I am using the pET SUMO cloning system from Invitrogen to purify proteins in E. coli. BL21(DE3) cells are used for expression of the protein. Following induction with IPTG, I find that the E. coli produce not only my fusion protein, but also the SUMO fusion partner by itself in high amounts. This happens for all of my proteins and it is inconvenient because the His tag is attached to the SUMO protein, so during purification with Ni-NTA resin, I always end up with a mixed sample of fusion protein and SUMO. I think that this might be due to endogenous proteases in E. coli acting on the SUMO protease cleavage site between my protein and the SUMO tag, resulting in a mix of production of the full fusion protein and SUMO by itself. Are there any adjustments I can make to the culture conditions to reduce the in vivo cleavage? I don't suppose the E. coli could be cultured in the presence of protease inhibitors...
Thanks!
Are you sure the degradation is happening in vivo during expression or during the purification ?
Besides lowering the induction and expression temperature of the culture (around 14 °C for BL21) , there isn't much you can do to reduce in vivo cleavage. You can stop the culture and pellet it as soon as it reaches the stationary phase. The protein can be immediately purified with protease inhibitor cockatil in a cold room or using buffers at 4 °C to prevent further degradation. If the fusion protein is unstable on it own, adding about 5-10% glycerol, 1-10 mM of a Magnesium salt or ATP in the buffer provides additional stability.
Certain bacterial strains like the Arctic Express allow for induction at lower temperatures and some other have completely different protease profiles. If this issue is stubborn in regular strains, you can consider switching the expression strain.
Thank you for the suggestions. I am sure it is happening in vivo as I have done pilot expressions on 5-10 mL cultures where I do SDS-PAGE on the whole pellets obtained from 1 mL aliquots of those cultures. I see increased production of my protein after adding IPTG but also the production of SUMO as a lower molecular weight band. I have confirmed it is SUMO by mass spec.
I work heavily with SUMO, both looking at the covalent and non-covalent interactions with other proteins. The full gel banding you see is very common as SUMO interacts with many proteins and often drives complex formation or bimolecular phase condensates. HIS tags bind non-specifically as well. A never ending mess!
To overcome this I would suggest using protease inhibitors in all of your in-vivo experiments. We use NEM, 2IAA, sigma protease inhibitor cocktail, and PMSF with our bacterial and mammalian cultures. This should help reduce the number of bands you see on your gels. Working in a cold room or keeping everything on ice should help too.
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