title: An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications.
abstract: Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega's luc2 for reporter and imaging applications.
authors.affiliation: Department of Chemistry, Connecticut College, New London, CT 06320, USA. Electronic address: brbra@conncoll.edu.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Branchini
authors.affiliation: Department of Chemistry, Connecticut College, New London, CT 06320, USA.
authors.forename: Tara L
authors.initials: TL
authors.lastname: Southworth
authors.affiliation: Department of Chemistry, Connecticut College, New London, CT 06320, USA.
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Fontaine
authors.affiliation: Department of Biology, Connecticut College, New London, CT 06320, USA.
authors.forename: Dawn
authors.initials: D
authors.lastname: Kohrt
authors.affiliation: Department of Chemistry, Connecticut College, New London, CT 06320, USA.
authors.forename: Munya
authors.initials: M
authors.lastname: Talukder
authors.affiliation: Department of Chemistry "G. Ciamician", University of Bologna, 40126 Bologna, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Michelini
authors.affiliation: Department of Chemistry "G. Ciamician", University of Bologna, 40126 Bologna, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Cevenini
authors.affiliation: Department of Chemistry "G. Ciamician", University of Bologna, 40126 Bologna, Italy.
authors.forename: Aldo
authors.initials: A
authors.lastname: Roda
authors.affiliation: Department of Biology, Connecticut College, New London, CT 06320, USA.
authors.forename: Martha J
authors.initials: MJ
authors.lastname: Grossel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004795:Enzyme Stability; D048888:Fireflies / Q000201:enzymology; D017930:Genes, Reporter / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D049409:Luciferases, Firefly / Q000235:genetics*; D008163:Luminescent Measurements; D057054:Molecular Imaging / Q000379:methods*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013696:Temperature
chemical_list: D011993:Recombinant Fusion Proteins; C485197:luciferase, Photinus; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly
keywords: ATP; Bioluminescence; Imaging; Luciferase; Reporter; luc2