Dear All,
I am expressing a GST fusion protein in Bacterial cells and on purification of the protein, there are a lot of contaminating nucleic acids. I tried various approaches to get rid off the nucleic acids. I use 1X PBS + 1% triton X-100 (supplemented with DTT and protease inhibitors) as my lysis buffer. Initially I was washing the protein bound GST beads with the same buffer but in order to remove the nucleic acids, I started washing several times with up to 1M salt. I elute the protein with 10mM Reduced Glutathoine in a buffer with 50mM Nacl, 5% glycerol and 0.5M EDTA and 20mM HEPES ph 7.9. After eluting the protein, I loaded it on Q-Sepharose column and strangely it elutes after the nucleic acids elute i.e. Nucleic acids elute at just above 800mM salt while my protein elutes at over 1M salt and even then it comes down with some nucleic acids.
Could you please suggest any alternate strategy.
Thank you
Ikram
Hi there,
Include DNase in your lysis buffer. If your protein is naturally binding nucleic acids then you might also try a chromatography on heparin (it mimicks nucleic acids and then compete with for binding the protein of interest).
Non-specific binding of GST-tagged protein?
You conclude that using two purification steps (Affinity column: GST beads and Exchange ion chromatography on monoQ sepharose to prepare your GST-tagged protein you always have Nucleic acids contamination.
I advise you to verify firstly that your fractions contains really N. Acids, you know that many simple molecules could also have absorption at 260 nm.
I believe that you have trace of nucleic acids in your GST-tagged protein fractions; your protocol is adapted to eliminate nucleic acids, except mono Q sepahrose showing that your protein is a strong acid protein eluted (at 0.8 M) near N. acids (1M).
In my view you have a lot of procedures to eliminate N. Acids:
- You may use a Gel filtration purification step. The gel should be adapted to the Molecular Weight of your GST-tagged protein. This method could avoid the co-elution of protein with N. acids.
- In a personnel experience, I have used an Ammonium sulfate precipitation as a step of a protein purification protocol. That step has eliminated the major part of N. acids. So you can further apply the two steps to purify the GST-tagged protein.
- In the case that you must eliminate the traces of N. acids, may be you applied a treatment at the lysis step by nucleases (Certainly I mean a very low concentration) and before apply the two purification steps. In that procedure we wish that you don’t obtain contamination by nucleases which may interfere with your protein activity.
As Domininque said add nuclease to your prep.
And have you run your samples on an agarose gel to make sure that DNA is present?
DNase is the easier step: perhaps a first level screen for you. But definitely AmSO4 pptn is a good elimination strategy. Ofcourse you would need to scale up your production to ensure you get sufficient yield of your GST-tagged protein in the end. I would go to Gel filtration only if AmSO4 doesn't completely do the trick. But you will have significant losses as you go through each step, so starting amount of your protein should be high.
Dear Dominique and Dear Nejib
I have already tried using DNAse I and Benzonase treatments. None of them worked enough to rid off the contaminating nucleic acids.
Dear Mark and Mansha
Yes, I loaded the samples on a 5 % Native Polyacrylamide gel and then stained with SYBR Gold. That was my confirmation for the contaminations.
I am currently trying another protocol to prepare the Lysate with a lysis buffer that contains 1M NaCl and 100mM CaCl2. A crystallographer friend recommended this. They elute the protein from the bound beads with an elution buffer that contains 500mM Nacl and 10mM CaCl2. Then perform Ion Exchange chromatography. I should have the results of that by Sunday. Hopefully, that works. If not then I will try Ammonium Sulphate precipitation and gel filtration.
Thank you all for your help, will keep you updated.
Ikram
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