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Proteogenix – Refolding
Inclusion bodies and refolding
Recombinant protein expression in E. Coli has the disadvantage of expressing a significant number of proteins in inclusion bodies or pellets. It is often a defense system of the bacteria that protects itself from overexpressed protein by enclosing them in these inclusion bodies which has the effect of making the protein insoluble.
In such situations, Proteogenix has to use detergents and / or chaotropes like urea to extract and solubilise proteins from these inclusion bodies and / or pellets. The disadvantage here is that these conditions have a distorting effect on the proteins obtained.
Dialysis, chromatography buffer exchange, rapid dilution or on-column refolding
Depending on the application or end use of the protein (structural or functional study example), it is sometimes necessary to remove the denaturant (eg urea) to refold the protein. Again, many options are possible, including dialysis, chromatography buffer exchange, rapid dilution (drip) or on-column refolding. For all these techniques, the use of various additives in the final buffer is also possible to improve protein solubility and prevent precipitation during the process of removing the denaturing agent. It may be, for example detergents (eg SDS), stabilizers (eg glycerol), chaotropes (eg, urea), salts (eg sodium citrate) or chelators (eg EDTA).