PURIFICATION AND PROPERTIES OF THE CELLULAR AND SCRAPIE HAMSTER PRION PROTEINS
PURIFICATION AND PROPERTIES OF THE CELLULAR AND SCRAPIE HAMSTER PRION PROTEINS
复制标题
DOI：
10.1111/j.1432-1033.1988.tb14246.x
复制
发表时间：
1988-09-01
期刊：
影响因子：
--
通讯作者：
PRUSINER, SB
中科院分区：
文献类型：
--
作者：
TURK, E;TEPLOW, DB;PRUSINER, SB
在羊瘙痒症感染期间，朊病毒蛋白（PrP）的异常亚型（命名为PrPSc）积累并发现与感染性共纯化;迄今为止，尚未发现羊瘙痒症特异性核酸。未感染和羊瘙痒病感染的细胞都合成PrP同种型，称为PrPC，其表现出与PrPSc不同的物理特性。使用与蛋白质-A. sbd. Avidgel交联的PrP特异性单克隆抗体通过免疫亲和色谱法纯化PrPC。通过去污剂提取、聚（乙二醇）沉淀和重复差速离心纯化PrPSc聚合物。发现两种PrP同种型具有相同的N-末端氨基酸序列，其开始于预测的信号肽切割位点。PrPC的前8个残基为KKXPKPGG，PrPSc的前29个残基为KKXPKPGGWNTGGSXYPGQGSPGGNPYPP。精氨酸残基3和15 PrPSc和3 PrPC中似乎被修改，因为没有可检测的信号（表示为X），发现在这些位置在气相测序。发现两种PrP亚型都含有分子内二硫键，连接Cys 179和214，其产生含有两个N-连接的糖基化位点的36个氨基酸的环。PrPC的纯化方案的发展应有利于比较两个PrP亚型，并导致了解如何PrPSc是从PrPC或前体合成。
During scrapie infection an abnormal isoform of the prion protein (PrP), designated PrPSc, accumulates and is found to copurify with infectivity; to date, no nucleic acid has been found which is scrapie-specific. Both uninfected and scrapie-infected cells synthesize a PrP isoform, denoted PrPC, which exhibits physical properties that differentiate it from PrPSc. PrPC was purified by immunoaffinity chromatography using a PrP-specific monoclonal antibody cross-linked to protein-A.sbd.Avidgel. PrPSc was purified by detergent extraction, poly(ethylene glycol) precipitation and repeated differential centrifugation of PrPSc polymers. Both PrP isoforms were found to have the same N-terminal amino acid sequence which begins at a predicted signal peptide cleavage site. The first 8 residues of PrPC were found to be KKXPKPGG and the first 29 residues of PrPSc were found to be KKXPKPGGWNTGGSXYPGQGSPGGNPYPP. Arg residues 3 and 15 in PrPSc and 3 in PrPC appear to be modified since no detectable signals (denoted X) were found at these positions during gas-phase sequencing. Both PrP isoforms were found to contain an intramolecular disulfide bond, linking Cys 179 and 214, which creates a loop of 36 amino acids containing the two N-linked glycosylation sites. Development of a purification protocol for PrPC should facilitate comparisons of the two PrP isoforms and lead to an understanding of how PrPSc is synthesized either from PrPC or a precursor.