Creation of a thermostable firefly luciferase with pH-insensitive luminescent color |
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Authors: | Kitayama Atsushi Yoshizaki Hiroaki Ohmiya Yoshihiro Ueda Hiroshi Nagamune Teruyuki |
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Institution: | Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Tokyo, Japan. |
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Abstract: | The thermal instability and pH-sensitive spectral property of firefly luciferase have hampered its use as a sensitive multicolor luminescent label or bioluminescent resonance energy transfer donor. With the intention of improving the thermostability of a previously found firefly Hotaria parvula luciferase mutant with minor pH-sensitive spectral change (V368A), further mutation (E356R) was introduced by taking a reportedly stabilized mutant of Photinus pyralis luciferase into account. The double mutant E356R/V368A showed significantly improved thermostability because > 90% activity remained after incubation for 1 h at 45 degrees C, with its specific activity being maintained. Unlike the wild type or V368A, E356R/V368A showed no change in the emission maximum of 568 nm even at pH 6.3, also implying a mutual relationship between thermostability and the proportion of yellow-green luminescent peak under acidic condition. |
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