I am trying to purify a GST tagged protein of 70kda ( with GST tag 96kda) expressed in Rosetta. The pI of protein is 5.2. I am using the GSTrap column and AKTA prime for purification. The lysis and binding buffer is PBS, pH 7.3, DTT 10mM. During lysis I add 0.15mM PMSF. Elution buffer: HEPES 25mM pH 7, NaCl 200mM, 10 % glycerol, DTT 10mM, reduced glutathione 20mM.
I get nice peak in elution gradient upto 300 mAu but the protein elutes in the form of slightly white precipitation and when I concentration the elution fraction most of the protein precipitate and I hardly get 1mg of protein.
I have tried increaseing concentration of NaCl (500mM, 1M) in binding butter by relacing PBS with HEPES and in elution buffer concentration of NaCl was increased upto 500mM. The protein is a glycoprotein and it is not a membrane protein. Please give your suggestion. Thanking you in advance.
there are a variety of solubility enhancing additive you could try, going from less to more harsh as follows:
1 glycerol
2 sugars
3 non-detergents sulfobetaines (NDSB)
4 sugar based detergents (higher cmc)
5 ionic or nonionic detergents (lower cmc)
6 chaotropes (GuHCl, or urea)
I wouldn't try anything past 3-4 unless extremely desperate, you can also attempt to refold on column with a mixture of ox/reduced glutathione, good luck!
Hi, like Sebastian Schmitt
and Edward Michelini said before and I agree, your protein is eluting in aggregate form. It,s important to be clear whats your purpose here (What will your protein be used for ?). Also remember that glycosilation in Rosetta does not occur, hence, your protein is´nt a glycoprotein. There are many tips you could try to prevent aggregation and to improve protein stability, including exchange the host (like Shuffle (DE3)which allows S-S bonds formation by the S-S isomerase (DsbC) improving folding) .Also, as recommended previously you could use some stabilizing agents. I see you use glycerol. You could either, increase it concentration on the elution buffer or to add 0.5 M arginine, or both. If it is not a problem, you could extract your protein with urea, and then remove it on-column.
Another thing you could try is adding anionic detergents which prevent protein-protein interactions. I have had good results using 0.1 % sarchosyl to prevent aggregation and to improve protein folding. I recommend you to start adding 0.1 % sarchosyl in both your binding and elution buffers (That depending on what´s your purpose) and then try the rest.
I hope this could help you
Have you tried using a larger NaCl gradient in the wash buffer from 200 mM to 1M ?
Drastic changes in salt levels seems to cause certain proteins to crash at higher concentrations. In my case, I had preciptation issues with a GST tagged protein during dialysis. The issue was that i had been reducing the salt concentrations too rapidly. The preciptation problem was resolved when I made multiple baths with reducing salt levels.
Hi Sanketkumar,
Your target protein aggregates at high concentrations (although under the condition of 10% glycerol), indicating that the property of this protein is unstable. If you plan to cut off the GST tag, this phenomenon may become more obvious. It is recommended to change the tag or expression system in time. In addition, Rosetta is not conducive to the expression of eukaryotic proteins that require glycosylation to be active.
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