I have been using a high pressure homogenizer from Constant Systems to homogenize E. coli cells expressing various proteins. My task has been to investigate why some proteins seem to go from "insoluble" (using SDS-PAGE on BugBuster lysed cells) to "soluble" (SDS-PAGE on centrifuged homogenate) without being properly active (e.g. His-tag does not enable binding to column).
I have found that lowering the pressure to as low as 400 bar seems to lyse the cells and keep more protein in the insoluble fraction (which in some cases is an advantage). However, the effect on the protein varies from protein to protein.
Do you have similar experience or relevant knowledge?
Just a few comments:
1. A hidden tag can result in not binding, this condition does not imply improper folding
2. The pellet must be properly washed before further analysis
3. SDS-PAGE will not be a proper tool to determine if solubilization is occurring
4. The hydrophobic effect is favored by high temperature conditions
5. Having inclusion bodies is never an advantage, refolding is a pain in the ass
Hi Omar
Thank you for your answer.
When using SDS-PAGE in this context I only estimate how much protein ended up in the pellet or the supernatant after centrifugation and the distribution depends on the homogenization pressure. But the proteins that end up in the soluble fraction in this way show different properties than if it was produced in a soluble way in the first place (other bacterial strain or different growth conditions). So maybe the inclusion bodies break up into smaller particles that cannot be settled by centrifugation?
Best,
Lasse
Hello Lassen,
I never experienced that high-pressure disruption led to rupture of inclusion bodies. I am not denying that it may be possible for some proteins, but are you sure that getting higher insoluble protein at 400 bar isn't due to to incomplete lysis ? Also, beware of BugBuster: I don't know its composition, but I experienced that it leads to the spurious precipitation of of some proteins that otherwise would be soluble. Do you perform high-pressure disruption after treating cells with BugBuster ? If so, could it be that the "solubilisation" that you observe may be due to the fragmentation of the BugBuster precipitate ?
Hi Pierre
Thank you for your answer. I do not perform homogenization on BugBuster treated cells, but it is possible that my insoluble proteins are indeed just precipitated and not inclusion bodies. Do you know a way to determine the status of the insoluble protein without fancy equipment like an electron microscope?
I have myself been wondering exactly how I can assure that my cells are properly lysed at low pressure. My solution has been to treat the pellet after homogenization with BugBuster and look for soluble proteins, but I do not find much, so it appears that intracellular soluble proteins are released during homogenization in the pressure range I have used (400 bar to 1500 bar).
Best,
Lasse
Hi Lassen,
We are new to use the high-pressure homogenizer. Wondering, do you have a curve between the pressures used and the yields of IB in your experiences? Thank you very much in advance.
Andrew
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