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.
- Du C, Martin PA, Nickerson KW (1994) Comparison of disulfide contents and solubility at alkaline pH of insecticidal and noninsecticidal Bacillus thuringiensis protein crystals. Appl Environ Microbiol 60:3847–3853Google Scholar
- Li H, Bouwer G (2014) Evaluation of the synergistic activities of Bacillus thuringiensis Cry proteins against Helicoverpa armigera (Lepidoptera: Noctuidae). J Invertebr Pathol 121:7–13Google Scholar
- Park HW, Ge B, Bauer LS, Federici BA (1998) Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence. Appl Environ Microbiol 64:3932–3938Google Scholar
- Peña-Cardeña A, Grande R, Sánchez J, Tabashnik BE, Bravo A, Soberón M, Gómez I (2018) The C-terminal protoxin domain of Bacillus thuringiensis Cry1Ab toxin has a functional role in binding to GPI-anchored receptors in the insect midgut. J Biol Chem 293:20263–20272Google Scholar
- Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806Google Scholar
- Sramala I, Uawithya P, Chanama U, Leetachewa S, Krittanai C, Katzenmeier G, Panyim S, Angsuthanasombat C (2000) Single proline substitutions of selected helices of the Bacillus thuringiensis Cry4B toxin affect inclusion solubility and larvicidal activity. J Biochem Mol Biol Biophys 4:187–193Google Scholar
- Tabashnik BE (1992) Evaluation of synergism among Bacillus thuringiensis toxins. Appl Environ Microbiol 58:3343–3346Google Scholar
- Yin J, Ding X, Xia L, Yu Z, Lv Y, Hu S, Huang S, Cao Z, Xiao X (2011) Transcription of gene in an acrystalliferous strain of Bacillus thuringiensis XBU001 positively regulated by the metalloprotease camelysin gene at the onset of stationary phase. FEMS Microbiol Lett 318:92–100CrossRefGoogle Scholar