title: Expression, purification and luminescence properties of coelenterazine-utilizing luciferases from Renilla, Oplophorus and Gaussia: comparison of substrate specificity for C2-modified coelenterazines.
abstract: 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.
authors.affiliation: Yokohama Research Center, JNC Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan. sinouye@jnc-corp.co.jp
authors.forename: Satoshi
authors.initials: S
authors.lastname: Inouye
authors.forename: Yuiko
authors.initials: Y
authors.lastname: Sahara-Miura
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Sato
authors.forename: Rie
authors.initials: R
authors.lastname: Iimori
authors.forename: Suguru
authors.initials: S
authors.lastname: Yoshida
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Hosoya
mesh_terms: D000818:Animals; D002384:Catalysis; D033342:Copepoda / Q000201:enzymology*; D004926:Escherichia coli; D007093:Imidazoles / Q000737:chemistry; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D008163:Luminescent Measurements; D011719:Pyrazines / Q000737:chemistry; D048790:Renilla / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D007093:Imidazoles; D011719:Pyrazines; C017144:coelenterazine; D049410:Luciferases, Renilla