Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris.
The luciferase secreted by the deep-sea shrimp Oplophorus consists of 19 and 35kDa proteins. The 19-kDa protein (19kOLase), the catalytic component of luminescence reaction, was expressed in Escherichia coli using the cold-shock inducted expression system. 19kOLase, expressed as inclusion bodies, was solubilized with 6M urea and purified by urea-nickel chelate affinity chromatography. The yield of 19kOLase was 16 mg from 400 ml of cultured cells. 19kOLase in 6M urea could be refolded rapidly by dilution with 50mM Tris-HCl (pH 7.8)-10mM EDTA, and the refolded protein showed luminescence activity. The luminescence properties of refolded 19kOLase were characterized, in comparison with native Oplophorus luciferase. Luminescence intensity with bisdeoxycoelenterazine as a substrate was stimulated in the presence of organic solvents. The 19kOLase is a thermolabile protein and is 98 % inhibited by 1muM Cu2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction.
Inouye
Satoshi
S
Yokohama Research Center, Chisso Corporation, Kanazawa-ku, Yokohama 236-8605, Japan. sinouye@chisso.co.jp
Sasaki
Satoko
S
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002478:Cells, Cultured; D033364:Decapoda / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049449:Luminescence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D011993:Recombinant Fusion Proteins; D008156:Luciferases