Hi,
I wonder why E.coli cell pellet freezing is recommended before the lysis during protein expression and purification process.
Most of the commercial protein purification protocols recommend it,
and I also have some experience that fluorescent protein-expressing E.coli pellet showed more vivid color when it was in the freezer longer time. (though I didn't quantitatively measure it)
I've tried to find the regarding report but I couldn't.
Does anyone knows the reason or have idea?
Thank you for your opinion in advance.
DEar Bu Gayun
i do not tihnk that cell freezing increase the protein expression level, since once the cells are frozen all the processes, ag protein expression are also frozen. This approach it is generally used as a long term biomass storage.
Is it possible that in some cases. it may improve the efficience of the cell lysis and protein extraction since freeze/thaw is, itelf a possible cell lysis approach.
Regaridng the GFP, i think that is not the protein amount but the fluorescence intensity that may change a little in function of the temperature and therefore the pellet it seems coloured.
best regards
Manuele
Hi there,
As already mentioned by Manuele freezing/thawing cycle is improving the lysis efficiency by destabilizing the cell membrane integrity. So it is improving protein extraction but has nothing to do with protein expression level.
sonication produces a lot of heat. In order to prevent protein fold, it is is subjected to freezing. Has nothing to do with protein expression.
As others have explained, freezing will damage the cell wall and membrane of the bacteria due to ice crystal formation, thereby increasing protein yield. This will also break down cellular organisation, i.e., you need to add protease inhibitors before freezing.
One of the reason is storage. In spare time we collect our pellet and store it so that we can purify our protein immediately when we need it. We need not to wait for expression and extraction. Generally the storage is for 15-30 days.
Second the freezing of pellet leads to it's cell lysis. Slowly your cell while lyse and when you proceed with real cell lysis like sonication, freeze-thaw method, homogenization this will help you in complete lysis of your cells and ultimately improving the yield of your protein.
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