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Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay

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Abstract

Two kinds of Ca2+-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (λmax = 390 nm) and the mutant Y139F emits greenish light (λ max = 498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones—follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca2+ solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay.

Two kinds of Ca2+-regulated photoprotein obelin with altered color of bioluminescence were obtained and applied in dual-color simultaneous immunoassay of two gonadotropic hormones.

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Acknowledgements

This work was supported by Russian Foundation for Basic Research grant 06-04-08076, and by the Molecular and Cellular Biology program of the Russian Academy of Sciences.

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Authors and Affiliations

  1. Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, 660036, Russia

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

  2. Siberian Federal University, pr. Svobodnyi, 79, Krasnoyarsk, 660041, Russia

    Ludmila A. Frank

Authors
  1. Ludmila A. Frank
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  2. Vasilisa V. Borisova
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  3. Svetlana V. Markova
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  4. Natalia P. Malikova
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  5. Galina A. Stepanyuk
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  6. Eugene S. Vysotski
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Correspondence to Ludmila A. Frank.

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Frank, L.A., Borisova, V.V., Markova, S.V. et al. Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay. Anal Bioanal Chem 391, 2891–2896 (2008). https://doi.org/10.1007/s00216-008-2223-5

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  • DOI: https://doi.org/10.1007/s00216-008-2223-5

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