Fluorescent Proteins 101: Luciferases

For most people, the most familiar source of bioluminescence is the firefly. Along with charming nighttime displays, these insects have contributed an important tool to the scientific community: luciferases, which have become common genetic reporters and alternatives to fluorescent proteins.

Figure 1: Firefly, from Wikimedia Commons.

The North American firefly, Photinus pyralis, catalyzes a chemical reaction in its abdomen to produce yellow-green light (Figure 2). Despite the title of this post, this reaction is not fluorescence! Chemiluminescence, which includes all bioluminescence, is the production of light by a chemical reaction. In contrast, fluorescence occurs when a molecule is excited by a high-energy photon and re-emits a lower-energy photon; no chemical reaction takes place.

Figure 2: Simplified chemical reaction catalyzed by luciferase, releasing light. Created with BioRender.com.

Bioluminescent reporters require a chemical substrate (and sometimes cofactors) to be provided and are generally dimmer than fluorescent proteins. On the other hand, they don’t require an excitation light source (usually a laser in fluorescence assays), which means luminescence assays have lower background levels and improved signal sensitivity. Another advantage: luciferase reporters aren’t susceptible to photobleaching — they’ll keep emitting light as long as their substrate is available. Table 1 compares some general advantages and disadvantages of each approach.

Table 1: Properties of luminescence versus fluorescence

	Luminescence	Fluorescence
Source of light	Chemical reaction	High-energy photons
Kinetics of photon generation	Slower	Faster
Cofactors/substrates	Required	Not required
Signal strength	Lower	Higher
Sensitivity	Higher	Lower
Background	Lower	Higher
Photobleaching/phototoxicity	Not susceptible	Susceptible
Subcellular imaging	Improving	Well-established
High-throughput assays	Available	Well-established

Natural and engineered luciferases

Although firefly luciferase was the first bioluminescent reporter to be cloned and remains popular, other luciferases have been identified in the sea pansy (Renilla reniformis), click beetle (Pyrophorus plagiophthalamus), deep-sea copepod (Gaussia princeps), Japanese firefly (Luciola cruciata), and more. These variant proteins offer different kinetics, substrate requirements, and photon emission wavelengths.

In addition, researchers have engineered a variety of improved luciferases. NanoLuc® Luciferase is the best known: it’s derived from the small (19 kDa) luminous shrimp (Oplophorus gracilirostris) luciferase and engineered to be extra bright. Other luciferases have been modified to change the wavelength of light they emit or to optimize their expression in mammalian systems. Our luciferase collection page has a detailed list of luciferase options.

Table 2: Properties of luciferase enzymes

Name	Natural or engineered	Peak emission (nm)	Size (kDa)	Substrate	Cofactors	Secreted
North American firefly (Photinus pyralis)	Natural or codon-optimized	562	61	Luciferin	ATP	No
Sea pansy (Renilla reniformis)	Natural	481	36	Coelenterazine	None	No
Click Beetle Red (CBR) (Pyrophorus plagiophthalamus)	Natural or codon-optimized	613 (luciferin), 730–743 (analogs)	64	Luciferin or naphthyl-luciferin analogs	ATP	No
Deep sea copepod (Gaussia princeps)	Natural	480	20	Coelenterazine	None	Yes
Japanese firefly (Luciola cruciata)	Natural	562	64	Luciferin	ATP	No
Luminous shrimp (Oplophorus gracilirostris)	Natural	455	19	Coelenterazine	None	Yes
NanoLuc® (derived from Oplophorus gracilirostris)	Engineered	462	19	Furimazine	None	Optional
AkaLuc (derived from Photinus pyralis)	Engineered	677	61	AkaLumine	ATP	No

Common uses for plasmids expressing luciferase

Luciferase reporter assays for monitoring gene regulation

Since firefly luciferase was first cloned in 1985, luciferase assays have become a gold standard in gene expression analysis to investigate the effect of regulatory elements — such as promoters, enhancers, or untranslated regions — on gene expression. The regulatory element is cloned upstream of the luciferase gene, and expression of luciferase is measured via the light produced. A similar approach can be used to analyze cell signaling pathways or miRNA knockdown.

BRET for the best of both (fluorescent and luminescent) worlds

Forster Resonance Energy Transfer (FRET) is the transfer of energy from an excited donor fluorophore to an acceptor fluorophore, which then emits it as light. Bioluminescence Resonance Energy Transfer (BRET) works the same way, except that energy is transferred from a luminescence reaction to a nearby fluorophore, which emits light. No excitation illumination needed — just the luminescent substrate. This approach preserves the advantages of luminescent assays (low background, no photobleaching) but provides flexibility by pairing the luciferase with a variety of fluorescent proteins that produce different colors or have other advantageous properties. This approach can improve live-cell imaging by avoiding autofluorescence and phototoxicity and even allowing multiple proteins to be visualized simultaneously.

Complementation systems for examining protein-protein interactions

Split luciferases, such as NanoBiT, are expressed in two subunits and only catalyze a luminescent reaction when they come together. By fusing each luciferase subunit to other proteins, researchers can test whether those proteins interact, since light is only produced if a binding interaction brings the two luciferase components together.

A similar approach can test protein-protein interactions using BRET. A luciferase and a fluorescent protein are each fused to one of the proteins being tested for interaction. If no interaction occurs, the luciferase emits its characteristic light. However, if a protein interaction brings the fluorescent protein close enough, energy is transferred by BRET and the fluorescent protein emits light of a different color instead. This approach has even been used in biosensors where BRET reports on the change in conformation of a single protein.

Considerations for luciferase assays

Luciferases can only produce light in the presence of a compatible substrate, which varies depending on the specific luciferase you choose. Many luciferase assays require cell lysis as the most efficient means to disrupt the cell membrane and deliver the substrate; however, secreted luciferase or alternative substrates can permit measurements of luminescence from live cells. Luciferase variants with incompatible substrates can also allow multiplexing with experiments that supply both substrates and measure luciferase output at different wavelengths.

Addgene provides empty backbones with the luciferase gene, a variety of expression vectors, and literally thousands of reporter constructs with a gene of interest already inserted. In addition, we have recently partnered with Promega to offer their Promega Plasmid Collection, including tools using NanoLuc®, NanoBiT, and more.