FIGURE 5.

Biochemical validation and properties of FNR_AF. (A) Western blot, antibodies prepared against FNR_EC were used to detect FNR_AF, which was overproduced from plasmid pET100/D-TOPO fnr_AF cloned into E. coli strain BL21. The gel shows the molecular mass marker (MM), crude cell free extract (approximately 10 μg of total protein) prepared prior to induction of fnr_AF expression with IPTG (lane 1), 10 μg crude cell free extract 1 h after induction of fnr_AF expression with IPTG (lane 2), and elution’s 1 to 4 of 10 μg purified FNR_AF protein after elution from the nickel column (lanes 3–6). (B) Overproduction and purification of FNR_AF from the pET11A fnr_AF plasmid as shown by SDS-PAGE analysis. Gel lane 1 shows crude cell free extract (approximately 10 μg of total protein) prepared prior to induction of fnr_AF expression with IPTG; lane 2, 10 μg of crude cell free extract 1 h after induction of fnr_AF expression with IPTG; and lane 3, 10 μg FNR_AF after the second cationic interchange chromatographic column. M, molecular mass marker (Mr are indicated). (C) Ultraviolet/visible absorption spectrum of a representative result of biological duplicates for purified FNR_AF from anaerobically grown At. ferrooxidans with an inset of the ultraviolet/visible spectrum of anaerobically purified FNR_EC taken from Lazazzera et al. (1993). (D) Calculation of the FNR_AF iron content by spectrophotometric assay.