Production and characterization of the(13)C/N-15 single-labeled insecticidal protein Cry1Ab/Ac using recombinantEscherichia coli

文献类型： 外文期刊

第一作者： Wang, Zibo

作者： Wang, Zibo;Sun, Yu;Jiang, Wei;Wu, Guogan;Pan, Aihu;Li, Peng;Tang, Xueming;Wang, Zibo;Hu, Cong;Sun, Yu;Jiang, Wei;Wu, Guogan;Pan, Aihu;Li, Peng;Tang, Xueming

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关键词： Cry1Ab; Ac protein; insecticidal activity; prokaryotic expression; stable isotope

期刊名称：MICROBIOLOGYOPEN （ 影响因子：3.139； 五年影响因子：3.503 ）

ISSN： 2045-8827

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收录情况： SCI

摘要： Synthetic Cry1Ab/Ac proteins expressed by genetically modified (GM) crops have a high potential to control insect pests without utilizing large amounts of chemical insecticides. Before these crops are used in agriculture, the environmental fate and interactions in the soil must be understood. Stable isotope-labeled Cry1Ab/Ac protein is a highly useful tool for collecting such data. We developed a protocol to produce(13)C/N-15 single-labeled Cry proteins. The artificially synthesized geneCry1Ab/AcofBtrice Huahui No. 1, which has been certified by the Chinese government to be safe for human consumption, was subcloned into pUC57, and the expression vector pET-28a-CryAb/Acwas constructed and transformed intoEscherichia coliBL21 (DE3) competent cells. Next, 0.2 mM isopropyl thiogalactoside (IPTG) was added to these cells and cultured at 37 degrees C for 4 h to induce the synthesis and formation of inclusion bodies in M9 growth media containing either [U-C-13] glucose (5%C-13-enriched) or [N-15] ammonium chloride (5%N-15-enriched). Then, Cry inclusion bodies were dissolved in urea and purified by affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The purities of(13)C/N-15-labeled Cry proteins reached 99% with amounts of 12.6 mg/L and 8.8 mg/L, respectively. The delta C-13 and a(15)N values of(13)C-labeled Cry protein and(15)N-labeled Cry protein were 3,269 parts per thousand and 2,854 parts per thousand, respectively. A bioassay test revealed that the labeled Cry1Ab/Ac proteins had strong insecticidal activity. The stable isotope-labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies on Cry proteins in soil and the characteristics of nitrogen (N) and carbon (C) transformations. Our findings may also be employed as a reference for elucidating the environmental behavior and ecological effects of BT plants and expressed products.

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