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.
Inouye
Satoshi
S
Yokohama Research Center, Chisso Corporation, 5-1 Okawa, Kanazawa, Yokohama 236-8605, Japan. sinouye@chisso.co.jp
D000331:Aequorin / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001059:Apoproteins / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D037402:Hydrozoa / Q000737:chemistry / Q000235:genetics*; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D001059:Apoproteins; D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C060014:apoaequorin; C046165:phialidin; D000331:Aequorin; D002118:Calcium