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Issue 9, 2008
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Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay

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Abstract

The recombinant coelenterazine-dependent luciferases (isoforms MLuc164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. colicells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc39) was obtained 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.

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

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Article information


Submitted
29 Apr 2008
Accepted
02 Jun 2008
First published
17 Jun 2008

Photochem. Photobiol. Sci., 2008,7, 1025-1031
Article type
Paper

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

V. V. Borisova, L. A. Frank, S. V. Markova, L. P. Burakova and E. S. Vysotski, Photochem. Photobiol. Sci., 2008, 7, 1025
DOI: 10.1039/B807271J

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