Hello everyone,
I am supervising a master thesis student whose goal is to compare different lysis methods for recombinant protein purification. We are working with E. coli pellet and he is interested in doing osmotic shock for cell lysis. I have never done such method as I usually use a homogenizer and lysozyme.
Has anyone ever done this method? How effective is the lysis?
I am open for inputs for the optimized methods that work for you!
The supernatant will be loaded and purified using IMAC.
Thank you in advance!
Best regards,
Lieke Widowati
I haven't tried osmotic shock, but if I were to try it, I would include multiple freeze-thaw steps and include lysozyme and DNase+Mg2+. My preferred method of breaking E. coli is with the French press.
Adam B Shapiro thank you for the suggestion! I have also never tried it or even heard of this method. I will research it further to evaluate if it worth to try since this is for a master thesis to evaluate the effectivity of different lysis methods. So now we used High Pressure Homogenizer and osmotic shock.
Since the peptidoglycan layer of E. coli serves to protect the cells from osmotic lysis, it would probably be helpful to treat the cells with a muramidase enzyme to break up the peptidoglycan. However, the cell wall is inaccessible because it is inside the outer membrane, so you would also have to destabilize the outer membrane. This may possibly be achieved by including EDTA, since the outer membrane is stabilized by Mg2+ ions. Once the cells are broken, the nucleic acids will be released, which will result in an increase in viscosity. The nucleic acids can be degraded by adding RNase and DNA, plus Mg2+ in excess of the EDTA concentration (DNase is Mg2+-dependent).
Dear Lieke Widowati
to my knowledge the osmotic shock is generally used as gentle lysis approach to extract the periplasmic protein when you are expressing you recombiant protein directly in the periplasm thanks to the addition of pelB or ompA leader peptide leader sequence in front of your GOI sequence, but i do not think that it could be used as alternative to sonication, french press or chemical/enzimatic lysis (e.g B-per, Celllytic) for protein purification from cytoplasm,
best
Manuele
Manuele Martinelli do you think it is effective enough for lysis of inclusion bodies as well? As I also am not sure about this method. I always do a combinational of physical lysis and using lysozyme.
Dear Lieke Widowati
in general inclusion bodies are strong citoplasmic bodies difficult to be resuspended. I think that osmotic shock could be a sort of pre-washing of inclusion bodies to wash away at least a part of soluble protein and extract from inclusion bodies a more pure unfolded protein, but i think that for protein extraction from IB you have to use denaturing reagents as urea or guanidinium those are not compatible with osmotic shock
best
Manuele
Dear Manuele Martinelli thank you for the explanation! I will research it more to find more info.
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