Purification of BRD4 Domains and Mutants

BRD4 domains and mutants were expressed essentially as described (17 (link)) using One Shot BL21(DE3)pLysS bacteria stimulated with 0.5 mm isopropyl-β-d-thiogalactopyranoside (Sigma). Bacteria expressing His₆-tagged proteins BRD4 BD1 or BD2 wild type were resuspended in 50 mm HEPES, pH 7.5 (Applichem), 500 mm NaCl (Sigma), 5% glycerol (Sigma), 5 mm imidazole (Sigma), 0.5 mm Tris(2-carboxyethyl)phosphine (Pierce), and EDTA-free Complete, and lysed using a Microfluidizer (Microfluidics). The proteins were then purified by affinity chromatography using nickel-nitrilotriacetic acid-agarose (Macherey-Nagel) or His-trap columns (GE Healthcare) followed by a size exclusion chromatography step using a Superdex S75 26/60 column (GE Healthcare). The mutated variants were purified by affinity chromatography using the nickel-nitrilotriacetic acid Fast Start Kit (Qiagen) following the instructions for purification under native conditions. A second purification step by size exclusion chromatography using a Superdex S75 16/60 column was then performed. The fractions were collected and monitored by SDS-polyacrylamide gel electrophoresis.