Hi, I am currently working on an eukaryotic transcription factor called Nuclear Factor I X. I generated a truncated construct of this protein to remove the intrinsically disordered C-terminal domain in order to get well-diffracting crystals. I expressed the recombinant protein in E.coli SHuffle cells as a fusion protein fused with an MBP-tag. The MBP tag is not used for purification purposes, since we found a better protocol that exploits heparin chromatography to purify the protein, but it is needed to increase its solubility.
After I perform the heparin chromatography I can get good amount of the fusion protein, but after incubation overninght at RT with thrombin in solution, I found heavy precipitates in the sample. The protein eluted with a NaCl gradient, then the salt concentration of the sample was around 650 mM, at a concentration of 2 mg/ml (more or less) in a volume of 8 ml. The buffer also contains 50 mM HEPES pH 7 and 2 mM DTT.
Do you have any clue of how to solve this problem? I thought that the concentration of the protein was too high, especially considering that after MBP tag is removed the solubility of the cleaved protein is reduced. So, should I dilute the sample before cleavage? And should I optimize the cleavage conditions, for example modifying the cleavage time and temperature? If you have any ideas it would be great. Thank you!
As you pointed out, the MBP fusion helps to increase the solubility of your protein, so cutting it off with thrombin leads to reduced solubility and precipitation. One solution is to lower the protein concentration before cleavage. Another is to alter the buffer conditions to improve the solubility of the cleaved protein. 650 mM is a very high NaCl concentration, so I would suggest starting by dialyzing to get rid of the salt. Also, consider the pH of the buffer. If it is close to the predicted pI of the cleaved protein, consider shifting it up or down by at least one unit. It might also be helpful to include a mild detergent, such as CHAPS, which can be removed later if necessary.
Hi Adam. Regarding the pI of the protein, it is around 9, so the pH of the buffer should be fine. As you said, the NaCl and the protein concentration too were probably too high. Do you think I can dilute the protein by adding buffer without salt, in such a way to dilute both the salt and the protein concentration at the same time?
Thank you so much Adam for your help!
It's a good idea, but if you want to just get rid of the salt without diluting the protein (much), dialysis is an easy way. There will be some dilution, however, because the volume of the sample in the dialysis bag/cassette will increase due to osmotic pressure.
Dear MIchele
it seems that as Adam suggest, the MBP was able to maintain in solution your protein which itself is not folded and it precipitate once the MBP support was lost.
if you are using the Shufle, i suspect that you protein contain cisteines?
Did you run the precipitate in SDS-page with and with-out reducing agent to check if the precipitate contain high MW aggregated formed to the aspecific formation of S-S?
best regards
Manuele
Dear Manuele, thank you for your answer. Might it be that the protein is not unfolded but just not very soluble? By the way, no I didn't run an SDS-page under non-reducing conditions, but the buffer always contains 2mM DTT, so (at least in theory) cysteines should be reduced and not forming disulphide bonds, right?
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