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Journal of Biochemistry Advance Access published online on February 22, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn024
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© 2008 The Japanese Biochemical Society

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

Satoshi Inouye*

Yokohama Research Center, Chisso Corporation, 5-1 Okawa, Kanazawa, Yokohama 236-8605, Japan.

*To whom correspondence should be addressed: Dr. Satoshi Inouye: Tel: 81-45-786-5518, Fax: 81-45-786-5512, E-mail: sinouye{at}chisso.co.jp

Received January 4, 2008; Accepted February 15, 2008


  Abstract

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 2L of cultured cells with purity greater than 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 Ca2+ 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.

Key Words: aequorin, coelenterazine, luminescence spectrum, protein secretion, isotype clytin


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