Amyloid fibrils—nanoscale fibrillar aggregates with high levels of order—are
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-?? peptide (A??) 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 A?? 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.
淀粉樣纖維——具有高度有序性的納米級(jí)纖維聚集體——在當(dāng)今一些無(wú)法**的人類疾病中具有致病性；然而，自然界中也存在許多具有生理功能的淀粉樣蛋白。淀粉樣蛋白的形成過(guò)程 形成通常是成核-伸長(zhǎng)依賴性的，例如與阿爾茨海默病相關(guān)的致病性淀粉樣肽（A）。蜘蛛絲是最堅(jiān)硬的生物材料之一，與淀粉樣蛋白具有相同的特征。在這項(xiàng)研究中，表明形成淀粉樣納米纖維是各種蜘蛛絲蛋白（蜘蛛絲蛋白）所保留的固有特性。蜘蛛絲蛋白和A?都被蜘蛛絲蛋白N和C末端結(jié)構(gòu)域覆蓋，可以組裝成宏觀的蜘蛛絲狀纖維，由直的天然蜘蛛絲中觀察到的平行于纖維軸的納米纖維
結(jié)果表明，蜘蛛絲蛋白獨(dú)特的**結(jié)構(gòu)已經(jīng)進(jìn)化到允許淀粉樣蛋白的功能特性，同時(shí)指導(dǎo)其纖維化途徑以避免形成細(xì)胞毒性中間體。
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 ??-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.
相關(guān)工藝：將構(gòu)建體分別轉(zhuǎn)化到大腸桿菌BL21（DE3）感受態(tài)細(xì)胞中。將細(xì)胞在37°C的LB培養(yǎng)基中孵育過(guò)夜，然后轉(zhuǎn)移到含有100μg mL-1氨芐青霉素的新鮮LB培養(yǎng)基上。當(dāng)OD600為0.8-1.0時(shí)，在25°C下用1mm（終濃度）異丙基-d-1-硫代吡喃半乳糖苷（IPTG）誘導(dǎo)蛋白質(zhì)表達(dá)12小時(shí)。通過(guò)離心（5000rpm，20分鐘）收集細(xì)胞，并將其重新懸浮在20mm Tris pH 8.0中。為了純化蛋白質(zhì)，使用高壓均質(zhì)機(jī)（美國(guó)PhD Technology LLC）裂解細(xì)胞并離心以獲得包涵體。然后，如前所述，通過(guò)凍融策略對(duì)包涵體進(jìn)行廣泛清洗和溶解。
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