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

Amit Jathoul<sup>1</sup>,5, Erica Law2, Olga Gandelman3,\* ,

Martin Pule1, Laurence Tisi<sup>3</sup> and Jim Murray<sup>4</sup>

*Cancer Institute, University College London, Illumina Inc., Chesterford Research Park, Lumora Ltd., Cambridgeshire Business Park, School of Biosciences, Cardiff University Institute of Biotechnology, Cambridge UK* 

#### **1. 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.

$$\text{LH}\_2 + \text{ATP} + \text{O}\_2 \rightarrow \text{LO} + \text{AMP} + \text{PP}\_i + \text{CO}\_2 + \text{l}v$$

**A** 

Fig. 1. Bioluminescent reaction of Fluc (A) and chemical structures of luciferin (LH2) (B) and aminoluciferin (ALH2) (C) eliciting intense bioluminescence.

Fluc has found intensive application in small animal *in vivo* bioluminescence imaging (BLI)

<sup>\*</sup> Corresponding Author
