ICAR-National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi, 110012, India.
J Mol Model. 2019 Apr 26;25(5):136. doi: 10.1007/s00894-019-3994-7.
Bacillus thuringiensis (Bt) strains produce Cry (crystal) and Cyt (cytolytic) proteins belonging to the group of bacterial toxins known as pore-forming toxins (PFTs), which interact with midgut cells of target insects to create pores, disruption of ion homeostasis and eventual death. PFTs have synergistic insecticidal activities and have been used as biopesticides against agriculturally important insects. Identification of new Cyt proteins is important because of their specific toxicity towards hemipteran pests, against which the Cry proteins are not effective. We have structurally characterized a cyt (cyt1007) gene from an Indian Bt isolate SK-1007. The presence of a "Bacillus thuringiensis toxin" domain and maximum identity of 36% with Cyt2Ca in the deduced amino acid sequence indicated Cyt1007 protein to be a new member of Cyt family. Three dimensional (3D) modeling (PMDB ID: PM0081490) revealed that it adopts a typical ferredoxin-like fold, and is composed of a single domain of α/β architecture, in which a single β sheet is surrounded by two α helical layers. The putative lipid binding site and probable mode of action of Cyt1007 protein were predicted through comparative analysis with other Cyt toxins and their distant homologs Evf (Erwinia virulence factor) and VVA2 (Volvatoxin A2). Heterologous expression of cyt1007 gene as a 25 kDa protein in Escherichia coli was achieved at high levels in both soluble and insoluble fractions. Affinity chromatography-based purification yielded 83.6% purified Cyt1007 protein, which can be used for downstream applications for the investigation of its toxicity. Graphical abstract Steps in the structural characterization and heterologous expression of a new cyt gene cloned from Bacillus thuringiensis.
苏云金芽孢杆菌（Bt）菌株产生 Cry（晶体）和 Cyt（细胞溶解）蛋白，属于被称为孔形成毒素（PFT）的细菌毒素群，这些蛋白与靶昆虫的中肠细胞相互作用，形成孔，破坏离子稳态，最终导致死亡。PFT 具有协同杀虫活性，已被用作防治农业重要昆虫的生物农药。鉴定新的 Cyt 蛋白很重要，因为它们对半翅目害虫具有特异性毒性，而 Cry 蛋白对这些害虫无效。我们从印度 Bt 分离株 SK-1007 中对一个 cyt（cyt1007）基因进行了结构表征。该基因的推导氨基酸序列中存在一个“苏云金芽孢杆菌毒素”结构域，与 Cyt2Ca 的最大同一性为 36%，表明 Cyt1007 蛋白是 Cyt 家族的新成员。三维（3D）建模（PMDB ID：PM0081490）显示，它采用典型的铁氧还蛋白样折叠，由单一的α/β结构域组成，其中一个单一的β片层被两个α螺旋层包围。通过与其他 Cyt 毒素及其远缘同源物 Evf（肠杆菌属毒力因子）和 VVA2（Volvatoxin A2）进行比较分析，预测了 Cyt1007 蛋白的假定脂结合位点和可能的作用模式。在大肠杆菌中高水平可溶性和不溶性表达 cyt1007 基因，可表达出 25 kDa 的蛋白。基于亲和层析的纯化可获得 83.6%的纯化 Cyt1007 蛋白，可用于下游应用，以研究其毒性。
