Production of new recombinant aequorin and its physiological application

新型重组水母发光蛋白的制备及其生理应用

基本信息

批准号：
13557004
负责人：
KURIHARA Satoshi
金额：
$ 4.03万
依托单位：
Jikei University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Native aequorin has been used for measurement of intracellular Ca^<2+> transients. However, it is difficult to get a large number of jellyfish that is used for extraction of aequorin. We intended to produce apoasquorin using E.coli and then recombinant aequoin began being produced by mixing the apoaequorin and celenterazine. We recorded a relation between pCa and the light signal normalized by the peak light at pCa 2, which was performed by instantaneously mixing the recombinant aequorin and solutions with various concentrations of Ca^<2+>. The recombinant aequorin was slightly sensitive to Ca^<2+> in a range between pCa5 and pCa6. The level of Ca^<2+>-independent light in recombinant aequorin was lower than that of native aequorin. Mg^<2+> inhibited the light signal of the recombinant aequorin. We altered pH in the solution and confirmed that the light of aequorin was not significantly sensitive to pH change. Then, we injected the recombinant aequorin into superficial cells of the left ventricular papillary muscles of mouse to confirm whether the recombinant aequorin can be used for the measurement of the intracellular Ca^<2+> transients (CaT) in cardiac muscles. The peak of CaT measured with the recombinant aequorin was larger than that with native aequorin. The tail of CaT in the recombinant aequorin was slightly faster than that in the native aequorin. Thus, the recombinant aequorin can be used for the measurement of intracellular Ca^<2+> concentration. We established the method to produce a large amount of the recombinant aequorin. We can expect various recombinant aequorin with different characteristics, which is useful for cellular physiology.

天然水母发光蛋白已用于测量细胞内Ca 2+ 瞬变。然而，很难获得大量用于提取水母发光蛋白的水母。我们打算使用大肠杆菌生产脱辅基水母发光蛋白，然后开始通过混合脱辅基水母发光蛋白和celenterazine 来生产重组水母发光蛋白。我们记录了pCa和通过pCa 2 处的峰值光归一化的光信号之间的关系，这是通过瞬时混合重组水母发光蛋白和具有不同浓度的Ca 2+ 的溶液来进行的。重组水母发光蛋白对范围在pCa5和pCa6之间的Ca 2+ 稍微敏感。重组水母发光蛋白中Ca 2+ 不依赖性光的水平低于天然水母发光蛋白的水平。 Mg 2+ 抑制重组水母发光蛋白的光信号。我们改变了溶液的 pH 值，并证实水母发光蛋白的光对 pH 值变化不显着敏感。然后，我们将重组水母发光蛋白注射到小鼠左心室乳头肌的表层细胞中，以证实重组水母发光蛋白是否可以用于心肌细胞内Ca^2+瞬变(CaT)的测量。用重组水母发光蛋白测量的CaT峰大于用天然水母发光蛋白测量的CaT峰。重组水母发光蛋白中的CaT尾部比天然水母发光蛋白中的CaT尾部稍快。因此，重组水母发光蛋白可用于测量细胞内Ca 2+ 浓度。我们建立了大量生产重组水母发光蛋白的方法。我们可以期待具有不同特性的各种重组水母发光蛋白，这对于细胞生理学是有用的。