I am purifying a GST tagged protein. However, upon cleavage of GST tag the protein tends to form higher order oligomers or aggregates and cleaved protein comes in void volume during size exclusion chromatography. how can I prevent the higher order oligomer formation???? I have tried Triton-x, glycerol, arginine but the protein still comes in void volume.
It is preferable to first deposition the enzyme in the form of protein using ammonium sulfate salt, and then passed on the chromatographic, and this attached research may benefit you
Article Partial Separation and Some Kinetic studies of Glutathione S...
Dear Aruna
Is your protein contains hydrofobic regions or many cysteines?
Because sometimes the tag mask intrinsic problem of the proteins before the removal and in this case you need or return to expression changing conditions as induction temperature or E.coli strains or in other cases also re-desing the domain to remove regions that can induce the aggregation.
best regards
Manuele
@Manuele Martinelli, yes the protein has 21 cysteine residues.
Since I need soluble expression of my protein, the culture conditions have been optimized. Also I have tried few strains of BL21 other than DE3.
It is not just the oligomerization post GST tag cleavage, but the soluble expression of protein is less as well.
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