While eluting two of my proteins with 250mM of Imidazole, I am getting two different bands (Size of the proteins are mentioned in the Image attached). The genes are cloned in pET22b without Signal sequence and Histag is added at C terminus. The protein in expressed well in BL21 DE3 codon plus RIL. Suggestions needed to remove the non specific band during purification.
You are probably getting some degradation of your protein - because it is still binding to the column you are losing some portion of the N-terminus. It is most likely this is the signal peptide, which is only partially cleaved, or half your protein is remaining in the cytosol and so not processed, while half is sent to the periplasm. You need to calculate the difference in size of the two bands and see if that matches the expected size of the signal (2kDa I think). You might be able to separate these proteins by gel filtration if you think it is necessary, (for crystallography for instance), but if it is for a biotransforamtion experiment it may not be necessary.
Alternatively there may be some further degradation, not related to the signal sequence. In which case you probably need to separate the two bands (cut out of the gel) and do N-terminal sequencing to find out where the cleavage is. It may not matter, depending on the experiment.
Hi there,
Your proteins undergo Nter degradation as they are eluted from NiNTA. Either try IEC or HIC as SEC won't resolve proteins with close MW. The alternative is to redo from start and add protease inhibitor to the lysis buffer and see what happens.
Heating IMAC elution samples (with high imidazole) in SDS sometimes results in strand breakage, its usually best to only heat to 37C. That said such a specific and efficient digestion looks like proteolysis.
@Ellis: the signal peptide was removed at the time of cloning itself.
@Liger: I am using Protease inhibitors in lysis, wash and elution buffer.
The protein is from a thermophile so is stable at room temperature. I keep my proteins at 4 degree Celsius so I don't think this is because of degradation.
Thanx for your responses.
If you want to be sure about the content of the lower band, run MS fingerprint on it. Working with thermophile proteins is not a guarantee against degradation or stability issues. I've been aware of such proteins precipitating when working/storage temperature was too low for instance.
It is posible that the protein is degraded. It is advisable to keep the temperature at 4 degrees celsius during the procedure
Your proteins may be inherently sensitive to proteases at one or more sites - you can minimise this by expressing in protease deficient bacteria and/or at lower temperatures, and induce expression for shorter times. This may reduce the amount of cleaved protein produced, with only modest reduction in yields - it may be worth losing some yield to get more full-length protein. In addition, try to establish where in the N-terminal half of the protein (roughly) the cleavage is occurring (try searching your sequence against a protease database such as http://web.expasy.org/peptide_cutter). You may then be able to calculate a pI difference between the full-length protein and the truncated one, which allows a further separation of the proteins by ion-exchange chromatography.
www.malariaresearch.eu
You have two protein bands, are you sure that both were yours? you eluted with 250 mM straight or did gradient? Occasionally, we detected co-contaminant around 45 kDa during purification of our protein preparation. could you check by western, for example, for the identity of the bands. use anti-His antibody if you don't have your protein target receptor.
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