Production and characterization of the (13) C/(15) N single-labeled insecticidal protein Cry1Ab/Ac using recombinant Escherichia coli.
Production and characterization of the (13) C/(15) N single-labeled insecticidal protein Cry1Ab/Ac using recombinant Escherichia coli.
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DOI：
10.1002/mbo3.1125
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发表时间：
2020-11
期刊：
影响因子：
3.4
通讯作者：
Tang X
中科院分区：
文献类型：
--
作者：
Wang Z;Hu C;Sun Y;Jiang W;Wu G;Pan A;Li P;Tang X
由转基因作物表达的人工合成Cry 1Ab/Ac蛋白在不使用大量化学杀虫剂的情况下具有很高的控制害虫的潜力。在这些作物用于农业之前，必须了解土壤中的环境命运和相互作用。稳定同位素标记的Cry 1Ab/Ac蛋白是收集此类数据的非常有用的工具。我们开发了一种产生13 C/15 N单标记Cry蛋白的方案。将人工合成的Bt水稻华恢1号的Cry 1Ab/Ac基因亚克隆到pUC 57中，构建表达载体pET-28 a-CryAb/Ac，转化大肠杆菌BL 21（DE 3）感受态细胞。接下来，将0.2 mM异丙基硫代半乳糖苷（IPTG）加入这些细胞中，并在37°C下培养4 h，以在含有[U-13 C]葡萄糖（富含5% 13 C）或[15 N]氯化铵（富含5% 15 N）的M9生长培养基中诱导包涵体的合成和形成。然后，将Cry包涵体溶解在尿素中并在变性条件下通过亲和层析纯化，通过透析复性，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western印迹进一步检测。13 C/15 N标记的Cry蛋白的纯度达到99%，含量分别为12.6 mg/L和8.8 mg/L。13 C标记的Cry蛋白和15 N标记的Cry蛋白的δ 13 C和ε 15 N值分别为3，269 ‰和2，854 ‰。生物活性测试表明，标记的Cry 1Ab/Ac蛋白具有较强的杀虫活性。本研究首次生产的稳定同位素标记杀虫Cry蛋白，将为今后土壤中Cry蛋白的代谢研究以及氮（N）和碳（C）转化特征提供实验基础。本研究结果也可为阐明BT植物及其表达产物的环境行为和生态效应提供参考。本研究建立了利用重组大肠杆菌菌株生产13 C/15 N单标记杀虫蛋白Cry 1Ab/Ac的方法。稳定同位素标记的Cry蛋白以包涵体形式存在，被成功地溶解、纯化和复性，显示出强的杀虫活性。研究结果为进一步研究Cry蛋白的代谢归宿、生物学功能和安全性评价奠定了基础。
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 13C/15N single‐labeled Cry proteins. The artificially synthesized gene Cry1Ab/Ac of Bt rice 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/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM isopropyl thiogalactoside (IPTG) was added to these cells and cultured at 37°C for 4 h to induce the synthesis and formation of inclusion bodies in M9 growth media containing either [U‐13C] glucose (5% 13C‐enriched) or [15N] ammonium chloride (5% 15N‐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 13C/15N‐labeled Cry proteins reached 99% with amounts of 12.6 mg/L and 8.8 mg/L, respectively. The δ 13C and ä 15N values of 13C‐labeled Cry protein and 15N‐labeled Cry protein were 3,269‰ and 2,854‰, 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. We established in this study a method to produce 13C/15N single‐labeled insecticidal protein Cry1Ab/Ac using a recombinant Escherichia coli strain. A stable isotope‐labeled Cry protein existing in the form of inclusion bodies was solubilized, purified, and refolded successfully, revealing strong insecticidal activity. Our results lay a foundation for the further study of the metabolic fate, biological function, and safety evaluation of Cry proteins.
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影响因子：
5.2
作者：
Li P;Ye S;Liu H;Pan A;Ming F;Tang X
通讯作者：
Tang X
影响因子：
3
作者：
Ben Hamadou-Charfi, Dorra;Sauer, Annette Juliane;Stephan, Dietrich
通讯作者：
Stephan, Dietrich
影响因子：
2
作者：
Nguyen, H. T.;Jehle, J. A.
通讯作者：
Jehle, J. A.
影响因子：
3.5
作者：
Haranahalli K;Tong S;Ojima I
通讯作者：
Ojima I
DOI：
10.1073/pnas.95.6.2767
发表时间：
1998-03-17
影响因子：
11.1
作者：
Cheng, XY;Sardana, R;Altosaar, I
通讯作者：
Altosaar, I