Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina.
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina; Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina; Integrative Program for Biological and Genome Sciences, University of North Carolina, Chapel Hill, North Carolina.
Biophys J. 2023 Jun 20;122(12):2500-2505. doi: 10.1016/j.bpj.2023.05.002. Epub 2023 May 6.
Lyophilization is promising for tackling degradation during the drying and storage of protein-based drugs. Tardigrade cytosolically abundant heat soluble (CAHS) proteins are necessary and sufficient for desiccation-tolerance in vivo and protein protection in vitro. Hydrated CAHS proteins form coiled-coil-based fine-stranded, cold-setting hydrogels, but the dried protein remains largely uncharacterized. Here, we show that dried CAHS D gels (i.e., aerogels) retain the structural units of their hydrogels, but the details depend on prelyophilization CAHS concentrations. Low concentration samples (<10 g/L) form thin (<0.2 μm) tangled fibrils lacking regular structure on the micron scale. Upon increasing the concentration, the fibers thicken and form slabs comprising the walls of the aerogel pores. These changes in morphology are associated with a loss in disorder and an increase in large β sheets and a decrease in α helices and random coils. This disorder-to-order transition is also seen in hydrated gels as a function of concentration. These results suggest a mechanism for pore formation and indicate that using CAHS proteins as excipients will require attention to initial conditions because the starting concentration impacts the lyophilized product.
冷冻干燥在解决蛋白质类药物干燥和储存过程中的降解问题方面具有广阔的应用前景。缓步动物细胞溶质丰富的热可溶性（CAHS）蛋白是体内干燥耐受性和体外蛋白质保护所必需和充分的。水合 CAHS 蛋白形成基于螺旋卷曲的细股、冷定型水凝胶，但干燥后的蛋白质仍在很大程度上未被表征。在这里，我们表明 CAHS D 凝胶（即气凝胶）保留了其水凝胶的结构单元，但细节取决于预冷冻干燥 CAHS 浓度。低浓度样品（<10 g/L）形成薄（<0.2 μm）的缠结原纤维，在微米尺度上缺乏规则结构。随着浓度的增加，纤维变厚并形成由气凝胶孔壁组成的薄片。这种形态的变化与无序向有序的转变有关，无序向有序的转变与大 β 片层的增加和 α 螺旋和无规卷曲的减少有关。这种无序到有序的转变也随着浓度的变化出现在水凝胶中。这些结果为孔形成提供了一种机制，并表明将 CAHS 蛋白用作赋形剂将需要关注初始条件，因为起始浓度会影响冻干产品。
