Upon incubation of my protein with O/N with TEV protease (in 50mM Tris, 0.5mM EDTA), in the morning i saw a white thready precipitated formed. After running the SDS gels, following coomassie staining, i do not see my actual protein (either uncleaved or cleaved), or even the TEV protease. I believe the proteins (my protein with TEV) might have formed a conglomerate and precipitated. IS this a possibility and how can i dissolve this and incubate again. Will Glycerol help? Suggestions are really appreciated.
TEV protease precipitating? - ResearchGate. Available from: https://www.researchgate.net/post/TEV_protease_precipitating [accessed Jun 29, 2016].
Possibly your protein did not fold correctly but was kept in solution by the GST tag. It is also possible that your protein naturally forms large oligomers and that oligomerization is prevented then the GST tag is attached.
You can try to add glycerol to the buffer or other additives or a different buffer/pH. Alternatively you could clone a sub-sequence of your protein (single domain) or to replace the protein with a homolog from a different organism. Or use a different expression system.
It would help to know which protein you are working with.
I have had a similar experience with a protein that is naturally inclined to aggregate over time, so certainly a long overnight incubation would be a problem. It may be your protein, though in my case our TEV construct was not of optimal stability or activity, so when we switched to the TEV S219V construct, it was much more stable and the activity was improved. This allowed us to incubate with TEV at room temperature for an hour or two to get efficient cleavage rather than overnight at 4C. However, I agree that this behavior is probably related to your protein, so your best option would be to try to keep your protein at as low of a concentration as possible (within reason, say 20mL from your GST purification, rather than concentrating it before TEV cleavage) to minimize aggregation potential and switching to a more active TEV construct to lower your incubation time. I would combine these strategies with the aforementioned recommendations.
Hi Konrad,
I am working on purification of a DNA polymerase Pol Mu. The construct was a gift from NIH. Unfortunately, they are the only people to have worked on purification of this particular protein with the construct. I did a pilot experiment (1g cell pellet) for the same and i saw no precipitation. Also, i saw ~90 % cleavage in my pilot. When i repeated the same for 10g pellet, with similar conditions, i saw this precipitation formation. I checked for the pH, which was ~7.8 (should be fine). The buffer conditions were same as used in the pilot experiment.
I added glycerol to the the solution containing the protein as precipitate, however, the solubility is still bad.
Hey Taylor,
I had about 12 ml of my protein in solution. And i think the concentration might be high. I did a bradford assay, for which the concentration was 35 mg/mL which seemed weird to be so high for 10 g pellet. Can it be so high?
It depends entirely on the protein you are purifying. We've gotten yield in that range before, but then again we had a very robust expression system that gave us very good induction for our soluble protein of interest. We even got an entire gram of protein in a single purification, though that was from closer to 15g of E coli pellet. The key is whether the concentration you are seeing from Bradford agrees with your SDS-PAGE gels. At that concentration, if your product is pure, in the lane it should be quite prominent (a few millimeters thick on your gel image, real size). If you're not getting a band that thick, it's possible your Bradford could be lying to you. If you don't standardize before every test and use relatively fresh Bradford solution you may be overestimating protein concentration.
35 mg/ml is very high and many proteins will precipitate at this concentration. I would agree with Taylor, try a lower concentration for the TEV cleavage.
Hi Konrad,
Yes, that seems to be the case! Thank you everyone for your suggestions. I will decrease the concentration to ~5mg/mL and update you with the result.
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