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.