Cloning, expression, purification and characterization of an isotype of clytin, a calcium-binding photoprotein from the luminous hydromedusa Clytia gregarium

The cDNA for an isotype of clytin, a calcium-binding photoprotein from the luminous jellyfish Clytia gregarium, was identified and named clytin-II. The histidine-tagged apoprotein of clytin-II expressed into the periplasmic space of Escherichia coli cells was isolated by nickel chelate affinity chromatography. Recombinant clytin-II regenerated from apoprotein by incubation with coelenterazine was purified. The yield of purified clytin-II was 13 mg from 2 l of cultured cells with purity >95%. The luminescence properties of clytin-II were characterized by comparison with clytin-I and aequorin, and semi-synthetic clytin-II was also prepared. The initial luminescence intensity of clytin-II triggered by Ca(2+) was 4.5 times higher than that of clytin-I and aequorin, but the luminescence capacity was close to clytin-I and aequorin. Thus, clytin-II is a useful protein, showing high sensitivity in the signal-to-noise ratio of luminescence intensity.