Gaussia luciferase for bioluminescence tumor monitoring in comparison with firefly luciferase

Gaussia luciferase (Gluc) is a secreted reporter, and its expression in living animals can be assessed by in vivo bioluminescence imaging (BLI) or blood assays. We characterized Gluc as an in vivo reporter in comparison with firefly luciferase (Fluc). Mice were inoculated subcutaneously with tumor cells expressing both Fluc and Gluc and underwent Fluc BLI, Gluc BLI, blood assays of Gluc activity, and caliper measurement. In Gluc BLI, the signal from the tumor peaked immediately and then decreased rapidly. In the longitudinal monitoring, all measures indicated an increase in tumor burden early after cell inoculation. However, the increase reached plateaus in Gluc BLI and Fluc BLI despite a continuous increase in the caliper measurement and Gluc blood assay. Significant correlations were found between the measures, and the correlation between the blood signal and caliper volume was especially high. Gluc allows tumor monitoring in mice and should be applicable to dual-reporter assessment in combination with Fluc. The Gluc blood assay appears to provide a reliable indicator of viable tumor burden, and the combination of a blood assay and in vivo BLI using Gluc should be promising for quantifying and localizing the tumors.     

Flavin binding by bacterial luciferase: Affinity chromatography of bacterial luciferase

Immobilized FMN covalently attached to Sepharose-6B-hexanoate binds bacterial luciferase. Immobilized flavin is also effective in its reduced form as a substrate in the light emitting reaction catalyzed by luciferase.     

Resveratrol inhibits firefly luciferase

The potential therapeutic value of resveratrol in age-related disease settings including cancer, diabetes, and Alzheimer's has emerged from a rapidly growing body of experimental evidence. Protection from oxidative stress appears to be a common feature of resveratrol that may be mediated through SirT1, though more specific molecular mechanisms by which resveratrol mediates its effects remain unclear. This has prompted an upsurge in cell-based mechanistic studies, often incorporating reporter assays for pathway elucidation in response to resveratrol treatment. Here, we report that resveratrol potently inhibits firefly luciferase with a K(i) value of 2microM, and caution that this confounding element may lead to compromised data interpretation.     

Structure of bacterial luciferase

The generation of light by living organisms such as fireflies, glow-worms, mushrooms, fish, or bacteria growing on decaying materials has been a subject of fascination throughout the ages, partly because it occurs without the need for high temperatures. The chemistry behind the numerous bioluminescent systems is quite varied, and the enzymes that catalyze the reactions, the luciferases, are a large and evolutionarily diverse group. The structure of the best understood of these intriguing enzymes, bacterial luciferase, has recently been determined, allowing discussion of features of the protein in structural terms for the first time.     

Luminous bacteria synthesize luciferase anaerobically

Four species of luminous bacteria, Photobacterium phosphoreum, P. leiognathi, P. fischeri and Beneckea harveyi (two strains of each), were shown to synthesize luciferase anaerobically. One of these, P. phosphoreum, produced as much luciferase anaerobically as it did aerobically, and all four species were found to grow almost equally rapidly under the two sets of conditions. Previous work with B. harveyi and P. fischeri had shown that aerobic luciferase synthesis can proceed only after an inhibitor in the complex medium has been removed and a species-specific autoinducer secreted. All strains tested also removed the inhibitor and secreted an autoinducer anaerobically. The small amount of luciferase produced anaerobically by some strains is thus apparently not due either to lack of removal of inhibitor or to insufficient production of autoinducer but may involve an oxygen-dependent control mechanism.Abbreviations LU light units - OD optical density     

Nucleoside triphosphate specificity of firefly luciferase

Twelve naturally occurring nucleoside triphosphates have been examined as substrates and inhibitors of the light-producing reaction of firefly luciferase. Deoxyadenosine 5'-triphosphate was 1.7% as effective relative to ATP as a substrate, whereas all others tested were less than 0.1% as effective as ATP. At concentrations normally present in mammalian cell extracts no interference with ATP measurements results from these nucleotides.     

Intermediates in the bacterial luciferase reaction

Rapid formation of an unstable, intermediary state of an enzyme complex, which is obligatory in the bacterial luciferase reaction, was observed on aerobic oxidation of the luciferase-FMNH₂ complex. The rate of decay of this intermediate in the absence of aldehyde was measured. The value obtained coincided with that estimated from the decay of the peak intensity of luminescence observed on addition of aldehyde at intervals after mixing the luciferase-FMNH₂ complex with O₂. A sequential mechanism of the reaction of bacterial luciferase is discussed.     

The construction of a glucose-sensing luciferase

A novel luminescence-based glucose-sensing molecule was created by combining a galactose-/glucose-binding protein (GGBP) with luciferase. The glucose-sensing luciferase (GlcLuc) was constructed using a GGBP fused with a large domain and a small domain of Firefly luciferase (Lluc and Sluc). The luminescence intensity-based analysis with E. coli recombinant protein showed that the GlcLuc had luciferase activity in glucose or galactose in a concentration-dependent manner (K_d = 3.9 μM for glucose and 11 μM for galactose), and that the increase in the activity saturated within one minute after the injection of the ligands. These results indicated that the conformation change of the GGBP moiety following the ligand binding effectively induced the reconstitution of the GGBP-fused split luciferase. The Asp459Asn mutation, which was expected to lead to a glucose specific binding ability, was then introduced into the GlcLuc. The GlcLuc mutant showed the luciferase activity increasing only with the increase of glucose concentration, but not with that of galactose. Our results demonstrate that the GGBP fused with a split luciferase, which is reconstituted rapidly and specifically in the presence of glucose, provides a novel glucose-sensing system based on luminescence and may also contribute to the construction of luminescence-based sensing molecules for other substrates using other PBPs.     

Firefly luciferase: Purification and immobilization

The state of the art of firefly luciferase research is reviewed with special emphasis on its purification and immobilization. The notion of bioluminescence and its role in APT monitoring is described. The need to purify luciferase and the advantages of immobilization are discussed. An insight into the existing methods of luciferase purification and immobilization is given. The scope of the bioluminescent assay is underlined.