Help with His-Tag protein purification - (Jul/20/2012 )

I have a protein which is, about 31.6 kDA with the Histag (with Theoretical pI between 6.05-6.15), has been purified using Talon (more or less). I feel that my protein is in inclusion body because every time I express and purify, most of the protein remain in the insoluble fraction after I spin it at 35000 rmp. So I adjusted my purification so initially after I pallet my cells from the media I use 6M urea with 50mM NaH2PO4 and 300mM NaCl (pH 7, lysozyme and protease inhibitor cocktail mix) to resuspend the cells. Then I incubate the mixture for an hour at 4 degrees followed by sonication for 20mins. Then I separate the mixture by centrifugation at 35000 rpm. Now when I run the sample in SDS PAGE it looks like I have a lot more protein in the soluble fraction. So I purify my protein using Talon and Imidazole. Now my protein is in an elution buffer containing 6M urea, 150mM imidazole, 50mM NaH2PO4 and 300mM NaCl (pH 7). However, every time I try to dialyse Urea out it starts to precipitate. I haven’t been doing stepwise dialysis as in 4M, 2M, 1M so on. I just dialyse my protein containing 6Murea in buffer containing just 50mM NaH2PO4 and 300mM NaCl (pH 7). Is that why my protein is precipitating? I need the protein to be at 1mg/ml concentration in buffer containing 50mM NaCl or less.
I really need this before I can continue with the rest of my project. I will appreciate any help/suggestions.

My main questions are
How can I prevent it from precipitating;
What buffer should I use and what pH should the buffer be at to keep my protein from precipitating;
Once I dialyse out the urea, how do I make sure that the protein refolded correctly
Thank you in advance. If you require more information let me know or email me any suggestions

Perhaps you can play with the pH in your dialysis buffer to prevent it from precipitating, try lower down to pH 5.5
I am not sure how you can make sure your protein refolded correctly. Perhaps you can try to assay your protein to see the activity?

Also, try reduce your inducer and reduce your temperature for a slower protein expression. This may help in reducing your protein being expressed in inclusion body.

In order to make sure that you refold your protein correctly, what I would do is to steadily decrease the amount of urea you've got. So I'd go from 6 to 5,3,1 and 0M. What you could do is to use a spin concentrator in order to get rid of the excess buffer, and then resuspend it in the buffer with a lower amount of urea. But then I leave my protein for an hour between each stage, just to give it time.

It is slightly time consuming, but it tends o work well.

Best of Luck!