Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.
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Research Article|
June 28 2006
Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance
G. H. Erica Law;
G. H. Erica Law
1
* Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, U.K.
1 To whom correspondence should be addressed (email E.Law@biotech.cam.ac.uk).
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Olga A. Gandelman;
Olga A. Gandelman
* Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, U.K.
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Laurence C. Tisi;
Laurence C. Tisi
* Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, U.K.
† Lumora Ltd, c/o Institute of Biotechnology, Tennis Court Road, Cambridge CB2 1QT, U.K.
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Christopher R. Lowe;
Christopher R. Lowe
* Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, U.K.
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James A. H. Murray
James A. H. Murray
* Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, U.K.
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Publisher: Portland Press Ltd
Received:
November 17 2005
Revision Received:
March 09 2006
Accepted:
March 21 2006
Accepted Manuscript online:
March 21 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 397 (2): 305–312.
Article history
Received:
November 17 2005
Revision Received:
March 09 2006
Accepted:
March 21 2006
Accepted Manuscript online:
March 21 2006
Citation
G. H. Erica Law, Olga A. Gandelman, Laurence C. Tisi, Christopher R. Lowe, James A. H. Murray; Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance. Biochem J 15 July 2006; 397 (2): 305–312. doi: https://doi.org/10.1042/BJ20051847
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