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Controlled nucleation and growth of protein crystals by solvent freeze-out

机译：通过溶剂冻结控制蛋白质晶体的成核和生长

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Solvent freeze-out technology has been developed as a new concept in the field of protein crystallization. This technology allows the separation of the nucleation and growth steps but requires an understanding of the thermodynamics of the complex mixture of protein, solvent, salt, and buffer at temperatures for which few accurate data currently exist. The phase diagram of the given protein system was systematically investigated and confirmed for the identification of optimal crystallization conditions for zone I, which is the best region of protein crystallization, by employing a preliminary screening. As an initial necessity for protein crystallization, the delicate balance between repulsive and attractive forces in the given protein system was found at pH 4.4 and 5 wt % NaCl. The precise value of the supersaturation level (S) of zone I was estimated to be 9.56 ≥ S ≥ 29.34 after a statistical analysis of the initial screening by a Linbro test. Next, the supersaturation levels for the metastable zone were identified to be 7.0 ≥ S < 17.1 from a statistical analysis of all of the experimental results from both the Linbro test and the individual crystal growth measurements. Protein crystallization in zone I by the freeze-out technology was carried out and evaluated. The key process variable levels (KPVLs) were operated within the boundary of the phase diagram that was confirmed by the preliminary screening. With a NaCl concentration of 5 wt % at pH 4.4, quite a good quality of tetragonal hen egg-white lysozyme (HEWL) crystals was produced as a result of proper tuning of the net surface charge of HEWL, even with a very low value of the initial protein concentration. The number of tetragonal HEWL crystals was increased by increasing the ice mass, since the cooling rate applied to the system determines the ice growth rate and therefore the nucleation and growth rates of the protein crystals. Hence, nucleation of the given protein system can be controlled by moderate adjustment of the ice growth rate.
机译：溶剂冻干技术已被开发为蛋白质结晶领域的新概念。这项技术可以分离成核步骤和生长步骤，但是需要了解蛋白质，溶剂，盐和缓冲液复杂混合物在当前几乎没有准确数据的温度下的热力学。系统地研究了给定蛋白质系统的相图，并通过初步筛选确定了I区的最佳结晶条件，该区是蛋白质结晶的最佳区域。作为蛋白质结晶的最初必要性，在给定的蛋白质系统中，在pH 4.4和5 wt％NaCl下，排斥力和吸引力之间存在微妙的平衡。通过Linbro测试对初始筛选进行统计分析后，I区的过饱和度（S）的精确值估计为9.56≥S≥29.34。接下来，通过对来自Linbro测试和单个晶体生长测量的所有实验结果的统计分析，确定了亚稳区的过饱和度为7.0≥S <17.1。通过冻结技术在I区进行了蛋白质结晶并进行了评估。关键过程变量水平（KPVL）在相图的边界内操作，该相图已通过初步筛选得以确认。在pH值为4.4的NaCl浓度为5 wt％的情况下，由于适当调整了HEWL的净表面电荷，即使在非常低的pH值下，也可以产生相当高质量的四方鸡蛋清溶菌酶（HEWL）晶体。初始蛋白质浓度。通过增加冰量可以增加四方HEWL晶体的数量，因为应用于系统的冷却速率决定了冰的生长速率，因此决定了蛋白质晶体的成核和生长速率。因此，可以通过适度调节冰的生长速度来控制给定蛋白质系统的成核。

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