I am trying to purify a cysteine protease to use in an activity-based assay. The gene has been sequenced. It is in a pET based vector and has a His6 and GFP tag. I express in BL21 DE3 and the intact cells have good expression at the expected molecular weight. When I run my post-sonication sample on a gel, I notice loss of protein. Protein loss seems to be quite rapid. I do all purification steps at pH 8. Samples are kept on ice during purification with pepstatin A, aprotinin and fresh PMSF added. I want to avoid adding cysteine protease inhibitors as I will be measuring cysteine protease activity and any remaining inhibitor may confound my results. I purified the sample on a Ni-NTA column and instead of GFP-tagged protease, I obtained a pure sample of GFP. By LC-MS, I can confirm that the primary protein product is GFP with a short piece of my cysteine protease attached. I believe I am seeing autolysis of the protease portion of the protein and only the stable GFP survives. A GST tagged protein behaved similarly except I saw complete degradation of the protein. This is a novel protease. I have purified other isoforms of this protease with little difficulty. I do not know what the substrate of this protease is so cannot add a substrate during purification to stabilize the protease. I am considering adjusting pH during purification to limit autolysis. Is high pH or low pH more likely to limit autolysis? Does anybody have any other suggestions that may help me purify this protease?
Hi there,
Play with pH during extraction/purification : thiolate is the general base for such enzymes (helped with imidazole side chain of histidine). If low pH is not detrimental for protein stability (pH below 6 to ensure full protonation of both thiol and imidazole ring), you should go for it.
Some cys proteases are calcium dependent therefore the use of calcium chelator should also help if it is the case for your specific case.
Dear Suzana
did you see in the flow though recovered form the IMAC any bands that may resemble the only protease (with out tag)?
Because it is possible that you have aspecific autolysis and complete protein degradation but it is also possible that you have specific auto-cleavage or cleavage on the linker beetween GFP and your protein.
in this case you can try to change the position of the his tag (at the C-instead the N-term) and see if you are able to purify the digested part. At this point probably the best approach will be try to obtan the N-term sequence of the purified protein and if the digestion happen in the link, you can use the protein, if the digestion happen inside the protein and you need the entire protein to work, you can try to mutate the digestion site to see if you protect it from the autolysis.
good luck
Manuele
Do not use EDTA or EGTA during IMAC - it will cause your metal to be eluted.
Thank you for the suggestions. Manuele Martinelli I do see some proteins in the flow through. I will need to determine if one of them is intact protease with the tags cleaved off. I can easily tag the C-term of the protease with a His6 tag and deal with His6-GFP contamination of the protease-His6.
I've looked through the literature and found two examples where this protease has been purified. In one case, cell-free protein expression was used. In the other, a GST-tagged protease was isolated from E coli but their gels seem to show proteolysis and I don't know if I can believe their results.
I agree that your easiest recourse would be to find a pH that is far from the pH optimum of this protease. It would help if you had a substrate even if it is not the endogenous substrate. You might try zymography on casein- or gelatin- containing gels as a quick way to determine pH and other conditions that would inhibit the protease. You mentioned that if you knew the substrate, you could stabilize the protease with substrate. You have a clue to the substrate specificity of this protease since you know the sequences of the GFP- and GST fusion proteins. From the size of the protease remaining after purification, you would know approximately where the cleavage sites were. A peptide encompassing that region might work as a substrate.
You do not know the substrate.
Do you know the product? Adding this to the mixture would help , too.
Hi Suzana,
Check whether the recombinant protease is going into inclusion bodies post sonication. Also alternately you can try lysing and purification of protein under denaturing conditions followed by refolding.
Perhaps preparing the protease by intein trans-splicing would help: Article A general purification platform for toxic proteins based on ...
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