Crystallization of proteins from aqueous solution based on the pressure induced liquid-liquid phase separation phenomenon

从蛋白质水溶液相平衡的最新研究出发，讨论了亚稳液-液分离的可能性，一些研究小组试图建立理论模型来描述这种相行为。我们认为压力可以作为一个合适的变量来控制蛋白质溶液的相行为。本项目的目标是发展制备蛋白质晶体的实验方法。1.测量蛋白质在水溶液中的晶体生长和蛋白质溶液的热物理性质。在高压下测量溶菌酶在水溶液中的晶体生长速率。研究发现，压力对增长率有很强的依赖性。在压力下测定了溶菌酶水溶液的热物理性质(粘度、密度、溶解度)，为理论分析提供了参数。溶液…的密度用玻璃比瓶法、粘度下降针法和从溶液中蛋白质浓度的时间变化来测定溶解度。从溶解度数据，我们可以准确地估计过饱和度。晶体生长速率随压力的增加而减小。通过对晶体生长的压力依赖性分析，发现晶体生长的速率决定过程应该是传质过程。2、晶核产生率的测定：在100 Mpa以下的高压下，用直接观察法测量了晶核数的产生率，生长速率随压力的增加而减小，与晶体生长速率基本一致。因此，如果在高压下生产溶菌酶晶体，可以得到数量少但尺寸大得多的晶体，实验证明这是正确的。研究发现，晶核形成的速率决定过程为传质过程。3蛋白质微晶实验方法的开发。以上两个主题均基于静压实验。通过静压，可以得到较大尺寸的蛋白质晶体。本课题利用喷嘴压力降等动态压力来促进蛋白质溶液的液液相分离，为此，我们选择了超临界流体溶液快速膨胀的方法，首先用萘或乙酰水杨酸等有机化合物对装置进行了测试。然后，蛋白质水溶液和超临界二氧化碳混合并从喷嘴膨胀。通过使用不同直径的喷嘴，水溶液在较宽的温度和压力范围内膨胀到室内。在最佳喷嘴条件和腔室温度、压力条件下，可得到溶菌酶微晶。从这些结果可以得出结论，在喷嘴内可以发生液-液分离诱导压力。较少

Summaries of this project are as follows.From recent studies for the phase equilibrium of aqueous protein solutions, the possibility of a metastable liquid-liquid phase separation is discussed and some groups tried to develop theoretical models to describe such kinds of phase behavior. We thought that pressure can be used as a suitable variable to control the phase behavior of protein solutions. The development of experimental method to produce protein crystal is the aim of this project.1 Measurements of crystal growth of protein from aqueous solutions and thermophysical properties of protein solution.The rate of crystal growth of lysozyme from aqueous solution were measured under high pressures up to 100MPa. It was found that the growth rate strongly depends on the pressure. The thermophysical properties (viscosity, density, solubility) of lyzosyme aqueous solutions were also measured under pressure, which were needed as the parameters for theoretical analysis. The density of solution … More were measured with the glass pycnometer method, the viscosity falling needle method, and the solubility from the temporal change of protein concentration in the solution. From the solubility data, we can estimate the supersaturation degree accurately. The crystal growth rate decreases with pressure increases. From the analysis of pressure dependence of crystal growth, it was found that rate determining process should be mass transfer process.2 Measurement of production rate of crystal nucleus.The production rate of crystal nucleus number was measured with the direct observation method under high pressures up to 100MPa, The growth rate decreases with pressure increases which is almost same as the crystal growth rate. Therefore, if lysozyme crystals were produced under high pressures, few number but much larger size crystal can be obtained, It was proved to be correct from the experiments. The rate determining process for the production of crystal nucleus was found to be mass transfer process.3 Development of experimental method for the micro-crystal of protein.The above 2 themes were based on the experiment using static pressure. By using static pressure, we can get large size of protein crystal. In this themes, dynamic pressure such as drastic pressure drop in nozzle was applied to promote the liquid-liquid phase separation of protein solutions, To do so, we selected the rapid expansion method with supercritical fluid solution, At first, organic compound such as naphthalene or acetyl salicylic acid were used to test the apparatus. Then, the aqueous protein solution and supercritical carbon dioxide was mixed and expanded from the nozzle. With the use of nozzles with different diameter, the aqueous solutions were expanded into the chamber over a wide range of temperature and pressure. The microcrystal of lyzosyme can be produced at the optimum conditions of nozzle and temperature and pressure conditions of chamber. From these results, we can conclude that liquid-liquid phase separation induced pressure can be occurred in the nozzle. Less