Advanced Centre of Biochemical Engineering, Department of Biochemical Engineering, University College London, UK.
Biotechnol Bioeng. 2011 Feb;108(2):322-32. doi: 10.1002/bit.22957.
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.
可逆沉淀可以作为一种有效的蛋白质纯化工具。此外，在生物加工和制药行业中，确定沉淀或聚集发生的条件至关重要，因为这有助于更好的配方设计，并防止色谱法中的聚集。我们已经评估了通过微孔板中的光散射来确定蛋白质沉淀的方法，作为高通量测定蛋白质沉淀热力学参数的工具，具有筛选配方添加剂和相关生物加工条件（如 pH 值）的潜力。这为现有的基于微孔板的蛋白质热稳定性测量提供了一种有用的补充技术。我们使用鸡卵清溶菌酶和醇脱氢酶作为模型蛋白，确定了在硫酸铵和氯化钠浓度下可逆沉淀的程度，并且还证明了对数据进行全局拟合，以生成一个模型，该模型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，还允许对导致每种蛋白质沉淀自由能的结构核的平均大小进行近似确定。与蛋白质热稳定性测量并行快速收集蛋白质沉淀的热力学参数，将为蛋白质配方提供一个强大的平台，并为基于特定蛋白质结构相互作用的分子建模对可逆沉淀的理论预测进行测试提供有用的数据集。
