Development of a pH-Tolerant Thermostable Photinus pyralis Luciferase for Brighter In Vivo Imaging
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Measurement of Michaelis Constants for ATP and Mg2+ in Bioluminescence Reaction of Luciferase by a Home-Made Luminometer
Effects of ATP and Mg2+ concentrations on bioluminescence reaction of luciferase (Photinus pyralis) were investigated by home-made luminometer. The Michaelis constants of the enzyme for ATP and Mg2+ obtained from the Lineweaver-Burk graph, were 61.9 mM ±3.3 mM and 251.6mM ± 39.0mM...
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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 ...
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SpyTag can spontaneously form a covalent isopeptide bond with its protein partner SpyCatcher. Firefly luciferase from Photinus pyralis was cyclized in vivo by fusing SpyCatcher at the N terminus and SpyTag at the C terminus. Circular LUC was more thermostable and alkali-tolerant than the wild type, without compromising the specific activity. Structural analysis indicated that the cyclized LUC i...
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Error-prone PCR was used to isolate Photinus pyralis luciferase mutants producing bright light in the red-orange region of the spectrum. All mutations were clustered in the beta5-alpha10-beta6 region of N-terminal subdomain B and appear to affect bioluminescence color by modulating the position of the Ser314-Leu319 mobile loop with respect to the putative active site. Two red variants (Q283R an...
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Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabd...
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