Hi all My name is David Vizarraga.
I am trying to purify a protein. But When i purify it, after a few days It begins to autoproteolyze. It forms the same fragments in all bach of protein purification and finally It is totally proteolyzed in many small MW fragments. These fragments are joining together because in size exclusion column only appear one peak.
the question is if how can I stop the autoproteolysis process?
buffer of size exclusion: TRIS 20mM pH:7,4 and NaCl: 150mM.
I added the proteases inhibitor in the Lysis buffer
thank you
Protease inhibitor is not working or is not proper. I do not understand the part of the fragments joining together.
Why are you emphasizing on autoproteolysis? It may just the protease is chopping off your protein with time and that's why you see fragments. There are a number of approaches, I will share two approaches to tackle this issue. Use cocktail of protease inhibitors in all steps of your purification and finish your experiments as soon as possible. Do not store your protein long enough, always use fresh proteins for your experiments. The second way is to do limited proteolysis experiments to know the stable fragments. limited proteolysis outcome may help you in designing new constructs, hopefully, stable enough for your experiments.
Dear David,
How did you know whether those fragments are degradation products of your target proteins or other host cell proteins and/or their degradation products? Did you run SDS-PAGE?
You need to detect the proteins from SEC chromatogram peak fractions by SDS-PAGE. Use an appropriate marker protein to track the molecular size of all the proteins in the fractions. It is also good to use fresh cell lysates and freshly purified proteins as a control. I hope this will help.
But if you have really a problem associated with protease-mediated degradation, I will share the recommendations given by Zeyaul Islam.
Hi there,
If it's autoproteolysis, there is not a lot you can do apart specifically inhibit the protease activity of your protein. If it's protease contamination then you may consider a further step in your purification process. But whatever the case, you should add protease inhibitor cocktail in all the buffer systems you use for purification (ie. not only to the lysis buffer).
Hello David Vizarraga,
Its been a few years, but here are some suggestions
1. add protease "cocktail" is generally expensive solution but sometimes it is the only way to go in a purification.
2. try eluting your product into a conc. [buffer salt] that will take your protease out of its typical activity pH range where you suspect that it is eating itself - provided it is soluble at an extreme of its range of pH in which it chews itself. This way you can depress autoproteolysis with simple technique. Check it does not denature at the pH range and in conc [buffer salt]
3. run all stages of purification in a cold room or cold cabinet
4. Does your protease fit into any one of the main four catagories? If so, add a known potent small molecule inhibitor for this family to suppress auto-proteolysis.
Do you have sequence at all to search database to assign the protease to a known family?
Add EDTA 1mM to your ellution buffer and try to purify your specific protein as much as possible. Keep the protein at 4 degrees always. Even doing that is almost impossible to avoi some kind of degradation with the time.... Entropy kills slowly
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