Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase |
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Authors: | Galina A Stepanyuk James Unch Natalia P Malikova Svetlana V Markova John Lee Eugene S Vysotski |
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Institution: | (1) Photobiology laboratory, Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, 660036, Russia;(2) Promega Biosciences, LLC, San Luis Obispo, CA 93401, USA;(3) Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA; |
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Abstract: | It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by
Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the
use of some “yellow” Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered
by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca2+-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v
bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the
coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate
for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays. |
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