Renilla luciferase

Fig. 15. Light emission from Renilla and Cypridina luciferin by the action of luciferases (Renilla luciferin 2-monooxygenase and Cypridina luciferin 2-monooxygenase)...

Fig. 3.3.1 Luminescence spectrum of coelenterazine catalyzed by the luciferase of the decapod Oplophorus in 15 mM Tris-HCl, pH 8.3, containing 50 mM NaCl (solid line). For comparison, the luminescence catalyzed by the luciferase of the anthozoan sea pansy Renilla is shown with dashed line (in 25 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl).

The product coelenteramide is not noticeably fluorescent in aqueous solutions, but is highly fluorescent in organic solvents and also when the compound is in the hydrophobic environment of a protein. When coelenterazine is luminesced in the presence of Oplophorus luciferase, the solution after luminescence (the spent solution) is not fluorescent, presumably due to the dissociation of coelenteramide from the luciferase that provided a hydrophobic environment at the time of light emission. An analogous situation exists in the bioluminescence system of Renilla (Hori et al., 1973). 

Anthozoa. Anthozoans are plant-shaped polyps, either solitary or colonial, completely lacking the medusoid stage. They are found along coastal waters and include the luminescent genera Renilla (the sea pansies), Cavernularia (the sea cactuses), and Ptilosarcus and Pennatula (the sea pens). Bioluminescent anthozoans emit light by a luciferin-luciferase reaction that involves coelenterazine as the... 

Renilla luciferase. The luciferase of Renilla reniformis has been purified and characterized by Karkhanis and Cormier (1971) and Matthews et al. (1977a). The purified luciferase has a molecular weight of 35,000, and catalyzes the luminescence reaction of coelenterazine. The luciferase-catalyzed luminescence is optimum at pH 7.4, at a temperature of 32°C, and in the presence of 0.5 M salt (such as NaCl or KC1). The luciferase has a specific activity of 1.8 x 1015 photons s"1mg"1, and a turnover number of 111/min. The luminescence spectrum shows a maximum at 480 nm. The absorbance A28O of a 0.1% luciferase solution is 2.1. The luciferase has a tendency to self-aggregate, forming higher molecular weight species of lower luminescence activities. 

The cDNA encoding the luciferase of Renilla reniformis has been obtained and expressed in Escherichia coli (Lorenz et al., 1991). The cDNA contained an open reading frame encoding a 314-amino acid sequence. The recombinant Renilla luciferase obtained had a molecular weight of 34,000, and showed an emission maximum at 480 nm in the luminescence reaction of coelenterazine, in good agreement with the data of natural Renilla luciferase. 

Quantum yield of luciferin. Various values of quantum yield have been reported for coelenterazine in the luminescence reaction catalyzed by Renilla luciferase 0.055 (Matthews et al., 1977a), 0.07 (Hart, et al., 1979), and 0.10-0.11 (with a recombinant form Inouye and Shimomura, 1997). The quantum yield is significantly increased in the presence of Renilla green fluorescent protein (GFP) see below. 

The spectra of the luminescence of coelenterazine catalyzed by recombinant Renilla luciferase in the presence and absence of Renilla GFP are shown in Fig. 4.6.3 (Lorenz et al., 1991). Note that the luminescence intensity at the emission peak is increased more than... 

Fig. 4.6.3 Bioluminescence emission spectra measured with coelenterazine plus 1 i.M Renilla luciferase in the absence (a) and presence (b) of 1 jlM Renilla GFP. From Lorenz et al., 1991.

Coelenterazine emits chemiluminescence when dissolved in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) containing a trace amount of base. It also emits bioluminescence in aqueous media in the presence of a coelenterazine luciferase, such as Renilla luciferase or Oplophorus luciferase. In both cases, the luminescence reactions require molecular oxygen. The capability of coelenterazine to produce luminescence is attributed to the presence of the imida-zopyrazinone structure in the molecule. 

There are many kinds of luminous organisms that utilize coelenterazine as their luciferin. These organisms possess luciferases to catalyze the luminescent oxidation of coelenterazine. Coelenterazine luciferases have been isolated from about 10 kinds of organisms, including the anthozoans Renilla and Ptilosarcus, the scyphozoan... 

All of the luciferases cause the emission of a bluish light when they catalyze the oxidation of coelenterazine. However, there are some marked differences between the decapod shrimp luciferases and the cnidarian luciferases (Matthews et al., 1977a,b). For example, the luminescence caused by the former (Amax about 452 nm) is bluer than that caused by the latter (7max 470-480 nm), and the optimum pH of the former, about 8.5, is significantly higher than that of the latter (Renilla, 7.4 Ptilosarcus, 7.0). The optimum temperature of the decapod shrimp luciferases (35°C) is higher than those of Ptilosarcus (23°C) and Renilla (32°C). 

Luciferase activity on e-coelenterazine. In the presence of Renilla luciferase, the luminescence intensity of e-coelenterazine is more than 5 times higher than that of coelenterazine under the same conditions... 

This luminous brittle star has been briefly studied recently (Mallefet and Shimomura, 2004, unpublished). The animal contained a high level of coelenterazine luciferase activity (4 x 1012 photons s-1g 1), which is comparable to those in the luminous antho-zoans such as the sea pansy Renilla and sea pen Ptilosarcus (Shimomura and Johnson, 1979b). There is no evidence for the presence of a photoprotein in this brittle star. Thus, the luminescence system of Amphiura filiformis is considered to be a coelenterazine-luciferase system, differing from that of Ophiopsila californica. The luciferase has a molecular weight of 23,000 on the basis of gel filtration on Superdex 200 Prep, and catalyzes the luminescence reaction of coelenterazine in the presence of oxygen the light emission (A.max 475 nm) is optimum at pH 7.2. 

Coelenterazine can be detected and measured with a coelenterazine luciferase, i.e. a luciferase specific to coelenterazine. As the coelenterazine luciferase, the luciferases from the sea pansy Renilla and the copepods Gaussia and Pleuromamma are commercially available. Certain kinds of decapod shrimps, such as Oplophoms and Heterocarpus, contain a large amount of luciferase, and the luciferases purified from them are most satisfactory for the assay of coelenterazine considering their high activities and high quantum yields. Even partially purified preparations of these luciferases are satisfactory for most measurements. The author routinely uses purified Oplophoms luciferase. 

Hart, R. C., Matthews, J. C., Hori, K., and Cormier, M. J. (1979). Renilla reniformis bioluminescence Luciferase-catalyzed production of nonradiating excited states from luciferin analogues and elucidation of the excited state species involved in energy transfer to Renilla green fluorescent protein. Biochemistry 18 2204-2210. 

Inouye, S., and Shimomura, O. (1997). The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate. Biochem. Biophys. Res. Commun. 233 349-353. 

Karkhanis, Y. D., and Cormier, M. J. (1971). Isolation and properties of Renilla reniformis luciferase, a low molecular weight energy conversion enzyme. Biochemistry 10 317-326. 

Kreiss, P., and Cormier, M. J. (1967). Inhibition of Renilla reniformis bioluminescence by light effects on luciferase and its substrates. Biochim. Biophys. Acta 141 181-183. 

Liu, J., O Kane, D. J., and Escher, A. (1997). Secretion of functional Renilla reniformis luciferase by mammalian cells. Gene 203 141-148. 

Lorenz, W. W., et al. (1996). Expression of the Renilla reniformis luciferase gene in mammalian cells./. Biolumin. Chemilumin. 11 31-37. 

Mayerhofer, R., Langridge, W. H. R., Cormier, M. J., and Szalay, A. A. (1995). Expression of recombinant Renilla luciferase in transgenic plants results in high levels of light emission. Plant. J. 7 1031-1038. 

The following procedure works well for the synthetic system developed by Sharp and colleagues (Doench et al., 2003). It uses a Renilla luciferase plasmid (based on pRL-TK, Promega, WI) harboring 4 concatenated, partially mismatched target sites for the artificial CXCR4 miR in its 3 UTR (p-RL-TK-4 sites). 

Prepare transfection mix A that contains 1 fig of Renilla luciferase target plasmid (e.g., p-R-luc-4 sites) and 100 /(I of optiMEM (Invitrogen, CA). This is enough transfection mixture for 1 well, and can be scaled up for multiple transfections. For cultures that are to receive miR duplex, add 2 nM (calculated for a final volume of 500 fiL for each well). 

Cells are lysed for Firefly and Renilla luciferase assays using the Dual-Luciferase Reporter Assay system (Promega), following the manufacturer s instructions. We use a multimode microplate reader with automatic injectors (FLUOROstar Optima from BMG Labtech, OfFenburg, Germany) for luminescence measurements. 

Co-transfection of an unrelated reference construct is a common practice to compensate for variation in transfection efficiency between cultures. In the systems described here, we use Renilla luciferase as a reporter for miR-mediated repression, and co-transfect Firefly luciferase as a reference for transfection efficiency. Renilla luciferase activity from each transfection is normalized to the corresponding Firefly luciferase measurement. Repression by a miR is then calculated by dividing the normalized Renilla luciferase activity without miR by the normalized Renilla luciferase activity in the presence of miR. 

As pDNA and mRNA transfection differ in both the timing of mRNA expression and the gross amount of mRNA delivered to the cell, it is important to identify a suitable time point to measure miR-mediated repression. We observe that at any time point after transfection, pDNA transfections have higher measurable levels of miR-mediated repression compared to mRNA transfections (Fig. 6.2C). This difference may, in part, reflect a time lag of active miR-protein-complex formation relative to the onset of translation of the transfected Renilla luciferase mRNA. For single time point experiments, we decided to measure miR-mediated repression in mRNA and DNA transfections at 16 and 24 h, respectively. 

The normalized luciferase activity is calculated by dividing the relative light units (RLU) of firefly luciferase activity-background luminescence by that of Renilla luciferase-background luminescence. Typically, the cell lysate background is around 200 to 300 RLU (similar to that of a buffer control) and about 104 to 105 and 5 to 20 x 103 RLU for firefly and Renilla luciferase luminescence, respectively. 

To quantify the amount of firefly and Renilla luciferase RNAs, total RNA is extracted from transfected neurons, reverse transcribed, and subjected to real-time PCR amplification using QuantiTech SYBR Green PCR mixture (Qiagen). 

Translation of Renilla luciferase mRNAs (luciferase synthesis)... 

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