**2.2 Construction of the x12 Fluc mutant and revertants**

Seven mutations were introduced sequentially onto the 5 luciferase gene in pET16b-luc5 (Law *et al.*, 2006) using the QuickChangeTM Site Directed Mutagenesis (SDM) kit (Stratagene) according to the manufacturer's protocol. The primers used for the seven rounds of SDM are as follow:

5'-GCAGTTGCGCCC**GTG**AACGAC-3' and 5'-GTCGTT**CAC**GGGCGCAACTGC-3' for A105V; 5'-CCCTATTTTCATTC**CTG**GCCAAAAGCACTC-3' and 5'- GAGTGCTTTTGGC**CAG**GAATGAAAATAGGG-3' for F295L; 5'- GGCTACAT**ACT**GGAGACATAGC-3' and 5'-GCTATGTCTCC**AGT**ATGTAGCC-3' for S420T;

5'-CAAATCAAACCG**GGT**ACTGCGATTTTAAG-3' and 5'- CTTAAAATCGCAGT**ACC**CGGTTTGATTTG-3' for D234G; 5'- CCGCATAGA**TGT**GCCTGCGTCAGATTC-3' and 5'- GAATCTGACGCAGGC**ACA**TCTATGCGG-3' for T214C;

5'-CACCC**CGC**GGGGAT**TAT**AAACCGGG-3' and 5'- CCCGGTTT**ATA**ATCCCC**GCG**GGGTG-3' (AvaI) for E354R and D357Y

ALH2 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 ALH2. This indicates that red emission is not an intrinsic property of ALH2, 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 ALH2, 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 LH2 and

*D*-LH2 potassium salt was obtained from Europa Bioproducts and *D*-ALH2 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-GloTM luciferase (UG) was purchased from Promega and all other chemicals were purchased from Melford

Seven mutations were introduced sequentially onto the 5 luciferase gene in pET16b-luc5 (Law *et al.*, 2006) using the QuickChangeTM Site Directed Mutagenesis (SDM) kit (Stratagene) according to the manufacturer's protocol. The primers used for the seven

5'-GCAGTTGCGCCC**GTG**AACGAC-3' and 5'-GTCGTT**CAC**GGGCGCAACTGC-3' for

GGCTACAT**ACT**GGAGACATAGC-3' and 5'-GCTATGTCTCC**AGT**ATGTAGCC-3' for

ALH2, and presents a great potential for *in vivo* applications with both substrates.

Laboratories Ltd. or Sigma-Aldrich unless otherwise specified.

A105V; 5'-CCCTATTTTCATTC**CTG**GCCAAAAGCACTC-3' and 5'- GAGTGCTTTTGGC**CAG**GAATGAAAATAGGG-3' for F295L; 5'-

CCCGGTTT**ATA**ATCCCC**GCG**GGGTG-3' (AvaI) for E354R and D357Y

5'-CAAATCAAACCG**GGT**ACTGCGATTTTAAG-3' and 5'- CTTAAAATCGCAGT**ACC**CGGTTTGATTTG-3' for D234G; 5'-

CCGCATAGA**TGT**GCCTGCGTCAGATTC-3' and 5'- GAATCTGACGCAGGC**ACA**TCTATGCGG-3' for T214C;

5'-CACCC**CGC**GGGGAT**TAT**AAACCGGG-3' and 5'-

**2.2 Construction of the x12 Fluc mutant and revertants** 

**2. Materials and methods** 

rounds of SDM are as follow:

**2.1 Materials** 

S420T;

Boldface type represents the mutated codon, underlined letters represent modified endonuclease site used to facilitate screening, and the endonuclease used for screening is shown in parentheses. *E. coli* BL21 (pLysS) (Edge Biosystems, Gaithersburg, MD, USA or XL2-Blue ultracompetent cells (Stratagene) were used as cloning hosts for the generation and selection of mutants from site-directed mutagenesis. Expression from colonies was induced by adsorbing colonies onto HybondTM-N nitrocellulose membranes (Amersham Biosciences Corp., Piscataway, NJ, USA) and transferring membranes onto fresh Luria Bertani (LB) agar plates containing 100 g/ml carbenicillin and 1 mM IPTG and incubating for 3 hours at room temperature (RT). Bioluminescence was initiated by spraying membranes with 1 mM LH2 or 500M ALH2 in 0.1 M citrate buffer (pH 5) and colony screening was carried out by photographing emitted light with Nikon D70S camera (Nikon Corp., Tokyo, Japan). After seven rounds of SDM, mutations introduced were confirmed by sequencing of the entire luciferase gene using a facility provided by the Department of Genetics, University of Cambridge.
