title: Removal of twelve C-terminal amino acids from firefly luciferase abolishes activity.
abstract: cDNA coding for the luciferase in the firefly Photinus pyralis was cloned using pcDV1 primer and Honjo linker containing SP6 RNA polymerase promoter. This enabled conditions to be established to produce mRNA, capped with m7 GpppG, in vitro and then translated to form light emitting protein. Full length recombinant luciferase produced by in vitro translation, was fully active, had the same isoelectric focusing point as the native enzyme and produced a similar, yellow emission. Removal of the coding sequence for the last 12 amino acids at the C terminus, containing the peroxisome signal peptide, by polymerase chain reaction resulted in greater than or equal to 99% loss in activity of the protein formed from mRNA in vitro. This has important implications for using this luciferase as an indicator or reporter gene in eukaryotic cells, and for identifying the active centre of the enzyme.
authors.affiliation: Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, U.K.
authors.forename: G
authors.initials: G
authors.lastname: Sala-Newby
authors.forename: N
authors.initials: N
authors.lastname: Kalsheker
authors.forename: A K
authors.initials: AK
authors.lastname: Campbell
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D014644:Genetic Variation; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
chemical_list: D015345:Oligonucleotide Probes; D008156:Luciferases