I'm expressing a his6-MBP-tagged membrane protein under the araBAD promoter in C43 cells, because I've shown that with the same construct growing in C43 pushes more of the protein into the soluble fraction compared to growing in standard strains like NEB 10-beta.
I normally add protease inhibitors to cell pellets while they're thawing before I put them on the probe sonicator. I'm having an unusual amount of trouble with proteolysis with this guy*. Possible reasons:
1) I've noticed that the freeze-thaw cycle alone releases more protein from C43 cells than other strains!
2) The Lon protease is re-activated in C43 cells** - haha that jerk.
Should I add protease inhibitors to the lysis buffer before I freeze cell pellets, or should I resuspend cells in half the normal volume, freeze them, then add more buffer with protease inhibitors immediately when I remove from the freezer?
*(Switching to a nice Roche protease inhibitor cocktail instead of PMSF didn't fix the problem. I'm clearly not adding protease inhibitors soon enough).
**see Kwon, Soon-Kyeong, et al. “Comparative Genomics and Experimental Evolution of Escherichia Coli BL21(DE3) Strains Reveal the Landscape of Toxicity Escape from Membrane Protein Overproduction.” Scientific Reports, vol. 5, Nov. 2015. PubMed Central, doi:10.1038/srep16076.
I was in the habit of resuspending my cells in the lysis buffer before freezing, which would have protease inhibitors in it.
If the Lon family are active then PMSF should do something as it is a serine protease inhibitor. PMSF needs to be "topped up" as the purification progresses.
The EDTA free Roche PI cocktails are good.
Did the 10-beat strain show lower levels of proteolysis? If so then sacrificing yield for quality of prep seems to be a better plan, as scaling of a expression/purification process is easier than developing a method to eliminate proteolytic degradation of your target protein.
If I were you I would try to go for another construction instead of trying to avoid proteolysis. In my hands, when proteolysis occurs, is very difficult to avoid.
I usually add Proteases inhibitors before sonication step and after desalting step, work at cold condition either in cold room (4C) or put stuff in ice box. Working in such conditions will minimize protolysis. Please remember that cell thawing is one method of cell breakage thus protease release is expected. Hope this helps.
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