I have DNA binding protein and I am getting a conc, of 50um from 50ml BL21 DE3 culture (I will be scaling this up to 1mM). The protein is Hexa-His tagged at C-terminus and to elute the protein from Ni-NTA column I am using 1M imidazole as even at lower concentrations [even at 500mM] protein does not elute out at lower concentrations of imidazole.
I am doing dialysis in two steps. 5ml of protein elute (1M imidiazole, 400mM NaCl, 20mM Tris, pH 8) against 1L of 500mM Imdiazole, 20mM phosphate, 400mM NaCl, pH 7.5, followed by second step of dialysis against 1L buffer containing 20mM phosphate, 400mM NaCl, 50mM EDTA, and 2mM DTT, pH 7.5. [I am adding EDTA and DTT because I read the imdidazole might leach out Ni2+ and removal of imidazole results in precipitation]
While concentrating the protein in Millipore centricon tubes protein precipitates out. I cannot increase salt beyond 500mM NaCl as this will cause problems with NMR experiments. Any suggestions on how I can modify my final step (or the rest of the protocol) to prevent protein precipitation will be greatly appreciated.
- I think you can use 20mM Tris, pH 8, 1mM EDTA, 2mM DTT and 100 mM KCl for dialysis.
- if you want to change the pH then do gradually.
- If your protein is too concentrated that also sometimes help in precipitation.
- Hope this will help you
I have trouble with this too, with a DNA binding protein with zinc fingers. You may want to leave a low concentration of imidazole in the last buffer. Some proteins will just not tolerate loss of the imidazole.
Other things to try:
- If your protein needs an ion like zinc, include that in the buffer (we use 5uM)
- Be sure to equilibrate the centricon first by spinning some buffer through
- I take the centricon out of the centrifuge at least every 5 min and mix the sample-- local increase in protein concentration at the bottom of the filter may cause precipitation
- For DNA binding proteins, try increasing DTT (2 mM) and decreasing EDTA (0.2 mM)
- You could also try including a small oligo in the purification with the protein's DNA binding site
You can also add some buffer additives, such as L-arginine, trehalose etc, to maintain your protein in solution
I work with DNA polymerases and beyond the above suggested changes in buffer components I would consider trying a different type of concentrator. Our lab used to use the centricon concentrators and saw similar precipitation regularly. Then we switched to the amicon concentrators and haven't had any problems with proteins crashing. I can gets upwards of 1-2 mM with some proteins easily, but could only get about half this with the old concentrators.
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