Supattapone S, Nguyen H O, Muramoto T, Cohen F E, DeArmond S J, Prusiner S B, Scott M
Institute for Neurodegenerative Diseases, University of California at San Francisco, San Francisco, California 94143, USA.
J Virol. 2000 Dec;74(24):11928-34. doi: 10.1128/jvi.74.24.11928-11934.2000.
An abridged PrP molecule of 106 amino acids designated PrP106 can form infectious miniprions in transgenic (Tg) mice (29). Addition of six-histidine (His(6)) affinity tags to selective sites within PrP106 resulted unexpectedly in new PrP proteins that spontaneously adopted protease-resistant conformations when expressed in neuroblastoma cells and Tg mice. Acquisition of protease resistance depended on the length, charge, and placement of the affinity tag. Introduction of the disease-linked mutation E200K into the sequence of PrP106(140/6His) increased the recovery of protease-resistant PrP fivefold, whereas introduction of the mutations C213A and Delta214-220 did not affect the recovery of protease-resistant PrP. Treatment of cultured cells expressing affinity-tagged PrP106 mutants with polypropyleneimine dendrimer rendered these proteins sensitive to protease digestion in a manner similar to wild-type PrP(Sc). We conclude that certain affinity-tagged PrP106 proteins spontaneously fold into conformations partially resembling, yet distinct from, wild-type PrP(Sc). These proteins might be useful tools in the identification of new disease-causing mutations as well as for screening compounds for therapeutic efficacy.
一种由106个氨基酸组成的截短型朊蛋白分子，命名为PrP106，可在转基因（Tg）小鼠中形成具有感染性的微小朊病毒（29）。在PrP106内的选择性位点添加六个组氨酸（His(6)）亲和标签，意外地产生了新的PrP蛋白，这些蛋白在神经母细胞瘤细胞和Tg小鼠中表达时会自发形成蛋白酶抗性构象。蛋白酶抗性的获得取决于亲和标签的长度、电荷和位置。将疾病相关突变E200K引入PrP106(140/6His)序列中，使蛋白酶抗性PrP的回收率提高了五倍，而引入突变C213A和Delta214 - 220则不影响蛋白酶抗性PrP的回收率。用聚丙烯亚胺树枝状大分子处理表达亲和标签的PrP106突变体的培养细胞，使这些蛋白对蛋白酶消化敏感，其方式类似于野生型PrP(Sc)。我们得出结论，某些带有亲和标签的PrP106蛋白会自发折叠成部分类似于野生型PrP(Sc)但又与之不同的构象。这些蛋白可能是鉴定新的致病突变以及筛选具有治疗效果的化合物的有用工具。
