I have a protein that is easy to aggregate when the temperature is lower than 4℃ or the concentration is more than 5 mg/ml. As we know, protein often is stored on ice or 4℃ during experiments.
Except for mutating amino acid sequence, how to protect proteins from aggregation? Does any one know the mechanisms for this kind of protein aggregation?
Thank you in advance.
Hello Honghui Liu
It could be that in a non-ideal buffer, the proteins are likely to unfold, resulting in aggregates of insoluble proteins. If additives are added, 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. Osmolytes like sucrose and detergents exert a stabilizing effect and prevents aggregation. It favors proteins in their native state. If proteins contain cysteine residues you can consider adding reducing agents like beta mercaptoethanol to prevent oxidation and protein aggregation.
Some ionic stabilizers like salts also enhance solubility of proteins. So, try using different salts to achieve protein stability and prevent aggregation.
I hope this may help.
Best.
Hi, Malcolm Nobre . Thanks for your answer.
I used two kinds of buffer. One is composed of 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. Another is composed of 2.7 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4. I will try to add additives like sucrose and trehalose into the buffer mentioned above.
In addition, there are two Cysteines in seq (130 AA). Do I need to mutate this Cysteines to another amino acid?
Thank you again.
Dear Honghui Liu
Are the 2 cysteines involved in the formation of an S-S bonds or are free cysteines?
Did you run the aggregate in reducing and non reducing SDS-page to see if the aggregation is induced by the cysteines?
best regards
Manuele
Hi Manuele Martinelli ,
Thanks for your answer. Actually, there are 4 cysteines involved in the formation of an S-S bonds in the molecular. So, I think the aggregate may not be caused by free cysteines.
Thank you.
I would suggest a buffer screen... systematically work your way through different Goods buffers (TRIS, HEPES, etc.), salts (KCl, NaCl, etc.), additives (gly-gly, osmolytes, BOG, Tween-20) and see which of the buffer combinations give the highest A280 reading. Alternately, you could directly follow a published protocol, (such as dx.doi.org/10.1002/0471140864.ps2809s79) or buy buffer screen or additive screen kits (https://shop.2bind.com/collections/buffer-screens-for-proteins , https://www.moleculardimensions.com/products/additive-screens ) Anecdotal evidence, but my PhD advisor's advisor insisted that all protein purification buffers contain BOG and glycerol and swore by those two in conjunction.
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