Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme.
Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light (lambda(max) = 480 nm). Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA encoding the Metridia luciferase. The cDNA is an 897-bp fragment with a 656-bp open reading frame, which encodes a 219-amino acid polypeptide with a molecular weight of 23,885. The polypeptide contains an N-terminal signal peptide of 17 amino acid residues for secretion. On expression of the Metridia luciferase gene in mammalian Chinese hamster ovary cells the luciferase is detected in the culture medium confirming the existence of a naturally occurring signal peptide for secretion in the cloned luciferase. The novel secreted luciferase was tested in a practical assay application in which the activity of A2a and NPY2 G-protein-coupled receptors was detected. These results clearly suggest that the secreted Metridia luciferase is well suited as a reporter for monitoring gene expression and, in particular, for the development of novel ultrahigh throughput screening technologies.
Markova
Svetlana V
SV
Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences Siberian Branch, Krasnoyarsk 660036, Russia.
Golz
Stefan
S
Frank
Ludmila A
LA
Kalthof
Bernd
B
Vysotski
Eugene S
ES
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003445:Crustacea / Q000201:enzymology / Q000235:genetics*; D003470:Culture Media / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D021382:Protein Sorting Signals; D013379:Substrate Specificity; D013997:Time Factors
D003470:Culture Media; D018076:DNA, Complementary; D010455:Peptides; D021382:Protein Sorting Signals; D000242:Cyclic AMP; D008156:Luciferases