Separation and Purification of Protein by Crystallization

开展了蛋白质结晶的基础研究。目的是将结晶法提升为高水平的蛋白质纯化方法。首先，利用嗜热菌蛋白酶和溶菌酶两种酶蛋白，研究了蛋白质结晶的机理。得到以下结果。 1)结晶逐步进行：形成直径为60nm的初级颗粒，然后形成由初级颗粒组成的大晶体。 2) 晶体生长速率取决于初始过饱和度。存在一个最佳过饱和比率，在该比率下生长速率变得最大并且晶体的数量变得最小。最佳过饱和率的存在可以通过上述结晶的连续机制来解释。 3)初级颗粒不含有作为杂质的第三蛋白质。这一结果表明，至少在蛋白质结晶的早期阶段，分子之间的吸引相互作用优先作用于同一分子。 其次，提出了一种通过结晶纯化蛋白质的新方法。该方法包括以下操作：1)分离出初级颗粒，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率的差异： 3) 利用目标蛋白质和杂质之间沉淀物尺寸的差异。

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.