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 ( 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.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) 1 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. 1-3), bacterial luciferase (for review, see Refs. 4 -6), the Ca 2ϩ -regulated photoprotein aequorin (for review, see Refs. 7-9), Renilla luciferase (for review, see Refs. 10 and 11), and GFP (for review, see . Although time course studies of gene expression have been determined by real time imaging with these reporter genes, intracellular assays present difficulties for continuous measurements in some cases. For instance, it is not easy to keep an intracellular concentration of luciferin at a constant level in the case of application of firefly and Renilla luciferases. In the case of FL, additional methods may be required to facilitate the passage of the substrate across the cell membrane. In addition, the intensity and stability of the bioluminescent response of FL in living cells are reported to be affected by concentrations of ATP, luciferin, and luciferin-luciferase complex (15, 16). Despite the fact that the GFP possesses excellent properties as a reporter, it has some short...

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The bioluminescence spectra from the Ca 2+ -regulated photoproteins aequorin (k max = 469 nm) and obelin (k max = 482 nm) differ because aequorin has an H-bond from its Tyr82 to the bound coelenteramide, not present in obelin at the corresponding Phe88. Substitutions of this Phe88 by Tyr, Trp, or His shifted the obelin bioluminescence to shorter wavelength with F88Y having k max = 453 nm. Removal of the H-bond by the substitution of Y82F in aequorin shifted its bioluminescence to k max = 501 nm. All mutants were stable with good activity and were expressible in mammalian cells, thereby demonstrating potential for monitoring multiple events in cells using multi-color detection.

Obelin from the hydroid Obelia longissima and aequorin are members of a subfamily of Ca(2+)-regulated photoproteins that is a part of the larger EF-hand calcium binding protein family. On the addition of Ca(2+), obelin generates a blue bioluminescence emission (lambda(max) = 485 nm) as the result of the oxidative decarboxylation of the bound substrate, coelenterazine. The W92F obelin mutant is noteworthy because of the unusually high speed with which it responds to sudden changes of [Ca(2+)] and because it emits violet light rather than blue due to a prominent band with lambda(max) = 405 nm. Increase of pH in the range from 5.5 to 8.5 and using D(2)O both diminish the contribution of the 405 nm band, indicating that excited state proton transfer is involved. Fluorescence model studies have suggested the origin of the 485 nm emission as the excited state of an anion of coelenteramide, the bioluminescence reaction product, and 405 nm from the excited neutral state. Assuming that the dimensions of the substrate binding cavity do not change during the excited state formation, a His22 residue within hydrogen bonding distance to the 6-(p-hydroxy)-phenyl group of the excited coelenteramide is a likely candidate for accepting the phenol proton to produce an ion-pair excited state, in support of recent suggestions for the bioluminescence emitting state. The proton transfer could be impeded by removal of the Trp92 H-bond, resulting in strong enhancement of a 405 nm band giving the violet color of bioluminescence. Comparative analysis of 3D structures of the wild-type (WT) and W92F obelins reveals that there are structural displacements of certain key Ca(2+)-ligating residues in the loops of the two C-terminal EF hands as well as clear differences in hydrogen bond networks in W92F. For instance, the hydrogen bond between the side-chain oxygen atom of Asp169 and the main-chain nitrogen of Arg112 binds together the incoming alpha-helix of loop III and the exiting alpha-helix of loop IV in WT, providing probably concerted changes in these EF hands on calcium binding. But this linkage is not found in W92F obelin. These differences apparently do not change the overall affinity to calcium of W92F obelin but may account for the kinetic differences between the WT and mutant obelins. From analysis of the hydrogen bond network in the coelenterazine binding cavity, it is proposed that the trigger for bioluminescence reaction in these Ca(2+)-regulated photoproteins may be a shift of the hydrogen bond donor-acceptor separations around the coelenterazine-2-hydroperoxy substrate, initiated by small spatial adjustment of the exiting alpha-helix of loop IV.