0, and protein overproduction was then triggered by 0.2 mM isopropyl-ß-d-thiogalactopyranoside BMN673 (IPTG). After incubation
for 16 h at 20°C, cells were harvested by centrifuging at 10,000 × g for 15 min. Fnr was then purified as follows: the bacterial pellet was resuspended in 120 ml of buffer C (25 mM Tris–HCl [pH 8], 1 mM dithiothreitol (DTT)) and incubated with 0.2 mg.ml-1 of lysozyme and 5 mM EDTA for 10 min at 30°C. Cells were lysed by ultrasonication for 3 min using a Vibra cell ultrasonifier (Fisher Bioblock Scientific). Cell debris and membrane particles were removed by centrifuging at 43,000 × g for 1 h, and the resulting supernatant was loaded on a 30 ml DEAE-cellulose column (DE52; Whatman) equilibrated with buffer C. The non-retained fraction was adjusted to pH 7 with 1 M KH2PO4 and then loaded onto a 20 ml hydroxyapatite column (HA Ultrogel; Pall Corporation) equilibrated with buffer D (50 mM KH2PO4 [pH 7], 1 mM DTT). The column was developed with a linear gradient from 50 to 200 mM KH2PO4 at a flow rate of 2 ml/min. Fractions
containing recombinant Fnr were pooled and concentrated by ultrafiltration through an Omega disc membrane (30 kDa cutoff, Ø 43 mm, Pall Corporation). A polishing step was then carried out by gel filtration on a column of Superdex SD200 (1.5 × 60 cm, Amersham Biosciences) equilibrated with buffer F (25 mM Tris–HCl [pH 7.5]1, 50 mM NaCl, 1 mM DTT). The purified Trametinib solubility dmso protein, >90% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Additional file 1), was stored as pellets in liquid nitrogen. PAK5 Recombinant expression and purification of resD and plcR were performed using previously described methods [11, 12]. Reconstitution of Fnr holoprotein The following procedure was carried out under anoxic conditions (O2 < 1 ppm) in a glove box maintained at 18°C (Jacomex, France). All buffers were degassed under argon and equilibrated for at least 16 h in the glove box before use. ApoFnr (2 g/L) was incubated with 1 μM cysteine desulfurase CsdA
from E. coli, 1 mM l-cysteine, and 1 mM Fe(NH4)2(SO4)2 (Sigma-Aldrich) in the presence of 4 mM DTT in buffer F. Formation of the cluster was monitored by UV-visible spectroscopy using a Uvikon spectrophometer (Kontron) connected through optic fibers to the cuvette holder inside the glove box. The reaction was initiated by adding CsdA, and reached completion after 2 h (no further increase in the absorption at 416 nm). The protein was run through a 10 ml Sephadex G25 column (Amersham Biosciences) equilibrated in buffer F to remove excess reagents, and then concentrated by ultrafiltration using a Nanosep device with a molecular cutoff of 30 kDa (Pall Corporation). Protein biochemical analyses Protein concentrations were determined by either a bicinchoninic acid (BCA) assay (Pierce) or a Biuret method insensitive to thiols . Bovine serum albumin (BSA) was used as a standard.