Renaturing inclusion bodies
scottjc at usa.net
Fri Jul 17 08:28:15 EST 1998
I have also had some problems using Guandine-HCl denaturation and refolding,
my protein was also inactive when finally tested.
I have just tried an alternative technique which uses Sarkosyl to solubilise
the protein followed by dialysis. I have completed checking if the protein
is functional yet but for initial signs it looks ok. The reference for the
protocol is Nguyen etal in Protein Expression and Purification 4:425-433.
Also as a review of inclusion body purification - Rudolph & Lilie in FASEB
Tai Man Louie wrote:
> Does anyone has any experience for renaturing inclusion bodies,
> solublized by 8M Guanidine-HCl? This enzyme has only 1 cysteine residue
> and therefore the native protein should not have any disulfide bond.
> Guanidine-HCl is gradually removed by dialyzing against refolding
> buffers contain decreasing concentrations of urea. The enzyme finally
> stays in solution with 100 mM Tris-HCl (pH 7.8) + 10% glycerol, but most
> of the activities are lost? Comment please.
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