Separation and Purification of Protein by Crystallization

对蛋白质结晶过程进行了基础性研究。首先，利用热溶酶和溶菌酶两种酶对蛋白质结晶的机理进行了研究。得到了以下结果。1)结晶分阶段进行：先形成直径为60 nm的初生颗粒，然后形成由初生颗粒组成的大晶体。2)晶体生长速度与初始过饱和度有关。存在一个最佳的过饱和度比，在该过饱和度时，生长速度最大，晶体数量最少。最佳过饱和度比的存在可以用上述连续结晶机理来解释。3)一次粒子中不含第三种蛋白质作为杂质。这一结果表明，至少在蛋白质结晶的早期，分子间的吸引相互作用在同一分子中优先起作用。第二，提出了一种新的蛋白质结晶纯化方法。该方法包括以下操作：1)分离初级颗粒，而不是最终得到的大晶体；2)利用目标蛋白质和杂质之间晶体生长速度的差异：3)利用目标蛋白质和杂质之间沉淀的大小差异。

The fundamental research on the crystallization of protein was carried out. The objective was to upgrade the crystallization to a high level of protein purification method.First, the mechanism of the crystallization of protein was investigated, by using two kinds of enzyme protein, thermolysin and lysozyme. The following results were obtained. 1) The crystallization proceeds stepwise : the formation of the primary particles with 60 nm in diameter and then the formation of the large crystal composing of the primary particles. 2) The rate of the crystal growth depends on the initial supersaturation ratio. There is an optimal supersaturation ratio at which the growth rate becomes maximal and the number of the crystals becomes minimal. The presence of the optimal supersaturation ratio can be explained by the consecutive mechanism of the crystallization described above. 3) The primary particles don t contain the third protein as the impurity. This result suggests that at least at the early stage of the crystallization of protein the attractive interaction between the molecules acts preferentially in the same molecules.Second, a novel method of the purification of protein by the crystallization was proposed. The method includes the following operations : 1) to separate the primary particles, instead of the large crystal finally obtained ; 2) to utilize the difference in the rate of crystal growth between the protein in interest and the impurities : 3) to utilize the difference in the size of the precipitates between the protein in interest and the impurities.