field against lepidopteran pests.
KeywordsBacillus thuringiensis Cry2Aa C-terminal half of Cry1Ac Chimeric protein Helicoverpa armigera Synergistic activity
XFQ and YJS designed the study and wrote the manuscript. XQL, XMR, RL, and BBW participated in the experiments. SSY, LLQ, and XTM contributed to the figures and tables. XZD and LQX analyzed the mass spectrometric data. All authors have read and approved the final manuscript.
This work was supported by the National Key Research and Development Program (2017YFD0201201), Key Research and Development Program of Hunan Province (2015NK3032), the Training Program of Young Key Teachers in Universities of Hunan Province, Graduate Teaching Reform Program of Hunan Normal University (12JG14), and the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), P. R. China.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This research does not contain any studies with human participants or animals.
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