Production of new recombinant aequorin and its physiological application

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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.