title: Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay.
abstract: The recombinant coelenterazine-dependent luciferases (isoforms MLuc164 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 (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.
authors.affiliation: Photobiology Lab, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, 660036, Russia.
authors.forename: Vasilisa V
authors.initials: VV
authors.lastname: Borisova
authors.forename: Ludmila A
authors.initials: LA
authors.lastname: Frank
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Markova
authors.forename: Ludmila P
authors.initials: LP
authors.lastname: Burakova
authors.forename: Eugene S
authors.initials: ES
authors.lastname: Vysotski