Overexpression and Purification of Wbl Proteins

Overexpression and purification of the proteins of interest from E. coli BL21-Gold (DE3) for structural and biochemical studies were done as described in our study of WhiB7 (26 (link)). Samples after each step of purification were analyzed by SDS-PAGE and by UV-Visible (UV-Vis) spectroscopy. Unless otherwise specified, the final purified proteins in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.2 mM tris(2-carboxyethyl)phosphine were stored in liquid nitrogen until use. UV-Vis spectra of the purified proteins were recorded using an HP 8452a diode array UV-Vis spectrophotometer (Agilent Technologies Inc). The absorption at 410 nm, characteristic of proteins containing [4Fe-4S]²⁺ clusters (43 (link), 44 (link)), was used to estimate the occupancy of the Fe–S cluster in the protein samples containing a Wbl protein. Protein concentrations were estimated either by the Pierce Bradford Assay Kit (Thermo Fisher Scientific) or UV-Vis absorption spectroscopy.