Production of new recombinant aequorin and its physiological application

天然水母发光蛋白已被用于测量细胞内Ca^2+瞬变。然而，用于水母发光蛋白提取的水母很难获得大量的水母。我们打算使用大肠杆菌生产脱辅基水母发光蛋白，然后通过混合脱辅基水母发光蛋白和celentreazine开始生产重组水母发光蛋白。我们记录了pCa和光信号之间的关系，该关系通过瞬时混合重组水母发光蛋白和具有不同浓度Ca^2+的溶液来进行，该光信号由pCa 2处的峰值光归一化。重组水母发光蛋白在pCa 5和pCa 6之间的范围内对Ca^<2+>稍微敏感。重组水母发光蛋白的非Ca^<2+>依赖性光水平低于天然水母发光蛋白。Mg^2+抑制重组水母发光蛋白的光信号。我们改变了溶液中的pH值，并证实水母发光蛋白的光对pH值的变化不显着敏感。然后，将重组水母发光蛋白注射到小鼠左心室乳头肌表面细胞中，以确定其是否可用于心肌细胞内Ca^2+瞬变（CaT）的测定。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。CaT在重组水母发光蛋白中的尾部比在天然水母发光蛋白中的尾部稍快。因此，重组水母发光蛋白可用于测量细胞内Ca^2+浓度。建立了大量生产重组水母发光蛋白的方法。我们可以期待各种不同特性的重组水母发光蛋白，这是有用的细胞生理。

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.