Hydrostatic pressure rescues native protein from aggregates
Hydrostatic pressure rescues native protein from aggregates
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DOI：
10.1002/(sici)1097-0290(19990605)63:5
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发表时间：
1999-06-05
影响因子：
3.8
通讯作者：
Robinson, AS
中科院分区：
文献类型：
--
作者：
Foguel, D;Robinson, CR;Robinson, AS
蛋白质的错误折叠和错误组装是生物技术工业、生物化学研究和人类疾病中的主要问题。在这里，我们描述了一种新的方法，通过应用静水压力逆转聚集和增加重折叠。使用P22尾刺蛋白作为模型系统，沿聚集途径的中间体沿着进行鉴定和定量的尺寸排阻高效液相色谱法（HPLC）。使尾钉聚集体经受2.4kbar（35，000psi）的流体静压。这种处理使尾穗聚集体解离，并且一旦释放压力，就导致天然三聚体的形成增加。在这些压力下重折叠的尾穗三聚体对于形成感染性病毒颗粒是完全活跃的。该技术可以促进聚集体转化为天然蛋白质，而无需添加离液剂、改变缓冲液或传统重折叠方法所需的试剂的大规模稀释。我们的研究结果还表明，在折叠和聚集途径之间的交界处的一个或多个中间体是压力敏感的。这一发现支持了这样的假设，即识别的特定决定因素存在于蛋白质聚集，这些决定因素与折叠到天然状态的决定因素相似。对这种特异性的进一步理解应该会导致改进的重折叠方法。(C)John Wiley & Sons，Inc.
Misfolding and misassembly of proteins are major problems in the biotechnology industry, in biochemical research, and in human disease. Here we describe a novel approach for reversing aggregation and increasing refolding by application of hydrostatic pressure. Using P22 tailspike protein as a model system, intermediates along the aggregation pathway were identified and quantitated by size-exclusion high-performance liquid chromatography (HPLC). Tailspike aggregates were subjected to hydrostatic pressures of 2.4 kbar (35,000 psi). This treatment dissociated the tailspike aggregates and resulted in increased formation of native trimers once pressure was released. Tailspike trimers refolded at these pressures were fully active for formation of infectious viral particles. This technique can facilitate conversion of aggregates to native proteins without addition of chaotropic agents, changes in buffer, or large-scale dilution of reagents required for traditional refolding methods. Our results also indicate that one or more intermediates at the junction between the folding and aggregation pathways is pressure sensitive. This finding supports the hypothesis that specific determinants of recognition exist for protein aggregation, and that these determinants are similar to those involved in folding to the native state. An increased understanding of this specificity should lead to improved refolding methods. (C) 1999 John Wiley & Sons, Inc.