Thammasittirong Anon, Thammasittirong Sutticha Na Ranong
Department of Science and Bioinnovation, Faculty of Liberal Arts and Science, Kasetsart University, Nakhon Pathom, 73140, Thailand.
Microbial Biotechnology Unit, Faculty of Liberal Arts and Science, Kasetsart University, Nakhon Pathom, 73140, Thailand.
Heliyon. 2023 Aug 28;9(9):e19458. doi: 10.1016/j.heliyon.2023.e19458. eCollection 2023 Sep.
Receptor binding is one of the crucial steps to exhibit the insecticidal activity of Cry toxins. In addition, binding to receptors is a determining step for the specificity of toxins. In this work, receptor binding domain II was cloned from the full-length Cry4Ba toxin and heterologously expressed in . The 21 kDa purified protein was characterized as Cry4Ba domain II using Western blotting and tandem mass spectrometry coupled to liquid chromatography. Circular dichroism revealed the correct folding of the isolated domain II fragment, similar to that found in the Cry4Ba protein. Binding analysis using an enzyme-linked immunosorbent assay revealed that the purified Cry4Ba-domain II had bound to the 54 kDa alkaline phosphatase cloned from (Aa-mALP) with a dissociation constant of approximately 116.27 ± 11.09 nM. The binding affinity of Cry4Ba-domain II to Aa-mALP was comparable to that of Cry4Ba domain III, suggesting that both domains II and III of the Cry4Ba contributed equally in binding to the Aa-mALP protein. Our findings should provide more valuable insight on the molecular mechanisms in the toxin-receptor interaction of the Cry4Ba toxin.
受体结合是展现Cry毒素杀虫活性的关键步骤之一。此外，与受体结合是毒素特异性的决定性步骤。在本研究中，从全长Cry4Ba毒素中克隆出受体结合结构域II，并在[具体表达宿主未给出]中进行异源表达。通过蛋白质免疫印迹法和液相色谱串联质谱法鉴定，纯化得到的21 kDa蛋白为Cry4Ba结构域II。圆二色光谱显示分离出的结构域II片段折叠正确，与Cry4Ba蛋白中的情况相似。利用酶联免疫吸附测定法进行的结合分析表明，纯化的Cry4Ba结构域II已与从[具体来源未给出]克隆的54 kDa碱性磷酸酶（Aa-mALP）结合，解离常数约为116.27±11.09 nM。Cry4Ba结构域II与Aa-mALP的结合亲和力与Cry4Ba结构域III相当，表明Cry4Ba的结构域II和III在与Aa-mALP蛋白结合中发挥的作用相当。我们的研究结果应为Cry4Ba毒素的毒素-受体相互作用分子机制提供更有价值的见解。
