Corsaro A, Thellung S, Russo C, Villa V, Arena S, D'Adamo M C, Paludi D, Rossi Principe D, Damonte G, Benatti U, Aceto A, Tagliavini F, Schettini G, Florio T
Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), Genoa, Italy.
Neurochem Int. 2002 Jul;41(1):55-63. doi: 10.1016/s0197-0186(01)00137-1.
Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases. Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin. The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.
朊病毒病是人和动物中枢神经系统的致命性神经退行性疾病，其特征为中枢神经系统海绵状变性、星形胶质细胞增生以及淀粉样蛋白在脑内沉积。目前认为，细胞糖蛋白（朊病毒蛋白，PrP(C)）转变为一种改变的异构体（朊病毒瘙痒蛋白，PrP(Sc)），因其对细胞内分解代谢具有抗性而在脑组织内蓄积，这一转变代表了引发这些疾病的病原体。合成或重组多肽常用于阐明神经退行性疾病相关蛋白质的机制。在此，我们描述了一种方法，该方法能够以天然折叠状态合成并纯化人朊病毒蛋白片段90 - 231，其对应于PrP(Sc)的蛋白酶抗性核心。我们合成了PrP野生型和E200K突变体异构体的90 - 231多肽。采用谷胱甘肽S - 转移酶（GST）融合蛋白方法，在大肠杆菌中表达后获得了毫克量的多肽。通过评估GST活性来监测纯化融合蛋白的回收率。通过用凝血酶直接切割，将PrP片段从固定在谷胱甘肽偶联琼脂糖树脂上的融合蛋白中释放出来。通过考马斯亮蓝染色的丙烯酰胺凝胶以及使用单克隆抗PrP抗体的免疫印迹法鉴定重组蛋白。通过圆二色性（CD）分析，经凝胶过滤色谱纯化的肽主要呈现α - 螺旋结构，且具有完整的二硫键。同样的方法也成功用于合成和纯化E200K突变体PrP片段。
