We purified a 50 KDa metalloprotease (Serralysin) from Serratia marcescens. We employed a DEAE cellulose in tris buffer and recovery it in a elution with 100 mM NaCl in the same tris buffer (20 mM pH 8). After overnight dialysis, we put the Eppendorf on ice. We saw the purified protein in SDS PAGE. In the afternoon (6 hours after) we measured protein concentration (Bradford) and the amount of protein decreased too much. So, I don't know exactaly if degradation is due to self proteolysis or instability. I know that the same happens when we use PBS buffer. The pI of the protease is 5.5. I could use EDTA, which inhibbits metalloprotease, but EDTA is not good to be used in vivo (mouse). The protease is inhibbited at 72 Celsius degrees for 30 minutes, but almost all is lost. Does somebody have an idea? Please...
Heat inactivation may be irreversible if the enzyme aggregates or precipitates. Perhaps the idea here is to release and remove the metal ion by heat and dialysis? If so, perhaps you could instead use EDTA and dialysis, rather than heat and dialysis. Mix the enzyme with enough EDTA to bind all the metal, then dialyze against an EDTA-free buffer. The result is inactive, metal-free and EDTA-free enzyme. Alternatively, if EDTA is sufficient to remove the metal from the enzyme, you could use a faster procedure than dialysis such as gel filtration (by gravity or spin column).
If autolysis is the problem, then inhibition of the enzyme by metal chelation should solve it. It would only take one molar equivalent of EDTA to give full inhibition, since EDTA binds metals ions very tightly. The amount of EDTA might be too low to harm the mice. Other chelators might also work, such as citrate or phenanthroline. To restore activity, you would only have to add the metal ions back in excess of the chelator concentration.
Dear professor Adam, thank you once more for answering my question. I can add EDTA, but I think that to use the protease in mouse and culture cells with EDTA will not be possible, because can produce different results. But maybe use EDTA while inactivating at 72 degrees and after make a dilialysis again. Do you think would it be a good idea?
You can check BRENDA (https://www.brenda-enzymes.info/) for an inactivator of your enzyme, inactivators permanently kill an enzyme and thus can be removed.
However, if your enzyme survived o/n dialysis, but was killed by a few hours (on ice?) I suspect that the problem is not auto-digestion. Again, look at BRENDA for better storage conditions.
Heat inactivation may be irreversible if the enzyme aggregates or precipitates. Perhaps the idea here is to release and remove the metal ion by heat and dialysis? If so, perhaps you could instead use EDTA and dialysis, rather than heat and dialysis. Mix the enzyme with enough EDTA to bind all the metal, then dialyze against an EDTA-free buffer. The result is inactive, metal-free and EDTA-free enzyme. Alternatively, if EDTA is sufficient to remove the metal from the enzyme, you could use a faster procedure than dialysis such as gel filtration (by gravity or spin column).
I would like to inactivate permantly the enzyme. I need to kill the enzyme before injecting in the mouse or in cultured cells. The culture medium, for instance, harbours zinc ansld calcium ions wich could activate the enzyme.
Adam B Shapiro Thank you for the explanation. I faced similar situation in my current research. I will try out the method you described.
The protein could refold into an active form after heat denaturation if it is not aggregated or precipitated. If it is aggregated or precipitated, it will not be very useful for raising antibodies, if that is your intention.
You might try denaturing the protein with sodium dodecylsulfate. This might inhibit refolding even after dilution if it binds tightly enough to the protein. There would be a limit to how much of this detergent would be compatible with mice or cell culture, of course.
If you don't require the protein to be intact, you could immunize with a mild serine protease digest of the EDTA-treated enzyme. This would likely destroy its catalytic activity, while leaving large enough pieces to generate antibodies.
A simpler approach would be to have peptides synthesized based on the amino acid sequence. There are programs that help to identify the continuous epitopes that are most likely to be good as antigens. The peptides should be conjugated to an immunogenic protein for raising antisera in vivo. If you know the gene sequence, you wouldn't even need to purify the protein.
If your goal is to inject mice with an epitope, agree with Adam: work with coupled--peptides. Cheap these days and very effective. Other option, if autolysis is indeed involved and you decide you need the whole protein, consider mutagenizing one of the catalytic residues and expressing it in your favorite system.
Good luck!
Dear prof @Sebastien. I purified the protease directly from the culture medium. But the suggestion is very interesting if I decide to use heterologous expression. Thank you.
Dear professor Engelbert. I checked Brenda and I found a paper which suggested inactivation with both EDTA and acid conditions. It resulted in small loss and no activity. Than k you