**2.5 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% CO2.

Development of a pH-Tolerant Thermostable

0 5 10 15 20 25 30 **Time (min**

Fig. 2. Thermal inactivation of x12 Fluc and subset mutants.

whole cell or animal imaging (Frullano *et al.*, 2010).

**3.2 Effect of pH on x12 Fluc activity** 

**LH2 and ALH2**

0

0.2

Buffer A Buffer B

0.4

0.6

0.8

1

1.2

*Photinus pyralis* Luciferase for Brighter *In Vivo* Imaging 125

**A B**  Thermal inactivation of x12 Fluc was determined at 55C 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 55C 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**)**.

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

**3.3 Bioluminescence spectra and kinetic properties of WT Fluc, x12 Fluc and UG with** 

The bioluminescence spectrum of WT Fluc is known to undergo a classic red bathochromic shift with LH2 at low pH, whereas x12 Fluc and UG maintained consistent yellow-green colours emission maximum of 557 nm and 560 nm respectively over the
