title: Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications.
abstract: Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. Here, we present studies leading to the production of a set of red- and green-emitting luciferase mutants with bioluminescent properties suitable for expanding the use of the P. pyralis system to dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Using a combination of mutagenesis methods, we determined that the Ser284Thr mutation was sufficient to create an excellent red-emitting luciferase with a bioluminescence maximum of 615 nm, a narrow emission bandwidth, and favorable kinetic properties. Also, we developed a luciferase, containing the changes Val241Ile, Gly246Ala, and Phe250Ser, whose emission maximum was blue-shifted to 549 nm, providing a set of enzymes whose bioluminescence maxima were separated by 66 nm. Model studies demonstrated that in assays using a set of optical filters, the luciferases could be detected at the attomole level and seven orders of magnitude higher. In addition, in the presence of the Ser284Thr enzyme serving as a control, green light emission could be measured over a 10,000-fold range. The results presented here with the P. pyralis mutants provide evidence that simultaneous multiple analyte assay development is feasible with these novel proteins that require only a single substrate.
authors.affiliation: Department of Chemistry, Connecticut College, New London, CT 06320, USA. brbra@conncoll.edu
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Branchini
authors.forename: Tara L
authors.initials: TL
authors.lastname: Southworth
authors.forename: Neelum F
authors.initials: NF
authors.lastname: Khattak
authors.forename: Elisa
authors.initials: E
authors.lastname: Michelini
authors.forename: Aldo
authors.initials: A
authors.lastname: Roda
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D048888:Fireflies / Q000201:enzymology* / Q000235:genetics; D049454:Firefly Luciferin / Q000737:chemistry; D017930:Genes, Reporter; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods; D017354:Point Mutation
chemical_list: D049454:Firefly Luciferin; D049409:Luciferases, Firefly