| abstract |
The present invention relates to establishment of a series of artificial luciferases based on artificial amino acid sequences extracted by amino acid alignment of copepod-derived luciferase sequences in a database based on amino acid similarity. The present invention provides high luminescence intensity, high luminescence stability, and a spectrum with increased wavelength as luminescence characteristics. In the present invention, the copepod-derived luciferase sequences in a database were aligned based on amino acid similarity, and amino acid sequences with high frequency for each domain of the corresponding sequence were mainly extracted, thereby constructing artificial amino acid sequences. In this manner, a series of artificial luciferases (ALuc) was established. In this method, the enzymatic activity regions present in the first and second portions of the amino acid sequence were also aligned based on the similarity, thereby increasing their amino acid similarity. The luminescence intensity or luminescence stability of each enzyme in the resulting luciferase (ALuc) group were evaluated, and amino acid sequences of excellent ALucs were fed back to the reconstruction of amino acid sequences, thereby synthesizing a group of high-performance luciferases. The group of ALucs has superior luminescence characteristics, such as an increase in luminescence intensity, an increase in luminescence stability, or an increase in wavelength of the luminescence spectrum, which were not obtained before. Further, by using the artificial luciferases (ALuc) of the present invention, it is possible to provide a novel, superior bioassay system, such as a bioluminescent probe, two-hybrid assay, a luminescent capsule, or the like having improved measurement function. |