MTMDKSELVQKAKLAEQAERYDDMAAAMKAVTEQGHELSNEERNLLSVAYKNVVGARRSSWRVISSIEQKTERNEKKQQMGKEYREKIEAELQDICNDVLELLDKYLIPNATQPESKVFYLKMKGDYFRYLSEVASGDNKQTTVSNSQQAYQEAFEISKKEMQPTHPIRLGLALNFSVFYYEILNSPEKACSLAKTAFDEAIAELDTLNEESYKDSTLIMQLLRDNLTLWTSENQGDEGENLYFQ
Buffers: Ni-NTA column purification - 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, 5 % Glycerol, 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 supernatent 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 hexahistidine-tagged construct, was over-expressed and purified in-house to a final concentration of 2.5 mg/ml.
Enzymatic treatment : Add 20 ml of the TEV protease was added to each fraction and left at 4°C overnight.
The following steps were carried out to remove the cleaved products and TEV protease: Change buffer from Elution Buffer to 50 mM Tris pH 8, 150 mM NaCl using a 10-kD cutoff concentrator. Place 200 µl of 50 % Ni-NTA agarose in a 1.5 ml eppendorf tubes, add 1ml of 50 mM Tris pH 8, 150 mM NaCl mix, spin down and remove buffer. Repeat this resin wash step once. Add the TEV treated protein sample to the resin and mix for 30 min. Finally spin down resin and collect the supernatant which contains the cleaved YWHABA.
Buffers: Ni-NTA column purification - 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, 5 % Glycerol, 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 supernatent 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 hexahistidine-tagged construct, was over-expressed and purified in-house to a final concentration of 2.5 mg/ml.
Enzymatic treatment : Add 20 ml of the TEV protease was added to each fraction and left at 4°C overnight.
The following steps were carried out to remove the cleaved products and TEV protease: Change buffer from Elution Buffer to 50 mM Tris pH 8, 150 mM NaCl using a 10-kD cutoff concentrator. Place 200 µl of 50 % Ni-NTA agarose in a 1.5 ml eppendorf tubes, add 1ml of 50 mM Tris pH 8, 150 mM NaCl mix, spin down and remove buffer. Repeat this resin wash step once. Add the TEV treated protein sample to the resin and mix for 30 min. Finally spin down resin and collect the supernatant which contains the cleaved YWHABA.
NMR Spectroscopy:
Data Collection:
Data Processing: