The cold-induced expression system in Escherichia coli is useful and we have applied this system to prepare the coelenterazine-utilizing luciferases including Renilla luciferase (RLase), a red-shifted variant of Renilla luciferase (RLase-547), the catalytic domain of Oplophorus luciferase (19kOLase) and Gaussia luciferase (GLase). The luminescence properties of the purified luciferases were characterized by using 10 kinds of C2-modified coelenterazine analogues as a substrate. The order of the maximal luminescence intensity for native coelenterazine was GLase (100%) > RLase (8.0%) > RLase-547 (0.73%) > 19kOLase (0.09%) under our assay conditions. The substrate specificities of coelenterazine-utilizing luciferases for the C2-modified analogues showed significant differences, but the emission peaks catalyzed by coelenterazine-utilizing luciferases were not affected by the C2-substituted coelenterazine. These results suggest that the catalytic environment for the oxygenation process of coelenterazine and the excited species of coelenteramide might be different among coelenterazine-utilizing luciferases. (c) 2012 Elsevier Inc. All rights reserved.