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 35 kDa 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 6 M urea and purified by urea–nickel chelate affinity chromatography. The yield of 19kOLase was 16 mg from 400 ml of cultured cells. 19kOLase in 6 M urea could be refolded rapidly by dilution with 50 mM Tris–HCl (pH 7.8)–10 mM 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 1 μM Cu2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction.