Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay

Описание

Тип публикации: статья из журнала

Год издания: 2008

Идентификатор DOI: 10.1039/b807271j

Ключевые слова: calcium dependent coelenterazine-binding protein, guanidine, obelin, photoprotein, protein, recombinant coelenterazine dependent luciferase 164, recombinant coelenterazine dependent luciferase 39, recombinant enzyme, unclassified drug, article, assay, bacterial cell, bioluminescence, biotinylation, cell inclusion, chemical modification, copepod, disulfide bond, Escherichia coli, heating, metridia longa, molecular interaction, nonhuman, nucleotide sequence, priority journal, protein expression, protein folding, synthesis, Amino Acid Sequence, Animals, Copepoda, Gene Expression, Isoenzymes, Kinetics, Luciferases, Molecular Sequence Data, Protein Folding, Recombinant Proteins, Sequence Alignment, Spectrometry, Fluorescence, Copepoda, Metridia longa, Metridia luciferase, Renilla muelleri

Аннотация: The recombinant coelenterazine-dependent luciferases (isoforms MLuc 164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. coli cells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc09) was obtПоказать полностьюained in an active monomeric form with a high yield. The luciferase bioluminescence is found to be initiated not only by free coelenterazine, but also by Ca2+-dependent coelenterazine-binding protein (CBP) of Renilla muelleri on Ca2+ addition. The use of CBP as a "substrate" provides higher light emission and simultaneously the lower level of background. The high purity MLuc39 can be detected down to attomol with a linear range extending over 5 orders of magnitude. The MLuc39 reveals also a high stability towards heating and chemical modification; the chemically synthesized biotinylated derivatives of the luciferase preserve 35-40% of the initial activity The luciferase applicability as an in vitro bioluminescent reporter is demonstrated in model tandem bioluminescent solid-phase microassay combining the Ca2+-regulated photoprotein obelin and the Metridia luciferase.

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Издание

Журнал: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES

Выпуск журнала: Vol. 7, Is. 9

Номера страниц: 1025-1031

ISSN журнала: 1474905X

Место издания: CAMBRIDGE

Издатель: ROYAL SOC CHEMISTRY

Авторы

  • Borisova V.V. (Photobiology Lab.,Institute of Biophysics,Russian Academy of Sciences)
  • Frank Ludmila A. (Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch; Siberian Fed Univ)
  • Markova S.V. (Photobiology Lab.,Institute of Biophysics,Russian Academy of Sciences)
  • Burakova L.P. (Photobiology Lab.,Institute of Biophysics,Russian Academy of Sciences)
  • Vysotski E.S. (Photobiology Lab.,Institute of Biophysics,Russian Academy of Sciences)

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