Kunlawatwimon Thanapon, Promdonkoy Boonhiang, Boonserm Panadda
Institute of Molecular Biosciences, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom 73170, Thailand.
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand.
J Invertebr Pathol. 2025 Nov;213:108429. doi: 10.1016/j.jip.2025.108429. Epub 2025 Aug 15.
Vip3A proteins produced by the Bacillus thuringiensis bacterium exhibit insecticidal activity against various crop-damaging lepidopteran pests. However, their limited thermal stability and short shelf life render them unsuitable for use as biopesticides. In this study, we employed rational protein design to enhance the thermal stability of Vip3A64 while minimizing its negative impact on insecticidal activity. This involves substituting potential amino acids based on structural data and using a computational tool (HoTMuSiC) to predict the effect of mutations on the protein's thermal stability as defined by the melting temperature (Tm). We then introduced eight single amino acid substitutions (V239T, V320K, A351C, A351F, D621I, N633Y, E754W, and Q771I) into the tetrameric core and solvent-exposed domains of Vip3Aa64. As determined by the protein thermal shift (PTS) assays, the N633Y and V239T mutants exhibit higher protein melting temperatures than those of the wild type, indicating their enhanced thermal stability. In contrast to other mutants, V239T and N633Y retained their insecticidal activity after one hour of exposure to the high temperature of 55 ˚C. Furthermore, after one month of storage at 37 ˚C, N633Y was the only mutant capable of killing Spodopteraexigua larvae, indicating that it is more stable than the other mutants and the wild type. This study highlights the potential of rational protein design to improve thermal stability and provides a framework for developing effective insecticidal proteins for sustainable agriculture.
苏云金芽孢杆菌产生的Vip3A蛋白对多种危害作物的鳞翅目害虫具有杀虫活性。然而，其有限的热稳定性和较短的保质期使其不适合用作生物农药。在本研究中，我们采用合理的蛋白质设计来提高Vip3A64的热稳定性，同时将其对杀虫活性的负面影响降至最低。这包括根据结构数据替换潜在的氨基酸，并使用一种计算工具（HoTMuSiC）来预测突变对由解链温度（Tm）定义的蛋白质热稳定性的影响。然后，我们在Vip3Aa64的四聚体核心和溶剂暴露结构域中引入了八个单氨基酸替换（V239T、V320K、A351C、A351F、D621I、N633Y、E754W和Q771I）。通过蛋白质热迁移（PTS）测定确定，N633Y和V239T突变体的蛋白质解链温度高于野生型，表明它们的热稳定性增强。与其他突变体不同，V239T和N633Y在暴露于55℃的高温一小时后仍保留其杀虫活性。此外，在37℃储存一个月后，N633Y是唯一能够杀死甜菜夜蛾幼虫的突变体，这表明它比其他突变体和野生型更稳定。本研究突出了合理蛋白质设计在提高热稳定性方面的潜力，并为开发用于可持续农业的有效杀虫蛋白提供了一个框架。
