I am purifying an enzyme with C-terminal, his tag is expressed in E.coli. I get a clear pure ptn after Ni-NTA elution with 200mM imidazole. I stored the fractions at 4° C for 24 hours and then dialyzed it against Tris Cl pH 7.3, 0.2M NaCl and 1mM DTT. The protein aggregated in the dialysis bag itself within 6h (ptn conc >2 mg/ml). The protein didn't seem to be precipitated irreversibly. I could redissolve a small amount of the pellet with the same buffer and the enzyme was active. However, a smooth whitish jelly-precipitate kept forming, even in the supernatant stored at 4° C. I've tried adding more NaCl, more DTT (ptn has cysteine at N-terminal) and 10% glycerol, but nothing worked. At what step should I optimize it?
I've tried the same conditions for purification and dialysis for another protein that is 60% homologous to this one. I dialyzed it soon after purification, and it has happily remained soluble in the dialysis buffer for 3-4 days (at 2 mg/ml)). I've been thinking it might be due to residual imidazole or the buffer salt/pH, what else could be wrong?
Hi. You can try increasing/decreasing the pH of your buffer. If you are close to the isoelectric point of the protein it can become less soluble, especially at high concentrations.
the problem could be due to the fact that you store the sample for 24 h in 200 mM imidazol. the imidazol is known to induce often protein destabilization. I suggest you to use a desalting column to change the buffer (or do a gel filtration) right after the first purification step.
Some proteins have this problem. Try to store in phosphate buffer with NaCl and glycerol. Other option is to store in buffer containing 20% or 50% glycerol. 2 mg/ml is not very high concentration.
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