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 exogenous 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 prospective 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

## **1. Introduction**

#### **1.1 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 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 autobioluminescent production [12].

## **1.2 Available luciferase systems for biotechnological applications**

Of the ~40 different bioluminescent systems known to exist in nature [13], relatively few are available for biotechnological applications. The primary reasons for this are the lack of elucidated functional units, similarities in performance characteristics (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 1**.

## **1.3 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 animal model systems that have significantly different physical and biochemical properties 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 alterative luciferin compounds, to produce altered signal output kinetics that are shorter


#### **Table 1.**

*Common luciferases available for biotechnological applications, their luciferin compound, and their output wavelength.*

**5**

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

**Technique Common uses**

Codon optimization Improve expression efficiency

*Common approaches for engineering improvements in luciferase functionality.*

applications and improved functionality.

**2. Firefly and click beetle luciferases**

their source organism [14, 15].

following sections.

Alternative luciferin supplementation

**Table 2.**

**2.1 Background**

**1.4 Common methods for engineering improvements**

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

Mutagenic PCR Wavelength shifting, thermostability improvement, improve signal

Rational sequence mutation Wavelength shifting, altering luciferin compatibility, altering signal

Circular permutation Thermostability improvement, improve expression efficiency, expand reporter functionality

Split luciferase complementation Alter signal output kinetics, expand reporter functionality

Wavelength shifting, altering signal output kinetics

output intensity

output kinetics Synthetic recapitulation Enable functionality in alternative hosts, improve expression efficiency, improve ease of use

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 2**. Examples of the use of these techniques can be found in each of the

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 magnesium (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

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*


#### **Table 2.**

*Bioluminescence - Analytical Applications and Basic Biology*

nescent production [12].

those listed in **Table 1**.

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-

Of the ~40 different bioluminescent systems known to exist in nature [13], relatively few are available for biotechnological applications. The primary reasons for this are the lack of elucidated functional units, similarities in performance characteristics (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

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 animal model systems that have significantly different physical and biochemical properties 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 alterative 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

*Common luciferases available for biotechnological applications, their luciferin compound, and their output* 

**1.2 Available luciferase systems for biotechnological applications**

**1.3 The necessity of engineering luciferase proteins**

**4**

**Table 1.**

*wavelength.*

*Common approaches for engineering improvements in luciferase functionality.*

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.

### **1.4 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 2**. Examples of the use of these techniques can be found in each of the following sections.

## **2. Firefly and click beetle luciferases**

### **2.1 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 magnesium (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].

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-luciferin's structure as 2-(4-hydroxybenzothiazol-2-yl)-2-thiazoline 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.

### **2.2 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 metabolites 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 10<sup>−</sup>17 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.

#### **2.3 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 modelrelevant 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 luminescence 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 experiments were then performed based on mutant sequences that produced increased

**7**

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

**2.4 Engineering alternative output wavelengths**

**2.5 Engineering alternative signal kinetics**

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 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 hemoglobin's 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 mutation. This variant produces a red-shifted output with a peak at 615 nm, a narrow emission 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.

It has been demonstrated that varying the concentrations of FLuc's substrates (D-luciferin, ATP, etc.) can alter its reaction kinetics. High or saturating concentrations 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 binding inhibitors preventing enzyme turnover, or inhibitor-based stabilization can increase activity when substrate levels are high enough to compete with the inhibitory 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 commercial reagents are now widely used to support different experimental needs [14]. Another strategy that has been applied to alter reaction kinetics is the modification 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.

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

*Bioluminescence - Analytical Applications and Basic Biology*

in biotechnological applications.

**2.2 Initial application and limitations**

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-luciferin's structure as 2-(4-hydroxybenzothiazol-2-yl)-2-thiazoline 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 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 metabolites 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 10<sup>−</sup>17 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

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

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 modelrelevant 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-

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 experiments were then performed based on mutant sequences that produced increased

participate in its bioluminescence reaction: CoA, AMP, and PPi [20].

uptake, which has prevented its use for reporting real-time expression.

**2.3 Engineering improved expression and output**

cence at low luciferin concentrations [24].

**6**

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.

## **2.4 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 hemoglobin's 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 mutation. This variant produces a red-shifted output with a peak at 615 nm, a narrow emission 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.

### **2.5 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 concentrations 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 binding inhibitors preventing enzyme turnover, or inhibitor-based stabilization can increase activity when substrate levels are high enough to compete with the inhibitory 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 commercial reagents are now widely used to support different experimental needs [14].

Another strategy that has been applied to alter reaction kinetics is the modification 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.

## **3.** *Renilla* **luciferase**

## **3.1 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 significantly 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 mammalian 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 validated 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.

#### **3.2 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 overcome *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 output 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 cysteine 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.

**9**

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

**3.3 Engineering alternative output wavelengths**

compared to an equal initial light flux from RLuc8.

promising route for enhancing RLuc functionality.

**3.4 Engineering split luciferase applications**

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 tissues. 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 encouraging 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

In addition to engineering the protein itself, synthetic coelenterazine substrate analogs have also been created to improve light output and/or yield red-shifted emission 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 bioluminescent 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

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 fragment 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 complementation during MyoD/Id interaction. This study also showed that the split RLuc

*Bioluminescence - Analytical Applications and Basic Biology*

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 significantly less efficient than FLuc and produces a reduced relative light output intensity

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 mammalian 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 validated 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.

The initial limitation for using RLuc as a reporter was its less-than-optimal expression efficiency within mammalian cellular hosts. This limitation was overcome *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 output 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 cysteine 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

**3.** *Renilla* **luciferase**

with a quantum yield of ~7% [31].

**3.2 Engineering improved expression and output**

**3.1 Background**

**8**

applications.

## **3.3 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 tissues. 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 encouraging 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 emission 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 bioluminescent 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.

## **3.4 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 fragment 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 complementation during MyoD/Id interaction. This study also showed that the split RLuc

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.

## **4.** *Gaussia* **luciferase**

## **4.1 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 decarboxylation 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.

### **4.2 Engineering improved expression and output**

To enable improved expression efficiency in biomedical applications, a humanized version of GLuc, hGLuc, was generated *via* codon optimization. This humanoptimized variant has been shown to produce 2000-fold higher bioluminescent signal than the wild-type variant when expressed in mammalian cells [11]. In addition 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 wildtype 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

**11**

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

showed 2-fold enhanced luciferase activity [61].

**4.3 Engineering alternative output wavelengths**

signal due to absorption.

**4.4 Engineering alternative signal kinetics**

**4.5 Engineering split luciferase applications**

signal intensity [60]. The I90L variant produced six times higher light output than the wild-type protein in mammalian cells. Using a directed molecular evolution approach, Degeling et al. also identified a variant (S16K/M43V/V159M) that

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 mutations (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

Wild-type GLuc catalyzes a flash-type bioluminescent reaction, meaning that the light signal decays rapidly following luciferin exposure. Practically, this necessitates 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 [61–63]. 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 wildtype kinetic limitations and enables their use in high-throughput assay formats.

Like RLuc, GLuc's small size (185 amino acids, 19.9 kDa) makes it a good candidate 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 protein-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 - Analytical Applications and Basic Biology*

**4.** *Gaussia* **luciferase**

**4.1 Background**

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.

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 decarboxylation 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

To enable improved expression efficiency in biomedical applications, a humanized version of GLuc, hGLuc, was generated *via* codon optimization. This humanoptimized variant has been shown to produce 2000-fold higher bioluminescent signal than the wild-type variant when expressed in mammalian cells [11]. In addition to mammalian systems, the GLuc gene sequence has also been codon optimized for efficient expression in the alga *Chlamydomonas reinhardtii* [56], the fungus

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 wildtype 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

research community through a variety of engineered improvements.

*Candida albicans* [57], mycobacteria [58], and *Salmonella enterica* [59].

**4.2 Engineering improved expression and output**

**10**

signal intensity [60]. The I90L variant produced six times higher light output than the wild-type protein in mammalian cells. Using a directed molecular evolution approach, Degeling et al. also identified a variant (S16K/M43V/V159M) that showed 2-fold enhanced luciferase activity [61].

## **4.3 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 mutations (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.

## **4.4 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 necessitates 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 [61–63]. 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 wildtype kinetic limitations and enables their use in high-throughput assay formats.

## **4.5 Engineering split luciferase applications**

Like RLuc, GLuc's small size (185 amino acids, 19.9 kDa) makes it a good candidate 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 protein-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].

## **5.** *Oplophorus* **luciferase**

## **5.1 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 tetramer 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 emission 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 luminescent 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].

#### **5.2 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 doubleedged 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 deubiquitinating 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.

**13**

functionality.

**6.2 Initial uses and limitations**

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

in living cells.

**6. Bacterial luciferase**

mous fashion at a wavelength of 490 nm.

**6.1 Background**

**5.3 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 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].

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 discussed 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 attachment 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-

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 complexity of this system relative to its monomeric counterparts, it was not exogenously expressed until the early 1980s. Even then, it was initially utilized through expression 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 structure of the bacterial luciferase heterodimer was determined [74], however, this structural knowledge has yet to be leveraged as a means for engineering improved

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

## **5.3 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].

## **6. Bacterial luciferase**

## **6.1 Background**

*Bioluminescence - Analytical Applications and Basic Biology*

still produce observable light output at 70°C [8].

**5.2 Engineering improved expression and output**

its signal penetration in mammalian cellular applications.

*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 tetramer 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 emission 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

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 luminescent 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].

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 doubleedged 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

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 deubiquitinating 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

**5.** *Oplophorus* **luciferase**

**5.1 Background**

**12**

used to monitor gene expression.

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 discussed 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 attachment 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 autonomous 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 complexity of this system relative to its monomeric counterparts, it was not exogenously expressed until the early 1980s. Even then, it was initially utilized through expression 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 structure of the bacterial luciferase heterodimer was determined [74], however, this structural knowledge has yet to be leveraged as a means for engineering improved functionality.

## **6.2 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 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 bioavailability 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 system 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.

#### **6.3 Engineering eukaryotic expression**

Although several early attempts were made to enable Lux functionality within eukaryotic hosts, none of these achieved significant success [81–83]. 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].

**15**

**7. Fungal luciferase**

**7.1 Background**

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

**6.4 Engineering increased light output**

To overcome Lux's low level of bioluminescent output in human cells the orientation 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

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 oxidoreductase gene, *frp*. These mutations resulted in both improved thermotolerance and a ~7 times increase in bioluminescent production relative to the wild-type sequence.

Just as it has been used extensively as a bioreporter in bacterial species, the engineering 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 compound 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 is the most recent luciferase system to be functionally elucidated and made available for biotechnological applications. At the core of

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, luciferinrequiring 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 producing an enhanced level of signal output relative to its previous incarnation [87].

**6.5 Engineering improved bioreporter functionality**

*Bioluminescence - Analytical Applications and Basic Biology*

**6.3 Engineering eukaryotic expression**

a flavin oxidoreductase gene, *frp*.

such as firefly luciferase [86].

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 bioavailability 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 system 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.

Although several early attempts were made to enable Lux functionality within eukaryotic hosts, none of these achieved significant success [81–83]. 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

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

**14**

## **6.4 Engineering increased light output**

To overcome Lux's low level of bioluminescent output in human cells the orientation 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, luciferinrequiring 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 producing 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 oxidoreductase gene, *frp*. These mutations resulted in both improved thermotolerance and a ~7 times increase in bioluminescent production relative to the wild-type sequence.

## **6.5 Engineering improved bioreporter functionality**

Just as it has been used extensively as a bioreporter in bacterial species, the engineering 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 compound 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.

## **7. Fungal luciferase**

### **7.1 Background**

Fungal luciferase is the most recent luciferase system to be functionally elucidated and made available for biotechnological applications. At the core of this system is a monomeric luciferase gene, *luz*. In addition to the luciferase, two luciferin synthesis genes: *hisps* and *h3h*, work together as a polyketide synthase and a 3-hydroxybenzoate 6-monooxygenase to supply the required luciferin, 3-hydroxyhispidin. 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 cassette to be genetically encoded and exogenously expressed to produce an autobioluminescent 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 *h3h*, 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.

## **7.2 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 autobioluminescent 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 exogenously applied. Using this strategy, it has been possible to observe luminescence in cultured human cells, *Xenopus laevis* embryos, and small animal models subcutaneously 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 production 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 organisms. 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 temperatures 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.

**17**

**9. Conclusion**

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

**7.3 Potential future engineering goals**

**8. Outlook for future developments**

There are ~100 fungal species that use this luciferase/luciferin pathway for bioluminescent 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 evolutionary cues that can be leveraged as starting points for biotechnological advancement. 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 thermostability 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.

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 autobioluminescent 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.

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 - Analytical Applications and Basic Biology*

and continuous light production in any host.

**7.2 Initial uses and limitations**

its luciferin specificity.

this system is a monomeric luciferase gene, *luz*. In addition to the luciferase, two luciferin synthesis genes: *hisps* and *h3h*, work together as a polyketide synthase and a 3-hydroxybenzoate 6-monooxygenase to supply the required luciferin, 3-hydroxy-

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 autobioluminescent 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 exogenously applied. Using this strategy, it has been possible to observe luminescence in cultured human cells, *Xenopus laevis* embryos, and small animal models subcutaneously 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

It is currently unknown if the lack of demonstrated autobioluminescent production 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 organisms. 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 temperatures 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.

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 cassette to be genetically encoded and exogenously expressed to produce an autobioluminescent 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 *h3h*, along with a tyrosine ammonia lyase, two 4-hydroxyphenylacetate 3-monooxygenase components and the 4′-phosphopantetheinyl transferase gene *npgA*, to support caffeic acid synthesis

**16**

## **7.3 Potential future engineering goals**

There are ~100 fungal species that use this luciferase/luciferin pathway for bioluminescent 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 evolutionary cues that can be leveraged as starting points for biotechnological advancement. 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 thermostability 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.

## **8. 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 autobioluminescent 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.

## **9. 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

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.

## **Author details**

Andrew Kirkpatrick1 , Tingting Xu<sup>2</sup> , 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

© 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.

**19**

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

Molecular and Cellular Biology.

Cell. 2005;**4**(9):1539-1549

2005;**11**(3):435-443

2010;**2**:443-493

[10] McNabb DS, Reed R, Marciniak RA. Dual luciferase assay system for rapid assessment of gene expression in *Saccharomyces cerevisiae*. Eukaryotic

[11] Tannous B, Kim D, Fernandez J, Weissleder R, Breakefield X. Codonoptimized *Gaussia* luciferase cDNA for mammalian gene expression in culture and *in vivo*. Molecular Therapy.

[12] Kotlobay AA, Sarkisyan KS, Mokrushina YA, Marcet-Houben M, Serebrovskaya EO, Markina NM, et al. Genetically encodable bioluminescent system from fungi. Proceedings of the National Academy of Sciences of the United States of America. 2018;**115**(50):12728-12732

[13] Haddock SH, Moline MA, Case JF. Bioluminescence in the sea. Annual Review of Marine Science.

[14] Thorne N, Inglese J, Auld DS. Illuminating insights into firefly luciferase and other bioluminescent

[15] Widder EA, Falls B. Review of bioluminescence for engineers and scientists in biophotonics. IEEE Journal of Selected Topics in Quantum Electronics. 2014;**20**(2):232-241

[16] Fraga H. Firefly luminescence: A historical perspective and recent developments. Photochemical & Photobiological Sciences.

[17] England CG, Ehlerding EB, Cai W. NanoLuc: A small luciferase

reporters used in chemical biology. Chemistry & Biology.

2010;**17**(6):646-657

2008;**7**(2):146-158

1987;**7**(2):725-737

[1] Lee J. Bioluminescence: The first 3000 years. Journal of Siberian Federal

[2] Dubois R. Note sur la physiologie des pyrophores. Comptes Redus des Seances de la Societe de Biologie et de ses Paris.

[3] McElroy WD. The energy source for bioluminescence in an isolated system. Proceedings of the National Academy of Sciences of the United States of America. 1947;**33**(11):342-345

[4] McElroy WD, Hastings JW, Sonnenfeld V, Coulombre J. The requirement of riboflavin phosphate for bacterial luminescence. Science.

[5] Belas R, Mileham A, Cohn D, Hilman M, Simon M, Silverman M. Bacterial bioluminescence: Isolation

and expression of the luciferase genes from *Vibrio harveyi*. Science.

[6] Kricka LJ, Leach FR. In memoriam Dr. Marlene DeLuca 1987 OM Smith Lecture. Firefly luciferase: Mechanism of action, cloning and expression of the active enzyme. Bioluminescence and Chemiluminescence. 1989;**3**:1-5

[7] Matthews JC, Hori K, Cormier MJ. Purification and properties of *Renilla reniformis* luciferase. Biochemistry. 1977;**16**(1):85-91

[8] Shimomura O, Masugi T, Johnson FH, Haneda Y. Properties and reaction mechanism of the bioluminescence system of the deep-sea shrimp

*Oplophorus gracilorostris*. Biochemistry.

[9] De Wet JR, Wood K, DeLuca M, Helinski DR, Subramani S. Firefly luciferase gene: Structure and expression in mammalian cells.

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**References**

1885;**8**(2):559-562

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

## **References**

*Bioluminescence - Analytical Applications and Basic Biology*

keep the pace of development strong in the future.

**Acknowledgements**

**Conflict of interest**

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

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.

S.R., G.S., and D.C. are board members in the for-profit entity 490 BioTech.

**18**

**Author details**

Andrew Kirkpatrick1

provided the original work is properly cited.

, Tingting Xu<sup>2</sup>

2 University of Tennessee, Knoxville, Tennessee, USA

\*Address all correspondence to: dan.close@490biotech.com

1 490 BioTech, Inc., Knoxville, Tennessee, USA

© 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,

, Steven Ripp2

, Gary Sayler1,2 and Dan Close1

\*

[1] Lee J. Bioluminescence: The first 3000 years. Journal of Siberian Federal University. 2008;**3**(1):194-205

[2] Dubois R. Note sur la physiologie des pyrophores. Comptes Redus des Seances de la Societe de Biologie et de ses Paris. 1885;**8**(2):559-562

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sensitive bioluminescent reporter for differential gene expression in *Candida albicans*. Journal of Bacteriology. 1996;**178**(1):121-129

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[36] Loening AM, Fenn TD, Wu AM, Gambhir SS. Consensus guided mutagenesis of *Renilla* luciferase yields enhanced stability and light output. Protein Engineering, Design & Selection. 2006;**19**(9):391-400

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[40] Zhao H, Doyle TC, Wong RJ, Cao Y, Stevenson DK, Piwnica-Worms D, et al. Characterization of coelenterazine analogs for measurements of *Renilla* luciferase activity in live cells and living animals. Molecular Imaging. 2004;**3**(1):43-54

[41] Nishihara R, Suzuki H, Hoshino E, Suganuma S, Sato M, Saitoh T, et al. Bioluminescent coelenterazine derivatives with imidazopyrazinone C-6 extended substitution. Chemical Communications. 2015;**51**(2):391-394

[42] Nishihara R, Abe M, Nishiyama S, Citterio D, Suzuki K, Kim SB. Luciferase-specific coelenterazine analogues for optical contaminationfree bioassays. Scientific Reports. 2017;**7**(1):908

[43] Paulmurugan R, Gambhir SS. Monitoring protein-protein interactions using split synthetic *Renilla* luciferase protein-fragmentassisted complementation. Analytical Chemistry. 2003;**75**(7):1584-1589

[44] Jiang Y, Bernard D, Yu Y, Xie Y, Zhang T, Li Y, et al. Split *Renilla* luciferase protein fragment-assisted complementation (SRL-PFAC) to characterize Hsp90-Cdc37 complex and identify critical residues in protein/protein interactions. The Journal of Biological Chemistry. 2010;**285**(27):21023-21036

[45] Wang J, Guo W, Long C, Zhou H, Wang H, Sun X. The split Renilla luciferase complementation assay is useful for identifying the interaction of Epstein-Barr virus protein kinase BGLF4 and a heat shock protein Hsp90. Acta Virologica. 2016;**60**(1):62-70

[46] Kim SB, Ozawa T, Watanabe S, Umezawa Y. High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split *Renilla* luciferase. Proceedings of the National Academy

**20**

*Bioluminescence - Analytical Applications and Basic Biology*

modification. Analytical Biochemistry.

[26] Branchini BR, Southworth TL, Khattak NF, Michelini E, Roda A. Redand green-emitting firefly luciferase mutants for bioluminescent reporter applications. Analytical Biochemistry.

[27] Branchini BR, Southworth TL, Khattak NF, Murtiashaw MH, Fleet SE, editors. Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence. In: Genetically Engineered and Optical Probes for Biomedical Applications II. San Jose, California, USA: International Society for Optics and Photonics; 2004

[28] Mofford DM, Reddy GR, Miller SC. Aminoluciferins extend firefly luciferase bioluminescence into the near-infrared and can be preferred substrates over D-luciferin. Journal of the American Chemical Society.

2014;**136**(38):13277-13282

2013;**42**(2):662-676

1978;**27**(4):389-396

1991;**88**(10):4438-4442

[29] Li J, Chen L, Du L, Li M. Cage the firefly luciferin!—A strategy for developing bioluminescent probes. Chemical Society Reviews.

[30] Ward WW, Cormier MJ. Energy

interaction in Renilla bioluminescence. Photochemistry and Photobiology.

Longiaru M, Cormier MJ. Isolation and expression of a cDNA encoding *Renilla reniformis* luciferase. Proceedings of the National Academy of Sciences of the United States of America.

[32] Srikantha T, Klapach A, Lorenz WW, Tsai LK, Laughlin LA, Gorman JA, et al. The sea pansy *Renilla reniformis* luciferase serves as a

transfer via protein-protein

[31] Lorenz WW, McCann RO,

2007;**366**(2):131-136

2005;**345**(1):140-148

is brightening up the field of bioluminescence. Bioconjugate Chemistry. 2016;**27**(5):1175-1187

[18] Close DM, Ripp S, Sayler GS. Reporter proteins in whole-cell optical bioreporter detection

2009;**9**(11):9147-9174

1985;**82**(23):7870-7873

2002;**59**(11):1833-1850

2012;**2**(3):e201204004

2018;**3**(3):2628-2633

systems, biosensor integrations, and biosensing applications. Sensors.

[19] De Wet JR, Wood KV, Helinski DR, DeLuca M. Cloning of firefly luciferase cDNA and the expression of active luciferase in *Escherichia coli*. Proceedings of the National Academy of Sciences of the United States of Amercia.

[20] Marques SM, Esteves da Silva JC. Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions. IUBMB Life. 2009;**61**(1):6-17

[21] Viviani VR. The origin, diversity, and structure function relationships of insect luciferases. Cellular and Molecular Life Sciences: CMLS.

[22] Wood KV, Lam YA, McElroy WD. Introduction to beetle luciferases and their applications. Journal of Bioluminescence and

Chemiluminescence. 1989;**4**(1):289-301

[24] Pozzo T, Akter F, Nomura Y, Louie AY, Yokobayashi Y. Firefly luciferase mutant with enhanced activity and thermostability. ACS Omega.

[25] Fujii H, Noda K, Asami Y, Kuroda A, Sakata M, Tokida A. Increase in bioluminescence intensity of firefly luciferase using genetic

[23] Koksharov MI, Ugarova NN. Approaches to engineer stability of beetle luciferases. Computational and Structural Biotechnology Journal.

of Sciences of the United States of America. 2004;**101**(32):11542-11547

[47] Kaihara A, Kawai Y, Sato M, Ozawa T, Umezawa Y. Locating a protein−protein interaction in living cells via split *Renilla* luciferase complementation. Analytical Chemistry. 2003;**75**(16):4176-4181

[48] Stefan E, Aquin S, Berger N, Landry CR, Nyfeler B, Bouvier M, et al. Quantification of dynamic protein complexes using *Renilla* luciferase fragment complementation applied to protein kinase a activities *in vivo*. Proceedings of the National Academy of Sciences. 2007;**104**(43):16916-16921

[49] Lund CH, Bromley JR, Stenbaek A, Rasmussen RE, Scheller HV, Sakuragi Y. A reversible *Renilla* luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus. Journal of Experimental Botany. 2015;**66**(1):85-97

[50] Hatzios SK, Ringgaard S, Davis BM, Waldor MK. Studies of dynamic protein-protein interactions in bacteria using *Renilla* luciferase complementation are undermined by nonspecific enzyme inhibition. PLoS One. 2012;**7**(8):e43175

[51] Tannous BA. *Gaussia* luciferase reporter assay for monitoring biological processes in culture and *in vivo*. Nature Protocols. 2009;**4**(4):582-591

[52] Chung E, Yamashita H, Au P, Tannous BA, Fukumura D, Jain RK. Secreted Gaussia luciferase as a biomarker for monitoring tumor progression and treatment response of systemic metastases. PLoS One. 2009;**4**(12):e8316

[53] Badr CE, Hewett JW, Breakefield XO, Tannous BA. A highly sensitive assay for

monitoring the secretory pathway and ER stress. PLoS One. 2007;**2**(6):e571

[54] Suzuki T, Usuda S, Ichinose H, Inouye S. Real-time bioluminescence imaging of a protein secretory pathway in living mammalian cells using *Gaussia* luciferase. FEBS Letters. 2007;**581**(24):4551-4556

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[62] Welsh JP, Patel KG, Manthiram K, Swartz JR. Multiply mutated *Gaussia* luciferases provide prolonged

Biochemical and Biophysical Research Communications. 2009;**389**(4):563-568

[63] Maguire CA, Deliolanis NC, Pike L, Niers JM, Tjon-Kon-Fat LA, Sena-Esteves M, et al. Gaussia luciferase variant for high-throughput functional screening applications. Analytical Chemistry. 2009;**81**(16):7102-7106

[64] Remy I, Michnick SW. A highly sensitive protein-protein interaction assay based on *Gaussia* luciferase. Nature Methods. 2006;**3**(12):977-979

[65] Luker KE, Luker GD. Split *Gaussia* luciferase for imaging ligand-receptor binding. Methods in molecular biology

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[68] Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chemical Biology. 2012;**7**(11):

(Clifton, NJ). 2014;**1098**:59-69

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Journal. 1975;**151**(1):9-15

Letters. 2000;**481**(1):19-25

and intense bioluminescence.

2013;**85**(5):3006-3012

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

*Bioluminescence - Analytical Applications and Basic Biology*

monitoring the secretory pathway and ER stress. PLoS One. 2007;**2**(6):e571

[54] Suzuki T, Usuda S, Ichinose H, Inouye S. Real-time bioluminescence imaging of a protein secretory pathway in living mammalian cells using *Gaussia* luciferase. FEBS Letters.

[55] Bruce BJ, Szent-Gyorgyi CS. Luciferases, fluorescent proteins, nucleic acids encoding the luciferases and fluorescent proteins and the use thereof in diagnostics, high throughput screening and novelty items. U.S. Patent

[56] Shao N, Bock R. A codon-optimized

[57] Enjalbert B, Rachini A, Vediyappan G, Pietrella D, Spaccapelo R, Vecchiarelli A, et al. A multifunctional, synthetic *Gaussia princeps* luciferase reporter for live imaging of *Candida albicans* infections. Infection and Immunity.

luciferase from *Gaussia princeps* facilitates the in vivo monitoring of gene expression in the model alga *Chlamydomonas reinhardtii*. Current Genetics. 2008;**53**(6):381-388

2009;**77**(11):4847-4858

[58] Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, et al. Optimisation of bioluminescent reporters for use with mycobacteria.

[59] Wille T, Blank K, Schmidt C, Vogt V, Gerlach RG. *Gaussia princeps* luciferase as a reporter for transcriptional activity, protein secretion, and protein-protein interactions in *Salmonella enterica* serovar typhimurium. Applied and Environmental Microbiology.

[60] Kim SB, Sato M, Tao H. Split *Gaussia* luciferase-based bioluminescence template for tracing protein dynamics in living cells. Analytical Chemistry.

PLoS One. 2010;**5**(5):e10777

2012;**78**(1):250-257

2009;**81**(1):67-74

2007;**581**(24):4551-4556

6,232,107. 2001

of Sciences of the United States of America. 2004;**101**(32):11542-11547

[47] Kaihara A, Kawai Y, Sato M, Ozawa T, Umezawa Y. Locating a protein−protein interaction in living cells via split *Renilla* luciferase complementation. Analytical Chemistry. 2003;**75**(16):4176-4181

[48] Stefan E, Aquin S, Berger N, Landry CR, Nyfeler B, Bouvier M, et al. Quantification of dynamic protein complexes using *Renilla* luciferase fragment complementation applied to protein kinase a activities *in vivo*. Proceedings of the National Academy of Sciences. 2007;**104**(43):16916-16921

[49] Lund CH, Bromley JR, Stenbaek A, Rasmussen RE, Scheller HV, Sakuragi Y. A reversible *Renilla* luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus. Journal of Experimental Botany. 2015;**66**(1):85-97

[50] Hatzios SK, Ringgaard S, Davis BM, Waldor MK. Studies of dynamic protein-protein interactions in bacteria using *Renilla* luciferase complementation are undermined by nonspecific enzyme inhibition. PLoS

[51] Tannous BA. *Gaussia* luciferase reporter assay for monitoring biological processes in culture and *in vivo*. Nature

Protocols. 2009;**4**(4):582-591

[52] Chung E, Yamashita H, Au P, Tannous BA, Fukumura D, Jain RK. Secreted Gaussia luciferase as a biomarker for monitoring tumor progression and treatment response of systemic metastases. PLoS One.

[53] Badr CE, Hewett JW, Breakefield XO, Tannous BA. A highly sensitive assay for

One. 2012;**7**(8):e43175

2009;**4**(12):e8316

**22**

[61] Degeling MH, Bovenberg MSS, Lewandrowski GK, de Gooijer MC, Vleggeert-Lankamp CLA, Tannous M, et al. Directed molecular evolution reveals *Gaussia* luciferase variants with enhanced light output stability. Analytical Chemistry. 2013;**85**(5):3006-3012

[62] Welsh JP, Patel KG, Manthiram K, Swartz JR. Multiply mutated *Gaussia* luciferases provide prolonged and intense bioluminescence. Biochemical and Biophysical Research Communications. 2009;**389**(4):563-568

[63] Maguire CA, Deliolanis NC, Pike L, Niers JM, Tjon-Kon-Fat LA, Sena-Esteves M, et al. Gaussia luciferase variant for high-throughput functional screening applications. Analytical Chemistry. 2009;**81**(16):7102-7106

[64] Remy I, Michnick SW. A highly sensitive protein-protein interaction assay based on *Gaussia* luciferase. Nature Methods. 2006;**3**(12):977-979

[65] Luker KE, Luker GD. Split *Gaussia* luciferase for imaging ligand-receptor binding. Methods in molecular biology (Clifton, NJ). 2014;**1098**:59-69

[66] Yamaguchi I. *Oplophorus* oxyluciferin and a model luciferin compound biologically active with *Oplophorus luciferase*. The Biochemical Journal. 1975;**151**(1):9-15

[67] Inouye S, Watanabe K, Nakamura H, Shimomura O. Secretional luciferase of the luminous shrimp *Oplophorus gracilirostris*: cDNA cloning of a novel imidazopyrazinone luciferase. FEBS Letters. 2000;**481**(1):19-25

[68] Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chemical Biology. 2012;**7**(11): 1848-1857

[69] Chen Y, Wang L, Cheng X, Ge X, Wang P. An ultrasensitive system for measuring the USPs and OTULIN activity using Nanoluc as a reporter. Biochemical and Biophysical Research Communications. 2014;**455**(3-4):178-183

[70] Lackner DH, Carré A, Guzzardo PM, Banning C, Mangena R, Henley T, et al. A generic strategy for CRISPR-Cas9-mediated gene tagging. Nature Communications. 2015;**6**:10237

[71] Zhao J, Nelson TJ, Vu Q, Truong T, Stains CI. Self-assembling NanoLuc luciferase fragments as probes for protein aggregation in living cells. ACS Chemical Biology. 2015;**11**(1):132-138

[72] Meighen EA. Molecular biology of bacterial bioluminescence. Microbiological Reviews. 1991;**55**:123-142

[73] Engebrecht J, Nealson K, Silverman M. Bacterial bioluminescence: Isolation and genetic analysis of functions from *Vibrio fischeri*. Cell. 1983;**32**:773-781

[74] Fisher AJ, Raushel FM, Baldwin TO, Rayment I. Three-dimensional structure of bacterial luciferase from *Vibrio harveyi* at 2.4. ANG. Resolution. Biochemistry. 1995;**34**(20):6581-6586

[75] Engebrecht J, Simon M, Silverman M. Measuring gene expression with light. Science. 1985;**227**(4692):1345-1347

[76] King JMH, Digrazia PM, Applegate B, Burlage R, Sanseverino J, Dunbar P, et al. Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegradation. Science. 1990;**249**(4970):778-781

[77] Shaw JJ, Kado CI. Development of a *Vibrio* bioluminescence gene set to monitor phytopathogenic bacteria during the ongoing disease process in a nondisruptive manner. Nature Biotechnology. 1986;**4**(6):560-564

[78] de Weger L, Dunbar P, Mahafee F, Lugtenberg B, Sayler G. Use of bioluminescence markers to detect *Pseudomonas* spp. in the rhizosphere. Applied and Environmental Microbiology. 1991;**57**:3641-3644

[79] Ripp S, Nivens DE, Ahn Y, Werner C, Jarrell J, Easter JP, et al. Controlled field release of a bioluminescent genetically engineered microorganism for bioremediation process monitoring and control. Environmental Science & Technology. 2000;**34**(5):846-853

[80] Kuklin NA, Pancari GD, Tobery TW, Cope L, Jackson J, Gill C, et al. Real-time monitoring of bacterial infection *in vivo*: Development of bioluminescent staphylococcal foreignbody and deep-thigh-wound mouse infection models. Antimicrobial Agents and Chemotherapy. 2003;**47**(9):2740-2748

[81] Almashanu S, Musafia B, Hadar R, Suissa M, Kuhn J. Fusion of *luxA* and *luxB* and its expression in *Escherichia coli*, *Saccharomyces cerevisiae* and *Drosophila melanogaster*. Journal of Bioluminescence and Chemiluminescence. 1990;**5**(1):89-97

[82] Kirchner G, Roberts JL, Gustafson GD, Ingolia TD. Active bacterial luciferase from a fused gene: Expression of a *Vibrio harveyi luxAB* translational fusion in bacteria, yeast and plant cells. Gene. 1989;**81**(2):349-354

[83] Olsson O, Koncz C, Szalay AA. The use of the *luxA* gene of the bacterial luciferase operon as a reporter gene. Molecular & General Genetics. 1988;**215**(1):1-9

[84] Gupta RK, Patterson SS, Ripp S, Sayler GS. Expression of the *Photorhabdus luminescens lux* genes (*luxA*, *B*, *C*, *D*, and *E*) in *Saccharomyces cerevisiae*. FEMS Yeast Research. 2003;**4**(3):305-313

[85] Patterson SS, Dionisi HM, Gupta RK, Sayler GS. Codon optimization of bacterial luciferase (*lux*) for expression in mammalian cells. Journal of Industrial Microbiology & Biotechnology. 2005;**32**(3):115-123

[86] Close DM, Hahn R, Patterson SS, Ripp S, Sayler GS. Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models. Journal of Biomedical Optics. 2011;**16**(4):e12441

[87] Xu T, Ripp S, Sayler G, Close D. Expression of a humanized viral 2A-mediated *lux* operon efficiently generates autonomous bioluminescence in human cells. PLoS One. 2014;**9**(5):e96347

[88] Gregor C, Gwosch KC, Sahl SJ, Hell SW. Strongly enhanced bacterial bioluminescence with the *ilux* operon for single-cell imaging. Proceedings of the National Academy of Sciences. 2018;**115**(5):962-967

[89] Welham PA, Stekel DJ. Mathematical model of the *Lux* luminescence system in the terrestrial bacterium *Photorhabdus luminescens*. Molecular BioSystems. 2009;**5**(1):68-76

[90] Yagur Kroll S, Belkin S. Upgrading bioluminescent bacterial bioreporter performance by splitting the *lux* operon. Analytical and Bioanalytical Chemistry. 2011;**400**(4):1071-1082

[91] Airth R, McElroy W. Light emission from extracts of luminous fungi. Journal of Bacteriology. 1959;**77**(2):249-250

[92] Oliveira AG, Stevani CV. The enzymatic nature of fungal bioluminescence. Photochemical & Photobiological Sciences. 2009;**8**(10):1416-1421

[93] Oliveira AG, Desjardin DE, Perry BA, Stevani CV. Evidence that a single

**25**

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

bioluminescent system is shared by all known bioluminescent fungal lineages. Photochemical & Photobiological Sciences. 2012;**11**(5):848-852

[94] Oba Y, Stevani CV, Oliveira AG, Tsarkova AS, Chepurnykh TV, Yampolsky IV. Selected least studied but not forgotten bioluminescent systems. Photochemistry and Photobiology.

2017;**93**(2):405-415

*Biotechnological Advances in Luciferase Enzymes DOI: http://dx.doi.org/10.5772/intechopen.85313*

*Bioluminescence - Analytical Applications and Basic Biology*

[85] Patterson SS, Dionisi HM, Gupta RK, Sayler GS. Codon optimization of bacterial luciferase (*lux*) for expression in mammalian cells. Journal of Industrial Microbiology & Biotechnology. 2005;**32**(3):115-123

[86] Close DM, Hahn R, Patterson SS, Ripp S, Sayler GS. Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models. Journal of Biomedical Optics. 2011;**16**(4):e12441

[87] Xu T, Ripp S, Sayler G, Close D. Expression of a humanized viral 2A-mediated *lux* operon efficiently generates autonomous bioluminescence

[88] Gregor C, Gwosch KC, Sahl SJ, Hell SW. Strongly enhanced bacterial bioluminescence with the *ilux* operon for single-cell imaging. Proceedings of the National Academy of Sciences.

[90] Yagur Kroll S, Belkin S. Upgrading bioluminescent bacterial bioreporter performance by splitting the *lux* operon. Analytical and Bioanalytical Chemistry.

[91] Airth R, McElroy W. Light emission from extracts of luminous fungi. Journal of Bacteriology. 1959;**77**(2):249-250

[92] Oliveira AG, Stevani CV. The enzymatic nature of fungal bioluminescence. Photochemical & Photobiological Sciences. 2009;**8**(10):1416-1421

[93] Oliveira AG, Desjardin DE, Perry BA, Stevani CV. Evidence that a single

in human cells. PLoS One.

2014;**9**(5):e96347

2018;**115**(5):962-967

[89] Welham PA, Stekel DJ. Mathematical model of the *Lux* luminescence system in the terrestrial bacterium *Photorhabdus luminescens*. Molecular BioSystems. 2009;**5**(1):68-76

2011;**400**(4):1071-1082

[78] de Weger L, Dunbar P, Mahafee F, Lugtenberg B, Sayler G. Use of bioluminescence markers to detect *Pseudomonas* spp. in the rhizosphere.

[79] Ripp S, Nivens DE, Ahn Y, Werner C, Jarrell J, Easter JP, et al. Controlled field release of a bioluminescent genetically engineered microorganism for bioremediation process monitoring and control. Environmental Science & Technology. 2000;**34**(5):846-853

[80] Kuklin NA, Pancari GD, Tobery TW, Cope L, Jackson J, Gill C, et al. Real-time monitoring of bacterial infection *in vivo*: Development of bioluminescent staphylococcal foreignbody and deep-thigh-wound mouse infection models. Antimicrobial Agents and Chemotherapy. 2003;**47**(9):2740-2748

[81] Almashanu S, Musafia B, Hadar R, Suissa M, Kuhn J. Fusion of *luxA* and *luxB* and its expression in *Escherichia coli*, *Saccharomyces cerevisiae* and *Drosophila melanogaster*. Journal of

[82] Kirchner G, Roberts JL, Gustafson GD, Ingolia TD. Active bacterial

luciferase from a fused gene: Expression of a *Vibrio harveyi luxAB* translational fusion in bacteria, yeast and plant cells.

[83] Olsson O, Koncz C, Szalay AA. The use of the *luxA* gene of the bacterial luciferase operon as a reporter gene. Molecular & General Genetics.

[84] Gupta RK, Patterson SS, Ripp S, Sayler GS. Expression of the *Photorhabdus luminescens lux* genes (*luxA*, *B*, *C*, *D*, and *E*) in *Saccharomyces cerevisiae*. FEMS Yeast Research.

Bioluminescence

1990;**5**(1):89-97

1988;**215**(1):1-9

2003;**4**(3):305-313

and Chemiluminescence.

Gene. 1989;**81**(2):349-354

Applied and Environmental Microbiology. 1991;**57**:3641-3644

**24**

bioluminescent system is shared by all known bioluminescent fungal lineages. Photochemical & Photobiological Sciences. 2012;**11**(5):848-852

[94] Oba Y, Stevani CV, Oliveira AG, Tsarkova AS, Chepurnykh TV, Yampolsky IV. Selected least studied but not forgotten bioluminescent systems. Photochemistry and Photobiology. 2017;**93**(2):405-415

**27**

**Chapter 2**

**Abstract**

developments are anticipated.

NanoLuc ternary technology

ings, and immunoassays.

**1. Introduction**

Protein-Protein Interaction Assays

Protein-protein interaction assays are fundamental to basic biology, drug discovery, diagnostics, screening, and immunoassays. Protein-fragment complementation (PCA) is one of such useful protein-protein interaction assays. PCA when performed using luciferase is a reversible approach, whereas when performed using green fluorescent protein analogs is an irreversible approach. The NanoLuc technology developed in 2012 utilizes a small and structurally robust luciferase that is capable of producing very bright luminescence. NanoLuc PCA has been used to detect many protein-protein interactions and for screening purposes. Methods developed from NanoLuc PCA include the HiBiT technology and NanoLuc ternary technology. These novel technologies are promising in various fields and further

**Keywords:** NanoLuc, PCA, NanoBiT, protein-protein interaction, HiBiT,

It is predicted that there are 150,000–650,000 protein-protein interactions in the human interactome [1–3]. Protein-protein interaction assays have been developed and used for studies on basic biology, drug discoveries, diagnostics, screen-

In 1994, the first protein-fragment complementation assay (PCA) was developed

using split ubiquitin [4]. PCA typically uses two-split reporter proteins that are fused to the target proteins. The interaction leads to the association of the fragments and the subsequent reconstitution of the full-length structure from the two fragments (**Figure 1**) [5–8]. More recently, fluorescent proteins and luciferase enzymes have been widely utilized for innovative PCAs. Reversible PCAs generally utilize enzymes, and the exceptions are two fluorescent proteins IFP1.4 and UnaG [9, 10]. Most other PCA systems that use fluorescent proteins, including green fluorescent protein (GFP) analogs, show irreversible behavior. In such irreversible assays, once the full-length structure is reconstituted, it is difficult to separate them into the two fragments when dissociation occurs after the interaction. On the contrary, in the reversible PCA systems, both interaction and dissociation can be detected. Therefore, PCA systems using enzymes, such as luciferase, are more suitable to detect the spatiotemporal dynamics of protein-protein interactions. However, until recently, the luminescent signal is significantly weaker than the fluorescent signal. Recently, a novel luciferase enzyme, NanoLuc, and its furimazine substrate were developed [11, 12]. NanoLuc is small (19 kDa) and structurally stable, and produces

Using Split-NanoLuc

*Yuki Ohmuro-Matsuyama and Hiroshi Ueda*

## **Chapter 2**

## Protein-Protein Interaction Assays Using Split-NanoLuc

*Yuki Ohmuro-Matsuyama and Hiroshi Ueda*

## **Abstract**

Protein-protein interaction assays are fundamental to basic biology, drug discovery, diagnostics, screening, and immunoassays. Protein-fragment complementation (PCA) is one of such useful protein-protein interaction assays. PCA when performed using luciferase is a reversible approach, whereas when performed using green fluorescent protein analogs is an irreversible approach. The NanoLuc technology developed in 2012 utilizes a small and structurally robust luciferase that is capable of producing very bright luminescence. NanoLuc PCA has been used to detect many protein-protein interactions and for screening purposes. Methods developed from NanoLuc PCA include the HiBiT technology and NanoLuc ternary technology. These novel technologies are promising in various fields and further developments are anticipated.

**Keywords:** NanoLuc, PCA, NanoBiT, protein-protein interaction, HiBiT, NanoLuc ternary technology

#### **1. Introduction**

It is predicted that there are 150,000–650,000 protein-protein interactions in the human interactome [1–3]. Protein-protein interaction assays have been developed and used for studies on basic biology, drug discoveries, diagnostics, screenings, and immunoassays.

In 1994, the first protein-fragment complementation assay (PCA) was developed using split ubiquitin [4]. PCA typically uses two-split reporter proteins that are fused to the target proteins. The interaction leads to the association of the fragments and the subsequent reconstitution of the full-length structure from the two fragments (**Figure 1**) [5–8]. More recently, fluorescent proteins and luciferase enzymes have been widely utilized for innovative PCAs. Reversible PCAs generally utilize enzymes, and the exceptions are two fluorescent proteins IFP1.4 and UnaG [9, 10]. Most other PCA systems that use fluorescent proteins, including green fluorescent protein (GFP) analogs, show irreversible behavior. In such irreversible assays, once the full-length structure is reconstituted, it is difficult to separate them into the two fragments when dissociation occurs after the interaction. On the contrary, in the reversible PCA systems, both interaction and dissociation can be detected. Therefore, PCA systems using enzymes, such as luciferase, are more suitable to detect the spatiotemporal dynamics of protein-protein interactions. However, until recently, the luminescent signal is significantly weaker than the fluorescent signal.

Recently, a novel luciferase enzyme, NanoLuc, and its furimazine substrate were developed [11, 12]. NanoLuc is small (19 kDa) and structurally stable, and produces

#### **Figure 1.**

*Basic principle of PCA. (A) The reporter protein (F) is separated into two fragments (N and C). (B) N and C are fused to target protein (left). When the interaction occurs, N and C move to the neighboring position, and the full-length reporter protein is reconstituted.*

very bright luminescence. Based on this attractive enzyme, PCA systems were developed [13, 14]. This innovation on the NanoLuc PCA improves the luminescent signal, which is markedly better than the conventional PCA signal obtained using other luciferases. Herein, we will focus on the new PCA technology and its application, and further discuss potential improvements in the system.

#### **2. PCA using NanoLuc**

Verhoef et al. constructed a PCA system using NanoLuc [14]. They made several pairs of NanoLuc fragments by cutting at several loop regions, and selected a pair comprised of the N-terminal 52-amino acid (aa) fragment and the C-terminal 119-aa fragment (**Figure 2A**). These fragments were used to successfully detect the interaction between the transactivation domain fragment of p53 and Mdm2.

At almost the same time, Dixon et al. developed another NanoLuc-based PCA system designated *Nano*Luc *Bi*nary *T*echnology (NanoBiT) [13]. This was devised by first identifying a dissection site from 90 candidate sites. An 18-kDa N-terminal fragment and 13-aa C-terminal fragment were selected. The *KD* value between these fragments was 6 μM. This low affinity was suitable for PCA, but their use was hampered by the very low stability of the N-terminal fragment. The sequence of the N-terminal fragment was optimized from an N-terminal library containing 15,000 variants. The optimization increased the luminescent signal by 300-fold when the two fragments were interacting, which was 37% that of the wild-type NanoLuc. However, the affinity between the N- and C-terminal fragments became too strong for PCA (*K*D = 900 nM). As a next step, the sequence of the C-terminal peptide was optimized from 350 variants. Finally, two fragments were obtained. They were designated LgBiT (18 kDa) and SmBiT (11 aa). These exhibited significantly low

#### **Figure 2.**

*Two systems of NanoLuc PCA. (A) Verhoef et al. separated NanoLuc into N-terminal 52-aa fragment and C-terminal 119-aa fragment. (B) Dixon et al. separated NanoLuc into the large fragment, LgBiT (18 kDa), and the small fragment, SmBiT (11 aa).*

**29**

*Protein-Protein Interaction Assays Using Split-NanoLuc DOI: http://dx.doi.org/10.5772/intechopen.86122*

affinity (*K*D > 10 μM) and high luminescent intensity. The very bright signal and remarkably high signal/background ratio obtained enabled the quantitative detection of several interactions. Furthermore, the luminescent signals were capable of rapid change and were reversible depending on changing interactions (**Figure 2B**).

In spite of recent appearance, NanoBiT has been already used to analyze several protein-protein interactions. Elevation of plasma triglycerides causes various metabolic diseases. These triglycerides are digested by lipoprotein lipase [15–19]. Chi et al. used NanoBiT to demonstrate the association between lipoprotein lipase and angiopoietin-like 3 (ANGPTL3) induced by ANGPTL8 [20]. They further described

Guanine nucleotide-binding (G) protein-coupled receptors (GPCRs) bind G proteins or β-arrestins, and initiate several cellular signaling events. Regulator of G protein signaling (RGS) proteins regulate G proteins. The regulation has been implicated in several disease states, including various cancers, Parkinson's disease, and cardiomyopathy [21–25]. Several reports have described the use of NanoBiT to analyze the mechanisms of GPCRs. These included the interaction of several sets of RGS proteins and G proteins [26] and the interaction between the galanin receptor 2 GPCR and β-arrestin2 [27]. Furthermore, the LgBiT-fused galanin receptor 2 was modified with a fluorescent dye, and the conformational changes of galanin receptor induced by the binding of ligands, including galanin, spexin, and Fmoc-dA4-dQ14, were analyzed by bioluminescence resonance energy transfer (BRET). Stome et al. applied NanoBiT to analyze the interaction between the GPCR adenosine receptor 3 and β-arrestin2, and observed that the 1-deoxy-1-[6-[[(3-iodophenyl)methyl] amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (2-CI-IB-MECA) agonist recruited β-arrestin2 [28]. In addition, the authors described the importance of the phosphorylation site of adenosine receptor 3 for the association. The site was implicated as a potential clinical target. Melanocortin receptors are also categorized as GPCRs. Melanocortin 4 receptor (MC4R) binds one of melanocortins α-melanocyte-stimulating hormone (α-MSH), which is considered is important in obesity. Habara et al. isolated melanocortin receptor 4 and its regulator Melanocortin 2 receptor accessory protein 2 (MRAP2) from cats and analyzed the heterodimerization of these proteins [29]. Leory et al. characterized several mutants of Janus Kinase 2 in signaling by a transmembrane cytokine receptor, erythropoietin receptor [30]. Interactions with other membrane proteins implicated as important drug targets were also analyzed by NanoBiT. Folding and steric hindrance are problematic for many membrane proteins. The small size of SmBiT could eliminate these problems. O'Neil et al. revealed the amino acids of NADPH that were important for the interaction with p22 using NanoBiT [31]. Chaudhri et al. applied NanoBiT to analyze the association between programmed death ligand 1 (PD-L1) and B7–1 [32]. Peptide hormone, a member of the relaxin family of peptides, participates in reproduction, food intake, stress response, and glucose homeostasis [33–36]. Hu et al. demonstrated the association between the relaxin family peptide receptors peptide 3 and peptide 4 using NanoBiT [37]. The same group further reported that the interaction is electrostatic by analyzing the association between several mutant ligands and their receptors [38]. Equilibrative nucleoside transporters regulate the levels of adenosine and hypoxanthine level, and are crucial in purinergic signaling in the central nervous system, cardiovascular and renal systems, and in pathophysiological conditions including myocardial ischemia, inflammation, and diabetic nephropathy [39–41]. Grañe-Boladeras et al. analyzed the homo- and

**3. Application of NanoBiT for analysis of protein-protein interaction**

that the association inhibits the digestion activity of lipoprotein lipase.

*Bioluminescence - Analytical Applications and Basic Biology*

very bright luminescence. Based on this attractive enzyme, PCA systems were developed [13, 14]. This innovation on the NanoLuc PCA improves the luminescent signal, which is markedly better than the conventional PCA signal obtained using other luciferases. Herein, we will focus on the new PCA technology and its applica-

*Basic principle of PCA. (A) The reporter protein (F) is separated into two fragments (N and C). (B) N and C are fused to target protein (left). When the interaction occurs, N and C move to the neighboring position, and* 

Verhoef et al. constructed a PCA system using NanoLuc [14]. They made several pairs of NanoLuc fragments by cutting at several loop regions, and selected a pair comprised of the N-terminal 52-amino acid (aa) fragment and the C-terminal 119-aa fragment (**Figure 2A**). These fragments were used to successfully detect the interaction between the transactivation domain fragment of p53 and Mdm2.

At almost the same time, Dixon et al. developed another NanoLuc-based PCA system designated *Nano*Luc *Bi*nary *T*echnology (NanoBiT) [13]. This was devised by first identifying a dissection site from 90 candidate sites. An 18-kDa N-terminal fragment and 13-aa C-terminal fragment were selected. The *KD* value between these fragments was 6 μM. This low affinity was suitable for PCA, but their use was hampered by the very low stability of the N-terminal fragment. The sequence of the N-terminal fragment was optimized from an N-terminal library containing 15,000 variants. The optimization increased the luminescent signal by 300-fold when the two fragments were interacting, which was 37% that of the wild-type NanoLuc. However, the affinity between the N- and C-terminal fragments became too strong for PCA (*K*D = 900 nM). As a next step, the sequence of the C-terminal peptide was optimized from 350 variants. Finally, two fragments were obtained. They were designated LgBiT (18 kDa) and SmBiT (11 aa). These exhibited significantly low

*Two systems of NanoLuc PCA. (A) Verhoef et al. separated NanoLuc into N-terminal 52-aa fragment and C-terminal 119-aa fragment. (B) Dixon et al. separated NanoLuc into the large fragment, LgBiT (18 kDa),* 

tion, and further discuss potential improvements in the system.

**2. PCA using NanoLuc**

*the full-length reporter protein is reconstituted.*

**Figure 1.**

**28**

**Figure 2.**

*and the small fragment, SmBiT (11 aa).*

affinity (*K*D > 10 μM) and high luminescent intensity. The very bright signal and remarkably high signal/background ratio obtained enabled the quantitative detection of several interactions. Furthermore, the luminescent signals were capable of rapid change and were reversible depending on changing interactions (**Figure 2B**).

## **3. Application of NanoBiT for analysis of protein-protein interaction**

In spite of recent appearance, NanoBiT has been already used to analyze several protein-protein interactions. Elevation of plasma triglycerides causes various metabolic diseases. These triglycerides are digested by lipoprotein lipase [15–19]. Chi et al. used NanoBiT to demonstrate the association between lipoprotein lipase and angiopoietin-like 3 (ANGPTL3) induced by ANGPTL8 [20]. They further described that the association inhibits the digestion activity of lipoprotein lipase.

Guanine nucleotide-binding (G) protein-coupled receptors (GPCRs) bind G proteins or β-arrestins, and initiate several cellular signaling events. Regulator of G protein signaling (RGS) proteins regulate G proteins. The regulation has been implicated in several disease states, including various cancers, Parkinson's disease, and cardiomyopathy [21–25]. Several reports have described the use of NanoBiT to analyze the mechanisms of GPCRs. These included the interaction of several sets of RGS proteins and G proteins [26] and the interaction between the galanin receptor 2 GPCR and β-arrestin2 [27]. Furthermore, the LgBiT-fused galanin receptor 2 was modified with a fluorescent dye, and the conformational changes of galanin receptor induced by the binding of ligands, including galanin, spexin, and Fmoc-dA4-dQ14, were analyzed by bioluminescence resonance energy transfer (BRET). Stome et al. applied NanoBiT to analyze the interaction between the GPCR adenosine receptor 3 and β-arrestin2, and observed that the 1-deoxy-1-[6-[[(3-iodophenyl)methyl] amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (2-CI-IB-MECA) agonist recruited β-arrestin2 [28]. In addition, the authors described the importance of the phosphorylation site of adenosine receptor 3 for the association. The site was implicated as a potential clinical target. Melanocortin receptors are also categorized as GPCRs. Melanocortin 4 receptor (MC4R) binds one of melanocortins α-melanocyte-stimulating hormone (α-MSH), which is considered is important in obesity. Habara et al. isolated melanocortin receptor 4 and its regulator Melanocortin 2 receptor accessory protein 2 (MRAP2) from cats and analyzed the heterodimerization of these proteins [29]. Leory et al. characterized several mutants of Janus Kinase 2 in signaling by a transmembrane cytokine receptor, erythropoietin receptor [30].

Interactions with other membrane proteins implicated as important drug targets were also analyzed by NanoBiT. Folding and steric hindrance are problematic for many membrane proteins. The small size of SmBiT could eliminate these problems. O'Neil et al. revealed the amino acids of NADPH that were important for the interaction with p22 using NanoBiT [31]. Chaudhri et al. applied NanoBiT to analyze the association between programmed death ligand 1 (PD-L1) and B7–1 [32]. Peptide hormone, a member of the relaxin family of peptides, participates in reproduction, food intake, stress response, and glucose homeostasis [33–36]. Hu et al. demonstrated the association between the relaxin family peptide receptors peptide 3 and peptide 4 using NanoBiT [37]. The same group further reported that the interaction is electrostatic by analyzing the association between several mutant ligands and their receptors [38]. Equilibrative nucleoside transporters regulate the levels of adenosine and hypoxanthine level, and are crucial in purinergic signaling in the central nervous system, cardiovascular and renal systems, and in pathophysiological conditions including myocardial ischemia, inflammation, and diabetic nephropathy [39–41]. Grañe-Boladeras et al. analyzed the homo- and

hetero-oligomerization of ENT1 and ENT2, and revealed that the phosphorylation by protein kinase C promotes oligomerization [42].

#### **4. Application of NanoBiT for screening**

Several groups have successfully used NanoBiT in highly accurate drug screening, including illegal drugs [43, 44]. In the latter studies, β-arrestin2 was fused to SmBiT, and the CB1 and CB2 GPCRs of cannabinoid (the neurologically active component of cannabis) were fused to LgBiT. As cannabinoid induces the interaction between β-arrestin2 and these receptors, the luminescent intensity was increased by adding synthetic cannabinoids and their metabolites. The synthetic cannabinoids and metabolites were detected in subnanomolar concentrations in authentic urine samples with an accuracy rate of 73%.

Next, the authors tried to detect synthetic opioids, which act similarly to heroin or morphine. The μ-opioid receptor and β-arrestin2, which interact in the presence of opioid, were fused to LgBiT and SmBiT, respectively [45]. The system was nearly 100% successful in detecting subnanomolar levels of the synthetic opioids in blood samples.

Aggregation of TDP (transactivating response region DNA binding protein)-43 occurs in approximately 95% of amyotrophic lateral sclerosis patients [46, 47]. Oberstadt et al. constructed a screening system for inhibitors of aggregation by the fusion between the LgBiT and SmBiT probes and TDP-43 [48]. Aurorafin, chelerythrine, and riluzole were identified as inhibitors from the Library of Pharmacologically Active Compounds (LOPAC1280).

Stomes et al. selected agonists of the interaction between adenosine receptor 3 and β-arrestin2 and revealed the structural features of the selected ligands [28].

The NanoBiT screening system is not only effective for drug screening but can be valuable to screen enzyme substrates. Peptide ligases, which can connect two polypeptides, are powerful tools for protein engineering [49–52]. Li et al. performed the screening of substrates of the peptide ligase Sortase A by fusing this enzyme to SmBiT and the candidate peptides to LgBiT [53]. In addition to known substrate sequences, they rapidly identified some previously unknown substrates with varying activities. In addition, the measurement was very stable, and the signal was maintained for more than 16 h.

#### **5. Application of NanoBiT using self-assembling NanoLuc fragments**

Self-assembling NanoLuc fragments have been used to detect protein aggregation, to detect the edited protein by CRISPR/Cas9, to monitor viral entry, release, and propagation, and to analyze clathrin-dependent internalization (**Figure 3**).

**Figure 3.**

*Scheme for protein fragments self-assembly. Since the affinity between N and C is high, the full-length reporter protein is reconstituted by just mixing N and C.*

**31**

**Figure 4.**

*Protein-Protein Interaction Assays Using Split-NanoLuc DOI: http://dx.doi.org/10.5772/intechopen.86122*

The first description of the use of self-assembling NanoLuc fragments was provided by Zhao et al [54]. NanoLuc was separated into two fragments, N65 (1–65 aa) and 66C (66–171 aa). NanoLuc was rapidly reconstituted when N65 and 66C were mixed. Next, N65 was fused to the target proteins. When the target protein was soluble, N65, which had the correct structure, could reassemble with 66C, resulting in recovery of the luminescence. On the other hand, the insoluble target protein did not induce the recovery of the luminescence, because the aggregated N65 could not assemble with 66C (**Figure 4**). The aggregations of amyloid-β mutants were assessed using the system. Similar monitoring systems of protein aggregation using split-GFP and conventional split-luciferase systems had been previously reported [55–60]. However, a time lag occurred for the chromophore formation in the split-GFP system, and other luciferases were relatively unstable compared with NanoLuc.

*Self-assembling Nluc fragment as a probe for protein aggregation. (A) N65 is fused to the target protein. When the fusion protein is not aggregated, NanoLuc is reconstituted by the addition of 66C. (B) When the target* 

*protein is aggregated, N65 is also aggregated, and, then, the reassembly does not occur.*

Zhao et al. succeeded the robust measurement of amyloid-β in this study.

the small size as further described below.

formation (**Figure 5B**) [62].

Other self-assembling NanoLuc fragments were described [13]. The SmBiT sequence was optimized using peptides with different affinities to LgBiT. Of the candidates, the HiBiT peptide displayed high affinity (*K*D = 700 pM) although the affinity of SmBiT was very low (*K*D > 100 μM). HiBiT (11 aa) and LgBiT assembled spontaneously, allowing the construction of NanoLuc. HiBiT is a useful tag due to

In one of the split-GFP systems, GFP was split into two fragments [57]. The C-terminal fragment of GFP contains 16 aa (GFP11). Waldo et al. found that this and the other fragment (GFP1–10) expressed in the cell assembled spontaneously, and the GFP fluorescence was recovered. Leonetti et al. described the synthesis of the donor DNA templates encoding GFP11 and the tagging of endogenous proteins using CRISPR/Cas9 (**Figure 5A**) [61]. The formation of full-length GFP was induced by coexpression with GFP1–10, and the tagging endogenous protein by GFP11 could be detected. Instead of GFP11, Schwinn et al. used HiBiT as the tag for endogenous proteins and were successful in achieving the highly efficient integration and monitoring of the expression dynamics of the tagging proteins without the time lag, which occurs in the split-GFP system due to the chromophore

Ryes-Alaraz et al. analyzed the internalization of galanin receptor 2, which is dependent on the binding of the endogenous ligand, using an HiBiT-fused galanin receptor 2 [27]. LgBiT could bind to the HiBiT-fused receptor on the cell surface.

*Protein-Protein Interaction Assays Using Split-NanoLuc DOI: http://dx.doi.org/10.5772/intechopen.86122*

**Figure 4.**

*Bioluminescence - Analytical Applications and Basic Biology*

by protein kinase C promotes oligomerization [42].

**4. Application of NanoBiT for screening**

samples with an accuracy rate of 73%.

Pharmacologically Active Compounds (LOPAC1280).

signal was maintained for more than 16 h.

*protein is reconstituted by just mixing N and C.*

hetero-oligomerization of ENT1 and ENT2, and revealed that the phosphorylation

Several groups have successfully used NanoBiT in highly accurate drug screening, including illegal drugs [43, 44]. In the latter studies, β-arrestin2 was fused to SmBiT, and the CB1 and CB2 GPCRs of cannabinoid (the neurologically active component of cannabis) were fused to LgBiT. As cannabinoid induces the interaction between β-arrestin2 and these receptors, the luminescent intensity was increased by adding synthetic cannabinoids and their metabolites. The synthetic cannabinoids and metabolites were detected in subnanomolar concentrations in authentic urine

Next, the authors tried to detect synthetic opioids, which act similarly to heroin or morphine. The μ-opioid receptor and β-arrestin2, which interact in the presence of opioid, were fused to LgBiT and SmBiT, respectively [45]. The system was nearly 100% successful in detecting subnanomolar levels of the synthetic opioids in blood samples. Aggregation of TDP (transactivating response region DNA binding protein)-43

occurs in approximately 95% of amyotrophic lateral sclerosis patients [46, 47]. Oberstadt et al. constructed a screening system for inhibitors of aggregation by the fusion between the LgBiT and SmBiT probes and TDP-43 [48]. Aurorafin, chelerythrine, and riluzole were identified as inhibitors from the Library of

**5. Application of NanoBiT using self-assembling NanoLuc fragments**

Self-assembling NanoLuc fragments have been used to detect protein aggregation, to detect the edited protein by CRISPR/Cas9, to monitor viral entry, release, and propagation, and to analyze clathrin-dependent internalization (**Figure 3**).

*Scheme for protein fragments self-assembly. Since the affinity between N and C is high, the full-length reporter* 

Stomes et al. selected agonists of the interaction between adenosine receptor 3 and β-arrestin2 and revealed the structural features of the selected ligands [28]. The NanoBiT screening system is not only effective for drug screening but can be valuable to screen enzyme substrates. Peptide ligases, which can connect two polypeptides, are powerful tools for protein engineering [49–52]. Li et al. performed the screening of substrates of the peptide ligase Sortase A by fusing this enzyme to SmBiT and the candidate peptides to LgBiT [53]. In addition to known substrate sequences, they rapidly identified some previously unknown substrates with varying activities. In addition, the measurement was very stable, and the

**30**

**Figure 3.**

*Self-assembling Nluc fragment as a probe for protein aggregation. (A) N65 is fused to the target protein. When the fusion protein is not aggregated, NanoLuc is reconstituted by the addition of 66C. (B) When the target protein is aggregated, N65 is also aggregated, and, then, the reassembly does not occur.*

The first description of the use of self-assembling NanoLuc fragments was provided by Zhao et al [54]. NanoLuc was separated into two fragments, N65 (1–65 aa) and 66C (66–171 aa). NanoLuc was rapidly reconstituted when N65 and 66C were mixed. Next, N65 was fused to the target proteins. When the target protein was soluble, N65, which had the correct structure, could reassemble with 66C, resulting in recovery of the luminescence. On the other hand, the insoluble target protein did not induce the recovery of the luminescence, because the aggregated N65 could not assemble with 66C (**Figure 4**). The aggregations of amyloid-β mutants were assessed using the system. Similar monitoring systems of protein aggregation using split-GFP and conventional split-luciferase systems had been previously reported [55–60]. However, a time lag occurred for the chromophore formation in the split-GFP system, and other luciferases were relatively unstable compared with NanoLuc. Zhao et al. succeeded the robust measurement of amyloid-β in this study.

Other self-assembling NanoLuc fragments were described [13]. The SmBiT sequence was optimized using peptides with different affinities to LgBiT. Of the candidates, the HiBiT peptide displayed high affinity (*K*D = 700 pM) although the affinity of SmBiT was very low (*K*D > 100 μM). HiBiT (11 aa) and LgBiT assembled spontaneously, allowing the construction of NanoLuc. HiBiT is a useful tag due to the small size as further described below.

In one of the split-GFP systems, GFP was split into two fragments [57]. The C-terminal fragment of GFP contains 16 aa (GFP11). Waldo et al. found that this and the other fragment (GFP1–10) expressed in the cell assembled spontaneously, and the GFP fluorescence was recovered. Leonetti et al. described the synthesis of the donor DNA templates encoding GFP11 and the tagging of endogenous proteins using CRISPR/Cas9 (**Figure 5A**) [61]. The formation of full-length GFP was induced by coexpression with GFP1–10, and the tagging endogenous protein by GFP11 could be detected. Instead of GFP11, Schwinn et al. used HiBiT as the tag for endogenous proteins and were successful in achieving the highly efficient integration and monitoring of the expression dynamics of the tagging proteins without the time lag, which occurs in the split-GFP system due to the chromophore formation (**Figure 5B**) [62].

Ryes-Alaraz et al. analyzed the internalization of galanin receptor 2, which is dependent on the binding of the endogenous ligand, using an HiBiT-fused galanin receptor 2 [27]. LgBiT could bind to the HiBiT-fused receptor on the cell surface.

**Figure 5.**

*Application of self-assembling fragment for Crispr-Cas9 system. (A) GFP11-encoding template is inserted at the end of the genome encoding target protein by Crispr-Cas9 system. GFP11-fused target protein is expressed. By the addition of GFP1–10, full-length GFP is reconstituted and the target protein was detected. (B) HiBiT is used instead of GFP11, and higher sensitivity is attained.*

#### **Figure 6.**

*Application of self-assembling fragment for the analysis of internalization. LgBiT can bind to HiBiT-galanin receptor 2 on the cell membrane, while it cannot access HiBiT-galanin receptor 2 in the endosome.*

However, LgBiT could not bind to the HiBiT-fused receptor in cells due to the impermeability of LgBiT to cells (**Figure 6**).

This technology has often been used to quantify targets. Oh-hashi et al. used HiBiT to quantify the expression of transcription factor ATF4 that was induced by endoplasmic reticulum stress [63]. Sasaki et al. developed a quantitative detection system of viral entry and release using HiBiT fused to subviral particles and flavivirus-like particles of West Nile virus [64]. Tamura et al. constructed recombinant viruses carrying HiBiT [65]. Viral amplification and propagation were rapid and comparable with the parental viruses, due to the small size of HiBiT. The techniques proved useful to study the viral life cycle and pathogenesis.

#### **6. NanoLuc ternary technology**

In PCA, the reporter protein is generally separated into two fragments. To our knowledge, PCA using 3-split reporter protein was first reported by Cabantous et al. [66]. In the study, GFP was split into two peptides, GFP10 and GFP11, and the remaining part. The two peptides were each fused to an interacting partner. When the interaction occurred, the peptides came into close proximity with one another and then assembled to form the full length of GFP with the remaining part (**Figure 7**).

Possibly inspired by this GFP ternary technology, Dixon et al. developed the NanoLuc ternary technology, NanoLuc is consisted of 11 β-strands [67]. The

**33**

*Protein-Protein Interaction Assays Using Split-NanoLuc DOI: http://dx.doi.org/10.5772/intechopen.86122*

*from GFP10, GFP11, and externally added GFP1–9.*

**Figure 7.**

**Figure 8.**

authors dissected two β-strands and the remaining part. Each strand was fused to a Fab fragment of an antibody and an ankyrin repeat protein, which bound to distinct areas of the cancer marker, HER2. When both antibodies recognized HER2, the two strands came close together and the full length of NanoLuc was reconstituted from the three fragments of NanoLuc (**Figure 8**). The sensitivity was similar to the

*Sandwich immunoassay based on NanoLuc ternary technology developed by Dixon et al. The two β-strands are fused to a Fab and an ankyrin repeat protein, respectively, which bind to two distant parts of Her2 protein.* 

*GFP ternary split technology for the detection of protein-protein interaction. The two small fragments GFP10 and GFP11 are fused to the interacting proteins, respectively. When the interaction occurs, GFP is reconstituted* 

(Perkin Elmer) and NanoBiT. Furthermore, the detectable concentration range of

At almost the same time, we developed the NanoLuc ternary technology for use as an open-sandwich immunoassay (OS-IA), because OS-IA could not be performed using NanoBiT [68]. For OS-IA, two antigen-binding regions, the heavy-chain variable region (VH) and the light-chain variable region (VL), were isolated from the full-length antibody. OS-IA is based on the antigen-dependent interaction affinity between VH and VL, which is dependent on the antigen (**Figure 9**) [69]. The advantage of OS-IA is that small antigens can be noncompetitively detected with high sensitivity. VH and VL were fused to LgBiT and SmBiT. However, the signal was not increased by the addition of the small peptide antigen (7 aa) named BGP-C7. We suspected that fusion with LgBiT sterically hindered the interaction, or prevented the folding of these antibody fragments

The next step was to split LgBiT in two. The C-terminal strand (11 aa) was named LcBiT, and the remaining part was named LnBiT. LcBiT and SmBiT were fused to VH and VL, respectively. When LnBiT, VH-LcBiT, and VL-SmBiT were mixed, the signal was increased depending on the concentration of BGP-C7. The

sensitivity detected using the commercially available AlphaLISA HER2 kit

*NanoLuc was reconstituted from the two strands and externally added remainder of NanoLuc.*

HER2 was broader compared to the range detected by NanoBiT.

due to the relatively large size of LgBiT.

*Protein-Protein Interaction Assays Using Split-NanoLuc DOI: http://dx.doi.org/10.5772/intechopen.86122*

**Figure 7.**

*Bioluminescence - Analytical Applications and Basic Biology*

However, LgBiT could not bind to the HiBiT-fused receptor in cells due to the

*receptor 2 on the cell membrane, while it cannot access HiBiT-galanin receptor 2 in the endosome.*

This technology has often been used to quantify targets. Oh-hashi et al. used HiBiT to quantify the expression of transcription factor ATF4 that was induced by endoplasmic reticulum stress [63]. Sasaki et al. developed a quantitative detection system of viral entry and release using HiBiT fused to subviral particles and flavivirus-like particles of West Nile virus [64]. Tamura et al. constructed recombinant viruses carrying HiBiT [65]. Viral amplification and propagation were rapid and comparable with the parental viruses, due to the small size of HiBiT. The techniques

*Application of self-assembling fragment for the analysis of internalization. LgBiT can bind to HiBiT-galanin* 

*Application of self-assembling fragment for Crispr-Cas9 system. (A) GFP11-encoding template is inserted at the end of the genome encoding target protein by Crispr-Cas9 system. GFP11-fused target protein is expressed. By the addition of GFP1–10, full-length GFP is reconstituted and the target protein was detected. (B) HiBiT is* 

In PCA, the reporter protein is generally separated into two fragments. To our knowledge, PCA using 3-split reporter protein was first reported by Cabantous et al. [66]. In the study, GFP was split into two peptides, GFP10 and GFP11, and the remaining part. The two peptides were each fused to an interacting partner. When the interaction occurred, the peptides came into close proximity with one another and then assembled to form the full length of GFP with the remaining

Possibly inspired by this GFP ternary technology, Dixon et al. developed the NanoLuc ternary technology, NanoLuc is consisted of 11 β-strands [67]. The

impermeability of LgBiT to cells (**Figure 6**).

*used instead of GFP11, and higher sensitivity is attained.*

**6. NanoLuc ternary technology**

proved useful to study the viral life cycle and pathogenesis.

**32**

part (**Figure 7**).

**Figure 5.**

**Figure 6.**

*GFP ternary split technology for the detection of protein-protein interaction. The two small fragments GFP10 and GFP11 are fused to the interacting proteins, respectively. When the interaction occurs, GFP is reconstituted from GFP10, GFP11, and externally added GFP1–9.*

#### **Figure 8.**

*Sandwich immunoassay based on NanoLuc ternary technology developed by Dixon et al. The two β-strands are fused to a Fab and an ankyrin repeat protein, respectively, which bind to two distant parts of Her2 protein. NanoLuc was reconstituted from the two strands and externally added remainder of NanoLuc.*

authors dissected two β-strands and the remaining part. Each strand was fused to a Fab fragment of an antibody and an ankyrin repeat protein, which bound to distinct areas of the cancer marker, HER2. When both antibodies recognized HER2, the two strands came close together and the full length of NanoLuc was reconstituted from the three fragments of NanoLuc (**Figure 8**). The sensitivity was similar to the sensitivity detected using the commercially available AlphaLISA HER2 kit (Perkin Elmer) and NanoBiT. Furthermore, the detectable concentration range of HER2 was broader compared to the range detected by NanoBiT.

At almost the same time, we developed the NanoLuc ternary technology for use as an open-sandwich immunoassay (OS-IA), because OS-IA could not be performed using NanoBiT [68]. For OS-IA, two antigen-binding regions, the heavy-chain variable region (VH) and the light-chain variable region (VL), were isolated from the full-length antibody. OS-IA is based on the antigen-dependent interaction affinity between VH and VL, which is dependent on the antigen (**Figure 9**) [69]. The advantage of OS-IA is that small antigens can be noncompetitively detected with high sensitivity. VH and VL were fused to LgBiT and SmBiT. However, the signal was not increased by the addition of the small peptide antigen (7 aa) named BGP-C7. We suspected that fusion with LgBiT sterically hindered the interaction, or prevented the folding of these antibody fragments due to the relatively large size of LgBiT.

The next step was to split LgBiT in two. The C-terminal strand (11 aa) was named LcBiT, and the remaining part was named LnBiT. LcBiT and SmBiT were fused to VH and VL, respectively. When LnBiT, VH-LcBiT, and VL-SmBiT were mixed, the signal was increased depending on the concentration of BGP-C7. The

#### **Figure 9.**

*Open sandwich immunoassay (OS-IA) and NanoLuc ternary technology. (A) Principle of OS-IA. Small antigens (MW < 1000) can be noncompetitively detected. (B) Noncompetitive detection of small antigen by NanoLuc ternary technology. Visual detection was possible when sufficient amount of antigen was present.*

background signal without BGP-C7 was lower than the background signal of VH-LgBiT and VL-SmBiT.

Next, the signal was enhanced by optimizing the sequence of SmBiT. The signal was increased 288-fold using the sequence, which has higher affinity to LgBiT. The enhancement was high enough to permit detection by the naked eye. The detection limit of BGP-C7 was comparable with the limit detected by OS-ELISA. Furthermore, the strong signal was maintained for more than 1 h.

The small tags of the NanoLuc ternary system have proven to be very useful when both target proteins have complex structures. Furthermore, the system exhibits a robust and bright signal.

#### **7. Discussion and conclusion**

The described NanoLuc binary and ternary technologies are superior compared with other PCAs using other luciferase enzymes. The signals obtained were almost the strongest among the signals of PCAs using luciferase enzymes. The most important advantage is the small size of the fusion tags, SmBiT and LcBiT.

As mentioned in Section 4, NanoBiT has been used as a screening tool. In several studies, SmBiT was fused to membrane proteins, which are important targets of drug discovery. The brightness of NanoLuc increased the hit ratio, and SmBiT was validated as a tag for the fusion with proteins having complex structures, such as membrane proteins. Although the measurements were very accurate, researchers should pay attention to the influence of low-molecular weight compounds on enzymatic activity [70–72]. Some compounds increase enzymatic activity, while others decrease it. The TurboLuc system reported by Audi et al. is somewhat smaller in molecular weight (16 kDa) than NanoLuc [73]. Compared with NanoLuc

**35**

mechanism [77].

**Acknowledgements**

*Protein-Protein Interaction Assays Using Split-NanoLuc DOI: http://dx.doi.org/10.5772/intechopen.86122*

luciferase is possible, but careful examinations are needed.

costly especially when larger scale screening is intended.

efficiency and stability of the reconstitution for its wider use.

and Firefly luciferase, the activity of TurboLuc was less affected by low-molecular weight compounds. Ho et al. examined the influence of 42,460 PubChem compounds on enzymatic activities of several luciferases [74]. NanoLuc, *Renilla* luciferase, firefly luciferase, and *Gaussia* luciferase were affected by 2.7, 10, 4, and 0.02% of the compounds, respectively. The relationship between the chemical similarity and the inhibition profile showed that the compounds varied depending on the luciferase used. While NanoLuc has several advantages for screening, in some cases, researchers should select other enzymes or more than two enzymes. Furthermore, we previously cautioned using a mathematical model that the comparison between the affinity of interacting proteins and the signal detected by luciferase-based PCA can cause misinterpretation of the quantitation [75]. In addition, we suggested that the geometry of the interacting proteins influences the luminescent signal. In other words, the structures of the interacting proteins can affect the reconstitution of luciferase. Quantitative measurement with PCA using

One of the other problems is the unstable luminescence in cells. When we use the standard furimazine ester for live cell assay, the light intensity decreases within 1 h, and it will not be suitable for large screenings in cellulo. Recently, live cell substrates with longer half-life (Vivazine and Endurazine, Promega) have become available. These substrates can maintain the luminescent signal for several hours, although the peak luminescent intensity is significantly lower compared with that detected using the conventional furimazine ester. However, the luminescent intensity of unmodified furimazine can be maintained for several hours in vitro. Oxygen in cells and the culture medium will also be an important factor for the stable luminescence because the NanoLuc-catalyzed reaction requires oxygen, especially when the light emitted is strong. The last problem is the prices of these substrates, which tend to be

Detection of the interaction among more than three proteins, and the simultaneous detection of more than two interactions will become more important in future. For the detection among three proteins, the combination of NanoBiT and BRET might be useful. The brightness of NanoLuc often disturbs simultaneous detection using both NanoLuc and other luciferases. Therefore, NanoLuc inhibitors were developed [76]. After the measurement of NanoLuc luminescence, the luminescence can be diminished by the inhibitors, which enables detection of the luminescence of another luciferase. For the simultaneous detection by multicolors, several color variants of eNano-Lantern (a fusion protein of NanoLuc and fluorescent protein) can be applied to NanoBiT. In eNano-Lantern, the luminescence at longer wavelength can be observed by the efficient intramolecular BRET

Dixon et al. and the authors developed a novel PCA using 3-split NanoLuc [67, 68]. The pair of the small tags is very effective to avoid misfolding and steric hindrance of target proteins. Our next challenge will be to further improve the

This work was supported partly by the Strategic International Collaborative Research Program (SICORP), Japan Science and Technology Agency, by JSPS KAKENHI, grant numbers JP18H03851 (to HU) and JP17K06920 (to YOM), from the Japan Society for the Promotion of Science, Japan, and by Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials from MEXT, Japan.

#### *Protein-Protein Interaction Assays Using Split-NanoLuc DOI: http://dx.doi.org/10.5772/intechopen.86122*

*Bioluminescence - Analytical Applications and Basic Biology*

background signal without BGP-C7 was lower than the background signal of VH-

*Open sandwich immunoassay (OS-IA) and NanoLuc ternary technology. (A) Principle of OS-IA. Small antigens (MW < 1000) can be noncompetitively detected. (B) Noncompetitive detection of small antigen by NanoLuc ternary technology. Visual detection was possible when sufficient amount of antigen was present.*

Next, the signal was enhanced by optimizing the sequence of SmBiT. The signal was increased 288-fold using the sequence, which has higher affinity to LgBiT. The enhancement was high enough to permit detection by the naked eye. The detection limit of BGP-C7 was comparable with the limit detected by OS-ELISA. Furthermore, the strong signal was maintained for more than 1 h. The small tags of the NanoLuc ternary system have proven to be very useful when both target proteins have complex structures. Furthermore, the system

The described NanoLuc binary and ternary technologies are superior compared with other PCAs using other luciferase enzymes. The signals obtained were almost the strongest among the signals of PCAs using luciferase enzymes. The most impor-

As mentioned in Section 4, NanoBiT has been used as a screening tool. In several studies, SmBiT was fused to membrane proteins, which are important targets of drug discovery. The brightness of NanoLuc increased the hit ratio, and SmBiT was validated as a tag for the fusion with proteins having complex structures, such as membrane proteins. Although the measurements were very accurate, researchers should pay attention to the influence of low-molecular weight compounds on enzymatic activity [70–72]. Some compounds increase enzymatic activity, while others decrease it. The TurboLuc system reported by Audi et al. is somewhat smaller in molecular weight (16 kDa) than NanoLuc [73]. Compared with NanoLuc

tant advantage is the small size of the fusion tags, SmBiT and LcBiT.

**34**

LgBiT and VL-SmBiT.

**Figure 9.**

exhibits a robust and bright signal.

**7. Discussion and conclusion**

and Firefly luciferase, the activity of TurboLuc was less affected by low-molecular weight compounds. Ho et al. examined the influence of 42,460 PubChem compounds on enzymatic activities of several luciferases [74]. NanoLuc, *Renilla* luciferase, firefly luciferase, and *Gaussia* luciferase were affected by 2.7, 10, 4, and 0.02% of the compounds, respectively. The relationship between the chemical similarity and the inhibition profile showed that the compounds varied depending on the luciferase used. While NanoLuc has several advantages for screening, in some cases, researchers should select other enzymes or more than two enzymes. Furthermore, we previously cautioned using a mathematical model that the comparison between the affinity of interacting proteins and the signal detected by luciferase-based PCA can cause misinterpretation of the quantitation [75]. In addition, we suggested that the geometry of the interacting proteins influences the luminescent signal. In other words, the structures of the interacting proteins can affect the reconstitution of luciferase. Quantitative measurement with PCA using luciferase is possible, but careful examinations are needed.

One of the other problems is the unstable luminescence in cells. When we use the standard furimazine ester for live cell assay, the light intensity decreases within 1 h, and it will not be suitable for large screenings in cellulo. Recently, live cell substrates with longer half-life (Vivazine and Endurazine, Promega) have become available. These substrates can maintain the luminescent signal for several hours, although the peak luminescent intensity is significantly lower compared with that detected using the conventional furimazine ester. However, the luminescent intensity of unmodified furimazine can be maintained for several hours in vitro. Oxygen in cells and the culture medium will also be an important factor for the stable luminescence because the NanoLuc-catalyzed reaction requires oxygen, especially when the light emitted is strong. The last problem is the prices of these substrates, which tend to be costly especially when larger scale screening is intended.

Detection of the interaction among more than three proteins, and the simultaneous detection of more than two interactions will become more important in future. For the detection among three proteins, the combination of NanoBiT and BRET might be useful. The brightness of NanoLuc often disturbs simultaneous detection using both NanoLuc and other luciferases. Therefore, NanoLuc inhibitors were developed [76]. After the measurement of NanoLuc luminescence, the luminescence can be diminished by the inhibitors, which enables detection of the luminescence of another luciferase. For the simultaneous detection by multicolors, several color variants of eNano-Lantern (a fusion protein of NanoLuc and fluorescent protein) can be applied to NanoBiT. In eNano-Lantern, the luminescence at longer wavelength can be observed by the efficient intramolecular BRET mechanism [77].

Dixon et al. and the authors developed a novel PCA using 3-split NanoLuc [67, 68]. The pair of the small tags is very effective to avoid misfolding and steric hindrance of target proteins. Our next challenge will be to further improve the efficiency and stability of the reconstitution for its wider use.

#### **Acknowledgements**

This work was supported partly by the Strategic International Collaborative Research Program (SICORP), Japan Science and Technology Agency, by JSPS KAKENHI, grant numbers JP18H03851 (to HU) and JP17K06920 (to YOM), from the Japan Society for the Promotion of Science, Japan, and by Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials from MEXT, Japan.

*Bioluminescence - Analytical Applications and Basic Biology*

## **Author details**

Yuki Ohmuro-Matsuyama and Hiroshi Ueda\* Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan

\*Address all correspondence to: ueda@res.titech.ac.jp

© 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.

**37**

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*Protein-Protein Interaction Assays Using Split-NanoLuc DOI: http://dx.doi.org/10.5772/intechopen.86122*

## **References**

*Bioluminescence - Analytical Applications and Basic Biology*

**36**

**Author details**

provided the original work is properly cited.

Yuki Ohmuro-Matsuyama and Hiroshi Ueda\*

Tokyo Institute of Technology, Yokohama, Kanagawa, Japan

\*Address all correspondence to: ueda@res.titech.ac.jp

© 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,

Laboratory for Chemistry and Life Science, Institute of Innovative Research,

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**43**

**Chapter 3**

**Abstract**

Bacteria

*and Elena N. Efremenko*

conditions is optimized.

biomonitoring

Biosensors Using Free and

Immobilized Cells of Luminous

*Anvar D. Ismailov, Leyla E. Aleskerova, Kristina A. Alenina* 

The technologies of receiving free and immobilized photobacteria cells for biomonitoring of toxins are considered. The mechanisms of interaction of toxins with photobacteria are observed. The main attention is paid to the immobilized procedures and structures of carriers. Data on poly(vinyl)alcohol (PVA) cryogel immobilization of different strains of photobacteria are presented*.* It is established that intensity and stability of light emission of PVA cells is competently controlled by: (1) intensity and persistence of a luminescent cycle using bacterial strain; (2) type of the carrier and the composition of the gel-formation medium; (3) freeze-thawing procedures; and (4) physical and chemical conditions of storage and application. The developed technology of cryogenic gel formation has kept the survival of luminous bacteria in the carrier practically at 100% without the introduction of additional cryoprotecting agents and procedures of a light induction. With storage at −80°C, bioluminescent activity remained without changes about 2 years. Using the immobilized preparations of biosensor, the discrete and continuous analysis of heavy metals, chlorophenols, and pesticides is carried out. The sensitivity of free and immobilized cells to the chosen toxicants is approximately identical. The continuous monitoring of toxicant

**Keywords:** photobacteria, biosensors, immobilization, poly(vinyl)alcohol,

The interaction mechanism was investigated only for certain groups of chemicals and mainly with the use of free cells. Toxic agents that suppressed the emission

poisons, aliphatic, aromatic and heterocyclic hydrocarbons, alcohols, ketones,

• On the type of targets: membrane active substances, specific inhibitors of the genetic apparatus, and inhibitors of the energy and lipid metabolism enzymes.

• On the chemical structure: heavy metals, electron acceptors, respiratory

acids, and others. The level of toxin hydrophobicity is important.

**1. Toxin action on the photobacteria light emission**

of photobacteria can be divided into classes rather conditionally.

## **Chapter 3**

*Bioluminescence - Analytical Applications and Basic Biology*

[75] Dale R, Ohmuro-Matsuyama Y, Ueda H, Kato N. Mathematical model of the firefly luciferase complementation assay reveals a non-linear relationship between the detected luminescence and the affinity of the protein pair being analyzed. PLoS One. 2016;**11**:e0148256. DOI: 10.1371/journal.pone.0148256

[76] Walker JR, Hall MP, Zimprich CA, Robers MB, Duellman SJ, Machleidt T, et al. Highly potent cell-permeable and impermeable NanoLuc luciferase inhibitors. ACS Chemical Biology. 2017;**12**:1028-1037. DOI: 10.1021/

[77] Suzuki K, Kimura T, Shinoda H, Bai GR, Daniels MJ, Arai Y, et al. Five colour variants of bright luminescent protein for real-time multicolour bioimaging. Nature Communications.

2016;**7**:13718. DOI: 10.1038/

ncomms13718

acschembio.6b01129

fragment complementation. Analytical Chemistry. 2018;**90**:3001-3004. DOI: 10.1021/acs.analchem.7b05140

[69] Ueda H, Tsumoto K, Kubota K, Suzuki E, Nagamune T, Nishimura H, et al. Open sandwich ELISA: A novel immunoassay based on the interchain interaction of antibody variable region. Nature Biotechnology. 1996;**14**:1714-1718.

DOI: DOI 10.1038/nbt1296-1714

[70] Thorne N, Auld DS, Inglese J. Apparent activity in high-throughput screening: Origins of compounddependent assay interference.

[71] Thorne N, Shen M, Lea WA, Simeonov A, Lovell S, Auld DS, et al. Firefly luciferase in chemical biology: A compendium of inhibitors, mechanistic

evaluation of chemotypes, and

suggested use As a reporter. Chemistry & Biology. 2012;**19**:1060-1072. DOI: 10.1016/j.chembiol.2012.07.015

[72] Auld DS, Southall NT, Jadhav A, Johnson RL, Diller DJ, Simeonov A, et al. Characterization of chemical libraries for luciferase inhibitory activity. Journal of Medicinal Chemistry. 2008;**51**:2372-2386. DOI:

[73] Auld DS, Narahari J, Ho PI, Casalena D, Nguyen V, Cirbaite E, et al. Characterization and use of TurboLuc luciferase as a reporter for high-throughput assays. Biochemistry. 2018;**57**:4700-4706. DOI: 10.1021/acs.

[74] Ho PI, Yue K, Pandey P, Breault L, Harbinski F, McBride AJ, et al. Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays. ACS Chemical Biology. 2013;**8**:1009-1017.

cbpa.2010.03.020

10.1021/jm701302v

biochem.8b00290

DOI: 10.1021/cb3007264

Current Opinion in Chemical Biology. 2010;**14**:315-324. DOI: 10.1016/j.

**42**
