For in vitro protein refolding, the addition of the reduced and oxidized glutathione (-GSH- and -GSSG-) is useful to facillitate the disulfide bond formation of the protein. However, some established protocols have no addition of the glutathione but it has shown successful refolding of the protein with retained biological activity. Before refolding, the disulfide bonds would have been destroyed during protein solubilization by strong denaturants (e.g urea). In this case, may I know how does the refolded protein still maintain its stability and activity without glutathione redox reaction? Thank you.
During inclusion body formation unspecific/uncorrect disulfide-bonds are formed (intermolecular). To completely unfold the inclusion body and allow for correct formation of intramolecular disulfides, a reducing agent is imperative.
My understanding is that disulfide formation in vitro in the absence of glutathione can be catalized by atmospheric oxygen; that's why there are so many protocols where Cys-containing proteins successfully undergo oxidative refolding in that condition. GSSG/GSH is supposed to simply accelerate/facilitate the process.
By the way, chaotropes (such as urea) do not break existing disulfide bonds. You need reducing agents (BMe, DTT, etc.) for that.
Hi Alejandro,
What about formation of a disulphide bond in an environment of already present chaotropes ( such as 8M urea)?
Hi Alejandro Martin !
In my study, I solubilize my protein with 8M of urea, this concentration breaks the disulfide bonds?
I did the refolding of my protein with reduced glutathione. Do I need to use oxidized glutamine together?
Because, I have read in the articles the use of the two glutathione (GSH and GSSG). However, the refolding method I used, only with reduced glutathione showed biological activity for my enzyme.
Camila Pansa, no, urea will not break already formed disulfide bonds (nor will it prevent the formation of new disulfides, to answer Devanshu Mehta's question -although I would expect the formation of a larger than usual proportion of scrambled bonds, with the protein being denatured and all. I'm sure somewhere buried in some obscure paper is hard data to prove/disprove my guess).
As to whether you need the GSSH/GSSG pair, my personal experience is that it is not an absolute requirement (as your own experiment proves), but the yields of refolded protein may increase (sometimes dramatically) when it is included, depending on the specific protein. And then there's the issue that oxidized glutathione is surprisingly expensive, to the point that even in a lab setting you may need to balance efficiency and cost...
Hope it helps!