Hi guys,
My protein is nuclear human protein and I am purifying recombinant protein from bacterial culture. when I performed ion exchange chromatography, my recovery was nil. thinking it is bound to DNA, I performed Heparin column purification. During the purification 80% of protein was found in Flow through and 15% in NaOH wash. I have used 20 mM tris buffer pH 7.5 with 0.2 M NaCl and 5mM DTT in it.I am not able to conclude whether protein is bound to DNA or it is not binding to the DNA. and I also found many contaminated bands even after gel filtration though i get single sharp peak in chromatogram. please help me in understanding.
Thanks in advance
Hi Manjula,
From your information I can say that your protein is not binding to the column.As Rolando suggested high salt concentration is not a good idea before loading on to IEX column. For that reason you are losing 80% protein in flow through high ionic strength buffer will not allow protein to bind properly. Try to maintain same buffer you mentioned except 50 or 75mM NaCl and allow your protein to bind on heparin column. Finally you can follow either step or gradient elution. Heparin column is sufficient to remove DNA from your protein because it serves as cation exchanger, charge repulsion with in the column will make DNA come down.
If still you are worrying that protein is bound with DNA, I would recommend either (Polyethyleneimine)PEI precipitation.I hope that your first column is NiNTA so you can include 1M NaCl in your column buffers.It will serve as dual purpose:DNA exclude and contaminant bands also eliminated(you mentioned about contaminated bands in SEC).
Hope these will help you.
In my opinion, your protein did not bind to the column or you are using bad washing buffer thats why you can find your protein in flow through otherwise it should not happen if it is really recombinant. Can you tell the detail of your tag and lysis buffer?
Hi there,
Chromatography on heparin column is a mix of cation exchange chromatography and affinity chromatography. So the ionic strength of the starting buffer is crucial : if too high you won't be able to see any binding. In your case, you use 0.2M NaCl which might already be too high. Try to lower it in order to favor interaction of your protein with the matrix.
I hope you are not doing the wash step with NaOH as mentioned in your question...
For any purification, it would be good to check the predicted pI of your protein and try a variety of buffer conditions to see if you can improve purification. If you are worried about bound DNA, you can add a DNase step. Also, try spinning your initial lysate down hard (even in an ultra, if you have one) and look to see if your protein is in the soluble liquid fraction or the insoluble pellet. If it is insoluble, it probably won't stick to your column no matter what!
I have used DNase in my lysis step and centrifuged at 20K RPM. pI of protein is nearly 6.0 therefore I have chosen pH 7.5 for purification. protein is with His tag and it is in soluble form. I have used the NaOH for column wash not for the purification.
The calculated pI of the protein may indeed be 6.0 but in my experience it is often necessary to screen several different pHs and ionic strengths to find good binding to heparin. You can do multiple tubes with batch absorption and simply look at protein by A280 or BCA if your protein is well expressed, otherwise SDS-PAGE or western can be used to follow binding. Good luck!
I agree with Dominique. It can perform also as a cation exchanger. I also agree that probably your ionic strength in the starting conditions is too high for binding, mostly considering that ionic interaction with this ligand could be lower than with other exchanger. In my experience with this matrix, I have found that sanitization or using of NaOH kills the resolution in the protein mixture I was working (several trials), although in the column datasheet is claimed that NaOH (the same concentration) can be used. Let say, I separated two peaks which were eluted with around 0.3 and 0.6 M NaCl, respectively. After washing the column with NaOH and under the same experimental conditions, the first peak went along with the unbound fraction and only the second was bound, yet eluted at lower ionic strength. It seems that the ability of heparin as cation exchanger is diminished after NaOH washing. Try an unwashed (no NaOH treatment) matrix, reduce the ionic strength in the starting condition, and do not use too much column volume in your elution protocol since the binding is not so strong.
Hi Manjula,
From your information I can say that your protein is not binding to the column.As Rolando suggested high salt concentration is not a good idea before loading on to IEX column. For that reason you are losing 80% protein in flow through high ionic strength buffer will not allow protein to bind properly. Try to maintain same buffer you mentioned except 50 or 75mM NaCl and allow your protein to bind on heparin column. Finally you can follow either step or gradient elution. Heparin column is sufficient to remove DNA from your protein because it serves as cation exchanger, charge repulsion with in the column will make DNA come down.
If still you are worrying that protein is bound with DNA, I would recommend either (Polyethyleneimine)PEI precipitation.I hope that your first column is NiNTA so you can include 1M NaCl in your column buffers.It will serve as dual purpose:DNA exclude and contaminant bands also eliminated(you mentioned about contaminated bands in SEC).
Hope these will help you.
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