Ni-NTA column purification. Buffers: Wash buffer I (WB1): 50 mM Hepes pH 8.0, 300 mM NaCl, 5 % Glycerol, 10 mM Imidazole pH 8.0. Wash Buffer II (WBII): 50 mM Hepes pH 8.0, 300mM NaCl, 30 mM Imidazole pH 8.0. Elution buffer (EB): 50 mM Hepes pH 8.0, 300 mM NaCl, 5 % Glycerol, 250 mM Imidazole pH 8.0.
Procedure: Total volume of Ni-NTA added to BioRad drip column: 4 mls (50%). Resin washed with 12.5 ml of WB1. The supernatant was applied to a column using 5 ml pipette and allowed to pass over the resin. The flow through was collected in a 50 ml falcon tube and applied once more to the column. Two wash steps followed. Wash with 12.5 ml of WBI. Wash with 12.5 ml column vols of WBII. Elute with 14 mls of EB into 7x2 ml fractions. At this stage the purity of the protein was greater than 95 % based on SDS-PAGE analysis. The C-terminal hexahistidine tag was removed by TEV protease treatment. The TEV protease, a hexa-histidine-tagged construct, was over-expressed and purified in-house to a final concentration of 2.5 mg/mL.
Extraction buffer (EX): 50 mM Hepes pH 8.0, 300 mM NaCl, 5 % Glycerol, 10 mM Imidazole pH 8.0. 1 tablet protein inhibitor in 10ml EX buffer was added to the 1L growth pellet. Total vol: 45 mL (estimate). Cell breakage: 5 passes through the Emulsiflex C5 high pressure homogeniser. Total vol: 50 mls (estimate). Centrifuge for 30 mins at 16000 rpm and 4°C to remove cell debris. Discard pellet.
Concentration:
Ligand
MassSpec:
Crystallization:The TEV protease cleaved YWHAB was concentrated to 28 mg/mL and distributed into 18x50 mL aliquots before being frozen at -80°C. Crystals grew from a 1:2 ratio mix of protein-to-reservoir (0.05 M magnesium chloride, 0.1 M HEPES pH 7.5, 30 %v/v polyethylene glycol MME 550)
NMR Spectroscopy:
Data Collection:
Data Processing: