BRD4 domain 2
PDB:2OUO
Revision
Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_055114.1
Entry Clone Source:FivePrime
SGC Clone Accession:Tag:Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) ; TEV-cleavable (*) N-terminal his6 tag
Host:BL21 (DE3)
Construct
Prelude:Sequence:mhhhhhhssgvdlgtenlyfqsmKDVPDS QQHPAPEKSSKVSEQLKCCSGILKEMFAK KHAAYAWPFYKPVDVEALGLHDYCDIIKH PMDMSTIKSKLEAREYRDAQEFGADVRLM FSNCYKYNPPDHEVVAMARKLQDVFEMRF AKMPDE
Vector:pNIC28-Bsa4
Growth
Medium:LB
Antibiotics:Procedure:Grow starter cultures from freshly transformed colonies in 10 ml LB, 50 mg/ml kanamycin. This started culture was diluted 1:1000 in fresh media and was grown at 37°C to a OD600 of 0.4 and than transferred to 18 degC. Expression was induced at an OD600 of 0.6 - 0.7 using 1 mM IPTG (final concentration). Cells were harvested after 4h by centrifugation (15min, 6500rpm on a JLA 8.100 rotor), transferred to 50-ml tubes, and frozen at -20 degC.
Purification
ProcedureColumn 1 : DE52/Ni-NTA
A DE52 column (10gr suspended in 100ml of 2.5M NaCl) was equilibrated with 100ml of Loading Buffer. A 5ml NiNTA column was equilibrated with 20ml of Loading Buffer. The lysed sample was applied to the DE-52 column and washed through with 50 ml loading buffer. The flow through was applied to the 5 ml Ni-NTA column which was washed with 2x10ml of wash buffer and eluted with elution buffer in 5 ml aliquots. (Step elution using 50, 100, 150, 200 and 250 mM imidazole in the Elution Buffer)
Enzymatic treatment : Treated the IMAC elution(s) with TEV protease overnight.
Column 2 : SEC (AKTA-prime)
Fractions containing BRD4A collected from IMAC and treated with TEV protease overnight (identified by SDS PAGE) were concentrated to about 1.5ml and directly applied to a S75 16/60 column equilibrated in 10 mM Hepes pH 7.5, 100 m NaCl. The flow rate was 1ml/min and the pure protein eluted at 76-88min.
Concentration : The combined samples from the SEC column (identified by SDS PAGE) were concentrated using centricons with 10 kDa cut off.
Extraction
ProcedureThe cell pellets (5 gr wet wt) were re-suspended in 50 ml extraction buffer containing a Protease Inhibitor Coctail tablet (Roche), and lysed in a high pressure cell disrupter. The supernatant was centrifuged for 45 minutes at 53k g in a JA 25.5 rotor.
Concentration:LigandMassSpec:LC-ESI-MStof confirmed the correct mass of 15036 expected for this construct after TEV treatment.
Crystallization:Crystals were grown at 4 degC in 150nl sitting drops mixing 75 nl of BRD4A (19 mg/ml in 10mM Hepes pH 7.5, 100mM NaCl ,10mM DTT) with 75 nl of a solution containing 0.1M MIB pH 7.0 and 30.0% PEG 1K. Cryo protection was achieved by adding ethylene glycole to the crystallization mix (20% final w/v).
NMR Spectroscopy:Data Collection:Resolution: 1.89Å; X-ray source: Rotating anode, Rigaku FR-E superbright.
Data Processing: