Purifying a strange native protein - (Aug/26/2009 )

Hi guys, I am currently purifying a tagless protein (DNA binding protein) and I have encountered some unforseen problems with anion/cation exchange purification.

Basically I tried running the protein on a heparin sepharose column (suppose to bind to DNA binding protein) and a Q sepharose (anion-exchanger) column at pH 8.4 and very little of it was bound (~2%). I then try SP sepharose column (cation column) at pH 8.4 again and very little of it was bound as well (~5%). Logically this then suggested that the protein could have a pI of around 8.4. However I then tried it on the Q sepharose column again this time at pH 7.5 and only 10% it was bound. Most protocol suggested to adjust the pH of the loaded protein +/-1 of the pI so in theory it should give me a much better binding?

I am in a big puzzle right now and any help would be greatly apprieciated

Thanks in advance,
Terence

My experience is that there is always a sizeable amount of protein in the flow through.
I always pH my buffer to 7.4.
Did you dialyze your sample? What is the NaCl concentration? Is it low enough for binding?
Also, run the column as slow as possible. I get much better binding the more patient I am.

if your pI is indeed around 8.4 then you should use the cation column at pH 7.5 rather than the anion column.

mikew on Aug 26 2009, 09:13 PM said:

I also use the same columns for purifying other proteins as well and the amount of protein bound for this one is definitely too low i.e. >95% in flow throught. I also tried loading the protein at 7.4 with 1ml/min flow rate and I had to literally load the flowthrough back into the column three times to get a decent binding I think roughly about 20% of protein would bind on each flow which gives me 80% yield. 80% yield isn't bad I suppose but since I will be doing this quite often I would like to have a more convenient purification step.
Its a real pain in the ass this thing.

mdfenko on Aug 26 2009, 09:29 PM said:

In fact I have tried pH 7.4 on a SP sepharose as well (cation) and only about 20% of the protein bound!

Terence on Aug 26 2009, 04:07 PM said:

Although it is generally suggested to try binding at +/- 1 pH unit above or below the protein´s pI, this is only a rough guide.
Binding to ion exchangers is dependent on the net SURFACE charge and the topographical distribution of the individual acidic and basic amino acids. For example, a calculated pI , based on aa sequence data alone, can be way off if folding results in a predominant display of either acidic or basic groups on the protein surface.

I usually start trial experiments at pH 5 for cation and pH 9 for anion exchangers. It is always reassuring to get good binding at one of these extremes of pHs and then adjust these initial conditions more to neutrality.

Protein purification by traditional methods is usually challenging but very rewarding when you finally get that single band!

Hope this helps

Did you overload the column by any chance?