Expression, purification and characterization of a photoprotein, clytin, from Clytia gregarium.
A novel histidine-tagged secretion vector in Escherichia coli was constructed and large amounts of highly pure clytin, a calcium-binding photoprotein, was prepared. The histidine-tagged apoclytin expressed into the periplasmic space in E. coli was purified by nickel chelate affinity chromatography. Recombinant clytin was regenerated from apoclytin by incubation with coelenterazine in the presence of dithiothreitol and then purified by anion-exchange chromatography and hydrophobic chromatography. The yield of recombinant clytin was 20mg from 2L of cultured cells with purity greater than 95%. Luminescence properties of recombinant clytin were identical to that of native clytin (phialidin). The Ca(2+) sensitivity of recombinant clytin is lower than that of aequorin and clytin is suited for measuring higher concentration of Ca(2+). Semi-synthetic clytins were also prepared with coelenterazine analogues, and the initial intensity, luminescence capacity and half decay time were characterized.
Inouye
Satoshi
S
Yokohama Research Center, Chisso Corporation, 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan. sinouye@chisso.co.jp
Sahara
Yuiko
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D037402:Hydrozoa / Q000235:genetics*; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013053:Spectrophotometry
D017931:DNA Primers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C046165:phialidin; D002118:Calcium