Hi,
I've been working upon the protein that has pI around 9.43 (expasy tool) and using sodium phosphate buffer (pH- 7.5) in Ni-NTA chromatography. I used to get precipitation with buffer exchange to HEPES (pH-7.5) during dialysis. I further do Gel filtration to maintain the homogeneity, after that I get very low yield. I skipped dialysis to avoid precipitation many times, but still I didn't get good yield of protein. I also added glycerol(2-5%) to stabilize it, but the final problem arises with concentration that doesn't exceed 10mg/ml and my desired concentration should be more than 20mg/ml for crystallisation. Please suggest in this regard and your suggestion would be highly appreciated.
Precipitation of your protein could be caused by a number of problems. The protein may not fold well or may become denatured during purification, causing it to aggregate and precipitate. Nickel ions leaching from the Ni-NTA column may cause aggregation by interacting with the His tags; this can be remedied by adding EDTA after elution from the Ni-NTA column. The protein may need to be kept with high (or low) ionic strength when highly concentrated to prevent aggregation. Changing the pH could be helpful.
Thank you Adam for your valuable suggestion. Actually my protein has average pI around 9 and some reports are claiming it as around 8. So, I'm still bit confused to adjust the pH. Because, Histidine has pKa = 6.0 and the pH of the buffer should be more than 6 to get more protonation of histidine and to bind it more efficiently to column bed. I had used pH=7.5 as well as 8. I would rather change the pH of Size Exclusion Chromatography (SEC) buffer. Because I used to buffer exchange the protein after Ni-NTA and somtimes uses dialysis. I would try adding EDTA also as you suggested.
Why don't you try to do differential scanning fluorimetry with different buffer conditions just to check what's better for your protein?Try possible ligands, different cations and/or anions just to figure out what does your protein like.
You will need very few protein in order to do it and you will find a condition were you protein will be more stable. It seems to be a problem related with the overall condition rather than the pH of the buffer.
Good Luck!
Ignore the calculated PI. It is rarely useful and a distraction during trouble shooting. How good is your gel filtration profile? If you can concentrate the fractions and re-inject does your peak heights reduce? My guess: Your protein may have hydrophobic patches that may be binding to column material/tubes. Your inability to concentrate the protein beyond 10mg/ml hints at this. At higher concentration it likes aggregates and comes out of solution. There is no easy fix though, you will have to explore various buffers and additives. How did other groups purify this protein? You mention some reports with this protein.
Good Luck
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