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Aquorin已用于测量细胞内Ca^<2+>瞬变。但是，很难获得大量用于提取eequorin的水母。我们打算使用大肠杆菌产生丙氨核蛋白酶，然后通过混合apoaequorin和celenterazine来产生重组aequoin。我们记录了PCA和通过PCA 2处的峰光标准化的光信号之间的关系，该信号是通过瞬时将重组燃料蛋白和溶液与不同浓度Ca^<2+>的溶液进行的。重组源源对pCA5和PCA6之间的CA^<2+>略微敏感。 Ca^<2+>的水平 - 重组源中的独立光低于天然Aequorin的水平。 Mg^<2+>抑制重组源素的光信号。我们改变了溶液中的pH值，并确认了Aquorin的光对pH变化不显着敏感。然后，我们将重组释放蛋白注入小鼠的左心室乳头肌的表面细胞中，以确认是否可以使用重组源自eequorin来测量心脏肌肉中的细胞内Ca^<2+>瞬变（CAT）。用重组释放蛋白测量的CAT的峰值大于天然化学蛋白。重组源中的猫的尾巴比天然eequorin中的猫略快。因此，重组源蛋白可用于测量细胞内Ca^<2+>浓度。我们确定了生产大量重组源蛋白的方法。我们可以预期具有不同特征的各种重组源，这对于细胞生理很有用。