Cloning and Expression of cDNA for a Luciferase from the Marine Copepod Metridia longa

Abstract

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 (λmax = 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. 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 (λmax = 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. Continuous monitoring of dynamic changes in gene expression from living cells in response to various stimuli provides important information about cell physiology. For this purpose bioluminescent and fluorescent reporters have been introduced as tools for sensitive and convenient monitoring of gene expression. A cDNA encoding a bioluminescent or fluorescent reporter such as a luciferase or green fluorescent protein (GFP) 1The abbreviations used are: GFPgreen fluorescent proteinFLfirefly luciferaseCHOChinese hamster ovaryMLucMetridia luciferaseIPTGisopropyl-β-d-thiogalactopyranosideNPY2human neuropeptide Y receptor 2TricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is fused to the promoter region of the target gene, and the construct is transfected to mammalian cells. The gene expression is monitored simply by measuring light emitted through an enzymatic reaction or fluorescence. To date several bioluminescent proteins have been widely and successfully used for optical monitoring of gene expression in living cells: firefly luciferase (FL) (for review, see Refs. 1Millar A.J. Short S.R. Chua N.H. Kay S.A. Plant Cell. 1992; 4: 1075-1087Google Scholar, 2Ando S. De Amicis F. Rago V. Carpino A. Maggiolini M. Panno M. Lanzino M. Mol. Cell. Endocrinol. 2002; 193: 121-128Google Scholar, 3Hough R.B. Avivi A. Davis J. Joel A. Nevo E. Piatigorsky J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8145-8150Google Scholar), bacterial luciferase (for review, see Refs. 4Kondo T. Strayer C.A. Kulkarni R.D. Taylor