*4.2. Bacterial Expression of Human CYB5 and Purification*

The cDNA of the open reading frame of human full length CYB5 was cloned in pET15b, as described in Nunez et al., 2010 [44], except that the full sequence was used instead of only the soluble part of it. The resulting plasmid was transformed into BL21-DE3 for expression. A single colony was grown in Terrific Browth medium containing 100 μg/mL ampicillin for 72 h with shaking at 22 ◦C. Cells were harvested by centrifugation at 4000× *g*, and the resulting pellet was resuspended and incubated for 30 min in 50 mM Tris-HCl, pH 7.4, containing 1 mM PMSF and 1 mg/mL lysozyme. Cells were lysed by sonication. Then 0.02 mg/mL RNase and 0.05 mg/mL DNase were added, and CYB5 was solubilized at 4 ◦C with 1% (*w*/*v*) sodium cholate, pH 7.4, for 1 h with moderate shaking. Supernatant was loaded onto a DEAE-cellulose anion-exchange column equilibrated with 0.2% (*w*/*v*) sodium cholate, 20 mM sodium/potassium phosphate buffer, pH 7.4. CYB5 was eluted with 0.5 M NaCl, 0.2% cholate, and 20 mM sodium/potassium phosphate buffer, pH 7.4. Fractions containing CYB5 were applied to a hydroxylapatite column equilibrated with 0.5 M NaCl and 20 mM sodium/potassium phosphate buffer, pH 7.4. Pure CYB5 was eluted with 0.1% (*w*/*v*) sodium cholate and 0.5 M sodium/potassium phosphate buffer and dialyzed against 0.1% (*w*/*v*) sodium cholate, 1 mM PMSF, and 20 mM sodium/potassium phosphate buffer. CYB5 content of samples was determined by spectrophotometric techniques as described previously [28,29]. CYB5 was concentrated and stored at −20 ◦C.

#### *4.3. Bacterial Co-Expression of Human CPR Mutants and CYPs*

CPR forms were expressed as a full-length membrane bound proteins using a dedicated *E. coli* host, the BTC strain, using the specialized bi-plasmid system adequate for co-expression of CPR with representative human CYP [26,27]. Plasmid pLCM\_*POR* [20] was used for the expression of the membrane-bound, full-length WT form and mutants of human CPR [24]. The expression of human CYP-isoforms in the cell model was accomplished with plasmid pCWori containing human wildtype CYP cDNA (pCWh\_1A2, pCWh\_2A6 or pCWh\_3A4) [45]. The pLCM\_*POR* and the CYP plasmids pCWh were transfected through standard electroporation procedures [22]. Each strain was cultured in TB medium supplemented with peptone (2 g/L), thiamine (1 μg/mL), ampicillin (50 μg/mL), kanamycin (15 μg/mL), chloramphenicol (10 μg/mL), trace elements solution [46] (0.4 μL/mL), IPTG (0.2 mmol/L) and δ-Ala (100 μmol/L), final concentrations. Cultures were started with −80 ◦C glycerol stocks and cells were grown for 16 h at 28 ◦C with moderate agitation. CYP content of bacterial whole-cells preparations was determined using standard CO-difference spectrophotometry [47].

#### *4.4. Whole-Cell Mutagenicity Assays*

The mutagenicity assays were performed as described previously [20,23,45,47] using the liquid pre-incubation assay technique [48,49]). Briefly, BTC bacteria were grown for 18 h in TB medium supplemented with peptone (2 g/L), thiamine (1 μg/mL), ampicillin (50 μg/mL), kanamycin (15 μg/mL), a mixture of trace elements solution [46] (0.4 μL/mL) and IPTG (0.2 μmol/L), final concentrations. Pre-incubation was performed for 45 min in an orbital shaker at 37 ◦C before plating. Incubation buffer contained 10 mM glucose. Stock solutions of carcinogens were freshly made in dimethyl sulfoxide (DMSO) and working solutions were obtained by dilution in water. DMSO concentration in preincubations were ≤1.3%. Experiments were performed at least in triplicate. Revertant colonies on L-Arg selector plates were counted after 48 h incubation at 37 ◦C. Revertant colonies were determined by ProtoCOL 3 colony counter (Synbiosis, Cambridge, UK) using ProtoCOL

V0 1.0.6 Software. CYP-mediated bioactivation was expressed in terms of mutagenic activity [L-arginine prototrophic (revertant) colonies per nmole of test compound, or in revertant colonies per μmole of test compound], determined from the slope of the linear portion of the dose–response curve.
