| Buffers used for denaturing IMAC were (AD) 50 mM Tris, 8 M urea, 1 M NaCl, 10% glycerol, 5 mM imidazole, pH 8.0 (adjusted with HCl); (BD) 50 mM Na2HPO4/NaH2PO4, 8 M urea, 1 M NaCl, 10% glycerol, 5mMimidazole, pH 6.1; and (ED) 0.2MEDTA, 8Murea, 1 M NaCl, 10% glycerol, pH 8.0 (adjusted with NaOH).
Precharged Sepharose NTA-Ni21 Superflow was
packed in water into a peak column (4.6 3 100 mm) and equilibrated with lysis buffer. The sample was loaded at a flow rate of 1 ml/min. A flow rate of 3 ml/min was used during the washing of the column. Washing was performed in three steps. 20 CV of AD buffer was applied followed by the step gradient of 30 CV, changing the composition of the mobile phase from
100% AD to 100% BD in 25% increments. In the third step 20 CV of BD buffer was run through the column.
Protein elution was carried out at a flow rate of 1
ml/min in buffer ED. Twenty-microliter aliquots of the collected 1-ml fractions were analyzed by SDS?PAGE under reducing conditions. The percentage of the recombinant protein in each fraction was determined by densitometric analysis of the gel stained with Coomassie
Brilliant Blue R250.
Buffers used for refolding process were (UB) 50 mM Tris, 1 M NaCl, 8 M urea, 3 mM GSH, pH 8.0 (adjusted with HCl); (RB) 50 mM Tris, 1 M NaCl, 1 mM GSH, 0.1mM GSSG, pH 8.0 (adjusted with HCl); and (E) 0.1 M EDTA, 1 M NaCl, pH 8.0 (adjusted with NaOH).
For sample preparation and purification process,
protocols described in the previous paragraph were modified as follows. Instead of the elution step, buffer BD was switched to buffer AD. A step gradient of 10% increments from 100% AD to 100% UB was applied to remove glycerol from the refolding matrix and immobilized protein. Subsequently, the refolding process was started with 9 CV of RB, followed by a gradient spanning from 100% RB to 100% UB in 0.2 CV at a flow
rate of 0.7 ml/min. The process was continued with 1 CV of 100% of UB, 1.5 CV of a gradient from 100% UB to 100% RB, and 9 CV of 100% RB at the end of the first cycle. During the second cycle the percentage of UB concentration was decreased from 100 to 96%, thus, the UB percentage difference between cycles was 4%.
The percentage of UB reached 28% in cycle 19 and was followed by 25% UB in cycle 20, 20% UB in cycle 21, 15% UB in cycle 22, and 10% UB in cycle 23. Renatured protein was then eluted with buffer E at a flow rate of
1 ml/min. |