Purification of VCB and Bromodomain Complexes

Wild-type and mutant versions of human proteins VHL (UniProt accession number: P40337), ElonginC (Q15369), ElonginB (Q15370), Brd2 (P25440), Brd3 (Q15059) and Brd4 (O60885) were used for all protein expression. For expression of VBC, N-terminally His₆-tagged VHL (54–213), ElonginC (17–112) and ElonginB (1–104) were co-expressed in Escherichia coli BL21(DE3) at 24 °C for 16 h using 3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). E. coli cells were lysed using a pressure cell homogenizer (Stansted Fluid Power) and lysate clarified by centrifugation. His₆-tagged VCB was purified on a HisTrapFF affinity column (GE Healthcare) by elution with an imidazole gradient. The His₆ tag was removed using TEV protease and the untagged complex dialysed into low concentration imidazole buffer. VCB was then flowed through the HisTrapFF column a second time, allowing impurities to bind as the complex eluted without binding. VCB was then additionally purified by anion exchange and size-exclusion chromatography using MonoQ and Superdex-75 columns (GE Healthcare), respectively. The final purified complex was stored in 20 mM Bis Tris, pH 7, 150 mM sodium chloride and 1 mM dithiothreitol (DTT). Brd2^BD1 (71–194), Brd2^BD2 (344–455), Brd3^BD1 (24–146), Brd3^BD2 (306–416), Brd4^BD1 (44–178) and Brd4^BD2 (333–460) as well as equivalent mutant constructs were expressed with an N-terminal His₆ tag in E. coli BL21(DE3) at 18 °C for 20 h using 0.2 mM IPTG. His₆-tagged BDs were purified on nickel Sepharose™ 6 fast flow beads (GE Healthcare) by elution with increasing concentrations of imidazole. For crystallography the His₆-tagged BD was cleaved with TEV protease and dialysed into low concentration imidazole buffer. The BD was then flowed over the nickel beads a second time to remove impurities and protease. BDs were then additionally purified by size-exclusion chromatography using a Superdex-75 column. For AlphaLISA, ITC and ubiquitination reactions, following elution of His₆-tagged BDs from the nickel beads, the BDs were purified by size-exclusion chromatography using a Superdex-75 column. The final purified proteins were stored in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5, 150 mM sodium chloride and 1 mM DTT. All chromatography purification steps were performed using Äkta FPLC purification systems (GE Healthcare) or glass econo-columns (Bio-Rad) at room temperature.