pathogenic in some today incurable human diseases; however, there are also
many physiologically functioning amyloids in nature. The process of amyloid
formation is typically nucleation-elongation-dependent, as exemplified by the
pathogenic amyloid-𝜷 peptide (A𝜷) that is associated with Alzheimer’s
disease. Spider silk, one of the toughest biomaterials, shares characteristics
with amyloid. In this study, it is shown that forming amyloid-like nanofibrils is
an inherent property preserved by various spider silk proteins (spidroins).
Both spidroins and A𝜷 capped by spidroin N- and C-terminal domains, can
assemble into macroscopic spider silk-like fibers that consist of straight
nanofibrils parallel to the fiber axis as observed in native spider silk 。
These results suggest that spidroins´ unique primary structures have evolved to
allow functional properties of amyloid, and at the same time direct their
fibrillization pathways to avoid formation of cytotoxic intermediates.
淀粉样纤维——具有高度有序性的纳米级纤维聚集体——在当今一些无法治愈的人类疾病中具有致病性；然而，自然界中也存在许多具有生理功能的淀粉样蛋白。淀粉样蛋白的形成过程 形成通常是成核-伸长依赖性的，例如与阿尔茨海默病相关的致病性淀粉样肽（A）。蜘蛛丝是最坚硬的生物材料之一，与淀粉样蛋白具有相同的特征。在这项研究中，表明形成淀粉样纳米纤维是各种蜘蛛丝蛋白（蜘蛛丝蛋白）所保留的固有特性。蜘蛛丝蛋白和A⼟都被蜘蛛丝蛋白N和C末端结构域覆盖，可以组装成宏观的蜘蛛丝状纤维，由直的天然蜘蛛丝中观察到的平行于纤维轴的纳米纤维
结果表明，蜘蛛丝蛋白独特的一级结构已经进化到允许淀粉样蛋白的功能特性，同时指导其纤维化途径以避免形成细胞毒性中间体。
The constructs were transformed into E. coli BL21 (DE3) competent cells individually. The cells were incubated at 37 °C in LB medium overnight and transferred into fresh LB medium with 100 μg mL−1 ampicillin. Protein expression was induced with 1 mm (final concentration) Isopropyl 𝛽-d-1-thiogalactopyranoside (IPTG) when OD600 was 0.8–1.0 for 12 h at 25 °C. The cells were collected by centrifuge (5000 rpm, 20 min) and resuspended in 20 mm Tris pH 8.0. For protein purification, the cells were lysed by using High Pressure Homogenizer (PhD Technology LLC, USA) and centrifuged to obtain the inclusion bodies. Then the inclusion bodies were extensively washed and solubilized by a freeze-thawing strategy as previously described.
相关工艺：将构建体分别转化到大肠杆菌BL21（DE3）感受态细胞中。将细胞在37°C的LB培养基中孵育过夜，然后转移到含有100μg mL-1氨苄青霉素的新鲜LB培养基上。当OD600为0.8-1.0时，在25°C下用1mm（终浓度）异丙基-d-1-硫代吡喃半乳糖苷（IPTG）诱导蛋白质表达12小时。通过离心（5000rpm，20分钟）收集细胞，并将其重新悬浮在20mm Tris pH 8.0中。为了纯化蛋白质，使用高压均质机（美国PhD Technology LLC）裂解细胞并离心以获得包涵体。然后，如前所述，通过冻融策略对包涵体进行广泛清洗和溶解。
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