I am facing another problem during my protein purification. I purify a 120 kDa protein which is overexpressed in pichia pastoris. The protein is His-tagged (C-terminus) and FLAG-tagged (N-terminus). To concentrate the sample volume and to get rid of some protein contaminants, I wanted to perform ammonium sulfate precipitation. The precipitation itself works perfectly. I started with a total protein concentration of 20 mg/ml and a volume of 50 ml after cell lysis and precipitated all of my protein (proved by Western Blot and Coomassie gel) by adding AS powder (I ground it before use in a mortar) to 30% saturation over 10 min. Before harvesting the protein pellet, I let the solution stir overnight at 4°C. I then wanted to continue the purification with Ni-NTA and therefore resuspended the protein pellet in 5 ml (1/10th of the original volume) sodium-phosphate buffer pH 8 (50 mM sodium dihydrogen phosphate, 1 M NaCl). The pellet didn't dissolve. The literature says, that there might be significant amounts of AS left in the precipitate and it should be dialysed out to get the protein back into solution. So I dialysed the 5 ml (dialysis tubing 12 kDa cutoff) against the loading buffer in three steps: 1L for 2h (RT), 1L for 4 h (RT) and 2L o/n (4°C). The next day the protein slurry was still not solubilized. As I wanted to continue my work I took the precipitate and diluted it step by step in 8M Urea buffer (8M urea, 50 mM sodium dihydrogen phosphate, 1 M NaCl, pH8) until the solution got clear. This happened at a volume of roughly 40 ml. Still there was some insoluble precipitate left after centrifugation of the "clear" 40 ml solution, which also contained my protein of interest to some degree (as seen on WB).
So finally I ended up at a volume nearly the starting volume before AS-precipitation, which was definitely not the goal.
The main question is: why did my protein not re-solubilize? Was there anything wrong with the protocol? Any suggestions on how to better re-solubilize the protein?
By the way the protein didn't really bind to the Ni-NTA resin. But that also happened without the initial AS precipitation (see my other question).
- First, why do you so much want to precipitate your protein ? You should rather directly apply your clarified lysate to the Ni-NTA resin with low imidazole concentration (10 to 20 mM) and see how it works...
- Second, what is the pH of your lysis buffer (and your sample) before Ni-NTA purification ? If you pH is under 7-7,5, this won't work due to histidines protonation...
- Another thing that can prevent your protein from binding to Ni-NTA is the fact that your His-Tag is burried in your protein structure, which can by the way also modify the structure of your protein. To overcome this problem, you can change the position of your tag (from Nter to Cter for example).
Hope this will help.
thanks for your help!
i already tried direct application. Unfortunately the protein doesn't bind in the presence of imidazole (and even without binding is pretty bad, check out my other question). In turn i "purify" many contaminating proteins with the crude lysate. i therefore wanted to pre-purify the protein and increase the initial concentration prior loading it on ni-nta. pH of the lysis and loading/solubilisation buffer is 8, i also tried 8.5 and 9... didn't work. In parallel to additional purification steps i am actually following your (and many others) suggestion concerning subcloneing the his tag to the n-terminus. anyways, i'm curious, why the protein is not dissolving after the precipitation...
AS precipitation is usually well tolerated and reversible. The only things that I can think of is that either your protein has free sulfhydryls or the 1M NaCl you use is keeping your protein "salted out".
Hi Christoph,
- Apparently you're not the first one having problems with Ni-NTA purification of glycosylated protein (See Borsig et al., 1997, Expression and purification of His-tagged beta-1,4-galactosyltransferase in yeast and in COS cells, BBRC).
If your problem is the same (binding impaired by a bulky glycosylation near the His-Tag), then switching to Cterminal Tag is likely to overcome the problem, unless the N- and Cterminal extremities of your protein are close one from the other, or if there is another glycosylation site near Cter...
- For your resolubilization problem, you said you precipitated 50mL at about 20mg/mL. So you've got roughly 1 g proteins to dissolve, wich is really a lot ! So if you want to dissolve this in only 5 mL of buffer, probably the quantity of protein is far to large to get it dissolved (most of the proteins aren't soluble by themselves at 200mg/mL).
- Moreover, if you want to "pre-purify" your protein by AS, you should make several steps of AS concentration (5%, 10%, 15%... 30%). Each time, you will have some proteins precipitating and some others not. Then, resuspend each pellet separately with a sufficently large amount of buffer (e.g. 50mL), and analyse in wich fraction is most of your protein to know wich one to apply to Ni-NTA.
- Anyway, you should rather dilute your carified lysate than concentrate it before Ni-NTA purification, as contaminating proteins may interact with your protein at high concentration, which may also prevent your protein from binding to Nickel. So your low binding problem can also come from this, and to overcome this you should rather dilute your sample than concentrate it, and you can also add Triton X-100 up to 0,5% and/or Glycerol up to 10% (to lower hydrophobic interactions), or increase salt concentration to 0,5-1M NaCl (to lower electrostatic interactions).
Hope this will help you !
It sounds as though your protein may have irreversibly precipitated. I have seen this happen with membrane proteins but don't have much experience with 'soluble' proteins doing this. You could try an alternative precipitant such as PEG 4000 or PEG 6000. Yann's comments should help too --- try resuspending in a bigger volume of buffer and use several different precipitant concentrations to stepwise fractionate the material.
Hi Christoph,
I had have the same problem with my precipitated solubilization. I want to do a step-salting out with increased A.S% so, when I finished the first stage (20% A.S, at 4°C, stirring) I spin it and kept the pellet and then I salting out the supernatant,....... I resuspend my pellet on buffer 100 mM Tris pH 8, 150 mM NaCl and 0.01% triton and the many (almost all) the proteins go to the pellet..... untill i did dialyzate it, they go to the pellet.
I dont know what i am doing wrong !
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