I am trying to purify his tag protein from a Gold strain of E coli, Most of the protein goes to the inclusion bodies at 16 degree induction. I tried 10 degree afterwards and it some how made the fraction of protein soluble. For having high amount of my protein I tried using 0.5mM IPTG and 16 degree induction which worked and more protein became soluble (before and after centrifuge inputs were almost equal). But, upon SDS gel the protein seems degraded (which did not happen in the first case). All the three elutions having two strong non specific bands and one faint actual protein band. I need to know the reason/s of protein degradation during the washing and elution and sample preparation steps. I have used 500mM imidazole in elution buffer.
Congratulations on successful optimisation to get some soluble protein production. Tell me, were the non-specific bands you observed smaller than your target protein? Or larger? A gel picture might help us identify a solution better.
Nonetheless, If the band's are smaller they may indeed be due to degradation. A Western Blot would help you confirm this if you have anti-6his or anti-target protein antibodies. Alternatively MS/MS or peptide mass fingerprint mass spec would help identify the non-specific bands.
Have you tried adding a protease inhibitor cocktail, assuming the degradatiin occurs during sample prep?
Good luck.
@ Tsepo Lebiletsa Tsekoa .. thank you for your comment.
The non specific is smallar than the actual length. In addition, I am planning to do western blot for anti his tag to confirm if these are degradation.
Regarding the protease inhibitor, I am afraid I only added PMSF to the lysis buffer not to the wash and elution buffers.
Hi Hameed,
I don’t know specific information about your target protein, so I can only give you some basic advice.
1. Prove with WB that it is indeed your target protein;
2. The purification process should be accelerated and kept at low temperature as much as possible, and protease inhibitors should be added to avoid degradation of the target protein;
3. N-His construction protein degradation occurs more frequently, especially for some proteins with unstable properties. It is recommended that try C-His or both ends of His-tags.
Hope it helps!
Roger Davis Sebastian Schmitt
thanks for your suggestions. The WB confirmed that it was degradation. I accelerated the protein purification process at low temperature and now I have some proteins in the elutions.- Badges
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