Recombinant Protein Expression for BRD Family

The pET-28a(+) plasmid containing the BRD3 D1 and BRD3 D2 were purchased from GenScript. The Escherichia coli strain BL21 star(DE3) was transformed with the BRD3 D1 and BRD3 D2 plasmids and plated onto an agar plates containing kanamycin (100 mg/L) and chloramphenicol (35 mg/L). The plates were incubated overnight at 37 °C. A 5 mL LB culture with kanamycin(100 mg/L) and chloramphenicol (35 mg/L) was inoculated using a single colony from each plate and grown overnight at 37 °C and shaking at 220 rpm. Four primary cultures were used to inoculate 1 L of LB media containing chloramphenicol (35 mg/L) and kanamycin (100 mg/L) until the optical density at 600 nm had reached 0.6–0.8. At this point, an equilibration time of 30 min at 20 °C and 220 rpm was followed by the addition of 1 mM IPTG to induce protein expression. The culture was shaken for 16 h at 20 °C and 220 rpm. Cells were pelleted by centrifugation at 8000g and stored at −20 °C until purification. BRD4 D1, BRD2 D1, and BRDT D1 were expressed and purified as previous reported.^{33 (link)}