I would like to IP LONP1 protease from MCF-7 cell. LONP1 is a mitochondrial serine protease.
My question is what protease inhibitor should I use to IP but still maintain my LONP1 activity? I know some of the protease inhibitors are irreversible and may kill my LONP1 activity. Should I just abandon all serine protease inhibitors? Or should I only use reversible serine protease inhibitors?
I would much appreciate your suggestion!
Hi Yiming,
The answer to your question depends on what LONP1 cleaves or in other words what is LONP1's substrate & how stable is LONP1 when it is active (or inactive)? If you don't expect it to cleave itself or the IP antibodies and it is stable in the absence of an inhibitor, it might be better to not use serine protease inhibitors because there would be no need to use them. However, if you expect that inhibition would prevent self-cleavage, improve IP'ing, or improve stability, then you should use protein- or peptide-based reversible serine protease inhibitors which can then be washed out or removed by other means. However, this may prove difficult. Although I'm not familiar with the properties of LONP1, here are a few common reversible trypsin inhibitors that you may find useful: aprotinin aka BPTI, SBTI, and leupeptin.
Erik
Hello Yiming,
To evaluate the protease activity I think that you should not add inhibitors. If the protease is unstable due to autocleaving activity I recommend to maintain the samples in a pH below the optimal pH for activity, of course you should check how the protease stability changes with the pH.
Anyway, if you need information about inhibitors I recommend search for this protease in the MEROPS database:
http://merops.sanger.ac.uk/
You will find there all the inhibitors reported for this enzyme, even when it sould be inhibited by the standard serine protease inhibitors.
I hope this help,
Regards,
Laritza
Hi Yiming, I agree with Laritza that you should not use proteinase inhibitors for measuring activity, but LONP1 is not a "classical" serine proteinase like trypsin, chymotrypsin and thrombin.
So, what do you mean by "testing its activity"?
I would be concerned that IP by itself would affect the activity. Has the activity profile of LONP1 in IPs been published?
Thank you all for the great answers!
For Steingrimur's question, I want to test LONP1 proteolytic activity using casenine as substrate under ATP-stimulated manner. There are only few papers have done LONP1 IP, but they are moe interested in the protein interactions vs. activity. So, as far as I know, the activity profile of LONP1 in IP has not been published. Previous papers have used recombinate LONP1 for activity testing.
Steingrimur, why do you think that IP by itself would affect the activity? and what do you mean "LONP1 is not a classical serine protease?" AAA+ vs. non AAA+?
I have only worked with LONP1 for several weeks and have a lot to learn.
Again, thank you for these wonderful answers.
IP involves Ab binding to LONP1. Does the Ab binding affect the proteinase activity?
What I mean by a "classical" serine proteinase is a single enzyme that hydrolyses its substrate via a specific amino acid triad in the catalytic site (His, Asp, Ser). There are no organic cofactors needed for this activity.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides