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Hi I express my recombinant protein, with H5 insect cells, directly in the medium (Express Five). During my IMAC purification from the medium, I get a contaminant most likely Ferritin. I can get rid of most of it during the gel filtration step. However a minority remains. Does anyone know a trick to get rid of this ferritin contaminant or prevent insect cells to secrete it??? Thanks in advance JM |
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Hi JM, do you apply the cell supernatant directly on the column? Fractionation with ammonium sulfate might help. Make sure you have no agents in your buffers which will strip metal ions from the IMAC column (like EDTA, phosphate etc) To prevent binding of ferritin to the NTA-colum (iron might compete with nickel) include some nickel salt in the protein solution that you apply on the column and/or replace nickel with other suitable ions like cobalt. The easiest way to remove ferritin that still co-purifies probably is a column with immobilized anti-ferritin antibodies (maybe difficult get for H5 donor insect ferritin. For large scale, chicken IgY is cheap if you need to plan a immunization). Other options are non-his affinity tags and more chromatography steps (ion exchange, HIC, affinity etc which you actually wanted to avoid with your His-tag) HTH Wo |
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thanks for the reply. Yes I add directly the supernatant to the Imac beads. I just adjusted the pH around 6.5 and usually it's working quiet well. If you increase too much the pH something in the medium precipitates (pluronic detergent i think). Btw if you know something that can prevent that will be great....... I will try to add some Nickel salt in the medium and see if it improve. JM |
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