**4. 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

Development of a pH-Tolerant Thermostable

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**5. Acknowledgements** 

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significant pH at the lower end. Additionally, it possesses sufficient thermal stability to be applicable to assays that require temperatures lower than 50C 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 ALH2 detection. The latter assays could benefit further from increased brightness.

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. Expression and example of *in vivo* imaging of Flucs in mammalian cells.

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 LH2 and ALH2, 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.
