I'm trying to purify a mettaloprotease, but I have 90% of loss.
We use ammomium sulfate precipition, dyalisis (tris 20mM pH8), deae cellulose column (tris 20mM pH 8 and elution with 0.1 NaCl) and dialysis. I'd like to ask you if you think it would be possible to inhibth the protease (EDTA) during the purification to increase protein recovery.
Do you think the low recovery is due to self-proteolysis by the metalloprotease you are trying to purify? This might be the case only if the protease is a substrate for its own degradation. An alternative is that the low yield is due to the purification process itself. Have you kept track of the yield at each step? Maybe the process can be improved.
Metal chelation, such as by EDTA, may inhibit the enzyme activity, and this would prevent proteolysis. Then you could remove the EDTA and add the metal back at the end of the purification. However, a risk is that the enzyme could have lower stability in the absence of the metal cofactor. Another downside is that you would not be able to keep track of the yield through the purification by an activity assay if the enzyme is inactivated.
I think the loss can be due to self-proteolysis mainly. I followed each step and the most important loss is in deae purification step and dialysis. I really don't need enzyme activity because I will use it as immune coadjuvant. To use in cell culture or animal model, I can remove EDTA.
Dear Marina
if you think that autoprpteolysis is tge problem, you can certanilly add edta in the lysis buffer. Also repl ce dialysis with desalting can be helpfull because the long time required for the dialysis is not compatible with autoprpteolysis.
you can found more details about desalting on my blog ProteoCool https://proteocool.blogspot.com
ProteoCool n°8 (Buffer Exchange Methods overview) and
ProteoCool n°11(Rapid buffer Exchange by PD-10) available at page3
however in my experience to obtain. very high recovery rate you have to use an affinity purification as imac instead ammonium sulfate and deae... but in this case you can add edta only after elution from imac. and you need to re-design everything ..so edta and desalting are certamilly the first way to try.
good luck
Mamuele
Dear Manuele, thank you for answering. I found that EDTA binds to DEAE and competes for the active site. I'm trying to get IMAC to purify it and we are not going to use steps of dialysis. The buffers in your blog will be very useful. We are hopeful. Best regards!