title: The smallest natural high-active luciferase: cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa.
abstract: Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 SS bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of ∼3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging.
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk, Russia; Chair of Biophysics, Siberian Federal University, Krasnoyarsk, Russia.
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Markova
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk, Russia; Chair of Biophysics, Siberian Federal University, Krasnoyarsk, Russia.
authors.forename: Marina D
authors.initials: MD
authors.lastname: Larionova
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk, Russia.
authors.forename: Ludmila P
authors.initials: LP
authors.lastname: Burakova
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk, Russia. Electronic address: eugene.vysotski@gmail.com.
authors.forename: Eugene S
authors.initials: ES
authors.lastname: Vysotski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D033342:Copepoda / Q000201:enzymology*; D057809:HEK293 Cells; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D061987:Sf9 Cells; D013997:Time Factors
chemical_list: D008156:Luciferases
keywords: Bioluminescence; Coelenterazine; Copepod luciferase; Mammalian expression; Real-time imaging