Solvent-dependent precipitation of prion protein

Kang Cai , Jeanette L.C. Miller , Christopher J. Stenland , Kevin J. Gilligan , Randal C. Hartwell , Jarrett C. Terry , Rosemary B. Evans-Storms , Richard Rubenstein , Stephen R. Petteway Jr. , Douglas C. Lee
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引用次数: 38

Abstract

The misfolded isoform of the prion protein (PrPSc) possesses many unusual physiochemical properties. Previously, we and others reported on the differential partitioning of PrPSc from plasma derived therapeutic proteins during their purification processes. To understand the driving force behind these partitioning differences, we investigated the effects of various solvent conditions on the precipitation of PrPSc. In a physiological buffer, PrPSc remained in the supernatant after low speed centrifugation. At pH 5, PrPSc precipitation was nearly complete regardless of the salt content. PrPSc could also be precipitated at pH 8 by adding ethanol, but this precipitation was salt dependent. Based on these observations, an empirical mathematical model was constructed in which the PrPSc precipitation trends were fully described as a function of solvent pH, salt, and ethanol concentration. This model consistently predicted PrPSc partitioning during cold ethanol precipitation steps used in plasma protein purification processes, as shown by experimentally determined distributions of PrPSc and transmissible spongiform encephalopathy (TSE) infectivity. These results indicate that pH, salt, and ethanol content are the major solvent factors determining the precipitation of the infectious PrPSc in these processes and may provide a useful tool for assessing the differential partitioning of PrPSc in a given solvent environment.

朊病毒蛋白的溶剂依赖性沉淀
错误折叠的朊病毒蛋白(PrPSc)具有许多不寻常的物理化学性质。之前,我们和其他人报道了血浆来源的治疗蛋白在纯化过程中对PrPSc的差异分配。为了了解这些分配差异背后的驱动力,我们研究了不同溶剂条件对PrPSc沉淀的影响。在生理缓冲液中,低速离心后PrPSc保留在上清液中。在pH为5时,无论含盐量如何,PrPSc的沉淀几乎完全完成。在pH为8的条件下,加入乙醇也能使PrPSc析出,但这种析出与盐有关。基于这些观测结果,构建了一个经验数学模型,其中PrPSc的沉淀趋势完全描述为溶剂pH、盐和乙醇浓度的函数。该模型一致地预测了血浆蛋白纯化过程中使用的冷乙醇沉淀步骤中PrPSc的分配,如实验确定的PrPSc分布和传染性海绵状脑病(TSE)传染性所示。这些结果表明,pH、盐和乙醇含量是决定这些过程中感染性PrPSc沉淀的主要溶剂因素,并可能为评估PrPSc在给定溶剂环境下的差异分配提供有用的工具。
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