Generation of thermostable monomeric luciferases from Photorhabdus luminescens |
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Authors: | Westerlund-Karlsson Annette Saviranta Petri Karp Matti |
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Institution: | Department of Biochemistry and Pharmacy, Abo Akademi University, FIN-20520, Turku, Finland. |
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Abstract: | Bacterial luciferases and the genes encoding these light-emitting enzymes have an increasing number of applications in biological sciences. Temperature lability and the heterodimeric nature of these luciferases have been the major obstacles for their widespread use, for instance, as genetic reporters. Escherichia coli expressing wild-type Photorhabdus luminescens luciferase was found to produce eight times more light than the corresponding Vibrio harveyi luciferase clone in vivo at 37 degrees C. Three monomeric luciferases were created by translationally fusing the two genes encoding luxA and luxB proteins of P. luminescens. These clones were equally active in producing light in vivo when cultivated at 37 degrees C compared to cultivation at 30 degrees C. The fusion containing the longest linker showed the highest activity. In vitro, the monomeric luciferases were less active having at best 20% of activity of the wild-type enzyme due to the partial formation of insoluble aggregates. The results suggest that P. luminescens luciferase and monomeric derivatives thereof should be more suitable than the corresponding V. harveyi enzyme to be used as reporters in cell types which need cultivation at elevated temperatures. |
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Keywords: | Recombinant DNA Bioluminescence Vibrio harveyi luxAB Translational fusion Peptide linker |
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