Luciferase

Luciferase, distinct from a photoprotein, is a generic term for a class of oxidative enzymes emitting bioluminescence, which are widely employed in biotechnology for microscopy and serving as reporter genes for many of the same applications as fluorescent proteins. However, luciferase is dissimilar to fluorescent proteins that require an external light source, it requires the addition of the consumable substrate luciferin. Luciferases from different animal species exhibit inherent variability in light emission, allowing for a combined use of two or more luciferase enzymes in multiplex analyses, such as cell viability, in vivo imaging, and single and dual-spectral luciferase reporter assays.

What’s more, luciferase reactions are considered as containing either flash or glow kinetics, thus exerting specific detecting sensitivities and durable emission times to tolerate different experimental purposes. Luciferase has a unique advantage achieving high signal-to-noise ratios since most cells are not luminescent, which makes it a particularly versatile reporter protein used in expression studies to determine promoter activities with high temporal resolution and reproducibility without sampling or perturbing the system under investigation. However, the requirement for molecular oxygen to induce luminescence is a limitation of luciferase reporters, which essentially precludes them to be applied as reporters in obligate anaerobic bacteria. The implication of energy ATP and reduced flavin mononucleotide (FMNH₂) in reactions catalyzed by firefly and bacterial luciferases might also complicate the measurement or analysis of promoter activity in metabolic studies.

Classic Examples

Various organisms manage their light production with different luciferases through a variety of light-emitting reactions. Most studied luciferases are present in animals, including fireflies, and marine animals such as jellyfish, copepods, and the sea pansy, while luciferases could be also found in luminous fungi, along with organisms in other kingdoms like luminous bacteria. Luciferases from firefly and bacteria are commonly utilized.

Firefly luciferase takes luciferin as a substrate to oxidize it into oxyluciferin utilizing molecular oxygen and ATP, and liberates light at 560 nm. The luciferases of fireflies that contain over 2000 species are so miscellaneous to be widespreadly employed in molecular phylogeny, and the oxygen required in fireflies is provisioned through a tube in abdomen called abdominal trachea. The luciferase of Photinus pyralis firefly has been well-studied, which has an optimum activity at pH 7.8.

Bacterial luciferase encoded by the luxCDABE operon usually from Vibrio harveyi, Vibrio fischeri, or Photorhabdus luminescens, could catalyze the oxidation of reduced flavin mononucleotide and myristyl aldehyde to myristic acid and flavin mononucleotide (FMN), respectively, and liberate light with a wavelength of 490 nm. Bacterial bioluminescence is observed in Vibrio fischeri, Vibrio haweyi, and Vibrio harveyi. Some bioluminescent bacteria emit light by utilizing 'antenna' to accept the energy from the primary excited state of the luciferase, which leads to an excited lulnazine chromophore that generates emission light with a shorter wavelength, while in others a yellow fluorescent protein with FMN is treated as the chromophore that emits light with a red-shift compared to that from luciferase.

The type selection of luciferase to use should take into consideration that the firefly enzyme involves an exogenous addition of the decanal substrate, whereas the bacterial luciferase can be engineered by inclusion of luxCDE in the operon to produce the substrate endogenously. The suitability of luciferase for the determination of promoter activity in real time depends on the instability of the enzyme.

Mechanism of Reaction

All luciferases are categorized into oxidoreductase group, signifying that they act on single donors accompanied with the incorporation of molecular oxygen. Since luciferases possess diversity of unrelated species, no unifying mechanism has been elaborated until now, while many mechanisms reside in the combination of luciferase and luciferin. Moreover, to date, all of the characterized luciferase-luciferin reactions require molecular oxygen at some stage.

The oxidative process catalyzed by bacterial luciferase has been verified in a following procedure:

where the reduced flavin mononucleotide conducts the oxidization of a long-chain aliphatic aldehyde to an aliphatic carboxylic acid. An excited hydroxyl flavin intermediate formed in the reaction is further dehydrated to give FMN emitting blue-green light. Nearly all of the energy input into the reaction is transformed into light with a conversion efficiency of 80% to 90%.

Applications

Genetic engineering is usually applied by lab to produce luciferases for a number of purposes. A few of organisms like mice, silkworms, and potatoes are intercalated with luciferase genes to obtain the protein. The action of luciferase on the appropriate luciferin substrate during the oxidoreduction process could emit light that can be sensed by light sensitive apparatus such as modified optical microscopes or luminometer, which makes observation of biological processes available. Since bioluminescence of luciferase does not need light excitation, autofluorescence and virtually background-free fluorescence are ignorable, and therefore sample as little as 0.02pg can still be precisely measured through a standard scintillation counter.

Luciferase in biological research commonly functions as a reporter to evaluate the transcriptional efficacy in cells transfected with luciferase gene under the control of a promoter of concern. Proluminescent molecules could be applied in the detection of enzyme activity in joint or two-step luciferase assays after being converted into luciferin through a specific enzyme. Luciferase as a heat-sensitive protein could also be used in protein denaturation investigation to test the protective potentialities of heat shock proteins.

In the evaluation of cell viability or kinase activity, luciferase can also be utilized to determine cellular level of ATP by acting as an ATP sensor protein through biotinylation which immobilizes luciferase on the cell-surface by binding wtih a streptavidin-biotin complex. This endows luciferase the ability of monitoring the efflux of ATP from the cell, which thus effectively demonstrates the real-time release of ATP through bioluminescence. The luminescence intensity as a reflection of the sensitivity for ATP detection can be enhanced by changing the sequence of certain amino acid residues in the protein.

Different types of cells engineered to express a luciferase could be non-invasively visualized inside a live animal through a sensitive charge-couple device camera, which could trace the whole procedure of tumorigenesis and the response of tumors to therapies in animal models. In addition to the amount of luciferase, the signal intensity measured by in vivo imaging may also connect with other various factors, such as blood flow, intracellular pH, D-luciferin absorption, available co-factors, cell membrane permeability, transparency of overlying tissue and so on.

Reference

1. Baldwin T O, Christopher J A, Raushel F M, Sinclair J F, Ziegler M M, Fisher A J, Rayment I. Structure of bacterial luciferase. Curr Opin Struc Biol, 1995, 5(6):798–809.