**3. Results and discussion**

#### **3.1 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).


Table 1. Mutations and their positions in thermostable Fluc mutants

The loss of activity of 12 Fluc was measured at 55C 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 55C, 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).

Three month old Beta2m-mice were tail vein injected with 1 x 106 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

**3.1 Construction, expression, purification and thermal inactivation of 12 Fluc and** 

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

**Mutation Mutants Location** x12 x11 x5 x4 x2

The loss of activity of 12 Fluc was measured at 55C 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 55C, 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

F14R + + + Surface L35Q + + + Internal A105V + + Surface V182K + + + Surface T214A + Internal T214C + + Internal I232A + Surface I232K + + + Surface D234G + + Surface F295L + + Internal E354R + + + Surface E354K + Surface D357Y + + + Surface S420T + + Surface F465R + + + Surface

(i.p.) injection of 200 l of sterile D-luciferin (Regis Technologies, IL, USA).

(Law *et al.*, 2006; White *et al.*, 1996; Tisi *et al.*, 2002; Squirrell *et al.*, 1999).

Table 1. Mutations and their positions in thermostable Fluc mutants

1hr), which are all more stable than WT Fluc (fully inactivated within 10 min).

**2.6** *In vivo* **imaging** 

**subset mutants** 

**3. Results and discussion** 

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**)**.

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