2.2. Prokaryotic UROS expression and UROS enzymatic activity

The cDNA for UROS variants were generated by site directed mutagenesis and inserted into the prokaryotic expression vector pET32a. The integrity of the constructs were checked by sequencing. Freshly transformed E. coli BL21 (DE3) cells were cultured in the presence of ampicillin (50 μg/mL) and chloramphenicol (34 μg/mL) and UROS protein expression was induced by adding 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside, Sigma-Aldrich, USA), during 3 h at 37 °C. Bacterial pellets were harvested by centrifugation and maintained at −80 °C until analysis. UROS activity was measured in bacterial lysates with the coupled-enzyme assay as described previously [10]. Specific activity is expressed as uroporphyrin (μmol/h/g) of total proteins in bacterial lysates.