Abstract：
CONCLUSIONS: Redesigning the N- and C-capping repeats of the native DARPin G3 significantly improved its stability, and may facilitate its purification from the total soluble proteins of high-temperature dried leaf materials of transplastomic plants. Designed ankyrin repeat proteins (DARPins) constitute a promising class of binding molecules that can overcome the limitations of monoclonal antibodies and enable the development of novel therapeutic approaches. Despite their inherent stability, detailed studies have revealed that the original capping repeats derived from natural ankyrin repeat proteins impair the stability of the initial DARPin design. Consequently, the development of thermodynamically stabilized antibody mimetics may facilitate the development of innovative drugs in the future. In this study, we replaced the original N- and C-capping repeats with improved caps to enhance the thermostability of native DARPin G3. Computational analyses suggested that the redesigned thermostable DARPin G3 structure possessed optimal quality and stability. Molecular dynamics simulations verified the stability of the redesigned thermostable DARPin G3 at high temperatures. The redesigned thermostable DARPin G3 was expressed at high levels in tobacco transplastomic plants and subsequently purified from high-temperature dried leaf materials. Thermal denaturation results revealed that the redesigned thermostable DARPin G3 had a higher Tm value than the native DARPin G3, with a Tm of 35.51 °C greater than that of native DARPin G3. The results of the in vitro bioassays confirmed that the purified thermostable DARPin G3 from high-temperature dried leaf materials maintained its binding activity without any loss of affinity and specifically bound to the HER2 receptor on the cell surface. These findings demonstrate the successful improvement in the thermostability of DARPin G3 without compromising its biological activity.
摘要：
结论：重新设计天然DARPinG3的N-和C-加帽重复序列显著提高了其稳定性，并且可以促进其从转体植物的高温干燥叶片材料的总可溶性蛋白中的纯化。设计的锚蛋白重复蛋白（DARPins）构成了一类有前途的结合分子，可以克服单克隆抗体的局限性，并能够开发新的治疗方法。尽管它们固有的稳定性，详细的研究表明，源自天然锚蛋白重复蛋白的原始加帽重复会损害初始DARPin设计的稳定性。因此,热力学稳定的抗体模拟物的开发可能会促进未来创新药物的开发。在这项研究中,我们用改进的帽代替了原始的N-和C-加帽重复,以增强天然DARPinG3的热稳定性.计算分析表明，重新设计的热稳定DARPinG3结构具有最佳的质量和稳定性。分子动力学模拟验证了重新设计的热稳定DARPinG3在高温下的稳定性。重新设计的热稳定DARPinG3在烟草转体植物中高水平表达，随后从高温干燥的叶片材料中纯化。热变性结果显示,重新设计的热稳定DARPinG3具有比天然DARPinG3更高的Tm值,具有比天然DARPinG3大35.51°C的Tm。体外生物测定的结果证实,来自高温干燥叶材料的纯化的热稳定的DARPinG3保持其结合活性而没有任何亲和力损失,并且特异性地结合细胞表面上的HER2受体。这些发现证明了DARPinG3热稳定性的成功改善而不损害其生物活性。
