The luciferase secreted by the deep-sea shrimp Oplophorits 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 mu M Cu2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction. (C) 2007 Elsevier Inc. All rights reserved.