Hello to everyone.
We have a protein which we think it is a metalloprotease, but the experiments that we did, they dont give any clear result.
1) In SDS-PAGE gel, the protein have severals cuts, but when we add EDTA the cuts don't appear.
2) We did a ICP-MS to obtain the metal, and in solution only have Zn but in a relation 1:40 ( metal:protein) maybe the protein lost the metal?
3) In purify process if we add an anion exchange step, apparently the degradation/proteolysis appear in less time. maybe can the salt concentration increase the degradation/proteolysis?
What prove can I do to certify the protease activity?
P.D: The protein still together in spite of the cuts.
P.D: We dont think there are any contamination protease.
Many thanks!
Your data strongly support the hypothesis of a contaminating protease. Btw, did you use protease inhibitors during purification?
Dear David
but it this proteins contain some conserved metal binding sites ( eg cxxxc or hxxhe or others that van suggest that is it a metalloprptease?
can you share the gel to check the puritu level of your sample?
if do yuo think that zinc is its substrate you can try to add it to che proteins and check if under zinc addiction do you have conformational changes in your protein. the simplest way to do it is dsf (thermofluor) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841139/ which if you have axcess to a rt-pcr instrument with rox filter is a very simple tool.
of course if you will see tm shift under zinc addiction the best way to validate you data is produce a mutants missing a possible aa involved in zinc binding and demostrate the is still folded ( tm of wt apo protein similar to tm of mutant) but mutant is not able to bind the metal or is inactive.
good luck
Manuele
hello Manuele, there arent any metalloproteasa motif (like hxxhe) but there are His and Glu very close together in the space.
in SDS-Page apparentluy the protein is pure
i think that you need at least prove that
- you protein is able to bind zinc in high ratio. if you cannot do dsf just add zinc to it, remove the excess of zinc by dialysis or desalting and repeat icp-ms to se if now ratio is close to 1:1
2) a mutant in one of this 2 residues is inactive
otherwise you have to dipport the this activity come from 1:40 (thr metallated fraction) of you protein instead an imputrity that you are not able to see in sds-pahe and i'm surw that the most of scientist will reply to you as engelbert. it is a nice hypotesis but you need to confirm it with solid data
good luck
Manuele
regarding your question:
maybe the protein lost the metal.
Generally metal binding affinity of metalloprotease is not so low and in case your protein is really a metalloprotease is more probable that it is not metallated because in the growth media and in the e.coli cell there will not be enough zinc available during the overexpression and it is quite common produce metallo protein in apo form and reconstitute it after purification. one alternative possibility is supplement the media with some zinc but in my expertise this strategy is more succesfull in case of expression of proteins in the periplams.
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