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, Recombinant Proteins, Sequence Alignment, Spectrometry, Fluorescence, 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.
Журнал: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES
Выпуск журнала: Vol. 7, Is. 9
Номера страниц: 1025-1031
ISSN журнала: 1474905X
Место издания: CAMBRIDGE
Издатель: ROYAL SOC CHEMISTRY