Abstract
Objectives
Firefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study was aimed at obtaining mutant luciferase enzymes resistant to the sodium chloride inhibition.
Results
We first obtained two mutant luciferase enzymes whose inhibition were alleviated and determined the mutations to be Val288Ile and Glu488Val. Under medical dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride.
Conclusions
The mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The luciferase CR may be extensively useful in any bioassay which includes firefly luciferase and is employed in the presence of sodium chloride.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Acknowledgements
Arakawa of DKK-TOA Corporation and Dr. H. Funabashi of Hiroshima University for their helpful discussion.
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This study was funded by DKK-TOA Corporation.
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S.Y. and K.N. developed the original concept of this study. S.Y., K.N. and A.S. performed the experiments. A.K. discussed the experimental data and supervised writing of the manuscript. All authors and contributed to manuscript writing and approve of its contents.
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S.Y., K.N. and A.S. are employees of DKK-TOA Corporation. A.K. has no conflicts of interest.
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Yawata, S., Noda, K., Shimomura, A. et al. Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride. Biotechnol Lett 43, 1585–1594 (2021). https://doi.org/10.1007/s10529-021-03109-x
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DOI: https://doi.org/10.1007/s10529-021-03109-x