I am working on an acetyltransferase that is highly unstable. Its pI is 6.65, and its molecular weight is around 18 kDa. The protein elutes at 1M imidazole and begins to precipitate immediately after elution. After testing various pH levels and salt concentrations, I have been able to stabilize it in MES buffer at pH 5.5 with 1M salt and 5% glycerol, by immediately diluting it after collection. The highest concentration I achieved was approximately 6.67 mg/mL, which was only possible by adding 50 mM EDTA post-elution. This addition seemed to stabilize the protein, but I am uncertain if this approach is optimal, and replicating the results has been challenging.
However, when I try to concentrate it using a concentrator, its concentration rapidly decreases after buffer exchange. I have tested the flow-through, and the protein is not present there. I have also tried flushing the concentrator membrane with buffer, but there is no protein stuck to the membrane either. Only a negligible amount is precipitated. I am unable to determine what is happening to the protein. My eventual goal is to crystallize the protein.
Thank you.
Have you tried including a substrate during and after the purification to help with stability? Keeping the protein concentration as low as practical should help reduce precipitation. For crystallization, it may not be necessary to have the concentration very high, since the protein seems to be prone to self-associate.
Adam B Shapiro I haven't tried including a substrate yet. Another thing I found out today was that the protein is also getting eluted at 2M imidazole. Is that normal? (My protein has 7 internal histidines very close together).
Including 100mM EDTA post elution is making it stable though.
For His-tagged proteins art least, 0.5 M imidazole is generally more than sufficient, in my experience.
100 mM EDTA is an extremely high concentration. Do lower concentrations also work?
Adam B Shapiro Yes, I have tried 20 mM and 50 mM imidazole as well, which also leads to a significant decrease in precipitation. Do you have any idea or insight on why the protein is still eluting at 2 M imidazole?
One possibility is that the particular configuration of 7 histidine residues you mentioned results in an extremely high affinity for the IMAC resin. Another idea is that the interaction with the IMAC resin is ionic, rather than due to the affinity of His residues for the rer than its competitionsin, so the elution by imidazole is due to its ionic strength rather than its specific recognition by the resin.