Department of Pathology, The University of Melbourne, Victoria, Australia.
Neurochem Int. 2012 Feb;60(3):318-26. doi: 10.1016/j.neuint.2011.12.006. Epub 2011 Dec 17.
Transmissibility and distinctive neuropathology are hallmark features of prion diseases differentiating them from other neurodegenerative disorders, with pathogenesis and transmission appearing closely linked to misfolded conformers (PrP(Sc)) of the ubiquitously expressed cellular form of the prion protein (PrP(C)). Given the apparent pathogenic primacy of misfolded PrP, the utilisation of peptides based on the prion protein has formed an integral approach for providing insights into misfolding pathways and pathogenic mechanisms. In parallel with studies employing prion peptides, similar approaches in other neurodegenerative disorders such as Alzheimer Disease, have demonstrated that differential processing of parent proteins and quite minor variations in the primary sequence of cognate peptides generated from the same constitutive processing (such as Aβ1-40 versus Aβ1-42 produced from γ-secretase activity) can be associated with very different pathogenic consequences. PrP(C) also undergoes constitutive α- or β-cleavage yielding C1 (residues 112-231 human sequence) or C2 (residues 90-231), respectively, with the full cell biological significance of such processing unresolved; however, it is noteworthy that in prion diseases, such as Creutzfeldt-Jakob disease (CJD) and murine models, the moderately extended C2 fragment predominates in the brain suggesting that the two cleavage events and the consequent C-terminal fragments may differ in their pathogenic significance. Accordingly, studies characterising biologically relevant peptides like C1 and C2, would be most valid if undertaken using peptides completely free of any inherent non-native sequence that arises as a by-product of commonly employed recombinant production techniques. To achieve this aim and thereby facilitate more representative biophysical and neurotoxicity studies, we adapted the combination of high fidelity Taq TA cloning with a SUMO-Hexa-His tag-type approach, incorporating the SUMO protease step. This technique consistently produced sufficient yields (∼10 mg/L) of high purity peptides (>95%) equating to C1 and C2 of exact native primary sequence in the α-helical conformation suitable for biological and biophysical investigations.
传染性和独特的神经病理学是朊病毒疾病的标志性特征，将它们与其他神经退行性疾病区分开来，发病机制和传播似乎与普遍表达的朊病毒蛋白（PrP(C)）的错误折叠构象（PrP(Sc)）密切相关。鉴于错误折叠 PrP 的明显致病性，基于朊病毒蛋白的肽的利用已成为深入了解错误折叠途径和发病机制的一种重要方法。与使用朊病毒肽的研究平行，在阿尔茨海默病等其他神经退行性疾病中采用类似方法表明，亲本蛋白的差异加工以及从相同组成型加工生成的同源肽的一级序列中非常小的变异（例如γ-分泌酶活性产生的 Aβ1-40 与 Aβ1-42）可能与非常不同的致病后果相关。PrP(C) 也经历组成型α或β切割，分别产生 C1（人序列 112-231 个残基）或 C2（90-231 个残基），这种加工的完整细胞生物学意义尚未解决；然而，值得注意的是，在朊病毒疾病（如克雅氏病（CJD）和鼠模型）中，中度扩展的 C2 片段在大脑中占主导地位，这表明两个切割事件和随后的 C 末端片段在其致病意义上可能不同。因此，如果使用完全不含任何固有非天然序列的肽进行研究，这些肽具有生物学相关性，如 C1 和 C2，则此类研究将是最有效的，因为这些固有非天然序列是常见重组生产技术的副产品。为了实现这一目标，从而促进更具代表性的生物物理和神经毒性研究，我们采用了高保真 Taq TA 克隆与 SUMO-Hexa-His 标签型方法的结合，其中包含 SUMO 蛋白酶步骤。该技术始终能够产生足够的产量（∼10 mg/L），得到高纯度的肽（>95%），其为 α-螺旋构象中的精确天然一级序列，适合用于生物学和生物物理研究。
