Separation and Purification of Protein by Crystallization

通过结晶分离和纯化蛋白质

• 批准号: 03650778
• 负责人: OOSHIMA Hiroshi
• 金额: $ 1.28万
• 依托单位: Osaka City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03650778/
• 关键词: Crystallization; Protein; Bioseparation; Precipitation; Purification; Thermolysin; Primary particles; Polycrystal; タンパク質の結晶化; サ-モライシン; 沈殿法; ハイオセパレ-ション; 自己分解; 分離・精製; タンパク質分解酵素

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.

进行了蛋白质结晶的基本研究。目的是通过使用两种酶蛋白质，热溶酶素和溶菌酶，研究了蛋白质结晶的最高蛋白质纯化方法。获得以下结果。 1）结晶进行逐步进行：直径为60 nm的主要颗粒形成，然后形成主要颗粒的大晶体组成。 2）晶体生长的速率取决于初始过饱和比。有一个最佳的过饱和率，即生长速率变得最大，并且晶体的数量变得最小。最佳过饱和比的存在可以通过上述结晶的连续机制来解释。 3）主要颗粒不包含第三个蛋白作为杂质。该结果表明，至少在蛋白质结晶的早期阶段，分子之间的有吸引力的相互作用优先在同一分子中起作用。该方法包括以下操作：1）将主要颗粒分开，而不是最终获得的大晶体； 2）利用蛋白质与杂质的蛋白质之间的晶体生长速率差异：3）利用蛋白质之间的沉淀物大小的差异和杂质。

项目成果

G.Sazaki,H.Ooshima,J.Kato,Y.Harano,N.Hirokawa: "Mechanism of Crystallization of Enzyme Protein Thermolysin" J.Crystal Growth. (1993)

G.Sazaki，H.Ooshima，J.Kato，Y.Harano，N.Hirokawa：“酶蛋白嗜热菌蛋白酶的结晶机制”J.Crystal Growth。