The heparin used in GE Healthcare’s HiTrap™ Heparin HP Columns comes from porcine intestinal mucosa; the molecular weight of the polymer is between 5000 and 30000. The heparin is covalently coupled to highly cross-linked agarose beads then packed into polypropylene (which is non-interactive with biomolecules) columns. The columns are then sealed to prevent leakage. There are 2 sizes of HiTrap™ Heparin HP Columns, 1 ml and 5 ml. Heparin agarose can be used to separate many proteins, such as DNA binding proteins, growth factors, coagulation protein synthesis factors and steroid receptors.
In my experiments, I have purified vascular endothelial growth factor (VEGF), which has a heparin binding domain in its carboxy terminus. For the first purification, I used mammalian-expressed recombinant human VEGF (rhVEGF) supernatant. After diafiltration with a Sartocon Slice 200 filter (retention size 10,000), I adjusted the pH to 7.0 and the conductivity to 5. I loaded about 100 ug rhVEGF (quantified by ELISA) into a 1 ml HiTrap™ Heparin HP Column. Surprisingly, rhVEGF did not bind to the heparin agarose beads. I detected my rhVEGF in the flow through. After analysis, I found that there was some heparin-like material in the cell culture medium, which blocked the VEGF heparin binding domain. Using diafiltration, I was unable to clear this heparin-like material from my sample. For my second attempt at purification, I used VEGF expressed in yeast. Without interference from any heparin-like material and after loading 1 mg of VEGF, about 2/3 protein bound to the HiTrap™ Heparin 1 ml column. SDS-PAGE and Western blot results showed that VEGF bound to the Heparin column.
Because heparin is stable in the pH range 5-10, NaOH cannot be used to regenerate this column. I was not able to find a procedure for regeneration on GE Healthcare’s website. I tried 2 M KCl and 70% ethanol which worked well. I just don’t know how many times it can be re-used.
Tricky point: Heparin binding is weak affinity binding. Because a lot of proteins have heparin binding domains, HiTrap™ Heparin HP Columns cannot be used alone to purify target proteins, nor should they be used as an initial purification step. Instead, they should be used as a middle or late step of protein purification.
You should also check for heparin-like material in your culture media before using this column.
Xiang Yang
Research Scientist
Georgetown University
Lombardi Cancer Center