Hire Ramesh S, Makde Ravindra D, Dongre Tanaji K, D'souza Stanislaus F
Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre (BARC), Trombay, Mumbai 400085, India.
Toxicon. 2009 Sep 15;54(4):519-24. doi: 10.1016/j.toxicon.2009.05.022. Epub 2009 May 30.
An indigenous strain HD-550 of Bacillus thuringiensis subsp. kenyae was found to be toxic to lepidopteran as well as dipteran insects. The cry2Aa gene (classified as cry2Aa14) from this isolate was cloned and expressed in Escherichia coli. Only a little amount of the expressed Cry2Aa14 protein was observed in soluble fraction under normal induction condition. The inclusions were non-toxic to test insects, whereas solubilized Cry2Aa14 was highly toxic to lepidopteran and dipteran insects. Cry2Aa14 protein was expressed as thioredoxin (trx) fusion protein for improving the yield of active protein. An enhancement of nearly 15% was observed in the yield of active Cry2Aa14. The TrxA-Cry2Aa14 protein purified from the solubilized fraction also showed toxicity profile similar to the wild-type protein. The LC(50) values of Cry2Aa14 and TrxA-Cry2Aa14 protein against Spodoptera litura was 694 and 696 ng/cm(2), respectively, while for Culex quinquefasciatus the LC(50) values were 894 and 902 ng/ml, respectively. The broad spectrum toxicity of the Cry2Aa14 thus indicates that this protein could be an important component in integrated pest management. Further, the trx tag clearly led to higher yield, which facilitates protein purification for biophysical and biochemical characterization.
一种苏云金芽孢杆菌肯尼亚亚种的本地菌株HD - 550被发现对鳞翅目和双翅目昆虫均有毒性。从该分离株中克隆出cry2Aa基因（分类为cry2Aa14）并在大肠杆菌中表达。在正常诱导条件下，仅在可溶性部分观察到少量表达的Cry2Aa14蛋白。包涵体对受试昆虫无毒，而溶解后的Cry2Aa14对鳞翅目和双翅目昆虫具有高毒性。Cry2Aa14蛋白作为硫氧还蛋白（trx）融合蛋白表达以提高活性蛋白产量。活性Cry2Aa14的产量提高了近15%。从溶解部分纯化的TrxA - Cry2Aa14蛋白也显示出与野生型蛋白相似的毒性特征。Cry2Aa14和TrxA - Cry2Aa14蛋白对斜纹夜蛾的LC(50)值分别为694和696 ng/cm(2)，而对致倦库蚊的LC(50)值分别为894和902 ng/ml。因此，Cry2Aa14的广谱毒性表明该蛋白可能是害虫综合管理中的重要组成部分。此外，trx标签明显提高了产量，这有助于蛋白质纯化以进行生物物理和生化特性分析。
