Expression, purification and luminescence properties of coelenterazine-utilizing luciferases from Renilla, Oplophorus and Gaussia: Comparison of substrate specificity for C2-modified coelenterazines

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• 作者：Satoshi Inouye^a ; ^{sinouye@jnc-corp.co.jp} ; Yuiko Sahara-Miura^a ; Jun-ichi Sato^a ; Rie Iimori^b ; Suguru Yoshida^c ; Takamitsu Hosoya^c ; ^{thosoya.cb@tmd.ac.jp}
• 关键词：Coelenterazine ; Coelenteramide ; Coelenteramine ; Renilla ; Gaussia ; Oplophorus
• 刊名：Protein Expression and Purification
• 出版年：2013
• 出版时间：March, 2013
• 年：2013
• 卷：88
• 期：1
• 页码：150-156
• 全文大小：404 K

文摘

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.