Purification of Bromodomain Proteins

Purified proteins VBC, SMACRC2^BD (bromodomain), BRD4^BD1 or BRD4^BD2 were expressed and purified according to the previously published protocols^{28 (link),31 (link)}. Briefly, purification of N-terminal His₆ tagged proteins (1) SMARCA2^BD 1373–1511 ∆1400–1417; (2) VHL (54-213), ElonginB (17-112) ElonginC (1-104); and (3) BRD4 (44-168) cells were lysed by 2 passes on microfluidizer and ultracentrifuged at 235,000 g for lysate clarification. Clarified lysate was mixed with Talon resin and nutated for an hour at 4 °C, washed and eluted with 250 mM imidazole. The His₆ tag was removed using TEV protease. Cleaved proteins were then purified using Superdex S-75 column (GE Healthcare). Purified protein samples were stored in 10 mM HEPES 7.5, 150 mM NaCl, 0.5 mM TCEP at -80 °C to be used for crystallization/SPR studies.