Development of a pH-Tolerant Thermostable Photinus pyralis Luciferase for Brighter In Vivo Imaging

Firefly luciferase (Fluc) catalyzes a bioluminescent reaction using the substrates ATP and beetle luciferin in the presence of molecular oxygen (Fig. 1A). Because of its use of ATP and the simplicity of the single-enzyme system, firefly luciferase is widely used in numerous applications, notably those involving detection of living organisms, gene expression or amplification in both in vivo and in vitro systems.


Introduction
Firefly luciferase (Fluc) catalyzes a bioluminescent reaction using the substrates ATP and beetle luciferin in the presence of molecular oxygen (Fig. 1A).Because of its use of ATP and the simplicity of the single-enzyme system, firefly luciferase is widely used in numerous applications, notably those involving detection of living organisms, gene expression or amplification in both in vivo and in vitro systems.Fluc has found intensive application in small animal in vivo bioluminescence imaging (BLI) in which the activity or state of labelled proteins, cells, tissues and organs may be localised

Development of a pH-Tolerant Thermostable
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 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-Glo TM (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 LH 2 until now.Emerging applications of luciferases in in vivo imaging of protease activity (Dragulescu-Andrasi et al., 2009) require a different substrate -ALH 2 , one of the very few LH 2 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 ALH 2 (Monsees et al., 1995) therefore the properties of different firefly luciferases and their mutants with ALH 2 may impact on the choice of enzymes applied.
The limited data on Ppy Fluc bioluminescence with ALH 2 as a substrate show that these properties are very different from those of LH 2 .The bioluminescence colour with ALH 2 has long been reported as pH-independent orange-red (max 605nm) (White et al., 1966), Km for ALH 2 is approximately 26-times lower and Vmax is 10 times lower than that of LH 2 (Shinde et al., 2006).There has been no further analysis of either red-shifted excited state emitter with www.intechopen.com Bioluminescence -Recent Advances in Oceanic Measurements and Laboratory Applications 122 ALH 2 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 ALH 2 .This indicates that red emission is not an intrinsic property of ALH 2 , 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 ALH 2 , 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 LH 2 and ALH 2 , and presents a great potential for in vivo applications with both substrates.

Materials
D-LH 2 potassium salt was obtained from Europa Bioproducts and D-ALH 2 from Marker Gene Technologies Inc. (Eugene, OR, USA).x2 Fluc was donated by Dr. Peter White (Dstl, Porton Down, Salisbury, UK) [White et al., 2002;Willey et al., 2001].Ultra-Glo TM luciferase (UG) was purchased from Promega and all other chemicals were purchased from Melford Laboratories Ltd. or Sigma-Aldrich unless otherwise specified.

Construction of the x12 Fluc mutant and revertants
Seven mutations were introduced sequentially onto the 5 luciferase gene in pET16b-luc5 (Law et al., 2006)

Expression and purification of 12 Fluc and revertants
His 10 -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 protocol, with BSA as the standard.

Luciferase activity assays, kinetic analysis, pH dependence of activity, thermal inactivation and bioluminescence spectra
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 MgSO 4 (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 variant PMT sensitivity as previously described (Law et al., 2006).

Mammalian cell culture, retrovirus production and transduction of cells
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% FCS and 1 % glutamax in 5% CO 2 .

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Bioluminescence -Recent Advances in Oceanic Measurements and Laboratory Applications 124

In vivo imaging
Three month old Beta2m-mice were tail vein injected with 1 x 10 6 Raji cells expressing WT or x11 Fluc.These were imaged using 30-60 s exposures in an IVIS 200 imager (Caliper, NJ, USA) at days 3 and 10 after anaesthesia with isofluorane and 15 min after intra-peritoneal (i.p.) injection of 200 l of sterile D-luciferin (Regis Technologies, IL, USA).

Construction, expression, purification and thermal inactivation of 12 Fluc and subset mutants
Seven additional mutations, previously shown to confer slower rates of thermal inactivation of Fluc, were sequentially added onto the 5 Fluc by SDM to create x12 Fluc, which was expressed in BL21(DE3)pLysS and purified to > 90 % homogeneity as previously described (Law et al., 2006;White et al., 1996;Tisi et al., 2002;Squirrell et al., 1999).The loss of activity of 12 Fluc was measured at 55C in two buffers, one of which allows direct comparison with the previously described 5 Fluc (Fig. 2A -buffer A) and the other, mimicking conditions used in BART (bioluminescent assay for monitoring nucleic acid amplification in real-time) (Fig. 2A -buffer B) (Tisi et al., 2002) .12 Fluc retained 80% of starting total activity after 30 min of treatment at 55C, whereas 5 Fluc had < 1% activity remaining after 5 min at the same temperature (results not shown).When compared to previous thermostable Fluc mutants (Table 1) containing subsets of x12 Fluc mutations, x12 Fluc was the most resistant to thermal inactivation at 40ºC (85-90% of initial activity after 1hr) (Fig. 2B), followed by x4 and x2 Fluc (both 75-80% after 1hr) and then x5 Fluc (20% after 1hr), which are all more stable than WT Fluc (fully inactivated within 10 min).

Effect of pH on x12 Fluc activity
Detailed investigation on the pH-dependence of luciferase mutant activity revealed a significant further improvement in pH-tolerant profile from that of 5 Fluc (Law et al., 2006).
The normalised pH-dependence of activity was shown to facilitate comparison of activity across the range of pH values (Fig. 3A).The non-normalised results for 12 Fluc and UG emphasize the increase in activity for the 12 Fluc relative to UG (Fig. 3B).The high level of activity ( 80 % of maximum activity) exhibited by 12 Fluc across a range of physiologically relevant pH values (6.6 -8.6) is likely to offer greater sensitivity and reliability when used in place of existing luciferase mutants in many applications, particularly those requiring a lower pH than the optimal for for Fluc or those that experience pH fluctuations such as whole cell or animal imaging (Frullano et al., 2010).

Bioluminescence spectra and kinetic properties of WT Fluc, x12 Fluc and UG with LH 2 and ALH 2
The bioluminescence spectrum of WT Fluc is known to undergo a classic red bathochromic shift with LH 2 at low pH, whereas x12 Fluc and UG maintained consistent yellow-green colours emission maximum of 557 nm and 560 nm respectively over the www.intechopen.com Bioluminescence -Recent Advances in Oceanic Measurements and Laboratory Applications 126 investigated pH range of 6.2-8.8,(Table 2).Neither x12 nor UG showed any significant widening of the spectra at lower pH.Overall, bioluminescence spectra of x12 were even less pH-dependant than those of UG.Both thermostable mutants would be expected to retain their colour under physiological conditions, which may be pertinent to multispectral imaging.The tolerance of x12 Fluc to low pH may give it further advantage in terms of signal strength.

A B
Luciferase activity (20 l at 0. Kinetic parameters including Michaelis-Menten constants (Km) and catalytic constant (Kcat) were measured for the WT, x12 and UG luciferases with LH 2 and ALH 2 .The Kms of x12 Fluc (6.8 M) and UG (3.7 M) for LH 2 were lower than that of WT Fluc (18M) (Table 3), indicating an increase in affinity of the thermostable enzymes for LH 2 relative to the WT.Even stabilising mutations far from the active site can affect tertiary structure and lead to increased substrate affinities and altered enzyme kinetics (Squirrell et al., 1999).Catalytic efficiency characterised by Kcat/Km ratio was significantly compromised in both thermostable luciferases as compared to the WT, with UG being more thermostable but less catalytically efficient than x12.In terms of specific activity x12 Fluc retained only 15% of the specific activity of the WT, exceeding that of UG by four-fold.
For in vivo applications involving imaging of protease activity where ALH 2 is to be detected instead of LH 2 .Therefore, bioluminescence properties of these mutants with ALH 2 were tested.WT Fluc is known to produce pH independent orange-red emission with ALH 2 (White et al., 1966)    In terms of kinetic parameters significant differences were observed in Km and Kcat values for LH 2 and ALH 2 between the WT and thermostable luciferases.In the WT both Km and Kcat for ALH 2 were significantly lower than for LH 2 , suggesting that WT Fluc has higher affinity for ALH 2 than for LH 2 (Shinde et al., 2006) Table 4. Bioluminescent spectra of the WT, Fluc mutants and UG with ALH 2 at different pH WT Fluc appears to provide stringent interactions dictating reaction kinetics with ALH 2 , but not those that govern the emitting species.This may also be indicated by larger halfbandwidths of bioluminescence spectra with ALH 2 than with LH 2 .Low K m values were proposed to correlate with more blue-shifted emission (Kutuzova et al., 1997), but this is not always true and it is possible for Km to be more linked to bioluminescence spectra halfbandwidth, i.e. the extent of vibrational freedom of oxyluciferin (Viviani et al., 2001).However, here, WT Fluc has wider spectral half-bandwidth with ALH 2 as compared to LH 2 , but lower Km.WT Fluc had only 10% of the specific activity with ALH 2 as compared to LH 2 at pH 7.8 (Table 5).On the contrary, x12 Fluc's activity with ALH 2 (6%) is only 2.5-fold lower than that with LH 2 (15%), while UG had as low as 4% with both substrates.Reasons for lower Vm and red-shifted emission of WT Fluc with ALH 2 have been postulated (Shinde et al., 2006;McCapra and Perring, 1985;Wada et al., 2007).

Properties of thermostable mutants containing subsets of x12 Fluc mutations
Elimination of bathochromic shift at low pH, increase in thermostability and pH-tolerance were associated with the significant reduction in specific activity and changes in the essential kinetic parameters.In an attempt to identify mutations responsible for the undesirable associated changes and to improve the understanding of kinetics and activity differences between WT Fluc and x12 Fluc with LH 2 and ALH 2 a number of thermostable enzymes with subsets of x12 Fluc mutations were investigated (Table 1).These were x2 Fluc (E354R/ D357Y) (Baggett et al., 2004), x4 Fluc (T214A/ I232A/ F295L/ E354K) (Tisi et al., 2002) and x5 Fluc (F14R/ L35Q/ V182K/ I232K/ F465R) (Law et al., 2006).Among the subset mutants only x2 showed bathochromic shift with LH 2 at low pH, similar to that www.intechopen.com

Development of a pH-Tolerant Thermostable
Photinus pyralis Luciferase for Brighter In Vivo Imaging 129 observed in the WT (Table 2).x4 and x5 Flucs resisted red-shift at low pH in line with x12.
With ALH 2 none of the subset mutants showed any significant shift in the bioluminescence across the investigated pH range, which was similar to the behaviour of x12, WT and UG with this substrate (Table 4).Kinetic parameters were derived from flash heights (Hanes, 1932) by varying ALH2 concentration in the range of 0.1-600 M used with 3mM ATP to saturate (see Materials and Methods).Specific activity was determined in the presence of 20.1 pmol enzyme by integrating light in 20 ms pulses over 12 s.PMT voltage 550 mV.

Mutants
Table 5. Kinetic parameters of WT, Fluc mutants and UG with ALH 2 In terms of kinetic parameters, if compared to the WT, the effect of mutations in x4 on Km and Kcat with LH 2 differed significantly from mutations in both x2 and x5 subsets (Table 3).
Mutations in x4 increased the Km, while in two other subsets decreased it, and reduced the Kcat, while in the other subsets hardly any change was observed.These results correlated well with the previously reported in literature (Law et al., 2006;Tisi et al., 2002;Prebble et al., 2001;Branchini et al., 2007).Similar trends were observed in the kinetic parameters for ALH 2 (Table 5).Mutations in x4 significantly increased its Km for this substrate (as in x12), while two other mutants slightly decreased it.Kinetics of the x4 mutant with ALH 2 exhibited slower rise and slower decay, reminiscent of the kinetics of x12, whereas x2 and x5 displayed a sharp flash that strongly resembled the WT.
The differences in kinetic parameters and flash kinetics suggested that an undesirable effect of mutations was common to x4 and x12 mutants, but not to x2 or x5, on catalysis with both LH 2 and ALH 2 .It was hypothesised that reversion of buried x12 Fluc mutations T214C and F295L might enhance its catalytic properties.

Properties of T214 and F295 revertant mutants of x12 Fluc
x12 Fluc mutations T214C and F295L were individually reverted and their bioluminescent properties, catalytic parameters and thermal stability investigated.Bioluminescence spectra of both revertants were measured and found to match those of x12 Fluc with both substrates (Table 6).F295 revertant showed no bathochromic shift with either substrate and retain the pH-tolerance previously observed in x12 Fluc.T214 revertant had a flash kinetics similar to Table 6.Bioluminescent spectra of x12 Fluc and its revertants at different pH with LH 2 and ALH 2 T214 revertant exhibited Kms and Kcats similar to x12 Fluc for LH 2 and its Kcat/Km ratio was not significantly different from that of x12 Fluc (Table 7).The Km of T214 for ALH 2 was slightly lower than x12 Fluc and Kcat elevated, resulting in the higher Kcat/Km ratio.T214 revertant had similar specific activities to x12 Fluc with both substrates.However, the Km of F295 revertant for both LH 2 and ALH 2 was lower and Kcat was higher than that of x12 Fluc resulting in a much higher Kcat/Km ratio.Specific activity of F295 revertant with both substrates was two-fold brighter than that of x12 Fluc.As a result of these findings F295 revertant afforded higher sensitivity to the flash-based detection of LH 2 and ALH 2 than x12 Fluc.It is possible to conclude that in x12 Fluc residue F295 contributes much more to catalysis than T214 and its reverse-mutation may significantly improve the performance of x12 Fluc providing it has no negative effect on the thermostability.Although T214C and F295L are thermostabilising mutations (Law et al., 2006;Tisi et al., 2002), revertants displayed resistance to thermal inactivation similar to that of x12 Fluc at 40 º C and 50 º C (Fig. 4).Therefore, the mutation F295L is not essential for practically useful

In vivo imaging
Human retrovirus encoding Ppy Fluc or x11 Fluc genes bearing a myc tag was used to transduce Raji cells, which were sorted to the same expression levels (Fig. 5A) by flow cytometric sorting for myc tag staining and cultured at 37 º C. As an example of in vivo imaging using x11 Fluc, one million Raji cells expressing similar levels of WT or x11 Flucs were injected into the tail veins of immunocompromised Beta2m-mice to induce systemic lymphoma (Chao et al., 2011) and imaged after i.p. administration of LH 2 .Images revealed light signals predominantly from brain, spine and hips.x11 Fluc appeared very bright in vivo (Fig. 5B) because of its high thermostability and pH tolerance along with favourable kinetic parameters demonstrated in characterisation.It is expected to perform equally well under the changing physiological pH conditions and in combination with aminoluciferin used for imaging of protease assays in vivo.Further work to codon-optimise and test this mutant in mammalian cells is underway and will be published shortly.

Conclusion
In the present study, we describe the construction and characterisation of the firefly luciferase mutant 12 Fluc based on the mutations previously identified as increasing thermostability of the enzyme.Detailed characterization of its bioluminescent and biochemical properties revealed that it is the only luciferase mutant reported to exhibit  80 % of total activity across a wide pH range of 6.6 -8.8 covering physiologically significant pH at the lower end.Additionally, it possesses sufficient thermal stability to be applicable to assays that require temperatures lower than 50C or in assays involving short lengths of higher temperature exposure.This mutant could be beneficial for in vivo imaging with luciferin, particularly in assays that experience pH fluctuations, and for bioluminescence protease assays or in vivo protease imaging, in which the sensitivity of detection may be dependent on the sensitivity of ALH 2 detection.The latter assays could benefit further from increased brightness. A.
B.  The brightness of x12 Fluc was improved by identifying the mutation at the 295 position as having the major negative impact on the bioluminescent characteristics, reverting it and creating x11 Fluc, which possessed all the desirable properties.x11 Fluc has remarkably high activity and catalytic efficiency with LH 2 and ALH 2 , coupled to a high resistance to thermal inactivation and pH-tolerance.Its highly advantageous properties in terms of stability and brightness in mammalian cells have been demonstrated using systemic lymphoma expressing the mutant in mice as an example, as there was no normalisation for engraftment and no statistical difference between WT and x11 in this small sample set.
www.intechopen.comDevelopment of a pH-Tolerant Thermostable Photinus pyralis Luciferase for Brighter In Vivo Imaging 125 Thermal inactivation of x12 Fluc was determined at 55C in two different conditions, namely 200 nM of 12 Fluc in Buffer A (50 mM phosphate buffer, pH 7.8, 10% glycerol (v/v), 2 mM DTT) and 86 nM of 12 enzyme in Buffer B (20 mM Tris.Cl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100 (v/v), 5% trehalose (w/v), 0.5% BSA (w/v), 0.4 mg/ml PVP, 10 mM DTT). 30 l aliquots of enzyme in the respective condition were incubated in water bath at 55C for varying lengths of time up to 30 min.Enzyme activity was assayed by the injection of 100 l of TEM, pH 7.8, 1 mM ATP, 200 M LH2 into wells containing 5 l of enzyme and the measurement of flash height.PMT voltages used were 760 mV and 1000 mV for experiment in Buffer A and B respectively.Results shown are mean values  S.E.M. for triplicate measurements (A).Flash-based activity with LH2 was compared in aliquots of 0.5 M enzyme incubated at set temperatures over time.Samples equilibrated to room temperature before dispensing 260 l 70 M LH2 and 1 mM ATP solution in TEM buffer (pH 7.8) into 40 l luciferase mutant (B).

FACS
histogram showing expression of WT Fluc and thermostable mutants in Raji cells.Cells obtained by fluorescence activated cell sorting were expanded in vitro and expression of transgenes was analysed using a Cyan flow cytometer using anti-myc.FITC staining.Non-transduced cells (blue line -filled) were compared to WT Fluc (red line) and x11 Fluc (blue line -dashed) (A).In vivo imaging of systemic lymphoma expressing WT Fluc (left) and x11 mutant (right).Mice with lymphomas expressing either WT Fluc or x11 Fluc were imaged in three groups of three mice (B).

Fig. 5 .
Fig. 5. Expression and example of in vivo imaging of Flucs in mammalian cells.

Table 1 .
Mutations and their positions in thermostable Fluc mutants and here it was seen that WT Fluc, x12 Fluc and UG were red-shifted to

Table 2 .
Bioluminescent spectra of the WT, Fluc mutants and UG with LH 2 at different pH

Table 3 .
Kinetic parameters of WT, Fluc mutants and UG with LH 2 . In thermostable x12 Fluc both Km and Kcat for ALH 2 were very close to those of LH 2 , while in UG Km for ALH 2 was higher and Kcat was lower than for LH 2 .The mutations that invoke thermostability increase the affinity of Fluc for LH 2 , but reduce it for ALH 2 .WT Fluc had nearly 10-fold higher catalytic efficiency (Kcat/Km) with LH 2 and ALH 2 than x12 Fluc.x12 demonstrated comparable Kcat/Km ratio for both substrates, favouring its use with ALH 2 while UG had a similar catalytic efficiency to x12 Fluc for LH 2 , but 23-times lower Kcat/Km for ALH 2 .Overall, x12 Fluc seems to be a mutant of choice to be used with ALH 2 . of each mutant was added to 100 µM ALH2 and 3 mM ATP in chilled 0.1 M TEM buffer, adjusted to a varying pH and spectra were measured after 30 s at ca. 25ºC.Half bandwidths are widths of spectra at half maximum intensity.Data were corrected for PMT spectral response.UG was assayed at pH 6.2 not 6.5.Standard error +2nm.

Table 7 .
Kinetic parameters of x12 Fluc revertants with LH 2 and ALH 2 Development of a pH-Tolerant Thermostable Photinus pyralis Luciferase for Brighter In Vivo Imaging 133 www.intechopen.com