The DNA fragment encoding the first bromo domain (residue 1-122) of TbBDF5 (Tb927.11.13400) from T. brucei was amplified by polymerase chain reaction (PCR) from T. brucei genomic DNA. The forward and reverse primers were 5’-GGAGATATACATATGAGTCAAAACCGGCAG-3’ and 5’-GTGGTGGTGCTCGAGGCCACCCACTTTCTG-3’, respectively. PCR products were then cloned into the vector pET-22(b) (Novagen) cleaved with NdeI/XhoI by infusion method. The recombinant vectors were transformed into Escherichia coli strain BL21 (DE3). The recombinant TbBDF5-bromo1 domain possesses 130 amino acids with a His-tag in the C-terminus (LEHHHHHH). To obtain isotopically labeled TbBDF5-bromo1 domain, the domain was overproduced in Escherichia coli BL21 (DE3) using minimal medium containing 2.5 g/L 99% 13C-glucose and 0.5 g/L 99% 15NH4Cl as the sole carbon and nitrogen source, respectively. Cells were cultured at 37℃ until OD600 = 0.8, then induced with 0.5 mM IPTG at 16℃ for 20 hours. The induced cells were harvested and resuspended in 35 mL lysis buffer (20 mM Tris, 500 mM NaCl, pH 7.8), then lysed by sonication on ice. The lysate was centrifuged at 11000×g and 4℃ for 30 min to remove precipitate. The protein was purified by Ni2+-NTA column. The labeled protein was eluted with 15 ml Tris NaCl buffer containing 500 mM imidazole. The eluted protein was further purified using a Superdex 75 column on an AKTA purification system. The purified proteins were dialyzed with buffer containing 25 mM NaH2PO4, 100 mM NaCl, 1 mM EDTA and 1 mM DL-Dithiothreitol (DTT) (pH 6.8) for 4 times and then concentrated to 500 µL with a concentration of 0.8 mM. The final NMR buffer contains 25 mM phosphate (pH 6.8), 100 mM NaCl, 1 mM EDTA, 1 mM DTT in 10:90% D2O: H2O.
All NMR data were collected at 293 K on a Bruker DMX 600 spectrometer. The following spectra were recorded to obtain backbone and side chain resonance assignments: 1H–15N HSQC, CBCA(CO)NH, CBCANH, HC(CO)NH, HBHA(CO)NH, H(CCO)NH, HNCO, 3D 15N-edited and 13C-edited NOESY. NMR data was processed using NMRpipe and NMRDraw software (Delaglio et al. 1995), and then analyzed with Sparky 3 (Goddard and Kneller 1993).