I am trying to purify 50 kDa recombinant protein with his6-tag on the c-terminal by using Ni-NTA chromatography. I am using 1 mL HisTrap columns, and I loaded my sample (E. coli lysate containing the overexpressed protein) at 1 mL/min to the column. I washed then with 25 mM NaPhosphate + 0.5M NaCl, followed by wash with 30 mM imidazole. I then eluted with 500 mM imidazole, and saw lots of protein in the chromatogram at 280 nm. However, after gel run I see lots of impurites on the gel (image attached) in all collected of fractions, and the NanoDrop I use for protein concentration measuremnets also says there is a DNA impurity present. What can be the cause of this, and what can I try to get better purity? I would need need at least 85-90% purity..
Try eluting with a step gradient (if you're doing it manually) or a linear gradient (if you're using a FPLC) of imidazole, instead of a single step from 30 to 500 mM.
You may also need to use a larger column.
When this happens to me, it's almost always for the same two reasons: The protein wasn't expressed or I used too much resin for the amount of protein. The nickel ions are weakly attracted to all the negatively charged amino acids, which makes off-target proteins bind to the column until they're displaced by the His tag or imidazole. So to me, it looks like you have too much resin and the column is holding on to the off-target proteins in the lysate.
To fix this you could use less resin. I typically use loose resin and a gravity column and my starting point is 500 mL of resin per liter of culture, the literature on the HisTrap columns should give you their capacity and you should aim to oversaturate the column. You can also use larger cultures make more concentrated lysate.
Alternatively, proteins sometimes fold in such a way that the His tag is not accessible to the resin. If reducing the volume of resin doesn't work, you could try moving the His tag to the opposite terminus to see if you get better results.
Good luck!
Dear John Virtanen x
can you provide some more details about you protocol.
The amount of cell pellet lisate and loaded?
How did you centrifuged the lisate?
which is the MW expected for your protein? 50KDa?
it seems that you need to optimize the loading and washing buffer as well as the amount of protein to be loaded in the coloumn.
Did You wash with a buffer containing also 30mM of imidazole (eg phospate, nacl, imidazole buffer) or only with 30mM of imidazole? at which pH? fow how many coloumn volumes?
Are you adding some imidazole in the lysis/binding buffer?
i suggest you to mantain 10mM of imidazole in the lysis/binding buffer and to increase the amount of e.coli lisate loaded (loading the surnatant and not the entire lisate)
in the following link you can find some hints for buffer preparation for IMAC chromatography
https://www.blogger.com/video.g?token=AD6v5dzP9DkeYt4nwIFl1pNVQuJw7_vnHlvCcbCTzad5M6URrbtpOEJ9Z5MJ-gTcfR6n06A5fXwQmD5qJtKayesvHrQtqzfE0mhrKxtPpHHuJO0u2FMq9kmvLSz60LTG_RLcHgTUWkY
best
Manuele
Thank you for answers, I will try gradient elution next. It is a human protein I'm expresing in bacteria, and therefore I dont expect the yield to be very big.
Dear Manuele Martinelli , I begin with 10 grams of E. coli pellet, which I resuspend into 50 mL of the washing buffer (25 mM NaPhosphate + 0.5M NaCl). I then add lysozyme to 0.2 mg/mL and lyse using high pressure homogenizer (avestin emulsiflex) until I see that the lysate looks clear.
Dear John Virtanen
You said that you dont expect the yield to be very big, but you are able to see the induction band comparing induced vs non induced culture?
Because a so small band from 10g of E.coli loaded in a 1ml coloumn suggest very low expression yield or low ability of the protein to bind to the coloumn (e.g due to protein aggregation)
i suggest to you to add at least 10mM of imidazole in the washing buffer (25 mM NaPhosphate + 0.5M NaCl) used for resuspension and perform the imidazole gradient.
check the pH of the imidazole buffer.
best
Manuele