An Essential Antioxidant in H. thermophilus Figure 6. Phylogenetic tree of proteins annotated as rubrerythrin (Rbr) or Rbr-like proteins. The sequences from thermophiles are indicated by gray boxes. Circles and triangles denote domain-swapping and non-domain-swapping, respectively. Open and closed symbols denote whether each conformation was predicted by sequence alignment or experimental analyses, respectively. 1, H. thermophilus; 2, S. tokodaii; 3, P. furiosus; and 4, D. vulgaris. Group I, two-domain Rpr-type proteins from mainly Clostridia and Bacteroidia; II, single-domain Rbrs from anaerobic organisms, including Euryarchaeota; III, single-domain Fpx-SuE-type proteins from aerobic organisms, including Actinobacteria, a-, b-, c-, and d- proteobacteria, and Crenarchaeota; IV, two-domain Rbrs from mainly Cyanobacteria; V, two-domain Rbrs from various organisms, including Thermotogae, Clostridia, Actinobacteria, d-proteobacteria, Euryarchaeota, and Crenarchaeota; VI, two-domain Rbrs from mainly Clostridia, Bacteroidia, and d-proteobacteria; and VII, two-domain Rbrs from mainly Clostridia, Bacteroidia, and e-proteobacteria. doi:10.1371/journal.pone.0034825.g006 with the primer pair, FpxD-F (59-CTTTCCTGCAGTTCCG- CAAAAACAG-39), containing a PstI site (underlined) and FpxD- R (59- GGGGGATCCCATAAACTACC -39), containing a BamHI site (italics). The htk gene and upstream promoter [39] was amplified by PCR using primer pair, HTK-F (59- AAAGG- TACCCGTTGACGG-39) and HTK-R (59- AAAGGTACCG- TAACCAACATGATTAACAAT-39), with introduced KpnI sites (underlined). The amplified DNA fragment carrying the fpx gene was digested with PstI, KpnI, and BamHI, and then ligated with a KpnI-digested DNA fragment carrying the htk gene and PstI- BamHI-digested pUC19, resulting in pFGD. Heterologous expression and purification of recombinant Fpx E. coli BL21 harboring pET-Fpx was cultivated aerobically in LB medium containing ampicillin at 37uC. When the optical density at 600 nm of the culture reached 0.6, 1 mM IPTG and 1 mM FeSO4 were added to the medium. After further cultivation for 3 h, cells were harvested at 10,0006g for 15 min, and washed once with 20 mM Tris-HCl buffer [pH 8.0]. The cells were resuspended in the same buffer, and then disrupted by sonication using an Ultrasonic disruptor (TOMY, Nerima, Tokyo, Japan) at 100 W using a 50% duty cycle for 15 min. The sonicated mixture was transferred into a vial, which was then sealed with a rubber septum and an aluminum cap, and the head space gas was replaced with argon. The vial was heat-treated at 80uC for 15 min. The heat-denatured proteins and cell debris were removed by centrifugation at 100,0006g for 30 min, and the resulting supernatant was used as CFE. Chromatography was performed aerobically with the A¨KTA purifier system (GE Healthcare) at RT. The purity of recombinant Fpx was monitored by the ratio of absorption at 370 nm to that at 280 nm (A370/A280) and SDS- PAGE analysis. The heat-treated extract was applied onto a Q Sepharose fast-flow column (10 mm610 cm; GE Healthcare) equilibrated with 20 mM Tris-HCl buffer [pH 8.0] (buffer A). Proteins were eluted with a linear gradient from 0 to 0.25 M NaCl PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34825