Creation of a thermostable firefly luciferase with pH-insensitive luminescent color.
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.
Kitayama
Atsushi
A
Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Tokyo, Japan.
Yoshizaki
Hiroaki
H
Ohmiya
Yoshihiro
Y
Ueda
Hiroshi
H
Nagamune
Teruyuki
T
D000818:Animals; D001483:Base Sequence; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D004247:DNA / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D049449:Luminescence; D016297:Mutagenesis, Site-Directed; D018462:Photobiology
D004247:DNA; D008156:Luciferases