HisTrap™ HP Columns are prepacked with precharged Ni Sepharose™, which consists of 34 um highly cross-linked agarose beads with an immobilized chelating group, ideal for purification of Histidine-tagged recombinant proteins.
I have used HisTrap™ HP Columns to purify yeast expressed VEGF (~46 kD). Expressed VEGF automatically forms an antiparallel homodimer, which increases the number of his-tags on each protein, thus increasing the binding of VEGF-his fusion proteins to the HisTrap™ Ni Sepharose beads. Although the protocol suggests including 20 to 50 mM imidazole in the binding buffer, I only used 10 mM. Using a linear elution method (from 0 to 500 mM imidazole) to analyze VEGF-his binding with the HisTrap™ HP Column in an AKTA™explorer HPLC, under the control of Unicon 5.0 software, I found that there was still some elution in 20 mM imidazole. To increase recovery, I chose to use 10 mM Imidazole and 0.5 M NaCl in the initial binding buffer.
The HisTrap™ HP Column capacity is reported to be at least 40 mg of 6-histidine-tagged protein/ml medium. In my experiments, when I used a 5 ml column to purify 2 to 5 mg VEGF-his from about 1000 ml of yeast expression medium, one-third of my protein did not bind to the column in the first loading. The loading speed I used was 3 ml/min.
In my first experiment, I chose reagent grade imidazole to save some money. This imidazole had high absorbance at OD280, which covered the signal of my eluted protein. Using this grade of imidazole was a waste of my time and treasured, expressed protein.
The HisTrap™ HP Column is very stable. It can tolerate 0.01 M HCl or 0.1 M NaOH for 1 week; 1 M NaOH or 70% acetic acid for 12 hours; 2% SDS for 1 hour and 30% 2-propanol for 30 min. I use 1.5 M NaCl, 1 M NaOH and 30% propanol for CIP (Clean In Place). After stripping the column with 20 mM sodium phosphate, 0.5 M NaCl, 50 mM EDTA, pH 7.4, I recharged it with 20 mM sodium phosphate, 0.1 M NiSO4. Then this column was used repeatedly and functioned the same as new column.
Tricky Point One: There are a lot small molecules which can bind to HisTrap™ Ni Sepharose beads. These molecules then block binding of the target proteins and decrease the purity of purification. I used diafiltration to solve this problem. The diafiltration system I used was Sartoflow Slice 200 Benchtop with a molecular exclusion size 10,000 filter. After this step, almost all of the small molecules disappeared in the purified protein preparation, as analyzed by SDS-PAGE and silver staining. Tricky Point Two: You should choose high quality imidazole, which has low OD280 absorbance.
Xiang Yang
Research Scientist
Georgetown University
Lombardi Cancer Center