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Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase

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Abstract

The Renilla bioluminescent system in vivo is comprised of three proteins—the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca2+-binding superfamily of proteins with only three of the EF-hand loops having the Ca2+-binding consensus sequences. There is weak sequence homology with the Ca2+-regulated photoproteins but only as a result of the necessary Ca2+-binding loop structure. In combination with Renilla luciferase, addition of only one Ca2+ is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.

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Authors and Affiliations

  1. Photobiology Lab, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, 660036, USA

    Maxim S. Titushin, Svetlana V. Markova, Ludmila A. Frank, Natalia P. Malikova, Galina A. Stepanyuk & Eugene S. Vysotski

  2. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA

    John Lee & Eugene S. Vysotski

Authors
  1. Maxim S. Titushin
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  2. Svetlana V. Markova
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  3. Ludmila A. Frank
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  4. Natalia P. Malikova
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  5. Galina A. Stepanyuk
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  6. John Lee
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  7. Eugene S. Vysotski
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Correspondence to Eugene S. Vysotski.

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This paper was published as part of the themed issue on bioluminescence

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Titushin, M.S., Markova, S.V., Frank, L.A. et al. Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase. Photochem Photobiol Sci 7, 189–196 (2008). https://doi.org/10.1039/b713109g

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  • DOI: https://doi.org/10.1039/b713109g

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