期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 88, 期 1, 页码 150-156出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2012.12.006
关键词
Coelenterazine; Coelenteramide; Coelenteramine; Renilla; Gaussia; Oplophorus
类别
资金
- The Noguchi Institute
- Kurata Grants
- JSPS KAKENHI [23710266, 24651257]
- Grants-in-Aid for Scientific Research [24651257, 23710266] Funding Source: KAKEN
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.
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