I have expressed a protein of C. jejuni in E.coli M15 cells using pQE30 vector. My protein carries a N-terminal hexa his tag. I have purified my recombinant protein using Ni-NTA columns and I have got three bands. Western blot confirmed that a strong band of 41 KDa (expected size) is the protein I needed but there are two more bands of strong intensity. These two bands are constantly coming in elutions even when I'm using 60mM imidazole in my wash buffer and elutions I am performing is using 150mM, 250mM imidazole. Could it be possible that my His-Tag protein of C. jejuni is interacting with some protein of E.coli and pulling down it together?
The His-tag in pQE30 in expressed N-terminally of your protein. I suspect the bands you see are incomplete synthesized proteins. If you move the tag to the C terminus you shouldn't see any other bands by western blot because you would purify only full length His-tagged protein. Martin suggested that you see dimers and other multimers in your blot. This is only possible if you do native PAGE instead of SDS-PAGE.
Another possibility is that the antibodies to your C. jejuni protein show some cross reactivity to some E.coli proteins. His-tag purification usually yields a number of contaminations due to the fact that other proteins contain clusters of histidines. As Sagarika mentioned this is especially a problem when you have a low expression level. A lot of people add an ion exchange chromatography step for polishing.
Hi Avinash,
First of all, what are the MW of the other two bands? Maybe they're in fact dimers or trimers of the protein. You can check this out in WB using an anti HisTag antibody.
If they´re actually contaminants, you should do an Imidazole gradient and check the procedure with Coomassie staining, You will find an Imidazole concentration that elutes the contaminants. Take account that you can lose some yield in this way, otherwise you should consider HPLC wich allows you to make a continuous imidazole gradient (high purity and yield).
Hope this can help you
The His-tag in pQE30 in expressed N-terminally of your protein. I suspect the bands you see are incomplete synthesized proteins. If you move the tag to the C terminus you shouldn't see any other bands by western blot because you would purify only full length His-tagged protein. Martin suggested that you see dimers and other multimers in your blot. This is only possible if you do native PAGE instead of SDS-PAGE.
Another possibility is that the antibodies to your C. jejuni protein show some cross reactivity to some E.coli proteins. His-tag purification usually yields a number of contaminations due to the fact that other proteins contain clusters of histidines. As Sagarika mentioned this is especially a problem when you have a low expression level. A lot of people add an ion exchange chromatography step for polishing.
Thank you all guys for your precious suggestions, your explanations gave me a direction to think for next level solution.
Hello Sharma in addition to all that has been said, your protein may be degrading so make sure your add the appropriate concentration of protease inhibitor. And also go through SEC. This is highly recommended.
Usually , we could not get one single band with 6 His-Tag , because we used 6His genes to combine with Ni-NTA stronger which could not be washed by low imidazole, but some His genes (maybe 1-5 His genes) at chromosome also expressed ,which could combine with Ni-NTA too,if so ,some strategies you can take.
1) Add 6His tag at 5' and 3' terminus both, with higher imidazole to wash;
2) Change the Tag with others,that maybe better;
3) If something can not chang,you shoule konw the isoelectric point ,that you will know the acid-base property ,come through with the ion of Yin-Yang colum;
4) Come through with molecular sieve.
Hope for help !
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