My protein is almost insoluble without high concentration of arginine-hydrogen chloride adding to the cell lysate in the expression test. It cannot bind to the Nickel affinity column with high concentration of arginine-hydrogen chloride. Are there any other protein purification methods which can be used in this case?
Thanks.
Definetly a problem. You can try a Gelfiltration column using the Arginine in the buffer. Collect fractions to identfy the fraction with your protein at highest purity. I´m not sure though whether Gelfiltration alone is sufficient to get a protein out of a cell lysate.
Cheers.
Dear Arshad,
Some additional information.
1. 66 KDa
2. 6-8g E. coli cell pellets, haven't got purified protein yet. It precipitates badly when remove high concentration of arginine-hydrogen chloride.
3. Tm should be more than 65, thermophilic.
Thanks for your reply.
Since your protein is from a thermostable organism, I would try heat treatment with a small amount of cell lysate. You can test to see if your protein is still active after performing increasing temperature experiments. You can also try ammonium sulfate precipitation steps. Because of the high salt, I agree with Arshad about using a sizing column. If you want to tag your protein, I would use GST or MBP.
A combination of thermal treatment (10 min at 60°C under shaking) and protein through ammonium sulphate could solve at least in part your problem. Goog luck!
If your protein is insoluble without arginine, it is possible to do a lysate dialysis using a buffer arginine free, with this condition your protein is going to precipitate (the rest of “normal” proteins will be soluble), after recovering the precipitate you can try to recuperate the protein solubility in your buffer with arginine high concentration. If some material remains insoluble just remove it by centrifugation. Other possibility is the use of a detergent (v.g. Triton X-100), ), in this condition your protein could be soluble without arginine, the niquel column works fine in the presence of neutral surfactants.
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