Luciferase reporter protein

Here we describe the construction and use of adenoviruses to express lucif-erase- or GFP-fusion proteins for high-throughput cell-based screens. We constructed adenoviruses that express a cyclin Bl-luciferase reporter protein or unmodified luciferase. This enabled us to screen for small molecules that specifically up-regulated levels of the cyclin B 1-luciferase fusion protein without affecting luciferase. A similar approach was used to construct adenoviruses that express cyclin A-GFP or cyclin Bl-GFP fusion proteins. These viruses were used in an imaging-based screen to identify small molecules that specifically upregulated these proteins or altered their subcellular localization. 

Fig. l.A sample plate from a screen to identify small molecules that upregulate the expression of a cyclin-luciferase reporter protein. HeLa cells were infected with an adenovirus expressing cyclin-luciferase, as described in the methods section. The graph shows the activity of all wells from a single 384-well plate. Several compounds that upregulate cyclin-luciferase expression were identified. 

Indoprofen In a high-throughput screen of ca. 47,000 compound library, authors [43] have converged on indoprofen as a molecule that enhanced production of an SMN2- vs. SMN1 -luciferase reporter protein. Indoprofen, a NSAID and cyclooxygenase (COX) inhibitor, afforded a 13% enhancement of Smn protein and a fivefold increase in the number of nuclear gems in fibroblasts from SMA patients. Notably, other tested NSAIDs or COX inhibitors were inactive in the assay. 

Molecular weight. The molecular weight of C. hilgendorfii luciferase reported in the past varies considerably across a range of 50,000-80,000 (Chase and Langridge, 1960 Shimomura etal., 1961, 1969 Tsuji and Sowinski, 1961 Tsuji et al., 1974) it appears most likely to be 60,000-70,000. The luciferase is an acidic protein with an isoelectric point of 4.35 (Shimomura et al., 1961). The absorption spectrum of luciferase is that of a simple protein without any prosthetic group, showing a peak at 280 nm. Absorbance value at 280 nm of a 0.1% luciferase solution is approximately 0.96 (Shimomura etal., 1969). 

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. 

Hardin We can rescue locomotor activity rhythms with a static per RNA level. What is important here are the protein levels. The other answer we need to find for this story is looking in antennae with anti-PER antibodies to see whether the protein is cychng. The other experiment is to use a luciferase reporter to see whether there are rhythms in PER expression. 

KeyWords Adenovirus reporter protein luciferase green fluorescent protein high-throughput cell-based screening. 

Plasmid DNA used contained the luciferase reporter gene (for in vitro experiments) including the CMV sequence. Plasmids present the normalized criteria of quality endotoxin level <20 EU/mg, supercoiled DNA >90%, E. coli-derived DNA contaminant <5%, RNA contaminant <5%, and protein contamination <1%. Such a plasmid preparation has been described (16). 

Fig. 2. Transfection of the cationic and anionic formulations using a plasmid encoding for the luciferase reporter gene in presence and absence of bafilomycin. The Y axis represents the level of luciferase expression per protein (p.g), mean values+SD. The background level of the untreated cells was removed for each sample...

The most common reporter proteins (coded by reporter genes) employed in biosensor systems include firefly luciferase (lucFF gene) (Justus and Thomas, 1999), bacterial luciferase (luxCDABE gene) (Belkin et al., 1996 Vollmer et al., 1997 Min et al.,... 

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