I am currently working on a thermostable polymerase which I am overexpressing in the BL21(DE3) strain. The polymerase has a histag at the N-terminus. However, during purification, I am not obtaining a single band but rather several bands of different molecular weights ranging from 5-85 kDa. The expected molecular weight of my polymerase is 92 kDa. The additional bands observed on the SDS-PAGE gel after purification on TALON or AKTA systems, some appear to have the histag (confirmed by westernblott). I have attempted to optimize the purification conditions by adjusting buffers, using protease cocktails, DMSO, Betaine, low induction temperatures with longer time, shorter time of induction, and optimised times and amplitude of sonication, but none of these measures have yielded the desired results. Do you have any suggestion what should I try?
Have you optimized the codons for E. coli?
E. coli produces tRNA to some codons in preference to others, and this can cause translation to terminate early if it runs into a codon that it makes in low abundance. The easiest solution is to use an E. coli strain like Rosetta from Novagen, which boosts the production of rare codons.
Alternatively, you can move the His-tag to the C-terminus so that only fully translated proteins can be purified.
Hi Ryan, we've checked the codon optimalisation at the begining of the experiment, and its optimised for E.coli. Also we are checking if our moving His-tag to C-terminus and periplasmatic production was succesfull. Anyway, thanks for the answer.
If moving His tag to the C-term does not work, possibly the bands are degradation products from your protein. Maybe select from a variety of protease inhibitors if you have not already. We previously observed different PIs protect the protein from digestion to different extents, and some don't work as well.
And I agree another option is to use a different cell line.
Usually, after the affinity chromatography, you should use polishing step like size exclusion chromatography, which should remove proteins of other sizes.
If there are still bands afterwards, which do not contain the His-tag, you need more stringent conditions to remove the contaminants.
If degradation is problem, one approach is to add second tag to the end of the protein (so in your case C-terminus) and purify it by 2 chromatography steps. By this approach, you should obtain only full-length proteins.
However, at some point, you have to decide, what is still necessary to purify and what is already sufficiently purified, even if it's not 100% your full-length proteins. Of course that depends on the application.
BTW I'd be more worried about having smaller protein than you should have than by smaller fragments.
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