I am purifying GAPDH protein, and with the wild type I don't have any problem, and I obtain good quantities but I have a mutant which has a change in one aminoacid, and when i want to purify it, the protein precipitates and form aggregates and I lose all .
The protein aggregates after of before purification? What is the aminoacid? Are there cysteine residues in the sequence? What purification technique are you using?
The aggregation i could see in the last step, when i dialize with the storage buffer. But I think that the aggregation starts after the lisis cause I have protein in the pellet with the cell debris.I did a quickchange w87F . Yes i have cys in my protein. I purify by Using Polyhistidine Affinity Tags .
Try changing the salt concentration and pH. The mutant protein may be less likely to aggregate in different buffer conditions. You can also consider adding a nonionic detergent to reduce hydrophobic interactions, or one of the substrates to improve stability.
Run on-column refolding, be sure to have some beta-mercaptoethanol along the purification.
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