Mutational optimization of the coelenterazine-dependent luciferase from Renilla

Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesi...

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Bibliographic Details
Published in:Plant methods 2008-09, Vol.4 (1), p.23-23
Main Authors: Woo, Jongchan, von Arnim, Albrecht G
Format: Article
Language:English
Subjects:
Anthozoa
Luciferase
Methodology
Physiological aspects
Properties
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Summary:Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesis of active site residue Pro220 extended the half-life of photon emission, yielding brighter luminescence in E. coli. Random mutagenesis uncovered two new mutations that stabilized and increased photon emission in vivo and in vitro, while ameliorating substrate inhibition. Further amended with a previously identified mutation, a new triple mutant showed a threefold improved kcat, as well as elevated luminescence in Arabidopsis. This advances the utility of RLUC as a reporter protein, biosensor, or resonance energy donor.
ISSN:1746-4811
1746-4811
DOI:10.1186/1746-4811-4-23