**6**

in which the activity or state of labelled proteins, cells, tissues and organs may be localised

<sup>\*</sup> Corresponding Author

Development of a pH-Tolerant Thermostable

2001).

*Photinus pyralis* Luciferase for Brighter *In Vivo* Imaging 121

protein engineering (Hall *et al.*, 1999; Tisi *et al.*, 2002; Branchini *et al.*, 2009). Enhanced thermostability greatly improves the brightness achievable *in vivo*, and such enzymes are just recently finding application in animals (Law *et al.*, 2006; Baggett *et al.*, 2004; Mezzanotte *et al.*, 2011; Michelini *et al.*, 2008). A combination of higher thermal stability with increased pH-tolerance of Fluc is a very much desired and favourable feature for *in vivo* imaging

Thermostability can be greatly improved by one amino acid change of FLuc and it has been observed that both changes in the enzyme core and on the protein surface can alter stability (Tisi *et al.*, 2002b). The substitutions A217I, L or V, identified by random mutagenesis increase the thermo- and pH-stability of *Luciola cruciata* and *L. lateralis* Flucs, and the equivalent substitution in *Photinus pyralis* (*Ppy)* Luc (A215L) also increases thermostability (Kajiyama and Nakano, 1993; Squirrell *et al.*, 1998). By random mutagenesis of *Ppy* Luc, and N-terminal surface loop-based substitutions, E354K or E354R have been identified to increase thermostability (White *et al.*, 1996). Combination of E354 with mutation D357 produced thermostable double mutants, of which E354I/ D357Y (x2 Fluc; Table 1) and E354R/ D357F were shown to be more stable than D357Y or E354K alone (Willey *et al.*,

Cumulative addition of such mutations further enhances thermostability. A typical example is a mutant containing T214C, I232A, F295L and E354K, named x4 Luc (Table 1) (Tisi *et al.*, 2002b). Non-conserved surface-exposed hydrophobic residues previously mutated to Ala (Tisi *et al.*, 2001; Prebble *et al.*, 2001) have also been substituted for polar ones (F14R, L35Q, V182K, I232K and F465R) to produce a mutant, named x5 Luc, displaying additively improved thermostability, solvent stability and pH-tolerance in terms of activity and resistance to red-shift; while retaining the same specific activity relative to WT luciferase (Law *et al.*, 2002; Law *et al*., 2006). The most thermostable mutant luciferase, Ultra-GloTM (UG) was created from *Photuris pennsylvanica* luciferase, and is commercially available for a

Majority of studies on improving thermostability, pH-tolerance and brightness of Fluc have been carried out using LH2 until now. Emerging applications of luciferases in *in vivo*  imaging of protease activity (Dragulescu-Andrasi *et al.*, 2009) require a different substrate - ALH2, one of the very few LH2 analogues with which firefly luciferase also produces bioluminescence of relatively high intensity (White *et al.*, 1966). The substitution of the 6' group extends the range of groups that can be conjugated to luciferin, for example to amino acids (Shinde *et al.*, 2006), peptides (Monsees *et al.*, 1995) and linear or bulky N-alkyl groups (Woodroofe *et al.*, 2008). Peptide-conjugated pro-luciferins allow the bioluminescent measurement of protease activity and in such applications ALH2 (Monsees *et al.*, 1995) therefore the properties of different firefly luciferases and their mutants with ALH2 may

The limited data on *Ppy* Fluc bioluminescence with ALH2 as a substrate show that these properties are very different from those of LH2. The bioluminescence colour with ALH2 has long been reported as pH-independent orange-red (max 605nm) (White *et al.*, 1966), Km for ALH2 is approximately 26-times lower and Vmax is 10 times lower than that of LH2 (Shinde *et al.*, 2006). There has been no further analysis of either red-shifted excited state emitter with

which is likely to be useful in other applications (Foucault *et al.*, 2010).

number of assays (Hall *et al.*, 1999; Woodroofe *et al.*, 2008).

impact on the choice of enzymes applied.

and quantified sensitively and non-invasively. Different models and procedures for BLI are well described (Kung, 2005; Zinn *et al.*, 2008). For example, BLI is routinely applied to serially detect the burden of xenografted tumours in mice. Using more complex techniques, such as Fluc re-complementation, protein interactions such as chemokine receptor dimerisation (Luker *et al.*, 2008) have been imaged in small animals.

Recently, BLI has also been adapted to the detection of small molecules *in vivo* (Van de Bittner *et al.*, 2010). *D*-LH2 is typically given intravenously or intraperitoneally to mice and has a broad biodistribution profile (Berger *et al.*, 2010). Cellular levels of Mg and ATP are sufficient to drive the reaction, though kinetics depends on substrate diffusion. Light emitted from labelled cells is detected using imagers which consist of CCD cameras in a dark box. This gives invaluable insight into the effects of experiments in the context of living organisms in real time.

The advantages of BLI over comparative techniques such as positron emission tomography (PET) include its simplicity, low cost, non-requirement for radiation and versatility. The other main optical imaging technique, fluorescence imaging (FLI), in which fluorescent small molecules or proteins are imaged in small animals, has lower signal to noise ratio than BLI due to the background signal in FLI from autofluorescence, quenching of signal due to endogenous tissue chromophores and also the requirement, and dependence on penetration, of an excitation light. Thus, BLI is approximately three orders of magnitude more sensitive than FLI and has a very large dynamic range (Wood, 1998).

All optical imaging techniques suffer from low resolution and from wavelength dependence of imaging due to photon scatter and signal attenuation by endogenous absorbing compounds. For example haemoglobin absorbs strongly below 590 nm. Therefore it is the red part of the spectra that is detected most efficiently (Caysa *et al.*, 2009).

Wild-type (WT) luciferase is highly thermolabile, inactivating and bathochromic shifting at even room temperature, and is sensitive to buffer conditions such as pH (Law *et al.*, 2006). The recombinant WT luciferase retains between 30 and 45% of activity at pH 7.0 relative to that at the optimal pH of 7.8 – 8.0, depending on whether flash heights or integrated light were measured (Law *et al.*, 2006). While many *in vitro* applications, such as those used to detect DNA amplification (Gandelman *et al.*, 2010) or ATP-assays (Strehler, 1968), take place at alkaline pH values of 8.0 or higher, *in vivo* applications such as medical or whole cell imaging must take place at neutral pH of 6.9 – 7.2, dependent on the exact cell type. At 37ºC, WT Fluc shows a bathochromic shift and thus emits predominantly red light. Though light of red and longer wavelength does penetrate tissues more readily, red-shift luciferase bioluminescence is usually accompanied by a significant reduction in quantum yield and is therefore as such undesirable (Seliger and McElroy, 1959; Seliger and McElroy, 1960) as fluctuating levels of luciferase activity make quantitative studies problematic. Furthermore, for multispectral purposes (Mezzanotte *et al.*, 2011), any shift is undesirable. Therefore thermostable enzymes of different colours, which resist bathochromic shift are preferable.

The ideal Fluc would be highly thermostable, resist bathochromic shift, bright, have favourable kinetics (such as high substrate affinity) and have increased pH-tolerance. To address the issues of thermostability and bathochromic shift a number of recombinant mutant luciferases with increased thermal stability, giving brighter and more stable signals at elevated temperatures and resistant to bathochromic shift have been developed using

and quantified sensitively and non-invasively. Different models and procedures for BLI are well described (Kung, 2005; Zinn *et al.*, 2008). For example, BLI is routinely applied to serially detect the burden of xenografted tumours in mice. Using more complex techniques, such as Fluc re-complementation, protein interactions such as chemokine receptor

Recently, BLI has also been adapted to the detection of small molecules *in vivo* (Van de Bittner *et al.*, 2010). *D*-LH2 is typically given intravenously or intraperitoneally to mice and has a broad biodistribution profile (Berger *et al.*, 2010). Cellular levels of Mg and ATP are sufficient to drive the reaction, though kinetics depends on substrate diffusion. Light emitted from labelled cells is detected using imagers which consist of CCD cameras in a dark box. This gives invaluable insight into the effects of experiments in the context of living

The advantages of BLI over comparative techniques such as positron emission tomography (PET) include its simplicity, low cost, non-requirement for radiation and versatility. The other main optical imaging technique, fluorescence imaging (FLI), in which fluorescent small molecules or proteins are imaged in small animals, has lower signal to noise ratio than BLI due to the background signal in FLI from autofluorescence, quenching of signal due to endogenous tissue chromophores and also the requirement, and dependence on penetration, of an excitation light. Thus, BLI is approximately three orders of magnitude

All optical imaging techniques suffer from low resolution and from wavelength dependence of imaging due to photon scatter and signal attenuation by endogenous absorbing compounds. For example haemoglobin absorbs strongly below 590 nm. Therefore it is the

Wild-type (WT) luciferase is highly thermolabile, inactivating and bathochromic shifting at even room temperature, and is sensitive to buffer conditions such as pH (Law *et al.*, 2006). The recombinant WT luciferase retains between 30 and 45% of activity at pH 7.0 relative to that at the optimal pH of 7.8 – 8.0, depending on whether flash heights or integrated light were measured (Law *et al.*, 2006). While many *in vitro* applications, such as those used to detect DNA amplification (Gandelman *et al.*, 2010) or ATP-assays (Strehler, 1968), take place at alkaline pH values of 8.0 or higher, *in vivo* applications such as medical or whole cell imaging must take place at neutral pH of 6.9 – 7.2, dependent on the exact cell type. At 37ºC, WT Fluc shows a bathochromic shift and thus emits predominantly red light. Though light of red and longer wavelength does penetrate tissues more readily, red-shift luciferase bioluminescence is usually accompanied by a significant reduction in quantum yield and is therefore as such undesirable (Seliger and McElroy, 1959; Seliger and McElroy, 1960) as fluctuating levels of luciferase activity make quantitative studies problematic. Furthermore, for multispectral purposes (Mezzanotte *et al.*, 2011), any shift is undesirable. Therefore thermostable enzymes of different colours, which resist bathochromic shift are preferable. The ideal Fluc would be highly thermostable, resist bathochromic shift, bright, have favourable kinetics (such as high substrate affinity) and have increased pH-tolerance. To address the issues of thermostability and bathochromic shift a number of recombinant mutant luciferases with increased thermal stability, giving brighter and more stable signals at elevated temperatures and resistant to bathochromic shift have been developed using

dimerisation (Luker *et al.*, 2008) have been imaged in small animals.

more sensitive than FLI and has a very large dynamic range (Wood, 1998).

red part of the spectra that is detected most efficiently (Caysa *et al.*, 2009).

organisms in real time.

protein engineering (Hall *et al.*, 1999; Tisi *et al.*, 2002; Branchini *et al.*, 2009). Enhanced thermostability greatly improves the brightness achievable *in vivo*, and such enzymes are just recently finding application in animals (Law *et al.*, 2006; Baggett *et al.*, 2004; Mezzanotte *et al.*, 2011; Michelini *et al.*, 2008). A combination of higher thermal stability with increased pH-tolerance of Fluc is a very much desired and favourable feature for *in vivo* imaging which is likely to be useful in other applications (Foucault *et al.*, 2010).

Thermostability can be greatly improved by one amino acid change of FLuc and it has been observed that both changes in the enzyme core and on the protein surface can alter stability (Tisi *et al.*, 2002b). The substitutions A217I, L or V, identified by random mutagenesis increase the thermo- and pH-stability of *Luciola cruciata* and *L. lateralis* Flucs, and the equivalent substitution in *Photinus pyralis* (*Ppy)* Luc (A215L) also increases thermostability (Kajiyama and Nakano, 1993; Squirrell *et al.*, 1998). By random mutagenesis of *Ppy* Luc, and N-terminal surface loop-based substitutions, E354K or E354R have been identified to increase thermostability (White *et al.*, 1996). Combination of E354 with mutation D357 produced thermostable double mutants, of which E354I/ D357Y (x2 Fluc; Table 1) and E354R/ D357F were shown to be more stable than D357Y or E354K alone (Willey *et al.*, 2001).

Cumulative addition of such mutations further enhances thermostability. A typical example is a mutant containing T214C, I232A, F295L and E354K, named x4 Luc (Table 1) (Tisi *et al.*, 2002b). Non-conserved surface-exposed hydrophobic residues previously mutated to Ala (Tisi *et al.*, 2001; Prebble *et al.*, 2001) have also been substituted for polar ones (F14R, L35Q, V182K, I232K and F465R) to produce a mutant, named x5 Luc, displaying additively improved thermostability, solvent stability and pH-tolerance in terms of activity and resistance to red-shift; while retaining the same specific activity relative to WT luciferase (Law *et al.*, 2002; Law *et al*., 2006). The most thermostable mutant luciferase, Ultra-GloTM (UG) was created from *Photuris pennsylvanica* luciferase, and is commercially available for a number of assays (Hall *et al.*, 1999; Woodroofe *et al.*, 2008).

Majority of studies on improving thermostability, pH-tolerance and brightness of Fluc have been carried out using LH2 until now. Emerging applications of luciferases in *in vivo*  imaging of protease activity (Dragulescu-Andrasi *et al.*, 2009) require a different substrate - ALH2, one of the very few LH2 analogues with which firefly luciferase also produces bioluminescence of relatively high intensity (White *et al.*, 1966). The substitution of the 6' group extends the range of groups that can be conjugated to luciferin, for example to amino acids (Shinde *et al.*, 2006), peptides (Monsees *et al.*, 1995) and linear or bulky N-alkyl groups (Woodroofe *et al.*, 2008). Peptide-conjugated pro-luciferins allow the bioluminescent measurement of protease activity and in such applications ALH2 (Monsees *et al.*, 1995) therefore the properties of different firefly luciferases and their mutants with ALH2 may impact on the choice of enzymes applied.

The limited data on *Ppy* Fluc bioluminescence with ALH2 as a substrate show that these properties are very different from those of LH2. The bioluminescence colour with ALH2 has long been reported as pH-independent orange-red (max 605nm) (White *et al.*, 1966), Km for ALH2 is approximately 26-times lower and Vmax is 10 times lower than that of LH2 (Shinde *et al.*, 2006). There has been no further analysis of either red-shifted excited state emitter with

Development of a pH-Tolerant Thermostable

Genetics, University of Cambridge.

protocol, with BSA as the standard.

FCS and 1 % glutamax in 5% CO2.

**inactivation and bioluminescence spectra** 

**2.3 Expression and purification of 12 Fluc and revertants** 

variant PMT sensitivity as previously described (Law *et al.*, 2006).

**2.5 Mammalian cell culture, retrovirus production and transduction of cells** 

*Photinus pyralis* Luciferase for Brighter *In Vivo* Imaging 123

Boldface type represents the mutated codon, underlined letters represent modified endonuclease site used to facilitate screening, and the endonuclease used for screening is shown in parentheses. *E. coli* BL21 (pLysS) (Edge Biosystems, Gaithersburg, MD, USA or XL2-Blue ultracompetent cells (Stratagene) were used as cloning hosts for the generation and selection of mutants from site-directed mutagenesis. Expression from colonies was induced by adsorbing colonies onto HybondTM-N nitrocellulose membranes (Amersham Biosciences Corp., Piscataway, NJ, USA) and transferring membranes onto fresh Luria Bertani (LB) agar plates containing 100 g/ml carbenicillin and 1 mM IPTG and incubating for 3 hours at room temperature (RT). Bioluminescence was initiated by spraying membranes with 1 mM LH2 or 500M ALH2 in 0.1 M citrate buffer (pH 5) and colony screening was carried out by photographing emitted light with Nikon D70S camera (Nikon Corp., Tokyo, Japan). After seven rounds of SDM, mutations introduced were confirmed by sequencing of the entire luciferase gene using a facility provided by the Department of

His10-tagged WT recombinant luciferase (WT) and mutants were expressed and purified according to the optimised protocol described in (Law *et al.*, 2006). Total protein concentrations were estimated by the method of Bradford (Bradford, 1976), using the Coomassie Blue protein assay reagent kit from Pierce according to the manufacturer's

**2.4 Luciferase activity assays, kinetic analysis, pH dependence of activity, thermal** 

Luciferase mutants were diluted from purified stock solutions into pre-chilled 0.1 M Tris/ acetate; pH 7.8, 2 mM EDTA and 10 mM MgSO4 (TEM) containing 2 mM DTT to obtain the required concentration, unless specified otherwise. Refer to caption accompanying each table or figure for method details. Bioluminescence spectra were captured using a Varian fluorometer (Palo Alto, Ca, USA). For measurements at differing pH values, TEM buffer at different pH values was used to dilute substrates and enzymes. Data were corrected for

The genes encoding WT Fluc and x11 Fluc from pET16b constructs were cloned into mammalian retroviral expression vector SFG fused to Myc tags. These constructs were triple transfected into 293T cells, cultured in IMDM (Lonza, Basel, Switzerland) with 10% fetal calf serum (FCS) (Hyclone Labs Inc., Logan, UT, USA) and 1 % glutamax (Invitrogen Corp., Groningen, The Netherlands), along with plasmids encoding retroviral envelope and gagpol genes to produce retrovirus, which was used to transduce Raji cells. Transduced cells were sorted by flow cytometry using a Moflo-XDP instrument (Beckman Coulter, CA, USA) by anti-myc.FITC (Santa Cruz Biotechnology Inc., CA, USA) staining of the same mean fluorescence intensity and were cultured in RPMI 1640 (Lonza, Basel, Switzerland) with 10%

ALH2 or its higher catalytic efficiency. From other studies it is known that there are luciferase isoforms from *Pyrophorus plagiopthalamus* that emit green light (*PpldGr*: 550 nm at pH 7.6) and yellow light (*PplvY*: 577 nm at pH 7.6) with ALH2. This indicates that red emission is not an intrinsic property of ALH2, but merely a consequence of enzymatic interactions and conformation of the active site (White *et al.*, 1966; Nakatsu et al., 2006; Branchini *et al.*, 2001; Sandalova and Ugarova, 1999). Thus, it should be possible to engineer luciferase mutants with advantageous properties with ALH2, such as altered emission colour, higher activity and/or kinetics beneficial for *in vivo* imaging of protease activity.

In this paper we report on the construction and characterisation of a further improved x12 mutant based on the x5 mutant and seven additional mutations. Each of these mutations has previously been shown to confer slower rates of thermal inactivation (White *et al*., 1996; Squirrell *et al*., 1999; Tisi *et al.*, 2002). We compared the performance of x12 mutant with the WT *Ppy* and UG luciferases and demonstrated its pH tolerance and increased thermostability. A reversion of one of the mutations in the x12 resulted in a simplified mutant, termed x11 Fluc. Herein, we present properties of this mutant, which is highly thermostable, pH-tolerant, has high activity and catalytic efficiency with both LH2 and ALH2, and presents a great potential for *in vivo* applications with both substrates.
