Dear members,
I am trying to express and purify a mouse-derived enzyme (rotamase A) having 7 X His-TEV cleavage site.
The protein overexpression at 37C (0.8M IPTG induction for 3 hrs) shows a clear band in SDS-PAGE. However, the purified protein (after Ni-NTA) shows autodegradation resulting two closely placed band in SDS-PAGE with identical intensity. I tried cell lysis with and without protease inhibitor cocktail + PMSF. Unfortunately, there is no difference and two bands are observed.
It will be a great help if someone could help me in finding the problem of such autodegradation. I look forward to your comments and suggestions.
Thanks and regards,
Bikash
Have you checked (using e.g. WB) that the second band is absent from the crude lysates? It would be great if you could post a picture of the lysate and the purified sample.
It is odd that despite your efforts the problem is not at least partially solved. If after working as clean and quick as possible, in a cold room, and using protease inhibitors, the degradation is not slowed down, I would say there is a good chance that the protein is being degraded in vivo instead, or that this is not a degradation issue at all. Do a trial run in which the lysis and Ni-NTA purification are performed with denaturing buffers (containing e.g. 6 M GuHCl), where proteases will mostly be inactive, to see if the problem goes away. If it does not, this could be a case of post-translational modification, premature termination, or simply degradation by the host.
Are you using an expression strain that is deficient in proteases, such as BL21(DE3)?
If you can determine the cleavage site, such as by C-terminal sequencing or intact protein mass spectrometry, you may be able to mutate one residue to prevent proteolysis.
Hi Bikash, have you confirmed that the 2 bands are your protein of interest? This can be easily done using a His-tag antibody or a specific antibody to your protein. Another option would be sequencing each band. If one of the bands is an unrelated protein you have to think about additional purification steps.
If both bands are your protein: its sounds like there is a post translational modification happening like cleavage of some amino acids. Sometimes changing the expression temperature does help in reducing cleavage of protein (go 30C or even lower). Also, as Adam suggested using a different bacterial strain or as Alejandro suggested, doing the protein purification at 4C reduces the degradation.
Another reason why some proteins do run differently in the gel is some residual structure that has not been disrupted during the SDS sample prep. Try adding more SDS/DTT to the sample and make sure you boil it properly before loading your gel.
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