I work with a protein that is normally well behaved, but I've recently developed the following intermittent issue: following cell lysis by sonication -- and multiple rounds of centrifugation at 16,000 x g and passing the supernatant through a 0.22-μm syringe filter -- the lysate still becomes cloudy after ~15 minutes and aggregates. This has led to debris ruining one of our affinity columns.
Why does this happen (intermittently) and what are the remedies?
Thank you.
Well, in such cases I am using 8M urea in the lysis buffer and do the SDS-PAGE to see if the protein of interest is present. Do you want to isolate protein from E.coli? 8M Urea can be used for His-tag purification and the protein can be easily refolded. Alternatively you can also try to add some detergents - like SDS, Triton-X100 or Sarkosyl. Give me some more details, what tag, what is the protein origin and which expression system are you using.. :-)
Hello William Ford
The proteins in the cells are subjected to differences in pH and ionic strength when the cells are lysed. They need to be protected during the time of cell lysis. If lysis is done in a non-ideal buffer, the proteins are likely to unfold, resulting in aggregates of insoluble proteins. If additives are added during cell lysis, they can either stabilize the proteins from partially unfolding, preventing protein-protein interaction or they can aid as chemical chaperones, leading to a properly folded and non-aggregated state.
Addition of additives can serve two purposes,
1) increase protein solubility either prevention of aggregation via stabilization or 2) assistance to the stable folded state.
So, buffer supplemented with small molecule additives like osmolytes may help overcome this problem of protein aggregation during cell lysis and protein purification.
Osmolytes like sucrose and detergents exert a stabilizing effect and prevents aggregation. It favors proteins in their native state. If your protein of interest contains cysteine residues you can consider adding reducing agents like beta mercapto ethanol to prevent oxidation and protein aggregation.
Some ionic stabilizers like salts enhance solubility of proteins. So, try using different salts to achieve protein stability and prevent aggregation.
Also, the pH of the solution is important. When the pH of the buffer is equal to the pI of protein, the protein has no net charge. So, adjusting the pH below or above the protein pI induces a positive charge when pHpI. This will help modulate the interactions that cause aggregation.
Hope this information will help.
Good Luck.
Petr Rathner & Malcolm Nobre I am purifying a His-tagged protein expressed in BL21(DE3) cells and grown in M9 minimal media. The lysis buffer contains 20 mM phosphate, 500 mM NaCl, 20 mM imidazole (pH 7.4), which is sufficiently far from the pI. I'm avoiding disulfide reducers because I'm labelling native cysteine residues with a fluorine probe. (Reducers inhibit the labelling). I've used this buffer for years with no issues. This is why I'm so puzzled.
William Ford try to add the 8M Urea and refold on the column by slow gradient using the lysis buffer. I am doing this in the Äkta system and it works fine :-).
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