Production of new recombinant aequorin and its physiological application

天然木犀草素已被用于细胞内钙离子瞬变的测量。然而，很难得到大量用于提取木犀草素的水母。我们打算用大肠杆菌生产芦荟素，然后将芦荟素和西替利嗪混合，开始生产重组芦荟素。我们记录了PCA与由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Aequorin和含有不同浓度的Ca~(2+)和Gt；的溶液来实现的。在pCa5~pCa6范围内，重组水飞蓟素对Ca~(2+)和Gt~(2+)有轻微的敏感性。重组木犀草素的钙非依赖性LIGH水平低于天然木犀草素。Mg~(2+)~(2+)可抑制重组马兜铃素的光信号。我们改变了溶液中的pH，证实了赤藜芦素的光对pH变化不是很敏感。然后，我们将重组水飞蓟素注射到小鼠左室乳头肌的表层细胞中，以证实重组水飞蓟素是否可以用于心肌细胞内钙瞬变(CAT)的测定。用重组马兜铃素测得的过氧化氢酶峰值比用天然马兜铃素测得的大。重组Aequorin的猫尾巴略快于天然Aequorin。因此，重组水飞蓟素可用于细胞内钙离子浓度的测定。我们建立了大量生产重组水飞蓟素的方法。我们可以期待具有不同特性的各种重组水飞蓟素，这对细胞生理学是有用的。

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.