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Biotechnological Advances in Luciferase Enzymes

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Chapter
Biotechnological Advances in
Luciferase Enzymes
AndrewKirkpatrick, TingtingXu, StevenRipp, GarySayler
and DanClose
Abstract
This chapter explores the history of the bioengineering advances that have
been applied to common luciferase enzymes and the improvements that have been
accomplished by this work. The primary focus is placed on firefly luciferase (FLuc),
Gaussia luciferase (GLuc), Renilla luciferase (RLuc), Oplophorus luciferase (OLuc;
NanoLuc), and bacterial luciferase (Lux). Beginning with the cloning and exog-
enous expression of each enzyme, their step-wise modifications are presented and
the new capabilities endowed by each incremental advancement are highlighted.
Using the historical basis of this information, the chapter concludes with a pro-
spective on the overall impact these advances have had on scientific research and
provides an outlook on what capabilities future advances could unlock.
Keywords: firefly luciferase (FLuc), Gaussia luciferase (GLuc),
Renilla luciferase (RLuc), Oplophorus luciferase (OLuc; NanoLuc),
bacterial luciferase (Lux), biotechnology
. Introduction
. Historical perspective on the discovery of luciferase enzymes
The bioluminescent phenotype, which is spread across a variety of different
insects, bacteria, fungi, and marine animals, has intrigued mankind since before
the dawn of the modern scientific era [1]. The discovery that proteins, which would
come to be known as luciferases, were responsible for bioluminescent production
can be traced to early experiments by Raphael Dubois, who was able to produce
bioluminescence in situ by mixing the contents of click beetle abdomens in cold
water and extracting the components required for light production [2]. However, it
was not until the late 1940s that the first luciferase protein was successfully purified
from fireflies [3]. Around that same time, bacterial luciferase was elucidated and
successfully expressed in situ [4]. However, despite the progress made with these
luciferases, it would be some time until biotechnology had advanced to the point
where the genes responsible for their expression could be cloned and exogenously
expressed, setting off the use of luciferases as tools for scientific discovery [5, 6].
Following the exogenous expression of the previously described firefly and
bacterial luciferases, Renilla luciferase was isolated from the sea pansy Renilla
reniformis [7] and Oplophorus luciferase was isolated from the deep-sea shrimp,
Oplophorus gracilirostris [8]. Shortly thereafter, firefly luciferase was successfully
Bioluminescence
expressed in mammalian cells [9] and it was demonstrated that different luciferases
could be used in tandem within a single host if they utilized different luciferin
compounds [10]. More recently, Gaussia luciferase has been isolated from the
marine copepod, Gaussia princeps [11], which was a notable discovery because,
unlike alternative luciferases, it is naturally secreted and thus could be monitored
without needing to sacrifice the host cell during luciferin treatment. Since the
discovery of Gaussia luciferase there has been rapid development of these enzymes
through genetic engineering, but little progress on the introduction of new systems.
However, this was recently changed with the introduction of fungal luciferase as
a novel luciferase system, which like bacterial luciferase is capable of genetically
encoding both the luciferase and luciferin pathway genes to support autobiolumi-
nescent production [12].
. Available luciferase systems for biotechnological applications
Of the ~40 different bioluminescent systems known to exist in nature [13], rela-
tively few are available for biotechnological applications. The primary reasons for
this are the lack of elucidated functional units, similarities in performance charac-
teristics (such as wavelength output) relative to existing systems, the entrenchment
of existing luciferase systems within the literature and as commercially-available
products, and the relatively high monetary and time costs required to explore novel
systems in depth relative to their ultimate utility as research tools. As a result of
these barriers, the luciferases available as research tools are generally limited to
those listed in Table.
. The necessity of engineering luciferase proteins
Despite the variety of different luciferases available, it is impossible to identify
just one that could fit the needs of every experimental design. Furthermore, it is
unfortunately frequent that no luciferase can be found to fit the needs of a given
experiment. As a result, there has been significant effort to engineer the existing
luciferase enzymes to improve their functionality, make them easier to use, and
expand their utility. This is especially true as the prevalence of luciferase usage has
increased in biomedical applications, which rely upon human cellular and small ani-
mal model systems that have significantly different physical and biochemical proper-
ties relative to the native host organisms from which these proteins were sourced.
These changes in physical properties and the constraints applied by the needs
of biomedical research have necessitated that luciferases be modified to express
at longer output wavelengths that better penetrate animal tissues or that can be
co-expressed with alternative luciferases, to produce light upon exposure to alter-
ative luciferin compounds, to produce altered signal output kinetics that are shorter
Luciferase Luciferin compound Output wavelength (nm)
Firefly luciferase (FLuc) D-luciferin 560
Bacterial luciferase (Lux) Tetradecanal 490
Renilla luciferase (RLuc)Coelenterazine 480
Oplophorus luciferase (OLuc)Coelenterazine 460
Gaussia luciferase (GLuc)Coelenterazine 470
Table 1.
Common luciferases available for biotechnological applications, their luciferin compound, and their output
wavelength.
Biotechnological Advances in Luciferase Enzymes
DOI: http://dx.doi.org/10.5772/intechopen.85313
or longer than their wild-type kinetics, to allow multimeric enzymatic structures
to function as monomers, to stabilize or destabilize protein structure within the
host, to make expression more efficient, and to increase output intensity so that it
is easier to detect the signal. Imparting these changes makes it possible to utilize
specialized versions of each luciferase that better fit the experimental needs of
the researcher. As the breadth of luciferase usage continues to grow, and as new
luciferase systems have been introduced over the years, the lessons learned from
these modifications are refined and re-applied in order to continuously unlock new
applications and improved functionality.
. Common methods for engineering improvements
To support the need for continued luciferase improvement, a number of
techniques have become commonplace for different engineering goals. The most
commonly utilized approaches and their common engineering endpoints are shown
in Table. Examples of the use of these techniques can be found in each of the
following sections.
. Firefly and click beetle luciferases
. Background
Firefly luciferase (FLuc) is perhaps the most well-known, well-studied, and
widely-used of all the luciferases. It, and its close relatives from click beetles,
both function through the ATP-dependent oxidation of reduced D-luciferin
(2-(4-hydroxybenzothiazol-2-yl)-2-thiazoline acid) in the presence of magne-
sium (Mg2+) and molecular oxygen (O2) to yield carbon dioxide (CO2), AMP,
inorganic pyrophosphate (PPi), and oxyluciferin. The resulting oxyluciferin is
initially produced in an excited state, and as it returns to its ground state energy
is released in the form of light. The naturally occurring peak emission wavelength
for FLuc (as commonly derived from Photinus pyralis) is ~560nm, while click
beetle luciferases, such as those from Pyrophorus plagiophthalamus and related
species, can produce a variety of wavelengths from 537 to 613nm depending on
their source organism [14, 15].
Technique Common uses
Mutagenic PCR Wavelength shifting, thermostability improvement, improve signal
output intensity
Rational sequence mutation Wavelength shifting, altering luciferin compatibility, altering signal
output kinetics
Synthetic recapitulation Enable functionality in alternative hosts, improve expression
efficiency, improve ease of use
Codon optimization Improve expression efficiency
Circular permutation Thermostability improvement, improve expression efficiency,
expand reporter functionality
Alternative luciferin
supplementation
Wavelength shifting, altering signal output kinetics
Split luciferase complementation Alter signal output kinetics, expand reporter functionality
Table 2.
Common approaches for engineering improvements in luciferase functionality.
Bioluminescence
Although FLuc and click beetle luciferase were among the first luciferases to be
studied [16], it was not until the mid-1900s that significant progress was made in
understanding the system at a level where it could be experimentally useful. At this
time, McElroy successfully extracted firefly luciferase from purified firefly lanterns
and determined that ATP was required for bioluminescence [17]. This led to the
determination of D-luciferins structure as 2-(4-hydroxybenzothiazol-2-yl)-2-thi-
azoline acid and its eventual chemical synthesis [16]. With these pieces in place,
chemists were able to isolate oxyluciferin as a purified product of the luminescence
reaction and validate its mechanism of action [18]. In 1985, FLuc cDNA was cloned
by DeLuca etal. [19]. This provided an alternative to the use of crude extracts of
beetles as a source of the luciferase enzyme and opened the door for widespread use
in biotechnological applications.
. Initial application and limitations
In its initial incarnation, FLuc was highly useful as a reporter in molecular biology
and bioimaging studies and for assaying the presence and quantification of the metab-
olites that participate in or are connected to the light reaction. The early discovery that
ATP concentration was proportional to light intensity in beetle luciferase reactions
made this assay the primary method for monitoring the cell’s main source of energy.
Further entrenching this technology was its exceptional sensitivity. FLuc-based
bioluminescent ATP assays display detection capabilities down to 1017mol [15]. This
sensitivity for measuring ATP concentrations has been used in several applications
including screening for microbial contamination in food industries, assessing cell
viability [20], and assaying enzymes involving ATP generation or degradation [21].
However, ATP concentrations found in living cells (1–10mM) are generally saturating
for FLuc and therefore it cannot be routinely used to assay intracellular ATP content
[15]. In a similar vein, FLuc has also been used to assay for the other metabolites that
participate in its bioluminescence reaction: CoA, AMP, and PPi [20].
The major limitation encountered during the use of FLuc or beetle luciferases
has been the requirement that the luciferin substrate be exogenously provided
for luminescence to occur. To date, there are no bacterial systems for generating
luciferin de novo, which necessitates chemical synthesis and results in potential
storage concerns due to the labile nature of the chemical [18]. Furthermore, this
often requires that the host cell harboring the luciferase be lysed to enable substrate
uptake, which has prevented its use for reporting real-time expression.
. Engineering improved expression and output
Applications of wild-type beetle luciferases can be limited due to structural and
functional stability issues or variations in the specific activity of the enzyme under
varying temperatures, pHs, ion concentrations, or inhibitors [22]. For instance,
wild-type FLuc protein has a half-life of only 15minutes at 37°C.This required that
more thermostable forms be developed to assay human and small animal model-
relevant temperature conditions [23]. Pozzo etal. sought to address this issue by
combining amino acid mutations shown to enhance thermostability with other
mutations reported to enhance catalytic activity, resulting in an eight amino acid
FLuc mutant that exhibited both improved thermostability and brighter lumines-
cence at low luciferin concentrations [24].
Similarly, Fujii etal. produced variants capable of producing 10-fold higher
luminescence than the wild-type enzyme by screening a mutant library of FLuc
proteins generated by random mutagenesis [25]. Site-directed mutagenesis experi-
ments were then performed based on mutant sequences that produced increased
Biotechnological Advances in Luciferase Enzymes
DOI: http://dx.doi.org/10.5772/intechopen.85313
luminescence. It was observed that the substitution of D436 with a non-bulky
amino acid, I423 with a hydrophobic amino acid, and L530 with a positively charged
amino acid all increased luminescence intensities relative to the wild-type enzyme.
They further demonstrated that combining the mutations at I423, D436, and
L530 resulted in an overall increase in affinity and turnover rate for the ATP and
D-luciferin substrates that resulted in high amplification of luminescence intensity.
Studies like this represent an emerging trend of combining alterations to specific
properties of firefly luciferases in order to enhance its overall practical utility.
. Engineering alternative output wavelengths
Engineering wavelength-shifted luciferases has become an intense area of study
to enable multi-color assays and improve the efficiency of in vivo bioimaging. Due to
hemoglobins absorbance of wavelengths below 600nm in mammalian tissues, the use
of wild-type firefly luciferase is relatively handicapped compared to more red-shifted
variants [15]. To overcome this limitation, mutagenic engineering approaches have
been successfully used to generate a variety of red-shifted versions [26, 27]. Notable
among this group is a variant developed by Branchini etal. containing a S284T muta-
tion. This variant produces a red-shifted output with a peak at 615nm, a narrow emis-
sion bandwidth, and improved kinetic properties [26]. However, this is by no means
the only option available. Today, the wide variety of available output wavelengths
enables researchers to choose the variant most well suited to their needs, or multiple
variants that can be simultaneously triggered upon exposure to D-luciferin.
. Engineering alternative signal kinetics
It has been demonstrated that varying the concentrations of FLuc’s substrates
(D-luciferin, ATP, etc.) can alter its reaction kinetics. High or saturating concentra-
tions produce flash-type kinetics that result in an intense initial signal followed by a
rapid decay, while low concentrations produce glow-type kinetics with a relatively
lower initial signal and a slower decay [18]. There are many possible inhibitors that
could be responsible for these changes. Under high substrate conditions, byproducts
of the reaction such as oxyluciferin and L-AMP can act as tight active-site bind-
ing inhibitors preventing enzyme turnover, or inhibitor-based stabilization can
increase activity when substrate levels are high enough to compete with the inhibi-
tory compound [14]. Commercial reagents containing micromolar concentrations
of components such as pyrophosphate and/or CoASH have been shown to convert
FLuc reactions from flash- to glow-type kinetics, possibly due to the breakdown of
oxidized luciferin-AMP via pyrophosphorolysis and thiolysis into the less potent
inhibitors oxidized luciferin and oxidized luciferin-CoA, respectively. These com-
mercial reagents are now widely used to support different experimental needs [14].
Another strategy that has been applied to alter reaction kinetics is the modifica-
tion of the luciferin substrate. Mofford etal. demonstrated that near-infrared light
emission can be increased >10-fold from wild-type FLuc by replacing D-luciferin
with synthetic analogues [28]. These synthetic analogues were designed to emit
longer wavelength light by incorporating an aminoluciferin scaffold. Nearly all the
aminoluciferins tested in their studies resulted in higher total near-IR (695–770nm)
photon flux from live cells under both high- and low-dose conditions. A more recent
substrate modification strategy has been to conjugate the luciferin with distinctive
functional groups. These so-called “caged” luciferins react when they are cleaved by
enzymes or bioactive molecules and subsequently freed [29]. This strategy allows for
specified monitoring of biological processes by linking light output to the activity
and/or concentration of enzymes or molecules reacting to cleave the caged luciferins.
Bioluminescence
. Renilla luciferase
. Background
Like FLuc, Renilla luciferase (RLuc) is another commonly used bioluminescent
reporter. Derived from the sea pansy Renilla reniformis, RLuc is a decarboxylating
oxidoreductase that uses coelenterazine as its substrate. During its bioluminescent
reaction, coelenterazine is converted to coelenteramide in the presence of molecular
oxygen, yielding blue light with an emission peak at 480nm [30]. In addition to
their substrate preferences, one other important differentiator between RLuc and
FLuc is that the RLuc bioluminescent reaction does not require ATP.It is also signifi-
cantly less efficient than FLuc and produces a reduced relative light output intensity
with a quantum yield of ~7% [31].
The RLuc protein was first purified and characterized in the late 1970s [7].
However, its cDNA sequence was not identified and cloned into Escherichia coli
until 1991 [31]. Following that accomplishment, the recombinant RLuc protein was
quickly expressed in other organisms, including yeast [32], plant [33], and mam-
malian cells [34] to serve as a gene expression reporter. The successful detection of
RLuc bioluminescence from mammalian cells was particularly important because
it represented the proof-of-principle demonstration of this enzyme as a reporter
target for in vivo animal imaging. And indeed, imaging of RLuc activity in living
mice was successfully validated just several years later [35]. In this demonstration,
Bhaumik and Gambhir showed that intraperitoneally implanted RLuc-expressing
cells could be detected following the injection of coelenterazine into the tail-vein
[35]. Similarly, when cells were injected via the tail-vein, bioluminescent signal
could be used to visualize cell trafficking to the liver and lungs. This study also vali-
dated that D-luciferin could not be used as a substrate, opening the door for future
studies to multiplex RLuc with FLuc as dual-reporters for in vivo applications.
. Engineering improved expression and output
The initial limitation for using RLuc as a reporter was its less-than-optimal
expression efficiency within mammalian cellular hosts. This limitation was over-
come via a codon-optimization strategy that modified the RLuc gene sequence
while maintaining the wild-type protein sequence. A synthetic humanized version
of the luciferase gene that utilizes this strategy, called hRLuc, is now commercially
available and has been shown to produce up to several 100-fold higher light out-
put in many mammalian cell lines. Further hampering the expression of RLuc in
cell culture and small animal imaging applications was its tendency to be rapidly
inactivated upon exposure to animal serum. In its wild-type orientation the half-life
of the enzyme under routine experimental conditions ranged from 30 to 60minutes
[36]. An early study by Liu and Escher showed that a single mutation from cyste-
ine to alanine at amino acid 124 (RLucC124A) increased serum resistance, while
simultaneously increasing overall light output [37]. Following this study, Loening
etal. employed a consensus sequence guided mutagenesis strategy to screen for
mutants with improved serum stability [36]. These efforts identified a variant
termed RLuc8, which harbored eight substations (A55T/C124A/S130A/L136R/
A143M/M185V/M253L/S287L). The RLuc8 variant was shown to be >200-fold more
stable in mouse serum than the native protein and displayed an improved half-life
of 281hours. Fortuitously, the RLuc8 mutant also exhibited a 4-fold improvement
in brightness. The improved stability and light output characteristics of RLuc8
make it a more favorable reporter than wild-type RLuc for mammalian imaging
applications.
Biotechnological Advances in Luciferase Enzymes
DOI: http://dx.doi.org/10.5772/intechopen.85313
. Engineering alternative output wavelengths
Despite the improvements made to increase expression efficiency and output,
RLuc’s 480nm output maximum remained problematic for in vivo animal imaging
applications because it was prone to absorption and attenuation in organs and tis-
sues. This was especially problematic for deep tissue imaging (below subcutaneous
layer), where only 3% of the emission spectra could efficiently penetrate animal
tissue for detection. Therefore, to improve RLuc’s in vivo utility, many efforts were
undertaken to red-shift its emission spectra. Loening etal. hypothesized that
modifying the active site of the luciferase could create a chemical environment
favorable to specific coelenteramide species (i.e., the pyrazine anion form) that
emit green (535–550nm) light upon returning from their excited state. To test
this hypothesis, they made site-specific mutations at 22 amino acid residues at the
predicted active site of RLuc8 and identified red-shifted light emissions (peaks
between 493 and 513nm) in variants with mutations at eight of these locations
[38]. Unfortunately, these red-shifted mutants also possessed substantially reduced
signal intensities. To restore light output, random mutagenesis was carried out on
the red-shifted mutants. This process identified several residues where mutations
increased light output or resulted in further red-shifting. Based on these encourag-
ing results, Loening and colleagues performed several more rounds of site-directed
mutagenesis and successfully engineered three promising variants RLuc8.6-535,
RLuc.6-545, and RLuc8.6-547, which peaked at 535, 545, and 547nm, respectively,
when using coelenterazine as the substrate. All three variants exhibited greater
light output than wild-type RLuc, with the most improved, RLuc8.6-535, showing
6-times greater intensity and similar stability to RLuc8. In practice, this translated
to roughly a 2.2-fold increase in transmitted signal from the lungs of living mice
compared to an equal initial light flux from RLuc8.
In addition to engineering the protein itself, synthetic coelenterazine substrate
analogs have also been created to improve light output and/or yield red-shifted emis-
sion spectra. The analog coelenterazine-v was first shown to shift the emission peak
of wild-type RLuc to 513nm [39] and later demonstrated to yield emission peaks at
570nm (yellow) and 588nm (orange) in the RLuc8.6-535 and RLuc8.6-547 variants,
respectively [38]. However, this substrate is currently not commercially available
due to high background activity and difficulty in purification. Other analogs, such
as coelenterazine-f, -h, and -e have been shown to increase signal intensity by 4- to
8-fold relative to coelenterazine in RLuc-expressing mammalian cells in vitro, but
each has failed to compete with the native coelenterazine in living animal imaging
[40]. Despite these setbacks, Nishihara etal. have reported that analogs with ethynyl
or styryl group substitutions at the C-6 position significantly increased biolumines-
cent output and signal stability in RLuc8 and RLuc8.6-535 [41, 42], which suggest
that the development of new synthetic coelenterazine analogs will continue to be a
promising route for enhancing RLuc functionality.
. Engineering split luciferase applications
Due to its small size (311 amino acids, ~36kDa) and monomeric orientation,
the RLuc protein is an attractive option for use in split luciferase complementation
assays aimed at monitoring real-time protein-protein interaction. In an early study
attempting to achieve this goal, Paulmurugan and Gambhir [43] created RLuc frag-
ment pairs at two split sites (I223/P224 and G229/K230) and fused the individual
fragments to either the MyoD or Id proteins. They then successfully demonstrated
that RLuc could properly re-fold and restore luciferase activity upon complemen-
tation during MyoD/Id interaction. This study also showed that the split RLuc
Bioluminescence
reporter signal could be modulated by using an inducible promoter (e.g., NFκB
promoter/enhancer) to regulate the expression level of one of the two fragments.
The fragment pair based on the G229/K230 split site was later used to characterize
interactions between heat shock protein 90 (Hsp90) and the co-chaperone protein
Cdc37 [44], between Hsp90 and the Epstein-Barr virus protein kinase GBLF4 [45],
and to visualize androgen receptor translocation in the brains of living mice [46].
Kaihara etal. similarly leveraged a variant of RLuc split between S91 and Y92 to
demonstrate the recovery of bioluminescent activity during insulin-stimulated
protein-protein interactions [47], and Stefen etal. created a split variant using
fragments separated between residues 110 and 111 fused to protein kinase A (PKA)
regulatory and catalytic subunits to quantify G protein-coupled receptor (GPCR)-
induced disassembly of the PKA complex in living cells [48]. These types of split
RLuc complementation assays have also been applied to profile protein-protein
interactions in the Golgi apparatus in planta [49] and to study protein dynamics
during chemotaxis in bacteria [50], making it a broadly applicable approach.
. Gaussia luciferase
. Background
Isolated from the marine copepod Gaussia princeps, Gaussia luciferase (GLuc)
is the smallest known luciferase. It is comprised of only 185 amino acids and has a
molecular weight of 19.9kDa. Like RLuc, GLuc catalyzes the oxidative decarboxyl-
ation of coelenterazine in an ATP-independent manner to produce blue light with
a peak wavelength around 480nm. Despite this relatively short wavelength, GLuc
is one of the brightest luciferases and is capable of generating light output several
orders of magnitude higher than FLuc and RLuc [11]. However, unlike FLuc and
RLuc, the GLuc protein is naturally secreted from the cells. In biotechnological
applications, this allows signal measurements to be performed on culture medium
without cell lysis and when using blood or urine samples obtained during animal
applications [51, 52]. Its secretory nature also enables unique applications such as
monitoring protein processing through the secretory pathway and drug-induced
endoplasmic reticulum (ER) stress [53, 54]. It was first isolated and cloned by Bruce
and Szent-Gyorgyi in 2001 [55], and since has enjoyed rapid adoption within the
research community through a variety of engineered improvements.
. Engineering improved expression and output
To enable improved expression efficiency in biomedical applications, a human-
ized version of GLuc, hGLuc, was generated via codon optimization. This human-
optimized variant has been shown to produce 2000-fold higher bioluminescent
signal than the wild-type variant when expressed in mammalian cells [11]. In addi-
tion to mammalian systems, the GLuc gene sequence has also been codon optimized
for efficient expression in the alga Chlamydomonas reinhardtii [56], the fungus
Candida albicans [57], mycobacteria [58], and Salmonella enterica [59].
Building on this codon optimization-based approach, which enhances light
output by improving protein expression in the host organism without modifying
the peptide sequence, mutagenetic approaches have similarly been successfully
applied to engineer variants that produce greater signal intensities than the wild-
type protein. In one such example, Kim etal. performed site-directed mutagenesis
to the hydrophilic core region of GLuc and identified that changing the isoleucine
at position 90 to leucine (I90L) was the major contributing factor for improved
Biotechnological Advances in Luciferase Enzymes
DOI: http://dx.doi.org/10.5772/intechopen.85313
signal intensity [60]. The I90L variant produced six times higher light output
than the wild-type protein in mammalian cells. Using a directed molecular evolu-
tion approach, Degeling etal. also identified a variant (S16K/M43V/V159M) that
showed 2-fold enhanced luciferase activity [61].
. Engineering alternative output wavelengths
One limitation of the native GLuc protein is that its relatively blue-shifted
emission wavelength is easily absorbed and scattered by pigmented molecules in
animal tissues. This limits its utility in in vivo animal imaging applications. Several
attempts have been made to engineer a red-shift towards increased wavelengths,
but these efforts have met with only moderate success. In one notable example, Kim
etal. engineered a variant, which they termed Monsta, that harbors four muta-
tions (F89W/I90L/H95E/Y97W) resulting in a shifted peak emission wavelength
of 503nm. This is ~20nm red-shifted compared to wild-type GLuc [60]. Similarly,
several alternative variants (L40P, L40S, and L30S/L40P/M43V) generated by
Degeling etal. show 10–15nm shifts in their emission peaks [61]. Despite the fact
that these red-shifted variants have not enjoyed similar success to those of RLuc,
GLuc’s relatively increased signal strength can often compensate for the loss of
signal due to absorption.
. Engineering alternative signal kinetics
Wild-type GLuc catalyzes a flash-type bioluminescent reaction, meaning that
the light signal decays rapidly following luciferin exposure. Practically, this neces-
sitates immediate signal reading after substrate addition and thus makes GLuc
unsuitable for the majority of high-throughput applications. To overcome this rapid
signal decay, researchers have successfully engineered mutants that emit more stable
bioluminescence [6163]. Noticeably, a L30S/L40P/M43V variant has been shown to
exhibit glow-type kinetics with only a 20% loss in signal intensity over 10minutes,
compared to the >90% loss in signal intensity after 1minute from the wild-type
enzyme [61]. GLuc mutants such as these have been demonstrated to function in
96- and 384-well plate formats, which effectively allows them to overcome the wild-
type kinetic limitations and enables their use in high-throughput assay formats.
. Engineering split luciferase applications
Like RLuc, GLuc’s small size (185 amino acids, 19.9kDa) makes it a good candi-
date for split luciferase complementation assays. In an early attempt at developing
this functionality, Remy and Michnick evaluated the ability of fragment pairs
generated from cut sites between amino acids 65–109 of a truncated hGLuc sequence
exclusive of the secretion signal to reconstitute luciferase activity upon rejoining
[64]. By fusing the respective 5 and 3 sequences of the split hGLuc gene to a GCN4
leucine zipper-coding sequence and co-expressing the resulting fusions in HEK293
cells they were able to show that hGLuc activity could be successfully reconstituted
by leucine zipper-induced complementation of the split fragments. Their study
determined that the optimal split site for complementation was between G93 and
E94. This fragment pair has since been further demonstrated to be inducible and
reversible, which allows it to function as a highly sensitive tool for quantifying pro-
tein-protein interactions in cells and living mice [65]. Similarly, Kim and colleagues
also developed a split GLuc variant dissected at Q105 and demonstrated its utility to
monitor calcium-induced calmodulin and M13 peptide interaction, phosphorylation
of the estrogen receptor, and steroid-receptor binding in living cells [60].
Bioluminescence

. Oplophorus luciferase
. Background
Oplophorus luciferase (OLuc) is a naturally-secreted luciferase isolated from
the decapod Oplophorus gracilorostris, a deep-sea shrimp that ejects OLuc from
the base of an antennae in a brightly luminous cloud when stimulated. It is one
of the more complex luciferase proteins, as it is a 106kDa heterodimeric tetra-
mer consisting of two regions, each comprised of a 35 and 19kDa subunit. Like
RLuc and GLuc, OLuc uses coelenterazine as a substrate and does not require
ATP for functionality [17]. It produces primarily blue light, with a peak emis-
sion wavelength of 462nm. Even in its wild-type form, OLuc possesses robust
biochemical and physical characteristics relative to alternative luciferases. It
exhibits relatively little change in quantum yield throughout a pH range from 6
to 10, maintains thermostability across a temperature range of 20–50°C, and can
still produce observable light output at 70°C [8].
OLuc was first discovered in 1975 [66], and shortly after in 1978 the mechanics
of its bioluminescent reaction were identified [8]. Inouye etal. were the first to
clone the OLuc cDNAs encoding the 35 and 19kDa subunit proteins, which led to
their discovery that the 19kDa protein was responsible for catalyzing the lumines-
cent oxidation of coelenterazine. Although this 19kDa protein was found to be the
smallest known protein capable of catalyzing bioluminescence, it was also found to
be poorly expressed and unstable without the support of its 35kDa partner [67].
. Engineering improved expression and output
The need to co-express the 19 and 35kDa subunits of OLuc made it problematic
for routine reporter usage. To overcome this, Hall etal. performed three rounds of
mutagenesis on the 19kDa subunit to produce a novel variant, which they termed
NanoLuc (NLuc). This variant showed improved structural stability as well as
increased bioluminescent activity and glow-type kinetics with a peak emission
wavelength of 460nm. Furthermore, it was shown that this variant could oxidize
an alternative luciferin, furimazine, which resulted in greater light intensity and
lower background autoluminescence than when coelenterazine was used. NLuc’s
19kDa size and absence of post-translational modifications made it more agile than
FLuc, while its naturally high tolerance to temperature and pH made it more robust.
In practice, this NLuc variant was shown to poses 150-fold greater specific activity
than either FLuc or RLuc [68]. However, these improvements proved to be a double-
edged sword. The high stability and glow-type kinetics made it difficult to employ
NLuc for transient reporting activities, while its highly blue-shifted output limited
its signal penetration in mammalian cellular applications.
Nonetheless, NLuc’s small size and efficient expression make it an excellent
choice for studying low-dynamic activities. In one such example, Chen etal.
developed a sensitive assay in which NLuc was used to study the activity of deubiq-
uitinating enzymes. In this work, NLuc was fused to the C-terminus of His-tagged
ubiquitin that was attached to Ni2+ agarose beads. This allowed NLuc to be released
as the α-peptide linkages were cleaved so that deubiquitination could be monitored
via NLuc luminescence [69]. Similarly, Lackner etal. [70] used a CRISPR-Cas9-
mediated strategy to tag three cytokine-inducible genes (DACT1, IFIT1, and EGR1)
with NLuc. This allowed cytokine-induced upregulation to be measured in HAP1
cells. Under this design, they were able to show that NLuc luminescence correlated
strongly with quantitative PCR data, demonstrating that NLuc could reliably be
used to monitor gene expression.
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Biotechnological Advances in Luciferase Enzymes
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. Engineering split and paired luciferase applications
Zhao etal. showed that a split luciferase-based system could be used to monitor
protein stability by tracking protein aggregation with NLuc-based luminescence
[71]. To accomplish this, they broke NLuc into two fragments, termed N65 and
66C, and demonstrated that, upon interaction, luminescence was modulated by the
solubility of the protein fused to the N65 fragment. This property was maintained
in both bacterial and mammalian systems, confirming its utility for sensitive
detection of protein solubility in a straightforward, high-throughput assay format
in living cells.
In addition to these traditional split luciferase applications, NLuc has also
been employed for paired luciferase applications that utilize an unfused variant to
provide the highest possible light intensity and sensitivity, a destabilized variant
with an appended degradation signal (e.g., NLuc-PEST) that allows rapid response
to dynamic changes in environment, and a secreted variant (e.g., secNLuc) [17].
. Bacterial luciferase
. Background
Unlike the monomeric luciferases discussed above, bacterial luciferase (Lux)
is a heterodimer of two genes, luxA and luxB, that must join together to form a
functional unit. It is also only one of two systems, along with the fungal system dis-
cussed below, that additionally has a known genetic pathway for luciferin synthesis.
In the case of bacterial luciferase, this pathway consists of three additional genes,
luxC, luxD, and luxE, that work together to produce a long chain fatty aldehyde
[72]. In this process, luxD transfers an activated fatty acyl group to water, forming
a fatty acid. The fatty acid is then passed off to luxC and activated via the attach-
ment of AMP to create a fatty acyl-AMP.The luxE gene finally reduces this fatty
acyl-AMP to an aldehyde [72]. The natural aldehyde for this reaction is tetradecanal,
however, the luciferase is also capable of functioning with alternative aldehydes
as substrates [72]. Along with these genetic components, the system requires two
cofactors: oxygen and reduced riboflavin phosphate. When all components of the
system are present, bacterial luciferase will produce bioluminescence in an autono-
mous fashion at a wavelength of 490nm.
Although this process has been most well-studied in marine bacteria from
the Vibrio genus, the genetic organization and biochemical underpinnings of the
system are consistent across all known bacterial phyla [18]. Due to the complex-
ity of this system relative to its monomeric counterparts, it was not exogenously
expressed until the early 1980s. Even then, it was initially utilized through expres-
sion of the luxA and luxB genes as a standalone luciferase [5] before subsequently
being employed as a fully functional cassette that was capable of functioning in
an autonomous fashion [73]. Shortly after these demonstrations the crystal struc-
ture of the bacterial luciferase heterodimer was determined [74], however, this
structural knowledge has yet to be leveraged as a means for engineering improved
functionality.
. Initial uses and limitations
Because Lux emits its bioluminescent signal without the need for external
stimulation, it quickly became a valuable tool for optical imaging. The low hanging
fruit for this system was the real-time monitoring of gene expression. This was first
Bioluminescence

demonstrated by Enbreghet etal. [75], who fused Lux to inducible promoters to
study the mechanics of IPTG and arabinose induction in E. coli. This proved to be a
valuable approach because it allowed samples to be continuously monitored in order
to track gene expression dynamics over time. Building upon this work, a variety
of instances have been described where Lux has been placed under the control of a
promoter with a known inducer to track compound bioavailability. Repeated use of
the system for this propose has demonstrated that it is capable of reporting bioavail-
ability in a dose/response fashion [76], which makes it a valuable tool for monitoring
contaminant levels in mixed environmental samples. At a higher level, it has been
used for in situ bacterial monitoring, such as the visualization of bacterial invasion
of leaf [77] and root structures [78]. Further, due to the absence of light production
from non-bioluminescent species, it was also used to track specific populations of
bacteria within mixed communities within unperturbed environments [79].
Despite the advantages offered by avoiding the need for external stimulation
concurrent with visualization, Lux was significantly handicapped by its inability
to function within eukaryotic cells. Because of this, it was not originally applicable
to most modern biotechnological and biomedical applications outside of tracking
bacterial infections [80]. Furthermore, as a consequence of encoding both the
luciferase and luciferin generation pathways this system required significantly more
foreign DNA to be introduced in order to function exogenously. This made the sys-
tem more difficult to work with at the molecular level; especially before the advent
of today’s more efficient genetic assembly tools. Similarly, the heterodimeric nature
of the luciferase enzyme is more cumbersome than the monomeric orientation of
its counterparts. Nonetheless, given its relative advantages over the other systems, it
continues to be engineered to overcome these detriments and expand its utility.
. Engineering eukaryotic expression
Although several early attempts were made to enable Lux functionality within
eukaryotic hosts, none of these achieved significant success [8183]. The first
major breakthrough came with the expression of the luciferase in S. cerevisiae [84].
This achievement was made possible by using luciferase genes from the terrestrial
bacterium, Photorhabdus luminescens, which showed higher thermal stability
than those of marine bacteria, and expressing the individual heterodimer genes
from a single promoter using an internal ribosomal entry site (IRES) to link them
together. Under this orientation the luciferase was able to properly express within
the cell and produce light upon exposure to an n-decanal substrate. This same
strategy was then expanded to incorporate the expression of IRES-linked luciferin
synthesis pathway genes from dual promoters. When expressed concurrently with
the luciferase genes, the cell produced a bioluminescent signal without external
stimulation. The functionality of the system was then further improved by shifting
the intracellular redox balance to a more reduced state through the introduction of
a flavin oxidoreductase gene, frp.
Despite this success in S. cerevisiae, the direct application of these changes was
not sufficient to permit similar bioluminescent production from human cellular
hosts. To achieve this, the genes were codon optimized for the human genome and
mammalian-optimized IRES elements were employed to improve expression of the
downstream genes in human cells [85]. It was also determined that the full pathway
could not be expressed from a single promoter using IRES elements, so the luciferin
synthesis pathway was encoded on a separate plasmid. This approached allowed for
functionality in human cells, but the overall level of bioluminescent production was
several orders of magnitude lower than that of alterative bioluminescent systems
such as firefly luciferase [86].
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. Engineering increased light output
To overcome Lux’s low level of bioluminescent output in human cells the orienta-
tion of the cassette was subjected to further engineering. It was determined that the
use of multiple plasmids was detrimental to achieving high level expression, and that
the use of IRES elements was inefficiently expressing the downstream genes in the
paired orientation. Therefore, the IRES elements were replaced with viral 2A linker
sequences. These sequences were significantly shorter than the IRES sequences they
replaced and allowed for each linker region to have a unique genetic code that reduced
the chance for unintended recombination events. As a result, the full bacterial
luciferase cassette, inclusive of the flavin oxidoreductase component, could be placed
under the control of a single promoter and expressed from a single plasmid. This new
orientation made it possible to express bacterial luciferase as a single genetic construct
similar to what was commonly done with the alternative monomeric, luciferin-
requiring luciferase systems. As a result, the bacterial luciferase system could be
expressed more easily across a larger number of cell types and was capable of produc-
ing an enhanced level of signal output relative to its previous incarnation [87].
In addition to engineering increased expression via improved expression efficiency,
work has also been performed to alter the peptide sequence of the bacterial luciferase
genes to make light output more efficient. Gregor etal. [88] used random mutation
to alter the coding sequence of Lux cassette and uncovered a series of 15 mutations
that improved light output and thermostability. Of these mutations, six were within
the luciferase genes (three each in luxA and luxB), six were in the luciferin synthesis
pathway (with all six located in the luxC gene), and three were located in the oxidore-
ductase gene, frp. These mutations resulted in both improved thermotolerance and a
~7 times increase in bioluminescent production relative to the wild-type sequence.
. Engineering improved bioreporter functionality
Just as it has been used extensively as a bioreporter in bacterial species, the engineer-
ing of bacterial luciferase to function in eukaryotic cells opened the door to this same
functionality under much broader applications. The transition of the Lux cassette to
function as a single open reading frame made it possible to replace the constitutive
promoter with an inducible promoter and regulate its expression in response to com-
pound bioavailability [87]. However, computational modeling aimed at calculating the
metabolism of the required substrates and cofactors for the reaction relative to their
intracellular availably suggested that that control of the system should be imparted at
the level of the aldehyde recycling pathway, with luxA and luxB expressed continuously,
and luxC, luxD, and luxE placed under the control of the inducible promoter [89]. This
model was later proven to be correct when direct comparisons were performed using
either single open reading frame constructs where the full cassette was controlled by the
inducible promoter, or split cassettes where the luciferase and luciferin pathway genes
were switched between inducible and constitutive promoters [90]. Together, these
results significantly improved the functionality of the bacterial luciferase system as a
bioreporter despite its relative complexity compared the other luciferases.
. Fungal luciferase
. Background
Fungal luciferase is the most recent luciferase system to be functionally
elucidated and made available for biotechnological applications. At the core of
Bioluminescence

this system is a monomeric luciferase gene, luz. In addition to the luciferase, two
luciferin synthesis genes: hisps and hh, work together as a polyketide synthase and
a 3-hydroxybenzoate 6-monooxygenase to supply the required luciferin, 3-hydroxy-
hispidin. In addition to these genetic components, the reaction also requires
molecular oxygen and NAD(P)H as co-factors [91, 92]. When all components of
the system are present, it produces a luminescent signal at 520nm. Like Lux, fungal
luciferase is notable in that the genetic sequence of all components required for
bioluminescent production is characterized. This allows the fungal luciferase cas-
sette to be genetically encoded and exogenously expressed to produce an autobiolu-
minescent phenotype [12]. However, for this to occur the host organism must either
be capable of naturally synthesizing caffeic acid to act as a precursor for luciferin
synthesis, or the necessary genes for caffeic acid synthesis must be co-expressed.
Under this strategy, it is possible to synthetically assemble a seven gene cassette
consisting of the fungal luciferase genes: luz, hisps, and hh, along with a tyrosine
ammonia lyase, two 4-hydroxyphenylacetate 3-monooxygenase components and
the 4-phosphopantetheinyl transferase gene npgA, to support caffeic acid synthesis
and continuous light production in any host.
. Initial uses and limitations
Unlike the previous luciferases that have been discussed, fungal luciferase
has only recently been elucidated as of the time of this chapter. As a result, there
have yet to be any reports of its functionality outside of its initial validation [12].
Regardless, the initial characterization of the system provides valuable insights into
its functionality and potential limitations. From a practical standpoint, it has been
demonstrated that the system can be fully recapitulated in yeast to achieve autobio-
luminescent signal production. At this time only one luciferin synthesis pathway has
been demonstrated, but because genes sourced from alternative organisms are used
to enable caffeic acid synthesis in hosts that do not natively support these reactions,
it is likely that alterative genes could be substituted for these parts of the pathway.
For more complex hosts, such as human cells, the functionality of the system
has been demonstrated only under non-autobioluminescent conditions. In this
case, only the luciferase was genetically encoded and the luciferin was exog-
enously applied. Using this strategy, it has been possible to observe luminescence
in cultured human cells, Xenopus laevis embryos, and small animal models sub-
cutaneously injected with labeled cells. These demonstrations bode well for the
use of the fungal luciferase in the types of experimental designs most commonly
associated with traditional luciferase reporters and provide researchers with a
novel imaging tool that can be differentiated from alternative luciferases based on
its luciferin specificity.
It is currently unknown if the lack of demonstrated autobioluminescent pro-
duction in hosts outside of yeast is incidental, or if it is the result of metabolic or
molecular limitations on the expression of the full cassette within these organ-
isms. One possible explanation is that the required culture temperatures were not
compatible with full cassette functionality. It has been shown that fungal luciferase
is temperature sensitive and begins to decrease its output signal at temperatures
>18°C.Relative light output is halved at room temperature (26°C) and is abolished
above 30°C.This is detrimental to the use of this luciferase in human cell culture
and small animal model systems, as they will require the maintenance of tempera-
tures above 30°C to avoid the introduction of secondary environmental effects.
Similarly, the luciferase is only ~50% efficient at pH7, which could be detrimental
to some experimental designs. The optimal pH is 8, with improved retention of
performance at increased pH relative to decreased pH.
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Biotechnological Advances in Luciferase Enzymes
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. Potential future engineering goals
There are ~100 fungal species that use this luciferase/luciferin pathway for biolu-
minescent production [93]. It is believed that fungal bioluminescence evolved only
once, but that evolutionary pressure led to uneven distribution of the phenotype
among species. While this simplifies the system by allowing development to focus on
only a single incarnation, it is also potentially limiting in that there are fewer evolu-
tionary cues that can be leveraged as starting points for biotechnological advance-
ment. Nonetheless, this system is clearly in its infancy and will benefit from the
copious knowledgebase developed through the engineering of alternative luciferases.
It is likely that the primary development target will be overcoming the thermostabil-
ity issues present in the current incarnation of the system. Beyond this, and similar to
Lux, it is likely that investigators will seek to streamline expression of the relatively
large cassette size to make it more manageable from a molecular biology standpoint.
Once these efforts are achieved, the autobioluminescent nature and somewhat
red-shifted output of the fungal system will make it a welcome addition for real-time
imaging applications that currently rely on only the bacterial luciferase system.
. Outlook for future developments
There are ~40 different bioluminescent systems known to exist in nature [13].
However, only seven different families have been well described and only five
of the six detailed in this chapter enjoy widespread use [94]. Despite the relative
wealth of unexplored systems, relatively few new systems have become available
in recent history. Within the last 10years, the most notable advancements have
been the engineering of the bacterial luciferase system to function in eukaryotic
organisms and the elucidation of the fungal luciferase genetic pathway. Despite this,
the considerable progress of incremental engineering for firefly luciferase and the
development of NanoLuc from Oplophorus luciferase have provided a clear roadmap
for continued progress within the field. Historically, the ability to alter luciferase
conformation or luciferin compatibility to enable alternative output wavelengths
that better penetrate tissue, allow for multiplexed imaging of multiple luciferases, or
pair with fluorescent reporters for BRET applications has enabled new experimental
designs that have led to important discoveries. With the emergence of autobiolu-
minescent capabilities from the bacterial and fungal systems, it is likely that the
barriers will again be pushed back. These systems will compete with the established
luciferases and encourage further development to keep them competitive within
an increasingly crowded marketplace. In parallel they can also leverage the decades
of previous development in the other luciferases to jumpstart their engineering of
alternative output wavelengths, expression kinetics, and luciferin compatibly. Paired
with improvements in bioluminescent detection hardware and modern synesthetic
biology engineering tools, it is likely that this renewed age of luciferase engineering
will continue to expand the application space for bioluminescent imaging and drive
further exploration into the untapped potential of underexplored luciferases.
. Conclusion
There are a variety of different luciferase systems available for biotechnological
applications that can help investigators achieve their experimental goals. The high
utility afforded by these enzymes is the result of a rich history of engineering that
has enabled them to become versatile research tools. Historically, significant shifts
Bioluminescence

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.
Author details
AndrewKirkpatrick1, TingtingXu2, StevenRipp2, GarySayler1,2 and DanClose1*
1 490 BioTech, Inc., Knoxville, Tennessee, USA
2 University of Tennessee, Knoxville, Tennessee, USA
*Address all correspondence to: dan.close@490biotech.com
in utility have occurred with the elucidation and introduction of new luciferases,
followed by slower, but steady, incremental improvements as they are iteratively
engineered to improve their ease of use and expand their functionality. In the
context of the historical achievements that have been made with firefly, Renilla,
Gaussia, and Oplophorus luciferase, the improvements being made to bacterial
luciferase and the recent introduction of fungal luciferase point to promising things
to come and give hope that new luciferase systems will continue to be introduced to
keep the pace of development strong in the future.
Acknowledgements
Research funding was provided by the U.S.National Institutes of Health
under award numbers NIGMS-1R43GM112241, NIGMS-1R41GM116622, NIEHS-
2R44ES022567, NIEHS-1R43ES026269, and NIMH-1R43MH118186, and the
U.S.National Science Foundation under award number CBET-1530953.
Conflict of interest
S.R., G.S., and D.C. are board members in the for-profit entity 490 BioTech.

Biotechnological Advances in Luciferase Enzymes
DOI: http://dx.doi.org/10.5772/intechopen.85313
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... Bacterial luciferase (lux) utilizes fatty aldehyde as luciferin and O 2 and FMNH 2 as co-factors. It is thermostable up to 42°C [9] but has been limited primarily to prokaryotic or single-cellular eukaryotic hosts [10]. Although it functions in mammalian cells [11,12], it displays reduced luminescent output relative to bioluminescent systems requiring external substrate application and has a peak output at 490 nm, which is relatively blue-shifted compared to the in vivo imaging optimum [13]. ...
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... Biochemical characterization of these systems has demonstrated that light emission is achievable via multiple metabolic routes 18 . Several luciferases have been used as reporters of biomolecular processes, most notably firefly luciferase, various marine luciferases, such as Renilla, Gaussia, Oplophorus and others, and bacterial luciferase 19 . They have proven to be enormously useful primarily because luminescence can be readily quantified with high sensitivity and precision. ...
Preprint
Full-text available
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Chapter
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Protein-protein interactions can regulate different cellular processes, such as transcription, translation, and oncogenic transformation. The split Renilla luciferase complementation assay (SRLCA) is one of the techniques that detect protein-protein interactions. The SRLCA is based on the complementation of the LN and LC non-functional halves of Renilla luciferase fused to possibly interacting proteins which after interaction form a functional enzyme and emit luminescence. The BGLF4 of Epstein-Barr virus (EBV) is a viral protein kinase that is expressed during the early and late stages of lytic cycles, which can regulate multiple cellular and viral substrates to optimize the DNA replication environment. The heat shock protein Hsp90 is a molecular chaperone that maintains the integrity of structure and function of various interacting proteins, which can form a complex with BGLF4 and stabilize its expression in cells. The interaction between BGLF4 and Hsp90 could be specifically detected through the SRLCA. The region of aa 250-295 of BGLF4 is essential for the BGLF4/Hsp90 interaction and the mutation of Phe-254, Leu-266, and Leu-267 can disrupt this interaction. These results suggest that the SRLCA can specifically detect the BGLF4/Hsp90 interaction and provide a reference to develop inhibitors that disrupt the BGLF4/Hsp90 interaction.
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The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.