Thermodynamic parameters for salt-induced reversible protein precipitation from automated microscale experiments.
Thermodynamic parameters for salt-induced reversible protein precipitation from automated microscale experiments.
复制标题
自动化微尺度实验中盐诱导可逆蛋白质沉淀的热力学参数。
DOI：
10.1002/bit.22957
复制
发表时间：
2011
影响因子：
3.8
通讯作者：
Ahmad SS
中科院分区：
文献类型：
--
作者：
Ahmad SS
可逆沉淀可用作蛋白质的有效纯化工具。此外，确定沉淀或聚集发生的条件在生物加工和制药工业中至关重要，因为这可以帮助更好地配制并阻止色谱中的聚集。我们已经评估了通过微板中的光散射测定的蛋白质沉淀，作为高通量测定蛋白质沉淀热力学参数的工具，具有筛选制剂添加剂和相关生物工艺条件（如pH值）的潜力。这为现有基于微板的蛋白质热稳定性测量提供了有用的补充技术。使用鸡蛋白色溶菌酶和乙醇脱氢酶作为模型蛋白质，我们已经确定了可逆沉淀的程度作为硫酸铵和氯化钠浓度的函数，并且还证明了数据的全局拟合以生成模型，其中可以预测任何给定条件下的沉淀分数。全局拟合提供了热力学参数，包括蛋白质沉淀的自由能，并且还允许近似确定结构核的平均大小，其有助于每种蛋白质的沉淀自由能。蛋白质沉淀的热力学参数的快速收集，与蛋白质热稳定性测量并行，将为蛋白质配制提供一个强大的平台，并且还导致用于测试基于特定蛋白质结构相互作用的分子建模的可逆沉淀的理论预测的数据集。Biotechnol. Bioeng. 2011;108：322-332.© 2010 Wiley Periodicals，Inc.
Reversible precipitation can be used as an efficient purification tool for proteins. In addition, identifying conditions under which precipitation or aggregation occurs is of key importance in the bioprocessing and pharmaceutical industry, as this can aid in better formulations and hinder aggregation in chromatography. We have evaluated the precipitation of proteins as determined by light scattering in microplates as a tool for the high‐throughput determination of thermodynamic parameters for protein precipitation, with the potential for screening of formulation additives and relevant bioprocess conditions such as pH. This provides a useful complementary technique to existing microplate‐based protein thermostability measurements. Using hen egg‐white lysozyme and alcohol dehydrogenase as model proteins we have determined the extent of reversible precipitation as a function of ammonium sulfate and sodium chloride concentrations, and also demonstrated global fitting of the data to generate a model where the fraction precipitated can be predicted for any given condition. The global fit provided thermodynamic parameters, including the free energy for protein precipitation, and also allowed an approximate determination of the average size of the structural nucleus that contributes to the free energy of precipitation for each protein. The rapid collection of thermodynamic parameters for protein precipitation, in parallel with protein thermostability measurements, will provide a powerful platform for protein formulation, and also lead to datasets useful for testing theoretical predictions of reversible precipitation based on the molecular modeling of specific protein structure interactions. Biotechnol. Bioeng. 2011;108: 322–332. © 2010 Wiley Periodicals, Inc.